Search

The opioid receptor-like 1 receptor is a novel member of the opioid receptor family and its endogenous peptide ligand has been termed nociceptin and orphanin FQ. Activation of the opioid receptor-like 1 receptor by nociceptin/orphanin FQ in vivo produces hyperalgesia when this peptide is given supraspinally but analgesia at the spinal level. Nociceptin/orphanin FQ also reverses stress-induced analgesia, suggesting that the peptide has anti-opioid properties. Nociceptin/orphanin FQ knockout mice show alterations in pain sensitivity and stress responses and display increased morphine dependence, suggesting an interaction of the nociceptin/orphanin FQ system with classical opioid receptor function. To determine if the behavioural phenotype of nociceptin/orphanin FQ knockout mice reflects changes in either opioid receptor-like 1 or classical opioid receptor expression, we have carried out quantitative autoradiography of the opioid receptor-like 1, mu-, delta- and kappa-opioid receptors in the brains of these animals. Receptor density was measured on coronal sections from wild-type, heterozygous and homozygous mice using [(3)H]nociceptin, [(3)H][D-Ala(2)-N-methyl-Phe(4)-Gly(5) ol] enkephalin, [(3)H]deltorphin-I, or [(3)H](-)-N-methyl-N-[7-(1-pyrrodinyl)-1-oxospiro[4,5]dec-8-yl]-4-benzofuranacetamide to label opioid receptor-like 1, mu-, delta- and kappa-receptors, respectively. A region-specific up-regulation of the opioid receptor-like 1 receptor (up to 135%) was seen in brains from homozygous mice. Mu-Receptors also showed significant differences between genotypes whilst changes in delta- and kappa- receptors were minor. In conclusion the region-specific up-regulation of the opioid receptor-like 1 receptor indicates a tonic role for nociceptin/orphanin FQ in some brain structures and may suggest the peptide regulates the receptor expression in these regions. The changes in the opioid receptor-like 1 receptor may relate to the anxiogenic phenotype of these animals but the observed change in mu-receptors does not correlate with altered morphine responses.

Until recently the opioid receptor family was thought to consist of only the mu-, delta- and kappa-receptors. The cloning of opioid receptor like receptor (ORL1) and its endogenous ligand nociceptin/orphanin FQ, which displayed anti-opioid properties, has raised the issue of functional co-operativity of this system with the classical opioid system. ORL1 receptor knockout mice have been successfully developed by homologous recombination to allow the issue of potential heterogeneity of this receptor and also of compensatory changes in mu-, delta- or kappa-receptors in the absence of ORL1 to be addressed. We have carried out quantitative autoradiographic mapping of these receptors in the brains of mice that are wild-type, heterozygous and homozygous for the deletion of the ORL1 receptor. ORL1, mu-, delta- and kappa-receptors were labelled with [(3)H] leucyl-nociceptin (0.4 nM), [(3)H] DAMGO (4 nM), [(3)H] deltorphin-I (7 nM), and [(3)H] CI-977 (2.5 nM) respectively. An approximately 50% decrease in [(3)H] leucyl-nociceptin binding was seen in heterozygous ORL1 mutant mice and there was a complete absence of binding in homozygous brains indicating the single gene encodes for the ORL1 receptor and any putative subtypes. No significant gross changes in the binding to other opioid receptors were seen across genotypes in the ORL1 mutant mice demonstrating a lack of major compensation of classical opioid receptors in the absence of ORL1. There were a small number of region specific changes in the expression of classical opioid receptors that may relate to interdependent function with ORL1.

The pharmacology of neurogenic trigeminovascular vasodilator responses in the dura mater is of interest for understanding the pathophysiology of migraine and to develop new therapies for this disabling common condition. Aminergic mechanisms have been implicated in migraine through direct study of amines in patients, and by inference from the pharmacology of many effective anti-migraine compounds, particularly preventative agents. This study used intravital microscopy to assess the role of aminergic transmission in neurogenic dural vasodilatation (NDV) by measuring directly the diameter of dural arteries in sodium pentobarbitone anaesthetised rats. Electrical stimulation of a closed cranial window produces, by local depolarisation of nerves, dural vessel dilation that is monitored continuously on-line using video-microscopy and a video dimension analyser. This dural vasodilatation was not affected by pre-treatment with an alpha1-adrenoceptor agonist (phenylephrine, 1 and 5 microg/kg), or antagonist (corynanthine, 1 and 2 mg/kg), nor by an alpha2-adrenoceptor agonist (UK14,304, 5 microg/kg) or antagonist (yohimbine, 1 and 3 mg/kg). Similarly, we saw no effect of beta-adrenoceptor blockade (propranolol, 1 and 3 mg/kg). The lack of an inhibitory effect of UK14,304 the model of neurogenic dural vasodilation contrasts with its effect in neurogenic dural plasma protein extravasation model. The lack of inhibition of beta-adrenoceptor antagonists in the neurogenic vasodilatation model contrasts with their usefulness as migraine prophylactics, and suggests that their mechanism of action in migraine is unlikely to be through sensory trigeminal fibre terminals at the neurovascular junction. Moreover, the data indicate that the adrenergic system does not play a significant role in neurogenic dural vasodilation.

The cerebral circulation is innervated by calcitonin gene-related peptide (CGRP) containing fibers originating in the trigeminal ganglion. During a migraine attack, there is a release of CGRP in conjunction with the head pain, and triptan administration abolishes both the CGRP release and the pain at the same time. In the search for a novel treatment of migraine, a non-peptide CGRP antagonist has long been sought. Here, we present data on a human cell line and human and guinea-pig isolated cranial arteries for such an antagonist, Compound 1 (4-(2-Oxo-2,3-dihydro-benzoimidazol-1-yl)-piperidine-1-carboxylic acid [1-(3,5-dibromo-4-hydroxy-benzyl)-2-oxo-2-(4-phenyl-piperazin-1-yl)-ethyl]-amide). On SK-N-MC cell membranes, radiolabelled CGRP binding was displaced by both CGRP-(8–37) and Compound 1, yielding pKi values of 8.9 and 7.8, respectively. Functional studies with SK-N-MC cells showed that CGRP-induced cAMP production was antagonised by both CGRP-(8–37) and Compound 1 with pA2 values of 7.8 and 7.7, respectively. Isolated human and guinea pig cerebral arteries were studied with a sensitive myograph technique. CGRP induced a concentration-dependent relaxation in human cerebral arteries which was antagonized by both CGRP-(8–37) and Compound 1 in a competitive manner. In guinea pig basilar arteries, CGRP-(8–37) antagonised the CGRP-induced relaxation while Compound 1 had a weak blocking effect . The clinical studies of non-peptide CGRP antagonists are awaited with great interest.

We have examined the activity of a range of kinins on recombinant human bradykinin receptors, using a high throughput functional assay which measures intracellular Ca 2+ responses. The most potent agonist for Chinese hamster ovary (CHO) cells stably expressing recombinant human bradykinin B 1 receptors were Des-Arg 9 -bradykinin (EC 50 =7.9 nM) and Des-Arg 10 -kallidin (EC 50 =8.6 nM), while the most potent agonist for CHO cells expressing human bradykinin B 2 receptors was bradykinin (EC 50 =2.0 nM). These findings confirm the validity of the recombinant system and the microtitre plate imaging-based characterization system when compared to known agonist properties of the native receptors. The concentration–response relationship for bradykinin at bradykinin B 2 receptors was potently inhibited by [ d -Arg 0 ,Hyp 3 ,β-(2-thienyl)-Ala 5 , d -Tic 7 ,Oic 8 ]-bradykinin (Hoe140) (IC 50 =71 nM), which was 500-fold more potent against the B 2 -expressing cells than the B 1 cells. Bradykinin B 1 receptor-mediated responses activated by Des-Arg 10 -kallidin were fully antagonized by Des-Arg 9 -[Leu 8 ]bradykinin (IC 50 =59 nM), Des-Arg 10 -Hoe140 (IC 50 =211 nM) and most potently by Lys-Lys-Arg-Pro-Hyp-Gly-Igl-Ser- d -Igl-Oic (B9858) (IC 50 =14 nM), none of which displayed any activity against the bradykinin B 2 receptor cell line up to 3 μM. None of the antagonists displayed partial agonism activity in these cell lines. All bradykinin B 1 and B 2 receptor antagonists tested acted in an apparently non-competitive manner that is likely to be due in part to their kinetics and to the nature of the functional assay used.

The anti-emetic profile of the novel brain penetrant tachykinin NK1 receptor antagonist MK-0869 (L-754,030) 2-(R)-(1-(R)-(3,5-bis(trifluoromethyl)phenylethoxy)-3-(S)-(4-fluor o)phenyl-4-(3-oxo-1,2,4-triazol-5-yl)methylmorpholine and its water soluble prodrug, L-758,298, has been examined against emesis induced by cisplatin in ferrets. In a 4 h observation period, MK-0869 and L-758,298 (3 mg/kg i.v. or p.o.) inhibited the emetic response to cisplatin (10 mg/kg i.v.). The anti-emetic protection afforded by MK-0869 (0.1 mg/kg i.v.) was enhanced by combined treatment with either dexamethasone (20 mg/kg i.v.) or the 5-HT3 receptor antagonist ondansetron (0.1 mg/kg i.v.). In a model of acute and delayed emesis, ferrets were dosed with cisplatin (5 mg/kg i.p.) and the retching and vomiting response recorded for 72 h. Pretreatment with MK-0869 (4-16 mg/kg p.o.) dose-dependently inhibited the emetic response to cisplatin. Once daily treatment with MK-0869 (2 and 4 mg/kg p.o.) completely prevented retching and vomiting in all ferrets tested. Further when daily dosing began at 24 h after cisplatin injection, when the acute phase of emesis had already become established, MK-0869 (4 mg/kg p.o. at 24 and 48 h after cisplatin) prevented retching and vomiting in three out of four ferrets. These data show that MK-0869 and its prodrug, L-758,298, have good activity against cisplatin-induced emesis in ferrets and provided a basis for the clinical testing of these agents for the treatment of emesis associated with cancer chemotherapy.

Modulating monoamine activity as a therapeutic strategy continues to dominate antidepressant research, with a recent emphasis on agents with multiple targets, including combined serotonin/noradrenaline re-uptake inhibitors and numerous serotonin receptor ligands. An important new development has been the emergence of potential novel mechanisms of action, notably modulation of the activity of neuropeptides substance P and corticotrophin-releasing factor, and the intracellular messenger cyclic adenosine monophosphate. Efforts in this area have recently been rewarded by the demonstration of antidepressant efficacy of the substance P receptor antagonist MK-0869.

Abnormal processing of amyloid precursor protein (APP), in particular the generation of beta-amyloid (Abeta) peptides, has been implicated in the pathogenesis of Alzheimer's disease. This study examined the consequences of deleting the APP gene on hippocampal synaptic plasticity, and upon the biophysical properties of morphologically identified neurones in APP-null mice. The hippocampus of APP-null mice had a characteristic increase in gliosis throughout the CA1 region and a disruption of staining for the dendritic marker MAP2 and the presynaptic marker synaptophysin. The disruption of MAP2 staining was associated with a significant reduction in overall dendritic length and projection depth of biocytin labeled CA1 neurones. In two groups of APP-null mice that were examined at 8-12 months, and 20-24 months of age, there was an impairment in the formation of long-term potentiation (LTP) in the CA1 region compared to isogenic age matched controls. This LTP deficit was not associated with an alteration in the amplitude of EPSPs at low stimulus frequencies (0.033 Hz) or facilitation during a 100 Hz stimulus train, but was associated with a reduction in post-tetanic potentiation. Paired-pulse depression of GABA-mediated inhibitory post-synaptic currents was also attenuated in APP-null mice. These data demonstrate that the impaired synaptic plasticity in APP deficient mice is associated with abnormal neuronal morphology and synaptic function within the hippocampus.

NMDA-mediated neurotoxicity and oxidative stress have been implicated in the etiology of a number of degenerative diseases including motor neuron disease. The present study examined the effect of chronic administration of the glycine/NMDA receptor antagonist L-701,324 and the monoamine oxidase B inhibitor (r)-deprenyl on the onset and rate of progression of neurological impairment in the motor neuron degeneration (mnd) mouse, a murine model of neurodegeneration. Neurological assessment of mnd mice revealed an onset of motor deficits at 6 months of age as observed by the loss of hindlimb reflex extension. By 7 months, balance was also markedly impaired as measured by deficits in rotarod performance and ability to remain on balancing beams. At 8 months of age mnd mice exhibited gross abnormalities in walking pattern; animals were unable to flex their hindlimbs and tended to walk in small labored movements. Daily administration of L-701,324 (10 mg/kg p.o.) or r(-)-deprenyl (1 mg/kg p.o.) to mnd mice from 4 to 8 months of age failed to delay the onset of symptoms or slow the rate of deterioration of motor performance. These findings suggest that excessive activation of NMDA receptors may not be involved in the pathological process leading to motor neuron dysfunction in mnd mice and do not suggest a protective effect of deprenyl on motor neurons in these mice.

The putative neurotrophic effects of the immunophilin ligand GPI-1046 were evaluated in established experimental systems of neuron survival and axon growth in vitro and in vivo. GPI-1046 marginally increased neurite outgrowth of chick dorsal root ganglia in culture under conditions where a very robust effect of nerve growth factor was seen. GPI-1046 failed to protect dopaminergic neurons from 1-methyl-4-phenylpyridinium in culture or to protect cultured cortical neurons from experimentally induced apoptosis in vitro. In adult rats in vivo, daily administration of GPI-1046 (10 mg/kg, s.c.) for three days enhanced the maximal regeneration distance of both motor and large myelinated sensory axons measured using an electrophysiological assay. However, detailed morphometric analysis of these animals failed to provide evidence for an increase in axon numbers in GPI-1046-treated animals. The ability of GPI-1046 to promote the recovery of dopaminergic function following unilateral 6-hydroxydopamine lesions of the substantia nigra was also tested in rats. In the first study, the duration of amphetamine (3 mg/kg, s.c.)-induced circling, but not the maximal number of rotations, was significantly reduced in animals treated with GPI-1046 for five days (10 mg/kg/day). In a second study, testing the effects of delayed GPI-1046 administration, chronic treatment with GPI-1046 (10 mg/kg/day) for two weeks, beginning one month after surgery, did not alter circling responses. Morphometric analysis failed to reveal any changes in either the density of tyrosine hyroxylase-positive fibres in dopaminergic target areas or in cell numbers in the substantia nigra in both experiments. Thus, while GPI-1046 produced marginal effects on neurite outgrowth in dorsal root ganglia cultures and on functional paramaters of nerve regeneration in vivo, we failed to obtain evidence in support of the notion of a general neuroprotective effect of the compound or for an effect on morphologic nerve regeneration in vivo.

Recent cloning studies have isolated receptors which confer specific responsiveness to calcitonin gene related peptide (CGRP) and the related peptide adrenomedullin. Using in situ hybridisation, we demonstrate the heterogenous distribution of the mRNAs of two proposed CGRP1 receptors (RDC-1 and calcitonin receptor-like receptor, CRLR) in the rat brain. Adrenomedullin receptor mRNA was weakly expressed, principally in the cerebellum. These findings may assist in the determination of the function of these largely uncharacterised receptors.

These studies in anaesthetised rats showed, using intravital microscopy, that the novel anti-migraine agent, rizatriptan, significantly reduced electrically stimulated dural vasodilation but had no effect on increases in dural vessel diameter produced by exogenous substance P or calcitonin gene-related peptide (CGRP). Rizatriptan also significantly inhibited dural plasma protein extravasation produced by high intensity electrical stimulation of the trigeminal ganglion. We suggest that rizatriptan inhibits the release of sensory neuropeptides from perivascular trigeminal nerves to prevent neurogenic vasodilation and extravasation in the dura mater. These prejunctional inhibitory effects may be involved in the anti-migraine action of rizatriptan.

This study investigated the properties of a novel piperidine ether-based tachykinin NK1 receptor antagonist L-733,060, ((2S,3S)-3-((3,5-bis(trifluoromethyl)phenyl)methyloxy)-2-phenyl piperidine and its 2R,3R-enantiomer L-733,061 on [Ca2+]i mobilisation in Chinese hamster ovary cells transfected with human tachykinin NK1 receptors, compared to their effects in rodent cardiovascular and neurogenic plasma extravasation assays. Using FURA-2-imaging techniques, L-733,060 inhibited substance P-induced [Ca2+]i mobilisation with an estimated affinity of 0.8 nM whereas L-733,061 (30-300 nM) did not. No significant effects of L-733,060 were observed on mean arterial blood pressure or heart rate in conscious or anaesthetised rats at doses of3000 micrograms kg-1 i.v. L-733,060 also stereoselectively inhibited neurogenic plasma extravasation in rat dura produced by electrical stimulation of trigeminal nerves with an ID50 of 212 +/- 19 micrograms kg-1 i.v. Thus, L-733,060 is a novel antagonist of human tachykinin NK1 receptors which stereoselectively inhibits neurogenic plasma extravasation at doses that do not cause adverse cardiovascular effects.

The expression of the messenger RNAs encoding N-methyl- d-aspartate receptor subunits in neurologically normal post-mortem human brain was studied by in situ hybridization. In the caudate, putamen and nucleus accumbens strong hybridization signals were observed for N-methyl- d-aspartate R1−1 messenger RNA but much weaker signals for N-methyl- d-aspartate R1–3 and N-methyl- d-aspartate R1–4. N-Methyl- d-aspartate R1–2 was not detectable. N-methyl- d-aspartate R2B was the only N-methyl- d-aspartate R2 subunit detected in these nuclei. In the hippocampus the messenger RNAs for both N-methyl- d-aspartate R1–1 and N-methyl- d-aspartate R1–4 were strongly expressed in the dentate gyrus, CA3-CA1 pyramidal cells, subiculum, entorhinal cortex and perirhinal cortex. Much lower expression was seen for N-methyl- d-aspartate R1–2 and N-methyl- d-aspartate R1–3. The messenger RNAs for both N-methyl- d-aspartate R2A and N-methyl- d-aspartate R2B, but not N-methyl- d-aspartate R2C, subunits were expressed in the hippocampus. In the temporal cortex all N-methyl- d-aspartate R1 isoforms were expressed (N-methyl- d-aspartate R1–1 and N-methyl- d-aspartate R1–4 being the most abundant) and N-methyl- d-aspartate R2A and N-methyl- d-aspartate R2B but not N-methyl- d-aspartate R2C were also moderately expressed. In the brain stem N-methyl- d-aspartate R1–4 was strongly expressed in various nuclei including the locus coeruleus, nucleus centralis superior and deep pontine nuclei. Only weak expression was seen for N-methyl- d-aspartate R1–1 and N-methyl- d-aspartate R1–3 but not N-methyl- d-aspartate R1–2; of the N-methyl- d-aspartate R2 subunits only N-methyl- d-aspartate R2C was found to be expressed in these nuclei. In the cerebellum all the N-methyl- d-aspartate 1 isoforms were expressed (mostly N-methyl- d-aspartate R1–4) in the Purkinje layer which also expressed N-methyl- d-aspartate R2A and N-methyl- d-aspartate R2C. In the molecular layer cells were found expressing N-methyl- d-aspartate R1–4 and N-methyl- d-aspartate R2B and cells in the granule layer were found to express N-methyl- d-aspartate R1−1, N-methyl- d-aspartate R1–3 and N-methyl- d-aspartate R1–4 and N-methyl- d-aspartate R2C only. Preliminary studies indicated that the messenger RNA for the N-methyl- d-aspartate R2D subunit was not expressed in the above areas of brain. These results give the first demonstration of the distribution of N-methyl- d-aspartate receptor subunit messenger RNAs in the human brain. The region-specific expression of subunit combinations suggests a heterogeneity of N-methyl- d-aspartate receptors with diverse physiological/pathophysiological roles and provides a rationale for the development of discriminatory N-methyl- d-aspartate receptor antagonists to target selective neuronal populations.

Insulin and insulin-like growth factors (IGF-I, IGF-II) are closely related polypeptides which are found in the CNS and which promote neuronal survival and neurite outgrowth. They are each associated with specific cell surface receptors and several soluble binding proteins (IGFBPs) which are involved in regulating function and availability. Two analogues of IGF-I were produced by site directed mutagenesis: [Gln3, Ala4, Tyr15, Leu16]IGF-1 (QAYL-IGF) and a B-chain mutant in which the first 16 amino acids of IGF-1 were replaced by the first 17 amino acids of insulin. These analogues have significantly reduced binding affinity for IGFBPs. Using glucose deprivation as a damaging stimulus and assaying lactate dehydrogenase released from cultures as a marker for cell death, we have investigated the effect of IGF analogues on cell death of cerebrocortical and cerebellar granule cell cultures. In the presence of IGF-I, QAYL-IGF or B-chain mutant, the amount of LDH released from cortical and cerebellar granule cell cultures was significantly reduced compared to control (no glucose), indicating that these molecules promote survival. Both QAYL and B-chain mutants, which have reduced affinity for IGFBPs, are as effective as IGF-I in promoting cell survival in conditions of glucose deprivation and their reduced affinity for IGFBPs has no apparent deleterious effect on their neuroprotective function. We also show that the neuroprotective effect of the IGF analogues is due to a direct effect on the neurones in these cultures and is independent of the presence of glia.

A time course study in untreated male Sprague-Dawley rats showed that there was no significant difference in the rate of regeneration of motor and sensory axons after a crush injury. Acidic fibroblast growth factor, given topically directly to the site of the crush injury (osmotic minipump for three days) or systemically (i.v. once daily for three days), stimulated the regeneration of motor axons and myelinated sensory axons in the sciatic nerve of the rat. Dose-dependent increases in regeneration distance were seen after 3.6, 36 or 360 ng/day were applied locally and 3 or 10 micrograms/kg per day were given systematically. The greatest effects were achieved with 36 ng/day locally or 10 micrograms/kg per day systematically, when the increase in regeneration distance over three days compared to untreated rats was 47% and 48%, respectively. Administration of heparin vehicle had no significant effect on regeneration. We conclude that since a crush injury has little effect on the endoneurial tubes and supporting cells, the stimulatory effects of acidic fibroblast growth factor on peripheral nerve regeneration seen in this study are likely to be due to a direct acceleration of axonal extension. This is in contrast with the axonal regeneration that occurs across a gap after nerve transection, where axonal extension may be secondary to stimulatory effects on non-neuronal cells providing a supporting "bridge" across the gap. These results suggest that acidic fibroblast growth factor may be clinically useful in the treatment of peripheral neuropathy in man, particularly since systemic treatment for short periods may be effective.

The cellular immediate early genes are involved in the transcriptional events associated with the dopaminergic regulation of neurotransmitter expression within neurons of the neostriatum. To characterize these events in detail, quantitative in situ hybridization histochemistry was used to assess the temporal effects of acute dopamine receptor blockade with eticlopride, a dopamine D2 receptor antagonist, on the messenger RNA expression of the immediate early genes and neurotransmitters/receptors in the caudate-putamen and ventral tegmental area/substantia nigra pars compacta of the rat. Groups of rats were injected with a single dose of either isotonic saline or eticlopride (0.5 mg/kg i.p.) and killed at various time intervals ranging from 5 min to 24 h and frozen brain sections processed by in situ hybridization histochemistry. Using computerized image analysis, the changes in messenger RNA expression for c-fos, c-jun, jun B, jun D, nerve growth factor I-A and nerve growth factor I-B and for neurotensin, glutamate decarboxylase, proenkephalin, the dopamine D1 receptor and the short and long isoforms of the D2 receptor were examined in the caudate-putamen. In the ventral tegmental area and substantia nigra pars compacta, the messenger RNA expression of the above early response genes and that for neurotensin, tyrosine hydroxylase, cholecystokinin and the D2 receptor isoforms were also examined. In the neostriatum, eticlopride caused a rapid increase in c-fos messenger RNA with significantly increased levels at 10 min (P < 0.01). The levels peaked at 30 min and thereafter declined to control levels. A similar profile was observed for jun B messenger RNA, although levels were still significantly (P < 0.01) elevated at 1 h and declined to basal levels thereafter. No significant changes were observed for c-jun, jun D, nerve growth factor I-A and nerve growth factor I-B messenger RNAs. In the dorsolateral neostriatum, there was an increase in proneurotensin messenger RNA 10 min after eticlopride, this increase becoming significant (P < 0.01) at 60 min. Levels were maximal at 2-6 h and decreased after 12 h to basal levels. There were small increases in proenkephalin messenger RNA, but these were not significant (P < 0.05) until 6 h after the injection. Eticlopride did not have any significant effects on the messenger RNA levels for glutamate decarboxylase, the D1 receptor and the short and long isoforms of the D2 receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

To study whether weaning stimulates the development of a subtype of the delta-opioid receptor we have measured antinociceptive activity of putative delta 1 (DELT I) and delta 2 (DSLET) agonists in weaned and non-weaned 25-day-old rats. In weaned rats, DSLET produced antinociception reversed by the delta 2-antagonist naltriben, but in non-weaned rats DSLET had no effect. Responses to DELT I were evident in both weaned and non-weaned rats and were not antagonised by naltriben. This is the first evidence that delta-receptor subtypes develop independently and suggests that weaning is the stimulus for the expression of the delta 2-subtype.

The 4th International Symposium on CGRP was expertly organized, at a difficult time, by Inger Jansen-Olesen and Lars Edvinsson and held on 28-30 September 2001 at the Royal Danish School of Pharmacy, Copenhagen, Denmark.

Recently we showed that delay of weaning can prevent the transition from μ- to δ- opioid receptor control of stress-induced antinociception between postnatal day 20 and day 25 (1). We hypothesised that weaning might be the stimulus for the developmental expression of a subtype of the δ-receptor since 25% of δ-receptors develop in the fourth postnatal week (2) and have therefore studied the antinociceptive activity of putative selective δ 1 and δ 2 -agonists in weaned and non-weaned 25 day old rats. Antinociceptive responses were measured after i.p. administration of [D-Ser 2 , Leu 5 , Th 6 ]-enkephalin (DSLET) (δ 2 ) and [D-Ala 2 ]-deltorphin I (DELT I) (δ 1 )

The tachykinin NK1 receptor antagonist CP-99,994 ((+)-(2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine) (0.3-3 mg/kg i.v.), but not its inactive enantiomer CP-100,263, attenuated the retching and vomiting induced by cisplatin (10 mg/kg i.v.) in the ferret. CP-99,994, 3 mg/kg i.v., prevented vomiting in all ferrets tested. Since substance P is the preferred ligand at the NK1 receptor subtype these data support a role for the release of this peptide during the emetic response induced by cytotoxic chemotherapeutic agents.

I am grateful for the interest shown in my TiPS article1xNK1 (substance P) receptor antagonists – why are they not analgesic in humans?. Hill, R.G. Trends Pharmacol. Sci. 2000; 21: 244–246Abstract | Full Text | Full Text PDF | PubMed | Scopus (254)See all References1; the letters from Villanueva and from Urban and Fox make pertinent and useful observations that extend the discussion initiated in my short paper.Villanueva raises the interesting prospect of developing reliable animal tests to predict analgesic efficacy in humans by avoiding the reductionist approach, which is almost universally used today (although this is usually due to the operational constraints of what is possible and practical). He advises a holistic approach using, for example, neural ensemble recording, which is a fertile area for research. Advances in the identification of which areas of the CNS are involved in the perception of different aspects of clinical pain, using functional imaging techniques2xPain-related neurons in human cingulate cortex. Hutchison, W.D. et al. Nat. Neurosci. 1999; 2: 403–405Crossref | PubMed | Scopus (377)See all References2, together with the availability of suitable hardware, suggest that these techniques should also be applied in animal experimentation. Imaging techniques are also relevant to the investigation of those intractable pain states that Villanueva mentions as having no apparent sensory system correlates and that might originate in the CNS. It is important to note, however, that treatment of clinical pain is not yet based on a full understanding of the mechanisms of pain and that although great advances are being made in developing mechanism-based pain taxonomy as a guide to treatment, it will be some time before this is possible. Max3xIs mechanism-based pain treatment attainable? Clinical trial issues. Max, M.B. J. Pain. 2000; 1: 2–9Abstract | Full Text | Full Text PDF | PubMed | Scopus (37)See all References3 recently commented that his group have failed to find any significant differences between responses of allodynia and ongoing pain in neuropathic pain patients treated with a range of drugs, despite the fact that basic science studies strongly point to differences in the mechanisms that give rise to these symptoms. When a particular drug (be it an antidepressant, opioid, adrenoceptor agonist or NMDA receptor blocker) was effective, most of the symptoms experienced by the patient were relieved3xIs mechanism-based pain treatment attainable? Clinical trial issues. Max, M.B. J. Pain. 2000; 1: 2–9Abstract | Full Text | Full Text PDF | PubMed | Scopus (37)See all References3, which suggests that mechanism-based differences in pain symptoms might often be small. This has obvious implications in deciding which animal tests might or might not be predictive.Urban and Fox re-address the issue of: (1) whether appropriate animal tests were used to select those tachykinin NK1 receptor antagonists that have so far been studied as potential analgesics in humans and (2) whether these agents were able to cross the blood-brain barrier. This second point is important and received only little attention in my recent article1xNK1 (substance P) receptor antagonists – why are they not analgesic in humans?. Hill, R.G. Trends Pharmacol. Sci. 2000; 21: 244–246Abstract | Full Text | Full Text PDF | PubMed | Scopus (254)See all References1; however, my colleagues and I have dealt with this issue extensively in other publications4.xIn vitro and in vivo predictors of the anti-emetic activity of tachykinin NK1 receptor antagonists. Rupniak, N.M.J. et al. Eur. J. Pharmacol. 1997; 326: 201–209Crossref | PubMed | Scopus (100)See all References, 5.xNeurokinin antagonists. Rupniak, N.M.J. and Hill, R.G. : 135–155See all References. I strongly agree with the general points that Urban and Fox make but disagree with the specific suggestion that the selection of MK869 for clinical study was in some way flawed because we did not establish that this compound was active in guinea-pig antinociception assays before starting human analgesia trials. Although this particular compound was not subjected to such experiments, we have shown that analogues of this compound were active in guinea-pig antinociception tests (see Fig. 2 in Ref. 6xDiscrepant results from preclinical and clinical studies on the potential of substance P–receptor antagonist compounds as analgesics. Boyce, S. and Hill, R.G. : 313–324See all References6) and that MK869 itself was brain penetrant and active in tests in a range of species, including ferret, gerbil and rabbit4.xIn vitro and in vivo predictors of the anti-emetic activity of tachykinin NK1 receptor antagonists. Rupniak, N.M.J. et al. Eur. J. Pharmacol. 1997; 326: 201–209Crossref | PubMed | Scopus (100)See all References, 5.xNeurokinin antagonists. Rupniak, N.M.J. and Hill, R.G. : 135–155See all References, 6.xDiscrepant results from preclinical and clinical studies on the potential of substance P–receptor antagonist compounds as analgesics. Boyce, S. and Hill, R.G. : 313–324See all References, 7.xThe novel NK1 receptor antagonist MK-0869 (L-754,030) and its water soluble phosphoryl prodrug, L-758,298, inhibit acute and delayed cisplatin-induced emesis in ferrets. Tattersall, F.D. et al. Neuropharmacology. 2000; 39: 652–663Crossref | PubMed | Scopus (104)See all References. Perhaps most importantly, it has been shown that similar doses of MK869 to those found to be inactive in analgesic tests in humans were effective in the treatment of cisplatin-induced emesis in humans. Such an anti-emetic action requires high levels of receptor occupancy and brain penetration4.xIn vitro and in vivo predictors of the anti-emetic activity of tachykinin NK1 receptor antagonists. Rupniak, N.M.J. et al. Eur. J. Pharmacol. 1997; 326: 201–209Crossref | PubMed | Scopus (100)See all References, 7.xThe novel NK1 receptor antagonist MK-0869 (L-754,030) and its water soluble phosphoryl prodrug, L-758,298, inhibit acute and delayed cisplatin-induced emesis in ferrets. Tattersall, F.D. et al. Neuropharmacology. 2000; 39: 652–663Crossref | PubMed | Scopus (104)See all References. I therefore do not believe that the guinea-pig is the ‘right species’ in which to evaluate NK1 receptor antagonists as potential analgesics or that agents that are active in this species will be more likely to have predictable analgesic effects in humans. However, I do agree that some NK1 receptor antagonists tested in human pain studies could have been predicted, on the basis of lack of brain penetration or short duration of action in animal tests, to be unlikely to produce analgesia in humans4xIn vitro and in vivo predictors of the anti-emetic activity of tachykinin NK1 receptor antagonists. Rupniak, N.M.J. et al. Eur. J. Pharmacol. 1997; 326: 201–209Crossref | PubMed | Scopus (100)See all References4. Reliable but negative human pain trial data exists only for very few compounds and I would be delighted if SDZNKT343 were to advance to clinical efficacy studies, as suggested by Urban and Fox, to help resolve the issue.I also agree that only a limited range of pain states has been studied so far with NK1 receptor antagonists and it would be helpful if a wider range of conditions could be examined. In particular, it can be argued that these agents might be more effective at controlling long-term hyperalgesia or progressive hypersensitivity than in reducing the intensity of existing pain8xNeurokinin antagonists as potential agents for use in pain management. Ma, Q-P. and Hill, R.G. Curr. Opin. CPNS Invest. Drugs. 1999; 1: 65–71See all References8. However, if one takes a nihilistic approach it could also be argued that the post-operative pain resulting from third molar extraction provides a reliably predictive assay and that any drug that is going to be clinically useful for treating pain should be active in this test. The choice of clinical paradigm for analgesic drug evaluation is clearly a pivotal topic and is now receiving appropriate consideration3xIs mechanism-based pain treatment attainable? Clinical trial issues. Max, M.B. J. Pain. 2000; 1: 2–9Abstract | Full Text | Full Text PDF | PubMed | Scopus (37)See all References3.