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Abstract

Substance P, a neuropeptide associated with pain and inflammation, has been implicated in migraine pathophysiology through its action within the trigeminovascular system. This narrative review summarizes current evidence on the synthesis, release, receptor binding and downstream effects of substance P. It integrates preclinical and clinical findings to reassess its therapeutic relevance. Substance P is released from primary afferent neurons and acts on neurokinin-1 receptors, which are widely expressed in both peripheral tissues and central pain-processing regions. In the meninges, substance P contributes to vasodilation, plasma protein extravasation, mast cell degranulation and immune cell recruitment, all of which facilitate neurogenic inflammation and possibly lower the activation threshold of meningeal nociceptors. In the central nervous system, substance P promotes excitatory neurotransmission, potentiates glutaminergic activity and attenuates inhibitory GABAergic signaling, cumulatively amplifying pain transmission. Preclinical studies consistently demonstrate that neurokinin-1 receptors antagonists inhibit substance P-induced responses, such as neurogenic inflammation and neuronal activation, supporting their therapeutic potential. However, randomized controlled trials with neurokinin-1 receptors antagonists were not superior to placebo in treating migraine. A re-appraisal of these trials reveal that the disappointing results might be due to methodologic shortcomings, including underpowered samples and suboptimal efficacy endpoints. A recent randomized, double-blind, placebo-controlled, two-way crossover study showed that intravenous infusion of substance P is potent inducer of headache and arterial dilation in healthy adults. These findings align with the established biological functions of substance P and warrant renewed therapeutic interest. Advances in translational research, particularly those emphasizing direct measurement of meningeal nociceptor activity and refined clinical trial design, might overcome past limitations and clarify the role of substance P in migraine. The dismissal of substance P signaling as a therapeutic target might thus have been premature. Renewed efforts might uncover novel therapeutic strategies, offering hope for patients with migraine who remain unresponsive to existing treatment.

Graphical abstract

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Keywords

drug target headache migraine neurokinin-1 receptor substance P

Introduction

Migraine is a disabling neurological disorder (1,2), characterized by recurrent attacks of moderate-to-severe headache and accompanying photophobia, phonophobia, nausea and vomiting (3). Fundamental to migraine pathogenesis is the activation of the trigeminovascular system (TVS), which comprises the trigeminal nerve and its axonal projections that innervate the meninges and its blood vessels (4,5). Activation of this system leads to the release of various neuropeptides from sensory nerve fibers, including but not limited to substance P and calcitonin gene-related peptide (CGRP) (6,7).

Substance P is an 11-amino-acid neuropeptide of the tachykinin family that has been extensively studied for its role in pain transmission and neurogenic inflammation (8,9). Early preclinical experiments implicated substance P in migraine mechanisms, suggesting that it contributes to vasodilation, plasma protein extravasation (PPE), mast cell degranulation and immune cell recruitment within the meninges (10). These insights led to the development of neurokinin-1 receptor (NK1R) antagonists as potential therapeutic agents in migraine (11). In addition to its role in pain transmission, substance P has also been implicated in mood regulation, likely due to its expression in limbic brain regions such as the amygdala and hippocampus (12 –14). These regions are associated with the affective-emotional components of pain and psychiatric comorbidities, including anxiety and depression (15,16), which are common in people with migraine (17,18).

Despite these mechanistic insights, the translation into effective therapies has proven challenging. A considerable proportion of individuals with migraine continue to experience inadequate relief from existing treatments, underscoring a persistent unmet need (19). The failure of clinical trials involving NK1-R antagonists, despite promising preclinical data (20 –28), has contributed to a shift in focus toward other neuropeptide systems (29). However, this abandonment might have been premature, as trial design limitations and evolving understanding of migraine pathogenesis warrant renewed investigation of substance P signaling.

Randomized controlled trials of NK1R antagonists, dapitant, lanepitant, GR205171 and L-758,298, did not prove superior to placebo in treating migraine (30 –34). This disconnect between robust preclinical findings and negative clinical trials illustrates the challenges in translating molecular insights into effective migraine treatments. Importantly, recent human experimental data might shift this perspective. A randomized, double-blind, placebo-controlled, two-way crossover study demonstrated that intravenous infusion of substance P reliably induced headache and arterial dilation in healthy adults (35). Notably, this represents the first study to investigate the role of substance P in headache generation in humans directly because previous studies with substance P had only reported headache as a side effect rather than examining it as a primary outcome (36 –38). These findings provide direct evidence of the ability of substance P to trigger headache in humans and align with its known pro-nociceptive and vasodilatory properties.

Together, this emerging evidence supports a renewed appraisal of substance P. It appears to be a mechanistically and clinically relevant drug target in migraine. This narrative review summarizes current evidence on the synthesis, release, receptor binding and downstream effects of substance P. It integrates preclinical and clinical findings to reassess its therapeutic relevance.

Methods

We conducted a literature search using the MEDLINE database (via PubMed) for articles published up until April 1, 2025. The search terms included combinations of: “Substance P”, “Neurokinin-1 Receptor”, “Migraine”, “Trigeminovascular System” and “Neurogenic Inflammation”. The reference lists of selected articles were also manually searched for additional relevant studies. No language restrictions were imposed during the literature search.

Substance P in the trigeminovascular system

The TVS is widely accepted as the anatomical and physiological substate from which nociceptive messaging originates and ultimately yields the perception of migraine headache (4). In migraine, nociceptive signals are posited to arise from pain-sensitive intracranial structures, such as the meninges and its blood vessels (39,40). These signals are detected by the peripheral processes of first-order sensory neurons, which include thinly myelinated Aδ fibers and unmyelinated C fibers (41). The cell bodies of these pseudounipolar neurons reside in the trigeminal ganglion (TG) (42).

The central axonal processes of these neurons enter the brain stem at the level of the pons and descend ipsilaterally within the spinal tract of the trigeminal nerve (43). They terminate in the spinal trigeminal nucleus, particularly its caudal subdivision known as the trigeminal nucleus caudalis (TNC) (44). The second-order TNC neurons extend into the upper cervical spinal cord segments (C1-C3), where they interface with dorsal horn neurons (45). Together, TNC neurons and upper cervical dorsal horn neurons constitute the trigeminocervical complex (TCC), a functional integrated hub in migraine pathophysiology (46).

From the TCC, second-order neurons decussate and ascend contralaterally as the trigeminothalamic tract, carrying nociceptive messages to higher-order processing centers. These ascending fibers primarily target the thalamus, which serves as the major relay for trigeminal sensory input (47,48). The third-order thalamic neurons project to multiple cortical regions involved in sensory-discriminative and affective-emotional dimensions of pain, including the somatosensory cortices, anterior cingulate cortex and insula (49).

The integrated activity of these pain-processing networks underlies not only the sensory perception of migraine headache, but also its emotional salience. Within this framework, substance P acts at multiple levels to modulate nociceptive messaging, possibly contributing to the initiation, amplification and persistence of migraine attacks.

Synthesis and transport of substance P

Substance P is synthesized by a plethora of cell types, including sensory neurons and immune cells (50). In the TVS, its production is located in the cell bodies of first-order sensory neurons within the TG (51). The synthesis beings with transcription of the preprotachykinin A gene, which encodes the precursor protein preprotachykinin A (52). This precursor undergoes post-translational processing to generate substance P and neurokinin A (53).

Once synthesized, substance P is packaged into large dense-core vesicles, which are then transported via axonal transport mechanisms to both peripheral and central nerve terminals (54). This bidirectional enables the rapid and localized release of substance P in response to neuronal activation. The synthesis, localization and role of substance P in TVS are explained in Figure 1.

Figure 1.

Role of substance P in the trigeminovascular system. Transcription of the TAC1 gene in pseudounipolar sensory neurons of the trigeminal ganglion leads to the production of preprotachykinin A, which is post-translationally processed into substance P. Once synthesized, substance P is stored in dense-core vesicles and transported to both peripheral and central nerve terminals. Upon release from peripheral terminals, it induces vasodilation, plasma protein extravasation and immune cell recruitment. These responses are considered to activate perivascular afferents of first-order trigeminal nociceptive neurons, which then relay signals to second-order sensory neurons in the brainstem.

Release mechanisms in the trigeminovascular system

The release of substance P within the TVS can be initiated by a range of noxious stimuli, including mechanical stress, chemical irritants, thermal inputs and inflammatory mediators (55,56). These triggers depolarize sensory neurons, leading to the opening of voltage-gated calcium channels and a subsequent influx of calcium ions (57 –59). Elevated intracellular calcium concentrations serve as a key signal for exocytosis, prompting the fusion of substance P-containing dense-core vesicles with the neuronal membrane and the release of substance P into the extracellular space (60).

Importantly, substance P is not released in isolation. It acts in concert with CGRP (61), with which it is co-localized in the presynaptic terminals of primary afferent neurons (62,63). Immunohistochemical studies have indeed demonstrated this co-expression (64), suggesting a synergistic relationship wherein both substance P and CGRP contribute to migraine mechanisms.

The release of substance P is regulated by multiple modulating factors. These include certain ion channels, receptor-mediated signaling pathways and interactions with other neurotransmitters that fine-tune both the timing and extent of substance P release (55,65).

Binding to neurokinin-1 receptors

Substance P binds to the G-protein coupled receptors of the neurokinin receptor family, including neurokinin 1 (NK1), neurokinin 2 (NK2) and neurokinin 3 (NK3) receptors (66). Among these, substance P exhibits the highest affinity for the NK1 receptor (NK1R) (52), which constitutes its primary functional target. NK1R is widely expressed across both the central and peripheral nervous systems, as well as in various non-neuronal cells such as endothelial cells and vascular smooth muscle cells (VSMCs) (67,68). Within the TVS, NK1R expression has been found in meningeal arteries, the TG and TCC, underscoring its potential relevance to migraine mechanisms.

Upon binding, NK1R undergoes conformational activation and initiates intracellular signaling cascades, most notably through the activation of phospholipase C (69). This activation leads to the generation of second messengers, inositol triphosphate and diacylglycerol, which in turn promote intracellular calcium release and protein kinase C activation. These signaling events mediate a range of downstream effects, including vasodilation, PPE, neurogenic inflammation, and modulation of neuronal excitability (70).

Although substance P preferentially targets NK1R due to its highest binding affinity, it can also bind to NK2 and NK3 receptors with considerably lower affinity (71). NK2Rs and NK3Rs are primarily activated by neurokinin A and neurokinin B, respectively, and their contribution to migraine pathophysiology remains poorly understood (70). However, it warrants mention that these receptors are also expressed in TG neurons and might therefore play a role in pain and inflammation (67).

Emerging evidence has refined the traditional view of NK1R as a plasma membrane-restricted receptor (72). Following ligand binding and activation by substance P, NK1R can undergo agonist-induced internalization into endosomal compartments (73). This endosomal signaling might help sustain or alter intracellular pathways under certain conditions, potentially extending the effects of substance P beyond the initial cell surface receptor interaction. In migraine, such prolonged signaling could theoretically play a role in maintaining pain during attacks.

Regulation of the activity of substance P

The activity of substance P is regulated by several factors (74,75). One primary mechanism of regulation involves enzymatic degradation by widely expressed cell surface peptidases, including neutral endopeptidase and angiotensin-converting enzyme (74). These enzymes cleave substance P into non-functional fragments (76), thereby limiting the duration of the action of substance P.

Beyond enzymatic degradation, receptor-level desensitization also plays a critical role. Prolonged exposure to substance P activates G protein-coupled receptor kinases, which in turn facilitate the recruitment of β-arrestins to the NK1R (75). This process involves receptor internalization, effectively removing NK1R from the cell surface and attenuating further responsiveness to extracellular substance P. Importantly, internalized receptors can continue to signal from endosomal compartments or be targeted for degradation via β-arrestin-mediated pathways, contributing to both sustained intracellular signaling and signal termination. Moreover, genetic and epigenetic factors can influence the expression levels of substance P and its NK1R (77), further shaping individual variability.

Peripheral actions of substance P

Cephalic vasodilation

One of the principal peripheral actions of substance P is the induction of dilation in cerebral and dural blood vessels (78). This process begins with NK1R activation on endothelial cells, which stimulate endothelial nitric oxide synthase and leads to the generation of nitric oxide (NO), a potent vasodilator (79 –82). NO then diffuses into adjacent VSMCs, where it activates soluble guanylate cyclase (83). This enzyme catalyzes the conversion of guanosine triphosphate into cyclic guanosine monophosphate, a second messenger that activates protein kinase G (PKG) (Figure 2(a)) (84).

Figure 2.

Hypothesized mechanisms of substance P action on meningeal nociceptors. (a) Substance P promotes cephalic vasodilation by activating neurokinin 1 (NK1) receptors on endothelial cells, stimulating nitric oxide (NO) production via endothelial nitric oxide synthase (NOS). NO diffuses to vascular smooth muscle cells, where it activates soluble guanylate cyclase (GC), elevating cyclic GMP (cGMP) levels and subsequently activating protein kinase G (PKG). This cascade triggers potassium efflux through ATPsensitive potassium (KATP) channels and large conductance calcium-activated potassium (BKCa) channels, leading to membrane hyperpolarization, reduced calcium influx, and smooth muscle relaxation. (b) Substance P increases vascular permeability by disrupting endothelial tight junctions, resulting in plasma protein extravasation (PPE), perivascular edema and neurogenic inflammation. It also enhances chemotaxis and stimulates the release of pro-inflammatory cytokines from macrophages, neutrophils and lymphocytes, contributing to immune activation and sensitization of meningeal nociceptors. (c) Activation of mast cells via Mas-related G-protein coupled receptor member X2 (MRGPRX2) receptors by substance P leads to the release of histamine, serotonin, and prostaglandins. These mediators promote vasodilation, PPE, and immune-driven sensitization, processes implicated in migraine pathophysiology. (d) Substance P could also directly modulate neuronal excitability by facilitating ion flux through P2X3 and transient receptor potential vanilloid 1 (TRPV1) channels in trigeminal neurons, potentially enhancing nociceptor responsiveness. Although these effects are mainly observed at supraphysiological concentrations, they suggest a possible direct mechanism of nociceptor activation.

PKG exerts its vasorelaxant effects by phosphorylating multiple downstream targets, including ATP-sensitive potassium (K_ATP) channels and large-conductance calcium-activated potassium (BK_Ca) channels (85). Phosphorylation of these channels facilitate their opening, allowing potassium ions to exit the cell, which hyperpolarizes the VSMC membrane and reduces the probability of voltage-gated calcium channel opening (86). As a result, intracellular calcium levels decrease, leading to diminished actin-myosin interactions and VSMC relaxation, thereby promoting vasodilation (87).

In the context of migraine, arterial dilation is hypothesized to activate perivascular meningeal nociceptors through both mechanical and chemical stimuli (4,88). The expansion of vessel diameter might stretch mechanosensitive nociceptors, potentially involving Piezo channels (89), whereas local increases in extracellular potassium might further depolarize neurons, lowering their activation threshold. Together, these processes might initiate or exacerbate migraine headache. Notably, NO donors, as well as pharmacological openers of K_ATP channels and BK_Ca channels, have been shown to induce migraine attacks in people with migraine but not in healthy adults (90,91). These human experimental findings underscore the pathophysiological relevance of this signaling cascade.

Increase in vascular permeability and plasma protein extravasation

In addition to promoting vasodilation, substance P increases vascular permeability by inducing cytoskeletal contraction and disrupting endothelial tight junctions (Figure 2(b)) (92). This leads to the formation of intercellular gaps, allowing plasma proteins and pro-inflammatory mediators to extravasate into the perivascular space (93). The resultant tissue edema and activation of complementary pathways contribute to neurogenic inflammation within the meninges (94). This inflammatory environment sensitizes nearby meningeal nociceptors (95), lowering their activation threshold and perpetuating cephalic pain. In this way, PPE serves as an important interface between vascular responses and sustained nociceptive messaging characteristics of migraine.

Activation of mast cells and immune responses

Substance P also plays a central role in immune modulation by directly activating mast cells via Mas-related G-protein coupled receptor member X2 (MRGPRX2) expressed on their surface (Figure 2(c)) (96). This activation results in the release of various pro-inflammatory mediators, including histamine, tryptase, prostaglandins, cytokines and chemokine (97). Among these, histamine is particularly relevant, as it promotes vasodilation and PPE (98), augmenting the actions initiated by substance P (99). In addition, histamine and certain prostaglandins are well-established molecular migraine triggers in humans with migraine (100,101), underscoring the likelihood of mast cell involvement in migraine pathogenesis.

Modulation of other immune cells

Beyond mast cells, substance P modulates the activity of various other immune cell types, including macrophages, neutrophils and lymphocytes (Figure 2(b)) (102). It promotes chemotaxis, enhancing the production of pro-inflammatory cytokines, and modulates immune cell function (103,104). This widespread impact on the immune system is thought to contribute to the maintenance of inflammation and sensitization within the TVS, perpetuating migraine attacks (47).

Direct activation of meningeal nociceptors

In addition to its well-established vascular and pro-inflammatory effects on non-neuronal cells, substance P might also exert direct excitatory actions on meningeal nociceptors (Figure 2(d)). Experimental work by Spiegelman et al. (105) demonstrated that application of substance P onto TG slices in vitro elicited neuronal depolarization. In support, preclinical data have shown that substance P, via NK1R-dependent mechanisms, enhances calcium influx through P2X3 receptors in trigeminal neurons (106). However, an important caveat is the concentration of substance P used in these mechanistic studies. The in vitro studies were conducted using micromolar doses, approximately one million times higher than the picomolar concentrations used in human provocation experiments that demonstrated headache induction (35). This disparity raises questions about the physiological relevance of observed effects, such as neuronal depolarization and P2X3 activation (106), under experimental conditions. These supraphysiological doses might elicit non-specific cellular responses that do not accurately reflect endogenous signaling in human migraine. Consequently, although mechanistically informative, such findings must be interpreted with caution when extrapolating to clinical settings.

Emerging evidence indicates that substance P modulates nociceptive signaling through functional interactions with transient receptor potential (TRP) channels, which are key regulators of neurogenic inflammation and pain transduction (107). Substance P appears to sensitize TRPV1 and TRPA1 channels via NK1R-dependent mechanisms (108,109), enhancing their responsiveness to noxious stimuli and promoting peripheral sensitization. TRPV1 channels, non-selective cation channels predominantly expressed in nociceptive neurons (108), are co-localized with substance P and NK1 receptors in trigeminal sensory ganglia (110,111). Furthermore, TRPM8, a cold-sensitive channel also co-expressed with substance P in subsets of trigeminal neurons (112), has been linked to NO donor- and CGRP-induced migraine-like behaviors in rodents (113). These findings support a model in which TRP channels serve as downstream effectors of substance P signaling, amplifying trigeminal excitability and sustaining migraine-associated pain.

Taken together, the direct excitatory effects of substance P on nociceptive neurons might act synergistically with its indirect pro-nociceptive actions mediated vascular and immune mechanisms. This combined influence could activate meningeal nociceptors and trigger nociceptive messaging in ascending trigeminal pain pathways.

Interaction with other signaling molecules

Substance P does not exert its effects in isolation; rather, it operates within a broader network of neuropeptides and signaling molecules that collectively appear to shape migraine pathophysiology (29). Among these, CGRP is of particular importance due to its well-established role in the neurobiological basis of migraine. Substance P and CGRP are co-released from trigeminal sensory neurons (62,63), and both peptides contribute to vasodilation and neurogenic inflammation (61). However, these actions are mediated via distinct receptor systems and intracellular signaling pathways, with substance P acting primarily via NK1R and CGRP through its own G protein-coupled receptors (70,114). The combined effects of substance P and CGRP might therefore amplify vascular and nociceptive responses, particularly in the meninges. Thus, targeting a single mediator might be insufficient to fully disrupt the cascade of events that drive migraine attacks. A better understanding of how these signaling pathways converge is essential for the development of more effective and possibly multi-targeted therapeutic agents.

Central actions of substance P

Facilitating excitatory neurotransmission

NK1Rs are widely expressed within the central pain matrix, including the trigeminal nucleus caudalis, thalamus, cortex and brainstem nuclei involved in descending pain modulation (115 –117). Interestingly, preclinical data have demonstrated that iontophoretic application of substance P increases spontaneous firing in second-order nociceptive neurons (118). This effect has been observed in both feline and rodent models (118,119). In addition to enhancing baseline neuronal excitability, substance P amplifies response to noxious stimuli (120), suggesting a possible role in central sensitization.

Substance P can also modulate glutaminergic signaling by facilitating NMDA receptor function (121,122). It promotes phosphorylation of NMDA receptor subunits, reducing the magnesium block and increasing calcium influx (123). This process amplifies excitatory synaptic transmission and might contribute to changes in gene expression associated with chronic pain.

Moreover, substance P can attenuate inhibitory transmission by reducing GABA release from interneurons (124). The combined effect of increased excitation and reduced inhibition shifts the balance towards hyperexcitable neurons, promoting the transmission of nociceptive messages within the central nervous system (CNS).

Interaction with descending modulatory pain pathways

Substance P also modulates descending pain pathways, which originate in key brainstem regions such as the periaqueductal gray and the rostroventromedial medulla (123). Findings from preclinical experiments indicate that substance P can augment local excitatory inputs and enhance endocannabinoid signaling within these neural circuits, potentially increasing analgesic tone (125). By contrast, direct administration of substance P to the rostroventromedial medulla has been shown to elicit thermal antinociception in rats, suggesting a context-dependent role in pain modulation (126). These insights highlight the bidirectional effects of substance P within CNS structures involved in descending pain modulation, which might depend on both anatomical site and physiological state.

Preclinical neurokinin-1 receptor antagonist testing

Building on the mechanistic evidence described earlier, researchers developed NK1R antagonists as potential migraine treatments (Table 1) (127). Initial preclinical studies in rodents showed that compounds such as RP67580 and GR82334 suppressed PPE in a dose-dependent manner following TG stimulation (20,21). These results supported the involvement of NK1R in neurogenic inflammation.

Table 1.

Summary of preclinical studies evaluating NK1R antagonists in experimental migraine models.

Study (author, year)	NK1R antagonist (dose, route)	Animal species and stock (n)	Experimental method	Measured outcomes	Key effects of NK1 antagonist
Shepheard et al. (1993) (20)	RP67580 (0.1–1000 μg/kg, IV)	Male Sprague–Dawley rats (n = 8–10/group)	TG stimulation; [¹²⁵I]-albumin assay	Plasma protein extravasation (dura, conjunctiva, eyelid, lip)	Reduced plasma protein extravasation in all tested tissues
O'Shaughnessy et al. (1994) (21)	GR82334 (20–200 μg/kg, IV)	Male Sprague–Dawley rats (n = 4–9/group); Male Dunkin-Hartley guinea pigs (n = 6–7/group)	TG stimulation; [¹²⁵I]-albumin assay	Plasma protein extravasation (dura)	Prevented dural plasma protein extravasation in both species
Cutrer et al. (1995) (22)	Dapitant (RPR100893) (1–100 μg/kg, IV)	Male Hartley guinea-pig (n = 6–12/group)	Intracisternal capsaicin injection; Immunohistochemistry	c-Fos expression (TCC)	Reduced capsaicin-induced c-Fos expression
Beattie et al. (1995) (23)	GR203040 (rabbits; 1–100 μg/kg, IV) (rats; 1–10 mg/kg, IV)	Male Albino rabbits (n = 14); male Sprague–Dawley rats (n = 13)	NK1 agonist-mediated carotid vasodilation model; a) in vivo hemodynamic study of carotid TG stimulation; b) [¹²⁵I]-albumin extravasation assay	a) Change in carotid vascular resistance b) Plasma protein extravasation (dura, conjunctiva, eyelid, lip)	Inhibited NK1 agonist-induced vasorelaxation and TG-stimulation induced plasma protein extravasation in all tested tissues
Shepheard et al. (1995) (24)	CP-99,994 (l-3000 μg/kg, IV)	Male Sprague–Dawley rats (n = 8–10/group)	TG stimulation; a) [¹²⁵I]-albumin extravasation assay, b) in situ hybridization	a) Plasma protein extravasation (dura) b) c-Fos mRNA expression (TCC)	Decreased plasma protein extravasation and c-Fos mRNA expression
Phebus et al. (1997) (25)	Lanepitant (LY-303870) (0.001–100 μg/kg, IV or PO)	Male Hartley guinea pigs (Sample size not specified)	TG stimulation; Evans Blue extravasation assay	Plasma protein extravasation (dura)	Inhibited plasma protein extravasation
Polley et al. (1997) (26)	GR205171 (rabbits; 0.3–3 μg/kg, IV) (rats; 0.1–1 mg/kg, IV)(guinea pigs; 1–100 μg/kg, IV)	New Zealand White rabbits (n = 14); male AH/A rats (n = 14); male Dunkin Hartley guinea-pigs (n = 5–6)	NK1 agonist-mediated carotid vasodilation model; a) in vivo hemodynamic study of carotid TG stimulation; b) [¹²⁵I]-albumin extravasation assay, c) Immunohistochemistry	a) Change in carotid vascular resistance b) Plasma protein extravasation (dura, lip), c) c-Fos immunoreactivity (TCC)	Inhibited the NK1 agonist-induced vasodilation, TG stimulation-induced dural plasma protein extravasation, and c-Fos expression
Bolay et al. (2002) (27)	L-733060 (1 mg/kg, IV)	Male Sprague–Dawley rats (n = 6)	Cortical spreading depolarization; Histological staining	Plasma protein extravasation in dura	Suppressed plasma protein extravasation
Ramachandran et al. (2014) (28)	L-733060 (1 mg/kg, 3 min, IV)	Male Sprague–Dawley rats (n = 5–6/group)	Nitroglycerin-induced acute migraine model (4 mg/kg/min, 20 min, IV); a) Immunohistochemistry, b) Western blot	a) c-Fos (TCC) and CGRP (dura) immunoreactivity b) nNOS expression (dura)	Inhibited nitroglycerin-induced c-Fos immunoreactivity, CGRP immunoreactivity and nNOS expression
Pedersen et al. (2015) (129)	L-733060 (1 mg/kg, 3 min, IV)	Male Sprague–Dawley rats (n = 7–9/group)	Nitroglycerin-induced acute migraine model (4 mg/kg/min, 20 min, IV); Histological staining	Mast cell degranulation	Did not inhibit nitroglycerin-induced mast cell degranulation
Akerman et al. (2021) (128)	GR205171 (0.2 mg/kg, IV)	Male Sprague–Dawley rats (n = 9/group)	Nitroglycerin-induced acute migraine model (10 mg/kg, 2–3 min, SC); In vivo electrophysiology	Spontaneous and evoked firing of TCC neurons with meningeal input	Did not inhibit nitroglycerin-induced increase in spontaneous and evoked firing

Abbreviations: PO = per oral; IV = intravenous; TG = trigeminal ganglion; TCC = trigeminocervical complex; CGRP = calcitonin gene-related peptide; nNOS = neuronal nitric oxide synthase; NK1 = neurokinin 1.

Subsequent experiments evaluated GR205171, a CNS-penetrant NK1R antagonist with high affinity for the human receptor (127). In rabbits, GR205171 inhibited substance P-induced vasodilation of the carotid artery (26). In the same study, it reduced dural PPE and attenuated c-Fos expression in second-order trigeminal neurons, comprising an established marker of neuronal activation, in rodents. Despite these promising effects in neurovascular and inflammatory models, Akerman et al. (128) demonstrated that GR205171 did not attenuate NO donor-induced hypersensitivity or spontaneous firing in neurons of the trigeminocervical complex in rats. This finding aligns with rodent data from Pedersen et al. (129), who reported that the NK1R antagonist L-733,060 failed to prevent mast cell degranulation following NO-donor infusion. Together, these studies seem to underscore a critical mechanistic distinction: NK1R signaling precedes NO production and is therefore unlikely to influence pathophysiological processes once NO-mediated cascades have been initiated.

Additional animal studies demonstrated similar effects with other NK1R antagonists. For example, GR203040 reduced vasodilation induced by NK1R agonism and inhibited dural PPE after trigeminal stimulation (23). Likewise, CP-99,994 decreased both PPE and c-Fos mRNA expression in second-order trigeminal neurons (24).

These findings were extended to lanepitant, which binds with high affinity to both human and guinea pig NK1Rs (130,131). In guinea pig models, lanepitant inhibited PPE following TG activation (25). Dapitant, a highly selective antagonist for human NK1Rs, further supported these results (132). It suppressed c-Fos expression in second-order sensory neurons after capsaicin, a TRPV1 agonist, was administered into the cisterna magna (22).

Collectively, these consistent preclinical findings demonstrate that NK1R antagonists inhibit key markers of migraine-related processes, including PPE and c-Fos expression. This robust body of evidence provided a strong rationale for advancing NK1R antagonists into clinical trials as candidate migraine treatments.

Clinical trials of neurokinin-1 receptor antagonists in migraine

NK1R antagonists have been investigated across a range of pain disorders, including osteoarthritis, postoperative pain, fibromyalgia and neuropathic pain (133). Despite promising preclinical data, these agents failed to demonstrate clinical efficacy by the late 1990s. Controlled trials of NK1R antagonists in migraine similarly produced negative or inconclusive results (Table 2). Pharmaceutical agents such as dapitant, lanepitant, GR205171 and L-758,298 were evaluated in randomized, double-blind, placebo-controlled studies, yet none were superior to placebo (30 –34).

Table 2.

Clinical trials evaluating NK1R antagonists for migraine: design, dosing and efficacy outcomes.

Study	Drug	Indication	Design	Sample Size	Dose (Route)	Efficacy Endpoint	Key Findings
Goldstein et al. (1997) (30)	Lanepitant	Abortive	Double-blind, placebo-controlled	40	30, 80, or 240 mg (PO)	Headache relief at 2 hours	No significant difference compared to placebo
Connor et al. (1998) (31)	GR205171	Abortive	Double-blind, placebo-controlled	63	25 mg (IV)	Headache relief at 2 hours	No significant difference compared to placebo
Norman et al. (1998) (32)	L-758,298	Abortive	Double-blind, placebo-controlled	72	20, 40, or 60 mg (IV)	Headache relief at 2 hours	No significant difference compared to placebo
Goldstein et al. (2001) (33)*	Lanepitant	Preventive	Double-blind, placebo-controlled	84	200 mg (PO), daily	≥50% reduction in migraine headache days from baseline to months 1–3	No significant difference over 3 months vs. placebo; significant reduction at Month 3 (40.6% vs. 17.9%)
Diener et al. (2003) (34)	Dapitant	Abortive	Double-blind, placebo-controlled	139	1 5, or 20 mg (IV)	Headache relief at 2 hours	No significant difference

Abbreviations: PO = oral; IV = intravenous.

The study by Goldstein et al. (33) contains an inconsistency in reporting of efficacy outcomes. Although some sections refer specifically to “migraine headache days”, others describe the outcome more broadly as “headache days”. It is unclear whether the analyses were limited to migraine-specific headaches or included all headache types.

Dapitant trial

Dapitant (RPR100893) is a non-peptide NK1R antagonist developed as a potential treatment for acute migraine attacks (22). Diener et al. (34) conducted a randomized, double-blind, placebo-controlled, parallel-group trial to assess the efficacy and tolerability of dapitant in 139 adults with migraine. Participants were randomized to treat a single migraine attack of moderate-to-severe pain intensity with one of three oral dapitant doses (1, 5 or 20 mg) or placebo. Headache intensity was rated using a four-point scale, ranging from 0 (no pain) to 3 (severe pain). The primary efficacy endpoint was headache relief, defined as a reduction in pain intensity from moderate or severe (scores of 2 or 3) to mild or none (scores of 1 or no) at two hours post-dose. However, none of the dapitant doses were superior to placebo. The most common adverse events were asthenia (3–8%) and somnolence (3–10%).

Lanepitant trials

Lanepitant (LY303870) is a non-peptide, competitive NK1R antagonist with high affinity for both the human and guinea pig receptors (25). Based on promising preclinical data, Goldstein et al. (30,33) performed two randomized clinical trials to assess lanepitant's efficacy in both acute and preventive migraine treatment.

The first clinical trial applied a randomized, double-blind, placebo-controlled, crossover design (30). Fifty-three participants with migraine were enrolled and received three oral doses of lanepitant (30, 80 and 240 mg) and placebo. Only 40 participants treated their attacks with all four interventions (i.e. three active doses and the placebo). The primary efficacy outcome was the responder rate, defined as the proportion of participants achieving headache relief at 120 minutes post-dose. However, no lanepitant dose showed superiority over placebo. Adverse events were similar across all interventions.

In a separate 12-week, double-blind, placebo-controlled, parallel-group trial, lanepitant was evaluated for migraine prevention (33). Eighty-four participants were randomized to receive either lanepitant 200 mg daily (n = 42) or placebo (n = 42). The primary efficacy endpoint was the proportion of participants achieving at least a 50% reduction in monthly migraine days from baseline through week 12. Although 41% of participants in the lanepitant group achieved at least a 50% reduction in monthly migraine days, compared to 22% in the placebo group, the difference was not statistically significant. Notably, by the third month, the lanepitant group exhibited a statistically significant reduction in monthly migraine days compared to placebo (40.6% vs. 17.4%).

GR205171 trial

GR205171 is a NK1R antagonist with high affinity for the human receptor. Connor et al. (31) conducted a randomized, double-blind, placebo-controlled trial to evaluate its efficacy in the acute treatment of migraine attacks. Sixty-three participants with migraine received an intravenous dose of GR205171 (25 mg; n = 31) or placebo (n = 32) to treat a single migraine attack. The primary endpoint was headache relief at two hours post-infusion, measured on a four-point scale ranging from 0 (no pain) to 3 (severe pain). GR205171 failed to provide superior headache relief, compared to placebo. The trial did not report any data on treatment-emergent adverse events.

L-758,298 trial

L-758,298 is a CNS-penetrant, selective NK1R antagonist (134). Its potential as an acute migraine treatment was evaluated in a double-blind, placebo-controlled, in-clinic trial involving 72 patients with migraine (32). Patients were randomly assigned to receive an intravenous dose of L-758,298 at 20 mg (n = 4), 40 mg (n = 4) or 60 mg (n = 39), or placebo (n = 25). The primary efficacy endpoint was headache relief at two hours post-dose. The 60-mg dose of L-758,298 showed no statistically significant difference from placebo in achieving this outcome (33% vs. 52%). The reported adverse events were generally mild and transient.

Analysis of clinical trial outcomes

Multiple design-related limitations likely contributed to the negative outcomes observed in clinical trials evaluating NK1R antagonists for migraine. In studies assessing acute treatment, the primary endpoint was typically headache relief at two hours post-dose (30 –32,34), a measure now considered suboptimal relative to the more stringent standard of headache freedom recommended in current international guidelines (135). In addition, trial protocols frequently omitted key methodological details. Sample size calculations were either absent or inadequately justified, with insufficient documentation of expected treatment and placebo response rates. Many studies failed to specify whether missing data were imputed, if an intention-to-treat analysis was implemented, or whether statistical adjustments were applied for multiple comparisons, all of which could have substantially influenced the validity of the reported findings.

The only preventive trial, which assessed lanepitant (33), enrolled participants exclusively with episodic migraine, thereby limiting generalizability to individuals with chronic migraine. Although a statistically significant reduction in monthly migraine frequency was observed at three months, 40.6% with lanepitant compared to 17.4% with placebo, this result did not correspond to the trial's predefined primary endpoint, which was an average ≥50% reduction from baseline to months 1–3. The failure to meet this benchmark likely reflects limited statistical power due to a small sample size, which could have biased the efficacy estimates. Secondary outcomes and responder analyses were also inadequately powered and inconsistently reported, contributing further to interpretive uncertainty.

Taken together, these methodological shortcomings constrain the interpretability of trial outcomes and raise the possibility that observed inefficacy might reflect flawed study design rather than intrinsic pharmacologic failure. A rigorous re-evaluation of NK1R antagonists, using contemporary endpoints, transparent statistical frameworks and a more representative participant population, could therefore be justified.

Translational challenges

The failed NK1R antagonist trials in migraine might suggest limited translational value of commonly used preclinical markers. For example, most animal studies relied solely on dural PPE after TG stimulation as a surrogate for migraine headache (20,21,23,25). Although widely used, dural PPE is only an indirect marker of trigeminovascular activation and does not fully capture complex nature of headache or migraine pain experienced in humans. Inhibiting PPE might not reliably predict clinical efficacy, as evidenced by the discrepancy between preclinical and clinical trial outcomes (136 –139). As an alternative, direct electrophysiological recordings of meningeal nociceptor activity could offer a more precise and translatable metric of trigeminovascular activation.

Likewise, some preclinical studies used c-Fos expression to indicate neuronal activation within the ascending trigeminal pain pathways (22,24). Although c-Fos levels rise with intense neuronal firing (140), their suppression does not consistently correlate with therapeutic benefit in animal models of migraine (141). Moreover, c-Fos expression is neither specific to meningeal afferents, nor to nociceptive neurons.

Moreover, most preclinical migraine studies have relied exclusively on male animals, despite the higher prevalence of migraine in females and evidence for sex-specific differences in nociceptive signaling (142). Incorporating both sexes in experimental designs will improve the generalizability of preclinical findings and better reflect the clinical population. Together, these issues underscore the need for more translatable preclinical approaches, such as electrophysiological recordings of meningeal nociceptor activity, that might better align with human migraine biology.

Future directions

Conclusions about the failure of NK1R antagonists in migraine appear somewhat premature. Most trials used small samples and outdated efficacy endpoints. Modest but inconsistent therapeutic benefits appeared in the lanepitant trial for migraine prevention (33), indicating a potential signal. New controlled trials should therefore consider using up-to-date efficacy metrics: two-hour pain freedom post-dose for acute treatments and ≥50% reduction in monthly migraine days for migraine prevention.

Novel evidence has also renewed interest in substance P signaling and its relationship to headache disorders. Our lab has recently demonstrated that intravenous infusion of substance P induces headache in 15 (71%) of 21 healthy adults compared to two (10%) after placebo. In addition, we found that substance P dilated the superficial temporal artery. Although intriguing, these experimental data warrant confirmation in placebo-controlled studies that enroll people with migraine (35,143).

Preclinical models must likewise evolve. Dural PPE and trigeminal c-Fos expression are indirect measures of cephalic pain and should only be considered complementary. Instead, researchers should record meningeal nociceptor firing, which directly maps trigeminovascular activation and offers a clearer mechanistic bridge to cephalic pain. Rigorous, mechanism-based endpoints at both bench and bedside will give NK1R antagonists a fairer test and might reveal a niche for this drug class in migraine treatment.

Conclusions

Accumulating preclinical and emerging human experimental evidence converges on substance P and NK1R signaling as a pathogenic contributor to trigeminovascular activation in migraine. Negative results from NK1R antagonist trials could reflect methodological shortcomings, including underpowered samples and outdated endpoints, rather than true pharmacological failure. Carefully designed studies are needed to determine whether targeting substance P or its NK1R offers therapeutic promise for migraine.

Article highlights

Substance P has recently been shown to trigger headache and arterial dilation in humans, reinforcing its role in migraine and its potential as a therapeutic target.

Despite supportive preclinical data, NK1R antagonists have failed in clinical trials—likely due to outdated designs, small sample sizes, and suboptimal endpoints.

Future research should adopt modern clinical efficacy endpoints and more translatable preclinical models, such as direct recordings from meningeal nociceptors.

Footnotes

Acknowledgments

Figures were created using BioRender.com.

Author contributions

HA and MA conceptualized the manuscript. BA conducted the article screening and drafted the main text. All authors reviewed and revised the manuscript for intellectual content.

Data availability

Upon reasonable request, the corresponding author will provide the necessary materials to interested researchers for the purpose of academic scrutiny, reproducibility and further scientific investigation.

Declaration of conflicting interests

RHC has received personal fees from Pfizer and Lundbeck, outside of the submitted work. HMA has received personal fees from Lundbeck and Pfizer, outside of the submitted work. MA has received personal fees from AbbVie, Astra Zeneca, Eli Lilly, GlaxoSmithKline, Lundbeck, Novartis, Pfizer and Teva, outside of the submitted work. MA also serves as an Associate Editor of Brain and The Journal of Headache and Pain. HA has received personal fees from AbbVie, Lundbeck, Pfizer and Teva, outside of the submitted work. HA also serves as an Editorial Board Member of The Journal of Headache and Pain. The remaining author (BA) reports no competing interest.

Funding

This manuscript received funding from the Lundbeck Foundation (R403-2022-1352 to HA). Lundbeck Foundation (grant number R403-2022-1352).

ORCID iDs

Berkay Alpay

Haidar M. Al-Khazali

Messoud Ashina

Håkan Ashina

Rune Häckert Christensen

References

Ferrari

Klever

Terwindt

, et al. Migraine pathophysiology: lessons from mouse models and human genetics. Lancet Neurol 2015; 14: 65–80. 2014/12/17.

Global Burden of Disease Collaborative Network. Global Burden of Disease Study 2021 (GBD 2021). Seattle: USIfHMaEI, 2024.

Headache Classification Committee of the International Headache S. The international classification of headache disorders, 3rd edition (beta version). Cephalalgia 2013; 33: 629–808. 2013/06/19.

Ashina

Hansen

, et al. Migraine and the trigeminovascular system-40 years and counting. Lancet Neurol 2019; 18: 795–804. 2019/06/05.

Mayberg

Zervas

Moskowitz

. Trigeminal projections to supratentorial pial and dural blood vessels in cats demonstrated by horseradish peroxidase histochemistry. J Comp Neurol 1984; 223: 46–56. 1984/02/10.

Christensen

Ashina

Amin

. Calcitonin Gene-Related Peptide (CGRP) and Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) in Migraine Pathogenesis. Pharmaceuticals (Basel) 2022; 15: 2022/10/28. DOI: https://doi.org/10.3390/ph15101189

Edvinsson

JCA

Grell

Warfvinge

, et al. Differences in pituitary adenylate cyclase-activating peptide and calcitonin gene-related peptide release in the trigeminovascular system. Cephalalgia 2020; 40: 1296–1309. 2020/06/04.

Furst

. Transmitters involved in antinociception in the spinal cord. Brain Res Bull 1999; 48: 129–141. 1999/05/07.

O'Connor

O'Connell

O'Brien

, et al. The role of substance P in inflammatory disease. J Cell Physiol 2004; 201: 167–180. 2004/08/31.

10.

Silberstein

Lipton

Ramadan

. From migraine mechanisms to innovative therapeutic drugs. Neurology 2005; 64: S1–S3.

11.

Beattie

Connor

Hagan

. Recent developments in tachykinin NK1 receptor antagonists: prospects for the treatment of migraine headache. Can J Physiol Pharmacol 1995; 73: 871–877. 1995/07/01.

12.

Dasgupta

Baby

Krishna

, et al. Substance P induces plasticity and synaptic tagging/capture in rat hippocampal area CA2. Proc Natl Acad Sci U S A 2017; 114: E8741–E8749. 2017/10/05.

13.

Hokfelt

Vincent

Dalsgaard

, et al. Distribution of substance P in brain and periphery and its possible role as a co-transmitter. Ciba Found Symp 1982: 84–106. 1982/01/01. DOI: https://doi.org/10.1002/9780470720738.ch6

14.

Jones

Olpe

. On the role of the baseline firing rate in determining the responsiveness of cingulate cortical neurons to iontophoretically applied substance P and acetylcholine. J Pharm Pharmacol 1984; 36: 623–625. 1984/09/01.

15.

Thomas

Drevets

Dahl

, et al. Amygdala response to fearful faces in anxious and depressed children. Arch Gen Psychiatry 2001; 58: 1057–1063. 2001/12/26.

16.

Soetanto

Wilson

Talbot

, et al. Association of anxiety and depression with microtubule-associated protein 2- and synaptopodin-immunolabeled dendrite and spine densities in hippocampal CA3 of older humans. Arch Gen Psychiatry 2010; 67: 448–457. 2010/05/05.

17.

El Malky

Moslem Hefny Mahmoud

. Anxiety and depression in migraine patients and associated risk factors: a cross - sectional study. International Journal of Science and Research (IJSR) 2019; 8: 1986–1988.

18.

Karimi

Wijeratne

Crewther

, et al. The migraine-anxiety comorbidity among migraineurs: a systematic review. Front Neurol 2020; 11: 613372. 2021/02/05.

19.

Bennett

Reed

Buse

, et al. Predictors of Inadequate Response to Medication in Episodic Migraine: Results from American Migraine Prevalence and Prevention Study (AMPP) (P1.154). Neurology 2016; 16 Supplement. DOI: https://doi.org/10.1212/WNL.86.16_supplement.P1.154.

20.

Shepheard

Williamson

Hill

, et al. The non-peptide neurokinin1 receptor antagonist, RP 67580, blocks neurogenic plasma extravasation in the dura mater of rats. Br J Pharmacol 1993; 108: 11–12. 1993/01/01.

21.

O'Shaughnessy

Connor

. Investigation of the role of tachykinin NK1, NK2 receptors and CGRP receptors in neurogenic plasma protein extravasation in dura mater. Eur J Pharmacol 1994; 263: 193–198. 1994/09/22.

22.

Cutrer

Moussaoui

Garret

, et al. The non-peptide neurokinin-1 antagonist, RPR 100893, decreases c-fos expression in trigeminal nucleus caudalis following noxious chemical meningeal stimulation. Neuroscience 1995; 64: 741–750. 1995/02/01.

23.

Beattie

Beresford

Connor

, et al. The pharmacology of GR203040, a novel, potent and selective non-peptide tachykinin NK1 receptor antagonist. Br J Pharmacol 1995; 116: 3149–3157. 1995/12/01.

24.

Shepheard

Williamson

Williams

, et al. Comparison of the effects of sumatriptan and the NK1 antagonist CP-99,994 on plasma extravasation in Dura mater and c-fos mRNA expression in trigeminal nucleus caudalis of rats. Neuropharmacology 1995; 34: 255–261. 1995/03/01.

25.

Phebus

Johnson

Stengel

, et al. The non-peptide NK-1 receptor antagonist LY303870 inhibits neurogenic dural inflammation in Guinea pigs. Life Sci 1997; 60: 1553–1561. 1997/01/01.

26.

Polley

Gaskin

Perren

, et al. The activity of GR205171, a potent non-peptide tachykinin NK1 receptor antagonist, in the trigeminovascular system. Regul Pept 1997; 68: 23–29. 1997/01/15.

27.

Bolay

Reuter

Dunn

, et al. Intrinsic brain activity triggers trigeminal meningeal afferents in a migraine model. Nat Med 2002; 8: 136–142. 2002/02/01.

28.

Ramachandran

Bhatt

Ploug

, et al. Nitric oxide synthase, calcitonin gene-related peptide and NK-1 receptor mechanisms are involved in GTN-induced neuronal activation. Cephalalgia 2014; 34: 136–147. 2013/09/04.

29.

Tajti

Szok

Majlath

, et al. Migraine and neuropeptides. Neuropeptides 2015; 52: 19–30. 2015/06/22.

30.

Goldstein

Wang

Saper

, et al. Ineffectiveness of neurokinin-1 antagonist in acute migraine: a crossover study. Cephalalgia 1997; 17: 785–790. 1997/12/17.

31.

Connor

Bertin

Gillies

, et al. Clinical evaluation of a novel, potent, CNS penetrating NK1 receptor antagonist in the acute treatment of migraine. Cephalalgia 1998; 18: 37–40.

32.

Norman

Panebianco

Block

. A placebo-controlled, in-clinic study to explore the preliminary safety and efficacy of intravenous L-758,298 (a prodrug of the NK1 receptor antagonist L-754,030) in the acute treatment of migraine. Cephalalgia 1998; 18: 1092–1093.

33.

Goldstein

Offen

Klein

, et al. Lanepitant, an NK-1 antagonist, in migraine prevention. Cephalalgia 2001; 21: 102–106. 2001/06/26.

34.

Diener

and Group RPRS. RPR100893, A substance-P antagonist, is not effective in the treatment of migraine attacks. Cephalalgia 2003; 23: 183–185. 2003/03/29.

35.

Al-Khazali

Christensen

Gozalov

, et al. Effects of substance P on headache induction and arterial dilation in healthy adults. Cephalalgia 2025; 45: 3331024251336132. 2025/05/15.

36.

Duner

Pernow

. Circulatory studies on substance P in man. Acta Physiol Scand 1960; 49: 261–266. 1960/07/15.

37.

Liljedahl

Mattsson

Pernow

. The effect of substance P on intestinal motility in man. Scand J Clin Lab Invest 1958; 10: 16–25. 1958/01/01.

38.

Loefstroem

Pernow

Wahren

. Vasodilating action of substance P in the human forearm. Acta Physiol Scand 1965; 63: 311–324. 1965/03/01.

39.

PENFIELD

McNAUGHTON

. DURAL HEADACHE AND INNERVATION OF THE DURA MATER. Archives of Neurology & Psychiatry 1940; 44: 43–75.

40.

May

Goadsby

. The trigeminovascular system in humans: pathophysiologic implications for primary headache syndromes of the neural influences on the cerebral circulation. J Cereb Blood Flow Metab 1999; 19: 115–127. 1999/02/23.

41.

Levy

Strassman

. Mechanical response properties of A and C primary afferent neurons innervating the rat intracranial dura. J Neurophysiol 2002; 88: 3021–3031. 2002/12/06.

42.

Messlinger

Russo

. Current understanding of trigeminal ganglion structure and function in headache. Cephalalgia 2019; 39: 1661–1674. 2018/07/11.

43.

Lazarov

. The neurochemical anatomy of trigeminal primary afferent neurons. In: Contreras

(ed) Neuroscience - Dealing With Frontiers. Rijeka: IntechOpen, 2012, pp.167–194.

44.

Liu

Broman

Edvinsson

. Central projections of sensory innervation of the rat superior sagittal sinus. Neuroscience 2004; 129: 431–437.

45.

Fernandes

Carlos-Ferreira

Luz

, et al. Processing of trigeminocervical nociceptive afferent input by neuronal circuity in the upper cervical lamina I. Pain 2022; 163: 362–375. 2021/05/15.

46.

Goadsby

Bartsch

. On the functional neuroanatomy of neck pain. Cephalalgia 2008; 28: 1–7. 2008/06/14.

47.

Noseda

Burstein

. Migraine pathophysiology: anatomy of the trigeminovascular pathway and associated neurological symptoms, cortical spreading depression, sensitization, and modulation of pain. Pain 2013; 154: S44–S53. 2013/07/31.

48.

Noseda

Jakubowski

Kainz

, et al. Cortical projections of functionally identified thalamic trigeminovascular neurons: implications for migraine headache and its associated symptoms. J Neurosci 2011; 31: 14204–14217. 2011/10/07.

49.

Benarroch

. Central Processing and Modulation of Pain. In: 2021.

50.

Suvas

. Role of substance P neuropeptide in inflammation, wound healing, and tissue homeostasis. J Immunol 2017; 199: 1543–1552. 2017/08/23.

51.

Moskowitz

. The neurobiology of vascular head pain. Ann Neurol 1984; 16: 157–168.

52.

Hokfelt

Pernow

Wahren

. Substance P: a pioneer amongst neuropeptides. J Intern Med 2001; 249: 27–40. 2001/02/13.

53.

Carter

Krause

. Structure, expression, and some regulatory mechanisms of the rat preprotachykinin gene encoding substance P, neurokinin A, neuropeptide K, and neuropeptide gamma. J Neurosci 1990; 10: 2203–2214. 1990/07/01.

54.

Bisby

Keen

. Axonal transport of substance P-like immunoreactivity in regenerating rat sciatic nerve. Brain Res 1985; 361: 396–399. 1985/12/30.

55.

Chang

Jiang

Chen

. Ion Channels Involved in Substance P-Mediated Nociception and Antinociception. Int J Mol Sci 2019; 20: 2019/04/03. DOI: https://doi.org/10.3390/ijms20071596

56.

White

. Mechanism of prostaglandin E2-induced substance P release from cultured sensory neurons. Neuroscience 1996; 70: 561–565. 1996/01/01.

57.

Zhai

Liskova

Kubatka

, et al. Calcium Entry through TRPV1: A Potential Target for the Regulation of Proliferation and Apoptosis in Cancerous and Healthy Cells. Int J Mol Sci 2020; 21: 2020/06/18. DOI: https://doi.org/10.3390/ijms21114177

58.

Linhart

Obreja

Kress

. The inflammatory mediators serotonin, prostaglandin E2 and bradykinin evoke calcium influx in rat sensory neurons. Neuroscience 2003; 118: 69–74. 2003/04/05.

59.

Bouskila

Bostock

. Modulation of voltage-activated calcium currents by mechanical stimulation in rat sensory neurons. J Neurophysiol 1998; 80: 1647–1652. 1998/10/17.

60.

Huang

Neher

. Ca(2+)-dependent exocytosis in the somata of dorsal root ganglion neurons. Neuron 1996; 17: 135–145.

61.

Pedersen-Bjergaard

Nielsen

Jensen

, et al. Calcitonin gene-related peptide, neurokinin A and substance P: effects on nociception and neurogenic inflammation in human skin and temporal muscle. Peptides 1991; 12: 333–337. 1991/03/01.

62.

Uddman

Edvinsson

Ekman

, et al. Innervation of the feline cerebral vasculature by nerve fibers containing calcitonin gene-related peptide: trigeminal origin and co-existence with substance P. Neurosci Lett 1985; 62: 131–136.

63.

Price

Flores

. Critical evaluation of the colocalization between calcitonin gene-related peptide, substance P, transient receptor potential vanilloid subfamily type 1 immunoreactivities, and isolectin B4 binding in primary afferent neurons of the rat and mouse. J Pain 2007; 8: 263–272. 2006/11/23.

64.

Lee

Kawai

Shiosaka

, et al. Coexistence of calcitonin gene-related peptide and substance P-like peptide in single cells of the trigeminal ganglion of the rat: immunohistochemical analysis. Brain Res 1985; 330: 194–196. 1985/03/18.

65.

Iversen

Lee

Gilbert

, et al. Regulation of neuropeptide release. Proc R Soc Lond B Biol Sci 1980; 210: 91–111. 1980/10/29.

66.

Harris

Faust

Gondin

, et al. Selective G protein signaling driven by substance P-neurokinin receptor dynamics. Nat Chem Biol 2022; 18: 109–115. 2021/10/30.

67.

Edvinsson

Reducha

Sheykhzade

, et al. Neurokinins and their receptors in the rat trigeminal system: differential localization and release with implications for migraine pain. Mol Pain 2021; 17: 17448069211059400. 2021/12/14.

68.

Satake

Kawada

. Overview of the primary structure, tissue-distribution, and functions of tachykinins and their receptors. Curr Drug Targets 2006; 7: 963–974. 2006/08/22.

69.

Rodríguez

Coveñas

. The neurokinin-1 receptor: structure dynamics and signaling. Receptors 2022; 1: 54–71.

70.

Steinhoff

von Mentzer

Geppetti

, et al. Tachykinins and their receptors: contributions to physiological control and the mechanisms of disease. Physiol Rev 2014; 94: 265–301. 2014/01/03.

71.

Cascieri

Huang

Fong

, et al. Determination of the amino acid residues in substance P conferring selectivity and specificity for the rat neurokinin receptors. Mol Pharmacol 1992; 41: 1096–1099.

72.

Hegron

Peach

Tonello

, et al. Therapeutic antagonism of the neurokinin 1 receptor in endosomes provides sustained pain relief. Proc Natl Acad Sci U S A 2023; 120: e2220979120. 2023/05/22.

73.

Bowden

Garland

Baluk

, et al. Direct observation of substance P-induced internalization of neurokinin 1 (NK1) receptors at sites of inflammation. Proc Natl Acad Sci U S A 1994; 91: 8964–8968. 1994/09/13.

74.

Figini

Emanueli

, et al. The control of microvascular permeability and blood pressure by neutral endopeptidase. Nat Med 1997; 3: 904–907. 1997/08/01.

75.

Leeman

Slack

, et al. A substance P (neurokinin-1) receptor mutant carboxyl-terminally truncated to resemble a naturally occurring receptor isoform displays enhanced responsiveness and resistance to desensitization. Proc Natl Acad Sci U S A 1997; 94: 9475–9480. 1997/08/19.

76.

Katayama

Nadel

Bunnett

, et al. Catabolism of calcitonin gene-related peptide and substance P by neutral endopeptidase. Peptides 1991; 12: 563–567. 1991/05/01.

77.

Ebrahimi

Javid

Alaei

, et al. New insight into the role of substance P/neurokinin-1 receptor system in breast cancer progression and its crosstalk with microRNAs. Clin Genet 2020; 98: 322–330.

78.

Faraci

Kadel

Heistad

. Vascular responses of dura mater. Am J Physiol 1989; 257: H157–H161. 1989/07/01.

79.

Fiscus

. Molecular mechanisms of endothelium-mediated vasodilation. Semin Thromb Hemost 1988; 14: 12–22. 1988/01/01.

80.

Stewart-Lee

Burnstock

. Actions of tachykinins on the rabbit mesenteric artery: substance P and [Glp6,L-Pro9]SP6-11 are potent agonists for endothelial neurokinin-1 receptors. Br J Pharmacol 1989; 97: 1218–1224. 1989/08/01.

81.

Whittle

Lopez-Belmonte

Rees

. Modulation of the vasodepressor actions of acetylcholine, bradykinin, substance P and endothelin in the rat by a specific inhibitor of nitric oxide formation. Br J Pharmacol 1989; 98: 646–652. 1989/10/01.

82.

Wallerstedt

Bodelsson

. Endothelium-dependent relaxation by substance P in human isolated omental arteries and veins: relative contribution of prostanoids, nitric oxide and hyperpolarization. Br J Pharmacol 1997; 120: 25–30. 1997/01/01.

83.

Liu

Kang

Chen

. Activation mechanism of human soluble guanylate cyclase by stimulators and activators. Nat Commun 2021; 12: 5492. 2021/09/19.

84.

Francis

Busch

Corbin

, et al. cGMP-dependent protein kinases and cGMP phosphodiesterases in nitric oxide and cGMP action. Pharmacol Rev 2010; 62: 525–563. 2010/08/19.

85.

Clement

Christensen

Jansen-Olesen

, et al. The ATP sensitive potassium channel (K(ATP)) is a novel target for migraine drug development. Front Mol Neurosci 2023; 16: 1182515. 2023/07/17.

86.

Schubert

Nelson

. Protein kinases: tuners of the BKCa channel in smooth muscle. Trends Pharmacol Sci 2001; 22: 505–512. 2001/10/05.

87.

Khalil

. Regulation of Vascular Smooth Muscle Function. San Rafael (CA), 2010.

88.

Christensen

Younis

Lindberg

, et al. Intradural artery dilation during experimentally induced migraine attacks. Pain 2021; 162: 176–183. 2020/07/24.

89.

Della Pietra

Gomez Dabo

Mikulenka

, et al. Mechanosensitive receptors in migraine: a systematic review. J Headache Pain 2024; 25: 6. 2024/01/15.

90.

Thomsen

Al-Karagholi

Hougaard

, et al. Investigations of the migraine-provoking effect of levcromakalim in patients with migraine with aura. Cephalalgia 2024; 44: 3331024241237247. 2024/03/19.

91.

Al-Karagholi

Ghanizada

Nielsen

CAW

, et al. Opening of ATP sensitive potassium channels causes migraine attacks with aura. Brain 2021; 144: 2322–2332. 2021/03/27.

92.

Gao

Bayraktutan

. Substance P reversibly compromises the integrity and function of blood-brain barrier. Peptides 2023; 167: 171048. 2023/07/01.

93.

Delepine

Aubineau

. Plasma protein extravasation induced in the rat dura mater by stimulation of the parasympathetic sphenopalatine ganglion. Exp Neurol 1997; 147: 389–400. 1997/11/05.

94.

Owen-Woods

Joulia

Barkaway

, et al. Local microvascular leakage promotes trafficking of activated neutrophils to remote organs. J Clin Invest 2020; 130: 2301–2318. 2020/01/24.

95.

Vezzani

Viviani

. Neuromodulatory properties of inflammatory cytokines and their impact on neuronal excitability. Neuropharmacology 2015; 96: 70–82. 2014/12/03.

96.

Suzuki

Miura

Liu

, et al. Substance P induces degranulation of mast cells and leukocyte adhesion to venular endothelium. Peptides 1995; 16: 1447–1452. 1995/01/01.

97.

Metcalfe

Baram

Mekori

. Mast cells. Physiol Rev 1997; 77: 1033–1079. 1997/11/14.

98.

Worm

Falkenberg

Olesen

. Histamine and migraine revisited: mechanisms and possible drug targets. J Headache Pain 2019; 20: 30. 2019/03/27.

99.

Kakizoe

Kobayashi

Gonda

, et al. Synergistic interactions between neuropeptide and histamine on the capillary permeability in rat skin: evaluation by reflectance spectrophotometry. Microvasc Res 1997; 54: 27–34. 1997/07/01.

100.

Lassen

Heinig

Oestergaard

, et al. Histamine inhalation is a specific but insensitive laboratory test for migraine. Cephalalgia 1996; 16: 550–553. 1996/12/01.

101.

Wienecke

Olesen

Ashina

. Prostaglandin I2 (epoprostenol) triggers migraine-like attacks in migraineurs. Cephalalgia 2010; 30: 179–190. 2009/07/21.

102.

Mashaghi

Marmalidou

Tehrani

, et al. Neuropeptide substance P and the immune response. Cell Mol Life Sci 2016; 73: 4249–4264. 2016/06/18.

103.

Ruff

Wahl

Pert

. Substance P receptor-mediated chemotaxis of human monocytes. Peptides 1985; 6: 107–111. 1985/01/01.

104.

Delgado

McManus

Chambers

. Production of tumor necrosis factor-alpha, interleukin 1-beta, interleukin 2, and interleukin 6 by rat leukocyte subpopulations after exposure to substance P. Neuropeptides 2003; 37: 355–361. 2003/12/31.

105.

Spigelman

Puil

. Ionic mechanism of substance P actions on neurons in trigeminal root ganglia. J Neurophysiol 1990; 64: 273–281. 1990/07/01.

106.

Park

Bae

Kim

, et al. Substance P sensitizes P2X3 in nociceptive trigeminal neurons. J Dent Res 2010; 89: 1154–1159. 2010/07/24.

107.

Naono-Nakayama

Sunakawa

Ikeda

, et al. Differential effects of substance P or hemokinin-1 on transient receptor potential channels, TRPV1, TRPA1 and TRPM8, in the rat. Neuropeptides 2010; 44: 57–61. 2009/11/21.

108.

Zhang

Cang

Kawasaki

, et al. Neurokinin-1 receptor enhances TRPV1 activity in primary sensory neurons via PKCepsilon: a novel pathway for heat hyperalgesia. J Neurosci 2007; 27: 12067–12077. 2007/11/06.

109.

Perin-Martins

Teixeira

Tambeli

, et al. Mechanisms underlying transient receptor potential ankyrin 1 (TRPA1)-mediated hyperalgesia and edema. J Peripher Nerv Syst 2013; 18: 62–74. 2013/03/26.

110.

Bae

Hwang

, et al. Expression of vanilloid receptor TRPV1 in the rat trigeminal sensory nuclei. J Comp Neurol 2004; 478: 62–71. 2004/08/31.

111.

Kim

Son

Kim

, et al. Expression of transient receptor potential ankyrin 1 (TRPA1) in the rat trigeminal sensory afferents and spinal dorsal horn. J Comp Neurol 2010; 518: 687–698. 2009/12/25.

112.

Wei

Kim

McKemy

. Transient receptor potential melastatin 8 is required for nitroglycerin- and calcitonin gene-related peptide-induced migraine-like pain behaviors in mice. Pain 2022; 163: 2380–2389. 2022/03/31.

113.

Yang

Jiang

, et al. TRPV1 Activity and substance P release are required for corneal cold nociception. Nat Commun 2019; 10: 5678. 2019/12/14.

114.

Tepper

. History and review of anti-calcitonin gene-related peptide (CGRP) therapies: from translational research to treatment. Headache 2018; 58: 238–275. 2018/09/23.

115.

Mantyh

Rogers

Ghilardi

, et al. Differential expression of two isoforms of the neurokinin-1 (substance P) receptor in vivo. Brain Res 1996; 719: 8–13. 1996/05/06.

116.

Wolf

Moody

Quirion

, et al. Biochemical characterization and autoradiographic localization of central substance P receptors using [125I]physalaemin. Brain Res 1985; 332: 299–307. 1985/04/22.

117.

Maeno

Kiyama

Tohyama

. Distribution of the substance P receptor (NK-1 receptor) in the central nervous system. Brain Res Mol Brain Res 1993; 18: 43–58. 1993/04/01.

118.

Andersen

Lund

Puil

. Enkephalin and substance P effects related to trigeminal pain. Can J Physiol Pharmacol 1978; 56: 216–222. 1978/04/01.

119.

Thomson

Terman

Zeng

, et al. Decreased substance P and NK1 receptor immunoreactivity and function in the spinal cord dorsal horn of morphine-treated neonatal rats. J Pain 2008; 9: 11–19. 2007/10/24.

120.

Sastry

. Substance P effects on spinal nociceptive neurones. Life Sci 1979; 24: 2169–2177. 1979/06/04.

121.

Urban

Nagy

. Is there a nociceptive carousel? Trends Pharmacol Sci 1997; 18: 223–224. 1997/07/01.

122.

Randic

Hecimovic

Ryu

. Substance P modulates glutamate-induced currents in acutely isolated rat spinal dorsal horn neurones. Neurosci Lett 1990; 117: 74–80. 1990/09/04.

123.

Zieglgansberger

. Substance P and pain chronicity. Cell Tissue Res 2019; 375: 227–241. 2018/10/05.

124.

Govindaiah

Wang

Cox

. Substance P selectively modulates GABA(A) receptor-mediated synaptic transmission in striatal cholinergic interneurons. Neuropharmacology 2010; 58: 413–422. 2009/09/30.

125.

Drew

Lau

Vaughan

. Substance P drives endocannabinoid-mediated disinhibition in a midbrain descending analgesic pathway. J Neurosci 2009; 29: 7220–7229. 2009/06/06.

126.

Hamity

White

Hammond

. Effects of neurokinin-1 receptor agonism and antagonism in the rostral ventromedial medulla of rats with acute or persistent inflammatory nociception. Neuroscience 2010; 165: 902–913. 2009/11/07.

127.

Griffante

Carletti

Andreetta

, et al. [3h]GR205171 displays similar NK1 receptor binding profile in gerbil and human brain. Br J Pharmacol 2006; 148: 39–45. 2006/02/28.

128.

Akerman

Holland

Lasalandra

, et al. Inhibition of trigeminovascular dural nociceptive afferents by ca(2+)-activated K(+) (MaxiK/BK(ca)) channel opening. Pain 2010; 151: 128–136. 2010/07/27.

129.

Pedersen

Ramachandran

Amrutkar

, et al. Mechanisms of glyceryl trinitrate provoked mast cell degranulation. Cephalalgia 2015; 35: 1287–1297. 2015/03/01.

130.

Gitter

Bruns

Howbert

, et al. Pharmacological characterization of LY303870: a novel, potent and selective nonpeptide substance P (neurokinin-1) receptor antagonist. J Pharmacol Exp Ther 1995; 275: 737–744. 1995/11/01.

131.

Iyengar

Hipskind

Gehlert

, et al. LY303870, A centrally active neurokinin-1 antagonist with a long duration of action. J Pharmacol Exp Ther 1997; 280: 774–785. 1997/02/01.

132.

Pradier

Habert-Ortoli

Emile

, et al. Molecular determinants of the species selectivity of neurokinin type 1 receptor antagonists. Mol Pharmacol 1995; 47: 314–321. 1995/02/01.

133.

Duffy

. Potential therapeutic targets for neurokinin-1 receptor antagonists. Expert Opin Emerg Drugs 2004; 9: 9–21. 2004/05/25.

134.

Sorbera

Castaǹer

Bayes

, et al. Aprepitant and L-758298: antiemetic, antidepressant, tachykinin NK1 antagonist. Drugs Future 2002; 27: 211–222.

135.

Tfelt-Hansen

Pascual

Ramadan

, et al. Guidelines for controlled trials of drugs in migraine: third edition. A guide for investigators. Cephalalgia 2012; 32: 6–38. 2012/03/03.

136.

Markowitz

Saito

Moskowitz

. Neurogenically mediated plasma extravasation in dura mater: effect of ergot alkaloids. A possible mechanism of action in vascular headache. Cephalalgia 1988; 8: 83–91. 1988/06/01.

137.

May

Gijsman

Wallnofer

, et al. Endothelin antagonist bosentan blocks neurogenic inflammation, but is not effective in aborting migraine attacks. Pain 1996; 67: 375–378. 1996/10/01.

138.

Waeber

Castanon

, et al. 5-Carboxamido-tryptamine, CP-122,288 and dihydroergotamine but not sumatriptan, CP-93,129, and serotonin-5-O-carboxymethyl-glycyl -tyrosinamide block dural plasma protein extravasation in knockout mice that lack 5-hydroxytryptamine1B receptors. Mol Pharmacol 1996; 49: 761–765. 1996/05/01.

139.

Roon

Olesen

Diener

, et al. No acute antimigraine efficacy of CP-122,288, a highly potent inhibitor of neurogenic inflammation: results of two randomized, double-blind, placebo-controlled clinical trials. Ann Neurol 2000; 47: 238–241. 2000/02/09.

140.

Bullitt

. Expression of c-fos-like protein as a marker for neuronal activity following noxious stimulation in the rat. J Comp Neurol 1990; 296: 517–530. 1990/06/22.

141.

Offenhauser

Zinck

Hoffmann

, et al. CGRP Release and c-fos expression within trigeminal nucleus caudalis of the rat following glyceryltrinitrate infusion. Cephalalgia 2005; 25: 225–236. 2005/02/04.

142.

Guzman

Kopruszinski

Barber

, et al. Chronification of migraine sensitizes to CGRP in male and female mice. Cephalalgia 2025; 45: 3331024251317446. 2025/02/13.

143.

Pellesi

Edvinsson

. Revisiting substance P in migraine: a methodological approach inspired by anti-CGRP and anti-PACAP success. J Headache Pain 2025; 26: 22. 2025/02/01 20:44.

Substance P and the trigeminovascular system: From preclinical mechanisms to human headache induction

Abstract

Keywords

Introduction

Methods

Substance P in the trigeminovascular system

Synthesis and transport of substance P

Release mechanisms in the trigeminovascular system

Binding to neurokinin-1 receptors

Regulation of the activity of substance P

Peripheral actions of substance P

Cephalic vasodilation

Increase in vascular permeability and plasma protein extravasation

Activation of mast cells and immune responses

Modulation of other immune cells

Direct activation of meningeal nociceptors

Interaction with other signaling molecules

Central actions of substance P

Facilitating excitatory neurotransmission

Interaction with descending modulatory pain pathways

Preclinical neurokinin-1 receptor antagonist testing

Clinical trials of neurokinin-1 receptor antagonists in migraine

Dapitant trial

Lanepitant trials

GR205171 trial

L-758,298 trial

Analysis of clinical trial outcomes

Translational challenges

Future directions

Conclusions

Article highlights

Footnotes

Acknowledgments

Author contributions

Data availability

Declaration of conflicting interests

Funding

ORCID iDs

References