Emma J. Carlson

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.

Enantioselective inhibition of the formalin paw late phase by the NK1 receptor antagonist L-733,060 in gerbils

&NA; Intravenous administration of the NK1 receptor antagonist l‐733,060 to gerbils 3 h before intraplantar injection of formalin caused a dose‐dependent and complete inhibition of the late, but not the early, phase nociceptive response (paw licking). The ID50 for l‐733,060 (0.17 mg/kg) revealed a greater than 50‐fold separation in potency over its less active enantiomer l‐733,061 (ID50 ≥ 10 mg/kg). In contrast, the non‐brain penetrant quaternary ketone NK1 receptor antagonist, l‐743,310 (3 mg/kg), did not attenuate the response to formalin, indicating that the antinociceptive effect of blockade of NK1 receptors by l‐733,060 in this assay is centrally‐mediated. These findings add to the preclinical evidence that NK1 receptor antagonists may be of therapeutic use as centrally‐acting analgesics.

Intra-amygdala injection of the substance P (NK1 receptor) antagonist L-760735 inhibits neonatal vocalisations in guinea-pigs

The involvement of the basolateral amygdala in mediating the inhibition of neonatal vocalisation by substance P (NK(1) receptor) antagonists was examined. These studies determined whether the time course for separation-induced vocalisations in guinea-pig pups coincided with NK(1) receptor internalisation (a marker of substance P release) in the amygdala, and whether vocalisations could be blocked by focal injection of the NK(1) receptor antagonist L-760735 into this brain region. The peak period for neonatal vocalisations occurred 5-10 min following maternal separation. This coincided with the peak increase in the number of cells in the basolateral amygdala exhibiting NK(1) receptor endocytosis, consistent with the proposal that substance P is released in the amygdala as a result of isolation stress. Focal injection of L-760735 (15 nmol per side) but not L-770765 (an analogue of L-760735 which has low NK(1) receptor affinity) into the basolateral amygdala attenuated separation-induced vocalisations. In contrast, injection of L-760735 (15 nmol per side) into the dorsal ventricular nucleus of the thalamus, a region with relatively low density of NK(1) receptors, had no effect on neonatal vocalisations. These findings are consistent with other evidence that the amygdala is one possible site of action for the inhibition of neonatal vocalisations by substance P antagonists.

Reversal of behavioural and electrophysiological correlates of experimental peripheral neuropathy by the NK1 receptor antagonist GR205171 in rats.

In adult rats response latencies to innocuous mechanical stimuli were found to be reduced and, in electrophysiological studies, the receptive fields of dorsal horn neurones were enlarged 7-14 days after chronic constriction injury of the sciatic nerve. The NK1 receptor antagonist GR205171 at 3 mg kg(-1) blocked responses to NK1 agonist evoked activity and reversed the mechanical hypersensitivity following nerve ligation in behavioural assays. GR205171 also reversed the receptive field expansion of spinal dorsal horn neurones caused by loose ligation of the sciatic nerve in an electrophysiological assay in anaesthetised rats. The less active enantiomer L-796,325 did not block NK1 agonist evoked activity at up to 10 mg kg(-1) and had no effect on behavioural or electrophysiological changes following nerve injury, indicating that the effects of GR205171 were attributable to selective NK1 receptor blockade. These data suggest that NK1 receptor antagonists may be useful for the treatment of certain types of neuropathic pain.

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.

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.

Fused bicyclic pyrrolizinones as new scaffolds for human NK1 antagonists.

Previous work on human NK(1) antagonists in which the core of the structure is a substituted pyrrolidine has been disclosed. These compounds showed good binding affinity and functional IP activity, however, many did not exhibit the necessary brain penetration for good in vivo activity. The discovery and preparation of a novel 5,5-fused pyrrolidine core is presented in this paper. This scaffold maintains the excellent binding affinity and functional IP activity of the previously reported compounds, but also exhibits excellent brain penetration as observed in a gerbil foot-tapping assay. The determination of the core structural stereochemistry, which eventually led to the final synthesis of a single active diastereomer, is described.

Potent, Brain-Penetrant, Hydroisoindoline-Based Human Neurokinin-1 Receptor Antagonists

3-[(3aR,4R,5S,7aS)-5-{(1R)-1-[3,5-bis(trifluoromethyl)phenyl]ethoxy}-4-(4-fluorophenyl)octahydro-2H-isoindol-2-yl]cyclopent-2-en-1-one (17) is a high affinity, brain-penetrant, hydroisoindoline-based neurokinin-1 (NK(1)) receptor antagonist with a long central duration of action in preclinical species and a minimal drug-drug interaction profile. Positron emission tomography (PET) studies in rhesus showed that this compound provides 90% NK(1) receptor blockade in rhesus brain at a plasma level of 67 nM, which is about 10-fold more potent than aprepitant, an NK(1) antagonist marketed for the prevention of chemotherapy-induced and postoperative nausea and vomiting (CINV and PONV). The synthesis of this enantiomerically pure compound containing five stereocenters includes a Diels-Alder condensation, one chiral separation of the cyclohexanol intermediate, an ether formation using a trichloroacetimidate intermediate, and bis-alkylation to form the cyclic amine.

Spirocyclic NK1 antagonists I:[4.5] and [5.5]-spiroketals

A series of novel spiroketal-based 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.