The Different Phytochemical Profiles of Salvia officinalis Dietary Supplements Labelled for Menopause Symptoms.

Phytochemical screening of four commercial products containing Salvia officinalis was carried out. Total phenolic content was estimated spectrophotometrically through the use of the Folin-Ciocalteau method, flavonoid content was measured through the use of aluminum chloride and 2,4-dinitrophenylhydrazine colorimetric assays, and isoflavones and α/ß-thujones were analyzed through the use of high-performance liquid chromatograph (HPLC) and the gas chromatographic method. The analyses revealed the absence of thujones and isoflavones (i.e., genistin, genistein, and daidzein) in all four different extracts. The content of polyphenolic compounds varied among the samples, with the extract T being richer in both polyphenols and flavonoids than the other products by 1.8-3.2 and 1.4-4.0 times, respectively (p-value < 0.05). These results highlight the importance of quality control in salvia-based products since a thujone-free extract rich in polyphenols and flavonoids could be a good candidate for further preclinical and clinical studies to identify an effective herbal approach suitable for the long-term therapy of menopausal symptoms.

Novel P2X7 Antagonist Ameliorates the Early Phase of ALS Disease and Decreases Inflammation and Autophagy in SOD1-G93A Mouse Model.

Amyotrophic lateral sclerosis (ALS) is a disease with a resilient neuroinflammatory component caused by activated microglia and infiltrated immune cells. How to successfully balance neuroprotective versus neurotoxic actions through the use of anti-inflammatory agents is still under debate. There has been a boost of awareness regarding the role of extracellular ATP and purinergic receptors in modulating the physiological and pathological mechanisms in the nervous system. Particularly in ALS, it is known that the purinergic ionotropic P2X7 receptor plays a dual role in disease progression by acting at different cellular and molecular levels. In this context, we previously demonstrated that the P2X7 receptor antagonist, brilliant blue G, reduces neuroinflammation and ameliorates some of the pathological features of ALS in the SOD1-G93A mouse model. Here, we test the novel, noncommercially available, and centrally permeant Axxam proprietary P2X7 antagonist, AXX71, in SOD1-G93A mice, by assessing some behavioral and molecular parameters, among which are disease progression, survival, gliosis, and motor neuron wealth. We demonstrate that AXX71 affects the early symptomatic phase of the disease by reducing microglia-related proinflammatory markers and autophagy without affecting the anti-inflammatory markers or motor neuron survival. Our results suggest that P2X7 modulation can be further investigated as a therapeutic strategy in preclinical studies, and exploited in ALS clinical trials.

Binding Mode Exploration of B1 Receptor Antagonists' by the Use of Molecular Dynamics and Docking Simulation-How Different Target Engagement Can Determine Different Biological Effects.

The kinin B1 receptor plays a critical role in the chronic phase of pain and inflammation. The development of B1 antagonists peaked in recent years but almost all promising molecules failed in clinical trials. Little is known about these molecules' mechanisms of action and additional information will be necessary to exploit the potential of the B1 receptor. With the aim of contributing to the available knowledge of the pharmacology of B1 receptors, we designed and characterized a novel class of allosteric non-peptidic inhibitors with peculiar binding characteristics. Here, we report the binding mode analysis and pharmacological characterization of a new allosteric B1 antagonist, DFL20656. We analyzed the binding of DFL20656 by single point mutagenesis and radioligand binding assays and we further characterized its pharmacology in terms of IC50, B1 receptor internalization and in vivo activity in comparison with different known B1 antagonists. We highlighted how different binding modes of DFL20656 and a Merck compound (compound 14) within the same molecular pocket can affect the biological and pharmacological properties of B1 inhibitors. DFL20656, by its peculiar binding mode, involving tight interactions with N114, efficiently induced B1 receptor internalization and evoked a long-lasting effect in an in vivo model of neuropathic pain. The pharmacological characterization of different B1 antagonists highlighted the effects of their binding modes on activity, receptor occupancy and internalization. Our results suggest that part of the failure of most B1 inhibitors could be ascribed to a lack of knowledge about target function and engagement.

The atypical receptor CCRL2 is required for CXCR2-dependent neutrophil recruitment and tissue damage.

CCRL2 is a 7-transmembrane domain receptor that shares structural and functional similarities with the family of atypical chemokine receptors (ACKRs). CCRL2 is upregulated by inflammatory signals and, unlike other ACKRs, it is not a chemoattractant-scavenging receptor, does not activate ß-arrestins, and is widely expressed by many leukocyte subsets. Therefore, the biological role of CCRL2 in immunity is still unclear. We report that CCRL2-deficient mice have a defect in neutrophil recruitment and are protected in 2 models of inflammatory arthritis. In vitro, CCRL2 was found to constitutively form homodimers and heterodimers with CXCR2, a main neutrophil chemotactic receptor. By heterodimerization, CCRL2 could regulate membrane expression and promote CXCR2 functions, including the activation of ß2-integrins. Therefore, upregulation of CCRL2 observed under inflammatory conditions is functional to finely tune CXCR2-mediated neutrophil recruitment at sites of inflammation.

A potassium channel agonist protects hearing function and promotes outer hair cell survival in a mouse model for age-related hearing loss.

Age-related hearing loss (ARHL) is the most common sensory impairment mainly caused by degeneration of sensory hair cells in the cochlea with no causal medical treatment available. Auditory function and sensory hair cell survival critically depend on the Kv7.4 (KCNQ4) channel, a voltage-gated potassium channel expressed in outer hair cells (OHCs), with its impaired function or reduced activity previously associated with ARHL. Here, we investigated the effect of a potent small-molecule Kv7.4 agonist on ARHL in the senescence-accelerated mouse prone 8 (SAMP8) model. For the first time in vivo, we show that Kv7.4 activation can significantly reduce age-related threshold shifts of auditory brainstem responses as well as OHC loss in the SAMP8 model. Pharmacological activation of Kv7.4 thus holds great potential as a therapeutic approach for ARHL as well as other hearing impairments related to Kv7.4 function.

Discovery of Novel TRPM8 Blockers Suitable for the Treatment of Somatic and Ocular Painful Conditions: A Journey through pKa and LogD Modulation.

Transient receptor potential melastatin 8 (TRPM8) is crucially involved in pain modulation and perception, and TRPM8 antagonists have been proposed as potential therapeutic approaches for pain treatment. Previously, we developed two TRPM8 antagonists and proposed them as drug candidates for topical and systemic pain treatment. Here, we describe the design and synthesis of these two TRPM8 antagonists (27 and 45) and the rational approach of modulation/replacement of bioisosteric chemical groups, which allowed us to identify a combination of narrow ranges of pKa and LogD values that were crucial to ultimately optimize their potency and metabolic stability. Following the same approach, we then pursued the development of new TRPM8 antagonists suitable for the topical treatment of ocular painful conditions and identified two new compounds (51 and 59), N-alkoxy amide derivatives, that can permeate across ocular tissue and reduce the behavioral responses induced by the topical ocular menthol challenge in vivo.

Publisher Correction: Novel selective, potent naphthyl TRPM8 antagonists identified through a combined ligand- and structure-based virtual screening approach.

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Novel selective, potent naphthyl TRPM8 antagonists identified through a combined ligand- and structure-based virtual screening approach.

Transient receptor potential melastatin 8 (TRPM8), a nonselective cation channel, is the predominant mammalian cold temperature thermosensor and it is activated by cold temperatures and cooling compounds, such as menthol and icilin. Because of its role in cold allodynia, cold hyperalgesia and painful syndromes TRPM8 antagonists are currently being pursued as potential therapeutic agents for the treatment of pain hypersensitivity. Recently TRPM8 has been found in subsets of bladder sensory nerve fibres, providing an opportunity to understand and treat chronic hypersensitivity. However, most of the known TRPM8 inhibitors lack selectivity, and only three selective compounds have reached clinical trials to date. Here, we applied two virtual screening strategies to find new, clinics suitable, TRPM8 inhibitors. This strategy enabled us to identify naphthyl derivatives as a novel class of potent and selective TRPM8 inhibitors. Further characterization of the pharmacologic properties of the most potent compound identified, compound 1, confirmed that it is a selective, competitive antagonist inhibitor of TRPM8. Compound 1 also proved itself active in a overreactive bladder model in vivo. Thus, the novel naphthyl derivative compound identified here could be optimized for clinical treatment of pain hypersensitivity in bladder disorders but also in different other pathologies.

An innovative cell-based assay for the detection of modulators of soluble guanylate cyclase.

Guanylate cyclase (GC) catalyzes the biosynthesis of cyclic guanosine 3',5'- monophosphate (cGMP) from GTP. GC exists in two isoenzyme forms: soluble and membrane-bound. The soluble GC (sGC) is a heterodimer composed of an alpha and a beta subunit, and it contains heme as a prosthetic group. The most important physiological activator of sGC is nitric oxide, which activates the enzyme upon binding to the heme moiety. By producing the second messenger cGMP, which regulates various effector systems such as phosphodiesterases, ion channels, and protein kinases, sGC plays an important role in different physiological processes, thus representing a very attractive pharmacological target. In fact, the pathogenesis of several diseases, especially those of the cardiovascular system, has been linked to inappropriate regulation of sGC. In order to find new modulators for this important enzyme, an innovative cell-based assay has been developed and optimized for the use in high-throughput screening. This luminescent assay, which is suitable for both 96- and 384-well plate formats, has been achieved by stably expressing the alpha and beta subunits of a mutated form of sGC in Chinese hamster ovary cells. The mutated form synthesizes cyclic adenosine 3',5'-monophosphate instead of cGMP, allowing the detection of enzymatic activity by a reporter gene approach. We demonstrated that this cell line responds to compounds typically used in the field of sGC research and that it represents an innovative and robust assay to screen for sGC modulators with high efficiency and high sensitivity by means of standard luminescence readers.

Cervical Posttraumatic Unilateral Locked Facets: Clinical, Radiologic, and Surgical Remarks on a Series of 33 Patients.

STUDY DESIGN: Radiographic and clinical analysis. OBJECTIVE: Review author's experience with anterior discectomy, interbody fusion, and anterior cervical plating in 33 patients with posttraumatic unilateral cervical locked facets. SUMMARY OF BACKGROUND DATA: Unilateral cervical locked facet syndrome is a relatively uncommon injury that often is misdiagnosed and therefore subject to a dangerous delay in surgery. Management of this trauma is controversial. MATERIALS AND METHODS: Thirty-three patients with radiologically proven diagnosis of postraumatic unilateral cervical locked facets were treated by skull traction and surgical operation from January 2005 to December 2009. All patients preoperatively were assessed for neurological examination and underwent x-rays, magnetic resonance imaging, and computed tomography evaluation of the cervical spine. RESULTS: The unilateral locked facet level was C4-C5 in 13 patients, C5-C6 in 10, C6-C7 in 8, and C3-C4 in 2 patients. After closed reduction attempt with Crutchfield system, the correct alignment was achieved in 30 patients, who underwent anterior discectomy with cage, interbody fusion, and anterior cervical plating. In 3 patients there was an overdistraction and therefore a closed reduction was not possible, so they were firstly operated by posterior approach with opened reduction of the facets, lateral mass screws, and posterolateral fusion. In 2 of these patients there was an anterior fragment of the disk in the canal, so was also performed an anterior approach with discectomy, cage, and plating. There were no surgery-related complications. Postoperative neurological status was unchanged in the 3 patients with tetraplegia and improved in 8 of the 10 patients with radiculopathy. Fusion was obtained in all patients, as showed in the clinical and radiologic follow-up. CONCLUSIONS: The authors conclude that an anterior approach provides a safe and effective alternative for the treatment of patients with posttraumatic unilateral cervical locked facet, when preoperatively the cervical alignment of the dislocation is achieved with a closed reduction.

FFA4/GPR120 agonists: a survey of the recent patent literature.

FFA4/GPR120, a member of the rhodopsin family of G-protein-coupled receptors (GPCRs), is becoming an important target for therapeutic intervention in several areas of disease, including metabolic diseases, inflammation and cancer. In the last few years several patents on original chemotypes have been generated by different companies. In this review an analysis of the patents in the FFA4 agonism field is presented, with an emphasis on the documents published between 2013 and mid-2015. A discussion of the biological methods used in the patents is included. The general interest in this area is growing fast as half of the existing patents on FFA4 agonists have been issued after 2013. There is, however, a need of further diversifying new chemical classes away form the current substrate-like, carboxylic acid-containing agonists.

Ca(2+) release-activated Ca(2+) channel inhibitors.

Ca(2+) release-activated Ca(2+) (CRAC) channels are becoming important targets for therapeutic intervention in several areas of disease, including immunology, allergy and cancer. In parallel to the progression towards reliable methods for measuring CRAC currents and their inhibition, patents have been generated by several companies. In this Patent Review, an analysis of the patents in the CRAC channel inhibition filed is presented. A discussion of the biological methods used in the patents is included. The general interest in this area is growing fast with almost 80% of the patents issued after 2010.