Relevant Potency Research Articles

P0879 The totality of evidence for FYB202 – an EU-approved and US-licensed biosimilar to reference ustekinumab

Abstract Background Ustekinumab is a fully human IgG1/kappa monoclonal antibody to interleukin (IL)-12/23. It is authorized for the treatment of moderate to severe plaque psoriasis, active psoriatic arthritis, and active inflammatory bowel disorder. FYB202 has been approved by both EMA and FDA in September 2024 as a biosimilar to reference ustekinumab based on extensive comparative analytical characterization data and the proof of equivalence in clinical performance. Methods A full set of critical quality attributes was investigated with a comprehensive set of analytical methods. Functional comparability of FYB202 to the reference product for the main mechanism of action was assessed with IL-12 and IL-23 binding and bioassays. Equivalence in PK and efficacy as well as similarity in safety, tolerability, and immunogenicity were evaluated in clinical trials in healthy subjects and psoriasis patients. Results FYB202 was found to be analytically similar to reference ustekinumab with respect to physicochemical and biological properties, including structure, function, purity, and potency. Most relevant potency results are shown in Figure 1. A 3-arm PK study in 491 healthy volunteers demonstrated PK equivalence between FYB202 and both EU-approved and US-licensed ustekinumab and between the two reference drugs (all 90% CIs were within the predefined equivalence margin of 80 to 125% (Balser1). FYB202 was shown to be equivalent to reference ustekinumab in 392 patients with moderate-to-severe plaque psoriasis, with the mean percent improvement in PASI score from baseline to week 12 within the predefined equivalence intervals for both the EU- and US-specific analyses. The similarity in response was maintained over the whole course of the study. Transitioning from reference product to FYB202 had no clinically relevant effect on efficacy, safety, or immunogenicity (Papp2). Conclusion FYB202 has been shown to have similar physicochemical and functional properties and equivalent clinical performance was shown in clinical studies. Based on a totality of evidence approach, proof of therapeutic equivalence in the sensitive psoriasis population allows for extrapolation to other indications, with similar clinical performance of FYB202 and reference ustekinumab assumed across all approved indications. FYB202 provides a safe and effective biosimilar alternative for patients in need of ustekinumab therapy.

Activity Assessment of Factor Replacement Therapies to Guide Infusion Rate Protocols in Patients with Bleeding Disorders

Abstract Introduction The development of institutional protocols guiding administration of clotting factor replacements in patients with bleeding disorders requires an understanding of product stability over infusion duration. Inclusion of new factor products on formulary at our institution raised consideration for optimal administration routes, including whether a bolus intravenous push versus infusion over 4-12h provided acceptable factor activities. Here we assessed the activity of two clotting factor replacement therapies over a 24h period to guide clinical care protocols. Method This study was performed in the Special Coagulation Lab as a collaboration between pharmacy, hematology, and laboratory medicine. Two vials each of Novoeight, a recombinant antihemophilic factor used in patients with factor VIII deficiency, and Vonvendi, a recombinant von Willebrand Factor (VWF) replacement approved for prophylaxis in adults with Type 3 von Willebrand Disease, were reconstituted to clinically relevant potencies (13 IU/ml, Novoeight; 27 IU/ml, Vonvendi). Products were spiked separately into factor VIII-deficient plasma (Novoeight) or assay diluent (Vonvendi), given inability to obtain VWF-deficient plasma, and tested for activity at various timepoints. Novoeight samples were assessed by traditional one-stage FVIII assays, while Vonvendi samples were assessed for VWF antigen, ristocetin cofactor activity (RCA), and glycoprotein Ib binding activity (GP1bM). Results Novoeight samples maintained similar FVIII activities at all timepoints tested, with differences falling within the coefficient of variation of the assay. Specifically, the two samples had FVIII levels of 108% and 95% at baseline and levels of 98% and 91%, respectively, at 12h, which was the timepoint of greatest interest given plans to allow 12h infusion rates. For Vonvendi samples, measurement of VWF antigen remained fairly stable throughout the 24h period. However, VWF activity decreased in both RCA and GP1bM assays over time, including by 4h. RCA values were 193% and 190% at baseline, 113% and 95% at 4h, and 63% and 63% at 12h. GP1bM was 140% and 133% at baseline, 92% and 91% at 4h, and 56% and 70% at 12h. Together, these results supported implementation of protocols allowing for extended (up to 12h) infusion of Novoeight and short (3-5min) bolus infusion of Vonvendi. Conclusion Our study demonstrated maintained Novoeight activity over the study period, confirming infusion rates of 12h should be efficacious. Conversely, Vonvendi yielded significantly decreased activity levels in as little as 4h, indicating the need for faster infusion rates. Based on our results, current practices at our institution were adapted to include extended infusion for Novoeight only. Drug activity studies may not directly reflect patient outcomes, and additional studies of factor activities following product infusion into patients and clinical hemostasis correlates are necessary. Nevertheless, the clinical lab should continue to play a primary role in testing, monitoring, and developing patient care protocols for therapeutic drugs through multidisciplinary collaboration.

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.

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.

An improved reporter identifies ruxolitinib as a potent and cardioprotective CaMKII inhibitor.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) hyperactivity causes cardiac arrhythmias, a major source of morbidity and mortality worldwide. Despite proven benefits of CaMKII inhibition in numerous preclinical models of heart disease, translation of CaMKII antagonists into humans has been stymied by low potency, toxicity, and an enduring concern for adverse effects on cognition due to an established role of CaMKII in learning and memory. To address these challenges, we asked whether any clinically approved drugs, developed for other purposes, were potent CaMKII inhibitors. For this, we engineered an improved fluorescent reporter, CaMKAR (CaMKII activity reporter), which features superior sensitivity, kinetics, and tractability for high-throughput screening. Using this tool, we carried out a drug repurposing screen (4475 compounds in clinical use) in human cells expressing constitutively active CaMKII. This yielded five previously unrecognized CaMKII inhibitors with clinically relevant potency: ruxolitinib, baricitinib, silmitasertib, crenolanib, and abemaciclib. We found that ruxolitinib, an orally bioavailable and U.S. Food and Drug Administration-approved medication, inhibited CaMKII in cultured cardiomyocytes and in mice. Ruxolitinib abolished arrhythmogenesis in mouse and patient-derived models of CaMKII-driven arrhythmias. A 10-min pretreatment in vivo was sufficient to prevent catecholaminergic polymorphic ventricular tachycardia, a congenital source of pediatric cardiac arrest, and rescue atrial fibrillation, the most common clinical arrhythmia. At cardioprotective doses, ruxolitinib-treated mice did not show any adverse effects in established cognitive assays. Our results support further clinical investigation of ruxolitinib as a potential treatment for cardiac indications.

Identification of Sesamin from Sesamum indicum as a Potent Antifungal Agent Using an Integrated in Silico and Biological Screening Platform.

Due to the limited availability of antifungal drugs, their relevant side effects and considering the insurgence of drug-resistant strains, novel antifungal agents are urgently needed. To identify such agents, we have developed an integrated computational and biological screening platform. We have considered a promising drug target in antifungal drug discovery (exo-1,3-β-glucanase) and a phytochemical library composed of bioactive natural products was used. These products were computationally screened against the selected target using molecular docking and molecular dynamics techniques along with the evaluation of drug-like profile. We selected sesamin as the most promising phytochemical endowed with a potential antifungal profile and satisfactory drug-like properties. Sesamin was submitted to a preliminary biological evaluation to test its capability to inhibit the growth of several Candida species by calculating the MIC/MFC and conducting synergistic experiments with the marketed drug fluconazole. Following the screening protocol, we identified sesamin as a potential exo-1,3-β-glucanase inhibitor, with relevant potency in inhibiting the growth of Candida species in a dose-dependent manner (MIC and MFC of 16 and 32 µg/mL, respectively). Furthermore, the combination of sesamin with fluconazole highlighted relevant synergistic effects. The described screening protocol revealed the natural product sesamin as a potential novel antifungal agent, showing an interesting predicted pharmacological profile, paving the way to the development of innovative therapeutics against fungal infections. Notably, our screening protocol can be helpful in antifungal drug discovery.

From the Integrity of Potency Assays to Safe Clinical Intervention: Legal Perspectives.

Potency assays associated with the efficacy of investigational pharmaceutical products are one of the critical quality attributes that need to be carefully monitored during advanced therapy medicinal product (ATMP) development projects. Ensuring integrity of relevant potency assays for stem cell-based ATMPs is of paramount importance for safety and efficacy of clinical interventions. Yet, due to the complex and heterogeneous nature of stem cell-based ATMPs, creation of an appropriate set of potency assays is associated with a number of specific challenges ranging from intrinsic and operational to legal and regulatory ones. This chapter provides an overview of the EU regulatory landscape for advanced therapies, highlighting important aspects that need to be taken into consideration when preparing a strategic plan to meet the EU regulatory requirements.

A microphysiological system-based potency bioassay for the functional quality assessment of mesenchymal stromal cells targeting vasculogenesis

Mesenchymal stromal cells (MSCs) continue to be proposed for use in clinical trials to treat various diseases due to their therapeutic potential to pleiotropically influence endogenous regenerative processes, such as vasculogenesis. However, the functional heterogeneity of MSCs has hampered their clinical success and poses a significant manufacturing challenge with respect to MSC quality control. Here, we evaluated and qualified a quantitative bioassay based on an enhanced-throughput, microphysiological system to measure the specific paracrine bioactivity of MSCs to stimulate vasculogenesis as a measure of MSC potency. Using this novel bioassay, MSCs derived from multiple donors at different passages were co-cultured with human umbilical vein endothelial cells (HUVECs) and exhibited significant heterogeneity in vasculogenic potency between donors and cell passage. Using our microphysiological system (MPS)-based platform, we demonstrated that variations in MSC vasculogenic bioactivity were maintained when assayed across laboratories and operators. The differences in MSC vasculogenic bioactivity were also correlated with the baseline expression of several genes involved in vasculogenesis (hepatocyte growth factor (HGF), angiopoietin-1 (ANGPT)) or the production of matricellular proteins (fibronectin (FN), insulin-like growth factor-binding protein 7 (IGFBP7)). These findings emphasize the significant functional heterogeneity of MSCs in vasculogenic bioactivity and suggest that changes in baseline gene expression of vasculogenic or matricellular protein genes during manufacturing may affect this bioactivity. The development of a reliable and functionally relevant potency assay for measuring the specific vasculogenic bioactivity of manufactured MSCs will help to reliably assure their quality when used in appropriate clinical trials.

Rapid In-Process Measurement of Live Virus Vaccine Potency Using Laser Force Cytology: Paving the Way for Rapid Vaccine Development.

Vaccinations to prevent infectious diseases are given to target the body’s innate and adaptive immune systems. In most cases, the potency of a live virus vaccine (LVV) is the most critical measurement of efficacy, though in some cases the quantity of surface antigen on the virus is an equally critical quality attribute. Existing methods to measure the potency of viruses include plaque and TCID50 assays, both of which have very long lead times and cannot provide real time information on the quality of the vaccine during large-scale manufacturing. Here, we report the evaluation of LumaCyte’s Radiance Laser Force Cytology platform as a new way to measure the potency of LVVs in upstream biomanufacturing process in real time and compare this to traditional TCID50 potency. We also assess this new platform as a way to detect adventitious agents, which is a regulatory expectation for the release of commercial vaccines. In both applications, we report the ability to obtain expedited and relevant potency information with strong correlation to release potency methods. Together, our data propose the application of Laser Force Cytology as a valuable process analytical technology (PAT) for the timely measurement of critical quality attributes of LVVs.

Novel Potency Assay for MSC Secretome-Based Treatment of Idiopathic Male Infertility Employed Leydig Cells and Revealed Vascular Endothelial Growth Factor as a Promising Potency Marker.

Idiopathic male infertility is a highly prevalent diagnosis in developed countries with no specific treatment options. Although empirical medical treatment is widely used to restore male fertility, its efficacy remains limited and inconclusively proven. Therefore, the development of novel therapeutic approaches in this field is a high-priority task. Since the failure of testicular microenvironment components might be involved in the pathogenesis of idiopathic male infertility, application of mesenchymal stromal cells (MSCs) as well as the MSC secretome is worth considering. Previously, we showed that the intratesticular injection of MSCs or the MSC secretome led to the recovery of spermatogenesis at least through replenishing the testicular microenvironment and its maintenance by MSC-secreted paracrine factors. However, the clinical use of such products has been limited to single trials to date. This may be due to the lack of relevant potency tests reflecting mechanisms of action of the MSC secretome in male infertility models. Based on the presumptive MSC secretome mode of action on the testicular microenvironment, we suggest a novel approach to test the potential efficacy of the MSC secretome for idiopathic male infertility treatment. It represents a potency assay based on evaluation of testosterone production by isolated Leydig cells. We demonstrated that the MSC secretome stimulated testosterone secretion by Leydig cells in vitro. We then hypothesized that among the major factors of the MSC secretome, vascular endothelial growth factor (VEGF) could be responsible for the observed effects, which we confirmed by the revealed correlation between the extent of stimulated testosterone production and VEGF concentration in the MSC secretome. The pilot results obtained from the doxorubicin-induced male infertility murine model also indicate the important impact of VEGF in the MSC secretome’s regenerative effects. Utilizing VEGF as a surrogate factor, a novel approach to study the potency of MSC secretome-based products for idiopathic male infertility treatment is suggested. Further validation is required for its implementation into the biopharmaceutical manufacturing process.

Harmonization of Multiple SARS-CoV-2 Reference Materials Using the WHO IS (NIBSC 20/136): Results and Implications.

BackgroundThere is an urgent need for harmonization between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serology platforms and assays prior to defining appropriate correlates of protection and as well inform the development of new rapid diagnostic tests that can be used for serosurveillance as new variants of concern (VOC) emerge. We compared multiple SARS-CoV-2 serology reference materials to the WHO International Standard (WHO IS) to determine their utility as secondary standards, using an international network of laboratories with high-throughput quantitative serology assays. This enabled the comparison of quantitative results between multiple serology platforms.MethodsBetween April and December 2020, 13 well-characterized and validated SARS-CoV-2 serology reference materials were recruited from six different providers to qualify as secondary standards to the WHO IS. All the samples were tested in parallel with the National Institute for Biological Standards and Control (NIBSC) 20/136 and parallel-line assays were used to calculate the relevant potency and binding antibody units.ResultsAll the samples saw varying levels of concordance between diagnostic methods at specific antigen–antibody combinations. Seven of the 12 candidate materials had high concordance for the spike-immunoglobulin G (IgG) analyte [percent coefficient of variation (%CV) between 5 and 44%].ConclusionDespite some concordance between laboratories, qualification of secondary materials to the WHO IS using arbitrary international units or binding antibody units per milliliter (BAU/ml) does not provide any benefit to the reference materials overall, due to the lack of consistent agreeable international unit (IU) or BAU/ml conversions between laboratories. Secondary standards should be qualified to well-characterized reference materials, such as the WHO IS, using serology assays that are similar to the ones used for the original characterization of the WHO IS.

Clofoctol inhibits SARS-CoV-2 replication and reduces lung pathology in mice.

Drug repurposing has the advantage of shortening regulatory preclinical development steps. Here, we screened a library of drug compounds, already registered in one or several geographical areas, to identify those exhibiting antiviral activity against SARS-CoV-2 with relevant potency. Of the 1,942 compounds tested, 21 exhibited a substantial antiviral activity in Vero-81 cells. Among them, clofoctol, an antibacterial drug used for the treatment of bacterial respiratory tract infections, was further investigated due to its favorable safety profile and pharmacokinetic properties. Notably, the peak concentration of clofoctol that can be achieved in human lungs is more than 20 times higher than its IC50 measured against SARS-CoV-2 in human pulmonary cells. This compound inhibits SARS-CoV-2 at a post-entry step. Lastly, therapeutic treatment of human ACE2 receptor transgenic mice decreased viral load, reduced inflammatory gene expression and lowered pulmonary pathology. Altogether, these data strongly support clofoctol as a therapeutic candidate for the treatment of COVID-19 patients.

Development of functionally relevant potency assays for monovalent and multivalent vaccines delivered by evolving technologies.

A potency or potency-indicating assay is a regulatory requirement for the release of every lot of a vaccine. Potency is a critical quality attribute that is also monitored as a stability indicator of a vaccine product. In essence, a potency measurement is a test of the functional integrity of the antigen and is intended to ensure that the antigen retains immunocompetence, i.e., the ability to stimulate the desired immune response, in its final formulation. Despite its central importance, there is incomplete clarity about the definition and expectation of a potency assay. This article provides a perspective on the purpose, value, and challenges associated with potency testing for vaccines produced by new technologies. The focus is on messenger RNA vaccines in the light of experience gained with recombinant protein-based vaccines, which offer the opportunity to directly correlate in vitro antigenicity with in vivo immunogenicity. The challenges with developing immunologically relevant in vitro assays are discussed especially for multivalent vaccine products, the importance of which has been reinforced by the ongoing emergence of SARS-CoV-2 variants of concern. Immunoassay-based release of multivalent vaccine products, such as those containing multiple antigens from different variants or serotypes of the same virus, require antibodies that are selective for each antigen and do not significantly cross-react with the others. In the absence of such exclusively specific antibodies, alternative functional assays with demonstrable correlation to immunogenicity may be acceptable. Initiatives for geographically distributed vaccine technology facilities should include establishing these assay capabilities to enable rapid delivery of vaccines globally.

Abstract P247: Evaluating TRKB activity of novel preclinical brain-penetrant ROS1 and ALK inhibitors

Abstract Oncogenic ROS1 and ALK rearrangements are detected in up to 3% and up to 5% of advanced non-small cell lung cancer (NSCLC), respectively, with up to 40% of patients presenting with brain metastases at diagnosis. Although there are FDA-approved ROS1 and ALK inhibitors, there is a need for next-generation brain-penetrant inhibitors that have activity against drug-resistance mutations while sparing tropomyosin kinase B (TRKB) function in the central nervous system (CNS). TRKB inhibition in the CNS has been implicated in adverse events such as cognitive impairment, mood disorders, sleep effects or sleep disturbances, dizziness, ataxia or gait/motor disturbances, and weight gain, which have been observed with FDA-approved dual TRK/ROS1 inhibitor entrectinib and FDA-approved ALK inhibitor lorlatinib. TRKB-related CNS adverse events present a key challenge for the development of next-generation ROS1 and ALK therapies. We previously reported the discovery of NVL-520 and NVL-655, preclinical inhibitors of ROS1 and ALK, respectively, which have demonstrated activity against common drug-resistance mutations, selectivity versus TRKB, and brain penetrance in preclinical experiments. An important part of the discovery process was generating reliable and relevant TRKB potency measurements to enable structure-activity relationship (SAR) efforts. Here we describe four in vitro TRKB potency assays and explain their utility in guiding selectivity optimization for new inhibitors. These assays include (1) biochemical, (2) cell viability, (3) cellular coupled-enzyme, and (4) direct cellular phosphorylation readouts. Although biological contexts vary, each assay exhibits good cross-correlations, and we recommend multi-assay testing to improve confidence in evaluating TRKB inhibition. Using these assays, we present a multi-assay analysis of the preclinical TRKB potency and selectivity of NVL-520 and NVL-655 alongside FDA-approved or experimental inhibitors of ROS1 (crizotinib, entrectinib, repotrectinib, and taletrectinib) and ALK (crizotinib, ceritinib, alectinib, brigatinib, lorlatinib, and ensartinib). In these preclinical assays, we observed entrectinib and lorlatinib to exhibit poor ROS1-vs-TRKB and ALK G1202R-vs-TRKB selectivity, respectively, which we believe is consistent with their CNS adverse events. In the same experiment, we observed NVL-520 and NVL-655 to exhibit selectivity across all assays, including for ROS1 G2032R and ALK G1202R/L1196M mutations, respectively. In conclusion, we have devised a multi-assay approach to evaluate TRKB inhibition and guide discovery of selective ROS1 and ALK inhibitors, with the goal of minimizing adverse events and driving durable responses for patients. Citation Format: Anupong Tangpeerachaikul, Joshua C. Horan, Henry E. Pelish. Evaluating TRKB activity of novel preclinical brain-penetrant ROS1 and ALK inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P247.

Novel Nanocombinations of l-Tryptophan and l-Cysteine: Preparation, Characterization, and Their Applications for Antimicrobial and Anticancer Activities.

Tungsten oxide WO3 nanoparticles (NPs) were prepared in a form of nanosheets with homogeneous size and dimensions in one step through acid precipitation using a cation exchange column. The resulting WO3 nanosheet surface was decorated with one of the two amino acids (AAs) l-tryptophan (Trp) or l-cysteine (Cys) and evaluated for their dye removal, antimicrobial, and antitumor activities. A noticeable improvement in the biological activity of WO3 NPs was detected upon amino acid modification compared to the original WO3. The prepared WO3-Trp and WO3-Cys exhibited strong dye removal activity toward methylene blue and safranin dyes with complete dye removal (100%) after 6 h. WO3-Cys and WO3-Trp NPs revealed higher broad-spectrum antibacterial activity toward both Gram-negative and Gram-positive bacteria, with strong antifungal activity toward Candida albicans. Anticancer results of the modified WO3-Cys and WO3-Trp NPs against various kinds of cancer cells, including MCF-7, Caco-2, and HepG-2 cells, indicate that they have a potent effect in a dose-dependent manner with high selectivity to cancer cells and safety against normal cells. The expression levels of E2F2 and Bcl-2 genes were found to be suppressed after treatment with both WO3-Cys and WO3-Trp NPs more than 5-FU-treated cells. While expression level of the p53 gene in all tested cells was up-regulated after treatment 5–8 folds more as compared to untreated cells. The docking results confirmed the ability of both NPs to bind to the p53 gene with relevant potency in binding to other tested gens and participation of cysteine SH-functional group in such interaction.

Pharmacokinetics under the COVID-19 storm.

AimsThe storm‐like nature of the health crises caused by COVID‐19 has led to unconventional clinical trial practices such as the relaxation of exclusion criteria. The question remains: how can we conduct diverse trials without exposing subgroups of populations to potentially harmful drug exposure levels? The aim of this study was to build a knowledge base of the effect of intrinsic/extrinsic factors on the disposition of several repurposed COVID‐19 drugs.MethodsPhysiologically based pharmacokinetic (PBPK) models were used to study the change in the pharmacokinetics (PK) of drugs repurposed for COVID‐19 in geriatric patients, different race groups, organ impairment and drug‐drug interactions (DDIs) risks. These models were also used to predict epithelial lining fluid (ELF) exposure, which is relevant for COVID‐19 patients under elevated cytokine levels.ResultsThe simulated PK profiles suggest no dose adjustments are required based on age and race for COVID‐19 drugs, but dose adjustments may be warranted for COVID‐19 patients also exhibiting hepatic/renal impairment. PBPK model simulations suggest ELF exposure to attain a target concentration was adequate for most drugs, except for hydroxychloroquine, azithromycin, atazanavir and lopinavir/ritonavir.ConclusionWe demonstrate that systematically collated data on absorption, distribution, metabolism and excretion, human PK parameters, DDIs and organ impairment can be used to verify simulated plasma and lung tissue exposure for drugs repurposed for COVID‐19, justifying broader patient recruitment criteria. In addition, the PBPK model developed was used to study the effect of age and ethnicity on the PK of repurposed drugs, and to assess the correlation between lung exposure and relevant potency values from in vitro studies for SARS‐CoV‐2.

N-Benzyl-linoleamide, a Constituent of Lepidium meyenii (Maca), Is an Orally Bioavailable Soluble Epoxide Hydrolase Inhibitor That Alleviates Inflammatory Pain.

Lepidium meyenii (maca), a plant indigenous to the Peruvian Andes, recently has been utilized globally for claimed health or recreational benefits. The search for natural products that inhibit soluble epoxide hydrolase (sEH), with therapeutically relevant potencies and concentrations, led to the present study on bioactive amide secondary metabolites found in L. meyenii, the macamides. Based on known and suspected macamides, 19 possible macamides were synthesized and characterized. The majority of these amides displayed excellent inhibitory potency (IC50 ≈ 20-300 nM) toward the recombinant mouse, rat, and human sEH. Quantitative analysis of commercial maca products revealed that certain products contain known macamides (1-5, 8-12) at therapeutically relevant total concentrations (≥3.29 mg/g of root), while the inhibitory potency of L. meyenii extracts directly correlates with the sum of concentration/IC50 ratios of macamides present. Considering both its in vitro efficacy and high abundance in commercial products, N-benzyl-linoleamide (4) was identified as a particularly relevant macamide that can be utilized for in vivo studies. Following oral administration in the rat, compound 4 not only displayed acceptable pharmacokinetic characteristics but effectively reduced lipopolysaccharide-induced inflammatory pain. Inhibition of sEH by macamides provides a plausible biological mechanism of action to account for several beneficial effects previously observed with L. meyenii treatments.

PTG-0861: A novel HDAC6-selective inhibitor as a therapeutic strategy in acute myeloid leukaemia

Dysregulated Histone Deacetylase (HDAC) activity across multiple human pathologies have highlighted this family of epigenetic enzymes as critical druggable targets, amenable to small molecule intervention. While efficacious, current approaches using non-selective HDAC inhibitors (HDACi) have been shown to cause a range of undesirable clinical toxicities. To circumvent this, recent efforts have focused on the design of highly selective HDACi as a novel therapeutic strategy. Beyond roles in regulating transcription, the unique HDAC6 (with two catalytic domains) regulates the deacetylation of α-tubulin; promoting growth factor-controlled cell motility, cell division, and metastatic hallmarks. Recent studies have linked aberrant HDAC6 function in various hematological cancers including acute myeloid leukaemia and multiple myeloma. Herein, we report the discovery, in vitro characterization, and biological evaluation of PTG-0861 (JG-265), a novel HDAC6-selective inhibitor with strong isozyme-selectivity (∼36× ) and low nanomolar potency (IC50 = 5.92 nM) against HDAC6. This selectivity profile was rationalized via in silico docking studies and also observed in cellulo through cellular target engagement. Moreover, PTG-0861 achieved relevant potency against several blood cancer cell lines (e.g. MV4-11, MM1S), whilst showing limited cytotoxicity against non-malignant cells (e.g. NHF, HUVEC) and CD-1 mice. In examining compound stability and cellular permeability, PTG-0861 revealed a promising in vitro pharmacokinetic (PK) profile. Altogether, in this study we identified a novel and potent HDAC6-selective inhibitor (∼4× more selective than current clinical standards – citarinostat, ricolinostat), which achieves cellular target engagement, efficacy in hematological cancer cells with a promising safety profile and in vitro PK.

Demonstration of In Vitro to In Vivo Translation of a TYK2 Inhibitor That Shows Cross Species Potency Differences

Translation of modulation of drug target activity to therapeutic effect is a critical aspect for all drug discovery programs. In this work we describe the profiling of a non-receptor tyrosine-protein kinase (TYK2) inhibitor which shows a functionally relevant potency shift between human and preclinical species (e.g. murine, dog, macaque) in both biochemical and cellular assays. Comparison of the structure and sequence homology of TYK2 between human and preclinical species within the ATP binding site highlights a single amino acid (I960 → V) responsible for the potency shift. Through TYK2 kinase domain mutants and a TYK2 980I knock-in mouse model, we demonstrate that this single amino acid change drives a functionally relevant potency difference that exists between human and all evaluated preclinical species, for a series of TYK2 inhibitors which target the ATP binding site.

Identification of Novel 3-Hydroxy-pyran-4-One Derivatives as Potent HIV-1 Integrase Inhibitors Using in silico Structure-Based Combinatorial Library Design Approach.

We describe herein the development and experimental validation of a computational protocol for optimizing a series of 3-hydroxy-pyran-4-one derivatives as HIV integrase inhibitors (HIV INIs). Starting from a previously developed micromolar inhibitors of HIV integrase (HIV IN), we performed an in-depth investigation based on an in silico structure-based combinatorial library designing approach. This method allowed us to combine a combinatorial library design and side chain hopping with Quantum Polarized Ligand Docking (QPLD) studies and Molecular Dynamics (MD) simulation. The combinatorial library design allowed the identification of the best decorations for our promising scaffold. The resulting compounds were assessed by the mentioned QPLD methodology using a homology model of full-length binary HIV IN/DNA for retrieving the best performing compounds acting as HIV INIs. Along with the prediction of physico-chemical properties, we were able to select a limited number of drug-like compounds potentially displaying potent HIV IN inhibition. From this final set, based on the synthetic accessibility, we further shortlisted three representative compounds for the synthesis. The compounds were experimentally assessed in vitro for evaluating overall HIV-1 IN inhibition, HIV-1 IN strand transfer activity inhibition, HIV-1 activity inhibition and cellular toxicity. Gratifyingly, all of them showed relevant inhibitory activity in the in vitro tests along with no toxicity. Among them HPCAR-28 represents the most promising compound as potential anti-HIV agent, showing inhibitory activity against HIV IN in the low nanomolar range, comparable to that found for Raltegravir, and relevant potency in inhibiting HIV-1 replication and HIV-1 IN strand transfer activity. In summary, our results outline HPCAR-28 as a useful optimized hit for the potential treatment of HIV-1 infection by targeting HIV IN.