Angiostatic kinase inhibitors to sustain photodynamic angio-occlusion.

Targeted angiostatic therapy receives major attention for the treatment of cancer and exudative age-related macular degeneration (AMD). Photodynamic therapy (PDT) has been used as an effective clinical approach for these diseases. As PDT can cause an angiogenic response in the treated tissue, combination of PDT with anti-angiogenic compounds should lead to improved therapy. This study was undertaken to test the clinically used small molecule kinase inhibitors Nexavar® (sorafenib), Tarceva® (erlotinib) and Sutent® (sunitinib) for this purpose, and to compare the results to the combination of Visudyne®-PDT with Avastin® (bevacizumab) treatment. When topically applied to the chicken chorioallantoic membrane at embryo development day (EDD) 7, a clear inhibition of blood vessel development was observed, with sorafenib being most efficient. To investigate the combination with phototherapy, Visudyne®-PDT was first applied on EDD11 to close all <100 μm vessels. Application of angiostatics after PDT resulted in a significant decrease in vessel regrowth in terms of reduced vessel density and number of branching points/mm(2) . As the 50% effective dose (ED50) for all compounds was approximately 10-fold lower, Sorafenib outperformed the other compounds. In vitro, all kinase inhibitors decreased the viability of human umbilical vein endothelial cells. Sunitinib convincingly inhibited the in vitro migration of endothelial cells. These results suggest the therapeutic potential of these compounds for application in combination with PDT in anti-cancer approaches, and possibly also in the treatment of other diseases where angiogenesis plays an important role.

ALK inhibitors in the treatment of advanced NSCLC.

Pharmacologic agents that target protein products of oncogenes in tumors are playing an increasing clinical role in the treatment of cancer. Currently, the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) represent the standard of care for patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) harboring activating EGFR mutations. Subsequently other genetic abnormalities with "driver" characteristics - implying transforming and tumor maintenance capabilities have been extensively reported in several small distinct subsets of NSCLC. Among these rare genetic changes, anaplastic lymphoma kinase (ALK) gene rearrangements, most often consisting in a chromosome 2 inversion leading to a fusion with the echinoderm microtubule-associated protein like 4 (EML4) gene, results in the abnormal expression and activation of this tyrosine kinase in the cytoplasm of cancer cells. This rearrangement occurs in 2-5% of NSCLC, predominantly in young (50 years or younger), never- or former-smokers with adenocarcinoma. This aberration most commonly occurs a independently of EGFR and KRAS gene mutations. A fluorescent in situ hybridization assay was approved by the US Food and Drug Administration (FDA) as the standard method for the detection of ALK gene rearrangement in clinical practice and is considered the gold standard. Crizotinib, a first-in-class dual ALK and c-MET inhibitor, has been shown to be particularly effective against ALK positive NSCLC, showing dramatic and prolonged responses with low toxicity, predominantly restricted to the gastro-intestinal and visual systems, and generally self-limiting or easily managed. However, resistance to crizotinib inevitably emerges. The molecular mechanisms of resistance are currently under investigation, as are therapeutic approaches including crizotinib-based combination therapy and novel agents such as Hsp90 inhibitors. This review aims to present the current knowledge on this fusion gene, the clinic-pathological profile of ALK rearranged NSCLC, and to review the existing literature on ALK inhibitors, focusing on their role in the treatment of NSCLC.

Heavy metal ions are potent inhibitors of protein folding.

Environmental and occupational exposure to heavy metals such as cadmium, mercury and lead results in severe health hazards including prenatal and developmental defects. The deleterious effects of heavy metal ions have hitherto been attributed to their interactions with specific, particularly susceptible native proteins. Here, we report an as yet undescribed mode of heavy metal toxicity. Cd2+, Hg2+ and Pb2+ proved to inhibit very efficiently the spontaneous refolding of chemically denatured proteins by forming high-affinity multidentate complexes with thiol and other functional groups (IC(50) in the nanomolar range). With similar efficacy, the heavy metal ions inhibited the chaperone-assisted refolding of chemically denatured and heat-denatured proteins. Thus, the toxic effects of heavy metal ions may result as well from their interaction with the more readily accessible functional groups of proteins in nascent and other non-native form. The toxic scope of heavy metals seems to be substantially larger than assumed so far.

The prolyl-aminodipeptidases and their inhibitors as therapeutic targets for fibrogenic disorders

Many biologically active peptides are protected from general proteolytic degradation by evolutionary conserved prolines (Pro), due to conformational constraints imposed by the Pro residue. Thus the biological importance of prolyl-specific peptidases points to a high potential for drug discovery for this family of enzymes. Panels of inhibitors have been synthesized and their effects, determined in biological models, suggest the inhibition of families of enzymes with similar activities. Prolyl-specific aminodipeptidases include dipeptidyl-aminodipeptidase IV (DPP IV)/CD26, DPP8, DPP9 and fibroblast activation protease-alpha (FAP-alpha)/seprase, able to release X-Pro dipeptides from the N-terminus of peptides. DPP IV inhibitors are in clinical use for type 2 diabetes. In this review, the expression and the potential functions of prolyl-aminodipeptidases are reviewed in diseases, and the inhibitors developed for these enzymes are discussed, with a specific focus on inhibitors able to discriminate between DPP IV and fibroblast activation protease-alpha (FAPalpha)/seprase as potential leads for the treatment of fibrogenic diseases.

siRNA-Mediated Knock-Down of P-Glycoprotein Expression Reveals Distinct Cellular Disposition of Anticancer Tyrosine Kinases Inhibitors.

Studies on the cellular disposition of targeted anticancer tyrosine kinases inhibitors (TKIs) have mostly focused on imatinib while the functional importance of P-glycoprotein (Pgp) the gene product of MDR1 remains controversial for more recent TKIs. By using RNA interference-mediated knockdown of MDR1, we have investigated and compared the specific functional consequence of Pgp on the cellular disposition of the major clinically in use TKIs imatinib, dasatinib, nilotinib, sunitinib and sorafenib. siRNA-mediated knockdown in K562/Dox cell lines provides a unique opportunity to dissect the specific contribution of Pgp to TKIs intracellular disposition. In these conditions, abrogating specifically Pgp-mediated efflux in vitro revealed the remarkable and statistically significant cellular accumulation of imatinib (difference in cellular levels between Pgp-expressing and silenced cells, at high and low incubation concentration, respectively: 6.1 and 6.6), dasatinib (4.9 and 5.6), sunitinib (3.7 and 7.3) and sorafenib (1.2 and 1.4), confirming that these TKIs are all substrates of Pgp. By contrast, no statistically significant difference in cellular disposition of nilotinib was observed as a result of MDR1 expression silencing (differences: 1.1 and 1.5) indicating that differential expression and/or function of Pgp is unlikely to affect nilotinib cellular disposition. This study enables for the first time a direct estimation of the specific contribution of one transporter among the various efflux and influx carriers involved in the cellular trafficking of these major TKIs in vitro. Knowledge on the distinct functional consequence of Pgp expression for these various TKIs cellular distribution is necessary to better appreciate the efficacy, toxicity, and potential drug-drug interactions of TKIs with other classes of therapeutic agents, at the systemic, tissular and cellular levels.

Oligonucleotide-polyethylenimine complexes targeting retinal cells: structural analysis and application to anti-TGFbeta-2 therapy.

PURPOSE: The aim of this study was to characterize oligonucleotide-polyethylenimine (ODN/PEI) complex preparation for potential transfection of retinal cells in vitro and in vivo. METHODS: The effect of medium preparation [HEPES-buffered saline (HBS), water] on particle size and morphology was evaluated. Cultured Lewis rat retinal Müller glial (RMG) cells were transfected using fluorescein isothiocyanate (FITC)-ODN/PEI complexes specifically directed at transforming growth factor beta (TGFbeta)-2. Efficacy of transfection was evaluated using confocal microscopy, and regulation of gene expression was assayed using quantitative real-time RT-PCR and ELISA assay. One, 24, and 72 h after injection of FITC-ODN/PEI complexes into the vitreous of rat eyes, their distribution was analyzed on eye sections. RESULTS: Complexes prepared in HBS were smaller than complexes prepared in pure water and presented a core-shell structure. These particles showed a high cellular internalization efficacy, along with a significant and specific down-regulation of TGFbeta-2 expression and production in RMG cells, correlating with specific inhibition of cell growth at 72 h. In vivo, complexes efficiently transfect retinal cells and follow a transretinal migration at 24 h. After 72 h, ODN seems to preferentially target RMG cells without inducing any detectable toxicity. CONCLUSIONS: Specific down-regulation of TGFbeta-2 expression using ODN/PEI complexes may have potential interest for the treatment of retinal diseases associated with glial proliferation.

Role of P-glycoprotein in the uptake/efflux transport of oral vitamin K antagonists and rivaroxaban through the Caco-2 cell model.

Vitamin K antagonists (VKAs) are prescribed worldwide and remain the oral anticoagulant of choice. These drugs are characterized by a narrow therapeutic index and a large inter- and intra-individual variability. P-glycoprotein could contribute to this variability. The aim of this study was to investigate the involvement of P-gp in the transport of acenocoumarol, phenprocoumon and warfarin using an in vitro Caco-2 cell monolayer model. These results were compared with those obtained with rivaroxaban, a new oral anticoagulant known to be a P-gp substrate. The transport of these four drugs was assessed at pH conditions 6.8/7.4 in the presence or absence of the P-gp inhibitor cyclosporine A (10 μM) and the more potent and specific P-gp inhibitor valspodar (5 μM). Analytical quantification was performed by LC/MS. With an efflux ratio of 1.7 and a significant decrease in the efflux (Papp B-A), in the presence of P-gp inhibitors at a concentration of 50 μM, acenocoumarol can be considered as a weak P-gp substrate. Concerning phenprocoumon, the results suggest that this molecule is a poor P-gp substrate. The P-gp inhibitors did not affect significantly the transport of warfarin. The efflux of rivaroxaban was strongly inhibited by the two P-gp inhibitors. In conclusion, none of the three VKAs tested are strong P-gp substrates. However, acenocoumarol can be considered as a weak P-gp substrate and phenprocoumon as a poor P-gp substrate.

Phosphorylation of phosphatidylinositol-3-kinase-protein kinase B and extracellular signal-regulated kinases 1/2 mediate reoxygenation-induced cardioprotection during hypoxia.

In vivo exposure to chronic hypoxia (CH) depresses myocardial performance and tolerance to ischemia, but daily reoxyenation during CH (CHR) confers cardioprotection. To elucidate the underlying mechanism, we tested the role of phosphatidylinositol-3-kinase-protein kinase B (Akt) and p42/p44 extracellular signal-regulated kinases (ERK1/2), which are known to be associated with protection against ischemia/reperfusion (I/R). Male Sprague-Dawley rats were maintained for two weeks under CH (10% O(2)) or CHR (as CH but with one-hour daily exposure to room air). Then, hearts were either frozen for biochemical analyses or Langendorff-perfused to determine performance (intraventricular balloon) and tolerance to 30-min global ischemia and 45-min reperfusion, assessed as recovery of performance after I/R and infarct size (tetrazolium staining). Additional hearts were perfused in the presence of 15 micromol/L LY-294002 (inhibitor of Akt), 10 micromol/L UO-126 (inhibitor of ERK1/2) or 10 micromol/L PD-98059 (less-specific inhibitor of ERK1/2) given 15 min before ischemia and throughout the first 20 min of reperfusion. Whereas total Akt and ERK1/2 were unaffected by CH and CHR in vivo, in CHR hearts the phosphorylation of both proteins was higher than in CH hearts. This was accompanied by better performance after I/R (heart rate x developed pressure), lower end-diastolic pressure and reduced infarct size. Whereas the treatment with LY-294002 decreased the phosphorylation of Akt only, the treatment with UO-126 decreased ERK1/2, and that with PD-98059 decreased both Akt and ERK1/2. In all cases, the cardioprotective effect led by CHR was lost. In conclusion, in vivo daily reoxygenation during CH enhances Akt and ERK1/2 signaling. This response was accompanied by a complex phenotype consisting in improved resistance to stress, better myocardial performance and lower infarct size after I/R. Selective inhibition of Akt and ERK1/2 phosphorylation abolishes the beneficial effects of the reoxygenation. Therefore, Akt and ERK1/2 have an important role to mediate cardioprotection by reoxygenation during CH in vivo.

More than cell death: caspases and caspase inhibitors on the move.

It is becoming clear that "apoptotic" caspases can effect cellular processes other than cell death. A recent paper in Cell points to a novel role of the Drosophila caspase inhibitor DIAP1 as a determinant of cell migration.

HMG CoA reductase inhibitors (statins) for preventing acute kidney injury after surgical procedures requiring cardiac bypass.

BACKGROUND: Acute kidney injury (AKI) is common in patients undergoing cardiac surgery among whom it is associated with poor outcomes, prolonged hospital stays and increased mortality. Statin drugs can produce more than one effect independent of their lipid lowering effect, and may improve kidney injury through inhibition of postoperative inflammatory responses. OBJECTIVES: This review aimed to look at the evidence supporting the benefits of perioperative statins for AKI prevention in hospitalised adults after surgery who require cardiac bypass. The main objectives were to 1) determine whether use of statins was associated with preventing AKI development; 2) determine whether use of statins was associated with reductions in in-hospital mortality; 3) determine whether use of statins was associated with reduced need for RRT; and 4) determine any adverse effects associated with the use of statins. SEARCH METHODS: We searched the Cochrane Renal Group's Specialised Register to 13 January 2015 through contact with the Trials' Search Co-ordinator using search terms relevant to this review. SELECTION CRITERIA: Randomised controlled trials (RCTs) that compared administration of statin therapy with placebo or standard clinical care in adult patients undergoing surgery requiring cardiopulmonary bypass and reporting AKI, serum creatinine (SCr) or need for renal replacement therapy (RRT) as an outcome were eligible for inclusion. All forms and dosages of statins in conjunction with any duration of pre-operative therapy were considered for inclusion in this review. DATA COLLECTION AND ANALYSIS: All authors extracted data independently and assessments were cross-checked by a second author. Likewise, assessment of study risk of bias was initially conducted by one author and then by a second author to ensure accuracy. Disagreements were arbitrated among authors until consensus was reached. Authors from two of the included studies provided additional data surrounding post-operative SCr as well as need for RRT. Meta-analyses were used to assess the outcomes of AKI, SCr and mortality rate. Data for the outcomes of RRT and adverse effects were not pooled. Adverse effects taken into account were those reported by the authors of included studies. MAIN RESULTS: We included seven studies (662 participants) in this review. All except one study was assessed as being at high risk of bias. Three studies assessed atorvastatin, three assessed simvastatin and one investigated rosuvastatin. All studies collected data during the immediate perioperative period only; data collection to hospital discharge and postoperative biochemical data collection ranged from 24 hours to 7 days. Overall, pre-operative statin treatment was not associated with a reduction in postoperative AKI, need for RRT, or mortality. Only two studies (195 participants) reported postoperative SCr level. In those studies, patients allocated to receive statins had lower postoperative SCr concentrations compared with those allocated to no drug treatment/placebo (MD 21.2 µmol/L, 95% CI -31.1 to -11.1). Adverse effects were adequately reported in only one study; no difference was found between the statin group compared to placebo. AUTHORS' CONCLUSIONS: Analysis of currently available data did not suggest that preoperative statin use is associated with decreased incidence of AKI in adults after surgery who required cardiac bypass. Although a significant reduction in SCr was seen postoperatively in people treated with statins, this result was driven by results from a single study, where SCr was considered as a secondary outcome. The results of the meta-analysis should be interpreted with caution; few studies were included in subgroup analyses, and significant differences in methodology exist among the included studies. Large high quality RCTs are required to establish the safety and efficacy of statins to prevent AKI after cardiac surgery.

Efficacy of tumor necrosis factor inhibitors in patients with ankylosing spondylitis

Objectives: This study aims to investigate the efficacy of tumor necrosis factor-alpha blockers such as infliximab, etanercept, and adalimumab in the treatment of ankylosing spondylitis. Patients and methods: The outcome of tumor necrosis factor-alpha blocker treatment was analyzed retrospectively in 59 patients with ankylosing spondylitis who were being treated in our clinic during last nine years. The patients' Assessment of SpondyloArthritis International Society (ASAS) 20 and ASAS 40 response rates, adverse drugs effects, and treatment compliance were evaluated. Results: ASAS 20 response was achieved by 89.8% of the patients in the third month, and by 93.2% in the sixth month. ASAS 40 response was achieved by 61% of the patients in the third and sixth month. No statistically significant difference was detected between the three tumor necrosis factor-alpha blockers with regards to the ASAS 40 response rates. Mild infections, observed in 31 of the patients, were the most common side effects. Serious side effect was observed in only one patient. The number of patients who withdrew from the treatment for various reasons was six.

Protease inhibitors to treat hepatitis C in the Swiss HIV Cohort Study: high efficacy but low treatment uptake.

OBJECTIVES: Direct-acting antiviral agents (DAAs) have become the standard of care for the treatment of chronic hepatitis C virus (HCV) infection. We aimed to assess treatment uptake and efficacy in routine clinical settings among HIV/HCV coinfected patients after the introduction of the first generation DAAs. METHODS: Data on all Swiss HIV Cohort Study (SHCS) participants starting HCV protease inhibitor (PI) treatment between September 2011 and August 2013 were collected prospectively. The uptake and efficacy of HCV therapy were compared with those in the time period before the availability of PIs. RESULTS: Upon approval of PI treatment in Switzerland in September 2011, 516 SHCS participants had chronic HCV genotype 1 infection. Of these, 57 (11%) started HCV treatment during the following 2 years with either telaprevir, faldaprevir or boceprevir. Twenty-seven (47%) patients were treatment-naïve, nine (16%) were patients with relapse and 21 (37%) were partial or null responders. Twenty-nine (57%) had advanced fibrosis and 15 (29%) had cirrhosis. End-of-treatment virological response was 84% in treatment-naïve patients, 88% in patients with relapse and 62% in previous nonresponders. Sustained virological response was 78%, 86% and 40% in treatment-naïve patients, patients with relapse and nonresponders, respectively. Treatment uptake was similar before (3.8 per 100 patient-years) and after (6.1 per 100 patient-years) the introduction of PIs, while treatment efficacy increased considerably after the introduction of PIs. CONCLUSIONS: The introduction of PI-based HCV treatment in HIV/HCV-coinfected patients improved virological response rates, while treatment uptake remained low. Therefore, the introduction of PIs into the clinical routine was beneficial at the individual level, but had only a modest effect on the burden of HCV infection at the population level.