Potency Assay Research Articles

Schiff base compounds constructed from pyrazole–acetamide: Synthesis, spectroscopic characterization, crystal structure, DFT, molecular docking and antioxidant activity

Two new Schiff base compounds, namely N-(2-((4-(dimethylamino)benzylidene)amino)phenyl)-2-(5-methyl-1H-pyrazol-3-yl)acetamide (DBPA) and N-(2-((4-methoxybenzylidene)amino)phenyl)-2-(5-methyl-1H-pyrazol-3-yl)acetamide (MBPA), have been synthesized through the condensation of N-(2-aminophenyl)-2-(5-methyl-1H-pyrazol-3-yl)acetamide (A) with aromatic aldehydes. Their structures have been elucidated on the basis on spectral data (1H NMR, 13C NMR ESI-MS) and confirmed by a single-crystal X-ray diffraction analysis. Thus, the crystallographic studies showed that the analysis of the crystal packing of DBPA and MBPA revealed 1D and 2D supramolecular architectures, respectively, via various hydrogen bonding interactions. Moreover, the evaluation of compounds A, DBPA and MBPA for their antioxidant activity against DPPH and ABTS radical scavenging assay, and reduction potency assay has been undertaken. using quercetin, Trolox and catechin as standard references respectively. The results obtained showed that DBPA appeared more hydrogen radical donor while MBPA appeared more electron donor. The comparison of the activities of the studied three molecules was carried out using both DFT and molecular docking calculations. Furthermore, ADME/T calculations have been made in order to show that these compounds can be used as potent drugs.

Abstract 29 Umbilical Cord-derived Mesenchymal Stromal Cells Suppress Microglia Activation Induced by Demyelination

Abstract Introduction White matter diseases are often accompanied by microglial activation and neuroinflammation. Mesenchymal stromal cells (MSCs) manufactured from umbilical cord tissue possess immunomodulatory properties, making them promising candidates for cell therapy in white matter disease. MSCs can interact with resident macrophages and modify the course of inflammation. However, the influence MSCs have on microglia, the resident macrophages of the central nervous system (CNS), and its implications for white matter disease remain uncertain. Objectives From the blood, MSCs traffic to lung and interact with resident macrophages to alter the trajectory of inflammation. We hypothesized that MSCs injected into the cerebrospinal fluid (CSF) would similarly suppress the activation of microglia. Our goals in this study were to test the efficacy MSCs have on microglia activation and develop a relevant potency assay. Methods In this study, we employed a range of assays, including an in vivo acute spinal cord demyelination model, organotypic brain slice cultures, and microglia activation assays with primary microglia and an immortalized cell line. Using these assays, we assessed the impact cord tissue derived MSCs have on microglial activation. Results In spinal cords and brain slices, lysophosphatidylcholine produced robust demyelination and microglia activation that was suppressed by MSCs. To determine if MSCs could directly impact microglia, we isolated primary microglia and activated them in culture with lipopolysachride (LPS), a toll-like receptor agonist. We screen 6 cytokines induced by LPS and determined that MSCs suppress the release of tumor necrosis factor (TNF) from LPS-activated microglia. We confirmed these results in vivo as MSCs blocked TNF production by spinal cord microglia after acute demyelination in the spinal cord. Additionally, we confirmed the ability of MSCs to suppress TNF in a simple assay with a microglia cell line (IMG). Discussion In this study, we demonstrated that cord tissue MSCs altered the immune response, suppressed microglia activation, and the release of TNF, a cytokine known to contribute to white matter damage. Among these assays, IMG could be standardized as an effective potency assay to determine the efficacy of MSC for the use in treatment of white matter disease.

Development of a New Cell-Based AP-1 Gene Reporter Potency Assay for Anti-Anthrax Toxin Therapeutics.

Anthrax toxin is a critical virulence factor of Bacillus anthracis. The toxin comprises protective antigen (PA) and two enzymatic moieties, edema factor (EF) and lethal factor (LF), forming bipartite lethal toxin (LT) and edema toxin (ET). PA binds cellular surface receptors and is required for intracellular translocation of the enzymatic moieties. For this reason, anti-PA antibodies have been developed as therapeutics for prophylaxis and treatment of human anthrax infection. Assays described publicly for the control of anti-PA antibody potency quantify inhibition of LT-mediated cell death or the ET-induced increase in c-AMP levels. These assays do not fully reflect and/or capture the pathological functions of anthrax toxin in humans. Herein, we report the development of a cell-based gene reporter potency assay for anti-PA antibodies based on the rapid LT-induced degradation of c-Jun protein, a pathogenic effect that occurs in human cells. This new assay was developed by transducing Hepa1c1c7 cells with an AP-1 reporter lentiviral construct and has been qualified for specificity, accuracy, repeatability, intermediate precision, and linearity. This assay not only serves as a bioassay for LT activity, but has applications for characterization and quality control of anti-PA therapeutic antibodies or other products that target the AP-1 signaling pathway.

Possibility of using mathematical analysis methods to evaluate the potency of antibodies to hepatitis B virus surface antigen in human immunoglobulin preparations

Scientific relevance. Anti-hepatitis B virus surface-antigen (HBsAg) immunoglobulins are used to prevent hepatitis B in adults and children after exposure and to treat mild to moderate acute viral hepatitis B. The clinical effectiveness of human immunoglobulin preparations is determined by their potency, which is assessed by the content of antibodies to hepatitis B virus surface antigen (anti-HBs antibodies). Currently, this assessment involves using immunoassay techniques, such as enzyme-linked immunosorbent assay (ELISA).Aim. This study examined several mathematical methods for analysing the experimental data obtained in ELISA-based anti-HBs antibody potency assays of human immunoglobulin preparations.Materials and methods. This study used the international standard for human anti-HBs immunoglobulin, two immunoglobulin preparations, and an ELISA test kit for the detection and quantification of anti-HBs antibodies in serum or plasma samples.Results. Using sandwich ELISA, the authors ascertained that the measured anti-HBs antibody concentration depended on the choice of calibration curve calculation method (i.e. manual analysis, parallel-line analysis using PARALINE software, linear regression, and 4-parameter logistic regression). The measured anti-HBs antibody concentrations varied by ± 19 IU/mL. According to the study results, an incorrectly selected method of data analysis can lead to an erroneous calculation of the analyte potency (concentration) in the test sample.Conclusions. The study demonstrated the need for improved mathematical methods for the evaluation of experimental data used to determine the anti-HBs antibody concentration in human immunoglobulin preparations. It is essential to switch from manual to automated calculation (for example, using a 4-parameter logistic model), taking into account the requirements for bioanalytical methods and the capabilities of the available equipment.

The CombE-IDMS Assay as an Alternate Potency Method for Adjuvanted Quadrivalent Influenza Vaccines.

The potency of all currently licensed inactivated influenza viral vaccines is assayed by the single radial immunodiffusion (SRID) method. SRID relies upon antisera and reference antigen reagents which are produced, standardized, and distributed in the mass quantities needed for vaccine manufacturers only after a significant amount of time has elapsed from the seasonal strain recommendations issued by the WHO; this time delay is exacerbated under conditions of an emerging pandemic. Previously, the limited trypsin digestion isotope dilution mass spectrometry (LTD-IDMS) method, which does not require antisera or reference antigens, demonstrated comparable quantitation of immunologically active hemagglutinin, the primary viral antigen, to SRID in stressed vaccine materials. Here, we demonstrate a streamlined improvement to the LTD-IDMS method by eliminating the need for its precipitation and washing steps, saving time and labor in the sample preparation process while paving the way for plate-based high-throughput analysis. This is accomplished using dissimilar proteases in the pretreatment (a combination of chymotrypsin and elastase) and analytical (trypsin) digestion steps so that any pretreatment digests will not cause interference while monitoring analytical tryptic digests by IDMS. The combination of enzymes (CombE)-IDMS method is tested alongside LTD-IDMS and SRID for the first time on MF59 adjuvanted seasonal cell-based quadrivalent influenza vaccines (aQIVc) under stressed conditions of heating, oxidation, lowered and elevated pH, and freeze-thaw. Overall, a correlation in the degradation trend is observed between CombE-IDMS and SRID in the four strains of the quadrivalent formulation, highlighting the method's stability indicating capability as a rapid alternate potency assay in a highly complex formulation of aQIVc.

Comparison of Cost and Potency of Human Mesenchymal Stromal Cell Conditioned Medium Derived from 2- and 3-Dimensional Cultures.

Mesenchymal stromal cell (MSC)-derived products, such as trophic factors (MTFs), have anti-inflammatory properties that make them attractive for cell-free treatment. Three-dimensional (3D) culture can enhance these properties, and large-scale expansion using a bioreactor can reduce manufacturing costs. Three lots of MTFs were obtained from umbilical cord MSCs produced by either monolayer culture (Monol MTF) or using a 3D microcarrier in a spinner flask dynamic system (Bioreactor MTF). The resulting MTFs were tested and compared using anti-inflammatory potency assays in two different systems: (1) a phytohemagglutinin-activated peripheral blood mononuclear cell (PBMNC) system and (2) a lipopolysaccharide (LPS)-activated macrophage system. Cytokine expression by macrophages was measured via RT-PCR. The production costs of hypothetical units of anti-inflammatory effects were calculated using the percentage of TNF-α inhibition by MTF exposure. Bioreactor MTFs had a higher inhibitory effect on TNF (p < 0.01) than monolayer MTFs (p < 0.05). The anti-inflammatory effect of Bioreactor MTFs on IL-1β, TNF-α, IL-8, IL-6, and MIP-1 was significantly higher than that of monolayer MTFs. The production cost of 1% inhibition of TNF-α was 11-40% higher using monolayer culture compared to bioreactor-derived MTFs. A 3D dynamic culture was, therefore, able to produce high-quality MTFs, with robust anti-inflammatory properties, more efficiently than monolayer static systems.

Variability of in vivo potency assays of whole-cell pertussis, inactivated polio, and meningococcal B vaccines

For the batch release of vaccines, potency release assays are required. Non-animal in vitro tests have numerous advantages and are preferred; however, several vaccines are still released using in vivo assays. Their major drawback is the inherent variability with its practical implications. We quantified the variability of in vivo potency release assays for whole-cell pertussis, inactivated polio and meningococcal B (MenB) vaccines which showed large CV (Coefficient of Variation) ranging from 34% to 125%. As inherent variability might potentially be attributed to the highly variable immune system between individual animals, we evaluated the antibody titres to four MenB antigens in 344 individual outbred mice. These varied strongly, with more than 100-fold differences in antibody titres in responsive mice. Furthermore, within individual mice there was generally no correlation between the strengths of the responses to the four antigens. A mouse with a very low or no response to one antigen in many cases exhibited a strong response to another antigen. The large differences between individual animals is likely a considerable contributor to the inherent variability of in vivo potency assays. Our data again support the notion that it is preferred to move away from in vivo potency assays for monitoring batch to batch consistency as part of vaccine batch release testing.

Self-assembled GLP-1/glucagon peptide nanofibrils prolong inhibition of food intake.

Oxyntomodulin (Oxm) hormone peptide has a number of beneficial effects on nutrition and metabolism including increased energy expenditure and reduced body weight gain. Despite its many advantages as a potential therapeutic agent, Oxm is subjected to rapid renal clearance and protease degradation limiting its clinical application. Previously, we have shown that subcutaneous administration of a fibrillar Oxm formulation can significantly prolong its bioactivity in vivo from a few hours to a few days. We used a protease resistant analogue of Oxm, Aib2-Oxm, to form nanfibrils depot and improve serum stability of released peptide. The nanofibrils and monomeric peptide in solution were characterized by spectroscopic, microscopic techniques, potency assay, QCM-D and in vivo studies. We show that in comparison to Oxm, Aib2-Oxm fibrils display a slower elongation rate requiring higher ionic strength solutions, and a higher propensity to dissociate. Upon subcutaneous administration of fibrillar Aib2-Oxm in rodents, a 5-fold increase in bioactivity relative to fibrillar Oxm and a significantly longer bioactivity than free Aib2-Oxm were characterized. Importantly, a decrease in food intake was observed up to 72-hour post-administration, which was not seen for free Aib2-Oxm. Our findings provides compelling evidence for the development of long-lasting peptide fibrillar formulations that yield extended plasma exposure and enhanced in vivo pharmacological response.

Functional design of experiment for potency assay optimization and in-silico simulation

For biotherapeutic analytics, robust and reliable potency assays are required. Design of experiment (DoE) approaches are used to investigate the impact of multiple assay parameters. Currently, specific assay features (e.g., half effective concentration) are modelled independently from each other. A joint interpretation of several assay features is thus difficult. In our functional DoE approach, we use the functional relationship of the assay features to describe the sigmoidal dose-response curve. With the composed functional form, the direct impact of assay parameters on the dose-response curve shape was modelled. Moreover, a multivariate desirability was defined and used for assay optimization. We believe that functional modelling contributes to understanding the joint influence of assay parameters and helps to design robust biotherapeutic analytics.

Bringing function to the forefront of cell therapy: how do we demonstrate potency?

Unlike conventional pharmaceuticals, biologics and Advanced Therapy Medicinal Products (ATMPs) are required to meet a standard of "potency" as part of the final release criteria at completion of manufacture. During early phase clinical trials, most regulatory agencies have been willing to accept very immature potency assays with an expectation that these will be improved, qualified and validated during the clinical development of the drug to Marketing Authorisation Application (MAA) or Biologics License Application (BLA) submission.This model of continuous development of potency assay in parallel with drug development has already led to at least two notable problem cases; namely Iovance and Mesoblast. Both companies completed successful phase III clinical trials but, in both cases, the initial BLA was rejected on the basis that their potency assay for drug product release was inadequate. Fortunately these issues appear to have been overcome in March of this year, with Mesoblast receiving acceptance of their BLA for Remestemcel and Iovance obtaining a rolling BLA approval for Lifileucel.

Development and Characterization of an In Vitro Cell-Based Assay to Predict Potency of mRNA-LNP-Based Vaccines.

Messenger RNA (mRNA) vaccines have emerged as a flexible platform for vaccine development. The evolution of lipid nanoparticles as effective delivery vehicles for modified mRNA encoding vaccine antigens was demonstrated by the response to the COVID-19 pandemic. The ability to rapidly develop effective SARS-CoV-2 vaccines from the spike protein genome, and to then manufacture multibillions of doses per year was an extraordinary achievement and a vaccine milestone. Further development and application of this platform for additional pathogens is clearly of interest. This comes with the associated need for new analytical tools that can accurately predict the performance of these mRNA vaccine candidates and tie them to an immune response expected in humans. Described here is the development and characterization of an imaging based in vitro assay able to quantitate transgene protein expression efficiency, with utility to measure lipid nanoparticles (LNP)-encapsulated mRNA vaccine potency, efficacy, and stability. Multiple biologically relevant adherent cell lines were screened to identify a suitable cell substrate capable of providing a wide dose-response curve and dynamic range. Biologically relevant assay attributes were examined and optimized, including cell monolayer morphology, antigen expression kinetics, and assay sensitivity to LNP properties, such as polyethylene glycol-lipid (or PEG-lipid) composition, mRNA mass, and LNP size. Collectively, this study presents a strategy to quickly optimize and develop a robust cell-based potency assay for the development of future mRNA-based vaccines.

Characterizing On-Chip Angiogenesis Induction in a Microphysiological System as a Functional Measure of Mesenchymal Stromal Cell Bioactivity.

Mesenchymal stromal cells (MSCs) continue to be proposed for clinical investigation to treat myriad diseases given their purported potential to stimulate endogenous regenerative processes, such as angiogenesis. However, MSC functional heterogeneity has hindered clinical success and still poses a substantial manufacturing challenge from a product quality control perspective. Here, a quantitative bioassay based on an enhanced-throughput is described, microphysiological system (MPS) to measure the specific bioactivity of MSCs to stimulate angiogenesis as a potential measure of MSC potency. Using this novel bioassay, MSCs derived from multiple donors at different passages are co-cultured with human umbilical vein endothelial cells and exhibit significant heterogeneity in angiogenic potency between donors and cell passage. Depending on donor source and cellular passage number, MSCs varied in their ability to stimulate tip cell dominant or stalk cell dominant phenotypes in angiogenic sprout morphology which correlated with expression levels of hepatocyte growth factor (HGF). These findings suggest that MSC angiogenic bioactivity may be considered as a possible potency attribute in MSC quality control strategies. Development of a reliable and functionally relevant potency assay for measuring clinically relevant potency attributes of MSCs will help to improve consistency in quality and thereby, accelerate clinical development of these cell-based products.

Rapid determination of influenza vaccine potency by an SPR-based method using subtype or lineage-specific monoclonal antibodies.

Potency testing and release of annual influenza vaccines require preparation, calibration, and distribution of reference antigens (RAs) and antisera every year, which takes an average of 8 to 12 weeks, and can be a major limiting factor in pandemic situations. Here we describe for the first time a robust Surface Plasmon Resonance (SPR)-based method that employs influenza subtype or lineage hemagglutinin (HA) specific monoclonal antibodies (mAbs) to measure the HA concentration in influenza multivalent vaccines. Implementing such an advanced test method will at the very least eliminate the rate-limiting and laborious efforts of making antisera reagents annually, and thus expedite the influenza vaccine delivery to the public by at least 6 weeks. Results demonstrate that the SPR-based method, developed using Biacore, is robust and not influenced by the type of RAs (inactivated whole virus, split, or subunit vaccine-derived materials), whether they are used as monovalent or multivalent preparations. HA concentrations obtained for monovalent drug substances (DS) or quadrivalent drug products (DP) of inactivated influenza split vaccine showed a tight correlation (the best fit value for the slope is 1.001 with R2 of 0.9815 and P-value <0.0001) with the corresponding values obtained by the current potency assay, Single Radial Immunodiffusion (SRID). Supplementary analysis of the results by the Bland-Altman plot demonstrated good agreement between the SPR and SRID methods, with no consistent bias of the SPR versus SRID method. We further demonstrate that the SPR-based method can be used to estimate HA concentrations in intermediates of the influenza vaccine manufacturing process containing varying matrices and impurity levels. Further, the results demonstrate that the method is sensitive to detecting degradation of HA caused by elevated temperature, low pH, and freezing. It is evident from this report and other published work that the advancement of analytical techniques and the early findings are encouraging for the implementation of alternate potency assays with far-reaching benefits covering both seasonal and pandemic influenza.

Assessing the stability-indicating properties of alternative potency assays for inactivated influenza vaccine

Determination of the potency of a vaccine is critical to ensuring that an appropriate dose is delivered, lot-to-lot consistency is maintained, and that the formulation is stable over the life of the vaccine. The potency of inactivated influenza vaccines is determined routinely by the Single Radial Immunodiffusion (SRID) assay. A number of alternative potency assays have been proposed and have been under evaluation in recent years. The aim of this study was to compare a surface plasmon resonance-based assay and two different enzyme linked immunoassays against the current potency assay, SRID, and against mouse immunogenicity when haemagglutinin antigen of the A(H1N1)pdm09 component of an inactivated influenza vaccine is stressed by elevated temperature, low pH and freezing. This analysis demonstrated that the alternative assays had good correspondence with SRID for samples from most stress conditions and that the immunogenicity in mice corresponded with potency in SRID for all stress samples. Subject to further analysis, the assays have been shown to have the potential to possibly replace, and at least complement, SRID.

Impact of aluminum adjuvants on the stability of pneumococcal polysaccharide-protein conjugate vaccines

Development of a vaccine drug product requires formulation optimization to ensure that the vaccine’s effectiveness is preserved upon storage throughout the shelf-life of the product. Although aluminum adjuvants have been widely used in vaccine formulations to safely and effectively potentiate an immune response, careful attention must be directed towards ensuring that the type of aluminum adjuvant does not impact the stability of the antigenic composition. PCV15 is a polysaccharide-protein conjugate vaccine comprising the pneumococcal polysaccharide (PnPs) serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F), each individually conjugated to the protein carrier CRM197. PCV15 was formulated with either amorphous aluminum hydroxyphosphate sulfate adjuvant (AAHS) or aluminum phosphate adjuvant (AP) and examined for both stability and immunogenicity. Using a collection of methods to evaluate vaccine stability, it was discovered that certain PCV15 serotypes (e.g., 6A, 19A, 19F) formulated with AAHS resulted in a reduction of immunogenicity in vivo and a reduction in recoverable dose as tested by an in vitro potency assay. The same polysaccharide-protein conjugates formulated with AP were stable regarding all measures tested. Moreover, the reduction in potency of certain serotypes correlated with chemical degradation of the polysaccharide antigen caused by the aluminum adjuvant as measured by reducing polyacrylamide gel electrophoresis (SDS-PAGE), High-Pressure Size Exclusion Chromatography coupled with UV detection (HPSEC-UV) and ELISA immunoassay. This study suggests a formulation, which includes AAHS, may negatively impact the stability of a pneumococcal polysaccharide-protein conjugate vaccine that contains phosphodiester groups. This decrease in stability would likely result in a decrease in the “active” concentration of antigen dose, and herein, it is shown that such instability directly compromised vaccine immunogenicity in an animal model. The results presented in this study help to explain critical degradation mechanisms of pneumococcal polysaccharide-protein conjugate vaccines.

Development and qualification of cell-based relative potency assay for a human respiratory syncytial virus (RSV) mRNA vaccine

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infections worldwide. A safe and effective RSV vaccine has been an elusive goal but recent advances in vaccine technology have improved the likelihood that a vaccine for the prevention of RSV could be licensed in near future. We have developed an RSV vaccine V171 consisting of four lipids and messenger ribonucleic acid (mRNA) encoding an engineered form of the RSV F protein stabilized in its prefusion conformation. The lipids form lipid nanoparticles (LNP) with mRNA encapsulated during process, which protects the mRNA from degradation and enables the mRNA to be delivered into mammalian cells. Once inside the cells, the mRNA then can be translated into RSV F protein and elicit both humoral and cellular immune responses. Preclinical results and Phase I clinical trial results indicate that this mRNA vaccine targeting RSV F protein is a promising RSV vaccine approach and should be further evaluated in clinical trials. We have developed a cell-based relative potency assay to support the Phase II development of this vaccine. Test articles and a reference standard are tested with serial dilutions in a 96-well plate pre-seeded with Hep G2 cells. Cells were incubated for 16–18 h after transfection and then permeabilized and stained with a human monoclonal antibody specific to RSV F protein, followed by a fluorophore-conjugated secondary antibody. The plate is then analyzed for percentage of transfected cells and relative potency of the test article is calculated by comparing its EC50 to that of a reference standard. This assay takes advantage of the fact that due to the inherent variability in biological test systems an absolute measure of potency is more variable than a measure of activity relative to a standard. Targeting testing relative potency range 25–250 %, our assay showed an R2 close to 1 for linearity, relative bias of 1.05–5.41 %, and intermediate precision of 11.0 %. The assay has been used for testing of process development samples, formulation development samples, as well as drug product intermediate (DPI) and drug product (DP) in support of Phase II development of our RSV mRNA vaccine.

Potency assays and biomarkers for cell-based advanced therapy medicinal products.

Advanced Therapy Medicinal Products (ATMPs) based on somatic cells expanded in vitro, with or without genetic modification, is a rapidly growing area of drug development, even more so following the marketing approval of several such products. ATMPs are produced according to Good Manufacturing Practice (GMP) in authorized laboratories. Potency assays are a fundamental aspect of the quality control of the end cell products and ideally could become useful biomarkers of efficacy in vivo. Here we summarize the state of the art with regard to potency assays used for the assessment of the quality of the major ATMPs used clinic settings. We also review the data available on biomarkers that may substitute more complex functional potency tests and predict the efficacy in vivo of these cell-based drugs.

Uncovering Manufacturing Challenges Behind Cell and Gene Therapy

Uncovering Manufacturing Challenges Behind Cell and Gene Therapy

Novel multitarget small molecule inhibitor LCI133 and synthetic lethality in endometrial carcinoma via CDK9/PI3K axis.

e15118 Background: Despite advances in molecular characterization of endometrial carcinoma (EC), options for effective targeted therapy against EC remain limited. PI3K/PTEN/AKT/mTOR signaling is the most frequently mutated pathway in EC, but targeted inhibition often results in resistance via compensatory signaling. In response, the Molecular Targeted Therapeutics Laboratory has designed multidimensional small molecules in silico to (1) target compensatory molecular signaling pathways to induce synthetic lethality, (2) circumvent combination therapy-induced toxicity, complicated pharmacokinetics, and undesirable drug-drug interactions by placing each inhibitory domain in a single target inhibitor, and (3) prevent acquired resistance by inhibiting kinome adaptation. The objective of this study is to assess the synthetic lethality response generated in EC by combinations of PI3K, BRD4, CDK4/6, and CDK9 inhibition through multitargeted small molecule inhibitors LCI132, LCI133, and LCI136. Methods: Following primary screening in a cell-free assays for potency against target proteins, LCI132, LCI133, and LCI136 were screened against five EC cell lines for 48 hours; and cell viability assays were performed. Two EC cell lines (AN3 CA and RL95-2) were chosen for subsequent mechanistic studies and were treated with multitarget or single agent inhibitor for 24 hours; and cells were assessed by flow cytometry for cytostatic or cytotoxic response, and lysates were prepared accordingly for Western blot (WB) analysis. EC cells were also treated as indicated with multitarget or single target inhibitors for time-course assessment of MYC, HEXIM1, and MCL1 relative gene expression by qRT-PCR. Results: EC cell lines showed differential sensitivity to multitarget inhibitors with LCI133 proving most broadly potent, particularly against AN3 CA (IC50 248 nM) and RL95-2 (105 nM). LCI133 induced significant G2 phase cell cycle arrest in AN3 CA ( pFDR&lt; 0.05) and the apoptotic response in AN3 CA ( pFDR&lt; 0.05)and RL95-2 ( pFDR&lt; 0.05). qRT-PCR revealed all LCI compounds decreased AN3 CA HEXIM1 and MCL1 gene expression in a time-dependent manner, with LCI133 inhibiting expression up to 24 hours post-treatment. In AN3 CA, LCI133 dose-dependently decreased phosphorylation of RNA polymerase II (S2) and Akt (S473) and decreased total MCL-1 protein levels as assessed by WB, suggesting on-target simultaneous inhibition. Further studies revealed that LCI133 induces apoptosis in a caspase-dependent manner, resulting in increased levels of cleaved PARP protein. Conclusions: Multitarget PI3K/CDK4/6/9 inhibitors LCI133 and LCI136 are nanomolar potent in AN3 CA and RL95-2 EC cell lines. LCI133 ablated expression of antiapoptotic protein MCL-1 and induced caspase-dependent apoptosis in AN3 CA, implicating PI3K and CDK9 inhibition as a synthetic lethality combination in PTEN/PI3K co-mutated EC.

Potency Assays for Mesenchymal Stromal Cell Secretome-Based Products for Tissue Regeneration.

Adult stem cells maintaining tissue homeostasis and regeneration are tightly regulated by their specific microenvironments or stem cell niches. The dysfunction of niche components may alter the activity of stem cells and ultimately lead to intractable chronic or acute disorders. To overcome this dysfunction, niche-targeting regenerative medicine treatments such as gene, cell, and tissue therapy are actively investigated. Here, multipotent mesenchymal stromal cells (MSCs), and particularly their secretomes, are of high interest due to their potency to recover and reactivate damaged or lost stem cell niches. However, a workflow for the development of MSC secretome-based products is not fully covered by regulatory authorities, and and this issue significantly complicates their clinical translation and has possibly been expressed in a huge number of failed clinical trials. One of the most critical issues in this regard relates to the development of potency assays. In this review, guidelines for biologicals and cell therapies are considered to be applied for the development of potency assays for the MSC secretome-based products that aim for tissue regeneration. Specific attention is paid to their possible effects on stem cell niches and to a spermatogonial stem cell niche in particular.