Genetic knockout and pharmacological blockade studies of the 5-HT7 receptor suggest therapeutic potential in depression.

The affinity of several antidepressant and antipsychotic drugs for the 5-HT7 receptor and its CNS distribution suggest potential in the treatment of psychiatric diseases. However, there is little direct evidence of receptor function in vivo to support this. We therefore evaluated 5-HT7 receptors as a potential drug target by generating and assessing a 5-HT7 receptor knockout mouse. No difference in assays sensitive to potential psychotic or anxiety states was observed between the 5-HT7 receptor knockout mice and wild type controls. However, in the Porsolt swim test, 5-HT7 receptor knockout mice showed a significant decrease in immobility compared to controls, a phenotype similar to antidepressant treated mice. Intriguingly, treatment of wild types with SB-258719, a selective 5-HT7 receptor antagonist, did not produce a significant decrease in immobility unless animals were tested in the dark (or active) cycle, rather than the light, adding to the body of evidence suggesting a circadian influence on receptor function. Extracellular recordings from hypothalamic slices showed that circadian rhythm phase shifts to 8-OH-DPAT are attenuated in the 5-HT7 receptor KO mice also indicating a role for the receptor in the regulation of circadian rhythms. These pharmacological and genetic knockout studies provide the first direct evidence that 5-HT7 receptor antagonists should be investigated for efficacy in the treatment of depression.

Animal models of depression: challenges from a drug development perspective.

Clinically effective antidepressant drugs have been available for many years but our understanding of how these drugs bring about their therapeutic effects, and how to develop more diverse, better treatments has progressed little. At a time when informed choices need to be taken early on in drug development programs in order to exploit the opportunities for innovation created through genomics, this article considers the strengths and weaknesses of behavioral pharmacology assays and their various roles in the drug discovery process. In the past, a widespread lack of confidence in animal models of depression, combined with the high failure rate of clinical trials and escalating costs of drug development, has stifled a more entrepreneurial approach to drug discovery. In order to encourage greater confidence in discovery programs, the gap between exploratory preclinical and clinical studies needs to be bridged. Functional pharmacology markers need to be developed in patient populations, and in normal volunteers and preclinical species, so that selection of new drug targets can be made with greater confidence at earlier stages of discovery programs. The use of functional brain imaging to quantify drug actions in the human CNS is developing rapidly and may provide powerful new techniques to filter active new drugs of the future from those that are less promising.

P-Glycoprotein efflux reduces the brain concentration of the substance P (NK1 receptor) antagonists SR140333 and GR205171: a comparative study using mdr1a-/- and mdr1a+/+ mice.

Investigation of the antidepressant-like actions of substance P (NK1 receptor) antagonists has been hindered by the few available compounds that bind with high affinity to the rat and mouse NK1 receptor, as these are the most commonly used preclinical species. The best available compounds for such studies are SR140333 and GR205171. However, SR140333 does not penetrate the central nervous system (CNS) after systemic administration, and GR205171 is active only at high doses, where unspecific pharmacological effects occur, so that changes in behaviour cannot be attributed to selective NK1 receptor blockade. These compounds may be substrates for P-glycoprotein (P-gp) and hence are actively excluded from the brain. The present studies used mdr1a-/- mice, a spontaneously occurring mutant that is deficient in P-gp, to examine the CNS penetration of SR140333 and GR205171. Following systemic administration of SR140333 and GR205171 (0.01-10 mg/kg i.v.), considerably higher drug concentrations were achieved in the brains of mdr1a-/- than in mdr1a+/+ mice, and this corresponded with a greater ability to inhibit NK1-agonist-induced behaviours in the mdr1a-/- mutants. Moreover, an NK1-receptor-specific inhibition of aggressive behaviour by GR205171 (10 mg/kg) could be demonstrated in mdr1a-/-, but not mdr1a+/+, mice. These findings suggest that P-gp deficient mice may have useful applications in behavioural pharmacology studies, especially when highly brain-penetrant compounds are not yet available.

The substance P (NK1) receptor antagonist L-760735 inhibits fear conditioning in gerbils.

The ability of the substance P (NK(1) receptor) antagonist (SPA) L-760735 to inhibit conditioned fear was assessed in gerbils using a four plate apparatus. Animals that had been treated with diazepam (3 mg/kg) or L-760735 (3 mg/kg) 30 min before a 3 min conditioning session in the apparatus exhibited a release of plate crossings during the retest session approximately 3 h later. Plate crossings were also increased when animals received diazepam or L-760735 30 min before the retest session. In contrast, fluoxetine and venlafaxine (30 mg/kg) did not exhibit anxiolytic-like effects. During the retest session, gerbils drummed their hind feet on the floor; this behaviour was not observed spontaneously in gerbils that were naïve to the apparatus. Foot drumming was abolished by pretreatment with L-760735 or diazepam (3 mg/kg) but was markedly increased following administration of fluoxetine or venlafaxine (30 mg/kg). Foot drumming elicited by aversive conditioning alone or in combination with fluoxetine was abolished by administration of L-760735 and by amygdala lesions involving the basolateral and lateral nuclei, indicating that this behaviour is an alarm signal or fear response mediated via release of substance P in brain circuits involving the amygdala. The observations provide further evidence for an anxiolytic-like profile of SPAs in preclinical assays and demonstrate a clear difference between the actions of SPAs and established antidepressant drugs.

Chronic substance P (NK1) receptor antagonist and conventional antidepressant treatment increases burst firing of monoamine neurones in the locus coeruleus.

The mechanism of action of conventional antidepressants (e.g. imipramine) has been linked to modulation of central monoamine systems. Substance P (NK1) receptor antagonists may have antidepressant and anxiolytic effects in patients with major depressive disorder and high anxiety but, unlike conventional antidepressants, are independent of activity at monoamine reuptake sites, transporters, receptors, or monoamine oxidase. To investigate the possibility that substance P receptor antagonists influence central monoamine systems indirectly, we have compared the effects of chronic administration of imipramine with that of the substance P receptor antagonist L-760735 on the spontaneous firing activity of locus coeruleus neurones. Electrophysiological recordings were made from brain slices prepared from guinea-pigs that had been dosed orally every day for 4 weeks with either L-760735 (3 mg/kg), imipramine (10 mg/kg), or vehicle (water), or naive animals. Chronic, but not acute, treatment with the substance P receptor antagonist L-760735, induced burst firing of neurones in the locus coeruleus. This effect resembles that of the conventional antidepressant imipramine. However, their effects are dissociable since, in contrast to chronic imipramine treatment, chronic L-760735 treatment does not cause functional desensitisation of somatic alpha2 adrenoceptors. The mechanism by which chronic substance P receptor antagonist or conventional antidepressant treatment influences the pattern of firing activity of norepinephrine neurones remains to be elucidated. However, an indirect action in the periphery or distant brain nuclei has been excluded by the use of the in vitro slice preparation, suggesting a local site of action in the locus coeruleus.

Comparison of the phenotype of NK1R-/- mice with pharmacological blockade of the substance P (NK1 ) receptor in assays for antidepressant and anxiolytic drugs.

The phenotype of NK1R-/- mice was compared with that of acute pharmacological blockade of the tachykinin NK1 receptor on sensorimotor function and in assays relevant to depressive illness and anxiety. The dose range for L-760735 and GR205171 that was associated with functional blockade of central NK1 receptors in the target species was established by antagonism of the behavioural effects of intracerebroventricular NK1 agonist challenge in gerbils, mice and rats. The caudal grooming and scratching response to GR73632 was absent in NK1R-/- mice, confirming that the receptor had been genetically ablated. There was no evidence of sedation or motor impairment in NK1R-/- mice or following administration of L-760735 to gerbils, even at doses in excess of those required for central NK1 receptor occupancy. In the resident-intruder and forced swim test, the behaviour of NK1R-/- mice, or animals treated acutely with L-760735 or GR205171, resembled that seen with the clinically used antidepressant drug fluoxetine. However, the effects of GR205171 were not clearly enantioselective in mice. In contrast, although NK1R-/- mice also exhibited an increase in the duration of struggle behaviour in the tail suspension test, this was not observed following pharmacological blockade with L-760735 in gerbils or GR205171 in mice, suggesting that this may reflect a developmental alteration in the knockout mouse. There was no effect of NK1 receptor blockade with L-760735 in guinea-pigs or GR205171 in rats, or deletion of the NK1 receptor in mice, on behaviour in the elevated plus-maze test for anxiolytic activity. These findings extend previous observations on the phenotype of the NK1R-/- mouse and establish a broadly similar profile following acute pharmacological blockade of the receptor. These studies also serve to underscore the limitations of currently available antagonists that are suitable for use in rat and mouse behavioural assays.

An orally active, water-soluble neurokinin-1 receptor antagonist suitable for both intravenous and oral clinical administration.

1-(5-[[(2R,3S)-2-([(1R)-1-[3,5-Bis(trifluoromethyl)phenyl]ethyl]oxy)-3-(4-fluorophenyl)morpholin-4-yl]methyl]-2H-1,2,3-triazol-4-yl)-N,N-dimethylmethanamine hydrochloride 3 is a high affinity, orally active, h-NK(1) receptor antagonist with a long central duration of action and a solubility in water of >100 mg/mL. The construction of the 5-dimethylaminomethyl 1,2,3-triazol-4-yl unit, which incorporates the solubilizing group of 3, was accomplished by thermal rearrangement of a propargylic azide in the presence of dimethylamine. Compound 3 is highly effective in pre-clinical tests that are relevant to clinical efficacy in emesis and depression.

5-hydroxytryptamine (5-HT)1A autoreceptor adaptive changes in substance P (neurokinin 1) receptor knock-out mice mimic antidepressant-induced desensitization.

Antagonists at substance P receptors of the neurokinin 1 (NK1) type have been shown to represent a novel class of antidepressant drugs, with comparable clinical efficacy to the selective serotonin (5-HT) reuptake inhibitors (SSRIs). Because 5-HT(1A) receptors may be critically involved in the mechanisms of action of SSRIs, we examined whether these receptors could also be affected in a model of whole-life blockade of NK1 receptors, i.e. knock-out mice lacking the latter receptors (NK1-/-). 5-HT(1A) receptor labeling by the selective antagonist radioligand [(3)H]N-[2-[4-(2-methoxyphenyl)1-piperazinyl]-ethyl]-N-(2-pyridinyl)-cyclohexanecarboxamide (WAY 100635) and 5-HT(1A)-dependent [(35)S]GTP-gamma-S binding at the level of the dorsal raphe nucleus (DRN) in brain sections, as well as the concentration of 5-HT(1A) mRNA in the anterior raphe area were significantly reduced (-19 to -46%) in NK1-/- compared with NK1+/+ mice. Furthermore, a approximately 10-fold decrease in the potency of the 5-HT(1A) receptor agonist ipsapirone to inhibit the discharge of serotoninergic neurons in the dorsal raphe nucleus within brainstem slices, and reduced hypothermic response to 8-OH-DPAT, were noted in NK1-/- versus NK1+/+ mice. On the other hand, cortical 5-HT overflow caused by systemic injection of the SSRI paroxetine was four- to sixfold higher in freely moving NK1-/- mutants than in wild-type NK1+/+ mice. Accordingly, the constitutive lack of NK1 receptors appears to be associated with a downregulation/functional desensitization of 5-HT(1A) autoreceptors resembling that induced by chronic treatment with SSRI antidepressants. Double immunocytochemical labeling experiments suggest that such a heteroregulation of 5-HT(1A) autoreceptors in NK1-/- mutants does not reflect the existence of direct NK1-5-HT(1A) receptor interactions in normal mice.

Anxiolytic actions of the substance P (NK1) receptor antagonist L-760735 and the 5-HT1A agonist 8-OH-DPAT in the social interaction test in gerbils.

The gerbil social interaction test has previously detected anxiolytic effects of nicotine and diazepam. In the present study, the high affinity substance P (NK(1)) receptor antagonist L-760735 (3 mg/kg) significantly increased the time spent in social interaction, whereas its low affinity analogue L-781773 (3 mg/kg) was without effect. Diazepam (0.1 mg/kg) and the 5-HT(1A) receptor agonist 8-OH-DPAT (0.003 and 0.01 mg/kg) also increased social interaction, whereas an acute dose of the selective serotonin re-uptake inhibitor fluoxetine (10 mg/kg) decreased the time spent in social interaction. Diazepam (0.1 mg/kg) significantly increased locomotor activity, but this effect was independent of the increase in social interaction. The other drugs tested were without effect on locomotor activity. The present findings suggest that the gerbil social interaction may well provide a useful assay for detecting both anxiolytic and anxiogenic compounds, and suggests that the high affinity NK(1) receptor antagonist L-760735 may prove to be useful as an anxiolytic therapy.

Intra-amygdala injection of the substance P [NK(1) 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.

Pharmacological blockade or genetic deletion of substance P (NK(1)) receptors attenuates neonatal vocalisation in guinea-pigs and mice.

The regulation of stress-induced vocalisations by central NK(1) receptors was investigated using pharmacological antagonists in guinea-pigs, a species with human-like NK(1) receptors, and transgenic NK1R-/- mice. In guinea-pigs, i.c.v. infusion of the selective substance P agonist GR73632 (0.1 nmol) elicited a pronounced vocalisation response that was blocked enantioselectively by the NK(1) receptor antagonists CP-99,994 and L-733,060 (0.1-10 mg/kg). GR73632-induced vocalisations were also markedly attenuated by the antidepressant drugs imipramine and fluoxetine (30 mg/kg), but not by the benzodiazepine anxiolytic diazepam (3 mg/kg) or the 5-HT(1A) agonist buspirone (10 mg/kg). Similarly, vocalisations in guinea-pig pups separated from their mothers were blocked enantioselectively by the highly brain-penetrant NK(1) receptor antagonists L-733,060 and GR205171 (ID(50) 3 mg/kg), but not by the poorly brain-penetrant compounds LY303870 and CGP49823 (30 mg/kg). Separation-induced vocalisations were also blocked by the anxiolytic drugs diazepam, chlordiazepoxide and buspirone (ID(50) 0.5-1 mg/kg), and by the antidepressant drugs phenelzine, imipramine, fluoxetine and venlafaxine (ID(50) 3-8 mg/kg). In normal mouse pups, GR205171 attenuated neonatal vocalisations when administered at a high dose (30 mg/kg) only, consistent with its lower affinity for the rat than the guinea-pig NK(1) receptor. Ultrasound calls in NK1R-/- mouse pups were markedly reduced compared with those in WT pups, confirming the specific involvement of NK(1) receptors in the regulation of vocalisation. These observations suggest that centrally-acting NK(1) receptor antagonists may have clinical utility in the treatment of a range of anxiety and mood disorders.

Analgesic and toxic effects of ABT-594 resemble epibatidine and nicotine in rats.

The present study directly compared the antinociceptive and toxic effects of the neuronal nicotinic receptor agonist ABT-594 ((R)-5-(2-azetidinylmethoxy)-2-chloropyridine) with (-)-nicotine and (+)-epibatidine. Like (-)-nicotine (0.8 and 1.6 mg/kg s.c.) and (+)-epibatidine (0.005 and 0.01 mg/kg s.c.), ABT-594 (0.05 and 0.1 mg/kg s.c.) increased response latencies in the hot-plate test in rats, indicating that it has antinociceptive activity. In contrast to (-)-nicotine and (+)-epibatidine, ABT-594 did not cause rotarod impairment at antinociceptive doses but did cause hypothermia and life-threatening adverse effects including seizures. ABT-594 (0.01 and 0.1 mg/kg i.v.) also produced a dose-dependent increase in blood pressure resembling that observed with (-)-nicotine (0.03, 0.1 and 0. 03 mg/kg i.v.) and (+)-epibatidine (0.001 and 0.003 mg/kg i.v.). Both the antinociceptive and toxic effects (convulsions and hypertension) were abolished by pretreatment with the brain penetrant neuronal nAChR antagonist mecamylamine (1 mg/kg s.c.; i.v. for cardiovascular studies), demonstrating that these actions of ABT-594 were mediated via activation of neuronal nicotinic receptors. Continuous infusion of ABT-594 (0.2 mg/kg per day s.c.) to rats for 7 days followed by challenge with mecamylamine (1 mg/kg i.p.) induced a nicotine-like abstinence syndrome suggesting that ABT-594 has nicotine-like dependence liability. These findings indicate that the acute safety profile of ABT-594 is not significantly improved over other nicotinic analgesics.

Phosphorylated morpholine acetal human neurokinin-1 receptor antagonists as water-soluble prodrugs.

The regioselective dibenzylphosphorylation of 2 followed by catalytic reduction in the presence of N-methyl-D-glucamine afforded 2-(S)-(1-(R)-(3, 5-bis(trifluoromethyl)phenyl)ethoxy)-3-(S)-(4-fluoro)phenyl-4-(5-(2- phosphoryl-3-oxo-4H,-1,2,4-triazolo)methylmorpholine, bis(N-methyl-D-glucamine) salt, 11. Incubation of 11 in rat, dog, and human plasma and in human hepatic subcellular fractions in vitro indicated that conversion to 2 would be expected to occur in vivo most readily in humans during hepatic circulation. Conversion of 11 to 2 occurred rapidly in vivo in the rat and dog with the levels of 11 being undetectable within 5 min after 1 and 8 mg/kg doses iv in the rat and within 15 min after 0.5, 2, and 32 mg/kg doses iv in the dog. Compound 11 has a 10-fold lower affinity for the human NK-1 receptor as compared to 2, but it is functionally equivalent to 2 in preclinical models of NK-1-mediated inflammation in the guinea pig and cisplatin-induced emesis in the ferret, indicating that 11 acts as a prodrug of 2. Based in part on these data, 11 was identified as a novel, water-soluble prodrug of the clinical candidate 2 suitable for intravenous administration in humans.

The substance P antagonist L-760,735 inhibits stress-induced NK(1) 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.

Novel strategies for pharmacotherapy of depression.

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.

Selective NMDA NR2B antagonists induce antinociception without motor dysfunction: correlation with restricted localisation of NR2B subunit in dorsal horn.

The present study investigated the regional distribution of the N-methyl-D-aspartate (NMDA) receptor containing the NR2B subunit protein in rat lumbar spinal cord and examined whether selective NR2B antagonists would exhibit antinociception with reduced side-effect liability than subtype non-selective NMDA antagonists and anticonvulsants. Immunocytochemical studies showed the NR2B subunit had a restricted distribution, with moderate labelling of fibres in laminas I and II of the dorsal horn suggesting a presynaptic location on primary afferent fibers and possible involvement in pain transmission. In the in vivo studies, the NMDA/glycine antagonists (MK-801, 0.02-1 mg/kg i.p., L-687,414 10-300 mg/kg i.p., and L-701,324 1-10 mg/kg i.p.) and the anticonvulsant, gabapentin (10-500 mg/kg p.o.), induced rotarod deficits at antinociceptive doses. In contrast, the selective NR2B antagonists, (+/-)-CP-101,606 (1-100 mg/kg p.o.) and (+/-)-Ro 25-6981 (3-100 mg/kg i.p.) showed a significant dose window. (+/-)-CP-101,606 caused no motor impairment or stimulation in rats at doses up to 100 mg/kg p.o., which is far in excess of those inhibiting allodynia in neuropathic rats (ID50 4.1 mg/kg, p.o.). (+/-)-Ro 25-6981 also showed a significant separation (ID50 allodynia 3.8 mg/kg, i.p.), however, some disruption of rotarod performance was observed at 100 mg/kg. The anticonvulsant lamotrigine (3-500 mg/kg p.o.) also showed a good dose window. These findings demonstrate that NR2B antagonists may have clinical utility for the treatment of neuropathic and other pain conditions in man with a reduced side-effect profile than existing NMDA antagonists.

Onset and progression of motor deficits in motor neuron degeneration (mnd) mice are unaltered by the glycine/NMDA receptor antagonist L-701,324 or the MAO-B inhibitor R(-)-deprenyl.

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.