Pro-inflammatory tachykinins form an important, expanding family of neuropeptides, containing 10- to 12-amino-acid peptides, such as substance P (SP), neurokinin A (NKA), neurokinin B (NKB), hemokinin-1 (HK-1) and endokinin A–D (EKA–EKD). These peptides activate G protein-coupled mammalian tachykinin receptors, NK1, NK2 and NK3, with varying affinity .
Tachykinins are involved in a broad range of biological actions, including pain transmission, inflammation, vasodilatation, platelet function, activation of the immune and endocrine systems, smooth muscle contraction and depression-like behaviour [2-5].
The discovery of SP, the first characterized neuropeptide, in 1931 has opened the research field of tachykinins. It is encoded by the preprotachykinin A (PPT-A, TAC1) gene and expressed predominantly in capsaicin-sensitive primary sensory neurons of the dorsal root and trigeminal ganglia, as well as in the central nervous system (CNS). Substance P preferentially binds to the NK1 receptors. Substance P has been proved to participate in various pathophysiological processes, such as vasodilatation, plasma protein extravasation, leucocyte infiltration, mast cell degranulation, pain, anxiety, depression, nausea and vomiting.
Neurokinin A is the other tachykinin product of the TAC1 gene. It is expressed mainly by capsaicin-sensitive sensory neurons and is a preferred binding ligand of the NK2 receptors, expressed predominantly on smooth muscle cells. The most important mediated physiological effect of NKA is smooth muscle contraction, mainly in the respiratory and gastrointestinal systems.
Neurokinin B is derived from the preprotachykinin B (PPT-B, TAC3) gene. Both NKB and NK3 receptors, which bind NKB with the highest affinity, are predominantly expressed in the CNS, where NKB exerts neural activation. There is some evidence that NK3 receptors also exist on the periphery, mainly in the joints , airways  and gastrointestinal tract .
In 2000, new members of the tachykinin family were discovered, encoded by the preprotachykinin C (PPT-C, TAC4) gene. The peptide products of the TAC4 gene are HK-1 in mice and their respective peptides, endokinins (EKA–EKD) in humans .
Hemokinin-1 and EKA–EKD differ from other tachykinins by their predominantly non-neuronal expression pattern . Remarkable expression of TAC4 mRNA has been reported in various tissues and cells of the immune system, such as T and B lymphocytes, macrophages, dendritic and endothelial cells, suggesting that they have an important role in the activation and differentiation of inflammatory and immune cells as well as the promotion of angiogenesis [9, 11-14].
Hemokinin-1 most closely resembles SP in sequence and also exhibits immunological cross-reactivity. Moreover, similar preference has been described for the NK1 receptor [10, 15-18]. However, several effects of HK-1 suggest the existence of presently unidentified receptors related to HK-1 .
NK1 receptor antagonists
CP-96,345 – the first NK1 receptor antagonist, discovered in 1991 [20, 21] – proved to be effective in several inflammatory conditions. In cerulein-induced pancreatitis of the rat, it was able to inhibit the pancreatic plasma extravasation and serum amylase increase ; in the zymosan-induced acute colitis of the rat, it decreased plasma extravasation ; while in murine experimental autoimmune encephalomyelitis (EAE), it could reduce the clinical and histological signs by stabilization of the blood–brain barrier and suppression of T-helper 1 immunity .
Effectiveness of two different NK1 receptor antagonists has been reported in complete Freund's adjuvant (CFA)-induced arthritis of the rat. WIN51708  and GR82334 decreased the mechanical hyperalgesia and destructive histological changes in the joint, when given intra-articularly .
Moreover, GR205171 also relieved mechanical hyperalgesia in CFA-induced arthritis in the rat, and it could inhibit joint swelling in animal models of neuropathic pain . Besides arthritic pain, GR205171 was able to attenuate reductions in carotid arterial vascular resistance evoked by the tachykinin NK receptor agonist SP methyl ester 1 and produced a dose-dependent inhibition of plasma protein extravasation in the dura mater .
The NK1 receptor antagonist L-703,606 was found to be effective in experimental animal models of carrageenin-induced arthritis. In rats, it was able to reduce the arthritic pain , as well as paw oedema .
RP67580 was tested in animal models of several different pathological conditions. Lam and Ng  reported that RP67580 was even able to improve the efficacy of dexamethasone in reducing arthritic pain and joint swelling in the rat adjuvant-induced arthritis model. Furthermore, administration of RP67580 resulted in abrogation of watery diarrhoea and reduction of colonic patch hypertrophy, leucocyte recruitment, tissue damage and mast cell infiltration when applied in a dinitro-fluorobenzene (DNFB)-induced colonic hypersensitivity model . In murine non-atopic airway inflammation, the development of both tracheal hyper-reactivity and neutrophil accumulation in the bronchoalveolar lavage fluid could be observed .
FK888 was also investigated in the inflammatory processes of the airways. Hirayama and colleagues found that FK888 was able to inhibit plasma exsudation but not bronchoconstriction induced by vagal stimulation in guinea-pigs .
The NK1 receptor antagonist SR140333 was described to decrease bodyweight loss, macroscopic and histological scores and reduced colonic myeloperoxidase (MPO) activity and tumour necrosis factor-α (TNF-α) tissue levels in dinitrobenzene sulfonic acid (DNBS)-induced colitis of the rat . Recently, it was also reported to reduce endotoxin-induced fever effectively in rats if administered by the intracerebroventricular route .
Another NK1 receptor antagonist, named MEN-11467, was observed to reduce the macroscopic damage, necrosis score and plasma protein extravasation in the early acute phase of acetic acid-induced rectocolitis in guinea-pigs .
CP-99,994 was described to abolish microvascular leakage in the rat trachea and main bronchi during toluene-induced respiratory tract irritation . It was also discovered to have a remarkable antiemetic effect .
Rolapitant (SCH 619734), a functionally competitive antagonist, was active in both acute and delayed emesis models in ferrets . In several different animal models, NK1 antagonists, such as aprepitant (MK-869), L-733060, CP-122721 and L-760735, have been shown to exhibit antidepressant-like and anxiolytic effects [40-44].
In contrast to the successful experiments with various animal models, NK1 receptor antagonists failed as anti-inflammatory compounds in most clinical trials. Early findings on the distribution and pharmacodynamics of SP and NK1 receptors triggered numerous pharmaceutical companies – including Eli Lilly, GlaxoSmithKline, Merck, Parke-Davis and Pfizer – to develop selective nonpeptide NK1 receptor antagonists for the therapy of painful conditions. However, these compounds did not exhibit significant analgesic activity in clinical trials and failed to reach the market for this indication. This story has been reviewed in detail by Hill  Urban and Fox  and Herbert and Holzer .
The early results showing that SP was more abundant in the dorsal horn of the spinal cord than in the ventral horn caught the attention of the pharmaceutical industry. Localization of SP in thin unmyelinated sensory fibres seemed to support involvement of the peptide in sensory neurotransmission. Substance P had a slow, but long-lasting, excitatory effect on dorsal horn sensory neurons. NK1 receptors are also present in dorsal horn neurons and dorsal root ganglia, such as in unmyelinated nerve fibres of the skin and dura mater .
Although the previous data presented strong evidence that SP participates in nociception, SP showed no algesic effect when injected intramuscularly or applied to the base of skin blisters. Substance P induced primary hyperalgesia in the knee joint of the rat and cat. Painful stimuli, inflammation and neuropathy were shown to influence SP content in primary afferent and dorsal root ganglion neurons .
In animal studies, NK1 antagonists showed no effect in acute pain models, such as hotplate and tail-flick tests, but they exerted antihyperalgetic effects in some animal models of inflammation and neuropathic pain .
The only clinical trial that has found significant analgesic effect was performed with CP-99994, involving patients who underwent surgical extraction of the third molar. In contrast, aprepitant and CP-122721 were ineffective for tooth extraction pain. Aprepitant and LY 303870 also failed to relieve neuropathic pain in patients with postherpetic neuralgia or diabetic neuropathy. LY 303870 exhibited no effect on osteoarthritic pain. GR205171 and L-758298 (water-soluble prodrug of aprepitant) could not improve various types of headache, including migraine .
Several theories have been proposed to explain the therapeutic failure of NK1 receptor antagonists as analgesics. Many of them have much higher affinity to the human NK1 receptor than to that of the rat. Consequently, preclinical studies involving high doses of NK1 antagonist applied to rats might have detected nonspecific effects, because functional magnetic resonance imaging and positron emission tomography could not be used to determine effective human doses of the drugs at that time. Species differences in the distribution of NK1 receptors at supraspinal sites have also been blamed .
The lack of effect of NK1 antagonists in osteoarthritis, diabetic neuropathy and various headache syndromes might be explained by involvement of multiple transmitters in these processes and a subordinate role of SP. Central and peripheral neurons of the nociceptive pathway contain multiple neurotransmitters. These are released together with SP and modulate the effect of each other. The peptide content of such neurons is influenced by inflammation, nerve injury or painful conditions. Some authors suggest that anxiolytic activity of NK1 receptor antagonists has been misinterpreted as antihyperalgesic activity in preclinical trials. Although an anxiolytic effect of NK1 antagonists has been described in animal models , L-759274 has recently failed to prove such efficacy in a placebo- and active-controlled, double-blind clinical trial .
NK1 receptor knockout mice became available after the disappointing clinical data had been obtained. These animals exhibit normal acute nociception and acute inflammatory processes, except that they show a slighter ‘wind-up’ phenomenon in the dorsal horn. TAC1 gene-deleted mice – lacking both SP and NKA – also have normal nociception. Earlier availability of genetically modified animals in the tachykinin field could have prevented pharmaceutical companies from investing vast resources into clinical development of NK1 antagonist analgesics [27, 45].
Human studies investigating NK1 receptor antagonists in airway inflammation were controversial. CP-99994 did not inhibit hypertonic saline-induced bronchoconstriction , in contrast to the results gained in animal experiments, but FK888 was capable to attenuate the recovery phase of exercise-induced airway constriction .
In contrast, based on previous promising results of animal experiments , NK1 receptor antagonists have been developed as a new effective group of antiemetics. The presence and functional role of tachykinins have been identified in the ferret, in the brainstem nuclei involved in nausea and vomiting. Nucleus tractus solitarii is the proposed site of action. Tachykinins are also transmitters of vagal afferents projecting to the area postrema, which is the chemoreceptor trigger zone. Substance P-induced neuronal excitation was shown to be prevented by NK1 antagonists with electrophysiological as well as positron emission tomographic examinations in animal studies .
Clinical trials confirmed the potent antiemetic effect of several NK1 antagonists in cancer-chemotherapy-induced nausea and vomiting and postoperative nausea and vomiting [52, 53]. Aprepitant and its injectible form, fosaprepitant dimeglumine (MK-0517), are in clinical use for the therapy of cancer-chemotherapy-induced nausea and vomiting and postoperative nausea and vomiting . Orally administered casopitant mesylate (GW679769B) was able to increase the efficacy of ondansetron in female patients at high risk for postoperative nausea and vomiting . Maropitant (Pfizer) has been approved by the USA Food and Drug Administration for the treatment of motion sickness-evoked nausea and vomiting in dogs.
Furthermore, on the basis of former animal studies [47, 56], aprepitant, L-759274 and casopitant were investigated as new pharmacological tools of antidepressant as well as anxiolytic therapy and were proved to be effective in major depression [57, 58], but not in anxiety disorder .
Combined receptor antagonists
A combination of SR140333 and SR48968 (NK1 and NK2 receptor antagonists, respectively) in endotoxin-induced acute pneumonitis was investigated by several groups. Veron and colleagues  found that it diminished neutrophil cell accummulation and matrix metalloproteinase-9 activity in the bronchoalveolar lavage fluid, while another research group described the reduction of neutrophil accummulation and attenuation of MPO activity and interleukin-1β production, suggesting the effectiveness of a combination of NK1 and NK2 receptor antagonists in airway inflammation .
The dual NK1/NK2 receptor antagonist FK224 showed no effect in asthmatic patients against NKA-induced bronchoconstriction [64, 65], although former human studies had revealed its protective effect against bradykinin-induced bronchoconstriction and cough . After the controversial results with FK224, further dual NK1/NK2 receptor antagonists, such as DNK-333, AVE-5883 and MEN11420, seemed to be effective in the attenuation of airway hyper-responsiveness in later studies [64, 66-68].
However, DNK-333 was also tested in diarrhoea-predominant irritable bowel syndrome, where the reduction of abdominal pain/discomfort and global symptoms of diarrhoea-predominant irritable bowel syndrome were observed .
The triple NK1/NK2/NK3 receptor antagonist CS-003 was able to decrease airway responsiveness in human bronchial asthma .
Calcitonin gene-related peptide antagonists
Regarding the potential therapeutic use of CGRP antagonists as anti-inflammatory agents, in a murine model of endotoxin-induced acute pneumonitis, CGRP(8–37) was shown to diminish neutrophil accumulation, MPO level and interleukin-1β production .
Olesen and colleagues  provided evidence that CGRP is a key element in the pathophysiology of migraine, by showing in a phase II trial that intravenous administration of a small-molecule CGRP receptor antagonist, olcegepant (BIBN4096BS), showed efficacy comparable to the response rate obtained with triptans .
Telcagepant (MK-0974) was the first orally available and highly selective CGRP receptor antagonist. It effectively reduced pain and accompanying symptoms, such as nausea, photophobia and phonophobia, in phase II and III trials .
MK-3207 was tested as a further CGRP receptor antagonist with a higher oral bioavailability and potency than telcagepant, but despite its effectiveness in acute migraine it was stopped because of a higher concentration of liver transaminases .
A phase II trial with the oral CGRP receptor antagonist BI 44370 TA was recently completed and showed that the compound was well tolerated and effective against migraine-like headache .
The anti-inflammatory effects of tachykinin and CGRP receptor antagonists are summarized in Tables 1 and 2.
Table 1. Anti-inflammatory effects of tachykinin and calcitonin gene-related peptide (CGRP) receptor antagonists in animal models
|Receptor||Antagonist||Anti-inflammatory effects in animal models|
|NK1||CP-96,345||Cerulein-induced pancreatitis in rats, zymosan-induced colitis in rats, EAE [22-24]|
|WIN51708||Adjuvant-induced arthritis in rats [25, 26]|
|L-703,606||Carrageenan-induced arthritis in rats |
|RP67580||Adjuvant-induced arthritis in rats, murine DNFB-induced colitis, murine non-atopic airway inflammation [30-32]|
|SR140333||DNBS-induced colitis in rats, endotoxin-induced fever in rats [34, 35]|
|MEN-11467||Acetic acid-induced colitis in guinea-pigs |
|GR205171||SP-induced plasma protein extravasation in rats |
|CP-99,994||Toluene-induced respiratory tract irritation in rats, anti-emetic effect in ferrets and dogs [37, 38]|
|Rolapitant (SCH 619734)||Anti-emetic effect in ferrets |
|Aprepitant (MK-869)||Anxiolytic and antidepressant effect in gerbils [40, 42-44]|
|L-733060||Anxiolytic and antidepressant effect in gerbils [42-44]|
|CP-122721||Anxiolytic and antidepressant effect in gerbils [42-44]|
|L-760735||Anxiolytic effect in gerbils |
|NK2||Saredutant (SR48968)||No effect in non-atopic , but protective effect in endotoxin-induced  murine airway inflammation|
|SR144190||Acetylcholine-induced airway hyper-reactivity in guinea-pigs |
|Nepadutant (MEN 11420)||Acetic acid-induced colitis in guinea-pigs, bacterial toxin-induced enteritis in mice [62, 63]|
|NK3||Talnetant (SB 223412-A)||Inflammatory thermal hyperalgesia in rats |
|Osanetant (SR142801)||Endotoxin-induced murine airway inflammation |
|Combination of NK1 and NK2 antagonists||SR140333 + SR48968||Endotoxin-induced murine airway inflammation [7, 59]|
|CALCRL-RAMP1||CGRP(8–37)||Endotoxin-induced acute pneumonitis in mice |
Table 2. Anti-inflammatory effects of tachykinin and calcitonin gene-related peptide (CGRP) receptor antagonists in human trials
|Receptor||Antagonist||Anti-inflammatory effects in human trials|
|NK1||CP-99,994||No inhibition of hypertonic saline-induced bronchoconstriction |
|FK888||Attenuation of the recovery phase of exercise-induced airway narrowing |
|Aprepitant (MK-869)||Acute and delayed chemotherapy-induced nausea and vomiting, postoperative nausea and vomiting [52, 53]|
|Fosaprepitant (MK-0517)||Chemotherapy-induced nausea and vomiting |
|Casopitant (GW679769B)||Postoperative nausea and vomiting |
|L-759274||Major depression [56-58]|
|NK2||Saredutant (SR48968)||Decreased bronchoconstriction on response to NKA |
|Dual NK1/NK2||FK224||No protective effect in NKA-induced bronchoconstriction |
|DNK-333||Decreased airway hyper-responsiveness, IBS-D [66, 69]|
|AVE-5883||Decreased airway responsiveness, inhibition of bronchoconstriction |
|MEN-48968||Decreased airway responsiveness |
|Triple NK1/NK2/NK3||CS-003||Decreased airway hyper-responsiveness in human bronchial asthma |
|CALCRL-RAMP1||Olcegepant (BIBN4096BS)||Human migraine |
|Telcagepant (MK-0974)||Human migraine |
|BI 4370 TA||Human migraine |