Current Pharmaceutical Biotechnology - Volume 21, Issue 9, 2020

In 1791, Galvani established that electricity activated excitable cells. In the two centuries that followed, electrode stimulation of neuronal, skeletal and cardiac muscle became the adjunctive method of choice in experimental, electrophysiological, and clinical arenas. This approach underpins breakthrough technologies like implantable cardiac pacemakers that we currently take for granted. However, the contact dependence, and field stimulation that electrical depolarization delivers brings inherent limitations to the scope and experimental scale that can be achieved. Many of these were not exposed until reliable in vitro stem-cell derived experimental materials, with genotypes of interest, were produced in the numbers needed for multi-well screening platforms (for toxicity or efficacy studies) or the 2D or 3D tissue surrogates required to study propagation of depolarization within multicellular constructs that mimic clinically relevant arrhythmia in the heart or brain. Here the limitations of classical electrode stimulation are discussed. We describe how these are overcome by optogenetic tools which put electrically excitable cells under the control of light. We discuss how this enables studies in cardiac material from the single cell to the whole heart scale. We review the current commercial platforms that incorporate optogenetic stimulation strategies, and summarize the global literature to date on cardiac applications of optogenetics. We show that the advantages of optogenetic stimulation relevant to iPS-CM based screening include independence from contact, elimination of electrical stimulation artefacts in field potential measuring approaches such as the multi-electrode array, and the ability to print re-entrant patterns of depolarization at will on 2D cardiomyocyte monolayers.

Growing evidence suggests that Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs) can be used as a new human cell-based platform to assess cardiac toxicity/safety during drug development. Cardiotoxicity assessment is highly challenging due to species differences and various toxicities, such as electrophysiological and contractile toxicities, which can result in proarrhythmia and heart failure. To explore proarrhythmic risk, the Multi-Electrode Array (MEA) platform is widely used to assess QT-interval prolongation and the proarrhythmic potential of drug candidates using hiPSC-CMs. Several consortiums, including the Comprehensive in vitro Proarrhythmia Assay (CiPA) and the Japanese iPS Cardiac Safety Assessment (JiCSA), have demonstrated the applicability of hiPSC-CMs/MEA for assessing the torsadogenic potential of drug candidates. Additionally, contractility is a key safety issue in drug development, and efforts have been undertaken to measure contractility by a variety of imaging-based methods using iPS-CMs. Therefore, hiPSC-CMs might represent a standard testing tool for evaluating the proarrhythmic and contractile potentials. This review provides new insights into the practical application of hiPSC-CMs in early or late-stage nonclinical testing during drug development.

Human hepatocytes are essential cell types for pharmacokinetics and the safety evaluation of pharmaceuticals. However, widely used primary hepatocytes with individual variations in liver function lose those functions rapidly in culture. Hepatic cell lines are convenient to use but have low liver functions. Human-Induced Pluripotent Stem (hiPS) cells can be expanded and potentially differentiated into any cell or tissue, including the liver. HiPS cell-derived Hepatocyte-Like Cells (hiPSHeps) are expected to be extensively used as consistent functional human hepatocytes. Many laboratories are investigating methods of using hiPS cells to differentiate hepatocytes, but the derived cells still have immature liver functions. In this paper, we describe the current uses and limitations of conventional hepatic cells, evaluating the suitability of hiPS-Heps to pharmacokinetics and the safety evaluation of pharmaceuticals, and discuss the potential future use of non-conventional non-monolayer culture methods to derive fully functional hiPS-Heps.

Neurotoxicity, as well as cardiotoxicity and hepatotoxicity, resulting from administration of a test article is considered a major adverse effect both pre-clinically and clinically. Among the different types of neurotoxicity occurring during the drug development process, seizure is one of the most serious one. Seizure occurrence is usually assessed using in vivo animal models, the Functional Observational Battery, the Irwin test or electroencephalograms. In in vitro studies, a number of assessments can be performed using animal organs/cells. Interestingly, recent developments in stem cell biology, especially the development of Human-Induced Pluripotent Stem (iPS) cells, are enabling the assessment of neurotoxicity in human iPS cell-derived neurons. Further, a Multi-Electrode Array (MEA) using rodent neurons is a useful tool for identifying seizure-inducing compounds. The Consortium for Safety Assessment using Human iPS Cells (CSAHi; http://csahi.org/en/) was established in 2013 by the Japan Pharmaceutical Manufacturers Association (JPMA) to verify the application of human iPS cell-derived neuronal cells to drug safety evaluation. The Neuro Team of CSAHi has been attempting to evaluate the seizure risk of compounds using the MEA platform. Here, we review the current status of neurotoxicity and recent work, including problems related to the use of the MEA assay with human iPS neuronal cell-derived neurons, and future developments.

In preclinical drug development, accurate prediction of drug effects on the human heart is critically important, whether in the context of cardiovascular safety or for the purpose of modulating cardiac function to treat heart disease. Current strategies have significant limitations, whereby, cardiotoxic drugs can escape detection or potential life-saving therapies are abandoned due to false positive toxicity signals. Thus, new and more reliable translational approaches are urgently needed to help accelerate the rate of new therapy development. Renewed efforts in the recovery of human donor hearts for research and in cardiomyocyte isolation methods, are providing new opportunities for preclinical studies in adult primary cardiomyocytes. These cells exhibit the native physiological and pharmacological properties, overcoming the limitations presented by artificial cellular models, animal models and have great potential for providing an excellent tool for preclinical drug testing. Adult human primary cardiomyocytes have already shown utility in assessing drug-induced cardiotoxicity risk and helping in the identification of new treatments for cardiac diseases, such as heart failure and atrial fibrillation. Finally, strategies with actionable decision-making trees that rely on data derived from adult human primary cardiomyocytes will provide the holistic insights necessary to accurately predict human heart effects of drugs.

Drug-induced toxicity remains one of the leading causes of discontinuation of the drug candidate and post-marketing withdrawal. Thus, early identification of the drug candidates with the potential for toxicity is crucial in the drug development process. With the recent discovery of human- Induced Pluripotent Stem Cells (iPSC) and the establishment of the differentiation protocol of human iPSC into the cell types of interest, the differentiated cells from human iPSC have garnered much attention because of their potential applicability in toxicity evaluation as well as drug screening, disease modeling and cell therapy. In this review, we expanded on current information regarding the feasibility of human iPSC-derived cells for the evaluation of drug-induced toxicity with a focus on human iPSCderived hepatocyte (iPSC-Hep), cardiomyocyte (iPSC-CMs) and neurons (iPSC-Neurons). Further, we CSAHi, Consortium for Safety Assessment using Human iPS Cells, reported our gene expression profiling data with DNA microarray using commercially available human iPSC-derived cells (iPSC-Hep, iPSC-CMs, iPSC-Neurons), their relevant human tissues and primary cultured human cells to discuss the future direction of the three types of human iPSC-derived cells.

Current cardiac safety assessment platforms (in vitro hERG-centric, APD, and/or in vivo animal QT assays) are not fully predictive of drug-induced Torsades de Pointes (TdP) and do not address other mechanism-based arrhythmia, including ventricular tachycardia or ventricular fibrillation, or cardiac safety liabilities such as contractile and structural cardiotoxicity which are another growing safety concerns. We organized the Consortium for Safety Assessment using Human iPS cells (CSAHi; http://csahi.org/en/) in 2013, based on the Japan Pharmaceutical Manufacturers Association (JPMA), to verify the application of human iPS/ES cell-derived cardiomyocytes for drug safety evaluation. The CSAHi HEART team focused on comprehensive screening strategies to predict a diverse range of cardiotoxicities using recently introduced platforms such as the Multi-Electrode Array (MEA), cellular impedance, Motion Field Imaging (MFI), and optical imaging of Ca transient to identify strengths and weaknesses of each platform. Our study showed that hiPS-CMs used in these platforms could detect pharmacological responses that were more relevant to humans compared to existing hERG, APD, or Langendorff (MAPD/contraction) assays. Further, MEA and other methods such as impedance, MFI, and Ca transient assays provided paradigm changes of platforms for predicting drug-induced QT risk and/or arrhythmia or contractile dysfunctions. In contrast, since discordances such as overestimation (false positive) of arrhythmogenicity, oversight, or opposite conclusions in positive inotropic and negative chronotropic activities to some compounds were also confirmed, possibly due to their functional immaturity of hiPS-CMs, hiPS-CMs should be used in these platforms for cardiac safety assessment based upon their advantages and disadvantages.

Background: Estrogen Receptors (ER) are members of the nuclear intracellular receptors family. ER once activated by estrogen, it binds to DNA via translocating into the nucleus and regulates the activity of various genes. Withaferin A (WA) - an active compound of a medicinal plant Withania somnifera was reported to be a very effective anti-cancer agent and some of the recent studies has demonstrated that WA is capable of arresting the development of breast cancer via targeting estrogen receptor. Objective: The present study is aimed at understanding the molecular level interactions of ER and Tamoxifen in comparison to Withaferin A using In-silico approaches with emphasis on Withaferin A binding capability with ER in presence of point mutations which are causing de novo drug resistance to existing drugs like Tamoxifen. Methods: Molecular modeling and docking studies were performed for the Tamoxifen and Withaferin A with the Estrogen receptor. Molecular docking simulations of estrogen receptor in complex with Tamoxifen and Withaferin A were also performed. Results: Amino acid residues, Glu353, Arg394 and Leu387 was observed as crucial for binding and stabilizing the protein-ligand complex in case of Tamoxifen and Withaferin-A. The potential of Withaferin A to overcome the drug resistance caused by the mutations in estrogen receptor to the existing drugs such as Tamoxifen was demonstrated. Conclusion: In-silico analysis has elucidated the binding mode and molecular level interactions which are expected to be of great help in further optimizing Withaferin A or design / discovery of future breast cancer inhibitors targeting estrogen receptor.

Aims: Biodegradable polymeric microneedles containing atorvastatin calcium were developed in order to improve the percutaneous absorption of the drug, useful for the treatment of hypercholesterolemia. Background: The use of physical enhancers like microneedles have shown good results to increase the delivery of drugs through the skin, the use of microneedles has very important advantages for transdermal drug delivery, for example, they are painless, easy to use and safe, they increase time interval of drug activity, dose, and reductions in adverse reactions, they also offer, the facility to remove the system instantly of the skin. Objective: Develop polymer microneedles loaded with a calcium atorvastatin and evaluate them by Differential Scanning Calorimetry (DSC), Scanning Electron Microscopy (SEM), bioadhesion, postwetting- bioadhesion, breaking strength, drug release test and in vitro percutaneous absorption studies to demonstrate the use of microneedles atorvastatin is able to cross the skin. Methods: The microneedles were made with poly (methyl vinyl ether-alt-maleic acid) as biodegradable polymer using the technique of casting in solution in a mold. After solidification these microneedles were characterized by Differential Scanning Calorimetry (DSC), Scanning Electron Microscopy (SEM), bioadhesion, post-wetting-bioadhesion, breaking strength, drug release test and in vitro percutaneous absorption studies. Results: In general, the performances were satisfactory for optimal formulation in terms of DSC with no interactions between drug and excipients, SEM shows microneedles with a conical shape, bioadhesion of 1570 g.f, post wetting-bioadhesion of 1503.4 g.f, breaking strength of 1566.7g.f that is sufficient to disrupt Stratum corneum, good drug release and a flux of 33.4 μg/cm2*h with a tLag of 15.14 h for the in vitro percutaneous absorption. Conclusion: The results indicate that it is possible to generate microneedles to increase the percutaneous absorption of calcium atorvastatin transdermally, with the potential to be used as an alternative to the oral route for the treatment of dyslipidemias.

Background and Objective: Dental peri-implantitis, which can be caused by several different microbial factors, is characterized by inflammatory lesions of the surrounding hard and soft tissues of an oral implant. Matrix Metalloproteinase 9 (MMP9) is thought to be involved in the pathogenesis of peri-implantitis. However, the regulatory mechanism of MMP9 in peri-implantitis has not been fully elucidated. In this study, we tried to evaluate the regulatory mechanism of MMP9 in peri-implantitis. Methods: We collected Peri-Implant Crevicular Fluid (PICF) from ten healthy implants and ten periimplantitis patients and compared their expression level of MMP9. We also cultured macrophages from the peripheral blood of healthy volunteers infected by Porphyromonas gingivalis to reveal the regulatory mechanism of MMP9 in peri-implantitis. Western blot, immunofluorescence staining and quantitative Polymerase Chain Reaction (RT-PCR) were used to better characterize the mechanism of MMP9. Results: The expression of MMP9 was up-regulated in peri-implantitis patient PICF and P. gingivalis infected human macrophages. LOX-1, not dectin-1, was found to mediate MMP9 expression in human macrophages with P. gingivalis infection. Expression of Erk1/2 was responsible for infection-induced MMP9 expression. Finally, use of a broad-spectrum metalloproteinase inhibitor impaired LOX-1 expression in infected macrophages. Conclusion: Our results demonstrate that MMP9 is involved in dental peri-implantitis and is regulated by LOX-1 and Erk1/2. This LOX-1/MMP9 signaling pathway may represent a potential drug target for peri-implantitis.

Objective: Amylases enzymes hydrolyze starch molecules to produce diverse products including dextrins, and progressively smaller polymers. These include glucose units linked through α-1- 1, α-1-4, α-1-6, glycosidic bonds. Methods: This enzyme carrying an (α /β) 8 or TIM barrel structure is also produced containing the catalytic site residues. These groups of enzymes possess four conserved regions in their primary sequence. In the Carbohydrate-Degrading Enzyme (CAZy) database, α-amylases are classified into different Glycoside Hydrolase Families (GHF) based on their amino acid sequence. The present objective was to study one such enzyme based on its molecular characterization after purification in our laboratory. Its main property of solid-natural starch degradation was extensively investigated for its pharmaceutical/ industrial applications. Results: Amylase producing bacteria Bacillus cereus sm-sr14 (Accession no. KM251578.1) was purified to homogeneity on a Seralose 6B-150 gel-matrix and gave a single peak during HPLC. MALDITOF mass-spectrometry with bioinformatics studies revealed its significant similarity to α/β hydrolase family. The enzyme showed an efficient application; favourable Km, Vmax and Kcat during the catalysis of different natural solid starch materials. Analysis for hydrolytic product showed that this enzyme can be classified as the exo-amylase asit produced a significant amount of glucose. Conclusion: Besides the purified enzyme, the present organism Bacillus cereus sm-sr14 could degrade natural solid starch materials like potato and rice up to the application level in the pharmaceutical/ industrial field for alcohol production.