Rod A. Porter

SB-334867-A: THE FIRST SELECTIVE OREXIN-1 RECEPTOR ANTAGONIST

The pharmacology of various peptide and non‐peptide ligands was studied in Chinese hamster ovary (CHO) cells stably expressing human orexin‐1 (OX1) or orexin‐2 (OX2) receptors by measuring intracellular calcium ([Ca2+]i) using Fluo‐3AM. Orexin‐A and orexin‐B increased [Ca2+]i in CHO‐OX1 (pEC50=8.38±0.04 and 7.26±0.05 respectively, n=12) and CHO‐OX2 (pEC50=8.20±0.03 and 8.26±0.04 respectively, n=8) cells. However, neuropeptide Y and secretin (10 pM – 10 μM) displayed neither agonist nor antagonist properties in either cell‐line. SB‐334867‐A (1‐(2‐Methyylbenzoxanzol‐6‐yl)‐3‐[1,5]naphthyridin‐4‐yl‐urea hydrochloride) inhibited the orexin‐A (10 nM) and orexin‐B (100 nM)‐induced calcium responses (pKB=7.27±0.04 and 7.23±0.03 respectively, n=8), but had no effect on the UTP (3 μM)‐induced calcium response in CHO‐OX1 cells. SB‐334867‐A (10 μM) also inhibited OX2 mediated calcium responses (32.7±1.9% versus orexin‐A). SB‐334867‐A was devoid of agonist properties in either cell‐line. In conclusion, SB‐334867‐A is a non‐peptide OX1 selective receptor antagonist.

Central Nervous System Drug Disposition: The Relationship between in Situ Brain Permeability and Brain Free Fraction

The dispositions of 50 marketed central nervous system (CNS) drugs into the brain have been examined in terms of their rat in situ (P) and in vitro apparent membrane permeability (Papp) alongside lipophilicity and free fraction in rat brain tissue. The inter-relationship between these parameters highlights that both permeability and brain tissue binding influence the uptake of drugs into the CNS. Hydrophilic compounds characterized by low brain tissue binding display a strong correlation (R2 = 0.82) between P and Papp, whereas the uptake of more lipophilic compounds seems to be influenced by both Papp and brain free fraction. A nonlinear relationship is observed between logPoct and P over the 6 orders of magnitude range in lipophilicity studied. These findings corroborate recent reports in the literature that brain penetration is a function of both rate and extent of drug uptake into the CNS.

Characterisation of the binding of [3H]-SB-674042, a novel nonpeptide antagonist, to the human orexin-1 receptor

This study characterises the binding of a novel nonpeptide antagonist radioligand, [3H]SB‐674042 (1‐(5‐(2‐fluoro‐phenyl)‐2‐methyl‐thiazol‐4‐yl)‐1‐((S)‐2‐(5‐phenyl‐(1,3,4)oxadiazol‐2‐ylmethyl)‐pyrrolidin‐1‐yl)‐methanone), to the human orexin‐1 (OX1) receptor stably expressed in Chinese hamster ovary (CHO) cells in both a whole cell assay and in a cell membrane‐based scintillation proximity assay (SPA) format. Specific binding of [3H]SB‐674042 was saturable in both whole cell and membrane formats. Analyses suggested a single high‐affinity site, with Kd values of 3.76±0.45 and 5.03±0.31 nM, and corresponding Bmax values of 30.8±1.8 and 34.4±2.0 pmol mg protein−1, in whole cell and membrane formats, respectively. Kinetic studies yielded similar Kd values. Competition studies in whole cells revealed that the native orexin peptides display a low affinity for the OX1 receptor, with orexin‐A displaying a ∼five‐fold higher affinity than orexin‐B (Ki values of 318±158 and 1516±597 nM, respectively). SB‐334867, SB‐408124 (1‐(6,8‐difluoro‐2‐methyl‐quinolin‐4‐yl)‐3‐(4‐dimethylamino‐phenyl)‐urea) and SB‐410220 (1‐(5,8‐difluoro‐quinolin‐4‐yl)‐3‐(4‐dimethylamino‐phenyl)‐urea) all displayed high affinity for the OX1 receptor in both whole cell (Ki values 99±18, 57±8.3 and 19±4.5 nM, respectively) and membrane (Ki values 38±3.6, 27±4.1 and 4.5±0.2 nM, respectively) formats. Calcium mobilisation studies showed that SB‐334867, SB‐408124 and SB‐410220 are all functional antagonists of the OX1 receptor, with potencies in line with their affinities, as measured in the radioligand binding assays, and with approximately 50‐fold selectivity over the orexin‐2 receptor. These studies indicate that [3H]SB‐674042 is a specific, high‐affinity radioligand for the OX1 receptor. The availability of this radioligand will be a valuable tool with which to investigate the physiological functions of OX1 receptors.

Receptor Occupancy and Brain Free Fraction

This study was designed to investigate whether brain unbound concentration (Cu,brain) is a better predictor of dopamine D2 receptor occupancy than total brain concentration, cerebrospinal fluid concentration (CCSF), or blood unbound concentration (Cu,blood). The ex vivo D2 receptor occupancy and concentration-time profiles in cerebrospinal fluid, blood, and brain of six marketed antipsychotic drugs were determined after oral administration in rats at a range of dose levels. The Cu,brain was estimated from the product of total brain concentration and unbound fraction, which was determined using a brain homogenate method. In conclusion, the Cu,brain of selected antipsychotic agents is a good predictor of D2 receptor occupancy in rats. Furthermore, Cu,brain seems to provide a better prediction of D2 receptor occupancy than CCSF or Cu,blood for those compounds whose mechanism of entry into brain tissue is influenced by factors other than simple passive diffusion.

Anorexia and weight loss in male rats 24 h following single dose treatment with orexin-1 receptor antagonist SB-334867

Acute systemic treatment with the selective orexin-1 receptor antagonist SB-334867 (30 mg/kg, i.p.) has been reported not only to inhibit food intake and to accelerate behavioural satiety in rats, but also to produce a significant loss of bodyweight over the 24 h period post-dosing. The present studies were designed to test the hypothesis that the inhibition of weight gain following acute treatment with SB-334867 is due to a persistent anorectic action of the compound. In Experiment 1, the acute effects of SB-334867 (30 mg/kg, i.p.) on food intake and behaviour in a 1 h test with palatable mash were assessed as a function of injection-test interval. Results confirmed that, when administered 30 min prior to testing, SB-334867 significantly suppressed mash intake and accelerated behavioural satiety. More importantly, significant anorexia and behavioural change were also observed when animals were tested 24 h, but not 48 h, post-dosing. As previously reported, all animals treated with the orexin-1 receptor antagonist lost bodyweight over the 24 h period following acute treatment. The generality of these findings was confirmed in Experiment 2, where acute treatment with SB-334867 (30 mg/kg, i.p.) significantly suppressed home cage chow consumption over the 24 h period post-dosing, an effect also accompanied by a significant loss of bodyweight. The results of Experiment 3 showed that, following i.p. administration of 30 mg/kg, SB-334867 has good CNS penetration, reaches peak plasma and brain concentrations at 30 min, and maintains good exposure over 4 h post-dosing. Overall, current data support the hypothesis that a persistent anorectic action contributes to the significant loss of bodyweight observed 24 h following acute dosing with SB-334867. As the compound is virtually undetectable in plasma or brain beyond 8 h post-dosing, and since nothing is known about potentially active metabolites, we consider the possibility that single dose treatment with SB-334867 results in enduring alterations to the orexin-1 receptor and/or downstream signalling pathways.

The hypocretins are weak agonists at recombinant human orexin-1 and orexin-2 receptors

The pharmacology of the orexin‐like peptides, hypocretin‐1 and hypocretin‐2, was studied in Chinese hamster ovary (CHO) cells stably expressing orexin‐1 (OX1) or orexin‐2 (OX2) receptors by measuring intracellular calcium ([Ca2+]i) using Fluo‐3AM. Orexin‐A and orexin‐B increased [Ca2+]i in CHO‐OX1 (pEC50=7.99±0.05 and 7.00±0.10 respectively, n=8) and CHO‐OX2 (pEC50=8.30±0.05 and 8.21±0.07 respectively, n=5). However, hypocretin‐1 and hypocretin‐2 were markedly less potent, with pEC50 values of 5.31±0.04 and 5.41±0.04 respectively in CHO‐OX2 cells (n=5). In CHO‐OX1 cells 10 μM hypocretin‐1 only elicited a 37.5±3.4% response whilst 10 μM hypocretin‐2 elicited a 18.0±2.1% response (n=8). Desensitisation of OX1 or OX2 with orexin‐A (100 nM) abolished the response to orexin‐A (10 nM) and the hypocretins (10 μM), but not to UTP (3 μM). In conclusion, the hypocretins are only weak agonists at the orexin receptors.

Discovery process and pharmacological characterization of a novel dual orexin 1 and orexin 2 receptor antagonist useful for treatment of sleep disorders.

The hypothalamic peptides orexin-A and orexin-B are potent agonists of two G-protein coupled receptors, namely the OX(1) and the OX(2) receptor. These receptors are widely distributed, though differentially, in the rat brain. In particular, the OX(1) receptor is highly expressed throughout the hypothalamus, whilst the OX(2) receptor is mainly located in the ventral posterior nucleus. A large body of compelling evidence, both pre-clinical and clinical, suggests that the orexin system is profoundly implicated in sleep disorders. In particular, modulation of the orexin receptors activation by appropriate antagonists was proven to be an efficacious strategy for the treatment of insomnia in man. A novel, drug-like bis-amido piperidine derivative was identified as potent dual OX(1) and OX(2) receptor antagonists, highly effective in a pre-clinical model of sleep.

N-1 substituted pyrimidin-4-ones: Novel, orally active inhibitors of lipoprotein-associated phospholipase A2

From two related series of 2-(alkylthio)-pyrimidones, a novel series of 1-((amidolinked)-alkyl)-pyrimidones has been designed as nanomolar inhibitors of human lipoprotein-associated phospholipase A2. These compounds show greatly enhanced activity in isolated plasma. Selected derivatives such as compounds 51 and 52 are orally active with a good duration of action.

New quinoline NK3 receptor antagonists with CNS activity.

Lead optimisation starting from the previously reported selective quinoline NK(3) receptor antagonists talnetant 2 (SB-223412) and 3 (SB-222200) led to the identification of 3-aminoquinoline NK(3) antagonist 10 (GSK172981) with excellent CNS penetration. Investigation of a structurally related series of sulfonamides with reduced lipophilicity led to the discovery of 20 (GSK256471). Both 10 and 20 are high affinity, potent NK(3) receptor antagonists which despite having different degrees of CNS penetration produced excellent NK(3) receptor occupancy in an ex vivo binding study in gerbil cortex.

Glycine transporter 1 (GlyT1) inhibitors exhibit anticonvulsant properties in the rat maximal electroshock threshold (MEST) test

Glycine can act as either an inhibitory neurotransmitter or as a potentiator of NMDA-dependent excitatory neurotransmission. There is some evidence that glycine can have both pro- and anticonvulsant properties in various rodent models of epilepsy. In the present study we tested several glycine transporter 1 (GlyT1) inhibitors including NFPS, SSR 504734, Lu AA21279, Org 25935, SB-710622, GSK931145, as well as the glycine agonist d-serine, in the maximal electroshock threshold (MEST) test in the rat. In a series of experiments, male Sprague-Dawley rats (n=12/group) were pre-treated with a compound of interest and then received an electric shock delivered via corneal electrodes. A cohort of satellite animals (n=3/group) was also used to measure blood and brain levels of Org 25935. All GlyT1 inhibitors increased seizure thresholds dose-dependently, indicative of anticonvulsant activity. SB-710622 and GSK931145 had lower minimum effective doses (MEDs) in the MEST test than other GlyT1 inhibitors. At estimated t(max), increases in dose administered were paralleled by increases in blood and brain concentrations of Org 25935. Thus, increasing extracellular concentration of glycine via inhibition of its uptake protects from electroshock-induced seizures in the rat. Whether strychnine-sensitive or strychnine-insensitive glycine binding sites are involved in this effect remains to be determined.