Clinical Drug Screening Research Articles

The future of cancer therapy: exploring the potential of patient-derived organoids in drug development.

Cancer therapy is on the brink of a significant transformation with the inclusion of patient-derived organoids (PDOs) in drug development. These three-dimensional cell cultures, directly derived from a patient's tumor, accurately replicate the complex structure and genetic makeup of the original cancer. This makes them a promising tool for advancing oncology. In this review, we explore the practical applications of PDOs in clinical drug screening and pharmacognostic assessment, as well as their role in refining therapeutic strategies. We provide insights into the latest advancements in PDO technology and its implications for predicting treatment responses and facilitating novel drug discoveries. Additionally, we address the operational challenges associated with incorporating PDOs into the drug development process, such as scaling up organoid cultures, ensuring consistent results, and addressing the ethical use of patient-derived materials. Aimed at researchers, clinicians, and key stakeholders in oncology, this article aims to succinctly present both the extraordinary potential and the obstacles to integrating PDOs, thereby shedding light on their prospective impact on the future of cancer treatment.

Clinical drug screening reveals clofazimine potentiates the efficacy while reducing the toxicity of anti-PD-1 and CTLA-4 immunotherapy

Emerging as the most potent and durable combinational immunotherapy, dual anti-PD-1 and CTLA-4 immune checkpoint blockade (ICB) therapy notoriously increases grade 3-5 immune-related adverse events (irAEs) in patients. Accordingly, attempts to improve the antitumor potency of anti-PD-1+CTLA-4 ICB by including additional therapeutics have been largely discouraged due to concerns of further increasing fatal toxicity. Here, we screened ∼3,000 Food and Drug Administration (FDA)-approved drugs and identified clofazimine as a potential third agent to optimize anti-PD-1+CTLA-4 ICB. Remarkably, clofazimine outperforms ICB dose reduction or steroid treatment in reversing lethality of irAEs, but unlike the detrimental effect of steroids on antitumor efficacy, clofazimine potentiates curative responses in anti-PD-1+CTLA-4 ICB. Mechanistically, clofazimine promotes E2F1 activation in CD8+ Tcells to overcome resistance and counteracts pathogenic Th17 cells to abolish irAEs. Collectively, clofazimine potentiates the antitumor efficacy of anti-PD-1+CTLA-4 ICB, curbs intractable irAEs, and may fill a desperate clinical need to improve patient survival.

A multicomponent LC-MS/MS method for drugs of abuse testing using volumetric DBS and a clinical evaluation by comparison with urine

BackgroundDrug testing commonly use urine as a specimen and immunoassays for screening. The need for supervised urine collection has led to an interest in alternative specimens and a need for using mass spectrometry methods already for screening. In addition, mass spectrometry methods allow for broad multipanel screening which of great value because of the increased number of substances that needs to be covered has increased over time. One alternative specimen of interest for drugs of abuse testing is dried blood spots (DBS) and this work aimed at developing multipanel screening methods based on selected reaction monitoring liquid chromatography - mass spectrometry for both urine and dried finger blood as specimens. Materials and MethodsThe urine method comprised 37 analytes and utilised salted out liquid/liquid extraction in 96-well format, respectively, and the blood method comprised 35 analytes, a 10 µL volumetric DBS device and a two-step solvent extraction procedure. In both cases stable isotope labelled internal standards were used for almost all analytes. ResultsThe methods were validated according to forensic standard. The lowest reporting limits were generally set at 100 ng/mL for urine and 1 ng/mL for blood and the accuracy and imprecision were within limits of 15 and 20%. The methods were applied in a clinical study on patients receiving methadone maintenance treatment for opioid dependence. Methadone was detected in all urine and DBS samples, for urine sometimes below the commonly applied screening cutoff limit of 300 ng/mL. In 20 out of 99 cases no other drug was detected in any specimen. The most commonly other detected substances were pregabalin, amphetamine, alprazolam, zopiclone and THCCOOH. Findings in urine and DBS generally agreed well but more positives were detected in DBS. ConclusionMultipanel methods using liquid chromatography – mass spectrometry suitable for clinical drug screening were successfully developed for urine and blood collected by finger-pricking and stored as DBS.

A Nano-Electroporation-DNA Tensioner Platform Enhances Intracellular Delivery and Mechanical Analysis Toward Rapid Drug Assessment.

In vitro, drug assessment holds tremendous potential to success in novel drug development and precision medicine. Traditional techniques for drug assessment, however, face remarkable challenges to achieve high speed, as limited by incubation-based drug delivery (>several hours) and cell viability measurements (>1d), which significantly compromise the efficacy in clinical trials. In this work, a nano-electroporation-DNA tensioner platform is reported that shortens the time of drug delivery to less than 3s, and that of cellular mechanical force analysis to 30min. The platform adopts a nanochannel structure to localize a safe electric field for cell perforation, while enhancing delivery speed by 103 times for intracellular delivery, as compared to molecular diffusion in coculture methods. The platform is further equipped with a DNA tensioner to detect cellular mechanical force for quantifying cell viability after drug treatment. Systematic head-to-head comparison, by analyzing FDA (food and drug administration)-approved drugs (paclitaxel, doxorubicin), demonstrated the platform with high speed, efficiency, and safety, showing a simple yet powerful tool for clinical drug screening and development.

In vitro oogenesis from murine premeiotic germ cells using a new three-dimensional culture system

A faithful reconstitution of the complete process of oogenesis in vitro is helpful for understanding the molecular mechanisms, genetics, and epigenetic changes related to gametogenesis; it can also be useful for clinical drug screening, disease research, and regenerative medicine. To this end, given the consensus that murine female germ cells initiate meiosis at E13.5, substantial works have reported the successful generation of fertile oocytes using E12.5 female gonads as starting materials. Nevertheless, our data demonstrated that murine germ cells at E12.5 have heterogeneously initiated a meiotic transcriptional program based on a measurement of pre‐mRNAs (unspliced) and mature mRNAs (spliced) at a single-cell level. Therefore, to establish a platform that faithfully recapitulates the entire process in vitro (from premeiotic murine germ cells to fully developed oocytes), we here report a novel three-dimensional organoid culture (3-DOC) system, which successfully induced fully developed oocytes from E11.5 premeiotic female germ cells (oogonia). Compared with 2D culture and other 3D culture methods, this new culture system is more cost-effective and can create high-quality oocytes similar to in vivo oocytes. In summary, our new culture platform provides an experimental model for future research in regenerative medicine and reproductive biology.

Integrated Acoustic Chip for Culturing 3D Cell Arrays.

Three-dimensional (3D) cell arrays provide an in vitro platform for clinical drug screening, but the bulky culture devices limit their application scenarios. Here, we demonstrate an integrated portable device that can realize contact-free construction of 3D cell spheroids. The interaction between the ultrasound generated by the portable device and the capillary results in periodic pressure nodes or anti-nodes, which lead to form a 3D cell array for cell culture. Such a 3D cell array pattern can be constructed in seconds and requires only 1 μL of cell samples. We further assessed the spheroids formed by the portable device and the impact of the acoustic field on spheroids and demonstrated the drug screening with assembled spheroids. More importantly, the integrated acoustic device can be further integrated with other components for more complex cell culture and all-round analysis. This portable and effective integrated device provides a new avenue for clinical biomedicine.

The design basis and application in urology of the tumor-on-a-chip platform.

Clinical research has been desperately looking for effective treatments for tumors. As the molecular mechanisms of tumors have not been thoroughly defined, and the problem of anti-tumor drug resistance and clinical drug screening are not effective, new ways are currently needed to explore and improve. Given the limitations of traditional cell culture models and preclinical animal models, we review the recently developed tumor chip, a micro-physiological platform based on microfluidic technology that maximizes replication of the human TEM. Here, we focus on the design basis of the tumor microarray and its application in urology oncology, summarizing the challenges facing future development in the field. In conclusion, this review demonstrates the wide range and application of tumor chips and the potential of these systems for the future treatment and management of urological tumors.

CALR frameshift mutations in MPN patient-derived iPSCs accelerate maturation of megakaryocytes.

SummaryCalreticulin (CALR) mutations are driver mutations in myeloproliferative neoplasms (MPNs), leading to activation of the thrombopoietin receptor and causing abnormal megakaryopoiesis. Here, we generated patient-derived CALRins5- or CALRdel52-positive induced pluripotent stem cells (iPSCs) to establish an MPN disease model for molecular and mechanistic studies. We demonstrated myeloperoxidase deficiency in granulocytic cells derived from homozygous CALR mutant iPSCs, rescued by repairing the mutation using CRISPR/Cas9. iPSC-derived megakaryocytes showed characteristics of primary megakaryocytes such as formation of demarcation membrane system and cytoplasmic pro-platelet protrusions. Importantly, CALR mutations led to enhanced megakaryopoiesis and accelerated megakaryocytic development in a thrombopoietin-independent manner. Mechanistically, our study identified differentially regulated pathways in mutated versus unmutated megakaryocytes, such as hypoxia signaling, which represents a potential target for therapeutic intervention. Altogether, we demonstrate key aspects of mutated CALR-driven pathogenesis dependent on its zygosity, and found novel therapeutic targets, making our model a valuable tool for clinical drug screening in MPNs.

Development and Application of 3D Bioprinted Scaffolds Supporting Induced Pluripotent Stem Cells.

Three-dimensional (3D) bioprinting is a revolutionary technology that replicates 3D functional living tissue scaffolds in vitro by controlling the layer-by-layer deposition of biomaterials and enables highly precise positioning of cells. With the development of this technology, more advanced research on the mechanisms of tissue morphogenesis, clinical drug screening, and organ regeneration may be pursued. Because of their self-renewal characteristics and multidirectional differentiation potential, induced pluripotent stem cells (iPSCs) have outstanding advantages in stem cell research and applications. In this review, we discuss the advantages of different bioinks containing human iPSCs that are fabricated by using 3D bioprinting. In particular, we focus on the ability of these bioinks to support iPSCs and promote their proliferation and differentiation. In addition, we summarize the applications of 3D bioprinting with iPSC-containing bioinks and put forward new views on the current research status.

Gabapentin prevalence: clinical and forensic experience in St. Louis, Missouri, USA

Gabapentin (Neurontin) is an anti-epileptic drug that has had wide off-label prescription use since market release due to presumed negligible abuse potential. However, trends in drug misuse have demonstrated that gabapentin misuse is occurring, particularly in those with a history of opioid misuse. This is concerning, because although gabapentin has no direct ligand activity at opioid receptors, it does potentiate the analgesic effect of opioids, and concurrent use of gabapentin and opioids may increase the risk of respiratory depressive effects of opioids. This study investigates the incidence of gabapentin detected in urine samples collected for clinical drug screening purposes in a local hospital emergency department and in postmortem samples submitted by medical examiners in the St. Louis metropolitan area. The prevalence of gabapentin and co-detected drugs in both populations is contrasted, compared, and discussed. This study found that 30% of urine samples collected from patients with suspected drug intoxication presenting to SSM Health Saint Louis University Hospital, a quaternary care medical center, were positive for gabapentin, and nearly two thirds of those were also positive for oxycodone. Over a 6-month period, the incidence of gabapentin positive postmortem cases increased from 18% to 20%. Nearly all gabapentin positive postmortem cases were also positive for an opioid, the most significant being fentanyl, suggesting that gabapentin misuse may be due to its potentiating effect of opioid drug action. This study also highlights the limited utility of immunoassay-based urine drug screens.

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting.

Microcarriers are beads with a diameter of 60-250 µm and a large specific surface area, which are commonly used as carriers for large-scale cell cultures. Microcarrier culture technology has become one of the main techniques in cytological research and is commonly used in the field of large-scale cell expansion. Microcarriers have also been shown to play an increasingly important role in in vitro tissue engineering construction and clinical drug screening. Current methods for preparing microcarriers include microfluidic chips and inkjet printing, which often rely on complex flow channel design, an incompatible two-phase interface, and a fixed nozzle shape. These methods face the challenges of complex nozzle processing, inconvenient nozzle changes, and excessive extrusion forces when applied to multiple bioink. In this study, a 3D printing technique, called alternating viscous-inertial force jetting, was applied to enable the construction of hydrogel microcarriers with a diameter of 100-300 µm. Cells were subsequently seeded on microcarriers to form tissue engineering modules. Compared to existing methods, this method offers a free nozzle tip diameter, flexible nozzle switching, free control of printing parameters, and mild printing conditions for a wide range of bioactive materials.

Inversely Calibrated Curvilinear Artificial Neural Network Model for Simultaneous Assay of Ternary Cardiovascular Drug Mixture

Novel chemometric design, tailored for pre-clinical multiple drug screening, goals for bioanalytical future scope. A highly sensitive, non-linear multivariate Artificial Neural Network (ANN) is developed and applied for simultaneous spectrophotometric determination of three commonly concomitant cardiovascular drugs in laboratory made mixtures and spiked human plasma samples. Ticagrelor, Irbesartan and Hydrochlorothiazide have been simultaneously quantified in the curvilinear ranges of 0-30 µg/mL, 0-10 µg/mL and 0-3 µg/mL respectively. Highly overlapping Near UV absorption spectra of three drugs, in the region of 215-280 nm, have been recorded 1-nm range in synthetic ternary mixtures and trained iteratively. By inversely relating the concentration matrix (x-block) with its corresponding absorption one (y-block), gradient-descent back-propagation ANN calibration could be computed and optimized. All proposed mathematical modeling was manipulated using MATLAB® 2007, reaching down to sixth order exponential Mean Square Error, MSE. To validate, independent set of ternary synthetic mixtures has been constructed and examined, where excellent recovery results has been obtained. Furthermore, application of suggested model to varying ratios synthetic ternary mixtures as well as spiked plasma samples has resulted in accurate, precise and robust estimations with no background interference. ANN method was compared to a reference HPLC method; Student’s t-test and F-variance ratio were calculated and showed insignificant difference. This chemometric approach is an eco-friendly green assay, time-saving, and economic method. It initiates a pathway for clinical drug screening through affordable spectroscopic instrumentation.

Three-dimensional culture and clinical drug responses of a highly metastatic human ovarian cancer HO-8910PM cells in nanofibrous microenvironments of three hydrogel biomaterials

BackgroundOvarian cancer is a highly aggressive malignant disease in gynecologic cancer. It is an urgent task to develop three-dimensional (3D) cell models in vitro and dissect the cell progression-related drug resistance mechanisms in vivo. In the present study, RADA16-I peptide has the reticulated nanofiber scaffold networks in hydrogel, which is utilized to develop robust 3D cell culture of a high metastatic human ovarian cancer HO-8910PM cell line accompanied with the counterparts of Matrigel and collagen I.ResultsConsequently, HO-8910PM cells were successfully cultivated in three types of hydrogel biomaterials, such as RADA16-I hydrogel, Matrigel, and collagen I, according to 3D cell culture protocols. Designer RADA16-I peptide had well-defined nanofiber networks architecture in hydrogel, which provided nanofiber cell microenvironments analogous to Matrigel and collagen I. 3D-cultured HO-8910PM cells in RADA16-I hydrogel, Matrigel, and collagen I showed viable cell proliferation, proper cell growth, and diverse cell shapes in morphology at the desired time points. For a long 3D cell culture period, HO-8910PM cells showed distinct cell aggregate growth patterns in RADA16-I hydrogel, Matrigel, and collagen I, such as cell aggregates, cell colonies, cell clusters, cell strips, and multicellular tumor spheroids (MCTS). The cell distribution and alignment were described vigorously. Moreover, the molecular expression of integrin β1, E-cadherin and N-cadherin were quantitatively analyzed in 3D-cultured MCTS of HO-8910PM cells by immunohistochemistry and western blotting assays. The chemosensitivity assay for clinical drug responses in 3D context indicated that HO-8910PM cells in three types of hydrogels showed significantly higher chemoresistance to cisplatin and paclitaxel compared to 2D flat cell culture, including IC50 values and inhibition rates.ConclusionBased on these results, RADA16-I hydrogel is a highly competent, high-profile, and proactive nanofiber scaffold to maintain viable cell proliferation and high cell vitality in 3D cell models, which may be particularly utilized to develop useful clinical drug screening platform in vitro.

A potential novel therapy for FGFR1-amplified pancreatic cancer with bone metastasis, screened by next-generation sequencing and a patient-derived xenograft model.

Effective therapies are limited for pancreatic cancer, particularly for those with distant tumour metastases. Therefore, more individualised drug screening is urgently required. Next-generation sequencing (NGS) is a powerful tool to investigate the genomic landscape of patients and the mechanism of drug response, which may provide a broader vision for potential clinical drug screening. Patient-derived xenograft (PDX) models may have a significant advantage in predicting clinical treatment response. In our previous study, a PDX of pancreatic cancer bone metastasis was established, and NGS was conducted to investigate the molecular information. In the present study, these data were further analysed and fibroblast growth factor receptor 1 (FGFR1) amplification was identified in a panel of 416 cancer-associated genes. Thus, AZD4547, an inhibitor against FGFR, was selected as a potential therapy, and was evaluated using the PDX model. AZD4547 was shown to exhibit antitumor activity by reducing the expression of FGFR1 and its targets. The present study also demonstrated the high potential of the novel NGS/PDX-based drug screening platform to improve individualised cancer treatment.

Biosynthesis of the Central Piperidine Nitrogen Heterocycle in Series a Thiopeptides

Summary of main observation and conclusionThiopeptides, arising from complex posttranslational modifications of a genetically encoded precursor peptide, are of great interest due to their structural complexity and important biological activities. All of these antibiotics share a macrocyclic peptidyl core that contains a central, six‐membered nitrogen heterocycle and are classified into five series a—e based on the oxidation state of the central nitrogenous ring. Here, we report that the biosynthesis of the central piperidine heterocycle of series a thiopeptides relies on the activity of homologues of an F420H2‐dependent reductase TppX4 by exploiting and characterizing the piperidine‐containing thiopeptin biosynthetic gene (tpp) cluster in Streptomyces tateyamensis. In vitro reconstruction of TppX4 activity demonstrated that the piperidine heterocycle of thiopeptins was transformed from a dehydropiperidine heterocycle, and TppX4 tolerated the changes in the C‐termini and macrocyclic peptidyl core of substrate and also tolerated dehyropiperidine‐containing monocyclic or bicyclic thiopeptides. The identification of TppX4 and its substrate tolerance enriches the biosynthetic toolbox for development of additional thiopeptide analogs for clinical drug screening.

Mechanism of Surface-Enhanced Raman Scattering Based on 3D Graphene-TiO2 Nanocomposites and Application to Real-Time Monitoring of Telomerase Activity in Differentiation of Stem Cells.

With a burst development of new nanomaterials for plasmon-free surface-enhanced Raman scattering (SERS), the understanding of chemical mechanism (CM) and further applications have become more and more attractive. Herein, a novel SERS platform was specially designed through electrochemical deposition of graphene onto TiO2 nanoarrays (EG-TiO2). The developed EG-TiO2 nanocomposite SERS platform possessed remarkable Raman activity using copper phthalocyanine (CuPc) as a probe molecule. X-ray photoelectron spectroscopy measurement revealed that the chemical bond Ti-O-C was formed at the interface between graphene and TiO2 in EG-TiO2 nanocomposites. Both experimental and theoretical results demonstrated that the obvious Raman enhancement was attributed to TiO2-induced Fermi level shift of graphene, resulting in effective charge transfer between EG-TiO2 nanocomposites and molecules. Taking advantage of a marked Raman response of the CuPc molecule on the EG-TiO2 nanocomposite surface as well as specific recognition of CuPc toward multiple telomeric G-quadruplex, EG-TiO2 nanocomposites were tactfully employed as the SERS substrate for selective and ultrasensitive determination of telomerase activity, with a low detection limit down to 2.07 × 10-16 IU. Interestingly, the self-cleaning characteristic of EG-TiO2 nanocomposites under visible light irradiation successfully provided a recycling ability for this plasmon-free EG-TiO2 substrate. The present SERS biosensor with high analytical performance, such as high selectivity and sensitivity, has been further explored to determine telomerase activity in stem cells as well as to count the cell numbers. More importantly, using this useful tool, it was discovered that telomerase activity plays an important role in the proliferation and differentiation from human mesenchymal stem cells to neural stem cells. This work has not only established an approach for gaining fundamental insights into the chemical mechanism (CM) of Raman enhancement but also has opened a new way in the investigation of long-term dynamics of stem cell differentiation and clinical drug screening.

Application of Bioartificial Liver Reactor with Modified Membrane in Drug-Screening

The objective is to observe the advantage of the poly(vinylidene difluoride)-modified (PVDF-modified) bioartificial liver (BAL) reactors in the drug screening of Chinese herbs quercetin and resveratrol. The active ingredients of quercetin (0.1 mmol/L) was used to acted on rat hepatic cells for 12 hours both in the PVDF-modified and in the unmodified bioartificial liver reactors, then cisplatin (0.2 mmol/L) was added for 24 hours. Another Chinese herb resveratrol (0.1 mmol/L) was used with the same treatment. Finally, the synthesis of urea and albumin, the activities of lactate dehydrogenase (LDH) and aminotransferase (AST) were detected in these two cultured hepatocytes. Significant difference of the synthesis of urea and albumin was obtained between the PVDF-modified and the unmodified BAL reactors, and a less significant difference was for the activities of LDH and AST. The results showed that the PVDF-modified BAL reactor was more sensitive than the unmodified in liver toxicity and in activity of the liver-protective medicine. Hence, the PVDF-modified BAL reactor is more suitable for clinical drug screening.

3 Randomized Discontinuation Designs in Early Clinical Drug Screening – how to Adapt if the Drug Works too well? The Cabozantinib Experience

3 Randomized Discontinuation Designs in Early Clinical Drug Screening – how to Adapt if the Drug Works too well? The Cabozantinib Experience

Pre-clinical transgenic mouse models of nervous system tumors

AbstractThe most common primary CNS tumors are gliomas, where other than a few subtypes such as oligodendrogliomas, the survival has remained unchanged despite advances in surgical, chemo- and radiation therapy, especially for the most malignant and common glioma; glioblastoma multiforme (GBM). Recent novel therapies like immuno- and gene therapy have shown some promise in existing pre-clinical models, but have failed to demonstrate therapeutic benefit in patients. The reason(s) for such failures include our incomplete understanding of the molecular pathogenesis of these tumors and also due to testing of novel biological therapies in less than ideal pre-clinical models, which for the most part have included xenografts established in mice from glioma cell lines or patient explants. Transgenic mouse models offers an opportunity to develop and utilize an easily replenished, reproducible, manipulated spontaneous and more appropriate pre-clinical model of human cancers. Here we highlight on how mouse models are generated using several techniques and how mouse models have come to the forefront to address several issues such as identifying novel tumour modifier genes of central and peripheral nervous system tumours. Lastly we discuss how mouse models may provide an invaluable tool in pre clinical drug screening and testing.