Evaluation Of Potency Research Articles

Evaluation of chemical composition, antioxidant, antibiofilm and antibacterial potency of essential oil extracted from gamma irradiated clove (Eugenia caryophyllata) buds

Evaluation of chemical composition, antioxidant, antibiofilm and antibacterial potency of essential oil extracted from gamma irradiated clove (Eugenia caryophyllata) buds

Anti-Oxidation and Anti-Inflammatory Potency Evaluation of Ferulic Acid Derivatives Obtained through Virtual Screening.

Various factors such as ultraviolet rays can cause a continuous threat to our skin, resulting in inflammation or oxidation problems. Ferulic acid (FA), with certain antioxidant and anti-inflammatory properties, is widely used in many cosmetics, even used to treat various diseases in the clinic. In this study, the FA structural skeleton was used to search for FA derivatives. Then, molecular docking, the rule of five, and Veber rules were performed to virtually screen compounds that can bind to proteins with a good drug likeness. DPPH and ABTS were used to evaluate their antioxidant potency and an MTT assay was employed to investigate the toxicities of the compounds, while Griess Reaction System and ELISA were used to judge the concentration variations of NO and different inflammatory factors (TNF-α, IL-1β, and IL-6). Western blotting featured nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression levels. The trend of the intracellular changes of reactive oxygen species (ROS) was detected by the DCFH-DA method and fluorescence staining. As a result, we found that the ferulic acid derivative S-52372 not only had certain scavenging effects on free radicals in biochemical experiments, but also prevented inflammation and oxidative stress in LPS-stimulated RAW264.7 cells in the cellular environment; intracellular ROS and inflammatory mediators, including iNOS, COX-2, TNF-α, and IL-6, were also suppressed. In a computer prediction, S-52372 owned better water solubility and lower toxicity than FA. This compound deserves further research to find an ideal FA derivative.

Identification, potency evaluation, and mechanism clarification of α-glucosidase inhibitors from tender leaves of Lithocarpus polystachyus Rehd

Lithocarpus polystachyus Rehd. known as Sweet Tea in China has attracted lots of interest for its good hypoglycemic effect and the potential as a hypoglycemic agent. Based on affinity separation-UPLC-Q-TOF-MS/MS, 54 potential α-glucosidase inhibitiors were identified and 44 were structurally determined. Out of them, 41 were identified for the first time from this plant including flavonoids, fatty acids, triterpenes, alkaloids, and coumarins. Enzyme assays revealed that flavonoids exhibited higher inhibitory activity against α-glucosidase than others with astilbin (IC50 = 6.14 μg·mL−1), morin (IC50 = 8.46 μg·mL−1), and naringenin (IC50 = 10.03 μg·mL−1) showing 2- to 4-fold higher potency than the positive control acarbose. They were proved as reversible inhibitors with mixed inhibition mechanism. Ki (Ki') values and molecular dockings strongly supported the potency order of astilbin, morin and naringenin that showed in the enzyme assays.

Effects of Gynostemma pentaphyllum on spinal cord motor neurons and microglial cells in vitro

The regenerative capability of spinal cord neurons is limited to impossible. Thus, experimental approaches supporting reconstruction/regeneration are in process. This study focused on the evaluation of the protective potency of an extract from Gynostemma pentaphyllum (GP), a plant used in traditional medicine with anti-oxidative and neuroprotective activities, in vitro on organotypic spinal cord cultures, the motor-neuron-like NSC-34 cell line and the microglial cell line BV-2. Organotypic cultures were mechanically stressed by the slicing procedure and the effect of GP on motor neuron survival and neurite sprouting was tested by immunohistochemistry. NSC-34 cells were neuronal differentiated by using special medium. Afterwards, cell survival (propidium iodide/fluorescein diacetate labeling), proliferation (BrdU-incorporation), and neurite sprouting were evaluated. BV-2 cells were stimulated with LPS/interferon γ and subjected to migration assay and nanoparticle uptake. Cell survival, proliferation and the expression pattern of different microglial activation markers (cFOS, iNOS) as well as transcription factors (PPARγ, YB1) were analyzed. In organotypic cultures, high-dose GP supported survival of motor neurons and especially of the neuronal fiber network. Despite reduced neurodegeneration, however, there was a GP-mediated activation of astro- and microglia. In NSC-34 cells, high-dosed GP had degenerative and anti-proliferative effects, but only in normal medium. Moreover, GP supported the neuro-differentiation ability. In BV-2 cells, high-dosed GP was toxic. In lower dosages, GP affected cell survival and proliferation when combined with LPS/interferon γ. Nanoparticle uptake, migration ability, and the transcription factor PPARγ, however, GP affected directly. The data suggest positive effects of GP on injured spinal motor neurons. Moreover, GP activated microglial cells. The dual role of microglia (protective/detrimental) in neurodegenerative processes required further experiments to enhance the knowledge about GP effects. Therefore, a possible clinical use of GP in spinal cord injuries is still a long way off.

Design, one-pot synthesis, molecular docking study, and antibacterial evaluation of novel 2H-chromene based imidazo[1,2-a]pyridine derivatives as potent peptide deformylase inhibitors

An efficient, environmentally friendly, one-pot three-component synthesis of a series of 2H-chromene-based imidazo[1,2-a]pyridines had been designed and were synthesized. This protocol was developed by the reaction of substituted 2H-chromene aldehydes and 2-aminopyridine in a mixture of nitroalkane and DMF under microwave irradiation at 60W, 100°C in 15 min with the presence of FeCl3 as the catalyst. The products were obtained in excellent yields with high functional group tolerance. All these compounds had been investigated further in vitro for evaluation of antibacterial potency by agar-well diffusion method against human pathogenic Gram-positive and Gram-negative bacteria, with the determination of minimum inhibitory concentration (MIC) values. Indeed, compound 13i strongly inhibited peptide deformylase (MIC = 16 μg/ml) in the Gram-negative Escherichia coli, in silico. From structure-activity relationships based on the biological and molecular docking results and the potent antibacterial activities, it could be stated that the selected synthetic compounds could be used as potent drugable antibacterial agents.

Abstract 2437: Potency and selectivity evaluation of TEAD covalent inhibitors in living cells by two complementary mass spectrometry methods

Abstract The HIPPO pathway plays an important role in cancer progression and among its essential components are the transcription factor proteins from the TEAD family. The TEAD family consists of 4 different members (TEAD1-TEAD4) who modulate gene expression through the interaction with co-activator proteins such as YAP1 and TAZ. Recent studies have shown that the central pocket of TEAD could be an alternative druggable approach for inhibiting YAP1. However, until recently, the irreversible binding mechanism of such a compound in living cells was not clearly established. Here, we report a mass spectrometry-based approach to demonstrate and quantitatively assess the target occupancy of TEADs covalent inhibitors both in vitro cellular and ex vivo tumor models. Applying our method to compounds from different chemical series confirmed that these inhibitors modulate the TEAD central pocket protein in cell cultures and led us to quantify their target occupancy for all TEADs expressed in our cellular models. We extended and strengthened the characterization of our inhibitors by implementing a proteomic LC-MS/MS cysteine profiling strategy - assessing the selectivity of inhibitors across free cysteine residues in the cellular proteome. As a result, we showed a high selectivity of the first inhibitors towards TEADs cysteine binding. To our knowledge, this is the first study proving by direct means and not by indirect biomarker and cell proliferation phenotypes that the central pocket of TEAD is actually available to ligand binding in cells, allowing direct measurement of target engagement for different TEAD proteins and finally demonstrating selectivity of inhibitors. These two-mass spectrometry-based methods developed here are very robust, powerful and can be currently used to help for the development of TEADs inhibitors Citation Format: Armelle Buzy, Pascal Paul, Stephanie Hamon, Olivier Venier, Jean-Claude Guillemot, Vincent Mikol, Jurgen Moll, Laurent Debussche, Iris Valtingojer. Potency and selectivity evaluation of TEAD covalent inhibitors in living cells by two complementary mass spectrometry methods [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2437.

Synthesis and Cytotoxic Potential of 3-oxo-19β-Trifluoroacetoxy-18αH-oleane-28-oic Acid

Trifluoroacetic acid-promoted Wagner-Meerwein rearrangement of betulonic acid carboxamide led to the formation of the expected 19β,28-lactam along with a new germanicane-type 3-oxo-19β-trifluoroacetoxy-18αH-oleane-28-oic acid. The structure of this triterpenoid was confirmed by 2D NMR analyses. A primary evaluation of biological potency revealed an anticancer activity with GI50 < 5 μM against leukemia, colon cancer, breast cancer, and prostate cancer cell lines, while the parent compounds were not active.

Characterization of the SARS-CoV-2 Neutralization Potential of COVID-19-Convalescent Donors.

The current SARS-CoV-2 pandemic has triggered the development of various SARS-CoV-2 neutralization tests. A wild-type virus (using African green monkey VeroE6 cells), a pseudovirus (using human Caco-2 cells), and a surrogate neutralization test platform were applied to characterize the SARS-CoV-2 neutralization potential of a cohort of 111 convalescent plasma donors over a period of seven months after diagnosis. This allowed an in-depth validation and assay performance analysis of these platforms. More importantly, we found that SARS-CoV-2 neutralization titers were stable or even increased within the observation period, which contradicts earlier studies reporting a rapid waning of Ab titers after three to four months. Moreover, we observed a positive correlation of neutralization titers with increasing age, number of symptoms reported, and the presence of the Rhesus Ag RhD. Validation of the platforms revealed that highest assay performances were obtained with the wild-type virus and the surrogate neutralization platforms. However, our data also suggested that selection of cutoff titers had a strong impact on the evaluation of neutralization potency. When taking strong neutralization potency, as demonstrated by the wild-type virus platform as the gold standard, up to 55% of plasma products had low neutralization titers. However, a significant portion of these products were overrated in their potency when using the surrogate assay with the recommended cutoff titer. In summary, our study demonstrates that SARS-CoV-2 neutralization titers are stable for at least seven months after diagnosis and offers a testing strategy for rapid selection of high-titer convalescent plasma products in a biosafety level 1 environment.

Evaluation of potency of the selected bioactive molecules from Indian medicinal plants with MPro of SARS-CoV-2 through in silico analysis

BackgroundThe recent outbreak of the novel SARS-CoV-2 across the globe and the absence of specific drug against this virus lead the scientific community to look into some alternative indigenous treatments. India as a hub of Ayurvedic and medicinal plants can shed light on its treatment using specific active bio-molecules from these plants. ObjectivesKeeping our herbal resources in mind, we were interested to inquire whether some phytochemicals from Indian spices and medicinal plants can be used as alternative therapeutic agents in contrast to synthetic drugs. Materials and methodsWe used insilico molecular docking approach to test whether bioactive molecules of herbal origin such as hyperoside, nimbaflavone, ursolic acid, 6-gingerol, 6-shogaol and 6-paradol, curcumin, catechins and epigallocatechin, α-Hederin, piperine could bind and potentially block the Mproenzyme of the SARS-CoV-2 virus. ResultsUrsolic acid showed the highest docking score (−8.7 kcal/mol) followed by hyperoside (−8.6 kcal/mol), α-Hederin (−8.5 kcal/mol) and nimbaflavone (−8.0 kcal/mol). epigallocatechin, catechins, and curcumin also exhibited high binding affinity (Docking score −7.3, −7.1 and −7.1 kcal/mol) with the Mpro. The remaining tested phytochemicals exhibited moderate binding and inhibitory effects. ConclusionThis finding provides a basis for biochemical assay of tested bioactive molecules on SARS-CoV-2 virus.

Discovery and Characterization of the Biflavones From Ginkgo biloba as Highly Specific and Potent Inhibitors Against Human Carboxylesterase 2.

Human carboxylesterase 2 (CES2), one of the most abundant hydrolases distributed in the small intestine, has been validated as a key therapeutic target to ameliorate the intestinal toxicity caused by irinotecan. This study aims to discover efficacious CES2 inhibitors from natural products and to characterize the inhibition potentials and inhibitory mechanisms of the newly identified CES2 inhibitors. Following high-throughput screening and evaluation of the inhibition potency of more than 100 natural products against CES2, it was found that the biflavones isolated from Ginkgo biloba displayed extremely potent CES2 inhibition activities and high specificity over CES1 (>1000-fold). Further investigation showed that ginkgetin, bilobetin, sciadopitysin and isoginkgetin potently inhibited CES2-catalyzed hydrolysis of various substrates, including the CES2 substrate-drug irinotecan. Notably, the inhibition potentials of four biflavones against CES2 were more potent than that of loperamide, a marketed anti-diarrhea agent used for alleviating irinotecan-induced intestinal toxicity. Inhibition kinetic analyses demonstrated that ginkgetin, bilobetin, sciadopitysin and isoginkgetin potently inhibited CES2-catalyzed fluorescein diacetate hydrolysis via a reversible and mixed inhibition manner, with K i values of less than 100 nM. Ensemble docking and molecular dynamics revealed that these biflavones could tightly and stably bind on the catalytic cavity of CES2 via hydrogen bonding and π-π stacking interactions, while the interactions with CES1 were awfully poor. Collectively, this study reports that the biflavones isolated from Ginkgo biloba are potent and highly specific CES2 inhibitors, which offers several promising lead compounds for developing novel anti-diarrhea agent to alleviate irinotecan-induced diarrhea.

RETRACTED: Development of a simple QSAR model for reliable evaluation of acetylcholinesterase inhibitor potency

With the aging of the western population, more and more people are affected by the neurodegenerative Alzheimer's and Parkinson's disease. Inhibitors of acetylcholinesterase (AChE) have proven to be effective in the treatment of disease symptoms. We report the QSAR regression model for the estimation of potency of a set of 94 structurally diverse compounds (oximes, N-hydroxyiminoacetamides, 4-aminoquinolines and flavonoids) to inhibit AChE, pKi (AChE). The model is based on three simple descriptors: the valence molecular connectivity index of the zero-order, 0χv, combined with the number of 10-membered rings (nR10) and number of hydroxyl groups in a molecule (nOH). QSAR model yielded r = 0.947, S.E. = 0.51 and S.E.cv= 0.53; the range of pKi (exp) = 6.03. It showed its stability when the set of 94 compounds was enlarged, comprising 184 compounds in total (r = 0.886, S.E. = 0.85 and S.E.cv = 0.88; the range of pKi (exp) = 10.21), resulting in regression parameters which were similar, although only for 0χv coefficients within the limits of S.E. (0.167(13) and 0.172(16) for the set with 94 and 184 compounds, respectively. The predictive power of the model was shown by the prediction of pKi values for 61 randomly chosen compounds (S.E.test = 0.86) from the calibration model made on the other 123 compounds (S.E. = 0.85), all taken from the pool of 184 compounds. QSAR descriptors 0χv, nR10 and nOH were well chosen for describing the interactions of the AChE active site (amino acid interaction) with ligands through the estimation of the inhibitory potency.

Evaluation of Secondary Metabolites and Antibacterial Potency of the Root Extract of Lemon balm (Lamiaceae) Vegetable against Multidrug Resistant Bacteria

Lemon balm (Lamiaceae) vegetable is known for its aromatherapeutic properties is being used in curring many diseases. The aim of this study was to scientifically screen the medicinally active secondary metabolites and antibacterial potency of Lemon balm vegetable grown in Nigeria. Gas chromatography-mass spectrometry (GCMS) was used to analyse the extract while the antibacterial screening was carried out using agar-well diffusion method. Thirty-four (34) therapeutically active organic compounds were identified in the root extract with terpenoids and phenolic compounds representing the major class, including 2,3-bis[(9E)-9- octadecenoyloxy]propyl (9E)-9-octadecenoate (24.6%) as the major component, methyl (11E)-11-octadecenoate (11.7%), dipalmitin (10.7%), α-methyl-D-galactopyranoside (7.7%), eudesm-7(11)-en-4-ol (4.4%), 1,7,7- trimethylbicyclo[2.2.1]heptan-2-ol (3.0%), isoborneol (2.9%) and 2,6-cresotaldehyde (2.7%) as well as other minor compounds. The highest inhibitory effect of the root extract was observed against Proteus mirabilis which showed a zone of inhibition of 19 mm. The result evidently showed that the sample have several therapeutically active secondary metabolites that can be applied in the treating bacteria diseases.

Next generation polyphosphazene immunoadjuvant: Synthesis, self-assembly and in vivo potency with human papillomavirus VLPs-based vaccine

Poly[di(carboxylatomethylphenoxy)phosphazene] (PCMP), a new member of polyphosphazene immunoadjuvant family, is synthesized. In vitro assessment of a new macromolecule revealed hydrolytic degradation profile and immunostimulatory activity comparable to its clinical stage homologue PCPP; however, PCMP was characterized by a beneficial reduced sensitivity to the ionic environment. In vivo evaluation of PCMP potency was conducted with human papillomavirus (HPV) virus-like particles (VLPs) based RG1-VLPs vaccine. In contrast with previously reported self-assembly of polyphosphazene adjuvants with proteins, which typically results in the formation of complexes with multimeric display of antigens, PCMP surface modified VLPs in a composition dependent pattern, which at a high polymer-to VLPs ratio led to stabilization of antigenic particles. Immunization experiments in mice demonstrated that PCMP adjuvanted RG1-VLPs vaccine induced potent humoral immune responses, in particular, on the level of highly desirable protective cross-neutralizing antibodies, and outperformed PCPP and Alhydrogel adjuvanted formulations.

Transcriptomic pathway and benchmark dose analysis of Bisphenol A, Bisphenol S, Bisphenol F, and 3,3',5,5'-Tetrabromobisphenol A in H9 human embryonic stem cells

Bisphenol A (BPA) is a chemical used in the manufacturing of plastics to which human exposure is ubiquitous. Numerous studies have linked BPA exposure to many adverse health outcomes prompting the replacement of BPA with various analogues including bisphenol-F (BPF) and bisphenol S (BPS). Other bisphenols are used in various consumer applications, such as 3,3′,5,5’-Tetrabromobisphenol A (TBBPA), which is used as a flame retardant. Few studies to date have examined the effects of BPA and its analogues in stem cells to explore potential developmental impacts. Here we used transcriptomics to investigate similarities and differences of BPA and three of its analogues in the estrogen receptor negative, human embryonic stem cell line H9 (WA09). H9 cells were exposed to increasing concentrations of the bisphenols and analyzed using RNA-sequencing. Our data indicate that BPA, BPF, and BPS have similar potencies in inducing transcriptional changes and perturb many of the same pathways. TBBPA, the least structurally similar bisphenol of the group, exhibited much lower potency. All bisphenols robustly impacted gene expression in these cells, albeit at concentrations well above those observed in estrogen-positive cells. Overall, we provide a foundational data set against which to explore the transcriptional similarities of other bisphenols in embryonic stem cells, which may be used to assess the suitability of chemical grouping for read-across and for preliminary potency evaluation.

Development of an In Vitro Potency Assay System for Quality Control of Anti- Human Epidermal Growth Factor Receptor 2 Antibody Admixtures

Although the physical/chemical stability and potential interactions of trastuzumab and pertuzumab in a single infusion bag before co-administration have been evaluated preliminarily, it is not easy to clarify which monoclonal antibodies changes when the admixtures were analyzed as a whole, especially for in vitro potency evaluation. In this study, take admixtures of pertuzumab and trastuzumab biosimilar products as samples, an in vitro potency assay system was developed that can monitor the changes of potency of each monoclonal antibodies as well as their admixtures. Development of the assay system included 3 steps, the first step is to develop protein quantification assay for each monoclonal antibodies by specific antigen based enzyme-linked immunosorbent assay, then a specified cell based potency assay that could be used to measure the biological potency of each monoclonal antibodies and their admixtures was developed. After that, the enzyme-linked immunosorbent assay based protein quantification assay and the specified cell based potency assay were qualified for accuracy and precision. Meanwhile, admixtures containing different amount ratios of pertuzumab and trastuzumab biosimilar products were prepared and measured by the well qualified assays. Finally, the potency test data generated from admixtures of different amount ratio were summarized as an analysis table. The analysis table plus protein quantification results were then used as the basic tool for further in vitro potency evaluation of unknown admixtures. Using this system, the change of potency of each monoclonal antibodies in admixtures could be monitored. The in vitro potency assay system was then qualified in evaluating forced degraded samples. This study represents a good example on thorough biological potency evaluation for monoclonal antibodies admixtures.

Evaluation and Comparison of Smear Layer Removal Potency of Three Different Irrigation Regimes – A Stereomicroscopic and Scanning Electron Microscopic Study

Background: Irrigation plays a pivotal role in pulp therapy owing to its flushing action and removal of the smear layer which if present prevents penetration of medicaments and sealers into the dentinal tubules. Aim: This study aims to evaluate and compare the smear layer removal potency of three commercial Irrigating agents. Materials and Methods: Single rooted 30 teeth were selected, decoronated, and randomly divided into three groups according to the irrigation regime to be used (Group I − 5% sodium hypochlorite [NaOCl] + ethylenediaminetetraacetic acid [EDTA], Group II – Chloraxid Gel + EDTA, Group III – Tween Kleen + 3% NaOCl). After recommended biomechanical preparation and irrigation, teeth were stained with 1% methylene blue dye for 24 h and sectioned in 2 halves. Stereomicroscopy was used to assess dye penetration and scanning electron microscope (SEM) analysis was done to detect dentinal surface changes. Data were analyzed using one-way ANOVA test and Tukey honestly significant difference test by statistical software SPSS version 20.0. Results: The highest dye penetration exhibiting smear layer removal for the apical third was seen in Group II (44.78) and for the middle third in Group I (64.73) which was statistically significant. SEM analysis showed maximum dentinal tubules visibility and patency in Group I and minimum in Group III. Conclusion: Newer irrigating materials (1-hydroxyethylidene-1, 1-bisphosphonate) exhibited weak potency for smear layer removal. The sequential use of gold standard NaOCl + EDTA gave satisfactory results, however, in apical third NaOCl Gel + EDTA was found to be more effective. Therefore, it can be prudent to use NaOCl Gels as a safer alternative to conventional means.

Establishment of a mouse- and egg-adapted strain for the evaluation of vaccine potency against H3N2 variant influenza virus in mice.

Sporadic spreads of swine-origin influenza H3N2 variant (H3N2v) viruses were reported in humans, resulting in 437 human infections between 2011 and 2021 in the USA. Thus, an effective vaccine is needed to better control a potential pandemic for these antigenically distinct viruses from seasonal influenza. In this study, a candidate vaccine strain with efficient growth capacity in chicken embryos was established through serial blind passaging of A/Indiana/08/2011 (H3N2)v in mice and chicken embryos. Seven amino acid substitutions (M21I in PA; A138T, N165K, and V226A in HA; S312L in NP; T167I in M1; G62A in NS1 proteins) were found in the passaged viruses without a major change in the antigenicity. This mouse- and egg-adapted virus was used as a vaccine and challenge strain in mice to evaluate the efficacy of the H3N2v vaccine in different doses. Antibodies with high neutralizing titers were induced in mice immunized with 100 µg of inactivated whole-virus particles, and those mice were significantly protected from the challenge of homologous strain. The findings indicated that the established strain in the study was useful for vaccine study in mouse models.

Prolonged cultured human hepatocytes as an in vitro experimental system for the evaluation of potency and duration of activity of RNA therapeutics: Demonstration of prolonged duration of gene silencing effects of a GalNAc-conjugated human hypoxanthine phosphoribosyl transferase (HPRT1) siRNA

We report here the evaluation of a novel in vitro experimental model, prolonged cultured human hepatocytes (PCHC), as an experimental system to evaluate the potency and duration of effects of oligonucleotide therapeutics. A novel observation was made on the redifferentiation of PCHC upon prolonged culturing based on mRNA profiling of characteristic hepatic differentiation marker genes albumin, transferrin, and transthyretin. Consistent with the known de-differentiation of cultured human hepatocytes, decreases in marker gene expression were observed upon culturing of the hepatocytes for 2 days. A novel observation of re-differentiation was observed on day 7 as demonstrated by an increase in expression of the marker genes to levels similar to that observed on the first day of culture. The expression of the differentiation marker genes was highest on day 7, followed by a gradual decrease but remained higher than that on day 2 for up to the longest culture duration evaluated of 41 days. The redifferentiation phenomenon suggests that PCHC may be useful for the evaluation of the duration of effects of oligonucleotide therapeutics on gene expression in human hepatocytes. A proof of concept study was thereby conducted with PCHC with a GalNAc-conjugated siRNA targeting human hypoxanthine phosphoribosyl transferase1 (HPRT1). HPRT1 mRNA expression in siRNA-treated cultures decreased to 21% of that in untreated hepatocytes on day 1, <10% from days 2 to 12, <20% from days 16 to 33, and eventually recovered to 64% by day 41. Our results suggest that PCHC represent a clinically-relevant cost- and time-efficient experimental tool to aid in the evaluation of GalNAc-siRNA silencing activity, providing information on both efficacy and duration of efficacy. PCHC may be applicable in the drug development setting as a species- and cell type-relevant experimental tool to aid the development of oligonucleotide therapeutics.

Recent achievements of bioluminescence imaging based on firefly luciferin-luciferase system

Bioluminescence imaging (BLI) is a newly developed noninvasive visual approach which facilitates the understanding of a plethora of biological processes invitro and invivo due to the high sensitivity, resolution and selectivity, low background signal, and the lack of external light excitation. BLI based on firefly luciferin-luciferase system has been widely used for the activity evaluation of tumor-specific enzymes, for the detection of diseases-related bioactive small molecules and metal ions, and for the diagnosis and therapy of diseases including the studies of drug transport, the research of immune response, and the evaluation of drug potency and tissue distribution. In this review, we highlight the recent achievements in luciferin derivatives with red-shifted emission spectra, mutant luciferase-luciferin pairs, and the diagnostic and therapeutic application of BLI based on firefly luciferin-luciferase system. The development and application of BLI will expand our knowledge of the occurrence and development of diseases and shed light on the diagnosis and treatment of various diseases.

Impact of Direction of Transport on the Evaluation of Inhibition Potencies of Multidrug and Toxin Extrusion Protein 1 Inhibitors.

Multidrug and toxin extrusion (MATE) transporters are expressed on the luminal membrane of renal proximal tubule cells and extrude their substrates into the luminal side of the tubules. Inhibition of MATE1 can reduce renal secretory clearance of its substrate drugs and lead to drug-drug interactions (DDIs). To address whether IC50 values of MATE1 inhibitors with regard to their extracellular concentrations are affected by the direction of MATE1-mediated transport, we established an efflux assay of 1-methyl-4-phenylpyridinium (MPP+) and metformin using the human embryonic kidney 293 model transiently expressing human MATE1. The efflux rate was defined by reduction of the cellular amount of MPP+ and metformin for 0.25 minutes shortly after the removal of extracellular MPP+ and metformin. Inhibition potencies of 12 inhibitors toward MATE1-mediated transport were determined in both uptake and efflux assays. When MPP+ was used as a substrate, 8 out of 12 inhibitors showed comparable IC50 values between assays (<4-fold). IC50 values from the efflux assays were higher for cimetidine (9.9-fold), trimethoprim (10-fold), famotidine (6.4-fold), and cephalexin (>3.8-fold). When metformin was used as a substrate, IC50 values of the tested inhibitors when evaluated using uptake and efflux assays were within 4-fold of each other, with the exception of cephalexin (>4.7-fold). IC50 values obtained from the uptake assay using metformin showed smaller IC50 values than those from the efflux assay. Therefore, the uptake assay is recommended to determine IC50 values for the DDI predictions. SIGNIFICANCE STATEMENT: In this study, a new method to evaluate IC50 values of extracellular added inhibitors utilizing an efflux assay was established. IC50 values were not largely different between uptake and efflux directions but were smaller for uptake. This study supports the rationale for a commonly accepted uptake assay with metformin as an in vitro probe substrate for multidrug and toxin extrusion 1-mediated drug-drug interaction risk assessment in drug development.