Abstract Neuropeptides and kinins are important messengers in the nervous system and— on the basis of their anatomical localisation and the effects produced when the substances themselves are administered, to animals or to human subjects—a signiﬁcant number of them have been suggested to have a role in pain and inﬂammation. Experiments in gene deletion (knock-out or null mutant) mice and parallel experiments with pharmacological receptor antagonists in a variety of species have strengthened the evidence that a number of peptides, notably substance P and calcitonin gene-related peptide (CGRP), and the kinins have a pathophysiological role in nociception. Clinical studies with non-peptide pharmacological antagonists are now in progress to determine if blocking the action of these peptides might have utility in the treatment of pain.
Keywords Peptides · Substance P · CGRP · Kinins · Bradykinin · Des-Arg bradykinin
Progress in neuroscience research over the last ﬁve decades has led to changes in our understanding of the neurochemical events that underlie the function of both central (CNS) and peripheral nervous systems. Neuropeptides, originally considered exclusively in terms of regulation of hypothalamic-pituitary func- tion, were found to be distributed all over the CNS and, therefore, likely to be involved in wider aspects of brain function (see Oliver et al. 2000). Several char- acteristics distinguish neuropeptides from biogenic amines. First, neuropep- tides demonstrate very high afﬁnity interactions with their receptors. Second, in neuropeptidergic systems, no speciﬁc uptake or catabolic systems have been demonstrated. Third, synapses are not usually of a classical type and peptides may act at a distance from their site of release. Fourth, peptides are synthe- sized on polyribosomes by messenger RNA (mRNA) molecules, transcribed from the genome, whilst biogenic amines are produced non-ribosomally under the control of biosynthetic enzymes (derived from cellular gene transcripts). As a result of genome structure, and particularly the involvement of intron regulation of tissue-speciﬁc gene transcription, two different peptides can result from the same gene. Two examples of such alternative splicing are cal- citonin/calcitonin gene-related peptide (Amara et al. 1982) and the tachykinin family (Nawa et al. 1984).
The observation that some peptides have a pro-nociceptive role has led to the suggestion that antagonising the action of such peptides may lead to analgesia. This is not a new idea and indeed, one of the ﬁrst hypotheses for explaining the analgesic action of aspirin was that this drug opposed the algogenic effect of the peptide bradykinin (Guzman et al. 1964), and the idea that substance P might have a special role in pain perception was suggested by Lembeck in the 1950s (cited in Salt and Hill 1983). The literature on neuropeptides in pain signalling is therefore very extensive and can only be covered in a single chapter by being extremely selective. After reviewing the topic in a general way, a more detailed treatment will be given to the roles of substance P, calcitonin gene-related pep- tide (CGRP) and bradykinin (together with its des-[Arg] analogues). This focus is a consequence of these peptides being the ones which have received most study and in particular because medicinal chemists have succeeded in making speciﬁc receptor antagonist drugs that block their actions. Some of these re- ceptor antagonist drugs have already been evaluated for efﬁcacy as analgesics in a clinical setting. In the previous volume of this Handbook series devoted to the topic of Pain and Analgesia (vol. 130: Dickenson and Besson 1997), peptides did receive some attention, with a chapter devoted to substance P, but because of the lack of clinical data it was not possible at that time for the authors to come to any ﬁrm conclusion on the therapeutic utility of antagonists.
There has been a great increase in our understanding of the process of noci- ception in the last decade (e.g. see reviews by Hill 2001; Hunt and Mantyh 2001;
Julius and Basbaum 2001) and this has enabled a more complete assessment of the role of peptide messengers. One special feature of the small unmyeli- nated ﬁbres which contain peptides is that they are able to release them from both peripheral and central arborisations and thus may impinge on sensory transmission both in the peripheral innervated tissues and in the dorsal horn of the spinal cord (see Salt and Hill 1983; Hunt and Mantyh 2001; Jang et al.
2004). A variety of chemical and other stimuli, including H+ ions, heat, intense mechanical pressure and kinins both stimulate peripheral peptide release (see Julius and Basbaum 2001) and generate impulses which travel rostrally to excite the central terminals within the dorsal horn of the spinal cord. The
details of the nociceptive transmission process are described at length in the chapter by J.N. Wood of this volume. One important feature of peptide re- lease from primary afferent ﬁbres is that it is likely to be a parallel event,
with glutamate and other transmitters being released in concert with one or more peptides (Salt and Hill 1983; Leah, Cameron and Snow 1985; Hill 2001). It has been suggested that neurokinin A, substance P and CGRP might be
released together as co-transmitters from primary afferent ﬁbres in the caudal trigeminal nucleus (Samsam et al. 2000). Antibody microprobe studies have shown that some neuropeptides can be detected at sites relatively distant to their site of release (see Duggan and Furmidge 1994) and that interaction be-
tween peptides (as competing peptidase substrates) may inﬂuence the levels that individual peptides reach in the dorsal horn of the spinal cord (DHSC). Gene array studies have also shown that spinal cord injury can result in an
up-regulation of peptidase genes (Tachibana et al. 2002). It is also apparent that peptides can regulate receptor expression and, for example, CGRP can regulate the expression of the NK1 receptors at which substance P acts (Sey- bold et al. 2003). The number and diversity of neuropeptides (see Oliver et al.
2000) is another complication of trying to ascribe a physiological or patho- logical role to any individual peptide, and a signiﬁcant number of peptides also have extra-neuronal functions (Hokfelt et al. 2000). At the present time there are over 65 known neuropeptides (excluding the chemokines), receptors for over 20 neuropeptides have been cloned and many neuropeptides activate more than one receptor subtype (Oliver et al. 2000). It is of particular interest that a noxious insult in a neonatal rat results in the up-regulation of genes in the DHSC coding (amongst other things) for the synthesis of neuropeptides that persists into adulthood (Ren et al. 2005). It has been suggested that pep- tides have a particular role when the nervous system is under stress (Hokfelt et al. 2000), and many studies have been made of the effects of nerve injury on peptide expression. The changes seen are not especially helpful although it has been shown, for example, that injury to sensory nerves down-regulates CGRP and substance P yet up-regulates expression of vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) and galanin, with inﬂammation producing a different expression pattern (Garry et al. 2004). Galanin is a 29- amino-acid peptide that is found both in dorsal root ganglion cells and in
DHSC interneurons and whose expression increases markedly after periph- eral nerve lesions (Liu and Hokfelt. 2002), leading to the suggestion that it may have a role in the processes leading to neuropathic pain. It has recently been shown that the small proteins collectively known as chemokines might have an important role in neuropathic pain, but this area has been considered to be outside the scope of the present chapter (see Kinloch and Cox 2005; White et al. 2005). The drug discovery scientist is faced with the dilemma of too many potential targets and no easy way to determine which might be the most productive for discovering a new analgesic drug (Boyce et al.
2001). Historically, discovery of a neuropeptide receptor was consequent on the discovery of the neuropeptide itself, but with the advent of modern tech- nologies this no longer necessarily applies. The rate-limiting factor in drug discovery today is not the lack of attractive targets; indeed, the advent and rapid development of molecular- and bioinformatics-related techniques has created the problem of how to select the best targets from a rich supply of receptor clones. So-called “orphan” G protein-coupled receptors (oGPCRs) are being identiﬁed at a proliﬁc rate, albeit with little information available yet as to their endogenous ligands, many of which are likely to be peptides (Oliver et al. 2000). There are neuropeptides for which we have as yet no identiﬁed receptor, and paradoxically—through novel receptor research—we have orphan receptors with unidentiﬁed ligands but which are structurally related to known peptide receptor families. The vast majority of receptors for neuropeptides discovered thus far are G protein coupled (GPCRs), with the common basic structure of seven transmembrane domains and an extracellu- lar 20- to 100-amino-acid residue extracellular tail. This receptor superfamily has been divided into three subfamilies based on receptor sequence analysis:
(1) rhodopsin or β-adrenoceptor-like, (2) secretin or glucagon-like and (3) metabotropic or chemosensor-like receptors. Peptide GPCRs are not struc- turally homogeneous and belong to both class 1 and class 2. Peptides that act through GPCRs can be small—such as enkephalins, bradykinin, calcitonin
gene-related peptide, substance P and vasoactive intestinal peptide—or large peptides, glycoproteins and hormones, or even enzymes such as trypsin and thrombin (see Oliver et al. 2000).
Much emphasis is now being placed on transgenic animals for the preclinical validation of novel drug targets, including neuropeptide receptors. Thus, the phenotype of a mouse resulting from a speciﬁc gene deletion or silencing may
provide information regarding the functional role of a speciﬁc receptor for which the deleted gene codes in the tissue of interest and thus may predict the action of a speciﬁc receptor antagonist. Transgenic technology has now been used by numerous groups to elucidate the role of neuropeptides in vivo,
although at times some knock-out mice appear phenotypically normal. This may be related to compensatory mechanisms occurring during development probably due to up-regulation of molecules functionally related to the deleted
gene (see later and Oliver et al. 2000).
Preclinical Studies with NK1 Receptor Antagonists and Transgenic Mice
Evidence supporting the role of substance P as a pain transmitter comes from anatomical and immunocytochemical studies showing substance P is expressed in small unmyelinated sensory ﬁbres (Nagy et al. 1981; Hokfelt et al.
2004) and it is released into the dorsal horn of the spinal cord following in- tense noxious stimulation (Duggan et al. 1987). In addition, substance P when applied onto the dorsal horn neurons produces prolonged excitation which
resembles the activation observed following noxious stimulation (Henry 1976) and when given intrathecally produces behavioural hyperalgesia (Cridland and Henry 1986). More recently, it has been shown that following peripheral
noxious stimulation NK1 receptors become internalised on dorsal horn neu- rons and that this effect can be blocked by NK1 receptor antagonists (Mantyh et al. 1995). In addition to its effects on spinal nociceptive processing, sub- stance P has also been implicated in migraine headache. Sensory afferents that innervate meningeal tissues contain substance P and other neuropeptides (e.g. calcitonin gene related peptide; CGRP see later) and it has been suggested that release of these neuropeptides causes a neurogenic inﬂammation which could lead to activation of nociceptive afferents projecting to the brain stem (Shepheard et al. 1995; Williamson and Hargreaves 2001). The expectation was that therefore centrally acting NK1 receptor antagonists would be anti- nociceptive in animals and analgesic in man and constitute a novel class of analgesic drug.
Compounds which have been optimised for activity at NK1 receptors ex- pressed in humans typically have low afﬁnity for rats or mice, species used for anti-nociceptive studies (see Boyce and Hill 2004). Consequently, early studies with the NK1 receptor antagonists which were performed in rats and mice required high doses to observe anti-nociceptive effects, and so interpretation of the data was confounded by potential off-target activity such as blockade of ion channels (Rupniak et al. 1993). There is now considerable evidence gen- erated from well-controlled studies using enantiomeric pairs of a number of antagonist structures (one enantiomer having high afﬁnity for the NK1 recep- tor and the other low afﬁnity) to control for non-speciﬁc effects. Analgesic tests have also been developed in appropriate species (gerbils, guinea-pigs) with similar NK1 receptor pharmacology to human such that it has been pos- sible to demonstrate unequivocally that NK1 receptor antagonists do possess anti-nociceptive effects in animals. Nociception tests which demonstrate re- producible analgesic effects only with opioids such as morphine (hot plate, tail or paw ﬂick and paw pressure tests) do not reveal anti-nociceptive ef- fects of NK1 receptor antagonists in any species even when these agents are
administered at high doses (Rupniak et al. 1993). Similarly, NK1 antagonists have little effect on baseline spinal nociceptive reﬂexes in anaesthetised ani- mals (Laird et al. 1993). However, NK1 receptor antagonists such as CP-96,345 and LY303870, but not their less active enantiomers, CP-96,344 and LY396155, were shown to be potent inhibitors of the excitation of dorsal horn neurons elicited by prolonged noxious mechanical or thermal peripheral stimulation or by iontophoretic application of substance P in cats (Radhakrishnan and Henry 1991; Radhakrishnan et al. 1998), indicating that these effects are due to a speciﬁc blockade of NK1 receptors. In addition, aprepitant (MK-0869) or CP-99,994, but not its less active enantiomer CP-100,263, inhibited the facili- tation or wind up of a spinal ﬂexion reﬂex produced by C-ﬁbre conditioning stimulation in decerebrate/spinalised rabbits (Boyce et al. 1993; see Boyce and Hill 2000), and RP67580, but not its less active enantiomer RP68651, inhib- ited facilitation of the hind limb ﬂexor reﬂex in anaesthetised/spinalised rats (Laird et al. 1993).
In conscious animals, the ﬁrst demonstration of a clear enantioselective analgesic effect came from studies using L-733,060, a highly selective and brain penetrant NK1 receptor antagonist with long duration of action. This compound, but not its less active enantiomer L-733061, was able to inhibit the late phase nociceptive responses to intraplantar injection of formalin in gerbils (Rupniak et al. 1996). The poorly brain penetrant compound L-743,310, a potent inhibitor of peripherally mediated NK1 receptor agonist-induced chro- modacryorrhoea, failed to inhibit the late phase response, indicating that the anti-nociceptive effect of L-733,060 was via blockade of central NK1 receptors. Consistent with a central anti-nociceptive action, intrathecal injection of CP-
96,345, but not its less active enantiomer CP-96,344, attenuated the late phase formalin test response in rats (Yamamoto and Yaksh 1991) as did LY303870 (Iyengar et al. 1997). Oral administration of CP-99,994, SDZ NKT 343 (Novar- tis) or LY303870 have also been shown to attenuate mechanical hyperalgesia induced by carrageenan in guinea-pigs (Patel et al. 1996; Urban et al. 1999). L-733,060, but not its inactive isomer L-733,061, also reversed carrageenan- induced mechanical hyperalgesia in guinea-pigs (Boyce and Hill 2000).
In addition to their effects in assays of inﬂammatory hyperalgesia, NK1 receptor antagonists are effective in a number of putative neuropathic pain assays. Urban and colleagues (1999) using partial sciatic nerve ligation in guinea-pigs showed that SDZ NKT 343 and LY303870 reduced established me- chanical hyperalgesia following either oral or intrathecal administration. In contrast, RPR 100,893 was only active following intrathecal administration (Urban et al. 1999) probably due to poor brain penetration (Rupniak et al.
1997a). Likewise, CI-1021 was effective in reversing mechanical hypersensitiv- ity (reduction in weight bearing) in guinea-pigs following sciatic nerve chronic constriction injury (CCI; Gonzalez et al. 2000) and it reduced mechanical hyper- sensitivity in rats following induction of diabetes by streptozotocin treatment (Field et al. 1998). Intrathecal administration of RP67580, but not its less active
enantiomer, also reduced mechanical hyperalgesia in streptozotocin diabetic rats (Coudore-Civiale et al. 2000). GR205171 which has nanomolar afﬁnity at rat NK1 receptors, reversed both mechanical hypersensitivity and the increase in receptive ﬁeld size of dorsal horn neurons in rats following CCI, effects which were not observed with its inactive enantiomer L-796,325 (Cumberbatch et al.
1998). In contrast, using the spinal nerve ligation (Chung) model, it was not possible to demonstrate an anti-algesic effect of GR205171 at doses that were effective in rats with CCI (M Sablad, A Hama and M Urban, personal commu- nication; Boyce and Hill 2004). In addition to the nerve damage associated with CCI, a marked neurogenic inﬂammation also develops (Daemen et al. 1998) which could contribute to the development of hyperalgesia and allodynia. The analgesic effects of NK1 antagonists in CCI and partial nerve ligation models may therefore relate to anti-inﬂammatory actions and not speciﬁcally to effects on neuropathic pain.
NK1 receptor antagonists are effective in models of visceral pain. CP-99994 inhibited the nociceptive reﬂex response (depressor effect) to jejunal distension in rats (McLean et al. 1998) and TAK-637, but not its less active enantiomer, and CP-99994 reduced the number of abdominal contractions induced by colorectal distension in rabbits following sensitisation to acetic acid (Okano et al. 2002). Intrathecal administration of TAK-637 and CP-99994 inhibited abdominal contractions. In contrast, Julia et al. (1994) demonstrated that the NK2 receptor antagonist SR-48,968, but not the NK1 receptor antagonists CP-
96345 or RP-67,580, inhibited abdominal contractions to rectal distension in
rats. However, the NK1 antagonists did prevent distension-induced inhibition of colonic motility in this study.
NK1 receptor antagonists have also been shown to be active in tests involving inﬂammation associated with arthritic changes. Similar to indomethacin, daily administration of the NK1 receptor antagonist L-760,735 (3 mg /kg s.c.) for
21 days reduced paw oedema and the associated thermal and mechanical
hyperalgesia in Freund’s adjuvant arthritic guinea-pigs (see Boyce and Hill
2000). Consistent with these ﬁndings, Binder et al. (1999) showed that repeated administration of GR205171, but not its less active enantiomer, also reduced arthritic joint damage (joint swelling, synovitis and bone demineralisation) caused by complete Freund’s adjuvant (CFA) in rats. These ﬁndings suggest that NK1 receptor antagonists may possess anti-inﬂammatory as well as anti- nociceptive activity. Other data suggest that NK1 receptor antagonists are extremely potent inhibitors of neurogenic inﬂammation. The NK1 receptor antagonists RP-67,580, CP-99,994, LY30380 and aprepitant block neurogenic plasma extravasation in the dura following trigeminal ganglion stimulation in rats or guinea-pigs (Shepheard et al. 1993; Shepheard et al. 1995; Phebus et al.
1997; Boyce and Hill 2000; Williamson and Hargreaves 2001). In addition to its actions on neurogenic extravasation, CP-99,994 has also been shown to reduce c-fos messenger RNA (mRNA) expression in the trigeminal nucleus caudalis in rats after trigeminal ganglion stimulation (Shepheard et al. 1995).
NK1 receptor knock-out (−/−) mice exhibit little or no changes in acute no- ciception tests such as the hot plate, thermal paw withdrawal or responses to von Frey ﬁlaments (De Felipe et al. 1998; Mansikka et al. 1999). Acute nocicep- tive responses to intraplantar injection of chemical stimuli such as formalin or capsaicin, as well as the resultant mechanical/heat hypersensitivity, are slightly attenuated in the NK1(−/−) mice (De Felipe et al. 1998; Laird et al. 2001; Man- sikka et al. 1999). However, these mice do develop hyperalgesia after induction of hind paw inﬂammation with CFA (De Felipe et a 1998), which contrasts with ﬁndings observed with NK1 receptor antagonists (see Binder et al. 1999; Boyce and Hill 2000). Martinez-Caro and Laird (2000) failed to demonstrate any dif- ference between responses of wild-type (WT) and NK1(−/−) mice following partial sciatic nerve ligation, whereas NK1 receptor antagonists were effec- tive in the same model in guinea-pigs (see above). Using the L5 spinal ligation (modiﬁed Chung) model, Mansikka et al. (2000) found that NK1(−/−) mice did not develop mechanical hypersensitivity but hyperalgesic responses to ther- mal stimuli (radiant heat or cooling) were unaltered in NK1(−/−) compared to WT mice.
Data from NK1(−/−) mice support a role of NK1 receptors in visceral pain, particularly associated with neurogenic inﬂammation. Thus, instilla- tion of capsaicin, which evokes neurogenic inﬂammation, into the colon of the NK1(−/−) mice produced fewer abdominal contractions than were ob- served in WT mice and they failed to develop referred hyperalgesia (Laird et al. 2000). Similarly, behavioural responses to cyclophosphamide and the acute nociceptive (pressor) reﬂex response or primary hyperalgesia following intracolonic acetic acid were impaired in NK1(−/−) mice. In contrast, noci- ceptive responses to intracolonic mustard oil which was found to evoke direct tissue damage were unchanged in the NK1(−/−) mice (Laird et al. 2000). Elec- trophysiological studies have also shown that the characteristic ampliﬁcation (‘wind up’) of spinal nociceptive reﬂexes to repetitive high-frequency electri- cal stimulation is absent in NK1(−/−) mice (De Felipe et al. 1998), which is in agreement with the ﬁndings from studies with NK1 receptor antag- onist drugs. The evidence obtained from studies with the NK1(−/−) mice supporting the role of substance P and NK1 receptors in pain is less com- pelling than has been reported for non-peptide antagonists. It is not clear why there should be differences in anti-nociceptive proﬁle seen with NK1 receptor antagonists and in the NK1(−/−) mice. Non-speciﬁc actions of NK1 recep- tor antagonists contributing to the anti-nociceptive effects can be ruled out, as the studies outlined above are well-controlled with many demonstrating marked enantioselective inhibition. These differences may relate to compen- satory changes in the knock-out mice asa result of the life-long absence of NK1 receptors.
In summary, there is abundant preclinical data supporting the analgesic potential of NK1 antagonists, particularly in pain conditions associated with inﬂammation or nerve injury.