Slowing Progression of Cardiovascular Calcification With SNF472 in Patients on Hemodialysis: Results of a Randomized Phase 2b Study.

BACKGROUND: The high cardiovascular morbidity and mortality in patients with end-stage kidney disease could be partially caused by extensive cardiovascular calcification. SNF472, intravenous myo-inositol hexaphosphate, selectively inhibits the formation and growth of hydroxyapatite. METHODS: This double-blind, placebo-controlled phase 2b trial compared progression of coronary artery calcium volume score and other measurements of cardiovascular calcification by computed tomography scan during 52 weeks of treatment with SNF472 or placebo, in addition to standard therapy, in adult patients with end-stage kidney disease receiving hemodialysis. Patients were randomized 1:1:1 to SNF472 300 mg (n=92), SNF472 600 mg (n=91), or placebo (n=91) by infusion in the hemodialysis lines thrice weekly during hemodialysis sessions. The primary end point was change in log coronary artery calcium volume score from baseline to week 52. The primary efficacy analysis combined the SNF472 treatment groups and included all patients who received at least 1 dose of SNF472 or placebo and had an evaluable computed tomography scan after randomization. RESULTS: The mean change in coronary artery calcium volume score was 11% (95% CI, 7-15) for the combined SNF472 dose group and 20% (95% CI, 14-26) for the placebo group (P=0.016). SNF472 compared with placebo attenuated progression of calcium volume score in the aortic valve (14% [95% CI, 5-24] versus 98% [95% CI, 77-123]; P<0.001) but not in the thoracic aorta (23% [95% CI, 16-30] versus 28% [95% CI, 19-38]; P=0.40). Death occurred in 7 patients (4%) who received SNF472 and 5 patients (6%) who received placebo. At least 1 treatment-emergent adverse event occurred in 86%, 92%, and 87% of patients treated with SNF472 300 mg, SNF472 600 mg, and placebo, respectively. Most adverse events were mild. Adverse events resulted in discontinuation of SNF472 300 mg, SNF472 600 mg, and placebo for 14%, 29%, and 20% of patients, respectively. CONCLUSIONS: Compared with placebo, SNF472 significantly attenuated the progression of coronary artery calcium and aortic valve calcification in patients with end-stage kidney disease receiving hemodialysis in addition to standard care. Future studies are needed to determine the effects of SNF472 on cardiovascular events. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02966028.

A Role for the GIRK3 Subunit in Methamphetamine-Induced Attenuation of GABAB Receptor-Activated GIRK Currents in VTA Dopamine Neurons.

Repeated exposure to psychostimulants induces locomotor sensitization and leads to persistent changes in the circuitry of the mesocorticolimbic dopamine (DA) system. G-protein-gated inwardly rectifying potassium (GIRK; also known as Kir3) channels mediate a slow IPSC and control the excitability of DA neurons. Repeated 5 d exposure to psychostimulants decreases the size of the GABAB receptor (GABABR)-activated GIRK currents (IBaclofen) in ventral tegmental area (VTA) DA neurons of mice, but the mechanism underlying this plasticity is poorly understood. Here, we show that methamphetamine-dependent attenuation of GABABR-GIRK currents in VTA DA neurons required activation of both D1R-like and D2R-like receptors. The methamphetamine-dependent decrease in GABABR-GIRK currents in VTA DA neurons did not depend on a mechanism of dephosphorylation of the GABAB R2 subunit found previously for other neurons in the reward pathway. Rather, the presence of the GIRK3 subunit appeared critical for the methamphetamine-dependent decrease of GABABR-GIRK current in VTA DA neurons. Together, these results highlight different regulatory mechanisms in the learning-evoked changes that occur in the VTA with repeated exposure to psychostimulants. SIGNIFICANCE STATEMENT: Exposure to addictive drugs such as psychostimulants produces persistent adaptations in inhibitory circuits within the mesolimbic dopamine system, suggesting that addictive behaviors are encoded by changes in the reward neural circuitry. One form of neuroadaptation that occurs with repeated exposure to psychostimulants is a decrease in slow inhibition, mediated by a GABAB receptor and a potassium channel. Here, we examine the subcellular mechanism that links psychostimulant exposure with changes in slow inhibition and reveal that one type of potassium channel subunit is important for mediating the effect of repeated psychostimulant exposure. Dissecting out the components of drug-dependent plasticity and uncovering novel protein targets in the reward circuit may lead to the development of new therapeutics for treating addiction.

The CALCIPHYX study: a randomized, double-blind, placebo-controlled, Phase 3 clinical trial of SNF472 for the treatment of calciphylaxis.

BACKGROUND: Calcific uraemic arteriolopathy (CUA; calciphylaxis) is a rare disease seen predominantly in patients receiving dialysis. Calciphylaxis is characterized by poorly healing or non-healing wounds, and is associated with mortality, substantial morbidity related to infection and typically severe pain. In an open-label Phase 2 clinical trial, SNF472, a selective inhibitor of vascular calcification, was well-tolerated and associated with improvement in wound healing, reduction of wound-related pain and improvement in wound-related quality of life (QoL). Those results informed the design of the CALCIPHYX trial, an ongoing, randomized, placebo-controlled, Phase 3 trial of SNF472 for treatment of calciphylaxis. METHODS: In CALCIPHYX, 66 patients receiving haemodialysis who have an ulcerated calciphylaxis lesion will be randomized 1:1 to double-blind SNF472 (7 mg/kg intravenously) or placebo three times weekly for 12 weeks (Part 1), then receive open-label SNF472 for 12 weeks (Part 2). All patients will receive stable background care, which may include pain medications and sodium thiosulphate, in accordance with the clinical practices of each site. A statistically significant difference between the SNF472 and placebo groups for improvement of either primary endpoint at Week 12 will demonstrate efficacy of SNF472: change in Bates-Jensen Wound Assessment Tool-CUA (a quantitative wound assessment tool for evaluating calciphylaxis lesions) or change in pain visual analogue scale score. Additional endpoints will address wound-related QoL, qualitative changes in wounds, wound size, analgesic use and safety. CONCLUSIONS: This randomized, placebo-controlled Phase 3 clinical trial will examine the efficacy and safety of SNF472 in patients who have ulcerated calciphylaxis lesions. Patient recruitment is ongoing.

Protein kinase CK2 modulates IL-6 expression in inflammatory breast cancer.

Inflammatory breast cancer is driven by pro-angiogenic and pro-inflammatory cytokines. One of them Interleukin-6 (IL-6) is implicated in cancer cell proliferation and survival, and promotes angiogenesis, inflammation and metastasis. While IL-6 has been shown to be upregulated by several oncogenes, the mechanism behind this phenomenon is not well characterized. Here we demonstrate that the pleotropic Serine/Threonine kinase CK2 is implicated in the regulation of IL-6 expression in a model of inflammatory breast cancer. We used siRNAs targeted toward CK2 and a selective small molecule inhibitor of CK2, CX-4945, to inhibit the expression and thus suppress the secretion of IL-6 in in vitro as well as in vivo models. Moreover, we report that in a clinical trial, CX-4945 was able to dramatically reduce IL-6 levels in plasma of an inflammatory breast cancer patient. Our data shed a new light on the regulation of IL-6 expression and position CX-4945 and potentially other inhibitors of CK2, for the treatment of IL-6-driven cancers and possibly other diseases where IL-6 is instrumental, including rheumatoid arthritis.

Unnatural amino acid mutagenesis of the GABA(A) receptor binding site residues reveals a novel cation-pi interaction between GABA and beta 2Tyr97.

The binding pockets of Cys-loop receptors are dominated by aromatic amino acids. In the GABA(A) receptor alpha1Phe65, beta2Tyr97, beta2Tyr157, and beta2Tyr205 are present at the beta2/alpha1 interface and have been implicated in forming an important part of the GABA binding site. Here, we have probed interactions of these residues using subtle chemical changes: unnatural amino acid mutagenesis was used to introduce a range of Phe analogs, and mutant receptors expressed in oocytes were studied using voltage-clamp electrophysiology. Serial mutations at beta(2)97 revealed a approximately 20-fold increase in EC50 with the addition of each fluorine atom to a phenylalanine, indicating a cation-pi interaction between GABA and this residue. This is the first example of a cation-pi interaction in loop A of a Cys-loop receptor. Along with previous studies that identified cation-pi interactions in loop B and loop C, the result emphasizes that the location of this interaction is not conserved in the Cys-loop family. The data further show that alpha(1)65 (in loop D) is tolerant to subtle changes. Conversely, mutating either beta2Tyr157 (in loop B) or beta2Tyr205 (in loop C) to Phe substantially disrupts receptor function. Substitution of 4-F-Phe, however, at either position, or 4-MeO-Phe at beta2Tyr157, resulted in receptors with wild-type EC50 values, suggesting a possible hydrogen bond. The molecular scale insights provided by these data allow the construction of a model for GABA docking to the agonist binding site of the GABA(A) receptor.

Methamphetamine-evoked depression of GABA(B) receptor signaling in GABA neurons of the VTA.

Psychostimulants induce neuroadaptations in excitatory and fast inhibitory transmission in the ventral tegmental area (VTA). Mechanisms underlying drug-evoked synaptic plasticity of slow inhibitory transmission mediated by GABA(B) receptors and G protein-gated inwardly rectifying potassium (GIRK/Kir(3)) channels, however, are poorly understood. Here, we show that 1 day after methamphetamine (METH) or cocaine exposure both synaptically evoked and baclofen-activated GABA(B)R-GIRK currents were significantly depressed in VTA GABA neurons and remained depressed for 7 days. Presynaptic inhibition mediated by GABA(B)Rs on GABA terminals was also weakened. Quantitative immunoelectron microscopy revealed internalization of GABA(B1) and GIRK2, which occurred coincident with dephosphorylation of serine 783 (S783) in GABA(B2), a site implicated in regulating GABA(B)R surface expression. Inhibition of protein phosphatases recovered GABA(B)R-GIRK currents in VTA GABA neurons of METH-injected mice. This psychostimulant-evoked impairment in GABA(B)R signaling removes an intrinsic brake on GABA neuron spiking, which may augment GABA transmission in the mesocorticolimbic system.

Anaesthetic impairment of immune function is mediated via GABA(A) receptors.

BACKGROUND: GABA(A) receptors are members of the Cys-loop family of neurotransmitter receptors, proteins which are responsible for fast synaptic transmission, and are the site of action of wide range of drugs. Recent work has shown that Cys-loop receptors are present on immune cells, but their physiological roles and the effects of drugs that modify their function in the innate immune system are currently unclear. We are interested in how and why anaesthetics increase infections in intensive care patients; a serious problem as more than 50% of patients with severe sepsis will die. As many anaesthetics act via GABA(A) receptors, the aim of this study was to determine if these receptors are present on immune cells, and could play a role in immunocompromising patients. PRINCIPAL FINDINGS: We demonstrate, using RT-PCR, that monocytes express GABA(A) receptors constructed of α1, α4, ß2, γ1 and/or δ subunits. Whole cell patch clamp electrophysiological studies show that GABA can activate these receptors, resulting in the opening of a chloride-selective channel; activation is inhibited by the GABA(A) receptor antagonists bicuculline and picrotoxin, but not enhanced by the positive modulator diazepam. The anaesthetic drugs propofol and thiopental, which can act via GABA(A) receptors, impaired monocyte function in classic immunological chemotaxis and phagocytosis assays, an effect reversed by bicuculline and picrotoxin. SIGNIFICANCE: Our results show that functional GABA(A) receptors are present on monocytes with properties similar to CNS GABA(A) receptors. The functional data provide a possible explanation as to why chronic propofol and thiopental administration can increase the risk of infection in critically ill patients: their action on GABA(A) receptors inhibits normal monocyte behaviour. The data also suggest a potential solution: monocyte GABA(A) receptors are insensitive to diazepam, thus the use of benzodiazepines as an alternative anesthetising agent may be advantageous where infection is a life threatening problem.

GABAB receptor coupling to G-proteins and ion channels.

GABA(B) receptors have been found to play a key role in regulating membrane excitability and synaptic transmission in the brain. The GABA(B) receptor is a G-protein coupled receptor (GPCR) that associates with a subset of G-proteins (pertussis toxin sensitive Gi/o family), that in turn regulate specific ion channels and trigger cAMP cascades. In this review, we describe the relationships between the GABA(B) receptor, its effectors and associated proteins that mediate GABA(B) receptor function within the brain. We discuss a unique feature of the GABA(B) receptor, the requirement for heterodimerization to produce functional receptors, as well as an increasing body of evidence that suggests GABA(B) receptors comprise a macromolecular signaling heterocomplex, critical for efficient targeting and function of the receptors. Within this complex, GABA(B) receptors associate specifically with Gi/o G-proteins that regulate voltage-gated Ca(2+) (Ca(V)) channels, G-protein activated inwardly rectifying K(+) (GIRK) channels, and adenylyl cyclase. Numerous studies have revealed that lipid rafts, scaffold proteins, targeting motifs in the receptor, and regulators of G-protein signaling (RGS) proteins also contribute to the function of GABA(B) receptors and affect cellular processes such as receptor trafficking and activity-dependent desensitization. This complex regulation of GABA(B) receptors in the brain may provide opportunities for new ways to regulate GABA-dependent inhibition in normal and diseased states of the nervous system.

The F-loop of the GABA A receptor gamma2 subunit contributes to benzodiazepine modulation.

GABA(A) receptors can be modulated by benzodiazepines, although these compounds do not directly activate or inhibit the receptors. The prototypic benzodiazepine, diazepam, potentiates responses to GABA in GABA(A) receptors that contain a gamma subunit. Here we have used mutagenesis, radioligand binding, voltage clamp electrophysiology, and homology modeling to probe the role of the F-loop residues Asp(192)-Arg(197) in the GABA(A) receptor gamma(2) subunit in diazepam potentiation of the GABA response. Substitution of all of these residues with Ala and/or a residue with similar chemical properties to the wild type residue decreased the level of diazepam potentiation, and one mutation (D192A) resulted in its complete ablation. None of the mutations changed the GABA EC(50) or the [(3)H]flumazenil binding affinity, suggesting they do not affect GABA or benzodiazepine binding characteristics; we therefore propose that they are involved in the diazepam-mediated conformational change that results in an increased response to GABA. Homology models of the receptor binding pocket in agonist-bound and unbound states suggest that the F-loop is flexible and has different orientations in the two states. Considering our data in relation to these models, we find that the F-loop residues could contribute to hydrogen bond networks and hydrophobic interactions with neighboring residues that change during receptor activation.

Mutagenesis and molecular modeling reveal the importance of the 5-HT3 receptor F-loop.

The 5-HT(3) receptor is a member of the Cys-loop family of ligand-gated ion channels. The extracellular domains of these proteins contain six amino acid loops (A-F) that converge to form the ligand binding site. In this study we have mutated 21 residues in or close to the 5-HT(3) receptor F-loop (Ile(192) to Gly(212)) to Ala or to a residue with similar chemical properties. Mutant receptors were expressed in HEK293 cells, and binding affinity was measured using [(3)H]granisetron. Two regions displayed decreases in binding affinity when mutated to Ala (Ile(192)-Arg(196) and Asp(204)-Ser(206)), but only one region was sensitive when mutated to chemically similar residues (Ile(192)-Val(201)). Homology modeling using acetylcholine-binding protein crystal structures with a variety of different bound ligands suggests there may be distinct movements of Trp(195) and Asp(204) upon ligand binding, indicating that these residues and their immediate neighbors have the ability to interact differently with different ligands. The models suggest predominantly lateral movement around Asp(204) and rotational movement around Trp(195), indicating the former is in a more flexible region. Overall our results are consistent with a flexible 5-HT(3) receptor F-loop with two regions that have specific but distinct roles in ligand binding.