Search Results (28)

Glioblastoma (GBM) remains one of the most challenging forms of cancer to treat, despite that extensive molecular profiling is now available. Indeed, intratumoral cellular heterogeneity, receptor redundancy, and adaptive resistance through compensatory signaling limit the impact of targeted therapies. Moreover, immunotherapies also underperform: checkpoint blockade and vaccine strategies did not obtain consistent benefits in a low mutational burden, poorly immunogenic tumor microenvironment (TME) dominated by immunosuppressive myeloid cells. In this article, we provide evidence that tumor-associated macrophages (TAMs), a form of CNS resident microglia and infiltrating macrophage, derived from bone marrow, adopt a spatially and transcriptionally distinct, non-binary continuum, shaped by tumor-derived signals and niche constraints, allowing glioma cells to resist to immune and pharmaceutical therapeutics. Metabolic rewiring, including hypoxia-linked glycolytic pressure, lactate signaling, and lipid-associated programs, determine immunosuppressive outputs and restrict plasticity, while epigenetic imprinting (DNA methylation, histone modifications, and chromatin regulators) stabilizes these programs and limits access to inflammatory loci. We discuss how stem cell secretome, and extracellular vesicles (EVs) and their cargo may act as tunable autocrine/paracrine inputs that may bias microglial regulatory control. Finally, we highlight major translational confounders, including EV operational definitions, blood–brain barrier (BBB) permeability and regional exposure, inconsistent dosing units, mixed myeloid compartments, and manufacturing dependent variability. Therefore, an exposure-aware framework that integrates product identity, delivery evidence, state-sensitive potency assays, and functional endpoints would be highly desirable. Full article

Background/Objectives: Selective inhibition of JAK1 remains a major challenge in cytokine-signaling therapeutics due to the high structural similarity of the JAK family. Here, we present an integrated computational framework that combines large-scale binding-site conformational analysis, ensemble docking, and protein–ligand interaction fingerprinting (PLIF) to elucidate the structural determinants of JAK1 selectivity and prioritize JAK1-biased scaffolds. Methods: A curated set of JAK1 and JAK2 catalytic-domain structures was clustered to capture binding-site diversity, and representative conformers were evaluated using >2300 annotated ligands. Docking performance was assessed via AUC, early enrichment metrics, and structural pose validation against experimentally resolved complexes. The workflow was subsequently applied to a library of ~6000 drug-like compounds to prioritize candidates with predicted JAK1 preference. Results: Across the ensemble, the most predictive features reliably separated active from inactive ligands (AUC = 0.78–0.82) and captured subtle, systematic rank shifts supporting the reported JAK1 bias. Interaction fingerprint analysis revealed a conserved hinge-binding motif required for potency, alongside a JAK1-enriched hotspot adjacent to Glu aD.55 that contributes to isoform discrimination. Applied to a library of ~6000 drug-like molecules, the workflow yielded 174 candidates predicted to exhibit preferential JAK1 recognition and reduced JAK2 engagement. Conclusions: These findings define the structural and physicochemical features underlying JAK1 selectivity and illustrate how ensemble-based modeling can guide the discovery of next-generation selective kinase inhibitors. Full article

Background: An accurate, sensitive, and robust potency assay is essential for the quality control of mRNA drug substances, which are characterized by complex manufacturing processes, intricate molecular structures, and high susceptibility to degradation. Currently, mRNA vaccine manufacturers use a variety of biological potency assays, often without systematic method development or rigorous evaluation. As a result, these assays may lack sufficient accuracy and robustness, making it difficult to reliably distinguish mRNA drug substance samples with different potency levels. Therefore, there is a need for a standardized, robust, and reliable potency assay for the evaluation of mRNA drug substance samples across a range of potencies. Methods: In this study, we developed a cell-based relative potency assay for a respiratory syncytial virus (RSV) mRNA drug substance encoding an engineered prefusion (PreF) form of the RSV type A (RSV-A) F protein, a recognized target for RSV vaccine development. The RSV mRNA drug substance was complexed with transfection reagents and introduced into cells in vitro to enable expression of the RSV-A PreF protein, which was then quantified using a double-antibody sandwich ELISA. Results: Systematic optimization showed that cell line, cell density, transfection reagent, mRNA-to-transfection reagent ratios, and transfection duration all influenced assay performance. Under optimized conditions, the assay demonstrated acceptable accuracy and precision, with relative bias values ranging from −25% to 13% across the potency range of 44~156%, measured-to-expected ratios within 0.8~1.2, and relative standard deviations of 18% and 16% for intra- and inter-assay precision, respectively. Furthermore, the optimized potency assay effectively distinguished mRNA drug substance samples with varying potency levels. Conclusions: This study provides a useful functional complement to physicochemical characterization and supports quality control and batch-to-batch consistency of RSV mRNA drug substances. In addition, the development strategy may also serve as a useful reference for the establishment of in vitro potency assays for other mRNA drug substances. Full article

Impacted mandibular third-molar surgery commonly causes early postoperative pain, swelling, and trismus. This randomized, controlled, three-arm parallel trial evaluated whether intra-alveolar corticosteroid delivery via an absorbable gelatin sponge improves postoperative recovery compared with a saline control. Fifty-five patients were assessed for eligibility; 37 healthy adults (18–35 years) undergoing standardized mandibular third-molar extraction were randomized to dexamethasone 8 mg (Decort^®), methylprednisolone 40 mg (Prednol^®), or control (saline), all applied intra-alveolarly using a gelatin sponge carrier. Doses were selected using standard systemic glucocorticoid equivalence tables as a pragmatic potency reference, acknowledging unknown intra-alveolar pharmacokinetics/bioavailability. The prespecified primary endpoint (used for sample size planning) was postoperative Day 1 VAS pain; key secondary endpoints were Day 1 analgesic consumption and Day 3 facial swelling. Pain (VAS), analgesic use, trismus, and facial swelling (tragus–pogonion, tragus–labial commissure, and angulus–canthus distances) were assessed on postoperative Days 1, 2, 3, and 7 by a blinded evaluator. Two participants in the methylprednisolone group did not attend postoperative visits. To address potential attrition bias, an Intention-to-Treat (ITT) sensitivity analysis using conservative control-median imputation was performed alongside the available-case analyses. A global False Discovery Rate (FDR) correction was also applied to control for multiplicity. In both analyses, the steroid groups showed lower Day 1 pain scores than the control group. Methylprednisolone was associated with lower Day 3 swelling values than control for the tragus–pogonion and angulus–canthus measurements. These findings should be interpreted as preliminary, given the small sample size, linear swelling measurements, and lack of blinding verification. Full article

Cell-based potency assays (CBPAs) are required for the potency testing and commercial release of botulinum neurotoxin (BoNT)-based drug products. These CBPAs must account for the toxin’s biological activities while meeting regulatory guidelines for precision and accuracy. Here, studies describe the characterization and qualification of the BoSapient CBPA and demonstrate that it is fit for use as a relative potency assay for BoNT/A-containing samples. The CBPA is operated in a 96-well plate format and relies upon the fluorescence emissions of a reporter that directly responds to BoNT/A activity. The BoSapient cell line expresses the BoNT/A-receptors SV2 and complex gangliosides, is responsive only to intact BoNT/A, and can robustly detect picomolar and sub-picomolar BoNT/A quantities, making the CBPA appropriate for quantifying BoNT/A-based drug products. The cell line was passaged 30 times without significant loss of reporter expression or BoNT/A sensitivity. Manual and semi-automated CBPA methods were developed and qualified according to regulatory guidelines and shown to have low bias (<4% from expected) and high precision (standard deviation < 8) across all test concentrations. Furthermore, the semi-automated method using the CBPA is demonstrated to improve intermediate precision by 39% compared to the manual method, while reducing operator dependency during method execution. Full article

Background: The three commercial Enterovirus 71 (EV71) inactivated vaccines which have effectively controlled the EV71 pandemic currently rely on inherent variable in vivo potency methods for batch release. To align with 3R (Replacement, Reduction, Refinement) principles and enhance quality control, this study referred to WHO guidelines and the European Pharmacopoeia to develop in vitro relative potency (IVRP) methods. Methods: Working standards tracing to phase 3 clinical vaccines were established. Manufacture-specific IVRP methods were developed and validated per ICH Q14/Q2(R2), utilizing conformational epitope-targeting neutralizing monoclonal antibodies (MAbs). One of the MAbs (CT11F9) recognition sites was clarified with Cryo-EM. Subsequently, the performance of IVRP was assessed using varied concentrations and heat-treated vaccines. The correlation between IVRP and in vivo methods was analyzed, followed by setting IVRP specifications. Results: The manufacturer-specific working standard exhibited ED50 values comparable to those of related phase 3 clinical vaccines. All IVRP methods achieved a relative bias/precision/total error ≤ 15%. The IVRP methods correlated with in vivo methods (p < 0.05, r > 0.9) can discriminate EV71 antigen concentrations (p < 0.01, r > 0.99) and indicate the stability of the vaccines. Cryo-EM was adopted to identify the epitopes recognized by CT11F9, revealing that this neutralizing antibody recognizes a conformational epitope spanning VP1-3 of the same protomer. Using 31–47 batches of commercial vaccines, IVRP specifications were proposed as 0.56–1.35, 0.58–1.40, and 0.54–1.50. Conclusions: Based on conformational epitope-targeting neutralizing MAbs, manufacturer-specific IVRP methods, which were sensitive to process variations and correlated with in vivo results, have been established. IVRP methods provide a reliable, animal-free alternative for EV71 vaccine batch release. Full article

Background: Alterations in the adrenergic system have been associated with the pathophysiology of Alzheimer’s disease (AD). A novel α_1A-adrenergic receptor (AR)-positive allosteric modulator (PAM), CCF219B, has been shown to outperform donepezil with rescue of AD cognition/memory deficits with a reduction in amyloid biomarkers and without cardiovascular side effects. Initial pharmacological analysis in transfected cell lines revealed a signal bias with increased efficacy (but not potency) of cAMP signaling and ligand selectivity for norepinephrine (NE). As most GPCR allosteric modulators change the potency of agonists, we hypothesized and now report that CCF219B induced additional aspects of its allosteric interactions with NE that may provide mechanistic insight. Methods: Using Rat-1 fibroblasts stably transfected with α_1A-AR, we determined the activation profile of pERK and p38 messengers by CCF219B in the presence of NE. Using membranes prepared from the stably transfected fibroblasts or from the brain of WT mice or the AD mouse model, hAPP(lon), equilibrium or kinetic radioligand-binding analyses were performed. Results: We identified p-ERK1/2 but not p38 as an additional signal pathway that is potentiated by CCF219B in the presence of NE. An analysis of binding studies of CCF219B in membranes derived from the brains of WT or hAPP(lon) mice revealed profiles that were time-dependent and resulted in an increase in α_1A-AR expression that was unaltered in the presence of cycloheximide or when performed at 37 °C. hAPP(lon) mice displayed a reduction in α_1A-AR-binding sites that were rescued upon prolonged incubation with CCF219B but also displayed a compensatory increase in α_1B/D-AR subtype expression. Binding kinetics reveal that CCF219B can decrease the association rate of ³H-NE but only in the presence of GTP. The association rate increased for the radiolabeled antagonist, ¹²⁵I-HEAT. There were no changes in the dissociation rate of either radiolabel. Conclusions: CCF219B affects the association but not the dissociation rate of NE and explains its ability to increase the active state of the receptor by promoting a pre-coupled conformation, consistent with increasing efficacy but not potency. Potentiation of pERK may contribute to CCF219B’s ability to confer neuroprotection and be pro-cognitive in AD. CCF219B’s ability to increase the expression of α_1A-AR provides a positive feedback loop and strengthens the hypothesis that α₁-AR subtypes may be involved in AD etiology and/or progression. Full article

Background/Objectives: Trastuzumab is an effective therapeutic intervention for treating HER2-positive breast cancers. The cost-effectiveness, global demand, and patent expiration of trastuzumab have led to the inflow of its biosimilars in the global market. With the rise of biosimilars in the biopharmaceutical market, it has become crucial to ensure that the biosimilar is at par with the original monoclonal antibody (mAb)in terms of efficacy, safety, and quality. Bioassay is one of the critical quality attributes (CQAs), hence developing a reliable and robust bioassay is essential for the evaluation of their biological activity and the harmonization of the quality of these biologics, supporting their safe and effective use in clinical practice. Methods: The present study aimed to develop a robust cell-based bioassay to assess the bioactivity of trastuzumab and its biosimilars for quality control testing. For this purpose, molecular characterization of different HER2-positive breast cancer cell lines of SKBR3, BT474, MDA-MD-453, MDA-MB-175, MCF-7, and MDA-MB-231 was performed to select a suitable cell line for the cell-based bioassay. Results: The SKBR3 cell line was found to express the HER2 receptors significantly higher in comparison to the other cell lines, and it was thereby selected for further bioassay optimization. The biological activity of trastuzumab was determined using the inhibition of proliferation (IOP) assay on the SKBR3, which was optimized based on the parameters of cell seeding density, drug dilution range, and incubation time, and it was further validated as per the compendial guidelines and found valid for the parameters of specificity, accuracy (% relative bias = 0.0067%), precision (repeatability: % GCV = 1.21%), linearity (R2 = 0.99), and range (50% to 200%). Additionally, the biological activity of different trastuzumab biosimilars was assessed using the validated IOP assay and compared to the HER2 binding assay performed by flow cytometry. The biological activity of different trastuzumab biosimilars was found to be comparable to the WHO primary reference standard of trastuzumab in terms of its relative potency using the IOP assay and binding assay by flow cytometry. Conclusions: Thus, an economic and robust cell-based bioassay method was successfully developed to assess the bioactivity of trastuzumab and its biosimilars. Full article

A systematic structure–activity and computational modeling analysis of a series of glucagon-like peptide-1 receptor (GLP-1R) agonists based upon an ultra-short GLP-1 peptide, H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Bip-Bip-NH2, was conducted. This highly potent 11-mer peptide led to a deeper understanding of the α-helical bias of strategic α-methylation within the linear parent template as well as optimization of GLP-1R agonist potency by 1000-fold. These data were correlated with previously reported co-structures of both full-length GLP-1 analogs and progenitor N-terminal GLP-1 fragment analogs related to such ultra-short GLP-1R agonist peptides. Furthermore, the development of a quantitative structure–activity relationship (QSAR) model to analyze these findings is described in this study. Full article

Background: Lichen sclerosus (LS) is a chronic, inflammatory dermatosis that affects both genital and extragenital sites. It is often difficult to treat and may lead to a variety of complications if not adequately treated. The mainstay of therapy involves topical corticosteroids, topical calcineurin inhibitors, and systemic immunomodulators. Although a variety of topical, oral, and procedural therapies are available, a review comparing relative efficacy is lacking. To this end, this systematic review aimed to summarize the literature regarding treatment modalities and their respective response rates in patients with genital LS. Methods: A literature search was conducted in accordance with PRISMA guidelines. Results: This review qualitatively summarizes information from 31 randomized controlled trials, encapsulating a total of 1507 patients with LS, the majority of which were female (n = 1374, 91%). Topical corticosteroids, the mainstay of therapy for LS, were discussed throughout the literature, and proved to be more efficient than topical calcineurin inhibitors, topical hormonal therapy, topical vitamin E oil and cold cream. However, other treatment modalities proved to be more efficient than topical corticosteroids, including CO₂ and Nd:YAG laser therapies, and the addition of polydeoxyribonucleotide intradermal injections, to steroid therapy. Finally, other modalities that proved to be efficient in the treatment of LS included silk undergarments, human fibroblast lysate cream, platelet-rich plasma, acitretin, and surgical intervention. The risk of bias was assessed using the Cochrane risk-of-bias tool for randomized trials. Limitations included the inclusion of only randomized controlled trials, moderate or high risk of bias, and heterogeneity in treatment regimens, among others. Conclusion: Although high-potency topical corticosteroids have validated efficacy in the management of LS, other treatment modalities, including steroid-sparing agents and/or procedural adjuncts, have been demonstrated to have a beneficial role in the treatment of LS. Full article

A two-dimensional “checkerboard” array employing systematic titration (e.g., serial two-fold dilutions) is a well-established in vitro method for exploring the antibacterial effects of novel drug combinations. Minimum inhibitory concentrations (MICs) on the checkerboard are isoeffective points at which the antibiotic potency is the same. Representations of checkerboard MIC curves for a β-lactam and β-lactamase inhibitor combination are used in hypothetical “thought experiments” and reveal the ways in which current practices can be improved. Because different types of response (i.e., independence vs. additivity vs. one effective agent; interaction vs. noninteraction) produce different MIC curves, data from different strains/isolates should not be pooled indiscriminately, as the composition of a pooled dataset will influence any derived pharmacokinetic/pharmacodynamic (PK/PD) index. Because the β-lactamase inhibitor threshold concentration (C_T) parameter is a function of the β-lactam partner dosing regimen, it is not possible to derive a universal PK/PD index target based on C_T. Alternative susceptibility testing methods represent different planes through the checkerboard; a fixed ratio method is less prone to bias for all β-lactam and β-lactamase inhibitor combinations. Susceptibility test MICs will often not reflect the sensitivity of the strain/isolate to the β-lactamase inhibitor, so the use of these MICs to normalize PK/PD indices is inappropriate. Full article

The human CC chemokine receptor 7 (CCR7) is activated by two natural ligands, CC chemokine ligand 19 (CCL19) and 21 (CCL21). The CCL19-CCL21-CCR7 axis has been extensively studied in vitro, but there is still debate over whether CCL21 is an overall weaker agonist or if the axis displays biased signalling. In this study, we performed a systematic analysis at the transducer level using NanoBRET-based methodologies in three commonly used cellular backgrounds to evaluate pathway and ligand preferences, as well as ligand bias and the influence of the cellular system thereon. We found that both CCL19 and CCL21 activated all cognate G proteins and some non-cognate couplings in a cell-type-dependent manner. Both ligands recruited β-arrestin1 and 2, but the potency was strongly dependent on the cellular system. Overall, CCL19 and CCL21 showed largely conserved pathway preferences, but small differences were detected. However, these differences only consolidated in a weak ligand bias. Together, these data suggest that CCL19 and CCL21 share mostly overlapping, weakly biased, transducer profiles, which can be influenced by the cellular context. Full article

That signaling bias is a n^th level of complexity in the understanding of G protein-coupled receptor (GPCR) activation is a first fact. That its exhaustive description, including the mode d’emploi of its quantitative measurement, remains a challenge is a second fact. That the use of this concept is promising for the design of drug candidates is a third fact. That the translation of signaling biases observed into in vivo specific effects is well documented is a fourth fact. However, the road to apply those aspects of receptology to a systematic description of a ligand and, a fortiori, of a drug candidate, still necessitates a huge body of studies. In the present commentary, the merits of the molecular description of receptor bias signaling are highlighted and the ligand induced-fit impact on GPCR structure, as well as on the functional repertoire of GPCRs, is discussed. An emphasis is given to the practical aspects during drug design, and, thus, the practical limitations of the current approaches, particularly in the context of as soon as the data are transferred to more integrated/living systems, might be a major limitation. Full article

Lichen sclerosus (LS) is a chronic inflammatory disease that mainly affects the anogenital area, with a higher incidence in post-menopausal women. In the long term, it can lead to loss of vulvar architecture or progress to squamous cell carcinoma. The evidence-based treatment involves high-potency topical corticosteroids in long regimens. However, second-line treatments are not well-established, including laser therapy. This current study aims to assess the level of evidence supporting this therapy. We conducted a search for primary-level studies published before April 2023 through MEDLINE/PubMed, Embase, Web of Science, Scopus, and CENTRAL, with no restrictions on the publication language or date. The methodological quality and risk of bias of the included studies were evaluated using the updated Cochrane Collaboration’s tool for assessing risk of bias (RoB-2). Six studies (177 patients) met our eligibility criteria. Laser therapy was compared to topical corticosteroid treatment in five out of six studies. No significant histological differences were found, except for an increase in collagen production in the laser group. A greater reduction in itching, pain, and dyspareunia at 1 and 3 months of treatment in the laser group, as well as in the Skindex-29 at 6 months, was reported. Patient satisfaction was significantly higher among those who received laser therapy. Tolerability was excellent. No significant differences were observed in any of the previous aspects in the study compared to the placebo. In conclusion, there is not enough evidence to recommend laser therapy as a standalone treatment. Full article

Cannabinoid Receptor 2 (CB2) is a promising target for treating inflammatory diseases. We designed derivatives of 3-carbamoyl-2-pyridone and 1,8-naphthyridin-2(1H)-one-3-carboxamide CB2-selective agonists with reduced lipophilicity. The new compounds were measured for their affinity (radioligand binding) and ability to elicit cyclic adenosine monophosphate (cAMP) signalling and β-arrestin-2 translocation with temporal resolution (BRET-based biosensors). For the 3-carbamoyl-2-pyridone derivatives, we found that modifying the previously reported compound UOSS77 (also known as S-777469) by appending a PEG2-alcohol via a 3-carbomylcyclohexyl carboxamide (UOSS75) lowered lipophilicity, and preserved binding affinity and signalling profile. The 1,8-naphthyridin-2(1H)-one-3-carboxamide UOMM18, containing a cis configuration at the 3-carboxamide cyclohexyl and with an alcohol on the 4-position of the cyclohexyl, had lower lipophilicity but similar CB2 affinity and biological activity to previously reported compounds of this class. Relative to CP55,940, the new compounds acted as partial agonists and did not exhibit signalling bias. Interestingly, while all compounds shared similar temporal trajectories for maximal efficacy, differing temporal trajectories for potency were observed. Consequently, when applied at sub-maximal concentrations, CP55,940 tended to elicit sustained (cAMP) or increasing (arrestin) responses, whereas responses to the new compounds tended to be transient (cAMP) or sustained (arrestin). In future studies, the compounds characterised here may be useful in elucidating the consequences of differential temporal signalling profiles on CB2-mediated physiological responses. Full article