Drug Screening Methods Research Articles

In Vitro Evaluation of the Combination of Melaleuca alternifolia (Tea Tree) Oil and Dimethyl Sulfoxide (DMSO) against Trophozoites and Cysts of Acanthamoeba Strains. Oxygen Consumption Rate (OCR) Assay as a Method for Drug Screening.

Ameobae belonging to the genus Acanthamoeba are responsible for the human diseases Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE). The treatment of these illnesses is hampered by the existence of a resistance stage (cysts). In an attempt to add new agents that are effective against trophozoites and cysts, tea tree oil (TTO) and dimethyl sulfoxide (DMSO), separately and in combination, were tested In Vitro against two Acanthamoeba isolates, T3 and T4 genotypes. The oxygen consumption rate (OCR) assay was used as a drug screening method, which is to some extent useful in amoebicide drug screening; however, evaluation of lethal effects may be misleading when testing products that promote encystment. Trophozoite viability analysis showed that the effectiveness of the combination of both compounds is higher than when either compound is used alone. Therefore, the TTO alone or TTO + DMSO in combination were an amoebicide, but most of the amoebicidal activity in the combination’s treatments seemed to be caused mainly by the TTO effect. In fact, DMSO alone seems to be a non-amoebicide, triggering encystment. Regarding cytotoxicity, these compounds showed toxicity in human corneal epithelial cells (HCEpiC), even at low concentrations when tested in combination. In conclusion, the use of TTO and DMSO, in combination or alone, cannot be recommended as an alternative for AK treatment until more cytotoxicity and cyst adhesion tests are performed.

Validation of a human-serum-based in vitro growth method for drug screening on juvenile development stages of Schistosoma mansoni.

BackgroundSchistosomiasis affects over 200 million people worldwide but only praziquantel is available for treatment and control. Drug discovery is often based on phenotypic drug screening, involving different parasite stages retrieved from infected mice. Aiming to reduce animal use, we validated an in vitro growth method for juvenile Schistosoma mansoni for the purpose of drug sensitivity assays.Methodology/Principal findingsWe compared inter–batch variability of serum, worm size and organ development, gender distribution, and drug sensitivity between in vitro and in vivo grown worms over different life stages. In vitro developed S. mansoni in Hybridoma medium supplemented with 20% human serum were similar in size as in vivo worms until 28 days of incubation (males 1.4 ± 0.2 mm, females 1.1 ± 0.5 mm long). qPCR analysis revealed similar gender distribution both on newly transformed schistosomula and worms grown for 21 days. Worms developed in vitro and in vivo were similarly sensitive to praziquantel from 7 to 35 days of development with the exception of 21 days of development, where a slightly lower activity was observed for the in vitro grown worms (IC50: 0.54 μM in vitro, 0.14 μM in vivo 72 hours post-incubation). The evaluation of five additional drugs revealed a similar sensitivity on worms developed for 21 days, with the exception of mefloquine, where we observed a 10-fold lower sensitivity on in vitro developed schistosomes when compared to in vivo grown (IC50: 4.43 μM in vitro, 0.48 μM in vivo).ConclusionA large number of juvenile S. mansoni worms can be grown in vitro, which show similar drug sensitivity, gender distribution, size and morphology as the worms recovered from rodents, supporting the use of this method in drug screening efforts.

3D Collagen Vascular Tumor-on-a-Chip Mimetics for Dynamic Combinatorial Drug Screening.

Disease models, including in vitro cell culture and animal models, have contributed significantly to developing diagnostics and treatments over the past several decades. The successes of traditional drug screening methods were generally hampered by not adequately mimicking critical in vivo features, such as a 3D microenvironment and dynamic drug diffusion through the extracellular matrix (ECM). To address these issues, we developed a 3D dynamic drug delivery system for cancer drug screening that mimicks drug dissemination through the tumor vasculature and the ECM by creating collagen-embedded microfluidic channels. Using this novel 3D ECM microsystem, we compared viability of tumor pieces with traditionally used 2D methods in response to three different drug combinations. Drug diffusion profiles were evaluated by simulation methods and tested in the 3D ECM microsystem and a 2D 96-well setup. Compared with the 2D control, the 3D ECM microsystem produced reliable data on viability, drug ratios, and combination indeces. This novel approach enables higher throughput and sets the stage for future applications utilizing drug sensitivity predicting algorithms based on dynamic diffusion profiles requiring only minimal patient tissue. Our findings moved drug sensitivity screening closer to clinical implications with a focus on testing combinatorial drug effects, an option often limited by the amount of available patient tissues.

In Situ Observation of mtDNA Damage during Hepatic Ischemia-Reperfusion.

Hepatic ischemia-reperfusion (IR) injury is a severe pathophysiological event during liver surgery or transplantation and could lead to liver failure or even death. The energy supply of mitochondria plays an essential role in preventing IR injury. Mitochondrial DNA (mtDNA) is involved in maintaining the balance of energy by participating in an oxidative phosphorylation process. However, the exact relationship between IR and mtDNA remains unclear by reason of the lack of an accurate real-time analysis method. Herein, we fabricated a mitochondria-targeting fluorescent probe (mtDNA-BP) to explore mtDNA stability and supervise the changes in mtDNA in IR liver. By virtue of pyridinium electropositivity and suitable size, mtDNA-BP could accumulate in mitochondria and insert into the mtDNA groove, which made mtDNA-BP fluoresce strongly. This is attributed to the reduction of the intramolecular rotation energy loss that is restricted by DNA. By in situ fluorescence imaging, we observed in real time that mtDNA damage was aggravated by deteriorating IR injury, so the ROS-mtDNA-mediated IR damage signal pathway was speculated. Furthermore, on the basis of mtDNA-BP real-time response capability for mtDNA, we established a drug-screening method for inhibiting IR injury and found superior therapeutic performance of two potential drugs: pioglitazone and salidroside. This work contributes to our understanding of mtDNA-related disease and provides a new drug analysis method.

Unbiased Phenotype-Based Screen Identifies Therapeutic Agents Selective for Metastatic Prostate Cancer.

In American men, prostate cancer is the second leading cause of cancer-related death. Dissemination of prostate cancer cells to distant organs significantly worsens patients’ prognosis, and currently there are no effective treatment options that can cure advanced-stage prostate cancer. In an effort to identify compounds selective for metastatic prostate cancer cells over benign prostate cancer cells or normal prostate epithelial cells, we applied a phenotype-based in vitro drug screening method utilizing multiple prostate cancer cell lines to test 1,120 different compounds from a commercial drug library. Top drug candidates were then examined in multiple mouse xenograft models including subcutaneous tumor growth, experimental lung metastasis, and experimental bone metastasis assays. A subset of compounds including fenbendazole, fluspirilene, clofazimine, niclosamide, and suloctidil showed preferential cytotoxicity and apoptosis towards metastatic prostate cancer cells in vitro and in vivo. The bioavailability of the most discerning agents, especially fenbendazole and albendazole, was improved by formulating as micelles or nanoparticles. The enhanced forms of fenbendazole and albendazole significantly prolonged survival in mice bearing metastases, and albendazole-treated mice displayed significantly longer median survival times than paclitaxel-treated mice. Importantly, these drugs effectively targeted taxane-resistant tumors and bone metastases – two common clinical conditions in patients with aggressive prostate cancer. In summary, we find that metastatic prostate tumor cells differ from benign prostate tumor cells in their sensitivity to certain drug classes. Taken together, our results strongly suggest that albendazole, an anthelmintic medication, may represent a potential adjuvant or neoadjuvant to standard therapy in the treatment of disseminated prostate cancer.

12621 Targeted Chemical-Genetic Screen Platform for Identifying Drug Modes-of-Action

ABSTRACT IMPACT: The key to advancing precision medicine is to deepen our understanding of drug modes-of-action (MOA). This project aims to develop a novel method for predicting MOA of potential drug compounds, providing an experimental and computational platform for more efficient drug discovery. OBJECTIVES/GOALS: To develop (1) a targeted CRISPR-Cas9 chemical-genetic screen approach, and (2) a computational method to predict drug mode-of-action from chemical-genetic interaction profiles. METHODS/STUDY POPULATION: Screening drugs against a gene deletion library can identify knockouts that modulate drug sensitivity. These chemical-genetic interaction (CGI) screens can be performed in human cell lines using a pooled lentiviral CRISPR-Cas9 approach to assess drug sensitivity/resistance of single-gene knockouts across the human genome. A targeted, rather than genome-wide, library can enable scaling these screens across many drugs.CGI profiles can be derived from phenotypic screen readouts. These profiles are analogous to genetic interaction (GI) profiles, which represent sensitivity/resistance of gene knockouts to a second gene knockout rather than a drug. To computationally predict a drug’s genetic target, we leverage the property that a drug’s CGI profile will be similar to its target’s GI profile. RESULTS/ANTICIPATED RESULTS: Five proof-of-principle screens will be conducted with compounds that have existing genome-wide profiles and well-characterized MOA. I will generate CGI profiles for these five compounds and identify genes that are drug-sensitizers or drug-suppressors. I will then evaluate whether targeted library screens can recapitulate the CGIs found in genome-wide screens. Finally, I will develop a computational tool to integrate these CGI profiles with GI profiles (derived from another project) to predict gene-level and bioprocess-level drug targets. These predictions (from both targeted and genome-wide profiles) will be benchmarked against a drug-target and drug-bioprocess standard. DISCUSSION/SIGNIFICANCE OF FINDINGS: This work will develop a scalable, targeted chemical-genetic screen approach to discovering how putative therapeutics work. The targeted screen workflow provides a method for higher-throughput drug screening. The computational pipeline provides a powerful tool for exploring the MOA of uncharacterized drugs or repurposing FDA-approved drugs.

A potential screening method for epigenetic drugs: uncovering stress-induced gene silencing in Chlamydomonas

Histone modifications and DNA methylation together govern promoter availability, thereby influencing gene expression. This study queries the unicellular chlorophyte, Chlamydomonas reinhardtii using a three step “epigenetic assay” design to phenotypically track the variegation of a randomly integrated Paromomycin resistance transgene(s) (PmR ). Based on its position of integration, the PmR gene expression hinged on two epigenetic hallmarks: the spreading of heterochromatin, and the transmissible memory of epigenetic states across generations. Using a spot-dilution analysis, the loss of antibiotic resistance phenotype was scored from 0 to 4, four being maximally silenced. Appropriate construct designs were used to demonstrate that the cis-spread of heterochromatin could be interfered with a stronger euchromatic barrier (TUB2 promoter). When assayed for metal ion stress, a combination of Mn deficiency with excess Cu or Zn stress was shown to induce gene silencing in Chlamydomonas. Cu stress resulted in the accumulation of intracellular ROS, while Zn stress elevated the sensitivity to ROS. As proof of functional conservation, mammalian epigenetic drugs demonstrably interfered with stress-induced gene silencing. Finally, a selected group of transgenic clones responsive to HDACi sodium butyrate, when tested in a gradient plate format showed similarity in phenotype to the plant-derived compound cinnamic acid. This indicated a possible commonality in their mode of action, unlike curcumin which might have a different mechanism. Thus, using binned libraries, based on a common set of responses to known drugs, a cost-effective high-throughput screening strategy for epigenetically active compounds from plants or other sources is described.

Predicting Potential SARS-COV-2 Drugs-In Depth Drug Database Screening Using Deep Neural Network Framework SSnet, Classical Virtual Screening and Docking.

Severe Acute Respiratory Syndrome Corona Virus 2 has altered life on a global scale. A concerted effort from research labs around the world resulted in the identification of potential pharmaceutical treatments for CoVID-19 using existing drugs, as well as the discovery of multiple vaccines. During an urgent crisis, rapidly identifying potential new treatments requires global and cross-discipline cooperation, together with an enhanced open-access research model to distribute new ideas and leads. Herein, we introduce an application of a deep neural network based drug screening method, validating it using a docking algorithm on approved drugs for drug repurposing efforts, and extending the screen to a large library of 750,000 compounds for de novo drug discovery effort. The results of large library screens are incorporated into an open-access web interface to allow researchers from diverse fields to target molecules of interest. Our combined approach allows for both the identification of existing drugs that may be able to be repurposed and de novo design of ACE2-regulatory compounds. Through these efforts we demonstrate the utility of a new machine learning algorithm for drug discovery, SSnet, that can function as a tool to triage large molecular libraries to identify classes of molecules with possible efficacy.

MDM2 inhibitors: Targeting p53-MDM2 interaction to anti-cancer

P53 is a recognized tumor suppressor gene, which mainly depends on the activity of its transfer factor to realize the tumor suppressor effect. Mouse two-minute 2 (MDM2) is an important inhibitor of p53. When combined with MDM2, the activity of p53 will be reduced, and the anti-cancer effect will be weakened. According to the mechanism between p53 and MDM2, researchers focus on the inhibitors to inhibit their binding. Through a large number of drug screening methods and means, this article has found many new inhibitors of p53-MDM2 interaction, most of which are still in the clinical research stage. Specifically, we classify the drugs based on their different action mechanisms. Firstly, some drugs combine with MDM2 to inhibit the p53-MDM2 interaction. They are Siremadlin (NVP-HDM201), RG7112, and NVP-CGM09. While some act on p53, they rely on their induction of p53 signalling and inhibition of tumour cell proliferation in p53 wild-type tumor cell lines, like MK-8242 and KRT-232(AMG-232). What’s more, one inhibitor’s action bases on P53 and MDM2 in T cells is APG-115. And last but not least, there are also several drugs that stable tumor suppressor TP53, leading to p53 activation and inducing cell cycle arrest and apoptosis, they are Idasanutlin (RG7388) and Milademetan (DS-3032/RAIN-32). Furthermore, clinical studies are finding that monotherapy does not deliver a powerful therapeutic effect. Various combination strategies are being explored with MDM2 inhibitors preclinically and in the clinic. This article will talk about some specific combinations: APG-115 combine with immune checkpoint inhibitor PD-1/PD-L1, MDM2 inhibitors combine with BCL-2 inhibitors, anti-CD20 therapeutic antibodies, and the last, combine with alkylating agents.

Artificial Intelligence-Based Application to Explore Inhibitors of Neurodegenerative Diseases.

Neuroinflammation is a common factor in neurodegenerative diseases, and it has been demonstrated that galectin-3 activates microglia and astrocytes, leading to inflammation. This means that inhibition of galectin-3 may become a new strategy for the treatment of neurodegenerative diseases. Based on this motivation, the objective of this study is to explore an integrated new approach for finding lead compounds that inhibit galectin-3, by combining universal artificial intelligence algorithms with traditional drug screening methods. Based on molecular docking method, potential compounds with high binding affinity were screened out from Chinese medicine database. Manifold artificial intelligence algorithms were performed to validate the docking results and further screen compounds. Among all involved predictive methods, the deep learning-based algorithm made 500 modeling attempts, and the square correlation coefficient of the best trained model on the test sets was 0.9. The XGBoost model reached a square correlation coefficient of 0.97 and a mean square error of only 0.01. We switched to the ZINC database and performed the same experiment, the results showed that the compounds in the former database showed stronger affinity. Finally, we further verified through molecular dynamics simulation that the complex composed of the candidate ligand and the target protein showed stable binding within 100 ns of simulation time. In summary, combined with the application based on artificial intelligence algorithms, we unearthed the active ingredients 1,2-Dimethylbenzene and Typhic acid contained in Crataegus pinnatifida and Typha angustata might be the effective inhibitors of neurodegenerative diseases. The high prediction accuracy of the models shows that it has practical application value on small sample data sets such as drug screening.

Cell membrane chromatography for the analysis of the interaction between chloroquine and hydroxychloroquine with ACE2 receptors

The recent emergence of the novel pathogenic coronavirus disease 2019 (COVID-19) is responsible for a worldwide pandemic. In sight of this, there has been growing interest in the use of chloroquine (CQ) and hydroxychloroquine (HCQ) as potential treatments. In this study, we use angiotensin converting enzyme 2 (ACE2) over-expressed cell membrane chromatography (CMC) to study the interaction of CQ and HCQ with ACE2 receptor. Both CQ and HCQ were retained on the ACE2/CMC column. Then we analyzed the binding character of CQ and HCQ to ACE2 by CMC frontal analysis, ionic force investigation and competitive binding experiment. Results showed that CQ and HCQ KD values obtained from the CMC frontal analysis method were 8.22(±0.61) × 10−7 M and 11.70(±2.44) × 10−7 M. Compare to CQ, HCQ has the weaker affinity with ACE2. The action force of CQ, HCQ and ACE2 is mainly ionic force. CQ and HCQ have different degrees of competitive binding relationship with ACE2. Our study revealed the interaction of CQ and HCQ with ACE2 receptor, which provides new insights for the use of CQ and HCQ in the treatment of COVID-19. Moreover, this biomimetic drug screening method is expected to open the door for rapid targeting and separating bioactive ingredients active towards ACE2 receptor.

Methods and techniques of capillary electrophoresis for drug screening

Capillary electrophoresis (CE) shows enormous potential for application in new drug research and development. Because of the aqueous medium employed as the running buffer in CE, drug screening can be carried out in an environment similar to that in physiological testing media. Drug screening methods based on CE are different from other instrumental measurements in vitro. CE can not only sustain the biological activity of the screened molecules and ligands, but also help evaluate the interactions between the receptors and the ligands. Based on these interactions, some important pharmacological parameters related to drug screening, such as the association constant Kb, bonding rate constant Kon, and dissociation rate constant Koff, can be determined by CE. Thus, CE is an effective tool for simulating and predicting the entire interaction process between receptors and drugs in vivo. In this review, the history of CE for drug screening is revisited. The theories, common methods for drug screening by CE, and some application examples and related technologies are reviewed. The methods of drug screening by means of affinity CE and kinetic CE are introduced. Some selected studies on different ligands at the molecular and cellular level are reported, along with examples several types of drugs. Techniques based on a combination of CE with mass spectrometry and chemiluminescence are reviewed, with focus on the screening of candidate drugs and active compounds from traditional Chinese medicine. The application prospect of drug screening by CE combined with a DNA-encoded compound library is introduced. This paper discusses the core of the fraction collection step in CE and emphasizes the significance of combining CE with systematic evolution of ligands by exponential enrichment. In conclusion, various optional methods for CE drug screening would pave the way for new concepts related to drug screening and evaluation in the future.

A two-dimensional multiwell cell culture method for the production of CYP3A4-expressing hepatocyte-like cells from HepaRG cells.

Cytochrome P450 enzymes (CYP) function in drug metabolism in the liver. To evaluate numerous drug candidates, a high‐content screening (HCS) system with hepatocyte‐like cells (HLCs) that can replace adult human hepatocytes is required. Human hepatocellular carcinoma HepaRG is the only cell line capable of providing HLCs with high CYP3A4 expression comparable to that in adult hepatocytes after cell differentiation. The aim of this study was to design an ideal multiwell culture system for HLCs using transgenic HepaRG cells expressing the EGFP coding an enhanced green fluorescent protein under CYP3A4 transcriptional regulation. HLCs were matured on five different types of 96‐well black plates. Culturing HLCs on glass‐bottom Optical CVG plates significantly promoted cell maturation and increased metabolic activity by twofold under two‐dimensional (2D) culture conditions, and these features were enhanced by 2% collagen coating. Three plates for three‐dimensional (3D) cell cultures with a gas‐exchangeable fabric or dimethylpolysiloxane membrane bottom formed multiple round colonies, whereas they were ineffective for CYP3A4 expression. Under optimized conditions presented here, HLCs lost responsiveness to nuclear receptor‐mediated transcriptional induction of CYP3A4, suggesting that CYP3A4 transcription has already been fully upregulated. Therefore, HepaRG‐derived HLCs will provide an alternative to human hepatocytes with high levels of CYP3A4 enzyme activity even under 2D culture conditions. This will improve a variety of drug screening methods.

Myelin detection in fluorescence microscopy images using machine learning

BackgroundThe myelin sheath produced by glial cells insulates the axons, and supports the function of the nervous system. Myelin sheath degeneration causes neurodegenerative disorders, such as multiple sclerosis (MS). There are no therapies for MS that promote remyelination. Drug discovery frequently involves screening thousands of compounds. However, this is not feasible for remyelination drugs, since myelin quantification is a manual labor-intensive endeavor. Therefore, the development of assistive software for expedited myelin detection is instrumental for MS drug discovery by enabling high-content image-based drug screens. New methodIn this study, we developed a machine learning based expedited myelin detection approach in fluorescence microscopy images. Multi-channel three-dimensional microscopy images of a mouse stem cell-based myelination assay were labeled by experts. A spectro-spatial feature extraction method was introduced to represent local dependencies of voxels both in spatial and spectral domains. Feature extraction yielded two data set of over forty-seven thousand annotated images in total. ResultsMyelin detection performances of 23 different supervised machine learning techniques including a customized-convolutional neural network (CNN), were assessed using various train/test split ratios of the data sets. The highest accuracy values of 98.84±0.09% and 98.46±0.11% were achieved by Boosted Trees and customized-CNN, respectively. Comparison with existing methodsOur approach can detect myelin in a common experimental setup. Myelin extending in any orientation in 3 dimensions is segmented from 3 channel z-stack fluorescence images. ConclusionsOur results suggest that the proposed expedited myelin detection approach is a feasible and robust method for remyelination drug screening.

Switching of cell fate through the regulation of cell growth during drug-induced intrahepatic cholestasis

Understanding the fundamental mechanisms that govern the fate of cells during drug-induced intrahepatic cholestasis provides strategies for the establishment of evaluation methods for drug screening. In the present study, the aggregates of a differentiated human hepatic cell line, HepaRG, were incubated in medium with Y27632 or bosentan to clarify the changes in the behavior of bile canaliculi (BC) with the growth of cells during drug-induced intrahepatic cholestasis. With elapsed exposure time, the aggregates in the culture with bosentan caused the dilation of BC, and the hepatocytes ultimately exhibited apoptotic death after the disruption of BC. Y27632 caused the disruption of BC in the aggregates after dilation. However, there was no change in the number of cells within the aggregates in the culture with Y27632, in spite of its cytotoxicity. After 144h from the start of Y27632 exposure, the aggregates showed the rearrangement of BC. To inhibit cell division, the aggregates exposed to Y27632, which exhibited disruption of BC, were treated with mitomycin C for 2h and continuously exposed to Y27632. The inhibition of cell division could not induce the rearrangement of BC within these aggregates, which was similar to the phenomenon observed in the aggregates exposed to bosentan. These findings indicate that growth is an important factor that influences the switching of cell fate toward survival or death in drug-induced intrahepatic cholestasis process. Thus, the autoregulation of growth is a major contributor to the rearrangement of BC within aggregates.

Comparative anti-inflammatory effect of curcumin at air-liquid interface and submerged conditions using lipopolysaccharide stimulated human lung epithelial A549 cells

BackgroundInhalation of aerosolized drugs is a promising entry route for rapid and non-invasive therapeutics delivery to the lung. Curcumin exhibits potent anti-inflammatory properties, which are effective for use in lung diseases. The anti-inflammatory properties of curcumin have been widely studied in vitro with cells cultured in submerged conditions, however, the effectiveness using air-liquid interface (ALI) exposure is currently unknown. MethodsThe anti-inflammatory effect of curcumin under both ALI and submerged conditions was investigated in the present study. Lipopolysaccharide (LPS) stimulated A549 cells were exposed to curcumin under ALI (10–100 μM) using a dose-controlled air-liquid interface cell exposure (ALICE)-CLOUD system and submerged cell culture conditions (1–20 μM). The expression of pro-inflammatory cytokines (interleukin (IL)-6, IL-8), cell viability and cytotoxicity were studied for each exposure scenario. The cellular uptake behaviour of curcumin was studied with an equipotent cell-based dose (200 pmol/106 cell) at various time points up to 24 h. ResultsThe ALI delivery profile proved to be rapid, efficient and reproducible. For the doses studied, no significant effect on cell viability and cytotoxicity were observed. ALI exposure of curcumin was more effective in reducing pro-inflammatory cytokines expression in lung epithelial cells compared with submerged cell cultures. Furthermore, rapid cellular uptake and higher intracellular doses were achieved by ALI conditions. ConclusionsThe ALICE-CLOUD system combined with lung epithelial cells cultured under ALI conditions offers a reliable and relevant in vitro method for preclinical aerosolized drug screening. Curcumin might be a promising anti-inflammatory candidate drug for inhalation therapy of lung diseases.

A Comprehensive Review on Antiulcer properties of Herbal drugs

Herbal medicines are very useful in treating various human ailments. The plants and herbs consumed in its natural state have proved to have lesser side effects and are cost effective too. Recognition of this fact has created a demand for herbal medicines, both in developing and developed countries across the globe. Peptic ulcer disease in clinical practice is one of the common gastrointestinal disorders where duodenal ulcer, gastric ulcer, NSAIDs triggered ulcer and stress ulcer are common forms of peptic ulcer. Gastric ulcers are commonly found in the geriatrics and in the lower socio-economic class of people. Most synthetic drugs are used to treat peptic ulcers, but they cause adverse effects of various kinds. For its ethnic, ethno botanical and ethno pharmacological use, herbal medicines are therefore exceptional. In this review, attempts were made to know the natural antiulcer drugs with their recommended drug portion, screening method and sort of extract used for appraisal and observations to show its usage. The reason for this assessment is to assemble literature and the antiulcer residences of the herbal medicines used frequently. Different plants such as Allium sativum, Centella asiatica, Lawsonia inermis, Ocimum sanctum, Sesbania grandiflora, Adansonia digitata, Ficus religiosa, Glycyrrhiza glabra, Cynodon dactylon, have been shown to be involved in antiulcer care.

Abstract A25: Evaluation of Drug Disposition in Supratentorial Ependymoma

Abstract Introduction: The majority of pediatric supratentorial (ST) ependymomas (EPN) is driven by distinct gene fusions between C11orf95 and RELA. The resultant molecular group of ST-EPN-RELA tumors is characterized by constitutive activation of NF-κB signaling and deregulation of the p53 pathway. In contrast to surgery and radiotherapy, chemotherapy has failed to demonstrate significant benefit in the management of affected children. Alternative strategies including enhanced drug delivery, combination treatments, or application of new selective compounds are needed to tackle this disease. Material and Methods: RNAi and drug screening methods were applied to identify potential therapeutic approaches using ST-EPN-RELA cell lines. In order to identify optimal dosing strategies of selected drugs and to assess effects of combinatorial treatment approaches on blood-brain barrier (BBB) penetration, cerebral microdialysis combined with ultraperformance liquid chromatography and tandem mass spectrometry (UPLC-MS/MS) was applied. This approach allowed for exact, continuous, and time-dependent drug quantification in tumors or healthy tissue in freely moving experimental mice. Patient-derived xenograft models of ST-EPN-RELA were treated to investigate toxicity and outcome parameters. Results: Regulation of p53 signaling and nuclear protein shuttling were identified as promising therapeutic approaches. While low-dose dactinomycin could successfully reestablish p53 function in ST-EPN-RELA cells in vitro, penetration of the drug across the BBB was found to be very poor and did not result in a survival benefit of tumor-bearing mice. Preliminary results of alternative strategies such as combination with efflux pump inhibitors, liposomal packaging, and inhibition of XPO1 being the sole nuclear exporter of p53 hold promise to overcome these constraints. Conclusion: Oncogenic dependencies of ST-EPN-RELA are currently difficult to target. Preclinical evaluation of effective drug disposition combined with long-term treatment studies may help to better select promising compounds and thereby increase success rates of early clinical trials in patients with ST-EPN-RELA in the future. Citation Format: Julia Benzel, Max Sauter, Norman Mack, Abigail Davis, Johanna Weiss, Philipp Uhl, Jürgen Burhenne, Kendra K. Maass, Jens-Martin Hübner, Hendrik Witt, Anang Shelat, Amar Gajjar, Santhosh A. Upadhyaya, Aylin Camgoz, Frank Buchholz, Sina Oppermann, Marcel Kool, Daisuke Kawauchi, Olaf Witt, Walter E. Haefeli, Stefan M. Pfister, Clinton Stewart, Kristian W. Pajtler. Evaluation of Drug Disposition in Supratentorial Ependymoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A25.

Ex vivo modelling of drug efficacy in a rare metastatic urachal carcinoma

BackgroundEx vivo drug screening refers to the out-of-body assessment of drug efficacy in patient derived vital tumor cells. The purpose of these methods is to enable functional testing of patient specific efficacy of anti-cancer therapeutics and personalized treatment strategies. Such approaches could prove powerful especially in context of rare cancers for which demonstration of novel therapies is difficult due to the low numbers of patients. Here, we report comparison of different ex vivo drug screening methods in a metastatic urachal adenocarcinoma, a rare and aggressive non-urothelial bladder malignancy that arises from the remnant embryologic urachus in adults.MethodsTo compare the feasibility and results obtained with alternative ex vivo drug screening techniques, we used three different approaches; enzymatic cell viability assay of 2D cell cultures and image-based cytometry of 2D and 3D cell cultures in parallel. Vital tumor cells isolated from a biopsy obtained in context of a surgical debulking procedure were used for screening of 1160 drugs with the aim to evaluate patterns of efficacy in the urachal cancer cells.ResultsDose response data from the enzymatic cell viability assay and the image-based assay of 2D cell cultures showed the best consistency. With 3D cell culture conditions, the proliferation rate of the tumor cells was slower and potency of several drugs was reduced even following growth rate normalization of the responses. MEK, mTOR, and MET inhibitors were identified as the most cytotoxic targeted drugs. Secondary validation analyses confirmed the efficacy of these drugs also with the new human urachal adenocarcinoma cell line (MISB18) established from the patient’s tumor.ConclusionsAll the tested ex vivo drug screening methods captured the patient’s tumor cells’ sensitivity to drugs that could be associated with the oncogenic KRASG12V mutation found in the patient’s tumor cells. Specific drug classes however resulted in differential dose response profiles dependent on the used cell culture method indicating that the choice of assay could bias results from ex vivo drug screening assays for selected drug classes.

Comprehensive Drug Screening of Whole Blood by LC-HRMS-MS in a Forensic Laboratory.

As the number of prescriptions, over-the-counter medications and drugs of abuse continue to increase, forensic laboratories are faced with the challenge of developing more comprehensive screening methods in order to detect them in whole blood samples. Another challenge faced by forensic laboratories is detecting and identifying novel synthetic compounds as they emerge and change. Traditional drug screening methods include enzyme immunoassay (EIA) and either gas or liquid chromatography paired with mass spectrometry (GC-MS or LC-MS-MS, respectively). While these methods are good, they have their disadvantages. For example, EIA requires special reagents for each drug class, GC-MS requires extensive sample preparation, and LC-MS-MS only detects drugs on a known inclusion lists of compounds of interest. Described here is the development of a robust and comprehensive screening method for drugs in whole blood samples that eliminates the aforementioned disadvantages of the traditional methods. Using a Q Exactive Focus™ liquid chromatography-high-resolution accurate mass spectrometer (LC-HRMS-MS), a method was developed that is capable of detecting ~200 drugs at a concentration of 2μg/L for most analytes. This method also employs a more automated data processing feature which reduces processing time. Finally, it has the added benefit of retroactive data analysis, which allows it to be used for unknown drug analysis as well. Used as an initial screening method, the comprehensive drug screen using LC-HRMS-MS has the potential to take on two of the most important challenges faced by forensic laboratories today.