The Role of Substance P within Traumatic Brain Injury and Implications for Therapy

Adam Safwat (first author; Department of Clinical Neurosciences, University of Cambridge, CB2 0QQ, UK; as3028@cam.ac.uk; +44 1223 217888), 
Adel Helmy (joint-supervising author; Division of Neurosurgery, Department of Clinical Neurosciences, University of Cambridge, CB2 0QQ, UK; aeh33@cam.ac.uk; +44 1223 216147), 
Arun Gupta (joint-supervising author; Neurosciences Critical Care Unit, Addenbrooke’s Hospital, CB2 0QQ, UK; akg36@medschl.cam.ac.uk; +44 7801 231016)

Correspondence to:
Adam Safwat
as3028@cam.ac.uk
Department of Clinical Neurosciences
Cambridge University

Keywords: substance P, neurokinin-1 receptor antagonist, traumatic brain injury, neuroinflammation, neurogenic inflammation

Abstract
This review examines the role of the neuropeptide substance P within the neuroinflammation that follows traumatic brain injury. It examines it in reference to its preferential receptor; the neurokinin-1 receptor and explores the evidence for antagonism of this receptor in traumatic brain injury with therapeutic intent. Expression of substance P increases following traumatic brain injury. Subsequent binding to the neurokinin-1 receptor results in neurogenic inflammation, a cause of deleterious secondary effects that include an increased intracranial pressure and poor clinical outcome. In several animal models of TBI, neurokinin-1 receptor antagonism has been shown to reduce brain oedema and the resultant rise in intracranial pressure. A brief overview of the history of substance P is presented, alongside an exploration into the chemistry of the neuropeptide with a relevance to its functions within the central nervous system. This review summarises the scientific and clinical rationale for substance P antagonism as a promising therapy for human TBI.  













Introduction
Trauma is a significant cause of death globally1, with traumatic brain injury (TBI) a leading cause of death and disability within the spectrum of traumatic injuries worldwide, especially in the younger populations (<45 years) of both developed and developing nations.1,2
Approximately 40% of those sustaining severe TBI will die from their injury, and 60% will have an unfavourable outcome (including death) based on the Glasgow Outcome Scale.3 Mortality increases based on the severity of the injury4, and those that survive may be left with debilitating physical and neurocognitive impairments that have a negative impact on the survivor’s daily living and ability to work, whilst also negatively impacting surrounding family and the greater healthcare and socioeconomic system that must cater for post-TBI disability.5
Despite an inordinate cost to healthy human life and the world economy, no significant breakthroughs in the clinical management for TBI have occurred in the past 40 years.6 While preventative measures in the form of road laws and mandatory safety devices have proven to be successful in reducing incidence of TBI,7,8 there has been no progress in developing neuroprotective pharmacological interventions. Well-designed clinical trials in TBI patients are difficult due to the heterogenous nature of the disease and patient cohorts, as well as a lack of appropriate early endpoints.3 This highlights the importance of phase II experimental medicine trials as a means of addressing dosage, timing, and pharmacokinetics of novel agents.
As our understanding of TBI pathophysiology has developed, the scientific literature has moved towards a molecular and mechanistic understanding of the underlying processes that drive cellular injury. In particular, the role of inflammation has been proposed as a key driver of injury and a potentially tractable therapeutic target. Several inflammatory processes, including both innate inflammation and adaptive immunity have been reported in human TBI. The initiators of inflammation that link directly with tissue trauma or deformation are an area of active study, and this has led the concept of ‘Neurogenic Inflammation’.
Neurogenic inflammation occurs as part of this secondary brain injury, a result of the neuropeptide substance P’s release from primary afferent neurons that, through a complex chain of events, leads to breakdown of the blood-brain barrier, generation of a classical inflammatory response and the development of cerebral oedema.9–11 This cerebral oedema results in a rise in intracranial pressure (ICP), a significant cause of mortality and poor outcomes in TBI patients.12,13
This review will investigate the role of the neuropeptide substance P in the development of cerebral oedema following traumatic brain injury and focus on the evidence for inhibition at its preferential receptor: the neurokinin-1 receptor. There has been growing interest in substance P and its potential involvement within neuroinflammation following TBI, with much of the literature accounting studies performed in animal models. This review will consolidate the current literature in animal and man, focusing on the documented uses of neurokinin-1 receptor antagonists that block the action of substance P and its effects.

Secondary Brain Injury following TBI
From a clinical and therapeutic perspective, it is secondary brain injury that is modifiable and therefore a tractable target for intervention. Secondary brain injury encompasses a range or processed including a combination of neurogenic and classical inflammatory cascades, release of excitotoxic amino acids and neurotransmitters, loss of ionic homeostasis, activation of matrix metalloproteinases (MMPs), and the generation of free radicals.9–11,14 These deleterious effects result in oxidative stress, neurotoxicity, and recruitment of inflammatory cells into the site of injury, all furthering cell injury. Hyperpermeability and breakdown of the blood-brain barrier (BBB) leads to the development of vasogenic oedema,9–11 with the extracellular nature of this fluid resulting in an increased brain volume.11,15 This results in an increased intracranial pressure (ICP), a major therapy target by neurocritical care due to the mortality and poor outcomes associated with its rise.12,13 This insidious process begins at the point of the primary injury, and continues to occur in the days to weeks following.11
Substance P is upregulated following TBI, with increased perivascular SP immunoreactivity.16,17 The presence of elevated serum substance P levels is associated with an increased mortality, and an indicator of severity of injury.18,19 With the initial opening of the BBB and the associated vasogenic oedema thought to invite and accentuate further classical inflammation.20–22

Neurogenic Inflammation
The concept of neurogenic inflammation was originally described in 1901 by Bayliss, when he explored vasodilation in the lower limbs of dogs.23 He theorised that the sensory nerve branches from the dorsal root ganglion that were supplying the blood vessels were responsible for the vasodilatory effect seen. Neurogenic inflammation is now described as an inflammatory response that is neurally elicited through stimulation of capsaicin-sensitive primary sensory neurons, and the subsequent release of neuropeptides that elicit a characteristic inflammatory response.24,25 Sensory neurons respond to changes in pH and temperature, compounds such as serotonin and histamine, as well as cytokines and direct mechanical injury.26 Released neuropeptides include neurokinin A and B (NKA and NKB), as well as calcitonin gene-related peptide (CGRP), and of most interest in this review, substance P.20 The resulting combination of vasodilation, protein extravasation, vascular permeability and leukocyte adhesion is termed neurogenic inflammation.24,27 CGRP is a potent vasodilator, while vascular permeability and protein extravasation is a result of NKA, NKB and chiefly SP, of which all belong to the tachykinin family of neuropeptides.28 CGRP and SP often coexist within primary sensory neurons.29 CGRP’s release, in addition to vasodilation, causes an upregulation of the neurokinin-1 receptor and competes with SP for catabolism by endopeptidases, resulting in an increased concentration of SP and potentiating its biological effects.30
Breakdown of the blood-brain barrier occurs as a consequence of increased vascular permeability and protein extravasation of the cerebral arteries. These arteries are surrounded by a dense supply of sensory neurons containing CGRP and SP.31 This blood-brain barrier dysfunction leads to an influx of water, termed vasogenic oedema, and in combination with cerebral vasodilation, a cause of raised ICP and its associated poor outcomes following TBI. Furthermore, SP is involved in augmenting the classical inflammatory cascade. It acts to produce nitric oxide and cytokines, activates mast cells, microglia and astrocytes, and facilitates the recruitment of peripheral immune cells to the site. There is also evidence SP acts to increase transcytosis through endothelial cells via caveolae transport, allowing shifts of large proteins like albumin which increase the osmotic gradient and result in the release of inflammatory cytokines through microglial and astrocytic activation (Figure 1).20 
The other distinct family of kinins implicated in the mediation of neurogenic inflammation is the slow acting kinin family. They consist of the peptides bradykinin and kallidin (Lys-bradykinin), which are also released after CNS injury.32 Kinin peptide release occurs in both tissue and plasma. Cell death and the release of lysosomal enzymes in tissue injury activates the low molecular weight precursor protein kininogen (LMWK), leading to the release of kallidin. In blood, the activation of Hageman Factor XII by damaged surfaces leads to the release of bradykinin from the high molecular weight kininogen (HMWK).32,33 Kinin B1 and B2 receptors are expressed throughout the CNS.34 These receptors are G-protein coupled receptors, where activation results in the conversion of phosphatidylinositol-4-5-bisphosphate to inositol 1,2,5-trisphosphate (IP3) and diacylglycerol (DAG), leading to a rise in intracellular calcium and initiation of secondary messenger systems.34 
Downstream effects of kinin receptor activation include the release of arachidonic acid through the activation of phospholipase A2, which is metabolised into prostaglandins and reactive oxygen species (ROS) which contribute to inflammation and oxidative stress.32,35 Glutamate release is also implicated in bradykinin binding, leading to intraneuronal rises of Ca2+ and activation of enzymes that include phospholipases, endonucleases, and proteases, leading to cellular damage, termed glutamate excitotoxicity.32 Another consequence is nitric oxide (NO) release, which causes vasodilation and further contributes to oxidative stress.32 B2 kinin receptor stimulation on vascular endothelial cells by bradykinin increases vascular permeability and plasma extravasation.35 Additionally, degranulation of mast cells is caused by both bradykinin and SP, resulting in the release of serotonin and histamine, and causing further vascular permeability and plasma extravasation.32
The kinin and tachykinin systems are believed to act in a synergistic manner in the way they initiate and maintain inflammatory responses, with evidence that endogenous kinins cause inflammatory responses by stimulating the release of CGRP, NKA, and SP.32,36,37  Despite the strong evidence for kinin involvement in secondary injury, no clear benefit has been identified in clinical trials assessing the use of bradykinin receptor antagonists in patients with traumatic brain injury.38,39

Substance P
Discovery
Substance P was originally discovered in 193140 by Ulf von Euler, a postdoctoral student in training, and John Gaddum, senior assistant to Henry Dale, when attempting to further describe the distribution of acetylcholine in various organs. They identified an extract in equine brain and intestine that would cause stimulation of smooth muscle and potent hypotension in atropinized rabbit, an effect therefore independent of acetylcholine. It was initially isolated from horse small intestine and was described as "Preparation P",40 only becoming publicly termed "Substance P" in 1934 by Gaddum and Schild41 after developing the private nickname in the lab from the purified extracted powder "substance" within the various stored bottles labelled P1, P2 etc.42
Work on substance P continued after Euler returned to Stockholm to become professor of physiology at The Karolinska Instituet. Bengt Pernow joined Euler’s lab as a junior assistant and published his doctoral thesis on the purification of SP in 1953, as well studies on its distribution and biological actions.43 Not until 40 years later would Substance P be isolated, and the undecapeptide amino acid structure identified by Chang and Leeman in 1971.44 It was successfully synthesised the same year by Tregear et al.,45 before being developed as a radioimmunoassay in 197346 and facilitating further studies into the distribution of the neuropeptide.47–51
The mammalian tachykinin family (of which substance P is an example) would later be established with the discovery of neurokinin A (formerly known as substance K) and neurokinin B (formerly neuromedin K) in 1983.52,53 The remaining neuropeptide K in 1985, neuropeptide gamma in 1988 and haemokinin-1 in 2000 would be soon to follow.54–56 Their designation as ‘tachykinins’ relates to their fast stimulant action on smooth muscle and hypotensive properties, in contrast to the slow-acting kinins – the “true bradykinins”.57

Structure
Chang et al. identified the amino acid structure of substance P in 1971 as the undecapeptide (comprising of 11 amino acids) Arg1-Pro2-Lys3-Pro4-Gln5-Gln6-Phe7-Phe8-Gly9-Leu10-Met11-NH2.44 It has a relative molecular mass of 1.3 kDa.58 All tachykinins are amidated at the carboxyl terminal methionine. Substance P is an amphiphilic peptide, with positively charged residues on the N-terminus and hydrophobic residues on the C-terminus. It carries a net positive charge at physiologic pH.59
The mammalian tachykinins are closely related, sharing a common C-terminal sequence: Phe7-X8-Gly9-Leu10-Met11-NH2 (X is Phe or Val).60 The common carboxyl-terminal domain interacts with the tachykinin receptors, while the amino-terminal sequence dictates receptor specificity.61 Neuropeptide K and neuropeptide gamma are elongated forms of neurokinin A.62

Synthesis
Substance P is encoded by the preprotachykinin-1 (PPT1/TAC1) gene, located on the long arm of chromosome 7 between positions 21 and 22 (7q21-q22).63 TAC1 is a 7 exon gene, with the sequence encoding SP contained within exon 3 (Figure 2).64 Other tachykinins that are encoded by the TAC1 gene include neurokinin A, neuropeptide K and neuropeptide gamma.63 The remaining neurokinin B and haemokinin-1 are encoded by TAC3 and TAC4 respectively.60 Four messenger ribonucleic acid (mRNA) isoforms result via alternative RNA splicing of the TAC1 gene transcript: α-PPT, β-PPT, γ-PPT and δ-PPT. All four encode the substance P precursor protein, while only γ-PPT and β-PPT encode for the precursor for neurokinin A, neuropeptide K and neuropeptide gamma.65 β-PPT is the predominant mRNA found within human brain tissue, approximately representing 80-85% of the total PPT mRNA. The remaining PPT mRNA is γ-PPT, with no detectable α-PPT.66 This is in contrast to a study investigating striatal preprotachykinin gene expression within Sprague-Dawley rats, where γ-PPT is found to predominate.67
Synthesis of substance P occurs within the ribosomes in the perikaryon.68 Following synthesis, SP is packed into synaptic vesicles and axonally transported to terminal regions,69 concentrated within the axon terminals of primary afferent neurons.70 
The expression of PPT mRNA increases following noxious stimulation, as discovered in rats by Noguchi and colleagues.71 Following injection of formalin into the right hindpaw, dorsal root ganglion neurons showed increased expression of PPT mRNAs. This mirrors previous studies72,73 that observed an increase in SP-immunoreactivity in the dorsal horn following chemogenic nociceptive stimulus and correlates the increased PPT gene expression with SP synthesis. Similar increases in SP-immunoreactivity have been observed following traumatic brain injury and spinal cord injury in humans.16,17 Prominent immunoreactivity has been observed in the cerebral cortical pyramidal cells, cortical neurons and astrocytes, as well as an extensive perivascular distribution of SP nerve endings demonstrated in the cerebral cortical microvasculature in post-mortem tissue of TBI patients. No SP immunoreactivity was observed within the microvascular endothelium.16

Metabolism
Metabolism of substance P has been documented to occur by a number of enzymes, including: angiotensin-converting enzyme (ACE),74 neutral endopeptidase (NEP),74–76 SP-degrading enzyme (SP-DE),77 cathepsin-D,78 cathepsin-E,79 post-proline endopeptidase (PEP),80 and dipeptidyl amiopeptide IV (DPIV).81 All of the above enzymes have been identified to cleave substance P in vitro, but NEP and/or ACE are more likely to result in SP cleavage in vivo due to their cellular localization.75 
Neutral endopeptidase has been shown to be the predominant peptidase in tachykinin metabolism in the brain, as well as acting with a significant role within the spinal cord.82,83 It preferentially cleaves hydrophobic amino acids, catalysing the hydrolysis of the peptide bonds Gln6-Phe7, Phe7-Phe8 and Gly9-Leu10 within the amino acid structure of substance P. This results in fragments that lack the carboxyl terminal region that are necessary for its binding to the tachykinin receptor, therefore inactivating it.84 NEP’s role in the degradation of SP has been further explored through the use of NEP -/- (knockout) mice, where the NEP gene was deleted by homologous recombination. Widespread plasma extravasation in postcapillary endothelia was observed and reversed by recombinant NEP and neurokinin-1 receptor (SP) antagonists.85
Angiotensin-converting enzyme has been shown to inactivate substance P within plasma in vivo. 86,87 ACE has been shown to catalyse the hydrolysis of the Phe8-Gly9 bond, leaving the peptide lacking its carboxyl terminal region required for binding to the tachykinin receptor.87 Following ACE-inhibitor administration in mice, significant increases in plasma extravasation of Evans blue dye in the airways, gut and pancreas was noted.88 Pre-treatment with a neurokinin-1 receptor antagonist prior to administration of the ACE-inhibitor resulted in markedly reduced plasma extravasation of the Evans blue dye, indicating the contribution of ACE to substance P’s effect of plasma extravasation.88
SP is rapidly degraded, with its half-life reported to be in the range of seconds to tens of minutes in blood and tissues. 59,74,89,90 It is able to interact with higher molecular weight binding proteins such as fibronectin due to structural similarities between regions of the NK-1R and the glycoprotein. The resulting complex allows SP to evade degradation and provides stability.91 This suggests a shorter half-life in tissues, as unbound substance P within the tissues can be degraded by the cell surface metallopeptide neutral endopeptidase. This has been evidenced by stability noted of endogenous substance P compared to an added exogenous substance P, and the majority of SP within the plasma identified as being associated with high molecular weight material in a bound complex.89

Localisation
Substance P can be found widely distributed throughout the central, peripheral and enteric nervous systems. Within the human nervous system, SP immunoreactivity has been demonstrated by immunohistochemistry92,93 and radioimmunoassay94 in the telencephalon, diencephalon, mesencephalon, metencephalon, myelencephalon, and spinal cord.
Within the telencephalon of the brain, the greatest immunoreactivity was found within the basal ganglia (striatum, putamen and internal globus pallidus), hippocampus, and septum. Lower concentrations were identified in the cortex, external globus pallidus and amygdala. The hypothalamus within the diencephalon showed immunoreactivity for SP, while none was detected in the thalamus. Within the mesencephalon, the substantia nigra (both pars reticular and compacta) revealed the highest immunoreactivity of SP within the CNS, and reduced levels within the hypothalamus. The pons and medulla oblongata of the metencephalon and myelencephalon show immunoreactivity to SP (Figure 3).92,93,95 Edvinsson and colleagues96 investigated the distribution of substance P-like immunoreactivity in the cerebral arteries, choroid plexus and dura mater.  SP-fibres were numerous in pial vessels, with few identified in the choroid plexus and dura mater (in man – compared to high levels found in cat, rabbit and guinea pig). Those SP-containing nerve fibres that were found were closely associated with blood vessels.96 Mai et al.97 examined the distribution of SP immunoreactivity within the human brain using immunohistochemistry. They observed SP-immunoreactive “tubules” that were assumed to correspond with dendrites, and single-stranded or beaded fibres with were assumed to correspond to varicosities and synaptic boutons along dendrites. Grouped and diffuse beaded processes were observed in association with perikarya and proximal dendrites.97
Within the spine, the substantia gelatinosa contains the highest levels of immunoreactive SP, with high levels continuing in extension to the nucleus proprius and into the dorsal roots. The spinal ganglia contain moderately high levels of SP immunoreactivity, with levels remaining similar across segments. SP immunoreactivity drops significantly in the ventral root, ventral horn and other areas of grey matter, and is undetectable within the white matter.94,98
On a cellular level, substance P is expressed by a plethora of cell types including neurons,99 microglia,100 astrocytes,101 endothelial102 and epithelial103 cells. Many cells of the immune system also express high levels of SP, including T cells,104 dendritic cells,105 macrophages106 and eosinophils.107 They are also expressed by stem cells108 and mesenchymal stem cells (MSCs).109 Sensory nerve fibres positive for SP are found surrounding most blood vessels throughout the body. Cerebral arteries have a rich supply of sensory neurons, with SP-immunoreactive nerve fibres in close association with blood vessels in the dura mater and choroid plexus.96 With the widespread expression of substance P throughout various cell types, especially neurones, it is no surprise that substance P is implicated in a wide range of physiological and pathophysiological functions.

The Neurokinin-1 Receptor
Three tachykinin receptors (TACR; also referred to as neurokinin receptors) of differing types according to ligand affinity are described: the neurokinin-1 receptor (NK-1R), neurokinin-2 receptor (NK2R) and neurokinin-3 receptor (NK3R). Their preferential ligand of affinity is substance P, neurokinin A, and neurokinin B respectively, though they are not exclusive in their ability to bind to their respective receptors – especially at high ligand concentration.61,110 SP’s receptor binding affinity to the NK-1R is 100- and 500-fold greater than NKA and NKB respectively.111 The neurokinin-1 receptor is encoded by the 5-exon TACR1 gene localised on the short arm of chromosome 2 (2p13.1-p12).111 There is significant sequence homology between species, as cloned neurokinin 1 receptors from human, mouse, rat and guinea pig reveal a difference in amino acid structure of less than 10%.112
There are two isoforms of the neurokinin-1 receptor: a 407-amino acid residue full length version (NK-1R-F; long isoform), and a 311-amino acid residue truncated version (NK-1R-Tr; short isoform). The length of the carboxyl-terminus tail is the only difference between the two, with the short isoform boasting a relative molecular mass of 37 kDa and the long isoform a relative molecular mass of 46 kDa. It is the structural basis for the difference in functional properties between the two isoforms.113–115 The long isoform is significantly more abundant within select regions of the brain, while the short isoform appears more prevalently throughout the central nervous system and peripheral tissues.116 The short isoform receptor responds to lower concentrations of SP and is resistant to desensitisation.114 However, it has been shown to have a binding affinity 10-fold less than the long isoform full length version of the NK-1R.113
Within the brain, NK-1R mRNA expression was found to be high in the striatum, moderate in the cortex, hippocampus and amygdala, and low in the thalamus and cerebellum. Peripheral tissues that expressed high levels of NK-1R mRNA expression included adipose tissue, the pituitary, the prostate, bone, spleen, skeletal muscle, intestine, heart and lungs.116 On a cellular level, the NK-1R is expressed by neurons,117 endothelial cells,118 epithelial cells,119 fibroblasts,120 smooth muscle cells,121 and immune cells such as lymphocytes,122 natural killer cells,123 dendrites,124 monocytes/macrophages,106 microglia,100,122,125 astrocytes,125 eosinophils and mast cells.126
The tachykinin receptor family belong to family 1 (rhodopsin-like) of G protein-coupled receptors (GPCRs). These receptors have seven hydrophobic transmembrane domains (TM I-VII), with three extracellular loops (EL 1-3), three intracellular loops (IL 1-3), an intracellular carboxyl-terminus and an extracellular amino-terminus.63 The binding site of NK-1R’s agonists involves the residues of the first and second extracellular loop (EL 1 & 2), as well as the second and seventh transmembrane domain (TM II & VII).127 The third intracellular loop (IL 3) is responsible for binding to protein G, and the intracellular carboxyl-terminus dictates desensitisation of the receptor following phosphorylation of its serine and threonine residues in response to repeated binding of its agonist.61 SP enters into the hydrophobic ligand binding pocket (TM II and VII) between the extracellular surface, lipid bilayer and transmembrane domain. The remainder of the molecule interacts with the amino acids on the extracellular surface.24
Following binding of substance P to the neurokinin-1 receptor, translocation of G-protein-coupled-receptor kinases (GRKs) and β-arrestins from the cell’s cytosol to the plasma membrane occurs. GRKs phosphorylate the SP/NK-1R complex, while β-arrestins interact with the phosphorylated SP/NK-1R complex.128,129 The SP/NK-1R-β-arrestin complex is internalised via the process of endocytosis, contributing to desensitisation of the cell to SP-signalling. Upon internalisation of the SP/NK-1R-β-arrestin complex, substance P is detached from the receptor and metabolised. The NK-1R is recycled to the cell surface, leading to resensitisation of the cell.130
The formation of the SP/NK-1R complex activates phospholipase C and adenylate cyclase.131 Phospholipase C catalyses the hydrolysis of phosphoinositides into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG).132 Adenylate cyclase activation results in the formation of cyclic adenosine monophosphate accumulation (cAMP).133 These secondary messengers signal to a PKC-Raf-MEKs/PKA-MEKs pathway that activate extracellular signal-related kinases 1/2 (ERK1/2), which translocate into the cell’s nucleus and mediates the expression of cytokines.134–137 This is achieved through the mammalian target of rapamycin (mTOR) signalling pathway, serine/threonine-specific protein kinases, and transcription factors such as nuclear factor kappa B (NF-κB)134,138 and activator protein 1 (AP-1).133 Activation of ERK was brisk (peak within 1-2 minutes) when the full length isoform NK-1R was bound, compared to a slower (peak within 20-30 minutes) activation at the truncated short isoform NK-1R.115 Interestingly, cells that expressed the truncated isoform of the NK-1R did not activate the NF-κB pathway when NK-1R-Tr was activated, and they appear to have an alternate downstream signal pathway.115

Functions
The widespread expression of substance P throughout the body and in a variety of differing cell types affirms its role in a diverse array of biological functions. These include neurotransmission,139 plasma extravasation,140 vasodilation,140 nociception,141 smooth muscle contraction and relaxation,142,143 memory modulation and reinforcement,144 emesis145, respiration,146 neurogenesis,147 stimulation of cell growth148 and inflammation.61,149 In terms of pathophysiology, SP has been implicated in asthma,150 eczema,151 cancer,152 HIV/AIDS,153 rheumatoid arthritis,154 inflammatory bowel disease,155 sickle cell disease,156 affective disorders,157 chemotherapy-induced emesis,158 cardiovascular disease,159 and the focus of this review, traumatic brain injury via neurogenic inflammation.

The Blood-Brain Barrier
Physiological functions of the BBB
A barrier between the central nervous system (CNS) and peripheral circulation is necessary to maintain a strict homeostatic environment required for normal brain function. This environment is formed by three physical interfaces: the blood-brain barrier, the blood-CSF barrier and the arachnoid epithelium that separates the blood from the subarachnoid CSF.160 The current focus will be on the BBB separating the blood circulation and the brain’s extracellular fluid and how its dysfunction is involved and exacerbates the secondary injury process in TBI.
The BBB is comprised of a monolayer of specialised endothelial cells termed ‘brain microvascular endothelial cells’ (BMECs), joined together by tight junctions (TJ) and adherens junctions.161 These tight junctions are comprised of numerous important proteins with differing functions, including claudins, occludins, junctional adhesion molecules and the zona occludens.162 The adherens junctions contain protein cadherins, vascular endothelial cadherins (VE-cadherins) in high levels and lower levels of neural (N-) and epithelial (E-) cadherins.163 Together they result in limited paracellular diffusion of hydrophilic molecules, sealing adjacent endothelial cells and mediating their association with surrounding pericytes.164 In addition, there is reduced transcytosis and restricted vesicle-mediated transcellular transport, necessitating additional and polarized transporters that vary between the luminal and abluminal sides of the endothelial cells.165 This gives tight control over movement of molecules into and out of the brain. Transporters on the luminal side of the endothelial cell monolayer transport lipophilic molecules and specific nutrients (e.g. glucose, amino acids, nucleosides) into the CNS, while transporters on the abluminal side remove specified waste products.166,167 
Astrocytic end-feet nearly completely ensheath the vascular tube, regulating water flow through colocalized aquaporin-4 channels and ionic control of extracellular K+ via Kir4.1 K+ channels.164 Through intracellular Ca2+ levels they regulate neuronal activity and cerebral blood flow. In addition, they have been shown to communicate via gap junctions, and it has been proposed their regulation of vasodilation and vasoconstriction is transmitted through this method.168 Finally, they are involved in strengthening the tight junctions between endothelial cells, modulating the polarized endothelial cell transporters, and promoting enzymatic systems.169
Between the astrocytic end-feet and the endothelial cells sit pericytes embedded within the basal membrane.166 They offer maintenance and stability to the endothelial cells via regulation of angiogenesis and the extracellular matrix, with BBB permeability seen to increase with a decrease in pericyte coverage.164,165 In response to neuronal activity they modulate cerebral blood flow and vessel diameter.164
The BBB serves as a barrier isolating the brain’s environment, restricting access to pathogens and xenobiotics that may cause harm. Unfortunately, this same defence creates challenging barriers for drug delivery to the CNS.170 
BBB Dysfunction and Vasogenic Oedema
Vasogenic oedema is a result of increased BBB permeability leading to an increase of fluid in the extracellular space that originates from the vasculature. An oncotic gradient forms as extravasated vascular components enter the extracellular space, and water follows this gradient to add to the brain water content.171 This in turn results in increased total brain volume and a raised ICP.172,173 Numerous contributing factors result in the increased BBB permeability seen after injury, many having been discussed above. These include mechanical disruption of the tight junction (TJ) proteins between endothelial cells, recruitment of local and peripheral inflammatory cells, release of proinflammatory cytokines and chemokines from glial cells, and upregulation of MMPs.174–176 Disruption of the TJs allows increased paracellular transport of molecules that were initially restricted, including inflammatory cells. In addition, increased transcytosis through endothelial cells of proteins such as albumin and other large molecules results in further osmotic shifts and water movement.177
Blood-brain barrier disruption in TBI appears to be biphasic, initially occurring acutely after injury with increased permeability lasting for a few hours before declining to more normal levels.178,179 A second disruption occurs again at around day 3, though no additional brain oedema was noted with this later opening on an Evans Blue study performed on Sprague-Dawley rats.178,180

Neurokinin-1 Receptor Antagonists
Animal Models
Repeated capsaicin administration results in substance P depletion from primary sensory neurons.181 In animals pre-treated with capsaicin that underwent TBI, no significant oedema development was noted in comparison to controls.182 This was associated with a reduction in BBB permeability assessed by Evans blue dye. Furthermore, animals treated with capsaicin prior to injury suffered a reduction in post-traumatic functional deficit.182 While this is unreasonable as a potential treatment, it offered insight into what antagonism of the NK-1R might achieve.
The NK-1 receptor antagonist ‘N-acetyl-L-tryptophan’ (NAT) was administered to male Sprague-Dawley rats 30 minutes after injury using the acceleration-induced impact TBI model and compared to vehicle controls.27 Donkin et al. found significantly reduced oedema as assessed by brain water content and Evans Blue dye extravasation. MRI showed evidence of an attenuation of oedema using diffusion-weighted imaging. Additionally, NAT-treated rats showed significantly reduced functional deficits using the rotarod test and Barnes maze for motor function and cognitive outcome respectively.27 The study was repeated in female Sprague-Dawley rats by Corrigan et al. to ensure results did not differ depending on gender, identifying a similar attenuation of BBB breakdown seen in males.183 Donkin et al. repeated the experiment 2 years later, looking to assess what occurs when administration is delayed to 12h.179 They found despite requiring administration of a BBB-permeable form of NAT (L-732,138) beyond 5h, inhibition of the NK-1 receptor resulted in functional improvement compared to vehicle-controls. In addition, attenuation of axonal injury was found.179 Finally, Li et al. examined the NK-1 receptor antagonist ‘L-733,060’ in TBI in adult male wild type C57BL/6J mice and compared to vehicle, sham and SP knock out (SP-KO) mice.184 Functional deficits, lesion volume, brain water content and BBB breakdown were attenuated in SP-KO and NK-1R antagonist-treated mice. Additionally, SP-KO and NK-1R antagonist-treated mice showed a reduction in cytochrome c release (associated with apoptosis), reduced inflammatory cytokines and indicators of oxidative stress, as well as reactive oxygen species production.184
NK-1R antagonism has also been explored within an ovine model. Sheep have a gyrencephalic brain akin to humans, unlike the rodent lissencephalic brain.185 Vink and colleagues examined the effect of NK-1 receptor antagonists on ICP in sheep following TBI.186 A humane stunner was used to induce impact-acceleration injury, and then an ICP monitor is inserted through a burr hole to measure ICP. An NK-1 receptor antagonist was administered 30 minutes after injury, while other sheep received vehicle or were sham controls. After 4h there was a significant increase in ICP (>30 mmHg) of sheep that received vehicle, while those that received a NK-1 receptor antagonist had an ICP comparable to the sham group.186
Hyperphosphorylation of tau occurs following traumatic brain injury, with evidence that suggests it results in an increased risk of later developing dementia.187 The postulated mechanism is an increase in kinase activity following substance P’s binding to the neurokinin-1 receptor, notably kinases associated with tau phosphorylation. These include the serine/threonine-specific protein kinase Akt, extracellular signal-related kinases ERK1/2 and c-Jun N-terminal (JNK). In a study by Corrigan and colleagues, the neurokinin-1 receptor antagonist “EU-C-001” was administrated to male Sprague-Dawley rats following sham procedures, single moderate-to-severe TBI or repeated mild TBI. The NK-1RA EU-C-001 significantly attenuated tau phosphorylation following both single moderate-to-severe TBI and repeated mild TBI.188 It is interesting to note, this was not previously seen following the administration of the NK-1 receptor antagonist n-acetyl L-tryptophan (NAT) in a prior study, suggesting EU-C-001’s ability to cross the intact blood-brain barrier to bind to the neuronal NK-1 receptor is necessary for an effect on tau phosphorylation.179,188 
Finally, an intriguing study exploring the use of ACE-inhibitors in mice following TBI was performed by Harford-Wright et al.189 ACE is integral to the metabolism of substance P, and by inhibiting ACE, one would expect to see increased levels of substance P and potentiation of its effects as a result. The study team identified as expected increased immunoreactivity of SP compared to controls, and the ACE-inhibitor treated mice experienced a worsening of motor deficits.189 While not a study on neurokinin-1 receptor antagonism, it offers further insights into the benefit that might be gained from blocking the action of substance P following TBI. 

Previous Human Studies of Substance P Inhibition
The use of neurokinin-1 receptor antagonists has not yet been explored within humans in the context of traumatic brain injury. However, there are currently licensed uses within clinical practice, as well as much evidence in clinical trials investigating the use of NK-1R antagonists in a variety of disease processes.
Currently, post-operative and chemotherapy-induced nausea and vomiting (CINV) are the only licensed uses for neurokinin-1 receptor antagonists. Aprepitant was the first oral NK-1R antagonist approved for clinical use, when it was found to significantly improve chemotherapy-induced nausea and vomiting following cisplatin therapy in a clinical trial performed by Navari et al. in 1999.190 Other NK-1R antagonists have since been developed, such as rolapitant,191 netupitant,192 fosaprepitant193 and fosnetupitant194 in a combination of oral and IV preparations. Fosaprepitant and fosnetupitant are prodrugs of aprepitant and netupitant respectively, where metabolism converts the drug into its pharmacologically active agent.195 NK-1R antagonists are often given in combination anti-emetic therapy in addition to dexamethasone and serotonin type 3 receptor antagonists (5HT3RAs), as NK-1R antagonists have been thought to play a role in both acute and delayed CINV through antagonism of SP in the vomiting centre of the brain.196,197 An NK-1R antagonist was similarly investigated in the treatment of PONV in 1999 by Diemunsch et al.198 and found to be efficacious, though the drug first approved for PONV would be aprepitant and not the investigated vofopitant.
There has been interest in the use of NK-1R antagonists within neuropsychiatric disease processes such as depression,199–204 generalised anxiety,205 PTSD,206,207 migraines208,209 and pain.210–215 Unfortunately, in comparison to PONV and CINV, the evidence for their efficacy is less forthcoming. Kramer et al.202 investigated MK-869 (aprepitant) in 1998 in patients with major depressive disorder. They observed a similar antidepressant profile compared to paroxetine, with improvements of insomnia and genital symptoms observed when using the NK-1R antagonist. Another clinical trial by Kramer et al. performed in 2003 observed antidepressant effects of the NK-1R antagonist ‘L-759274’ versus placebo based on clinical depression scales.201 However, in a clinical trial by Keller et al.199 comparing aprepitant, paroxetine and placebo in major depressive disorder, the NK-1R antagonist showed no significant effect over placebo, despite PET imaging indicating appropriate high level receptor blockade. Another study by Ball et al. examining aprepitant against paroxetine and placebo achieved the same no efficacy result.200 The theory as to why results appeared inconclusive was investigated by Ratti et al.,203 whose hypothesis was that antidepressant effects would only be observed if full central neurokinin-1 receptor blockade is achieved. This was investigated using positron emission tomography, where >99% receptor occupancy was achieved, and efficacy was noted in one of two studies (one not meeting statistical significance based on the Hamilton Depression Rating scale). Further discussion on the topic is well summarised in a review article by Rupniak.204
There has been investigation into NK-1R antagonists in the respiratory system, with studies investigating chronic cough, asthma and COPD. Smith et al.216 observed significant improvement of chronic cough following orvepitant administration, as defined by objective cough frequency, cough severity visual analog scale (VAS) score and quality of life. The recovery of exercised-induced airway narrowing in asthma patients following administration of the NK-1R antagonist FK-888 was documented by Ichinose et al.217 A single inhaled dose of the NK-1R antagonist AVE5883 protected against neurokinin A-induced bronchoconstriction in asthma patients, though no effect was seen with seven days of pre-treatment by Boot et al.218 The perception of breathlessness in patients with COPD was not observed with NK-1R antagonism.219
Efficacy of NK-1R antagonists has also been explored within disorders causing pruritis. Clinical trials investigating their use in chronic refractory pruritis,220 atopic dermatitis,221,222 EGFRI-induced pruiritis223 and epidermolysis bullosa224 have been performed with mixed, generally unfavourable results.
Lastly, studies that explored treatment of overactive bladder,225 tinnitus,226 gastric motor function227 and the prevention of post-ERCP pancreatitis228 showed no significant effect with NK-1R antagonism.

Human Studies in TBI
Studies exploring the effects of neurokinin-1 receptor antagonists in animal TBI models have shown promise. They have been observed to reduce post-TBI oedema development, reduce ICP, attenuate BBB breakdown, as well improve functional outcomes.27,179,182,184,186 Together with clinical trials of NK-1R antagonists in humans have indicated a safety profile that demonstrate that these drugs are generally well tolerated, without significant adverse effects observed in studied population samples, the next logical step is translation into a clinical trial of a NK-1R antagonist in human TBI. One would hypothesize that neurokinin-1 receptor antagonism in human TBI patients, akin to animal models, would result in an attenuation of BBB breakdown and therefore a reduction in post-TBI cerebral oedema, mitigating the resultant intracranial pressure rise and poor clinical outcomes that follow.
A multi-centre phase II clinical trial of an investigational medicinal product (CTIMP) is planned, assessing the efficacy, tolerability and safety of EU-C-001 (a novel NK-1R antagonist) in ICU patients with moderate to severe traumatic brain injury. It includes both open-label and randomized double-blind placebo-controlled phases to address both pharmacokinetics and putative efficacy. Efficacy will be evaluated through assessment of ICP and Therapy Intensity Level (TIL), as well as other surrogate outcomes including CT and MR imaging, biomarker and cytokine data, as well as functional outcome measures. Once safety is examined in a smaller sample size, a larger phase III randomized-control trial is planned to better assess drug efficacy.

Conclusion
Traumatic brain injury remains a considerable global health issue. Despite significant morbidity and mortality, no noteworthy pharmacological advances have been made towards its management since the introduction of hypertonic saline for ICP control in 1988.229 The growing evidence for substance P’s role in neuroinflammation and resultant development of cerebral oedema following TBI provides a promising novel therapeutic target. Animal studies provide promising results when the action of substance P is blocked at its preferential receptor, the neurokinin-1 receptor.
Despite the current lack of evidence of NK-1R antagonist use in human TBI in the literature, the numerous clinical trials that have been performed in humans to date show NK-1R antagonists to be safe and well tolerated. No significant adverse events were noted in the previously described studies. Considering the apparent safety of NK-1R antagonists in human populations and the promising results seen in animal models of TBI, it would be prudent to investigate NK-1R antagonism in human TBI patients.





Transparency, Rigor, and Reproducibility
This review paper is not comprised of original research and has appropriately researched sources using PubMed’s search engine for MEDLINE. Sourced papers are appropriately cited in the references section.

Acknowledgements
Adel Helmy: Medical Research Council/Royal College of Surgeons of England Clinical Research Training Fellowship (Grant no. G0802251), the NIHR Biomedical Research Centre and the NIHR Brain Injury MedTech Co-operative.

Authorship contribution statement
Adam Safwat: investigation, writing – original draft, visualization
Adel Helmy: conceptualization (equal), supervision (equal), writing – review & editing (equal), funding acquisition (equal)
Arun Gupta: conceptualization (equal), supervision (equal), writing – review & editing (equal), funding acquisition (equal)

Authors’ disclosures
The authors would like to disclose that PresSura Neuro (Melbourne, Australia) are paying to cover staff and study costs for the PANGEA drug study.




References

1. Iaccarino C, Carretta A, Nicolosi F, et al. Epidemiology of severe traumatic brain injury. J Neurosurg Sci 2018;62(5):535–541; doi: 10.23736/s0390-5616.18.04532-0.
2. Rubiano AM, Carney N, Chesnut R, et al. Global neurotrauma research challenges and opportunities. Nature 2015;527(7578):S193–S197; doi: 10.1038/nature16035.
3. Rosenfeld JV, Maas AI, Bragge P, et al. Early management of severe traumatic brain injury. Lancet 2012;380(9847):1088–1098; doi: 10.1016/s0140-6736(12)60864-2.
4. Braakman R, Gelpke GJ, Habbema JD, et al. Systematic selection of prognostic features in patients with severe head injury. Neurosurgery 1980;6(4):362–70.
5. Lefkovits AM, Hicks AJ, Downing M, et al. Surviving the “silent epidemic”: A qualitative exploration of the long-term journey after traumatic brain injury. Neuropsychol Rehabil 2020;1–25; doi: 10.1080/09602011.2020.1787849.
6. Maas AIR, Menon DK, Adelson PD, et al. Traumatic brain injury: integrated approaches to improve prevention, clinical care, and research. Lancet Neurology 2017;16(12):987–1048; doi: 10.1016/s1474-4422(17)30371-x.
7. Servadei F, Begliomini C, Gardini E, et al. Effect of Italy’s motorcycle helmet law on traumatic brain injuries. Injury Prev 2003;9(3):257; doi: 10.1136/ip.9.3.257.
8. Chiu W-T, Huang S-J, Tsai S-H, et al. The impact of time, legislation, and geography on the epidemiology of traumatic brain injury. J Clin Neurosci 2007;14(10):930–935; doi: 10.1016/j.jocn.2006.08.004.
9. Gaetz M. The neurophysiology of brain injury. Clin Neurophysiol 2004;115(1):4–18; doi: 10.1016/s1388-2457(03)00258-x.
10. Sulhan S, Lyon KA, Shapiro LA, et al. Neuroinflammation and blood–brain barrier disruption following traumatic brain injury: Pathophysiology and potential therapeutic targets. J Neurosci Res 2020;98(1):19–28; doi: 10.1002/jnr.24331.
11. Sorby-Adams AJ, Marcoionni AM, Dempsey ER, et al. The Role of Neurogenic Inflammation in Blood-Brain Barrier Disruption and Development of Cerebral Oedema Following Acute Central Nervous System (CNS) Injury. Int J Mol Sci 2017;18(8):1788; doi: 10.3390/ijms18081788.
12. Balestreri M, Czosnyka M, Hutchinson P, et al. Impact of intracranial pressure and cerebral perfusion pressure on severe disability and mortality after head injury. Neurocrit Care 2006;4(1):8–13; doi: 10.1385/ncc:4:1:008.
13. Barone DG, Czosnyka M. Brain Monitoring: Do We Need a Hole? An Update on Invasive and Noninvasive Brain Monitoring Modalities. Sci World J 2014;2014:1–6; doi: 10.1155/2014/795762.
14. O’leary RA, Nichol AD. Pathophysiology of severe traumatic brain injury. J Neurosurg Sci 2018;62(5):542–548; doi: 10.23736/s0390-5616.18.04501-0.
15. Turner R, Vink R. Inhibition of neurogenic inflammation as a novel treatment for ischemic stroke. Drug News Perspect 2007;20(4):221; doi: 10.1358/dnp.2007.20.4.1103527.
16. Kleindienst A, Brabant G, Morgenthaler NG, et al. Brain Edema XIV. Acta Neurochir Suppl 2009;106:221–224; doi: 10.1007/978-3-211-98811-4_41.
17. Leonard AV, Manavis J, Blumbergs PC, et al. Changes in substance P and NK1 receptor immunohistochemistry following human spinal cord injury. Spinal Cord 2014;52(1):17–23; doi: 10.1038/sc.2013.136.
18. Lorente L, Martín MM, Pérez-Cejas A, et al. Persistently High Serum Substance P Levels and Early Mortality in Patients with Severe Traumatic Brain Injury. World Neurosurg 2019;132:e613–e617; doi: 10.1016/j.wneu.2019.08.064.
19. Zhou Y, Ye H, Lu W. Serum Substance P Concentration in Children With Traumatic Brain Injury: A First Report. World Neurosurg 2021;147:e200–e205; doi: 10.1016/j.wneu.2020.12.009.
20. Corrigan F, Mander KA, Leonard AV, et al. Neurogenic inflammation after traumatic brain injury and its potentiation of classical inflammation. J Neuroinflamm 2016;13(1):264; doi: 10.1186/s12974-016-0738-9.
21. Beaumont A, Marmarou A, Hayasaki K, et al. The permissive nature of blood brain barrier (BBB) opening in edema formation following traumatic brain injury. 2000;125–129; doi: 10.1007/978-3-7091-6346-7_26.
22. Barzó P, Marmarou A, Fatouros P, et al. Brain Edema X, Proceedings of the Tenth International Symposium San Diego, California, October 20–23, 1996. 1997;119–122; doi: 10.1007/978-3-7091-6837-0_37.
23. Bayliss WM. On the origin from the spinal cord of the vaso‐dilator fibres of the hind‐limb, and on the nature of these fibres1. J Physiology 1901;26(3–4):173–209; doi: 10.1113/jphysiol.1901.sp000831.
24. Harrison S, Geppetti P. Substance P. Int J Biochem Cell Biology 2001;33(6):555–576; doi: 10.1016/s1357-2725(01)00031-0.
25. Black PH. Stress and the inflammatory response: A review of neurogenic inflammation. Brain Behav Immun 2002;16(6):622–653; doi: 10.1016/s0889-1591(02)00021-1.
26. Corrigan F, Vink R, Turner RJ. Inflammation in acute CNS injury: a focus on the role of substance P. Brit J Pharmacol 2016;173(4):703–715; doi: 10.1111/bph.13155.
27. Donkin JJ, Nimmo AJ, Cernak I, et al. Substance P is Associated with the Development of Brain Edema and Functional Deficits after Traumatic Brain Injury. J Cereb Blood Flow Metabolism 2009;29(8):1388–1398; doi: 10.1038/jcbfm.2009.63.
28. Brain SD, Williams TJ. Interactions between the tachykinins and calcitonin gene‐related peptide lead to the modulation of oedema formation and blood flow in rat skin. Brit J Pharmacol 1989;97(1):77–82; doi: 10.1111/j.1476-5381.1989.tb11926.x.
29. Quartu M, Diaz G, Floris A, et al. Calcitonin gene-related peptide in the human trigeminal sensory system at developmental and adult life stages: Immunohistochemistry, neuronal morphometry and coexistence with substance P. J Chem Neuroanat 1992;5(2):143–157; doi: 10.1016/0891-0618(92)90040-w.
30. Seybold VS, McCarson KE, Mermelstein PG, et al. Calcitonin Gene-Related Peptide Regulates Expression of Neurokinin 1 Receptors by Rat Spinal Neurons. J Neurosci 2003;23(5):1816–1824; doi: 10.1523/jneurosci.23-05-01816.2003.
31. Uddman R, Edvinsson L, Owman C, et al. Perivascular Substance P: Occurrence and Distribution in Mammalian Pial Vessels. J Cereb Blood Flow Metabolism 1981;1(2):227–231; doi: 10.1038/jcbfm.1981.24.
32. Walker K, Perkins M, Dray A. Kinins and kinin receptors in the nervous system. Neurochem Int 1995;26(1):1–16; doi: 10.1016/0197-0186(94)00114-a.
33. Albert-Weissenberger C, Mencl S, Hopp S, et al. Role of the kallikrein–kinin system in traumatic brain injury. Front Cell Neurosci 2014;8:345; doi: 10.3389/fncel.2014.00345.
34. Thornton E, Ziebell JM, Leonard AV, et al. Kinin Receptor Antagonists as Potential Neuroprotective Agents in Central Nervous System Injury. Molecules 2010;15(9):6598–6618; doi: 10.3390/molecules15096598.
35. Raidoo DM, Bhoola KD. Pathophysiology of the Kallikrein-Kinin System in Mammalian Nervous Tissue. Pharmacol Therapeut 1998;79(2):105–127; doi: 10.1016/s0163-7258(98)00011-4.
36. Bertrand C, Geppetti P. Tachykinin and kinin receptor antagonists: therapeutic perspectives in allergic airway disease. Trends Pharmacol Sci 1996;17(7):255–259; doi: 10.1016/0165-6147(96)10027-4.
37. Geppetti P. Sensory neuropeptide release by bradykinin: mechanisms and pathophysiological implications. Regul Peptides 1993;47(1):1–23; doi: 10.1016/0167-0115(93)90268-d.
38. Shakur H, Andrews P, Asser T, et al. The BRAIN TRIAL: a randomised, placebo controlled trial of a Bradykinin B2 receptor antagonist (Anatibant) in patients with traumatic brain injury. Trials 2009;10(1):109; doi: 10.1186/1745-6215-10-109.
39. Ker K, Blackhall K. Bradykinin beta‐2 receptor antagonists for acute traumatic brain injury. Cochrane Db Syst Rev 2008;(1):CD006686; doi: 10.1002/14651858.cd006686.pub2.
40. Euler US v., Gaddum JH. An unidentified depressor substance in certain tissue extracts. J Physiology 1931;72(1):74–87; doi: 10.1113/jphysiol.1931.sp002763.
41. Gaddum JH, Schild H. Depressor substances in extracts of intestine. J Physiology 1934;83(1):1–14; doi: 10.1113/jphysiol.1934.sp003206.
42. Lembeck F, Zetler G. Substance P: A Polypeptide of Possible Physiological Significance, Especially Within the Nervous System. Int Rev Neurobiol 1962;4:159–215; doi: 10.1016/s0074-7742(08)60022-7.
43. Hökfelt T, Pernow B, Wahren J. Substance P: a pioneer amongst neuropeptides. J Intern Med 2001;249(1):27–40; doi: 10.1046/j.0954-6820.2000.00773.x.
44. CHANG MM, LEEMAN SE, NIALL HD. Amino-acid Sequence of Substance P. Nat New Biology 1971;232(29):86–87; doi: 10.1038/newbio232086a0.
45. TREGEAR GW, NIALL HD, POTTS JT, et al. Synthesis of Substance P. Nat New Biology 1971;232(29):87–89; doi: 10.1038/newbio232087a0.
46. POWELL D, LEEMAN S, TREGEAR GW, et al. Radioimmunoassay for Substance P. Nat New Biology 1973;241(112):252–254; doi: 10.1038/newbio241252a0.
47. Emson PC, Jessell T, Paxinos G, et al. Substance P in the amygdaloid complex, bed nucleus and stria terminalis of the rat brain. Brain Res 1978;149(1):97–105; doi: 10.1016/0006-8993(78)90590-5.
48. Robinson SE, Schwartz JP, Costa E. Substance P in the superior cervical ganglion and the submaxillary gland of the rat. Brain Res 1980;182(1):11–17; doi: 10.1016/0006-8993(80)90826-4.
49. Takahashi T, Konishi S, Powell D, et al. Identification of the motoneuron-depolarizing peptide in bovine dorsal root as hypothalamic substance P. Brain Res 1974;73(1):59–69; doi: 10.1016/0006-8993(74)91007-5.
50. Skrabanek P, Cannon D, Kirrane J, et al. Circulating immunoreactive substance P in man. Irish J Med Sci 1976;145(1):399; doi: 10.1007/bf02938979.
51. Brownstein MJ, Mroz EA, Kizer JS, et al. Regional distribution of substance P in the brain of the rat. Brain Res 1976;116(2):299–305; doi: 10.1016/0006-8993(76)90907-0.
52. Kangawa K, Minamino N, Fukuda A, et al. Neuromedin K: A novel mammalian tachykinin identified in porcine spinal cord. Biochem Bioph Res Co 1983;114(2):533–540; doi: 10.1016/0006-291x(83)90813-6.
53. KIMURA S, OKADA M, SUGITA Y, et al. Novel Neuropeptides, Neurokinin α and β, Isolated from Porcine Spinal Cord. Proc Jpn Acad Ser B 1983;59(4):101–104; doi: 10.2183/pjab.59.101.
54. Tatemoto K, Lundberg JM, Jörnvall H, et al. Neuropeptide K: Isolation, structure and biological activities of a novel brain tachykinin. Biochem Bioph Res Co 1985;128(2):947–953; doi: 10.1016/0006-291x(85)90138-x.
55. Kage R, McGregor GP, Thim L, et al. Neuropeptide‐γ: A Peptide Isolated from Rabbit Intestine that Is Derived from γ‐Preprotachykinin. J Neurochem 1988;50(5):1412–1417; doi: 10.1111/j.1471-4159.1988.tb03024.x.
56. Zhang Y, Lu L, Furlonger C, et al. Hemokinin is a hematopoietic-specific tachykinin that regulates B lymphopoiesis. Nat Immunol 2000;1(5):392–397; doi: 10.1038/80826.
57. Erspamer V. Biogenic Amines and Active Polypeptides of the Amphibian Skin. Annu Rev Pharmacolog 1971;11(1):327–350; doi: 10.1146/annurev.pa.11.040171.001551.
58. Anonymous. PubChem Compound Summary for CID 36511, Substance P. n.d.
59. Mashaghi A, Marmalidou A, Tehrani M, et al. Neuropeptide substance P and the immune response. Cell Mol Life Sci 2016;73(22):4249–4264; doi: 10.1007/s00018-016-2293-z.
60. Almeida TA, Rojo J, Nieto PM, et al. Tachykinins and Tachykinin Receptors: Structure and Activity Relationships. Curr Med Chem 2004;11(15):2045–2081; doi: 10.2174/0929867043364748.
61. O’Connor TM, O’Connell J, O’Brien DI, et al. The role of substance P in inflammatory disease. J Cell Physiol 2004;201(2):167–180; doi: 10.1002/jcp.20061.
62. Severini C, Improta G, Falconieri-Erspamer G, et al. The Tachykinin Peptide Family. Pharmacol Rev 2002;54(2):285–322; doi: 10.1124/pr.54.2.285.
63. Pennefather JN, Lecci A, Candenas ML, et al. Tachykinins and tachykinin receptors: a growing family. Life Sci 2004;74(12):1445–1463; doi: 10.1016/j.lfs.2003.09.039.
64. Carter M, Krause J. 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(7):2203–2214; doi: 10.1523/jneurosci.10-07-02203.1990.
65. Nakanishi S. Substance P precursor and kininogen: their structures, gene organizations, and regulation. Physiol Rev 1987;67(4):1117–1142; doi: 10.1152/physrev.1987.67.4.1117.
66. Bannon MJ, Poosch MS, Haverstick DM, et al. Preprotachykinin gene expression in the human basal ganglia: characterization of mRNAs and pre-mRNAs produced by alternate RNA splicing. Mol Brain Res 1992;12(1–3):225–231; doi: 10.1016/0169-328x(92)90088-s.
67. Haverstick DM, Jeziorski M, Bannon MJ. Developmental Profile of Striatal Preprotachykinin Gene Expression. J Neurochem 1990;55(3):764–768; doi: 10.1111/j.1471-4159.1990.tb04557.x.
68. Harmar A, Schofield JG, Keen P. Cycloheximide-sensitive synthesis of substance P by isolated dorsal root ganglia. Nature 1980;284(5753):267–269; doi: 10.1038/284267a0.
69. Brimijoin S, Lundberg JM, Brodin E, et al. Axonal transport of substance P in the vagus and sciatic nerves of the guinea pig. Brain Res 1980;191(2):443–457; doi: 10.1016/0006-8993(80)91293-7.
70. Takahashi T, Otsuka M. Regional distribution of substance P in the spinal cord and nerve roots of the cat and the effect of dorsal root section. Brain Res 1975;87(1):1–11; doi: 10.1016/0006-8993(75)90774-x.
71. Noguchi K, Morita Y, Kiyama H, et al. A noxious stimulus induces the preprotachykinin-A gene expression in the rat dorsal root ganglion: a quantitative study using in situ hybridization histochemistry. Mol Brain Res 1988;4(1):31–35; doi: 10.1016/0169-328x(88)90015-0.
72. Kantner RM, Kirby ML, Goldstein BD. Increase in substance P in the dorsal horn during a chemogenic nociceptive stimulus. Brain Res 1985;338(1):196–199; doi: 10.1016/0006-8993(85)90268-9.
73. Randić M, Miletić V. Effect of substance P in cat dorsal horn neurones activated by noxious stimuli. Brain Res 1977;128(1):164–169; doi: 10.1016/0006-8993(77)90245-1.
74. Skidgel RA, Engelbrecht S, Johnson AR, et al. Hydrolysis of substance P and neurotensin by converting enzyme and neutral endopeptidase. Peptides 1984;5(4):769–776; doi: 10.1016/0196-9781(84)90020-2.
75. Nadel JA. Neutral endopeptidase modulates neurogenic inflammation. European Respir J 1991;4(6):745–54.
76. Matsas R, Kenny AJ, Turner AJ. The metabolism of neuropeptides. The hydrolysis of peptides, including enkephalins, tachykinins and their analogues, by endopeptidase-24.11. Biochem J 1984;223(2):433–440; doi: 10.1042/bj2230433.
77. Probert L, Hanley MR. The immunocytochemical localisation of ‘substance-P-degrading enzyme’ within the rat spinal cord. Neurosci Lett 1987;78(2):132–137; doi: 10.1016/0304-3940(87)90621-5.
78. Azaryan AV, Galoyan AA. Substrate specificity of cerebral cathepsin D and high‐Mr aspartic endopeptidase. J Neurosci Res 1988;19(2):268–271; doi: 10.1002/jnr.490190213.
79. KAGEYAMA T. Rabbit procathepsin E and cathepsin E. Eur J Biochem 1993;216(3):717–728; doi: 10.1111/j.1432-1033.1993.tb18191.x.
80. Blumberg S, Teichberg VI, Charli JL, et al. Cleavage of substance P to an N-terminal tetrapeptide and a C-terminal heptapeptide by a post-proline cleaving enzyme from bovine brain. Brain Res 1980;192(2):477–486; doi: 10.1016/0006-8993(80)90898-7.
81. Heymann E, Mentlein R. Liver dipeptidyl aminopeptidase IV hydrolyzes substance P. Febs Lett 1978;91(2):360–364; doi: 10.1016/0014-5793(78)81210-1.
82. Hooper NM, Turner AJ. Isolation of two differentially glycosylated forms of peptidyl-dipeptidase A (angiotensin converting enzyme) from pig brain: a re-evaluation of their role in neuropeptide metabolism. Biochem J 1987;241(3):625–633; doi: 10.1042/bj2410625.
83. Sakurada T, Tan-No K, Yamada T, et al. Phosphoramidon potentiates mammalian tachykinin-induced biting, licking and scratching behaviour in mice. Pharmacol Biochem Be 1990;37(4):779–783; doi: 10.1016/0091-3057(90)90563-w.
84. Maria GUD, Bellofiore S, Geppetti P. Regulation of airway neurogenic inflammation by neutral endopeptidase. Eur Respir J 1998;12(6):1454–1462; doi: 10.1183/09031936.98.12061454.
85. Lu B, Figini M, Emanueli C, et al. The control of microvascular permeability and blood pressure by neutral endopeptidase. Nat Med 1997;3(8):904–907; doi: 10.1038/nm0897-904.
86. Wang L, Ahmad S, Benter IF, et al. Differential processing of substance P and neurokinin A by plasma dipeptidyl(amino)peptidase IV, aminopeptidase M and angiotensin converting enzyme. Peptides 1991;12(6):1357–1364; doi: 10.1016/0196-9781(91)90220-j.
87. Cascieri MA, Bull HG, Mumford RA, et al. Carboxyl-terminal tripeptidyl hydrolysis of substance P by purified rabbit lung angiotensin-converting enzyme and the potentiation of substance P activity in vivo by captopril and MK-422. Mol Pharmacol 1984;25(2):287–93.
88. Emanueli C, Grady EF, Madeddu P, et al. Acute ACE Inhibition Causes Plasma Extravasation in Mice That is Mediated by Bradykinin and Substance P. Hypertension 1998;31(6):1299–1304; doi: 10.1161/01.hyp.31.6.1299.
89. Corbally N, Powell D, Tipton KF. The binding of endogenous and exogenous substance-P in human plasma. Biochem Pharmacol 1990;39(7):1161–1166; doi: 10.1016/0006-2952(90)90257-l.
90. McGregor GP, Bloom SR. Radioimmunoassay of substance P and its stability in tissue. Life Sci 1983;32(6):655–662; doi: 10.1016/0024-3205(83)90211-4.
91. Rameshwar P, Joshi DD, Yadav P, et al. Mimicry between neurokinin-1 and fibronectin may explain the transport and stability of increased substance P immunoreactivity in patients with bone marrow fibrosis. Blood 2001;97(10):3025–3031; doi: 10.1182/blood.v97.10.3025.
92. Sutoo D, Yabe K, Akiyama K. Quantitative imaging of substance P in the human brain using a brain mapping analyzer. Neurosci Res 1999;35(4):339–346; doi: 10.1016/s0168-0102(99)00101-7.
93. McRitchie DA, Töurk I. Distribution of substance P‐like immunoreactive neurons and terminals throughout the nucleus of the solitary tract in the human brainstem. J Comp Neurol 1994;343(1):83–101; doi: 10.1002/cne.903430107.
94. Przewłocki R, Gramsch C, Pasi A, et al. Characterization and localization of immunoreactive dynorphin, α-neo-endorphin, met-enkephalin and substance P in human spinal cord. Brain Res 1983;280(1):95–103; doi: 10.1016/0006-8993(83)91177-0.
95. Emson PC, Arregui A, Clement-Jones V, et al. Regional distribution of methionine-enkephalin and substance P-like immunoreactivity in normal human brain and in Huntington’s disease. Brain Res 1980;199(1):147–160; doi: 10.1016/0006-8993(80)90237-1.
96. Edvinsson L, Rosendal-Helgesen S, Uddman R. Substance P: Localization, concentration and release in cerebral arteries, choroid plexus and dura mater. Cell Tissue Res 1983;234(1):1–7; doi: 10.1007/bf00217397.
97. Mai JK, Stephens PH, Hopf A, et al. Substance P in the human brain. Neuroscience 1986;17(3):709–739; doi: 10.1016/0306-4522(86)90041-2.
98. Cuello AC, Polak JM, Pearse AGE. SUBSTANCE P: A NATURALLY OCCURRING TRANSMITTER IN HUMAN SPINAL CORD. Lancet 1976;308(7994):1054–1056; doi: 10.1016/s0140-6736(76)90968-5.
99. Pickel VM, Reis DJ, Leeman SE. Ultrastructural localization of substance P in neurons of spinal cord. Brain Res 1977;122(3):534–540; doi: 10.1016/0006-8993(77)90463-2.
100. Lai J-P, Zhan G-X, Campbell DE, et al. Detection of substance P and its receptor in human fetal microglia. Neuroscience 2000;101(4):1137–1144; doi: 10.1016/s0306-4522(00)00398-5.
101. Michel J-P, Sakamoto N, Bouvier R, et al. Substance P-immunoreactive astrocytes related to deep white matter and striatal blood vessels in human brain. Brain Res 1986;377(2):383–387; doi: 10.1016/0006-8993(86)90886-3.
102. Milner P, Bodin P, Guiducci S, et al. Regulation of substance P mRNA expression in human dermal microvascular endothelial cells. Clin Exp Rheumatol 2004;22(3 Suppl 33):S24-7.
103. Watanabe M, Nakayasu K, Iwatsu M, et al. Endogenous Substance P in Corneal Epithelial Cells and Keratocytes. Jpn J Ophthalmol 2002;46(6):616–620; doi: 10.1016/s0021-5155(02)00617-2.
104. Lai J-P, Douglas SD, Ho W-Z. Human lymphocytes express substance P and its receptor. J Neuroimmunol 1998;86(1):80–86; doi: 10.1016/s0165-5728(98)00025-3.
105. Lambrecht BN, Germonpré PR, Everaert EG, et al. Endogenously produced substance P contributes to lymphocyte proliferation induced by dendritic cells and direct TCR ligation. Eur J Immunol 1999;29(12):3815–3825; doi: 10.1002/(sici)1521-4141(199912)29:12<3815::aid-immu3815>3.0.co;2-#.
106. Ho WZ, Lai JP, Zhu XH, et al. Human monocytes and macrophages express substance P and neurokinin-1 receptor. J Immunol Baltim Md 1950 1997;159(11):5654–60.
107. Weinstock JV, Blum A, Walder J, et al. Eosinophils from granulomas in murine schistosomiasis mansoni produce substance P. J Immunol Baltim Md 1950 1988;141(3):961–6.
108. Li Y, Douglas SD, Ho W. Human Stem Cells Express Substance P Gene and Its Receptor. J Hematoth Stem Cell 2000;9(4):445–452; doi: 10.1089/152581600419107.
109. Cho KJ, Trzaska KA, Greco SJ, et al. Neurons Derived From Human Mesenchymal Stem Cells Show Synaptic Transmission and Can Be Induced to Produce the Neurotransmitter Substance P by Interleukin‐1α. Stem Cells 2005;23(3):383–391; doi: 10.1634/stemcells.2004-0251.
110. Garcia-Recio S, Gascón P. Biological and Pharmacological Aspects of the NK1-Receptor. Biomed Res Int 2015;2015:495704; doi: 10.1155/2015/495704.
111. Gerard NP, Garraway LA, Eddy RL, et al. Human substance P receptor (NK-1): organization of the gene, chromosome localization and functional expression of cDNA clones. Biochemistry-us 1991;30(44):10640–10646; doi: 10.1021/bi00108a006.
112. Gerard NP, Bao L, Xiao-Ping H, et al. Molecular aspects of the tachykinin receptors. Regul Peptides 1993;43(1–2):21–35; doi: 10.1016/0167-0115(93)90404-v.
113. Fong TM, Anderson SA, Yu H, et al. Differential activation of intracellular effector by two isoforms of human neurokinin-1 receptor. Mol Pharmacol 1992;41(1):24–30.
114. Li H, Leeman SE, Slack BE, 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 National Acad Sci 1997;94(17):9475–9480; doi: 10.1073/pnas.94.17.9475.
115. Lai J-P, Lai S, Tuluc F, et al. Differences in the length of the carboxyl terminus mediate functional properties of neurokinin-1 receptor. Proc National Acad Sci 2008;105(34):12605–12610; doi: 10.1073/pnas.0806632105.
116. Caberlotto L, Hurd YL, Murdock P, et al. Neurokinin 1 receptor and relative abundance of the short and long isoforms in the human brain. Eur J Neurosci 2003;17(9):1736–1746; doi: 10.1046/j.1460-9568.2003.02600.x.
117. Whitty CJ, Paul MA, Bannon MJ. Neurokinin receptor mRNA localization in human midbrain dopamine neurons. J Comp Neurol 1997;382(3):394–400; doi: 10.1002/(sici)1096-9861(19970609)382:3<394::aid-cne6>3.0.co;2-z.
118. Greeno EW, Mantyh P, Vercellotti GM, et al. Functional neurokinin 1 receptors for substance P are expressed by human vascular endothelium. J Exp Medicine 1993;177(5):1269–1276; doi: 10.1084/jem.177.5.1269.
119. Böckmann S. Substance P (NK1) receptor expression by human colonic epithelial cell line Caco-2. Peptides 2002;23(10):1783–1791; doi: 10.1016/s0196-9781(02)00135-3.
120. Liu J, Hu J, Zhu Q, et al. Substance P receptor expression in human skin keratinocytes and fibroblasts. Brit J Dermatol 2006;155(4):657–662; doi: 10.1111/j.1365-2133.2006.07408.x.
121. Maghni K, Michoud M-C, Alles M, et al. Airway Smooth Muscle Cells Express Functional Neurokinin-1 Receptors and the Nerve-Derived Preprotachykinin-A Gene. Am J Resp Cell Mol 2003;28(1):103–110; doi: 10.1165/rcmb.4635.
122. Mantyh PW, Johnson DJ, Boehmer CG, et al. Substance P receptor binding sites are expressed by glia in vivo after neuronal injury. Proc National Acad Sci 1989;86(13):5193–5197; doi: 10.1073/pnas.86.13.5193.
123. Feistritzer C, Clausen J, Sturn DH, et al. Natural killer cell functions mediated by the neuropeptide substance P. Regul Peptides 2003;116(1–3):119–126; doi: 10.1016/s0167-0115(03)00193-9.
124. Marriott I, Bost KL. Expression of authentic substance P receptors in murine and human dendritic cells. J Neuroimmunol 2001;114(1–2):131–141; doi: 10.1016/s0165-5728(00)00466-5.
125. Burmeister AR, Johnson MB, Chauhan VS, et al. Human microglia and astrocytes constitutively express the neurokinin-1 receptor and functionally respond to substance P. J Neuroinflamm 2017;14(1):245; doi: 10.1186/s12974-017-1012-5.
126. Kleij HPM van der, Ma D, Redegeld FAM, et al. Functional Expression of Neurokinin 1 Receptors on Mast Cells Induced by IL-4 and Stem Cell Factor. J Immunol 2003;171(4):2074–2079; doi: 10.4049/jimmunol.171.4.2074.
127. Seelig A, Alt T, Lotz S, et al. Binding of Substance P Agonists to Lipid Membranes and to the Neurokinin-1 Receptor †. Biochemistry-us 1996;35(14):4365–4374; doi: 10.1021/bi952434q.
128. McConalogue K, Corvera CU, Gamp PD, et al. Desensitization of the Neurokinin-1 Receptor (NK1-R) in Neurons: Effects of Substance P on the Distribution of NK1-R, Gαq/11, G-Protein Receptor Kinase-2/3, and β-Arrestin-1/2. Mol Biol Cell 1998;9(8):2305–2324; doi: 10.1091/mbc.9.8.2305.
129. Nishimura K, Warabi K, Roush ED, et al. Characterization of GRK2-Catalyzed Phosphorylation of the Human Substance P Receptor in Sf9 Membranes †. Biochemistry-us 1998;37(5):1192–1198; doi: 10.1021/bi972302s.
130. Grady EF, Garland AM, Gamp PD, et al. Delineation of the endocytic pathway of substance P and its seven-transmembrane domain NK1 receptor. Mol Biol Cell 1995;6(5):509–524; doi: 10.1091/mbc.6.5.509.
131. Takeda Y, Blount P, Sachais BS, et al. Ligand Binding Kinetics of Substance P and Neurokinin A Receptors Stably Expressed in Chinese Hamster Ovary Cells and Evidence for Differential Stimulation of Inositol 1,4,5‐Trisphosphate and Cyclic AMP Second Messenger Responses. J Neurochem 1992;59(2):740–745; doi: 10.1111/j.1471-4159.1992.tb09430.x.
132. Koizumi H, Yasui C, Fukaya T, et al. Substance P induces inositol 1,4,5‐trisphosphate and intracellular free calcium increase in cultured normal human epidermal keratinocytes. Exp Dermatol 1994;3(1):40–44; doi: 10.1111/j.1600-0625.1994.tb00264.x.
133. Christian C, Gilbert M, Payan DG. Stimulation of Transcriptional Regulatory Activity by Substance P. Neuroimmunomodulat 1994;1(3):159–164; doi: 10.1159/000097156.
134. Koon H-W, Zhao D, Zhan Y, et al. Substance P-Stimulated Interleukin-8 Expression in Human Colonic Epithelial Cells Involves Protein Kinase Cδ Activation. J Pharmacol Exp Ther 2005;314(3):1393–1400; doi: 10.1124/jpet.105.088013.
135. Guo C-J, Lai J-P, Luo H-M, et al. Substance P up-regulates macrophage inflammatory protein-1β expression in human T lymphocytes. J Neuroimmunol 2002;131(1–2):160–167; doi: 10.1016/s0165-5728(02)00277-1.
136. Fiebich BL, Schleicher S, Butcher RD, et al. The Neuropeptide Substance P Activates p38 Mitogen-Activated Protein Kinase Resulting in IL-6 Expression Independently from NF-κB. J Immunol 2000;165(10):5606–5611; doi: 10.4049/jimmunol.165.10.5606.
137. Derocq J-M, Ségui M, Blazy C, et al. Effect of substance P on cytokine production by human astrocytic cells and blood mononuclear cells: characterization of novel tachykinin receptor antagonists. Febs Lett 1996;399(3):321–325; doi: 10.1016/s0014-5793(96)01346-4.
138. Lieb K, Fiebich BL, Berger M, et al. The neuropeptide substance P activates transcription factor NF-kappa B and kappa B-dependent gene expression in human astrocytoma cells. J Immunol Baltim Md 1950 1997;159(10):4952–8.
139. Otsuka M, Yoshioka K. Neurotransmitter functions of mammalian tachykinins. Physiol Rev 1993;73(2):229–308; doi: 10.1152/physrev.1993.73.2.229.
140. Louis SM, Jamieson A, Russell NJW, et al. The role of substance P and calcitonin gene-related peptide in neurogenic plasma extravasation and vasodilatation in the rat. Neuroscience 1989;32(3):581–586; doi: 10.1016/0306-4522(89)90281-9.
141. Koninck YD, Henry JL. Substance P-mediated slow excitatory postsynaptic potential elicited in dorsal horn neurons in vivo by noxious stimulation. Proc National Acad Sci 1991;88(24):11344–11348; doi: 10.1073/pnas.88.24.11344.
142. Honda I, Kohrogi H, Yamaguchi T, et al. Enkephalinase Inhibitor Potentiates Substance P- and Capsaicin-induced Bronchial Smooth Muscle Contractions in Humans. Am Rev Respir Dis 1991;143(6):1416–1418; doi: 10.1164/ajrccm/143.6.1416.
143. Mhanna MJ, Dreshaj IA, Haxhiu MA, et al. Mechanism for substance P-induced relaxation of precontracted airway smooth muscle during development. Am J Physiol-lung C 1999;276(1):L51–L56; doi: 10.1152/ajplung.1999.276.1.l51.
144. Huston JP, Hasenöhrl RU, Boix F, et al. Sequence-specific effects of neurokinin substance P on memory, reinforcement, and brain dopamine activity. Psychopharmacology 1993;112(2–3):147–162; doi: 10.1007/bf02244906.
145. Hornby PJ. Central neurocircuitry associated with emesis. Am J Medicine 2001;111(8):106–112; doi: 10.1016/s0002-9343(01)00849-x.
146. Bonham AC. Neurotransmitters in the CNS control of breathing. Resp Physiol 1995;101(3):219–230; doi: 10.1016/0034-5687(95)00045-f.
147. Park S-W, Yan Y-P, Satriotomo I, et al. Substance P is a promoter of adult neural progenitor cell proliferation under normal and ischemic conditions. J Neurosurg 2007;107(3):593–599; doi: 10.3171/jns-07/09/0593.
148. Reid TW, Murphy CJ, Iwahashi CK, et al. Stimulation of epithelial cell growth by the neuropeptide substance P. J Cell Biochem 1993;52(4):476–485; doi: 10.1002/jcb.240520411.
149. Palma C, Manzini S. Substance P induces secretion of immunomodulatory cytokines by human astrocytoma cells. J Neuroimmunol 1998;81(1–2):127–137; doi: 10.1016/s0165-5728(97)00167-7.
150. Pavón-Romero GF, Serrano-Pérez NH, García-Sánchez L, et al. Neuroimmune Pathophysiology in Asthma. Frontiers Cell Dev Biology 2021;9:663535; doi: 10.3389/fcell.2021.663535.
151. Zhan M, Zheng W, Jiang Q, et al. Upregulated expression of substance P (SP) and NK1R in eczema and SP-induced mast cell accumulation. Cell Biol Toxicol 2017;33(4):389–405; doi: 10.1007/s10565-016-9379-0.
152. Muñoz M, Coveñas R. Involvement of substance P and the NK-1 receptor in cancer progression. Peptides 2013;48:1–9; doi: 10.1016/j.peptides.2013.07.024.
153. Li Y, Douglas SD, Song L, et al. Substance P enhances HIV-1 replication in latently infected human immune cells. J Neuroimmunol 2001;121(1–2):67–75; doi: 10.1016/s0165-5728(01)00439-8.
154. Okamura Y, Mishima S, Kashiwakura J, et al. The dual regulation of substance P-mediated inflammation via human synovial mast cells in rheumatoid arthritis. Allergol Int 2017;66:S9–S20; doi: 10.1016/j.alit.2017.03.002.
155. Patel M, Subas SV, Ghani MR, et al. Role of Substance P in the Pathophysiology of Inflammatory Bowel Disease and Its Correlation With the Degree of Inflammation. Cureus 2020;12(10):e11027; doi: 10.7759/cureus.11027.
156. Brandow AM, Wandersee NJ, Dasgupta M, et al. Substance P is increased in patients with sickle cell disease and associated with haemolysis and hydroxycarbamide use. Brit J Haematol 2016;175(2):237–245; doi: 10.1111/bjh.14300.
157. Ebner K, Singewald N. The role of substance P in stress and anxiety responses. Amino Acids 2006;31(3):251–272; doi: 10.1007/s00726-006-0335-9.
158. Park HS, Won HS, An HJ, et al. Elevated serum substance P level as a predictive marker for moderately emetogenic chemotherapy‐induced nausea and vomiting: A prospective cohort study. Cancer Med-us 2020;10(3):1057–1065; doi: 10.1002/cam4.3693.
159. Mistrova E, Kruzliak P, Dvorakova MC. Role of substance P in the cardiovascular system. Neuropeptides 2016;58:41–51; doi: 10.1016/j.npep.2015.12.005.
160. Serlin Y, Shelef I, Knyazer B, et al. Anatomy and physiology of the blood–brain barrier. Semin Cell Dev Biol 2015;38:2–6; doi: 10.1016/j.semcdb.2015.01.002.
161. Xu L, Nirwane A, Yao Y. Basement membrane and blood–brain barrier. Stroke Vasc Neurology 2018;4(2):78–82; doi: 10.1136/svn-2018-000198.
162. Ballabh P, Braun A, Nedergaard M. The blood–brain barrier: an overview Structure, regulation, and clinical implications. Neurobiol Dis 2004;16(1):1–13; doi: 10.1016/j.nbd.2003.12.016.
163. Stamatovic SM, Johnson AM, Keep RF, et al. Junctional proteins of the blood-brain barrier: New insights into function and dysfunction. Tissue Barriers 2016;4(1):e1154641; doi: 10.1080/21688370.2016.1154641.
164. Obermeier B, Verma A, Ransohoff RM. Chapter 3 The blood–brain barrier. Handb Clin Neurology 2016;133:39–59; doi: 10.1016/b978-0-444-63432-0.00003-7.
165. Rhea EM, Banks WA. Role of the Blood-Brain Barrier in Central Nervous System Insulin Resistance. Front Neurosci-switz 2019;13:521; doi: 10.3389/fnins.2019.00521.
166. Daneman R, Prat A. The Blood–Brain Barrier. Csh Perspect Biol 2015;7(1):a020412; doi: 10.1101/cshperspect.a020412.
167. Abbott NJ, Rönnbäck L, Hansson E. Astrocyte–endothelial interactions at the blood–brain barrier. Nat Rev Neurosci 2006;7(1):41–53; doi: 10.1038/nrn1824.
168. Alvarez JI, Katayama T, Prat A. Glial influence on the blood brain barrier. Glia 2013;61(12):1939–1958; doi: 10.1002/glia.22575.
169. Wong AD, Ye M, Levy AF, et al. The blood-brain barrier: an engineering perspective. Frontiers Neuroeng 2013;6:7; doi: 10.3389/fneng.2013.00007.
170. Su Y, Sinko PJ. Drug delivery across the blood–brain barrier: why is it difficult? how to measure and improve it? Expert Opin Drug Del 2006;3(3):419–435; doi: 10.1517/17425247.3.3.419.
171. Donkin JJ, Vink R. Mechanisms of cerebral edema in traumatic brain injury: therapeutic developments. Curr Opin Neurol 2010;23(3):293–299; doi: 10.1097/wco.0b013e328337f451.
172. Michinaga S, Koyama Y. Pathogenesis of Brain Edema and Investigation into Anti-Edema Drugs. Int J Mol Sci 2015;16(5):9949–9975; doi: 10.3390/ijms16059949.
173. Unterberg AW, Stover J, Kress B, et al. Edema and brain trauma. Neuroscience 2004;129(4):1019–1027; doi: 10.1016/j.neuroscience.2004.06.046.
174. Chiu C-C, Liao Y-E, Yang L-Y, et al. Neuroinflammation in animal models of traumatic brain injury. J Neurosci Meth 2016;272:38–49; doi: 10.1016/j.jneumeth.2016.06.018.
175. Abdul-Muneer PM, Pfister BJ, Haorah J, et al. Role of Matrix Metalloproteinases in the Pathogenesis of Traumatic Brain Injury. Mol Neurobiol 2016;53(9):6106–6123; doi: 10.1007/s12035-015-9520-8.
176. Jha RM, Kochanek PM, Simard JM. Pathophysiology and treatment of cerebral edema in traumatic brain injury. Neuropharmacology 2018;145(Pt B):230–246; doi: 10.1016/j.neuropharm.2018.08.004.
177. Cash A, Theus MH. Mechanisms of Blood–Brain Barrier Dysfunction in Traumatic Brain Injury. Int J Mol Sci 2020;21(9):3344; doi: 10.3390/ijms21093344.
178. Prakash R, Carmichael ST. Blood−brain barrier breakdown and neovascularization processes after stroke and traumatic brain injury. Curr Opin Neurol 2015;28(6):556–564; doi: 10.1097/wco.0000000000000248.
179. Donkin JJ, Cernak I, Blumbergs PC, et al. A Substance P Antagonist Reduces Axonal Injury and Improves Neurologic Outcome When Administered Up to 12 Hours after Traumatic Brain Injury. J Neurotraum 2011;28(2):217–224; doi: 10.1089/neu.2010.1632.
180. Başkaya MK, Rao AM, Doğan A, et al. The biphasic opening of the blood–brain barrier in the cortex and hippocampus after traumatic brain injury in rats. Neurosci Lett 1997;226(1):33–36; doi: 10.1016/s0304-3940(97)00239-5.
181. Kashiba H, Ueda Y, Senba E. Systemic capsaicin in the adult rat differentially affects gene expression for neuropeptides and neurotrophin receptors in primary sensory neurons. Neuroscience 1996;76(1):299–312; doi: 10.1016/s0306-4522(96)00334-x.
182. Nimmo AJ, Cernak I, Heath DL, et al. Neurogenic inflammation is associated with development of edema and functional deficits following traumatic brain injury in rats. Neuropeptides 2004;38(1):40–47; doi: 10.1016/j.npep.2003.12.003.
183. Corrigan F, Leonard A, Ghabriel M, et al. A Substance P Antagonist Improves Outcome in Female Sprague Dawley Rats Following Diffuse Traumatic Brain Injury. Cns Neurosci Ther 2012;18(6):513–515; doi: 10.1111/j.1755-5949.2012.00332.x.
184. Li Q, Wu X, Yang Y, et al. Tachykinin NK1 receptor antagonist L-733,060 and substance P deletion exert neuroprotection through inhibiting oxidative stress and cell death after traumatic brain injury in mice. Int J Biochem Cell Biology 2019;107:154–165; doi: 10.1016/j.biocel.2018.12.018.
185. Vink R, Bahtia KD, Reilly PL. The relationship between intracranial pressure and brain oxygenation following traumatic brain injury in sheep. Acta Neurochir Suppl 2008;189–192; doi: 10.1007/978-3-211-85578-2_37.
186. Gabrielian L, Helps SC, Thornton E, et al. Brain Edema XV. Acta Neurochir Suppl 2013;118:201–204; doi: 10.1007/978-3-7091-1434-6_37.
187. Collins-Praino LE, Corrigan F. Does neuroinflammation drive the relationship between tau hyperphosphorylation and dementia development following traumatic brain injury? Brain Behav Immun 2017;60:369–382; doi: 10.1016/j.bbi.2016.09.027.
188. Corrigan F, Cernak I, McAteer K, et al. NK1 antagonists attenuate tau phosphorylation after blast and repeated concussive injury. Sci Rep-uk 2021;11(1):8861; doi: 10.1038/s41598-021-88237-0.
189. Harford-Wright E, Thornton E, Vink R. Angiotensin-converting enzyme (ACE) inhibitors exacerbate histological damage and motor deficits after experimental traumatic brain injury. Neurosci Lett 2010;481(1):26–29; doi: 10.1016/j.neulet.2010.06.044.
190. Navari RM, Reinhardt RR, Gralla RJ, et al. Reduction of Cisplatin-Induced Emesis by a Selective Neurokinin-1–Receptor Antagonist. New Engl J Medicine 1999;340(3):190–195; doi: 10.1056/nejm199901213400304.
191. Hesketh PJ, Schnadig ID, Schwartzberg LS, et al. Efficacy of the neurokinin‐1 receptor antagonist rolapitant in preventing nausea and vomiting in patients receiving carboplatin‐based chemotherapy. Cancer 2016;122(15):2418–2425; doi: 10.1002/cncr.30054.
192. Aapro M, Rugo H, Rossi G, et al. A randomized phase III study evaluating the efficacy and safety of NEPA, a fixed-dose combination of netupitant and palonosetron, for prevention of chemotherapy-induced nausea and vomiting following moderately emetogenic chemotherapy. Ann Oncol 2014;25(7):1328–1333; doi: 10.1093/annonc/mdu101.
193. Weinstein C, Jordan K, Green S, et al. Single-dose fosaprepitant for the prevention of chemotherapy-induced nausea and vomiting in patients receiving moderately emetogenic chemotherapy regimens: a subgroup analysis from a randomized clinical trial of response in subjects by cancer type. Bmc Cancer 2020;20(1):918; doi: 10.1186/s12885-020-07259-5.
194. Hata A, Okamoto I, Inui N, et al. Randomized, Double-Blind, Phase III Study of Fosnetupitant Versus Fosaprepitant for Prevention of Highly Emetogenic Chemotherapy-Induced Nausea and Vomiting: CONSOLE. J Clin Oncol 2022;40(2):180–188; doi: 10.1200/jco.21.01315.
195. Navari RM, Schwartzberg LS. Evolving role of neurokinin 1-receptor antagonists for chemotherapy-induced nausea and vomiting. Oncotargets Ther 2018;11:6459–6478; doi: 10.2147/ott.s158570.
196. Li Q, Wu Y, Wang W, et al. Effectiveness and safety of combined neurokinin-1 antagonist aprepitant treatment for multiple-day anthracycline-induced nausea and vomiting. Curr Prob Cancer 2019;43(6):100462; doi: 10.1016/j.currproblcancer.2019.01.003.
197. Zhang Y, Yang Y, Zhang Z, et al. Neurokinin-1 Receptor Antagonist-Based Triple Regimens in Preventing Chemotherapy-Induced Nausea and Vomiting: A Network Meta-Analysis. Jnci J National Cancer Inst 2016;109(2):djw217; doi: 10.1093/jnci/djw217.
198. Diemunsch P, Schoeffler P, Bryssine B, et al. Antiemetic activity of the NK1 receptor antagonist GR205171 in the treatment of established postoperative nausea and vomiting after major gynaecological surgery. Bja Br J Anaesth 1999;82(2):274–276; doi: 10.1093/bja/82.2.274.
199. Keller M, Montgomery S, Ball W, et al. Lack of Efficacy of the Substance P (Neurokinin1 Receptor) Antagonist Aprepitant in the Treatment of Major Depressive Disorder. Biol Psychiat 2006;59(3):216–223; doi: 10.1016/j.biopsych.2005.07.013.
200. Ball WA, Snavely DB, Hargreaves RJ, et al. Addition of an NK1 receptor antagonist to an SSRI did not enhance the antidepressant effects of SSRI monotherapy: results from a randomized clinical trial in patients with major depressive disorder. Hum Psychopharmacol Clin Exp 2014;29(6):568–577; doi: 10.1002/hup.2444.
201. Kramer MS, Winokur A, Kelsey J, et al. Demonstration of the Efficacy and Safety of a Novel Substance P (NK1) Receptor Antagonist in Major Depression. Neuropsychopharmacol 2004;29(2):385–392; doi: 10.1038/sj.npp.1300260.
202. Kramer MS, Cutler N, Feighner J, et al. Distinct Mechanism for Antidepressant Activity by Blockade of Central Substance P Receptors. Science 1998;281(5383):1640–1645; doi: 10.1126/science.281.5383.1640.
203. Ratti E, Bettica P, Alexander R, et al. Full central neurokinin-1 receptor blockade is required for efficacy in depression: evidence from orvepitant clinical studies. J Psychopharmacol 2013;27(5):424–434; doi: 10.1177/0269881113480990.
204. Rupniak NMJ, Kramer MS. NK1 receptor antagonists for depression: Why a validated concept was abandoned. J Affect Disorders 2017;223:121–125; doi: 10.1016/j.jad.2017.07.042.
205. Michelson D, Hargreaves R, Alexander R, et al. Lack of efficacy of L-759274, a novel neurokinin 1 (substance P) receptor antagonist, for the treatment of generalized anxiety disorder. Int J Neuropsychoph 2013;16(1):1–11; doi: 10.1017/s1461145712000065.
206. Mathew SJ, Vythilingam M, Murrough JW, et al. A selective neurokinin-1 receptor antagonist in chronic PTSD: A randomized, double-blind, placebo-controlled, proof-of-concept trial. Eur Neuropsychopharm 2011;21(3):221–229; doi: 10.1016/j.euroneuro.2010.11.012.
207. Kwako LE, George DT, Schwandt ML, et al. The neurokinin-1 receptor antagonist aprepitant in co-morbid alcohol dependence and posttraumatic stress disorder: a human experimental study. Psychopharmacology 2015;232(1):295–304; doi: 10.1007/s00213-014-3665-4.
208. Goldstein D, Wang O, Saper J, et al. Ineffectiveness of neurokinin‐1 antagonist in acute migraine: a crossover study. Cephalalgia 1997;17(7):785–790; doi: 10.1046/j.1468-2982.1997.1707785.x.
209. Goldstein D, Offen W, Klein E, et al. Lanepitant, an NK‐1 antagonist, in migraine prevention. Cephalalgia 2001;21(2):102–106; doi: 10.1046/j.1468-2982.2001.00161.x.
210. Dionne RA, Max MB, Gordon SM, et al. The substance P receptor antagonist CP‐99,994 reduces acute postoperative pain. Clin Pharmacol Ther 1998;64(5):562–568; doi: 10.1016/s0009-9236(98)90140-0.
211. Goldstein DJ, Wang O, Todd LE, et al. Study of the analgesic effect of lanepitant in patients with osteoarthritis pain. Clin Pharmacol Ther 2000;67(4):419–426; doi: 10.1067/mcp.2000.105243.
212. Chizh BA, Göhring M, Tröster A, et al. Effects of oral pregabalin and aprepitant on pain and central sensitization in the electrical hyperalgesia model in human volunteers. Bja Br J Anaesth 2007;98(2):246–254; doi: 10.1093/bja/ael344.
213. Tillisch K, Labus J, Nam B, et al. Neurokinin‐1‐receptor antagonism decreases anxiety and emotional arousal circuit response to noxious visceral distension in women with irritable bowel syndrome: a pilot study. Aliment Pharm Therap 2012;35(3):360–367; doi: 10.1111/j.1365-2036.2011.04958.x.
214. Goldstein DJ, Wang O, Gitter BD, et al. Dose-Response Study of the Analgesic Effect of Lanepitant in Patients with Painful Diabetic Neuropathy. Clin Neuropharmacol 2001;24(1):16; doi: 10.1097/00002826-200101000-00004.
215. Sindrup SH, Graf A, Sfikas N. The NK1‐receptor antagonist TKA731 in painful diabetic neuropathy: A randomised, controlled trial. Eur J Pain 2006;10(6):567–567; doi: 10.1016/j.ejpain.2005.08.001.
216. Smith J, Allman D, Badri H, et al. The Neurokinin-1 Receptor Antagonist Orvepitant Is a Novel Antitussive Therapy for Chronic Refractory Cough Results From a Phase 2 Pilot Study (VOLCANO-1). Chest 2020;157(1):111–118; doi: 10.1016/j.chest.2019.08.001.
217. Ichinose M, Miura M, Yamauchi H, et al. A neurokinin 1-receptor antagonist improves exercise-induced airway narrowing in asthmatic patients. Am J Resp Crit Care 1996;153(3):936–941; doi: 10.1164/ajrccm.153.3.8630576.
218. Boot JD, Haas S de, Tarasevych S, et al. Effect of an NK1/NK2 Receptor Antagonist on Airway Responses and Inflammation to Allergen in Asthma. Am J Resp Crit Care 2007;175(5):450–457; doi: 10.1164/rccm.200608-1186oc.
219. Mahler DA, Gifford AH, Gilani A, et al. Antagonism of substance P and perception of breathlessness in patients with chronic obstructive pulmonary disease. Resp Physiol Neurobi 2014;196:1–7; doi: 10.1016/j.resp.2014.02.008.
220. He A, Alhariri JM, Sweren RJ, et al. Aprepitant for the Treatment of Chronic Refractory Pruritus. Biomed Res Int 2017;2017:4790810; doi: 10.1155/2017/4790810.
221. Welsh SE, Xiao C, Kaden AR, et al. Neurokinin‐1 receptor antagonist tradipitant has mixed effects on itch in atopic dermatitis: results from EPIONE, a randomized clinical trial. J Eur Acad Dermatol 2021;35(5):e338–e340; doi: 10.1111/jdv.17090.
222. Lönndahl L, Holst M, Bradley M, et al. Substance P Antagonist Aprepitant Shows no Additive Effect Compared with Standardized Topical Treatment Alone in Patients with Atopic Dermatitis. Acta Dermato Venereol 2018;98(3):324–328; doi: 10.2340/00015555-2852.
223. Vincenzi B, Trower M, Duggal A, et al. Neurokinin-1 antagonist orvepitant for EGFRI-induced pruritus in patients with cancer: a randomised, placebo-controlled phase II trial. Bmj Open 2020;10(2):e030114; doi: 10.1136/bmjopen-2019-030114.
224. Chiou AS, Choi S, Barriga M, et al. Phase 2 trial of a neurokinin-1 receptor antagonist for the treatment of chronic itch in patients with epidermolysis bullosa: A randomized clinical trial. J Am Acad Dermatol 2020;82(6):1415–1421; doi: 10.1016/j.jaad.2019.09.014.
225. Haab F, Braticevici B, Krivoborodov G, et al. Efficacy and safety of repeated dosing of netupitant, a neurokinin‐1 receptor antagonist, in treating overactive bladder. Neurourol Urodynam 2014;33(3):335–340; doi: 10.1002/nau.22406.
226. Roberts C, Inamdar A, Koch A, et al. A Randomized, Controlled Study Comparing the Effects of Vestipitant or Vestipitant and Paroxetine Combination in Subjects With Tinnitus. Otol Neurotol 2011;32(5):721–727; doi: 10.1097/mao.0b013e318218a086.
227. MADSEN JL, FUGLSANG S. A randomized, placebo‐controlled, crossover, double‐blind trial of the NK1 receptor antagonist aprepitant on gastrointestinal motor function in healthy humans. Aliment Pharm Therap 2008;27(7):609–615; doi: 10.1111/j.1365-2036.2008.03618.x.
228. Shah TU, Liddle R, Branch MS, et al. Pilot Study of Aprepitant for Prevention of Post-ERCP Pancreatitis in High Risk Patients: A Phase II Randomized, Double-Blind Placebo Controlled Trial. Jop J Pancreas 2012;13(5):514–518; doi: 10.6092/1590-8577/855.
229. Worthley LIG, Cooper DJ, Jones N. Treatment of resistant intracranial hypertension with hypertonic saline: Report of two cases. J Neurosurg 1988;68(3):478–481; doi: 10.3171/jns.1988.68.3.0478.
 
















Figure 1: Diagrammatic representation of neurogenic inflammation at the blood-brain interface. Substance P binds to the neurokinin-1 receptor, causing neurogenic inflammation. Potentiation of classical inflammation can be seen to occur with the activation of microglia and astrocytes, as well as recruitment of peripheral immune cells. Adapted from “Neurogenic inflammation after traumatic brain injury and its potentiation of classical inflammation.” by Corrigan et al.20 Created with BioRender.com.






Figure 2: A representation of the synthesis of the tachykinins. The TAC1 gene is responsible for the substance P (SP), neurokinin A (NKA), neuropeptide K (NPK) and neuropeptide gamma (NPγ) peptides, with the corresponding sequences contained within the exons involved in encoding labelled. The TAC3 and TAC4 genes are responsible for the peptides neurokinin B (NKB) and haemokinin-1 (HK-1) respectively. Created with BioRender.com.
	


Figure 3: A representation of the distribution of SP immunoreactivity within the different regions of the brain, from higher concentrations to lower concentrations. Created with BioRender.com.










Figure Legends

Figure 1: Diagrammatic representation of neurogenic inflammation at the blood-brain
interface. Substance P binds to the neurokinin-1 receptor, causing neurogenic inflammation.
Potentiation of classical inflammation can be seen to occur with the activation of microglia
and astrocytes, as well as recruitment of peripheral immune cells. Adapted from
“Neurogenic inflammation after traumatic brain injury and its potentiation of classical
inflammation.” by Corrigan et al.20 Created with BioRender.com.

Figure 2: A representation of the synthesis of the tachykinins. The TAC1 gene is responsible
for the substance P (SP), neurokinin A (NKA), neuropeptide K (NPK) and neuropeptide
gamma (NPγ) peptides, with the corresponding sequences contained within the exons
involved in encoding labelled. The TAC3 and TAC4 genes are responsible for the peptides
neurokinin B (NKB) and haemokinin-1 (HK-1) respectively. Created with BioRender.com.

Figure 3: A representation of the distribution of SP immunoreactivity within the different
regions of the brain, from higher concentrations to lower concentrations. Created with
BioRender.com.


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image2.png

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