Trends Pharmacol Sci Research Articles

High Throughput Drug Synergy Testing in a Clinical Trial of Panobinostat, Carfilzomib, Dexamethasone to Define Response Biomarkers for Relapsed/Refractory Multiple Myeloma

Background: The treatment of multiple myeloma (MM) is optimized by use of combination regimens consisting of agents with different mechanisms of action. Panobinostat is a pan-inhibitor of histone deacetylases types I,II, and IV. Panobinostat, bortezomib, dexamethasone was shown to be an effective regimen (San Miguel et al Lancet Hematol 2016; Richardson et al Blood 2016), leading to the FDA approval of panobinostat for patients with relapsed/refractory MM. Carfilzomib is a proteasome inhibitor that was FDA approved in relapsed/refractory MM with the advantage of minimal neuropathy. Panobinostat and carfilzomib has also been shown to be a highly active regimen in relapsed/refractory MM with an overall response rate of up to 75% (Berdeja et al, Haematologica, 2015). With the heterogeneity of MM, individual patients exhibit wide variability in responses to drug combinations. A test that could predict patient responses to specific agents might enable clinicians to optimize therapy for patients, improving outcomes. We developed an in vitro high throughput drug sensitivity assay with formal synergy testing to predict response. In this ongoing trial, Panobinostat with Carfilzomib and Dexamethasone for Relapsed/Refractory Multiple Myeloma: Correlation with In Vitro Chemosensitivity Testing (NCT03256045), we will correlate individual patient in vitro sensitivity assay results with individual clinical response to the same triple drug regimen. Study Design and Methods: This study's objective is to directly demonstrate the utility of a high throughput drug sensitivity assay in determining biomarkers (e.g. individual IC50s, AUCs and/or synergy scores) to accurately predict response to combination therapy that was given prospectively to all enrolled patients. We are enrolling patients with relapsed/refractory MM by IMWG criteria with measurable disease defined by the detection of a quantifiable monoclonal protein in the urine or serum or an abnormal serum free light chain ratio. Additionally, patients must have adequate blood counts and organ function. Patients who have had prior autologous or allogeneic transplants or CAR-T cell therapy are eligible. The regimen consists of panobinostat 20 mg orally on days 1,3,5,15,17,19; carfilzomib 20 mg/m2/dose IV on days 1,2 of cycle 1, then dose escalation up to 45 mg/m2/dose days 8,9,15,16 and all days for subsequent cycles; and dexamethasone 20 mg orally on days of carfilzomib. Dose reductions of all three drugs are permitted per patient tolerance to allow continuation on study treatment. Up to 12 cycles of treatment are permitted. Patients are monitored by serial electrocardiograms and assessments of cardiac function. Safety parameters including adverse events are recorded. CD138+ plasma cells are procured from the patient bone marrow (aspiration and biopsy) and blood (when present) by magnetic bead separation. Cells are then added to 384-well plates and incubated overnight before the drugs are added. Cells are exposed to 8 concentrations (spanning 4 logs) of panobinostat, carfilzomib, or dexamethasone as singlet, doublet and triplet combinations for 72 hours. Cell viability is determined using CellTiter-Glo and IC50 and AUC values are are calculated by fitting data using least squares method to the standard four-parameter logistic model. Curve fitting is performed using IDBS XLFit software. The combination index is calculated by the method described by Chou and Talalay, Trends Pharmacol Sci 1983;4:450-4. Concentrations of Drug1 and Drug2 (that is, panobinostat and dexamethasone or panobinostat and carfilzomib) alone or in combinations are determined that give rise to 90% growth inhibition. At 90% Growth Inhibition, the Combination Index or CI = ([D1] in the combination / [D1] alone) + ([D2] in the combination / [D2] alone). All patients are treated with panobinostat, carfilzomib, and dexamethasone and evaluated for response using the IMWG response criteria. At the completion of enrollment at 35 patients, we plan to correlate the in vitro testing data with in vivo clinical response to determine appropriate biomarkers. This will be done by correlating the IC50s and AUCs for the individual drugs for responders vs. non-responders (including degree of response VGPR vs PR vs SD), as well as correlations of the synergy scores for each of the pairs of drugs in the responders vs. non-responders. Enrollment was initiated in April 2018. Disclosures Becker: Accordant Health Services/Caremark: Consultancy; AbbVie, Amgen, Bristol-Myers Squibb, Glycomimetics, Invivoscribe, JW Pharmaceuticals, Novartis, Trovagene: Research Funding; The France Foundation: Honoraria. Libby:Abbvie: Consultancy; Pharmacyclics and Janssen: Consultancy; Akcea: Consultancy; Alnylam: Consultancy. Cowan:Juno: Research Funding; Abbvie: Research Funding; Sanofi: Consultancy; Janssen: Consultancy, Research Funding; Cellectar: Consultancy; Celgene: Consultancy, Research Funding. Hammer:Glycomimetics: Consultancy.

19th European Symposium on Radiopharmacy and Radiopharmaceuticals (ESRR\u201918)

Aim: Arginase catalyzes the hydrolysis of arginine to ornithine and urea, being the final enzyme of the urea cycle, which is a ubiquitous pathway to excrete toxic ammonia from organisms. Lately, it also has emerged as a key regulator of nitric oxide by competing with NO synthase for the same substrate. Therefore, arginase overexpression has been associated with a series of pathogenic processes that can go from cardiovascular, immune-mediated or inflammatory conditions to tumor cell metabolism.(1) Several research groups have been involved in the development of small-molecule arginase inhibitors reaching to data holding great promise. However, only recently the drug development industry is attaining clinical trials.(2)Since the association to PET labeling techniques has great relevance not only for the evaluation and characterization of some of this molecules, but also to increase the library of radiotracers available, our goal with this work was to synthesize and evaluate the [18F]fluorinated equivalent of a late-generation arginase inhibitor.(3) Methods: An arylboronic acid pinacol ester-derived precursor was synthesized in view of a Cu-mediated nucleophilic [18F]fluorination. Radiochemical yield for the conversion of the intermediate species was assessed by TLC-SG and/or radioHPLC. Deprotection of the amino acid moiety was achieved by hydrolysis and the final radiotracer was purified by semi-preparative HPLC and obtained, after reformulation, as a physiological and injectable solution. Further stability and distribution-coefficient studies were performed. Results: In summary, radiochemical yield of the conversion of the arylboronic ester-derived precursor reached ~80% when using 60 to 15 μmol (lower amounts brought significant losses in yield). Final [18F]fluorinated compound was obtained with radiochemical purity ≥95% in an overall yield of 11% (d.c., non-automated synthesis). The radiotracer showed stability in solution up to 4 h and an experimental log D of -0.67±0.05. Conclusion: The [18F]fluorinated arginase inhibitor was efficiently labeled with fluorine-18 in good yield. Preliminary in vitro studies using R22v1 cell lines, which express Arg2, revealed a cellular uptake of the radiotracer susceptible of being blocked after treatment with arginase inhibitors. Further studies are currently being performed in different cell lines which either overexpress Arg1 or Arg2 to evaluate the potential of the developed radiotracer towards arginase mapping.

Effects of isoflurane on dopaminergic neurone synaptic vesicle exocytosis

Despite widespread clinical use of anaesthetics, their pharmacological mechanisms of action are poorly understood. Volatile anaesthetics such as isoflurane induce a reversible state of amnesia, unconsciousness, and immobility in response to painful stimuli.1 At the cellular level, volatile anaesthetics exert prominent effects on synaptic transmission. While these synaptic effects involve predominately postsynaptic actions, data from our laboratory support presynaptic mechanisms as well.2 At presynaptic sites, anaesthetics differentially inhibit synaptic vesicle (SV) exocytosis depending on neurone phenotype, with greater inhibition of excitatory glutamatergic SV release than of inhibitory GABAergic SV release from hippocampal neurones.3,4 However, anaesthetic effects on dopamine release from dopaminergic (DA) neurones have not been studied, even though dopaminergic mechanisms have been implicated in emergence from anaesthesia.5 We investigated the effects of isoflurane on SV release in DA neurones (identified by post hoc tyrosine hydroxylase immunoreactivity) from rat ventral tegmental area (VTA) neurones using live-cell imaging to measure SV exocytosis and Ca2+ influx. We employed a pH-sensitive variant of eGFP (pHluorin) fused to the luminal domain of the vesicular monoamine transporter (vMAT) to measure exocytosis and the cell permeant fluorescent Ca2+ indicator Fluo-5F to measure intracellular Ca2+ concentration. Isoflurane differentially inhibited SV exocytosis and Ca2+ influx induced by electrically stimulated action potentials from both DA (by 29 ± 4%, n = 13, p < 0.0001 and 42 ± 3%, n = 6, p < 0.0001, respectively) and non-DA neurones (by 16 ± 5%, n = 10, p < 0.0055 and 58 ± 5%, n = 8, p < 0.0001, respectively). In contrast to other neurotransmitter phenotypes, isoflurane also inhibited SV exocytosis evoked by elevated K+ in DA neurones, suggesting a voltage-gated Na+ channel (Nav) independent mechanism of anaesthetic action in this neuronal population. This contrasts with SV exocytosis from glutamate or GABA releasing neurones, which requires Nav. SV release is known to be tightly coupled to Ca2+ entry6. The degree of inhibition of SV exocytosis by isoflurane in DA neurons was proportional to the reduction in Ca2+ influx, and could be mimicked by reducing extracellular Ca2+ concentration. Use of subtype-specific voltage-gated Ca2+ channel toxins revealed that SV exocytosis in DA neurones was solely mediated by P/Q-type and N-type Ca2+ channels. Isoflurane inhibited SV exocytosis mediated by both P/Q-type and N-type Ca2+ channels by a Nav-independent mechanism, supporting a role for inhibition of these Ca2+ channel subtypes in the presynaptic effects of isoflurane in DA neurones. These findings shed light on the presynaptic targets of isoflurane in DA neurones, and provide a molecular target for isoflurane induced unconsciousness and emergence from isoflurane anaesthesia. Funding: US NIH grant GM58055. 1.Hemmings HC Jr, Akabas MH, Goldstein PA, et al. Trends Pharmacol Sci. 2005; 26: 503–10.2.Hemmings HC Jr, Yan W, Westphalen RI, et al. Mol Pharmacol. 2005; 67: 1591–9.3.Westphalen RI, Hemmings HC Jr. J Pharmacol Exp Ther. 2006; 316: 216–23.4.Baumgart JP, Zhou ZY, Hara M, et al. Proc Natl Acad Sci USA. 2015; 112: 11959–64.5.Solt K, Van Dort CJ, Chemali JJ, et al. Anesthesiology. 2014; 121: 311–9.6.Dodge FA Jr, Rahamimoff R. J Physiol. 1967; 193: 419–32.

Electrochemical Potential Gradient As a Quantitative Test Platform for Cellular Oxidative Stress

Oxidative stress (OS), involved in many biological phenomena, such as aging, carcinogenesis and inflammation, is defined as an imbalance between reactive oxygen species (ROS) and organism’s antioxidant defenses. The consequences of this imbalance lead to the disruption of cellular redox homeostasis and oxidative damage to DNA, proteins and lipids.(1) One metric of the relative strengths of oxidizers implicated in causing pathogenic oxidative damage via ROS production is the effective redox potential experienced by the cells. Therefore, establishing the relationship between the strength of the oxidative action, characterized by redox potential, and the biomolecular consequences is critical to elucidating the thermodynamic framework of oxidative damage. Experiments with OS in the lab environment require subjecting the study object to a given level of oxidative bias relative to biosystem’s natural equilibrium, preferably in a well-defined, reproducible and quantitative fashion. Popular in vitro OS inducers include exogenous chemical agents (hydrogen peroxide, paraquat, menadione, and t-butyl hydroperoxide), ionizing radiation or heat shock. In addition to disrupting redox homeostasis, each of these agents will also have a specific chemical interaction with cell components, which have to be accounted for during interpretation.(2) Here, we describe a novel way to create an OS environment for surface attached Chinese Hamster Ovary (CHO) cells by exposing them to an electrochemical potential gradient. With this technique we can apply a stable linear potential profile over a cell monolayer, thus providing an efficient method to screen live cell response to an exogenous redox background. It is based on a bipolar electrode configuration (3), adapted for a rectangular cell growth flask. A constant current is produced in cell media, thus generating an electric field and potential drop over the mammalian cell monolayer. Surface attached cells experience varying electrochemical potential, depending on their location along the current axis. Subsequently, cell viability gradients were produced within a range of treatment levels. The live-dead assay fluorescence image of the flask bottom slide shows the CHO cell condition following incubation under the redox potential gradient for a fixed time interval (see Figure). Images contain a rather uniform live, dead and a narrow changeover region ( magnified view in the lower panel of Figure ), consistent with a commencement of cell death at increasingly positive solution redox potential. The cell viability ratio was calculated from integrated pixel density using a custom ImageJ software macro. Therefore, the sigmoidal cell viability curve shape, produced when cells are exposed to the electrochemical potential gradient , is consistent with a two state conversion model and could be numerically characterized by a half maximum effective potential. This concept is widely used to describe dose-response curves in toxicology. Such numerical values would be useful to compare cell viability when subjected to oxidative stress in diverse experimental circumstances. That way, the higher effective potential value would indicate the increase in cell viability or the efficacy of the antioxidant. We have compared CHO cell response when exposed to three hydrogen peroxide concentrations (20 mM, 50 mM, and 100 mM) with the exposure to electrochemically induced potential gradient. Redox potential values of these H2O2 preparations were measured in separate experiments 1h after hydrogen peroxide was diluted into the cell growth media. Remarkably, cell viability vs solution redox potential dependencies are very close suggesting that exogenous hydrogen peroxide causes CHO cell OS largely by a positive shift in the solution redox potential, at least in this concentration range. Our test platform allows for direct imaging and analysis of cell viability, exposed to a range of electrochemical potentials. The slide vessel system can readily be adapted for other cellular assays such as ROS detection, apoptosis, etc. The system is particularly useful for providing a linear range of oxidative and reductive potentials in a single experiment. By replacing the vessel contents before during and after treatment a wide range of solution conditions can be tested and cell images can be acquired during oxidative treatment. This is particularly useful for in vitro testing of the effectiveness of additives such as anti-oxidants as it enables monitoring a real time cellular response. In addition, various cell types including cancer cell lines can be compared in their resistance to oxidative potentials and effectiveness of various drugs. References (1) Halliwell, B. Trends Pharmacol Sci 2013, 34, 301. (2) Buettner, G. R.; Wagner, B. A.; Rodgers, V. G. J. Cell Biochem Biophys 2013, 67, 477. (3) Fleischmann, M.; Ghoroghchian, J.; Rolison, D.; Pons, S. J Phys Chem-Us 1986, 90, 6392. Figure 1

The amyloid cascade hypothesis: are we poised for success or failure?

The first description of Alzheimer's disease (AD) was made in 1907 by Alois Alzheimer (Allgemeine Zeitschrift fur Psyciatrie und Psychisch-Gerichtliche Medizin 64, 3, 1907), although other contemporary physicians had made similar, and rather more complete, assessments of the neuropathological changes present in the AD brain (Fischer, Monatsschr Psychiat Neurol 22, 17, 1907). Our knowledge of AD has increased dramatically and continues to accelerate. This year is 25years after the publication of a series of papers that, in various ways, articulated the amyloid cascade hypothesis (ACH) for AD (Beyreuther and Masters, Brain Pathol 1, 241-251, 1991; Hardy and Allsop, Trends Pharmacol Sci 12, 383-388, 1991; Selkoe, Neuron 6, 487-498, 1991; Hardy and Higgins, Science 256, 184-185, 1992). This review will cover some familiar territory, but we shall also place the ACH into a wider context, compare it with other hypotheses for AD, explore the evolution of the hypothesis to encompass new findings, and determine, irrespective of the merits of the hypothesis itself, whether it has been useful for the research field, both in academia and in industry. Finally, we shall review how the ACH has led to a number of therapeutic approaches, all of which have, to date, failed to reach their primary efficacy end-points in clinical trials and reflect upon what the future may hold. We review the amyloid cascade hypothesis (ACH) and compare it with other hypotheses that have been posited to explain the initiation and progression Alzheimer's disease. We document the data that support the ACH, and also reflect upon its deficiencies. We list the recent clinical failures of amyloidocentric drugs and anticipate the results that new therapeutic approaches may deliver. This article is part of the60th Anniversary special issue.

Electrochemical Potential Gradient As a Quantitative Test Platform for Cellular Oxidative Stress

Oxidative stress (OS), involved in many biological phenomena, such as aging, carcinogenesis and inflammation, is defined as an imbalance between reactive oxygen species (ROS) and organism’s antioxidant defenses. The consequences of this imbalance lead to the disruption of cellular redox homeostasis and oxidative damage to DNA, proteins and lipids.(1) One metric of the relative strengths of oxidizers implicated in causing pathogenic oxidative damage via ROS production is the effective redox potential experienced by the cells. Therefore, establishing the relationship between the strength of the oxidative action, characterized by redox potential, and the biomolecular consequences is critical to elucidating the thermodynamic framework of oxidative damage. Experiments with OS in the lab environment require subjecting the study object to a given level of oxidative bias relative to biosystem’s natural equilibrium, preferably in a well-defined, reproducible and quantitative fashion. Popular in vitro OS inducers include exogenous chemical agents, ionizing radiation or heat shock. In addition to disrupting redox homeostasis, each of these agents will also have a specific chemical interaction with cell components, which have to be accounted for during interpretation.(2) Here, we describe a novel way to create an OS environment for surface attached Chinese Hamster Ovary (CHO) cells by exposing them to an electrochemical potential gradient. With this technique we can apply a stable linear potential profile over a cell monolayer, thus providing an efficient method to screen live cell response to an exogenous redox background. It is based on a bipolar electrode configuration (3), adapted for a rectangular cell growth flask. A constant current is produced in cell media, thus generating an electric field and potential drop over the mammalian cell monolayer. Surface attached cells experience varying electrochemical potential, depending on their location along the current axis. Subsequently, cell viability gradients were produced within a range of treatment levels. The live-dead assay fluorescence image of the flask bottom slide shows the CHO cell condition following incubation under the redox potential gradient for a fixed time interval (see Figure). Images contain a rather uniform live, dead and a narrow changeover region ( magnified view in the lower panel of Figure ), consistent with a commencement of cell death at increasingly positive solution redox potential. The cell viability ratio was calculated from integrated pixel density. Therefore, the sigmoidal cell viability curve shape, produced when cells are exposed to the electrochemical potential gradient , is consistent with a two state conversion model and could be numerically characterized by a half maximum effective potential. This concept is widely used to describe dose-response curves in toxicology. Such numerical values would be useful to compare cell viability when subjected to oxidative stress in diverse experimental circumstances. That way, the higher effective potential value would indicate the increase in cell viability or the efficacy of the antioxidant. We have compared CHO cell response when exposed to three hydrogen peroxide concentrations (20 mM, 50 mM, and 100 mM) with the exposure to electrochemically induced potential gradient. Redox potential values of these H2O2 preparations were measured in separate experiments 1h after hydrogen peroxide was diluted into the cell growth media. Remarkably, cell viability vs solution redox potential dependencies are very close suggesting that exogenous hydrogen peroxide causes CHO cell OS largely by a positive shift in the solution redox potential, at least in this concentration range. Our test platform allows for direct imaging and analysis of cell viability, exposed to a range of electrochemical potentials. The slide vessel system can readily be adapted for other cellular assays such as ROS detection, apoptosis, etc. The system is particularly useful for providing a linear range of oxidative and reductive potentials in a single experiment. By replacing the vessel contents before during and after treatment a wide range of solution conditions can be tested and cell images can be acquired during oxidative treatment. This is particularly useful for in vitro testing of the effectiveness of additives such as anti-oxidants as it enables monitoring a real time cellular response. In addition, various cell types including cancer cell lines can be compared in their resistance to oxidative potentials and effectiveness of various drugs. Figure 1. Overlay of the fluorescence images of the cell growth flask bottom following the application of the electrochemical potential gradient and live-dead staining (left-live, right-dead). References (1) Halliwell, B. Trends Pharmacol Sci 2013, 34, 301. (2) Buettner, G. R.; Wagner, B. A.; Rodgers, V. G. J. Cell Biochem Biophys 2013, 67, 477. (3) Fleischmann, M.; Ghoroghchian, J.; Rolison, D.; Pons, S. J Phys Chem-Us 1986, 90, 6392. Figure 1

Abstract B07: Targeting an MT1-MMP/MMP2 axis in melanoma by a novel MT1-MMP/MMP2 inhibitor

Abstract Metastatic melanoma remains the deadliest of all skin cancers with a survival rate at five years of less than 15%. Matrix metalloproteases (MMPs) are among the proteolytic enzymes responsible for the degradation/ digestion of the extracellular matrix (ECM) and have been associated with the aggressiveness of a variety of cancers including melanoma (1,2). MT1-MMP is a membrane associated matrix metalloproteinase that controls pericellular proteolysis and is an important, invasion-promoting, pro-tumorigenic MMP in cancer. We have recently demonstrated that MT1-MMP plays a key role in melanoma metastasis (3). MT1-MMP expression increases with disease progression and is associated with poor melanoma patient outcome, underscoring a pivotal role of MT1-MMP in melanoma pathogenesis. Indeed, we showed that MT1-MMP is required for melanoma cells to metastasize, as cells deprived of MT1-MMP fail to form distant metastasis in an orthotopic mouse melanoma model. Mechanistically, we demonstrated for the first time that MT1-MMP affects cell invasion and motility by activating an MMP2-RAC1 signaling axis in melanoma cells (3). These findings imply MT1-MMP/MMP2 represent good molecular targets to thwart melanoma metastasis. However, the targeting of MMPs has not met with very successful stories mostly because the use of broad-spectrum inhibitors have been accompanied by severe side effects, such as musculoskeletal pain and inflammation (4,5) due to the targeting of both the “good” and the “bad” MMPs (6-8). Here we present new data in support of a novel, specific MT1-MMP/MMP2 inhibitor. ND-322 is a slow binding selective inhibitor of MT1-MMP and MMP2 (9). Data in our laboratory indicate ND-322 inhibits cell growth, migration and invasion of several melanoma cell lines similarly to the knock down of MT1-MMP and/or MMP2; it decreases RAC1 activity; and importantly, significantly reduces in vivo tumor growth and metastasis in an orthotopic mouse melanoma model. Preliminary data in vitro also show ND-322 in combination with vemurafenib decreases melanoma cell viability in a synergistic manner. Given these results and considering that this compound is very well tolerated in vivo (10), we believe ND-322 represent a promising novel, safe addition to the current standard of care for melanoma patients with advanced disease. Literature Cited 1 Bartolome, R.A., Ferreiro, S., Miquilena-Colina, M.E., Martinez-Prats, L., Soto- Montenegro, M.L., Garcia-Bernal, D., Vaquero, J.J., Agami, R., Delgado, R., Desco, M., Sanchez-Mateos, P., &amp; Teixido, J. Am J Pathol 174 (2), 602-612 (2009). 2 Moro, N., Mauch, C., &amp; Zigrino, P. Eur J Cell Biol 93 (1-2), 23-29 (2014). 3 Shaverdashvili k., W.P., Ma J., Zhang K., Osman I., Bedogni B. Pigment Cell and melanoma Res In Press (2013). 4 Skiles, J.W., Gonnella, N.C., &amp; Jeng, A.Y. Curr Med Chem 11 (22), 2911-2977 (2004). 5 Drummond, A.H., Beckett, P., Brown, P.D., Bone, E.A., Davidson, A.H., Galloway, W.A., Gearing, A.J., Huxley, P., Laber, D., McCourt, M., Whittaker, M., Wood, L.M., &amp; Wright, A. Ann N Y Acad Sci 878, 228-235 (1999). 6 Zucker, S. &amp; Cao, J. Cancer Biol Ther 8 (24), 2371-2373 (2009). 7 Hua, H., Li, M., Luo, T., Yin, Y., &amp; Jiang, Y. Cell Mol Life Sci 68 (23), 3853-3868 (2011). 8 Dufour, A. &amp; Overall, C.M. Trends Pharmacol Sci 34 (4), 233-242 (2013). 9 Gooyit, M., Lee, M., Schroeder, V.A., Ikejiri, M., Suckow, M.A., Mobashery, S., &amp; Chang, M. J Med Chem 54 (19), 6676-6690 (2011). 10 Cui, J., Chen, S., Zhang, C., Meng, F., Wu, W., Hu, R., Hadass, O., Lehmidi, T., Blair, G.J., Lee, M., Chang, M., Mobashery, S., Sun, G.Y., &amp; Gu, Z. Mol Neurodegener 7, 21 (2012). Citation Format: Khvaramze Shaverdashvili, Poki Wong, Jun Ma, Keman Zhang, Iman Osman, Barbara Bedogni. Targeting an MT1-MMP/MMP2 axis in melanoma by a novel MT1-MMP/MMP2 inhibitor. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B07.

Diterpenic compounds with selective antiproliferative activity in multidrug resistant cancer cells

The failure of chemotherapeutics in cancer is mainly imputed to multidrug resistance (MDR). Among several proposed approaches to overcome MDR, the modulation of P-glycoprotein (Pgp) is the most highlighted [1]. Another valuable strategy is to take advantage of the collateral sensitivity effect, where some compounds may selectively exert a more pronounced cytotoxicity on cells with MDR phenotype than in the parental ones [1]. Macrocyclic diterpenes isolated from Euphorbia species, have been recognized as potent Pgp modulators and as having a collateral sensitivity effect on MDR cell variants [2]. Our main goal is to explore these two approaches, since tackling more than one MDR mechanism simultaneously might be a solution to control MDR in its complexity. The fractionation of the methanol extract of the aerial parts of E. piscatoria led to the isolation of a macrocyclic diterpene that allowed to build a small library of derivatives [3]. In this work, eleven compounds were evaluated for their potential collateral sensitivity effect on nine cell lines, gastric, pancreatic and colon cancer cell models (drug sensitive and drug resistant sublines), using the SRB assay. Most of the derivatives exhibited significant collateral sensitivity effect towards the MDR gastric cell line (with Pgp overexpression) and the MDR colon cells. The most significant results were obtained with a benzoyl derivative that demonstrated ability to target different cellular contexts with concomitant high antiproliferative activity. These compounds were previously assessed as Pgp modulators, at non-cytotoxic doses, on MDR1-mouse lymphoma cells [3]. A regression analysis between the antiproliferative activity presented herein and the previously assessed Pgp modulatory effect showed a strong relation between the compounds that presented both high Pgp modulation and cytotoxicity. Acknowledgements: FCT, Portugal (PTDC/QUI-QUI/099815/2008; PTDC/QEQ-MED/0905/2012; SFRH/BD/72915/2010).

Targeting protein–protein interactions to rescue Δf508‐cftr: a novel corrector approach to treat cystic fibrosis

Cystic fibrosis (CF) is one of the most prevalent life‐threatening autosomal recessive disorders in the Western World, with an estimated incidence of 1 in 2000–3000 Caucasian newborns and a median age of survival of 40 years. The first report of CF as a distinct pathology was published in 1938 by Dorothy Andersen, a physician at the Babies & Children's Hospital of Columbia University, who differentiated its diagnosis from celiac disease in the pancreas (Andersen, 1938). This seminal description was followed by other contributions that linked CF to altered Cl− permeability in epithelial cells in the gastrointestinal and hepato‐billiary systems, respiratory tract, reproductive system and sweat glands (Kreindler, 2010); and culminated with the discovery of the gene responsible of the disease, referred to as the cystic fibrosis transmembrane regulator (CFTR) (Riordan et al, 1989). The CFTR is a cAMP‐regulated chloride and bicarbonate channel that controls anion conductance in epithelial cells located in mucosal surfaces. Functional alteration of CFTR leads to the accumulation of mucous secretions that cannot be cleared favouring inflammation and infection. Cumulative evidence shows a strong correlation between the severity of the CF phenotype and the degree of CFTR dysfunction, being stronger for those patients where the channel function is absent (Kreindler, 2010). The CF phenotype is caused by more than 1000 mutations of the CFTR gene including missense, such as R117H or G551D that significantly reduce channel activity, and nonsense like G542X, R553X or W1282X, which abrogate protein expression (Kreindler, 2010). However, the most prevalent mutation in CF patients (70%) is the in‐frame deletion of Phenylalanine at position 508 (ΔF508‐CFTR) (Kreindler, 2010; Rowe & Verkman, 2013). This deletion produces a partially unfolded CFTR protein that is retained in the endoplasmic reticulum and diverted to degradation by the proteosome, resulting in a virtually absent CFTR activity …

THU0072 Prophylactic Treatment with Anti-MIL-21 Ameliorates Disease in Mice with Collagen-Induced Arthritis

BackgroundInterleukin 21 (IL-21), a type I cytokine, signals through a composite receptor consisting of a unique private receptor (IL-21R) and the common cytokine receptor γ chain (1). The immune-mediated and...

Substrate binds in the S1 site of the F253A mutant of LeuT, a neurotransmitter sodium symporter homologue

LeuT serves as the model protein for understanding the relationships between structure, mechanism and pharmacology in neurotransmitter sodium symporters (NSSs). At the present time, however, there is a vigorous debate over whether there is a single high-affinity substrate site (S1) located at the original, crystallographically determined substrate site or whether there are two high-affinity substrates sites, one at the primary or S1 site and the other at a second site (S2) located at the base of the extracellular vestibule. In an effort to address the controversy over the number of high-affinity substrate sites in LeuT, one group studied the F253A mutant of LeuT and asserted that in this mutant substrate binds exclusively to the S2 site and that 1 mM clomipramine entirely ablates substrate binding to the S2 site. Here we study the binding of substrate to the F253A mutant of LeuT using ligand binding and X-ray crystallographic methods. Both experimental methods unambiguously show that substrate binds to the S1 site of the F253A mutant and that binding is retained in the presence of 1 mM clomipramine. These studies, in combination with previous work, are consistent with a mechanism for LeuT that involves a single high-affinity substrate binding site.

Variation: use it or misuse it–replication and its variants

Key Points in a previous article ([1][1]), we discussed variation between individuals, termed variance. When measurements are taken of subjects in separate groups, the variance of the measurements within the group can be compared with the variance that exists between groups. This allows us to

Erratum to: Neonicotinoids in bees: a review on concentrations, side-effects and risk assessment

There is an incorrect text in the original publication of the article. The authors would like to correct the error, and the corrected sentence should read as given below. Within the different insecticide classes, the neonicotinoid insecticides, which include imidacloprid, acetamiprid, clothianidin, thiamethoxam, thiacloprid, dinotefuran and nitenpyram, are an important group of neurotoxins specifically acting as agonists of the insect nicotinic acetylcholine receptors (nAChR) (Matsuda et al. Trends Pharmacol Sci 22:573–580, 2001; Elbert et al. Pest Manag Sci 64:1099–1105, 2008).

Selective Glucocorticoid Receptor Agonists for Treatment of Inflammatory Bowel Disease – in vitro studies

Selective Glucocorticoid Receptor Agonists for Treatment of Inflammatory Bowel Disease – in vitro studies

Abstract 4319: A novel tool to study apoptosis in living mice

Abstract INTRODUCTION. The limitations of cellular models and xenograft for the identification of anti-neoplastic drugs are well known. The purpose of our study is to test the validity of a novel methodology to identify apoptotic cells in living mice genetically modified or pharmacologically treated to develop cancer. METHODS AND RESULTS. The method presented here relies on the combined use of reporter mice engineered to express the luciferase gene ubiquitously (1-2) and modified luciferase substrates (Glo technology) where a pro-luciferin (VivoGlo Caspase-3/7) is converted into a luminogenic substrate only in the presence of the apoptotic enzymes Caspase-3/7 (3-4). The novel methodology was tested by inducing liver apoptosis in luciferase reporter mice by a single i.p. injection of D-galactosamine (D-GalN; 800mg/kg) and Lipopolysaccharide (LPS; 100μg/kg) shown to increase the number of apoptotic cells in the liver of 5-10 fold compared to vehicle-treated animals (5); 6 hours after LPS/D-GalN or vehicle administration, mice were treated i.p. with increasing doses (17-150mg/kg) of the VivoGlo Caspase-3/7 substrate (Z-DEVD-Aminoluciferin, Sodium Salt) and subjected to the bioluminescence in vivo imaging procedure. The in vivo imaging data obtained clearly show a dose-dependent increase of photon emission in the hepatic area of mice treated with D-GalN/LPS. No photon emission was observed in organs not affected by the apoptotic treatment. A complete analysis of pro-apoptotic effects induced by the treatment was carried out by ex vivo imaging acquisition of photon emission in several dissected tissues. This investigation confirmed that light emission observed in vivo was produced selectively by liver and adipose tissues. Finally, the presence of increased apoptotic cells in liver was also confirmed by western blot analysis and Caspase-3/7 enzymatic activity assay carried out on protein extracts. CONCLUSIONS. The present study, by showing the power of modified luciferin substrates to label apoptotic cells in living animals, provides an important advancement over current imaging methodologies based on fluorescence, nuclear or magnetic resonance including: virtually null background, high sensitivity and simple instrumentation needed for the in vivo imaging measurement of the Caspase-3/7 activity. The present application carried out in reporter mice genetically engineered to develop specific cancers will open the way to novel, more predictive approaches for the study of anti-cancer treatments that will enable, for the first time, to measure drug efficacy in space and time providing relevant information to be rapidly translated to human therapy. REFERENCES 1- Maggi et al. Trends Pharmacol Sci. 2004. 2- Maggi A and Ciana P Nat Rev Drug Discov. 2005. 3- Meisenheimer PL et al. Promega Notes 2008. 4- Kizaka-Kondoh S et al. Clin Cancer Res. 2009. 5- Nakama T et al. Hepatology. 2001. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4319.

Arterial Stiffness and Endothelial Function

Functional assessment of the blood pressure waveform helps us to obtain an estimation of the arterial stiffness or its converse, arterial elasticity.1 Several epidemiological studies have clearly demonstrated that arterial stiffness is a significant predictor of cardiovascular disease incidence in the general population.2 There is clear evidence now that decreased arterial elasticity or increased arterial stiffness in normotensive and prehypertensive subjects induces increased risk for the development of arterial hypertension. In vitro and in vivo experiments have shown that the vascular endothelium plays a crucial role in vascular tone and consequently in arterial stiffness. However, there are no data available regarding the endothelial pathways involved in the adaptation of arterial mechanics during changes in blood flow. The elegant study by Bellien et al3 in this issue of Hypertension studied the endothelial pathways involved in the adaption of arterial mechanics during changes in blood flow. Besides identifying the “endothelial-derived relaxing factor” nitric oxide (NO) as important in this biological process, a unique aspect found in their study was the “endothelial-hyperpolarizing factor” EDHF. Endothelium-dependent relaxation/vasodilation in response to neurohumoral mediators and physical forces, such as the shear stress caused by blood flow, is generally attributed to the release of NO and/or prostacyclin. However, NO and prostacyclin cannot fully explain these endothelium-mediated vasodilator responses. The relaxations observed in the presence of NO synthase and cyclooxygenase inhibitors are often associated with hyperpolarization of the vascular smooth …

NO Way to Relax

The roles of nitric oxide (NO) in the heart have been studied intensely ever since the first report nearly 2 decades ago showing that endogenous NO modulates cardiac myocyte function.1 All 3 NO synthase (NOS) isoforms have been found in mammalian heart tissues (see reviews in Massion et al2 and Belge et al3). A complex array of myocyte and nonmyocyte cells in the heart express NOS isoforms, and the local generation of NO2,3 and reactive oxygen species (ROS)4 may exert both autocrine and paracrine effects on cellular function. Not only is the endothelial isoform of NOS (eNOS, or NOS3) robustly expressed in the endothelial cells of the cardiac vasculature, but eNOS is also present within cardiac myocytes, where the enzyme associates with the scaffolding/regulatory protein caveolin-3 in T tubules in plasmalemmal caveolae.5 The neuronal NOS (nNOS, or NOS1) is also expressed in cardiac myocytes, where the enzyme appears to be localized in the sarcoplasmic reticulum and modulates phospholamban phosphorylation.6 Although both eNOS and nNOS appear to be physiologically expressed in cardiac tissues, the inflammation-related NOS isoform (iNOS, or NOS2) only appears in the heart after immunoactivation; iNOS may modulate the decline in myocardial function seen in sepsis.7 There have been innumerable studies of the cardiac effects of various NOS inhibitors, NO-donating drugs, and NO itself.2 The cardiac phenotypes of NOS “knockout” mouse models have been characterized extensively, and mice lacking 1, 2, or all 3 NOS isoforms have been generated and characterized.8 In addition, the roles of NOS substrates, cofactors, and allosteric modulators have been explored exhaustively in diverse cardiac model systems. Emerging from this broad range of experimental evidence is an increasingly clear view that NO and NOS are key determinants of cardiac function. Yet the molecular mechanisms and …

Angiotensin II type-1 receptor regulates RhoA and Rho-kinase/ROCK activation via multiple mechanisms. Focus on “Angiotensin II induces RhoA activation through SHP2-dependent dephosphorylation of the RhoGAP p190A in vascular smooth muscle cells”

angiotensin ii, a crucial regulator of the cardiovascular system, is not only required for physiological functions, such as maintenance of blood pressure and plasma sodium concentration but also is involved in pathophysiological conditions such as hypertension and atherosclerosis (26). Angiotensin II exerts its effect by mainly binding to the angiotensin II type-1 (AT1) receptor. As its seven transmembrane structure suggests, the AT1 receptor couples to several heterotrimeric G proteins including Gq. In addition, the activation of the AT1 receptor leads to increased reactive oxygen species; activation of various protein tyrosine and serine/threonine kinases; and of small G proteins such as Ras, Rac, and RhoA, which may require heterotrimeric G protein-dependent and -independent mechanisms (6, 7, 11, 19).

Editorial Comment on: Basic Mechanisms of Urgency: Preclinical and Clinical Evidence

[87] Peters SLM, Schmidt M, Michel MC. Rho kinase: a target for treating urinary bladder dysfunction? Trends Pharmacol Sci 2006;27:492–7. [88] Avelino A, Cruz F, TRPV1. (vanilloid receptor) in the urinary tract: expression, function and clinical applications. Naunyn-Schmiedeberg’s Arch Pharmacol 2006;373:287–99. [89] Silva C, Silva J, Ribeiro M-J, Avelino A, Cruz F. Urodynamic effect of intravesical resiniferatoxin in patients with neurogenic detrusor overactivity of spinal origin: results of a double-blind randomized placebo-controlled trial. Eur Urol 2005;48:650–5.

Successful Smoking Cessation and Improvement of Negative Symptoms With Varenicline in a Stable Schizophrenia Patient

Back to table of contents Previous article Next article LETTERFull AccessSuccessful Smoking Cessation and Improvement of Negative Symptoms With Varenicline in a Stable Schizophrenia PatientIon Anghelescu M.D.,Ion Anghelescu M.D.Search for more papers by this author,Published Online:1 Jan 2009AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InEmail To the Editor: The selective nicotinic partial agonist varenicline is approved for smoking cessation 1 in more than 30 countries. Freedman 2 has reported a case of a patient with a schizophrenic exacerbation after initiation of varenicline for smoking cessation. Here we present a case of successful smoking cessation and substantial improvement of negative symptoms in a clinically stable patient with schizophrenia. Case ReportMr. A is a 27-year-old former student with the diagnosis of schizophrenia, paranoid subtype for 4 years. His last admission as an inpatient took place 2 years ago and was due to delusions, visual and tactile hallucinations, and ideas of reference. He had no insight into his disorder and ideas of grandiosity (“the creator and guardian of the light”). By then he had also been a cannabis user (on average 0.5 g per day). No somatic disorders had been reported. He was then put on depot medication which he received every 14 days (risperidone, 37.5 mg). He smoked cigarettes for 5 years, increasing to 30 cigarettes a day over the last 6 months. His Positive and Negative Syndrome Scale (PANSS) negative symptoms score fluctuated between 42 and 45 over this period of time, with a PANSS positive symptoms score of only 8 points. Quitting smoking strategies like group counseling and nicotine replacement therapy via patch were altogether unsuccessful. Therefore, a trial with varenicline added to risperidone was started. The drug was titrated to 2 mg per day over a period of 1 week. It was tolerated well, and he experienced no nausea. After 2 weeks, complete abstinence was achieved (that was confirmed via CO measurement in the expiration air) and maintained for 6 months. Moreover, his PANSS negative score decreased from the second week on varenicline to 22 points. Risperidone and 9-hydroxy-risperidone concentrations remained unchanged.Discussion We report for the first time a successful trial of smoking cessation in a patient with very mild positive but marked negative symptoms which substantially improved during the treatment with varenicline without exacerbation of psychotic symptoms. We interpret this clinical observation by the indirect dopamine releasing properties of varenicline. 3 Schizophrenia patients seem to have altered nicotinic acetylcholine receptors and stimulation by nicotine has a positive effect on cognition and other symptoms of schizophrenia, which is thought a transient effect. 4 Nicotine as a full agonist on nicotinic acetylcholine receptors has a very high affinity for these receptors and a tolerance inducing effect that has not been described for varenicline yet. Since the patient still received risperidone he was probably sufficiently protected from the development of new positive symptoms. Systematic controlled studies of schizophrenia patients with different predominant symptoms treated with varenicline seem warranted to evaluate its possible risks and advantages in this population.Department of Psychiatry and Psychotherapy, Charité, University Medicine Berlin, Germany, Campus Benjamin Franklin, GermanyReferences1 . Cahill K, Stead LF, Lancaster T: Nicotine receptor partial agonists for smoking cessation. Cochrane Database Syst Rev 2007; CD00103Google Scholar2 . Freedman R: Exacerbation of schizophrenia by varenicline. Am J Psychiatry 2007; 164:1269Google Scholar3 . Rollema H, Coe JW, Chambers LK, et al: Rationale, pharmacology and clinical efficacy of partial agonists of alpha4beta2 nACh receptors for smoking cessation. Trends Pharmacol Sci 2007; 28:316–325Google Scholar4 . Adams CE, Stevens KE: Evidence for a role of nicotinic acetylcholine receptors in schizophrenia. Front Biosci 2007; 12:4755–4772Google Scholar FiguresReferencesCited byDetailsCited ByFrontiers in Psychiatry, Vol. 9Varenicline disrupts prepulse inhibition only in high-inhibitory ratsProgress in Neuro-Psychopharmacology and Biological Psychiatry, Vol. 53EPA Guidance on Tobacco Dependence and Strategies for Smoking Cessation in People with Mental Illness30 January 2014 | European Psychiatry, Vol. 29, No. 2Acta Neuropsychiatrica, Vol. 23, No. 5Expert Opinion on Pharmacotherapy, Vol. 12, No. 11Journal of the Korean Society for Research on Nicotine and Tobacco, Vol. 1, No. 2 Volume 21Issue 1 Winter, 2009Pages 102-103 Metrics PDF download History Published online 1 January 2009 Published in print 1 January 2009