Angela R. Williams

Effects of the bradykinin B1 receptor antagonist des-Arg9[Leu8]bradykinin and genetic disruption of the B2 receptor on nociception in rats and mice

Abstract The contributions of B1 and B2 bradykinin receptors to acute and chronic inflammatory hyperalgesia were examined using the peptide B1 receptor antagonist des‐Arg9[Leu8]bradykinin and transgenic Bk2r‐/‐ mice. In normal rats and mice, des‐Arg9[Leu8]bradykinin (30 nmol/kg i.v. or s.c.) inhibited carrageenan‐induced hyperalgesia and the late phase nociceptive response to formalin. The active dose range was narrow, suggesting partial agonist activity of this peptide. In rats with monoarthritis, des‐Arg9[Leu8]bradykinin (up to 30 nmol/kg i.v.) failed to reduce the number of vocalisations elicited by gentle flexion and extension of the inflamed limb; however, hyperalgesia was exacerbated by administration of the B1 receptor agonist des‐[Arg9]bradykinin (100 nmol/kg i.v.), consistent with other evidence for local induction of B1 receptors during adjuvant‐induced arthritis. The nociceptive response to intraplantar injection of bradykinin (10 nmol) and hyperalgesia induced by carrageenan (0.6 mg) were absent in Bk2r‐/‐ mice, indicating that stimulation of B2 receptors is an essential step in the initiation of some nociceptive and inflammatory reactions. However, the nociceptive response to formalin (2.5% intraplantar), including inhibition of the late phase by des‐Arg9[Leu8]bradykinin (0.3 nmol), and induction of thermal hyperalgesia by Freunds adjuvant (0.1%) appeared intact in Bk2r‐/‐ mice. These findings support other evidence for an involvement of B1 receptors in inflammatory hyperalgesia and suggest that B1 receptor antagonists may be clinically useful as anti‐inflammatory and analgesic drugs.

In vitro and in vivo predictors of the anti-emetic activity of tachykinin NK1 receptor antagonists

The ability of tachykinin NK1 receptor antagonists to inhibit GR73632 (D-Ala-[L-Pro9,Me-Leu8]substance P-(7-11))-induced foot tapping in gerbils was employed as an indirect measure of brain penetration and this was compared with their ability to prevent acute emesis induced by cisplatin in ferrets. (+)-GR203040 ((2S,3S and 2R,3R)-2-methoxy-5-tetrazol-1-yl-benzyl-(2-phenyl-piperidin- 3-yl)-amine), CP-99,994 ((2S,3S)-cis-3-(2-methoxybenzylamino)-2-phenyl piperidine) dihydrochloride), and L-742,694 (2-(S)-(3,5-bis(trifluoromethyl)benzyloxy)-3-(S)-phenyl-4-(5-(3-oxo-1,2, 4-triazolo)methylmorpholine) potently inhibited GR73632-induced foot tapping (ID50 < or = 0.85 mg/kg), and acute retching induced by cisplatin (ID50 < or = 0.18 mg/kg). RPR100893 ((3aS,4S,7aS)-7,7-diphenyl-4-(2-methoxyphenyl)-2-[(S)-2-(2-m ethoxyphenyl)proprionyl] perhydroisoindol-4-ol) was not a potent antagonist of retching (ID50 4.1 mg/kg) or foot tapping (ID50 > 10 mg/kg). High doses (3-10 mg/kg) of CGP49823 ((2R,4S)-2-benzyl-1-(3,5-dimethylbenzoyl)-N-[(4-quinolinyl)methyl] -4-piperineamine) dihydrochloride), FK888 (N2-[(4R)-4-hydroxy-1-(1-methyl-1H-indol-3-yl)carbonyl-L-propyl]-N-methy l-N-phenylmethyl-L-3-(2-naphthyl)-alaninamide), and LY303870 ((R)-1-[N-(2-methoxybenzyl)acetylamino]-3-(1H-indol-3-yl)-2-[N-(2-(4-(pi peridinyl)piperidin-1-yl)acetyl)amino]propane) were required to inhibit foot tapping; these agents were not anti-emetic in this dose range. SR140333 ((S)-1-[2-[3-(3,4-dichlorphenyl)-1 (3-isopropoxyphenylacetyl)piperidin-3-yl] ethyl]-4-phenyl-1 azaniabicyclo [2.2.2]octane; 3-10 mg/kg) failed to inhibit foot tapping or emesis. Affinities for the human and ferret tachykinin NK1 receptor were highly correlated (r = 0.93, P = 0.0008). Inhibition of foot tapping in gerbils, but not NK1 receptor binding affinity, predicted anti-emetic activity in ferrets (r = 0.75, P < 0.01). These findings confirm that the anti-emetic activity of tachykinin NK1 receptor antagonists is dependent on brain penetration.

The substance P antagonist L-760,735 inhibits stress-induced NK1 receptor internalisation in the basolateral amygdala

The distribution of NK(1) receptor immunoreactivity in the amygdaloid complex, induction of NK(1) receptor endocytosis in the amygdala following immobilisation stress, and the ability of pretreatment with the substance P antagonist L-760,735 or imipramine to block this response were examined in gerbils, a species with human-like NK(1) receptor pharmacology. Highest levels of immunolabelling were observed in the anterior, amygdalo-hippocampal and medial nuclei. Less dense labelling was observed in the basolateral nucleus, where it was possible to clearly visualise the distal dendrites of NK(1) immunoreactive neurones and quantify the effect of immobilisation stress on NK(1) receptor endocytosis morphology, a marker of local substance P release. Immobilisation for 1 h caused an approximately 60% increase in the number of dendritic processes undergoing NK(1) receptor endocytosis in the basolateral amygdala that was inhibited by acute pretreatment of animals with L-760,735 (3 mg/kg), but not by imipramine (10 mg/kg). These findings are consistent with other evidence that the amygdala represents a possible site of action for the antidepressant and anxiolytic efficacy of substance P antagonists.

Differential inhibition of foot tapping and chromodacryorrhoea in gerbils by CNS penetrant and non-penetrant tachykinin NK1 receptor antagonists

The inhibition of GR73632-induced foot tapping in gerbils by central nervous system (CNS) penetrant of tachykinin NK1 receptor antagonists was investigated. Intracerebroventricular infusion of the highly selective tachykinin NK1 receptor agonist GR73632 (3 pmol) induced a vigorous repetitive hind foot tapping response which was inhibited by CP-99,994 (ID50 = 0.06 mg/kg i.v.) but not by its less active enantiomer, CP100,263 (10 mg/kg i.v.). Similarly, the poorly CNS penetrant quaternised compound, L-743,310, failed to inhibit foot tapping at doses up to 3 mg/kg i.v. In contrast, all three compounds inhibited chromodacryorrhoea induced by systemic administration of GR73632 (0.5 nmol i.v.) (ID50 = 0.06, 2.95 and 0.004 mg/kg i.v., respectively). These findings confirm that foot tapping and chromodacryorrhoea in gerbils provide simple assays for the central and peripheral activation of tachykinin NK1 receptors.

2-Aryl Indole NK1 receptor antagonists: optimisation of indole substitution

The synthesis and biological evaluation of a series of 2-aryl indoles with high affinity for the human neurokinin-1 (hNK1) receptor are reported, concentrating on optimisation of the indole substitution.

Comparison of the functional blockade of rat substance P (NK1) receptors by GR205171, RP67580, SR140333 and NKP-608

Extensive screening of compound libraries was undertaken to identify compounds with high affinity for the rat NK(1) receptor based on inhibition of [(125)I]-substance P binding. RP67580, SR140333, NKP-608 and GR205171 were selected as compounds of interest, with cloned rat NK(1) receptor binding K(i) values of 0.15-1.9 nM. Despite their high binding affinity, NKP-608 and GR205171 exhibited only a moderate functional antagonism of substance P-induced inositol-1-phosphate accumulation and acidification rate at 1 microM using cloned or native rat NK(1) receptors in vitro. The ability of the compounds to penetrate the CNS was determined by inhibition of NK(1) agonist-induced behaviours in gerbils and rats. GR205171 and NKP-608 potently inhibited GR73632-induced foot drumming in gerbils (ID(50) 0.04 and 0.2 mg/kg i.v., respectively). In contrast, RP67580 and SR140333 were poorly brain penetrant in gerbils (no inhibition at 10 mg/kg i.v.) and were not examined further in vivo. In rats, only high doses of GR205171 (10 or 30 mg/kg s.c.) inhibited NK(1) agonist-induced sniffing and hypertension, whilst NKP-608 (1 or 10 mg/kg i.p.) was without effect. GR205171 (3-30 mg/kg s.c.) caused only partial inhibition of separation-induced vocalisations in rat pups, a response that is known to be NK(1) receptor mediated in other species. These observations demonstrate the shortcomings of currently available NK(1) receptor antagonists for rat psychopharmacology assays.

2-Aryl indole NK1 receptor antagonists: optimisation of the 2-Aryl ring and the indole nitrogen substituent

Novel 2-aryl indole hNK1 receptor ligands were prepared utilising palladium cross-coupling chemistry of a late intermediate as a key step. Compounds with high hNK1 receptor binding affinity and good brain penetration (e.g., 9d) were synthesised.

Antinociceptive activity of the tachykinin NK1 receptor antagonist, CP-99,994, in conscious gerbils.

1 The ability of CP‐99,994, and its less active enantiomer, CP‐100,263, to inhibit spontaneous behaviours and hyperalgesia induced by central infusion of the NK1 receptor agonist, GR73632 or intraplantar injection of formalin was investigated in rats and gerbils 2 GR73632 (3 pmol, i.c.v.)‐induced foot tapping in gerbils was dose‐dependently inhibited by CP‐99,994 (0.1‐1 mg kg−1, s.c), but not by CP‐100,263 (10 mg kg−1, s.c.) using pretreatment times up to 60 min. The centrally active dose‐range for CP‐99,994 was increased to 1 −10 mg kg−1 s.c. with a higher challenge dose of GR73632 (30 pmol, i.e.v.) 3 In gerbils, intrathecal (i.t.) injection of GR73632 (30 pmol) elicited behaviours (licking, foot tapping or flinching and face washing) which closely resembled, but which was less specifically localized than, behaviours seen in animals injected with formalin (0.1–5%) into one hindpaw 4 In rats, CP‐100,263, but not CP‐99,994 (up to 30 mg kg−1), inhibited the early phase response to intraplantar injection of 5% formalin (ID50= 13.9 mg kg−1. The late phase was inhibited by both compounds (ID50 values 36.3 and 20.9 mg kg−1, respectively). In gerbils, there was marginal evidence for enantioselective inhibition of the early phase induced by formalin (2%). The ID50 values were 6.2 mg kg−1 for CP‐99,994 and 13.4 mg kg−1 for CP‐100,263 5 Intrathecal injection of GR73632 (30 pmol) caused thermal hyperalgesia in gerbils which was inhibited enantioselectively by s.c. administration of CP‐99,994 (ID50 = 2.46 mg kg−1, but not by CP‐100,263 (30 mg kg−1) 6 In gerbils, intraplantar injection of formalin (0.1%) caused thermal hyperalgesia which was inhibited by CP‐99,994 (ID50= 1.1 mg kg−1, s.c). There was a nonsignificant trend for an anti‐algesic effect of CP‐100,236 (estimated ID50 = 8.2 mg kg−1, s.c) 7 These findings support the proposal that NKi receptor antagonists may be useful in the clinical management of pain and reinforce the need to dissociate specific and nonspecific antinociceptive effects of available compounds.

Spirocyclic NK1 antagonists II: [4.5]-spiroethers

A series of novel spiroether-based neurokinin-1 (NK(1)) antagonists is described. The effect of modifications to the spiroether ring and aromatic substituents are discussed, leading to the identification of compounds with high affinity and excellent CNS penetration.

2-Aryl indole NK1 antagonists: optimisation of the amide substituent

The in vivo properties of a series of 2-arylindole NK(1) antagonists have been improved, by modification of the amide substituent. The 1-(2-methoxyphenyl)piperazine amide was identified as a major area of metabolism in the lead compound 1. Replacement of this amine moiety by a 4-benzyl-4-hydroxypiperidine resulted in a compound 18 with reduced clearance and improved central duration of action.