A novel peptide, AMG 416 (USAN: velcalcetide), has been identified which acts as an agonist of the calcium-sensing receptor (CaSR). This report summarizes the in vitro and in vivo characterization of AMG 416 activity and the potential clinical utility of this novel compound. AMG 416 activates the human CaSR in vitro, acting by a mechanism distinct from that of cinacalcet, the only approved calcimimetic, since it can activate the CaSR both in the presence or absence of physiological levels of extracellular calcium. Administration of AMG 416 in vivo into either normal or renally compromised rats results in dose-dependent reductions in parathyroid hormone (PTH) levels and corresponding decreases in serum calcium, regardless of the baseline level of PTH. Treatment of 5/6 nephrectomized rats with AMG 416 resulted in dramatic improvements in their metabolic profile, including lower PTH and serum creatinine levels, reduced amounts of vascular calcification, attenuated parathyroid hyperplasia and higher expression of the parathyroid gland regulators CaSR, Vitamin D receptor and FGF23 receptor compared with vehicle-treated animals. No drug accumulation was observed under this dosing regimen, and the terminal half-life of AMG 416 was estimated to be 2 to 4.5 hours. As a long-acting CaSR agonist, AMG 416 is an innovative new therapy for the treatment of hemodialysis patients with secondary hyperparathyroidism.

Phosphodiesterase (PDE) 4 inhibition is a well known anti-inflammatory mechanism, but the development of PDE4 inhibitors has been hampered by side effects such as nausea and emesis. Local delivery of a PDE4 inhibitor to the site of inflammation may overcome these issues. The purpose of this study was to assess the therapeutic potential of E6005, a novel PDE4 inhibitor developed as a topical agent for atopic dermatitis (AD). E6005 potently and selectively inhibited human PDE4 activity with an IC50 of 2.8 nM, and suppressed the production of various cytokines from human lymphocytes and monocytes with IC50 values ranging from 0.49 to 3.1 nM. In mice models, the topical application of E6005 produced an immediate anti-pruritic effect as well as an anti-inflammatory effect with reduced expression of cytokines/adhesion molecules. Based on these observed effects, topical E6005 ameliorated the appearance of atopic dermatitis-like skin lesions in two types of AD models, hapten- and mite-elicited models, exhibiting inhibitory effects comparable to that of tacrolimus. The use of (14)C-labeled E6005 showed rapid clearance from the blood and low distribution to the brain, contributing to the low emetic potential of this compound. These results suggest that E6005 may be a promising novel therapeutic agent with anti-pruritic activity for the treatment of AD.

The aim with the present study is to develop a blood-brain barrier (BBB) permeability model, which is applicable in the drug discovery phase. The BBB ensures proper neural function but it restricts many drugs from entering the brain and this complicates the development of new drugs against central nervous system diseases. Many in vitro models have been developed to predict BBB permeability but the permeability characteristics of the human BBB are notoriously complex and hard to predict. Consequently, one single suitable BBB permeability screening model, which is generally applicable in the early drug discovery phase, does not yet exist. A new refined ex vivo insect based BBB screening model that uses an intact, viable whole brain under controlled 'in vitro' like exposure conditions is presented. This model uses intact brains from dessert locusts, which are placed in a well containing the compound solubilized in an insect buffer. After a limited period of time the brain is removed and the compound concentration in the brain is measured by conventional LC-MS. The data presented here includes 25 known drugs and the data shows that the ex vivo insect model can be used to measure the brain uptake over the hemolymph barrier of drugs and that the brain uptake shows linear correlation with in situ perfusion data obtained in vertebrates. Moreover, this study shows that the insect ex vivo model is able to identify Pgp substrates and the model allows differentiation between low permeability compounds and compounds that are Pgp substrates.

The ovarian cycle affects susceptibility to behavioral and neurological conditions. The molecular mechanisms underlying these changes are poorly understood. Deficits in cyclical fluctuations in steroid hormones and receptor plasticity play a central role in physiological and pathophysiological menstrual conditions. It has been suggested that synaptic GABA-A receptors mediate phasic inhibition in the hippocampus and extrasynaptic receptors mediate tonic inhibition in the dentate gyrus. In this study, we report a novel role of extrasynaptic, delta-containing GABA-A receptors as crucial mediators of the estrous cycle-related changes in neuronal excitability in mice, with hippocampus subfield specificity. In molecular and immunofluorescence studies, there was significant increase in delta-subunit, but not alpha4- and gamma2-subunits, in the dentate gyrus during diestrus stage. However, delta-subunit upregulation was not evident in the CA1 region. The delta-subunit expression was undiminished by age, ovariectomy, and in mice lacking progesterone receptors, but was significantly reduced by finasteride, a neurosteroid synthesis inhibitor. Electrophysiological studies confirmed greater potentiation of GABA currents by progesterone-derived neurosteroid allopregnanolone in dissociated dentate gyrus granule cells in diestrus than in CA1 pyramidal cells. The baseline conductance and allopregnanolone potentiation of tonic currents in dentate granule cells from hippocampal slices were higher than in CA1 pyramidal cells. In behavioral studies, susceptibility to hippocampus kindling epileptogenesis was lower in mice during diestrus stage. These results demonstrate the estrous cycle-related plasticity of neurosteroid-sensitive, delta-containing GABA-A receptors that mediate tonic inhibition and seizure susceptibility. These findings may provide novel insight on molecular cascades of menstrual disorders like catamenial epilepsy, premenstrual syndrome and migraine.

20-Hydroxyeicosatetraenoic acid (20-HETE) contributes to the migration and proliferation of vascular smooth muscle cells (VSMC) in vitro, but there are few studies that address its effects on vascular remodeling in vivo. The present study determined whether inhibition of 20-HETE production attenuates intimal hyperplasia (IH) and vascular remodeling following balloon-injury (BI). Sprague Dawley rats (SD) underwent BI of the common carotid artery and were treated with vehicle, 1-Aminobenzotriazole (ABT, 50 mg/kg intraperitoneal, once daily), or HET0016 (2 mg/kg, subcutaneous injection, twice daily) for 14 days. Fourteen days after BI and treatment, the animals underwent carotid angiography and the arteries were harvested for morphometric, enzymatic and immunohistochemical analysis. There was a 96% reduction of angiographic stenosis in the rats treated with 1-ABT. There was a 61% and 66% reduction of the intima/media area ratios in the 1-ABT and HET0016 treated rats compared with the vehicle-treated group. 20-HETE levels were elevated in BI carotid arteries and the levels were markedly suppressed in the 1-ABT and HET0016 treated groups (p<0.001). Immunostaining indicated that the expression of CYP4A enzyme was markedly increased in the neointima of BI arteries and it colocalized with the expression of smooth muscle-specific actin, indicating increased proliferation of VSMC. An increase in the expression of CYP4A and the production of 20-HETE contributes to neointimal growth in BI rat carotid arteries. Systemic administration 1-ABT or HET0016 prevents the increase in 20-HETE levels and attenuates VSMC migration and proliferation resulting in a marked reduction in IH and vascular remodeling following endothelial injury.

Nociceptin/Orphanin FQ peptide receptor (NOP) agonists produce antinociceptive effects in animal models following spinal administration and potentiate mu-opioid receptor (MOP)-mediated antinociception. The aim of this study was to determine the antinociceptive effects of spinally administered bifunctional NOP/MOP ligands and the antinociceptive functions of spinal NOP and MOP receptors in mice. Antinociceptive effects of bifunctional NOP/MOP ligands BU08028 and SR16435 were pharmacologically compared with the putative bifunctional ligand buprenorphine, selective NOP agonist SCH221510 and selective MOP agonist morphine in neuropathic and inflammatory pain models. Additionally, the degree of tolerance development to the antiallodynic effects of SR16435 and buprenorphine were determined following repeated intrathecal administration. Our data indicated that BU08028 and SR16435 were more potent than morphine and SCH221510 in attenuating nerve injury-induced tactile allodynia and inflammation-induced thermal hyperalgesia. Co-administration of receptor-selective antagonists further revealed that both NOP and MOP in the spinal cord mediated the antiallodynic effects of BU08028 and SR16435, but intrathecal buprenorphine-induced antiallodynic effects were primarily mediated by MOP. Repeated intrathecal administration of SR16435 resulted in reduced and slower development of tolerance to its antiallodynic effects compared to buprenorphine. In conclusion, both NOP and MOP receptors in the spinal cord independently drive antinociception in mice. Spinally administered bifunctional NOP/MOP ligands not only can effectively attenuate neuropathic and inflammatory pain, but also have higher antinociceptive potency with reduced tolerance development to analgesia. Such ligands therefore display a promising profile as spinal analgesics.

Ca(2+)e relaxation of isolated, phenylephrine (PE)-contracted mesenteric arteries is dependent on an intact perivascular sensory nerve network that expresses the Ca(2+)-sensing receptor (CaSR). Activation of the receptor stimulates an endocannabinoid vasodilator pathway, which is dependent on cytochrome P450 and phospholipase A2 but largely independent of the endothelium. In the present study, we determined the role of nitric oxide in perivascular nerve CaSR-mediated relaxation of PE-contracted mesenteric resistant arteries isolated from mice. Using Automated Wire Myography, we studied the effects of NOS gene knockout (NOS-/-) and pharmacologic inhibition of NOS on Ca(2+)e-induced relaxation of PE-contracted arteries. eNOS-/- up-regulates but nNOS-/- down-regulates CaSR expression. NOS-/- reduced maximum Ca(2+)e-induced relaxation with no change in EC50 values with eNOS-/- having the largest effect. The responses of vessels to Calindol and Calhex 231 indicate that the CaSR mediates relaxation. L-NIO reduced Ca(2+)e-induced relaxation of PE-contracted arteries from C57BL/6 control mice by ≈ 38% but had lesser effect in vessels from eNOS-/- mice. 7-NI had no significant effect on relaxation of arteries from NOS-/- mice, but both L-NAME and L-NMMA significantly reduced the relaxation maxima in all groups. Interestingly, the nNOS-selective inhibitor, S-MeTC significantly increased the EC50 value by ≈ 60% in tissues from C57BL/6 mice but reduced the maximum response by ≈ 80% in those from nNOS-/- mice. BK channels play a major role in the process as demonstrated by the effect of iberiotoxin. We conclude that CaSR signaling in mesenteric arteries stimulates eNOS and NO production that regulates Ca(2+)e-induced relaxation.

The G protein coupled receptor 55 (GPR55) is a lysophosphatidylinositol (LPI) receptor that is also responsive to certain cannabinoids. Although GPR55 has been implicated in several (patho)physiological functions, its role is still enigmatic mainly owing to the lack of selective GPR55 antagonists. Here we show that the compound CID16020046 ((4-[4-(3-hydroxyphenyl)-3-(4-methylphenyl)-6-oxo-1H,4H,5H,6H-pyrrolo [3,4-c] pyrazol-5-yl] benzoic acid) is a selective GPR55 antagonist. In yeast cells expressing human GPR55, CID16020046 antagonized agonist-induced receptor activation. In HEK293 cells stably expressing human GPR55 (HEK-GPR55), the compound behaved as an antagonist on LPI-mediated Ca2+ release and extracellular signal-regulated kinases (ERK1/2) activation, but not in HEK293 cells expressing cannabinoid receptor 1 or 2 (CB1 or CB2). CID16020046 concentration-dependently inhibited LPI-induced activation of nuclear factor of activated T-cells (NFAT), nuclear factor kappa of activated B cells (NF-κB) and serum response element (SRE), translocation of NFAT and NF-κB, and GPR55 internalization. It reduced LPI-induced wound healing in primary human lung microvascular endothelial cells (HMVEC-L) and reversed LPI-inhibited platelet aggregation, suggesting a novel role for GPR55 in platelet and endothelial cell function. CID16020046 is therefore a valuable tool to study GPR55-mediated mechanisms in primary cells and tissues.

The blood-testis barrier (BTB) prevents the entry of many xenobiotic compounds into seminiferous tubules thereby protecting developing germ cells. Understanding drug transport across the BTB may improve drug delivery into the testis. Members of one class of drug, nucleoside reverse transcriptase inhibitors (NRTIs), do penetrate the BTB, presumably through interaction with physiological nucleoside transporters. By investigating the mechanism of nucleoside transport, it may be possible to design other drugs to bypass the BTB in a similar manner. We present a novel ex vivo technique to study transport at the BTB that employs isolated, intact seminiferous tubules. Using this system, we found that over 80% of total uptake by seminiferous tubules of the model nucleoside uridine could be inhibited by 100 nM nitrobenzylmercaptopurine riboside (NBMPR), a concentration that selectively inhibits equilibrative nucleoside transporter 1 (ENT1) activity. In primary cultured rat Sertoli cells, 100 nM NBMPR inhibited all transepithelial transport and basolateral uptake of uridine. Immunohistochemical (IHC) staining showed ENT1 to be located on the basolateral membrane of human and rat Sertoli cells, whereas ENT2 was located on the apical membrane of Sertoli cells. Transepithelial transport of uridine by rat Sertoli cells was partially inhibited by the NRTIs zidovudine, didanosine, and tenofovir, consistent with an interaction between these drugs and ENT transporters. These data indicate that ENT1 is the primary route for basolateral nucleoside uptake into Sertoli cells and a possible mechanism for nucleosides and nucleoside-based drugs to undergo transepithelial transport.

An X-ray crystal structure of cytochrome P450 2B4 in complex with the drug paroxetine was solved at 2.14 Å resolution. The structure revealed a conformation intermediate to that of the recently solved complex with amlodipine and of the more compact complex with 4-(4-chlorophenyl)imidazole in terms of the placement of the F-G cassette. Moreover, the comparison of the new structure with 15 previously solved structures of P450 2B4 revealed some new insights into determinants of active site size and shape. The 2B4-paroxetine structure is nearly superimposable on a previously solved closed structure in a ligand free state. Despite the overall conformational similarity among multiple closed structures, the active site cavity volume of the paroxetine complex is enlarged. Further analysis of the accessible space and binding pocket near the heme reveals a new sub-chamber that resulted from the movement of secondary structural elements and rearrangements of active site side chains. Overall, the results from the comparison of all the 16 structures of P450 2B4 demonstrate a cluster of protein conformations that were observed in the presence or absence of various ligands.