Part III Compounds in Preclinical Development

16 May

References

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

Introduction

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).

Substance P

2.1

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.

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