Pharmacological Window Research Articles

Bougainvillea glabra Choisy bracts extract in free and liposomal forms reduce hyperplasia induced by let-60gain-of-function in Caenorhabditis elegans.

In this study, we evaluated the toxicological and antiproliferative effects of B. glabra Choisy bract extract (BGCE) in its free and loaded into liposomes forms administered to C. elegans mutants with let-60 gain-of-function (gf). Our results demonstrated that the concentration up to 75 μg CAE/mL of BGCE was safe for the worms. Notably, we developed BGCE-loaded liposomes to extend the pharmacological window up to 100 μg CAE/mL without toxicity. In addition, the extract and liposomes reduced the number and area of the multivulva formed in let-60 gf mutants. There was also an increase in the apoptotic signaling in the germline cells and increased longevity mediated through DAF-16 nuclear translocation with GST-4 activation in the treated animals. Our findings demonstrated that the BGCE-loaded liposomes possess antitumoral effects due to the activation of the apoptotic signaling and DAF-16 nuclear translocation.

Caloxin-derived peptides for the inhibition of plasma membrane calcium ATPases

Plasma membrane calcium ATPases (PMCAs) are a family of transmembrane proteins responsible for the extrusion of cytosolic Ca2+ to the extracellular milieu. They are important players of the calcium homeostasis possibly implicated in some important diseases. The reference inhibitors of PMCA extruding activity are on one hand ortho-vanadate (IC50 in the 30 mM range), and on the other a series of 12- to 20-mer peptides named caloxins (IC50 in the 100 µM scale). As for all integral membrane proteins, biochemistry and pharmacology are difficult to study on isolated and/or purified proteins. Using a series of reference blockers, we assessed a pharmacological window with which we could study the functionality of PMCAs in living cells. Using this system, we screened for alternative versions of caloxins, aiming at shortening the peptide backbone, introducing non-natural amino acids, and overall trying to get a glimpse at the structure-activity relationship between those new peptides and the protein in a cellular context. We describe a short series of equipotent 5-residue long analogues with IC50 in the low µM range.

Natural Products That Changed Society.

Until the end of the 19th century all drugs were natural products or minerals. During the 19th century chemists succeeded in isolating pure natural products such as quinine, morphine, codeine and other compounds with beneficial effects. Pure compounds enabled accurate dosing to achieve serum levels within the pharmacological window and reproducible clinical effects. During the 20th and the 21st century synthetic compounds became the major source of drugs. In spite of the impressive results achieved within the art of synthetic chemistry, natural products or modified natural products still constitute almost half of drugs used for treatment of cancer and diseases like malaria, onchocerciasis and lymphatic filariasis caused by parasites. A turning point in the fight against the devastating burden of malaria was obtained in the 17th century by the discovery that bark from trees belonging to the genus Cinchona could be used for treatment with varying success. However isolation and use of the active principle, quinine, in 1820, afforded a breakthrough in the treatment. In the 20th century the synthetic drug chloroquine severely reduced the burden of malaria. However, resistance made this drug obsolete. Subsequently artemisinin isolated from traditional Chinese medicine turned out to be an efficient antimalarial drug overcoming the problem of chloroquine resistance for a while. The use of synthetic analogues such as chloroquine or semisynthetic drugs such as artemether or artesunate further improved the possibilities for healing malaria. Onchocerciasis (river blindness) made life in large parts of Africa and South America miserable. The discovery of the healing effects of the macrocyclic lactone ivermectin enabled control and partly elimination of the disease by annual mass distribution of the drug. Also in the case of ivermectin improved semisynthetic derivatives have found their way into the clinic. Ivermectin also is an efficient drug for treatment of lymphatic filariasis. The serendipitous discovery of the ability of the spindle toxins to control the growth of fast proliferating cancer cells armed physicians with a new efficient tool for treatment of some cancer diseases. These possibilities have been elaborated through preparation of semisynthetic analogues. Today vincristine and vinblastine and semisynthetic analogues are powerful weapons against cancer diseases.

Systematic Quantification of Synapses in Primary Neuronal Culture.

SummaryMost neurological disorders display impaired synaptic connectivity. Hence, modulation of synapse formation may have therapeutic relevance. However, the high density and small size of synapses complicate their quantification. To improve synapse-oriented screens, we analyzed the labeling performance of synapse-targeting antibodies on neuronal cell cultures using segmentation-independent image analysis based on sliding window correlation. When assessing pairwise colocalization, a common readout for mature synapses, overlap was incomplete and confounded by spurious signals. To circumvent this, we implemented a proximity ligation-based approach that only leads to a signal when two markers are sufficiently close. We applied this approach to different marker combinations and demonstrate its utility for detecting synapse density changes in healthy and compromised cultures. Thus, segmentation-independent analysis and exploitation of resident protein proximity increases the sensitivity of synapse quantifications in neuronal cultures and represents a valuable extension to the analytical toolset for in vitro synapse screens.

Verapamil and Alzheimer's Disease: Past, Present, and Future.

Verapamil is a phenylalkylamine class calcium channel blocker that for half a century has been used for the treatment of cardiovascular diseases. Nowadays, verapamil is also considered as a drug option for the treatment of several neurological and psychiatric disorders, such as cluster headache, bipolar disorders, epilepsy, and neurodegenerative diseases. Here, we review insights into the potential preventive and therapeutic role of verapamil on Alzheimer’s disease (AD) based on limited experimental and clinical data. Pharmacological studies have shown that verapamil has a wide therapeutic spectrum, including antihypertensive, anti-inflammatory, and antioxidative effects, regulation of the blood-brain barrier function, due to its effect on P-glycoprotein, as well as adjustment of cellular calcium homeostasis, which may result in the delay of AD onset or ameliorate the symptoms of patients. However, the majority of the AD individuals are on polypharmacotherapy, and the interactions between verapamil and other drugs need to be considered. Therefore, for an appropriate and successful AD treatment, a personalized approach is more than necessary. A well-known narrow pharmacological window of verapamil efficacy may hinder this approach. It is therefore important to note that the verapamil efficacy may be conditioned by different factors. The onset, grade, and brain distribution of AD pathological hallmarks, the time-sequential appearances of AD-related cognitive and behavioral dysfunction, the chronobiologic and gender impact on calcium homeostasis and AD pathogenesis may somehow be influencing that success. In the future, such insights will be crucial for testing the validity of verapamil treatment on animal models of AD and clinical approaches.

High-throughput microscopy exposes a pharmacological window in which dual leucine zipper kinase inhibition preserves neuronal network connectivity

Therapeutic developments for neurodegenerative disorders are redirecting their focus to the mechanisms that contribute to neuronal connectivity and the loss thereof. Using a high-throughput microscopy pipeline that integrates morphological and functional measurements, we found that inhibition of dual leucine zipper kinase (DLK) increased neuronal connectivity in primary cortical cultures. This neuroprotective effect was not only observed in basal conditions but also in cultures depleted from antioxidants and in cultures in which microtubule stability was genetically perturbed. Based on the morphofunctional connectivity signature, we further showed that the effects were limited to a specific dose and time range. Thus, our results illustrate that profiling microscopy images with deep coverage enables sensitive interrogation of neuronal connectivity and allows exposing a pharmacological window for targeted treatments. In doing so, we revealed a broad-spectrum neuroprotective effect of DLK inhibition, which may have relevance to pathological conditions that ar.e associated with compromised neuronal connectivity.

Co-delivery of 5-Fluorouracil and Curcumin Nanohybrid Formulations for Improved Chemotherapy Against Oral Squamous Cell Carcinoma.

The chemotherapeutics agent, 5-fluorouracil (5-FU), and curcumin (Cur), a natural antioxidant, has a wide pharmacological window to treat oral carcinoma; however, both drugs have limited bioavailability. This research study designs to develop a nanoemulsions (NEs) formulation by combining 5-FU and Cur to improve anticancer activity against oral cavity squamous cell carcinoma (OSCC) cells from the diversified origin for in vitro analysis, SCC090 (human tongue) and SCC152 (human hypo-pharynx). NEs formulated through homogenization, applying high-energy ultrasonication technique. The prepared 5-FUNE/Cur-NE/5-FU-Cur-NE were characterized and optimized by different in vitro assays to evaluate release system and treatment of OSCC cells to monitor cellular acceptability, such as in vitro anticancer activity by MTT assay, cell uptake studies and protein expression associated apoptotic study. 5-FUNE/Cur-NE/5-FU-Cur-NE successfully formulated and show mean-value of the particle size (150-200nm), surface charge (-25.70 to -37.91mV), and PDI (0.194). In vitro release of 5-FUNE/Cur-NE/5-FU-Cur-NEs was monitored over a course of 04days, where acidic pH shows higher release as compared to alkaline pH, along with acceptable stability data. Cytotoxicity study has shown higher-dose-dependent anticancer effect with a reduced IC50 value of NEs as compared to BLNE. Cellular uptake study of 5-FUNE/Cur-NE/5-FU-Cur-NEs upgraded many folds, comparatively BLNE and show potential cell arrest. Additionally, the cell protein (Blc2, Bax, P53, and P21) expression was revised and raised cell apoptosis. The combinational loaded, 5-FU and Cur in nanoformulation system have proven their potency to deliver improved anticancer activity, against oral cancer.

Cre-inducible human CD59 mediates rapid cell ablation after intermedilysin administration.

Cell ablation is a powerful tool for studying cell lineage and/or function; however, current cell-ablation models have limitations. Intermedilysin (ILY), a cytolytic pore-forming toxin that is secreted by Streptococcus intermedius, lyses human cells exclusively by binding to the human complement regulator CD59 (hCD59), but does not react with CD59 from nonprimates. Here, we took advantage of this feature of ILY and developed a model of conditional and targeted cell ablation by generating floxed STOP-CD59 knockin mice (ihCD59), in which expression of human CD59 only occurs after Cre-mediated recombination. The administration of ILY to ihCD59+ mice crossed with various Cre-driver lines resulted in the rapid and specific ablation of immune, epithelial, or neural cells without off-target effects. ILY had a large pharmacological window, which allowed us to perform dose-dependent studies. Finally, the ILY/ihCD59-mediated cell-ablation method was tested in several disease models to study immune cell functionalities, hepatocyte and/or biliary epithelial damage and regeneration, and neural cell damage. Together, the results of this study demonstrate the utility of the ihCD59 mouse model for studying the effects of cell ablation in specific organ systems in a variety of developmental and disease states.

Photoprotective efficiency of PLGA-curcumin nanoparticles versus curcumin through the involvement of ERK/AKT pathway under ambient UV-R exposure in HaCaT cell line

Curcumin (Cur) has been demonstrated to have wide pharmacological window including anti-oxidant and anti-inflammatory properties. However, phototoxicity under sunlight exposure and poor biological availability limits its applicability. We have synthesized biodegradable and non-toxic polymer-poly (lactic-co-glycolic) acid (PLGA) encapsulated formulation of curcumin (PLGA-Cur-NPs) of 150 nm size range. Photochemically free curcumin generates ROS, lipid peroxidation and induces significant UVA and UVB mediated impaired mitochondrial functions leading to apoptosis/necrosis and cell injury in two different origin cell lines viz., mouse fibroblasts-NIH-3T3 and human keratinocytes-HaCaT as compared to PLGA-Cur-NPs. Molecular docking studies suggested that intact curcumin from nanoparticles, bind with BAX in BIM SAHB site and attenuate it to undergo apoptosis while upregulating anti-apoptotic genes like BCL2. Real time studies and western blot analysis with specific phosphorylation inhibitor of ERK1 and AKT1/2/3 confirm the involvement of ERK/AKT signaling molecules to trigger the survival cascade in case of PLGA-Cur-NPs. Our finding demonstrates that low level sustained release of curcumin from PLGA-Cur-NPs could be a promising way to protect the adverse biological interactions of photo-degradation products of curcumin upon the exposure of UVA and UVB. Hence, the applicability of PLGA-Cur-NPs could be suggested as prolonged radical scavenging ingredient in curcumin containing products.

An Acute, Double‐Blind, Placebo‐Controlled Crossover Study of 320 mg and 640 mg Doses of a Special Extract of Bacopa monnieri (CDRI 08) on Sustained Cognitive Performance

Standardized extracts of the traditional Ayurvedic medicine Bacopa monnieri (BM) (Brahmi) have been recently shown to have cognitive enhancing effects in chronic administration studies. Pre-clinical work has also identified a number of acute anxiolytic, nootropic, and cardiovascular effects of BM. There has, however, been little research on the acute effects of BM on cognitive function. The current study aimed to assess the acute effects of a specific extract of BM (KeenMind®-CDRI 08) in a double-blind, placebo-controlled study in normal healthy participants who completed a cognitively demanding series of tests. Twenty-four healthy volunteers completed six repetitions of the Cognitive Demand Battery (CDB) after consuming a placebo, 320 mg BM or 640 mg of BM in a cross-over design and provided cardiovascular and mood assessments before and after treatment. Change from baseline scores indicated that the 320 mg dose of BM improved performance at the first, second, and fourth repetition post-dosing on the CDB, and the treatments had no effect upon cardiovascular activity or in attenuating task-induced ratings of stress and fatigue. It was concluded that assessment of an earlier pharmacological window and use of less memory-specific cognitive tests together with more temporally sensitive measures of brain activity may improve our understanding of the acute neurocognitive properties of BM.

Kolaviron protects apoptotic cell death in PC12 cells exposed to Atrazine

Kolaviron (KV), a natural biflavonoid obtained from the seeds of Garcinia kola, has been documented for its wide pharmacological window, including anti-apoptotic activities. However, the underlying mechanisms are poorly understood at the cellular level. This study investigates the anti-apoptotic activity of KV in PC12 cells, a rat pheochromocytoma, exposed to endocrine disruptor-atrazine (ATZ). KV (60 μM) treatment for 24 h shows significant anti-apoptotic responses in PC12 cells exposed to ATZ (232 μM) for 24 h. KV treatment recovers the ATZ-induced levels of malondialdehyde, reactive oxygen species (ROS), caspase-3 activity and depleted levels of glutathione and catalase activity. However, KV was found to be ineffective to restore the ATZ-induced expression (mRNA) and activity of glutathione-peroxidase (GSH-Px) and glutathione reductase (GR). KV treatment also demonstrates significant restoration in ATZ-induced alterations in the expression of apoptosis markers viz., p53, Bax, Bcl2, caspase-3, caspase-9, cyclooxygenase-2 (COX-2), c-Jun and c-fos. Flow cytometric analysis confirms the involvement of ROS in the mediation of ATZ-induced apoptosis in PC12 cells. Together, these data suggest that KV has the therapeutic potential against chemical-induced apoptotic cell death in the neuronal system.

Protective potential of trans-resveratrol against 4-hydroxynonenal induced damage in PC12 cells

The role of 4-hydroxynonenal (4-HNE), a byproduct of membrane lipid peroxidation has been suggested in neurodegeneration. However, the underlying mechanisms are poorly understood. The investigations were carried out to study the preventive potential of trans-resveratrol against 4-HNE induced damage in PC12 cells. Trans-resveratrol, a natural compound obtained from grape skin and found in red wine, is reported to have wide pharmacological window. Cells pretreated with trans-resveratrol (5, 10 and 25 μM) for 24 h were exposed to 4-HNE (25 μM) for 2 h. Pre-treatment of trans-resveratrol was found to be significantly effective in countering the cytotoxic responses of 4-HNE. Significant reduction in reactive oxygen species generation, restoration of intracellular glutathione, and lipid peroxidation levels suggest the improved antioxidant defense system in the cells pretreated with trans-resveratrol. Further, 4-HNE induced alterations in the protein expression of mitochondria-mediated apoptosis markers (Bax, Bcl-2 and Caspase-3) were significantly restored by pre-treatment of trans-resveratrol suggesting the protective potential of trans-resveratrol in PC12 cells against 4-HNE induced oxidative damage. Together these data show the prophylactic potential of trans-resveratrol in oxidative stress mediated apoptotic neurodegeneration.

Developmental striatal critical period of activity-dependent plasticity is also a window of susceptibility for haloperidol induced adult motor alterations

Experience-dependent plasticity during critical periods of postnatal (PN) development shapes the adult brain anatomy and function. In rat motor system, there is a critical period of activity-dependent plasticity in the striatum (PN30-37). In this period, motor activity of running in a circular path induced in the Circling Training test (CT), elicits several plasticity changes on striatal synapses. It has been recently proposed that developmental critical periods might represent a unique pharmacological window of vulnerability to induce life-lasting behavioral modifications. In this paper we tested the hypothesis of existence of a pharmacological susceptibility to induce adult alterations on motor behavior during the striatal critical period. Due to its main action on the striatum and developmental motor behavioral effects, we applied the prototypical antipsychotic haloperidol to male rats (i.p. 0.7 or 2.5 mg/kg/day) before, during or after the period of plasticity (PN20-27, PN30-37 or PN40-47 respectively). Then, in the adulthood (PN80), we evaluated induced motor activity in the CT. The results showed that only rats exposed to the D2R blocker during the period PN30-37 increased the CT activity in comparison to control rats. Moreover, only these animals also showed an increase in the spontaneous locomotor activity at the open field test. These behavioral alterations were not accompanied by permanent striatal changes either on the number of D2R binding sites or on its mRNA expression levels. In conclusion, we have shown a pharmacological susceptibility of inducing adult motor behavior alterations by haloperidol during a natural critical period of activity-dependent plasticity (PN30-37) in rat striatum development. These results also emphasize the importance of behavioral screening for pharmacological agents to be used in developmental stages of maturation.

The Relative Binding Affinities of PDZ Partners for CFTR: A Biochemical Basis for Efficient Endocytic Recycling

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial chloride channel mutated in patients with cystic fibrosis. Its expression and functional interactions in the apical membrane are regulated by several PDZ (PSD-95, discs large, zonula occludens-1) proteins, which mediate protein-protein interactions, typically by binding C-terminal recognition motifs. In particular, the CFTR-associated ligand (CAL) limits cell-surface levels of the most common disease-associated mutant DeltaF508-CFTR. CAL also mediates degradation of wild-type CFTR, targeting it to lysosomes following endocytosis. Nevertheless, wild-type CFTR survives numerous cycles of uptake and recycling. In doing so, how does it repeatedly avoid CAL-mediated degradation? One mechanism may involve competition between CAL and other PDZ proteins including Na (+)/H (+) exchanger-3 regulatory factors 1 and 2 (NHERF1 and NHERF2), which functionally stabilize cell-surface CFTR. Thus, to understand the biochemical basis of WT-CFTR persistence, we need to know the relative affinities of these partners. However, no quantitative binding data are available for CAL or the individual NHERF2 PDZ domains, and published estimates for the NHERF1 PDZ domains conflict. Here we demonstrate that the affinity of the CAL PDZ domain for the CFTR C-terminus is much weaker than those of NHERF1 and NHERF2 domains, enabling wild-type CFTR to avoid premature entrapment in the lysosomal pathway. At the same time, CAL's affinity is evidently sufficient to capture and degrade more rapidly cycling mutants, such as DeltaF508-CFTR. The relatively weak affinity of the CAL:CFTR interaction may provide a pharmacological window for stabilizing rescued DeltaF508-CFTR in patients with cystic fibrosis.