WAY-318068: a novel, potent and selective noradrenaline re-uptake inhibitor with activity in rodent models of pain and depression.

BACKGROUND AND PURPOSE: Antidepressants, which raise the CNS concentrations of 5-HT and noradrenaline, are frequently used in the treatment of chronic pain; however, it is not known if increasing CNS noradrenaline levels alone is sufficient for efficacy, in part resulting from a lack of small molecules with sufficient selectivity. EXPERIMENTAL APPROACH: In this report, we present the in vitro pharmacological and in vivo pharmacokinetic and pharmacological properties of the novel, orally available and CNS penetrant inhibitor of the noradrenaline transporter (NET), WAY-318068 (1-[(1S,2R)-1-(3,5-difluorophenyl)-2-hydroxy-3-(methylamino)propyl]-7-fluoro-3,3-dimethyl-1,3-dihydro-2H-indol-2-one). KEY RESULTS: WAY-318068 is a potent and effective inhibitor of the NET with a K(i) of 8.7 nM in a binding assay, and an IC(50) of 6.8 nM in an assay of transporter function, without significant binding to the dopamine transporter. Furthermore, the compound has only weak activity at the 5-HT transporter, leading to a functional selectivity of greater than 2500-fold. It is orally bioavailable with substantial quantities of the compound found in the CNS after oral dosing. As measured by microdialysis in rats, the compound causes a robust and significant increase in cortical noradrenaline levels without affecting 5-HT. WAY-318068 was effective in models of acute, visceral, inflammatory, osteoarthritic, neuropathic, diabetic and bone cancer pain, as well as in traditional models of depression at doses that do not cause motor deficits. CONCLUSIONS AND IMPLICATIONS: Collectively, the present results support the conclusion that selectively increasing CNS levels of noradrenaline is sufficient for efficacy in models of depression and pain.

Inhibition of osteoclasts prevents cartilage loss and pain in a rat model of degenerative joint disease.

OBJECTIVE: To investigate the relationship between efficacy of a bisphosphonate, pain and extent of joint damage in the monosodium iodoacetate (MIA) model of painful degenerative joint disease. METHODS: Zoledronate treatment was initiated prior to and at various times following model induction, including late time points representing advanced disease. Radiographic and histological structural parameters were correlated with pain as measured by weight bearing. RESULTS: Intraarticular (IA) MIA resulted in a progressive loss of bone mineral density (BMD) and chondrocytes, thinning of cartilage, loss of proteoglycan, resorption of calcified cartilage and subchondral bone, as well as pain. This was completely prevented by pre-emptive chronic zoledronate treatment with joint sections being histologically indistinguishable from saline-injected controls. When initiation of treatment was delayed efficacy was reduced. In animals with advanced joint degeneration, treatment partially restored BMD and had a significant, but limited, effect on pain. We confirmed these radiographic and behavioral findings in the medial meniscal tear model. To understand the mechanism-of-action of zoledronate we investigated an early time point 4 days post-model induction when chondrocytes were histologically viable, with minor loss of proteoglycan and generalized synovitis. Osteoclast-mediated resorption of the calcified cartilage was observed and was prevented by two doses of zoledronate. CONCLUSION: Subchondral bone remodeling plays an important role in nociception and the pathobiology of the MIA model with osteoclasts being implicated in both bone and cartilage resorption. Inhibition of osteoclastic activity when initiated early leads to improved efficacy.

Hyperpolarization-activated cyclic nucleotide-gated channel mRNA and protein expression in large versus small diameter dorsal root ganglion neurons: correlation with hyperpolarization-activated current gating.

Hyperpolarization-activated cyclic nucleotide-gated channels (HCN) are responsible for the functional hyperpolarization-activated current (I(h)) in dorsal root ganglion (DRG) neurons. We studied HCN1-4 channel mRNA and protein expression and correlated these findings with I(h) functional properties in rat DRG neurons of different size. Quantitative RT-PCR (TaqMan) analysis demonstrated that HCN2 and HCN1 mRNAs were more abundantly expressed in large diameter (55-80 microm) neurons, while HCN3 mRNA was preferentially expressed in small diameter (20-30 microm) neurons. HCN4 mRNA expression was very low in neurons of all sizes. At the protein level, subunit-selective polyclonal antibodies and immunofluorescence indicated that HCN1 and HCN3 are present in large diameter neurons and small diameter neurons. Staining in small diameter neurons was in IB4-positive (non-peptidergic) and IB4-negative (peptidergic) cells. HCN2 immunofluorescent staining was heterogeneous and predominantly in large diameter neurons and in small diameter IB4-negative neurons. HCN4 was poorly expressed in all neurons. Functionally, I(h) amplitude and density were significantly larger, and activation kinetics faster, in large diameter neurons when compared with small neurons. I(h) activation rates in small and large diameter DRG neurons were consistent with the relative abundance of HCN subunits in the respective cell type, considering the reported HCN channel activation rates in heterologous systems (HCN1>HCN2 approximately HCN3>HCN4), suggesting exclusivity of roles of different HCN subunits contributing to the excitability of DRG neurons of different size. Additionally, a functional role of I(h) in small DRG neuron excitability was evaluated using a computational model.

Species-specific in vitro pharmacological effects of the cannabinoid receptor 2 (CB2) selective ligand AM1241 and its resolved enantiomers.

BACKGROUND AND PURPOSE: Racemic (R,S) AM1241 is a cannabinoid receptor 2 (CB(2))-selective aminoalkylindole with antinociceptive efficacy in animal pain models. The purpose of our studies was to provide a characterization of R,S-AM1241 and its resolved enantiomers in vitro and in vivo. EXPERIMENTAL APPROACH: Competition binding assays were performed using membranes from cell lines expressing recombinant human, rat, and mouse CB(2) receptors. Inhibition of cAMP was assayed using intact CB(2)-expressing cells. A mouse model of visceral pain (para-phenylquinone, PPQ) and a rat model of acute inflammatory pain (carrageenan) were employed to characterize the compounds in vivo. KEY RESULTS: In cAMP inhibition assays, R,S-AM1241 was found to be an agonist at human CB(2), but an inverse agonist at rat and mouse CB(2) receptors. R-AM1241 bound with more than 40-fold higher affinity than S-AM1241, to all three CB(2) receptors and displayed a functional profile similar to that of the racemate. In contrast, S-AM1241 was an agonist at all three CB(2) receptors. In pain models, S-AM1241 was more efficacious than either R-AM1241 or the racemate. Antagonist blockade demonstrated that the in vivo effects of S-AM1241 were mediated by CB(2) receptors. CONCLUSIONS AND IMPLICATIONS: These findings constitute the first in vitro functional assessment of R,S-AM1241 at rodent CB(2) receptors and the first characterization of the AM1241 enantiomers in recombinant cell systems and in vivo. The greater antinociceptive efficacy of S-AM1241, the functional CB(2) agonist enantiomer of AM1241, is consistent with previous observations that CB(2) agonists are effective in relief of pain.

Modulation of A-type potassium channels by a family of calcium sensors.

In the brain and heart, rapidly inactivating (A-type) voltage-gated potassium (Kv) currents operate at subthreshold membrane potentials to control the excitability of neurons and cardiac myocytes. Although pore-forming alpha-subunits of the Kv4, or Shal-related, channel family form A-type currents in heterologous cells, these differ significantly from native A-type currents. Here we describe three Kv channel-interacting proteins (KChIPs) that bind to the cytoplasmic amino termini of Kv4 alpha-subunits. We find that expression of KChIP and Kv4 together reconstitutes several features of native A-type currents by modulating the density, inactivation kinetics and rate of recovery from inactivation of Kv4 channels in heterologous cells. All three KChIPs co-localize and co-immunoprecipitate with brain Kv4 alpha-subunits, and are thus integral components of native Kv4 channel complexes. The KChIPs have four EF-hand-like domains and bind calcium ions. As the activity and density of neuronal A-type currents tightly control responses to excitatory synaptic inputs, these KChIPs may regulate A-type currents, and hence neuronal excitability, in response to changes in intracellular calcium.

Association and colocalization of the Kvbeta1 and Kvbeta2 beta-subunits with Kv1 alpha-subunits in mammalian brain K+ channel complexes.

The differential expression and association of cytoplasmic beta-subunits with pore-forming alpha-subunits may contribute significantly to the complexity and heterogeneity of voltage-gated K+ channels in excitable cells. Here we examined the association and colocalization of two mammalian beta-subunits, Kvbeta1 and Kvbeta2, with the K+ channel alpha-subunits Kv1.1, Kv1.2, Kv1.4, Kv1.6, and Kv2.1 in adult rat brain. Reciprocal coimmunoprecipitation experiments using subunit-specific antibodies indicated that Kvbeta1 and Kvbeta2 associate with all the Kv1 alpha-subunits examined, and with each other, but not with Kv2.1. A much larger portion of the total brain pool of Kv1-containing channel complexes was found associated with Kvbeta2 than with Kvbeta1. Single- and multiple-label immunohistochemical staining indicated that Kvbeta1 codistributes extensively with Kv1.1 and Kv1.4 in cortical interneurons, in the hippocampal perforant path and mossy fiber pathways, and in the globus pallidus and substantia nigra. Kvbeta2 codistributes extensively with Kv1.1 and Kv1.2 in all brain regions examined and was strikingly colocalized with these alpha-subunits in the juxtaparanodal region of nodes of Ranvier as well as in the axons and terminals of cerebellar basket cells. Taken together, these data provide a direct demonstration that Kvbeta1 and Kvbeta2 associate and colocalize with Kv1 alpha-subunits in native tissues and provide a biochemical and neuroanatomical basis for the differential contribution of Kv1 alpha- and beta-subunits to electrophysiologically diverse neuronal K+ currents.

Generation and characterization of subtype-specific monoclonal antibodies to K+ channel alpha- and beta-subunit polypeptides.

Molecular characterization of mammalian voltage-sensitive K+ channel genes and their expression became possible with the cloning of the Shaker locus of Drosophila. However, analysis of the expression patterns and subunit composition of native K+ channel protein complexes requires immunological probes specific for the individual K+ channel gene products expressed in excitable tissue. Here, we describe the generation and characterization of monoclonal antibodies (mAbs) against eight distinct mammalian K+ channel polypeptides; the Kv1.1, Kv1.2, Kv1.4, Kv1.5 and Kv1.6 Shaker-related alpha-subunits, the Kv2.1 Shab-related alpha-subunit, and the Kv beta 1 and Kv beta 2 beta-subunits. We characterized the subtype-specificity of these mAbs against native K+ channels in mammalian brain and against recombinant K+ channels expressed in transfected mammalian cells. In addition, we used these mAbs to investigate the cellular and subcellular distribution of the corresponding polypeptides in rat cerebral cortex, as well as their expression levels across brain regions.