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Aims: Polyethylene glycol (PEG) is used in many applications including drug development. Due to exposure to environmental products, there is a high prevalence of preexisting anti-PEG antibodies in the global human population. The presence of anti-PEG antibodies is a concern for potentially reducing the efficacy of therapeutics after administration and represents a risk of safety events after exposure to PEGylated drug products. We developed and validated a creative and sensitive method for the detection of anti-PEG antibodies in human serum to support clinical programs for PEGylated drugs., Methods: In this method, biotin-PEG streptavidin beads were used to extract anti-PEG antibodies from human serum for analysis in an anti-PEG ELISA assay. The same serum sample was analyzed in an anti-drug antibody assay., Results: The anti-PEG antibody assay was validated with a screening cut point of 1.41 normalized signal, confirmatory cut point of 32.2% inhibition, sensitivity of 7.81 ng/mL and sufficient reproducibility, selectivity, and drug tolerance in accordance with the FDA 2019 Immunogenicity guidance., Conclusion: This method of removal of anti-PEG antibodies enables the use of a single sample to detect anti-drug and anti-PEG antibodies to support drug development programs.

Quality controls (QCs) are the primary indices of assay performance and an important tool in assay lifecycle management. Inclusion of QCs in the testing process allows for the detection of system errors and ongoing assessment of the reliability of the assay. Changes in the performance of QCs are indicative of changes in the assay behavior caused by unintended alterations to reagents or to the operating conditions. The focus of this publication is management of QC life cycle. A consensus view of the ligand binding assay (LBA) community on the best practices for factors that are critical to QC life cycle management including QC preparation, qualification, and trending is presented here.

The accuracy of reported sample results is contingent upon the quality of the assay calibration curve, and as such, calibration curves are critical components of ligand binding and other quantitative methods. Regulatory guidance and lead publications have defined many of the requirements for calibration curves which encompass design, acceptance criteria, and selection of a regression model. However, other important aspects such as preparation and editing guidelines have not been addressed by health authorities. The goal of this publication is to answer many of the commonly asked questions and to present a consensus and the shared views of members of the ligand binding assay (LBA) community on topics related to calibration curves with focus on providing recommendations for the preparation and editing of calibration curves.

Exposure to ionizing radiation disrupts metabolic pathways and causes oxidative stress, which can lead to organ damage. In this study, urinary metabolites from mice exposed to high-dose and low-dose whole-body irradiation (WBI HDR, WBI LDR) or partial-body irradiation (PBI BM2.5) were analyzed using targeted and untargeted metabolomics approaches. Significant metabolic changes particularly in oxidative stress pathways were observed on Day 2 post-radiation. By Day 30, the WBI HDR group showed persistent metabolic dysregulation, while the WBI LDR and PBI BM2.5 groups were similar to control mice. Machine learning models identified metabolites that were predictive of the type of radiation exposure with high accuracy, highlighting their potential use as biomarkers for radiation damage and oxidative stress. [ABSTRACT FROM AUTHOR]

This White Paper is focused on the technical aspects regarding quantifying pharmaceutically derived inorganic elements in biomatrices in support of GLP nonclinical and clinical studies using inductively coupled plasma (ICP) techniques. For decades ICP has been used in support of Environmental Protection Agency analyses and has more recently been applied for use in the pharmaceutical industry. Current bioanalytical method validation and sample analysis regulatory guidance applies to chromatographic platforms used for analysis of large- and small-molecule PK and TK assessments; however, it is not directly applicable to all aspects of various ICP techniques. Increasingly, quadrupole and high-resolution ICP-MS methods of analysis are being used to quantify inorganic elements contained in pharmaceutical compounds and biomatrices. Many elements occur endogenously in biomatrices, affecting quantification of blanks, standard curve samples, QC samples, and the selection of appropriate levels for the LLOQ.

Previously disclosed HIV (human immunodeficiency virus) attachment inhibitors, exemplified by BMS 806 (formally BMS378806, 1), are characterized by a substituted indole or azaindole ring linked to a benzoylpiperazine via a ketoamide or sulfonamide group. In the present report, we describe the discovery of a novel series of potent HIV entry inhibitors in which the indole or azaindole ring of previous inhibitors is replaced by a heterobiaryl group. Several of these analogues exhibited IC(50) values of less than 5 nM in a pseudotyped antiviral assay, and compound 13k was demonstrated to exhibit potency and selectivity similar to those of 1 against a panel of clinical viral isolates. Moreover, current structure-activity relationship studies of these novel biaryl gp120 inhibitors revealed that around the biaryl, a fine crevice might exist in the gp120 binding site. Taken in sum, these data reveal a hitherto unsuspected flexibility in the structure-activity relationships for these inhibitors and suggest new avenues for exploration and gp120 inhibitor design.

Background: Enfuvirtide is a potent inhibitor of systemic HIV-1 replication, but its penetration into the human central nervous system (CNS) has not been analysed. Here, we define cerebrospinal fluid (CSF) enfuvirtide pharmacokinetics and present a case illustrating the use of enfuvirtide as a probe to trace the origins of CSF HIV-1 quasispecies., Methods: Enfuvirtide CSF pharmacokinetics were assessed in 18 CSF and plasma sample pairs from four HIV-1-infected individuals. Enfuvirtide levels were measured by liquid chromatography tandem mass spectrometry using known standards and controls that included spiked CSF samples from untreated, HIV-negative individuals. A segment of the gp41 coding region encompassing the heptad repeat HR-1 and HR-2 domains was amplified from selected CSF and plasma samples and independent clones sequenced to assess resistance-associated mutations., Results: CSF and plasma samples obtained between 2 and 20 h after enfuvirtide injection showed plasma concentrations similar to previous reports (mean 3.687 SD +/- 1.828 mg/ml) with prolonged decay. By contrast, enfuvirtide in all CSF samples was below the assay detection limit of 0.025 mg/ml. In one individual, who developed a transient increase in CSF HIV-1 RNA, seven of seven CSF and plasma clones had identical enfuvirtide resistance-associated V38A mutations, suggesting that the CSF quasispecies derived from that of blood., Conclusions: Enfuvirtide penetration into CSF is negligible; thus, in clinical settings, where direct CNS drug exposure is crucial, this drug Is not likely to directly contribute to the local therapeutic effect. Enfuvirtide can be used as a tool to dissect the origin of the CNS virus.

The Third AAPS/FDA Bioanalytical Workshop, entitled "Quantitative Bioanalytical Methods Validation and Implementation: Best Practices for Chromatographic and Ligand Binding Assays" was held on May 1-3, 2006 in Arlington, VA. The format of this workshop consisted of presentations on bioanalytical topics, followed by discussion sessions where these topics could be debated, with the goal of reaching consensus, or identifying subjects where addition input or clarification was required. The discussion also addressed bioanalytical validation requirements of regulatory agencies, with the purpose of clarifying expectations for regulatory submissions. The proceedings from each day were reviewed and summarized in the evening sessions among the speakers and moderators of the day. The consensus summary was presented back to the workshop on the last day and was further debated. This communication represents the distillate of the workshop proceedings and provides the summary of consensus reached and also contains the validation topics where no consensus was reached.

Despite major advances in modern drug discovery and development, the number of new drug approvals has not kept pace with the increased cost of their development. Increasingly, innovative uses of biomarkers are employed in an attempt to speed new drugs to market. Still, widespread adoption of biomarkers is impeded by limited experience interpreting biomarker data and an unclear regulatory climate. Key differences preclude the direct application of existing validation paradigms for drug analysis to biomarker research. Following the AAPS 2003 Biomarker Workshop (J. W. Lee, R. S. Weiner, J. M. Sailstad, et al. Method validation and measurement of biomarkers in nonclinical and clinical samples in drug development. A conference report. Pharm Res 22:499-511, 2005), these and other critical issues were addressed. A practical, iterative, "fit-for-purpose" approach to biomarker method development and validation is proposed, keeping in mind the intended use of the data and the attendant regulatory requirements associated with that use. Sample analysis within this context of fit-for-purpose method development and validation are well suited for successful biomarker implementation, allowing increased use of biomarkers in drug development.

This study was performed to assess the feasibility of administering 1843U89, a potent, noncompetitive inhibitor of thymidylate synthase that does not require polyglutamation for activity, as a 2-min i.v. infusion daily for 5 days every 3 weeks, to determine whether folic acid supplementation ameliorates the toxic effects of 1843U89 and permits further dose escalation, and to recommend doses of 1843U89 administered without and with folic acid for further clinical evaluations. The study also sought to characterize the pharmacokinetic behavior of 1843U89 and to seek preliminary evidence of anticancer activity. Patients with advanced solid malignancies were treated with escalating doses of 1843U89 as a 2-min i.v. infusion daily for 5 days every 3 weeks. Initially, patients were treated in the absence of high-dose folic acid until dose-limiting toxicity was consistently noted. Next, patients were treated with escalating doses of 1843U89 preceded by 1000 mg of folic acid administered p.o. 30 min before each of the 5 daily doses of 1843U89. Patients (32) received 101 total courses of 1843U89 at doses ranging from 1 to 6 mg/m(2)/day with and without folic acid. At the 2 mg/m(2)/day dose level without folic acid, 2 of 7 new patients experienced dose-limiting toxicity, principally neutropenia, mucositis, and malaise in 3 of 11 courses. 1843U89 doses were further increased with folic acid to 6 mg/m(2)/day, but repetitive treatment was not feasible at this dose level because of an unacceptable high incidence of severe neutropenia and mucositis. Other toxicities included thrombocytopenia, rash, and fever. In contrast, repetitive treatment at the 5 mg/m(2)/day dose level was feasible. The pharmacokinetics of 1843U89 were neither dose dependent nor affected by folic acid. On day 1, clearance, terminal half-life, and steady-state volume of distribution values averaged 47.1 +/- 21.7 ml/min/m(2), 7.72 +/- 4.09 h, and 16.7 +/- 8.8 liter/m(2)/h, respectively. The results of the study indicate that the administration of 1843U89 as a 2-min infusion daily for 5 days every 3 weeks without and with folic acid is feasible at 1843U89 doses as high as 2 and 5 mg/m(2)/day, respectively. Because folic acid pretreatment results in no diminution of the antitumor activity of 1843U89 in preclinical studies and ameliorates the toxic effects of 1843U89 in both preclinical models and cancer patients, the therapeutic index of 1843U89 may be enhanced by folic acid pretreatment and, therefore, the development of 1843U89 with folic acid is warranted. However, the question of whether to administer 1843U89 at a dose of 2 mg/m(2)/day with folic acid, which is associated with negligible toxicity, or at its highest feasible dose with folic acid, 5 mg/m(2)/day, should be addressed in appropriately designed trials.

Objective: To characterize the disposition of total and free serum digoxin following the administration of digoxin Fab antibody in patients with varying degrees of renal function., Design: Observational study of pharmacokinetics and pharmacodynamics., Setting: Critical care and telemetry units of two university-affiliated teaching institutions, Hartford Hospital and Henry Ford Hospital., Patients: Fourteen digoxin-intoxicated patients (baseline total digoxin > 3.2 nmol/mL) with mean (+/- SD) serum creatinine of 380.1 +/- 212.2 mumol/L who received digoxin Fab antibody therapy., Measurements: Serum was drawn every 12 to 24 hours for 80 to 327 hours after Fab administration. Total and free digoxin were assayed in serum by fluorescence polarization immunoassay or modified immunofluorometric assay., Results: Before Fab was administered, total digoxin ranged from 3.5 to 10.5 nmol/mL. After treatment with Fab, total digoxin increased rapidly to a mean (+/- SD) maximum of 51.8 +/- 22.7 nmol/mL and decreased to 7.2 +/- 4.7 nmol/mL at the last measurement. Total digoxin was eliminated in a two-phase fashion. The half-life of the initial phase of total digoxin decline was 11.6 +/- 4.1 hours, and the half-life of the second or terminal elimination phase was 118 +/- 57 hours. Free digoxin levels decreased rapidly following Fab therapy, to a mean nadir of 0.6 +/- 1.1 nmol/mL, but rebounded to a mean maximum free digoxin concentration of 1.7 +/- 1.3 nmol/mL in 77 +/- 46 hours. The time to maximum free digoxin rebound occurred later in patients with end-stage renal disease (n = 4) compared with other patients (127 +/- 40 hours compared with 55 +/- 28 hours)., Conclusion: Elimination of digoxin following Fab therapy is prolonged in digoxin-toxic patients with renal dysfunction. In addition, rebound of free digoxin is delayed in anephric patients. Monitoring free digoxin following the administration of Fab may be of value in selected patients to guide additional Fab dosing, confirm possible rebound toxicity, or guide the reinitiation of digoxin therapy.

The analysis of toxic and essential elements in human matrices is used in clinical diagnostics and for biomonitoring of different populations to study related health outcomes. This work aimed to develop fast and reliable methods for the analysis of a broad range of elements in liquid human matrices, such as whole blood, serum, and urine, with a similar setup for the three matrices and different analysis needs. An easy and fast‐forward dilute‐and‐shoot method for 33 elements (i.e., Ag, Al, As, B, Ba, Be, Bi, Cd, Ce, Co, Cr, Cu, Hg, I, Li, Mn, Mo, Ni, Pb, Pd, Pt, Sb, Se, Sn, Sr, Te, Th, Tl, U, V, W, Zn, and Zr) was developed. 200 µL of either sample material was diluted with an alkaline reagent to a volume of 4 mL in total. Sample dilution and preparation of matrix‐matched calibration standards were performed in 48‐well plates by an automated liquid handler. Diluted samples were analyzed by inductively coupled plasma mass spectrometry on a Perkin Elmer NexIon 300D ICP‐MS instrument equipped with an ESI‐FAST SC2DX autosampler in kinetic energy discrimination mode with helium as cell gas at either 4.8 mL or 5.7 mL and 1600 W RF generator power. The method validation results showed good accuracy for fresh human samples from an external quality assessment scheme with measured concentrations within the assigned concentration ranges. Good precision and reproducibility for most elements were demonstrated with variation coefficients below or far below 8% and 15% for whole blood, 8% and 10% for serum, and 10% and 10% for urine, respectively. The developed reagent and instrumental setup were applicable to all three matrices. This minimizes the risk of human errors when switching between analyses of the different sample matrices and allows a rapid and easy analysis of whole blood, serum, and urine within one day if needed. The method demonstrated robustness over time, withstanding minor changes in the preparation of working solutions and samples, instrumental analysis, and setup. Analysis of human real samples showed the method's applicability for 33 toxic and essential elements in whole blood, serum, and urine and at concentrations relevant to clinical diagnostics as well as biomonitoring. [ABSTRACT FROM AUTHOR]

Therapeutic nucleic acids are an emerging class of therapy for treating various diseases through immunomodulation, protein replacement, gene editing, and genetic engineering. However, they need a vector to effectively and safely reach the target cells. Most gene and cell therapies rely on ex vivo gene delivery, which is laborious, time-consuming, and costly; therefore, devising a systematic vector for effective and safe in vivo delivery of therapeutic nucleic acids is required to target the cells of interest in an efficient manner. Synthetic nanoparticle vector poly beta amino ester (PBAE), a class of degradable polymer, is a promising candidate for in vivo gene delivery. PBAE is considered the most potent in vivo vector due to its excellent transfection performance and biodegradability. PBAE nanoparticles showed tunable charge density, diverse structural characteristics, excellent encapsulation capacity, high stability, stimuli-responsive release, site-specific delivery, potent binding to nucleic acids, flexible binding ability to various conjugates, and effective endosomal escape. These unique properties of PBAE are an essential contribution to in vivo gene delivery. The current review discusses each of the components used for PBAE synthesis and the impact of various environmental and physicochemical factors of the body on PBAE nanocarrier., (© 2022 Wiley Periodicals LLC.)

Background: Biomarkers are an integral component in the drug development paradigm. According to the US Food and Drug Administration (FDA), a biomarker is "a defined characteristic that is measured as an indicator of normal biological processes, pathogenic processes, or biological responses to an exposure or intervention, including therapeutic intervention" (FDA-NIH Biomarker Working Group. BEST (Biomarkers, EndpointS, and other Tools) Resource [Internet]. Silver Spring (MD): Food and Drug Administration (US); 2016-. Glossary. 2016 [Updated 2021 Nov 29, cited 2024 Apr 14]. Available from: https://www.ncbi.nlm.nih.gov/books/NBK338448/ Co-published by National Institutes of Health (US), Bethesda (MD)). The European Medicines Agency (EMA) defines a biomarker as "an objective and quantifiable measure of a physiological process, pathological process or response to a treatment (excluding measurements of how an individual feels or functions" European Medicines Agency (EMA). Biomaker. 2020a. Available from: https://www.ema.europa.eu/en/glossary-terms/biomarker#:~:text=Biomarker-,Biomarker,an%20individual%20feels%20or%20functions . Several clinical biomarkers are well-documented and have been used routinely for decades in health care settings and have long been accepted as valid endpoints for drug approval (for example, blood pressure measurement as a biomarker for cardiovascular health) (European Medicines Agency (EMA). Assessment report, TAGRISSO. 2016. Available from: https://www.ema.europa.eu/en/documents/assessment-report/tagrisso-epar-public-assessment-report_en.pdf . Accessed 15 Apr 2024). Recently, novel biomarkers have been identified and validated to accelerate developing innovative therapies indicated for serious human diseases, for example targeted/immune therapies of cancer (Chen in Med Drug Discov 21:100174, 2024). As indicators of the efficacy of new pharmacological treatments or therapeutic interventions, biomarkers can improve clinical trial efficacy and reduce uncertainty in regulatory decision making (Bakker et al. in Clin Pharmacol Ther 112:69-80, 2022; Califf in Exp Biol Med 243:213-221, 2018; Parker et al. in Cancer Med 10:1955-1963, 2021)., Methodology: This article describes case studies of recent drug approvals that successfully leveraged validated and non-validated biomarkers (i.e., tofersen for the neurodegenerative disease amyotrophic lateral sclerosis (ALS) in adults; and osimertinib for treatment of patients with metastatic epidermal growth factor receptor (EGFR) T790M mutation-positive non-small cell lung cancer (NSCLC))., Conclusions: Best practices for biomarker selection and strategies for health authority biomarker qualification programs are presented along with an overview of current limitations and challenges to optimizing biomarker applications along the drug development continuum from regulatory, translational, and operational perspectives., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Ethical Approval: The authors (HH, SS, CL, AQ) have no financial or material disclosures to make nor conflicts of interest to declare. This submitted work is an original manuscript and has not been submitted nor published elsewhere in any form or language, partially or in full., (© 2025. The Author(s), under exclusive licence to The Drug Information Association, Inc.)

Torin1, a selective kinase inhibitor targeting the mammalian target of rapamycin (mTOR), remains widely used in autophagy research due to its potent autophagy-inducing abilities, regardless of its unspecific properties. Recognizing the impact of mTOR inhibition on metabolism, our objective was to develop a reliable and thorough untargeted metabolomics workflow to study torin1-induced metabolic changes in mouse embryonic fibroblast (MEF) cells. Crucially, our quality assurance and quality control (QA/QC) protocols were designed to increase confidence in the reported findings by reducing the likelihood of false positives, including a validation experiment replicating all experimental steps from sample preparation to data analysis. This study investigated the metabolic fingerprint of torin1 exposure by using liquid chromatography—high resolution mass spectrometry (LC-HRMS)-based untargeted metabolomics platforms. Our workflow identified 67 altered metabolites after torin1 exposure, combining univariate and multivariate statistics and the implementation of a validation experiment. In particular, intracellular ceramides, diglycerides, phosphatidylcholines, phosphatidylethanolamines, glutathione, and 5′-methylthioadenosine were downregulated. Lyso-phosphatidylcholines, lyso-phosphatidylethanolamines, glycerophosphocholine, triglycerides, inosine, and hypoxanthine were upregulated. Further biochemical pathway analyses provided deeper insights into the reported changes. Ultimately, our study provides a valuable workflow that can be implemented for future investigations into the effects of other compounds, including more specific autophagy modulators. [ABSTRACT FROM AUTHOR]

Introduction: Fusion of the fragment crystallizable (Fc) to protein therapeutics is commonly used to extend the circulation time by enhancing neonatal Fcreceptor (FcRn)-mediated endosomal recycling and slowing renal clearance. This study applied kinetic modeling to gain insights into the cellular processing contributing to the observed pharmacokinetic (PK) differences between the novel recombinant ADAMTS13 fragment (MDTCS) and its Fcfusion protein (MDTCS-Fc). Methods: For MDTCS and MDTCS-Fc, their plasma PK profiles were obtained at two dose levels following intravenous administration of the respective proteins to mice. The plasma PK profiles of MDTCS were fitted to a kinetic model with three unknown protein-dependent parameters representing the fraction recycled (FR) and the rate constants for endocytosis (kup, for the uptake into the endosomes) and for the transfer from the plasma to the interstitial fluid (kpi). For MDTCS-Fc, the model was modified to include an additional parameter for binding to FcRn. Parameter optimization was done using the Cluster Gauss-Newton Method (CGNM), an algorithm that identifies multiple sets of approximate solutions ("accepted" parameter sets) to nonlinear least-squares problems. Results: As expected, the kinetic modeling results yielded the FR of MDTCS-Fc to be 2.8-fold greater than that of MDTCS (0.8497 and 0.3061, respectively). In addition, MDTCS-Fc was predicted to undergo endocytosis (the uptake into the endosomes) at a slower rate than MDTCS. Sensitivity analyses identified the association rate constant (kon) between MDTCS-Fc and FcRn as a potentially important factor influencing the plasma half-life in vivo. Discussion: Our analyses suggested that Fc fusion to MDTCS leads to changes in not only the FR but also the uptake into the endosomes, impacting the systemic plasma PK profiles. These findings may be used to develop recombinant protein therapeutics with extended circulation time. [ABSTRACT FROM AUTHOR]

The use of non-coding RNAs (ncRNAs) as drug targets is being researched due to their discovery and their role in disease. Targeting ncRNAs, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), is an attractive approach for treating various diseases, such as cardiovascular disease and cancer. This seminar discusses the current status of ncRNAs as therapeutic targets in different pathological conditions. Regarding miRNA-based drugs, this approach has made significant progress in preclinical and clinical testing for cardiovascular diseases, where the limitations of conventional pharmacotherapy are evident. The challenges of miRNA-based drugs, including specificity, delivery, and tolerability, will be discussed. New approaches to improve their success will be explored. Furthermore, it extensively discusses the potential development of targeted therapies for cardiovascular disease. Finally, this document reports on the recent advances in identifying and characterizing microRNAs, manipulating them, and translating them into clinical applications. It also addresses the challenges and perspectives towards clinical application. [ABSTRACT FROM AUTHOR]

Simple Summary: Oral ondansetron is commonly prescribed to treat nausea and vomiting in dogs, but no studies have evaluated how well it is absorbed in clinical patients after oral administration. The objective of this study was to evaluate how well oral ondansetron is absorbed in a population of client-owned dogs with naturally occurring nausea. Twenty-four dogs were randomly assigned to receive one of the following doses of oral ondansetron, which are all within the currently recommended dose range: 0.5 mg/kg q8h, 0.5 mg/kg q12h, 1 mg/kg q8h, and 1 mg/kg q12h. Blood samples were collected for ondansetron measurement at various time points after administration of the first dose of ondansetron, and nausea scores were recorded. Ondansetron blood concentrations averaged over an 8 h time period were not significantly different between dose groups. The mean nausea scores at baseline were similar among all groups and decreased over time. Blood ondansetron concentrations were below the limit of detection in 44% (32/72) of all samples collected and were not detected at any timepoint in 25% (6/24) of the dogs. These results raise the concern that orally administered ondansetron at the current recommended dosages may not be absorbed into the bloodstream effectively. The purpose of this study was to evaluate plasma ondansetron (OND) concentrations in a population of dogs with naturally occurring nausea after oral OND administration. Twenty-four dogs were randomly assigned to receive one of the following doses of oral OND: 0.5 mg/kg q8h, 0.5 mg/kg q12h, 1 mg/kg q8h, or 1 mg/kg q12h. Blood samples for plasma OND measurements were collected at baseline and 2, 4, and 8 h after administration of the first dose of OND. OND concentrations averaged over an 8 h time period were not significantly different between dose groups (0.5 mg/kg group: median 8.5 ng/mL [range 1–96.8 ng/mL], 1 mg/kg group: median 7.4 ng/mL [range 1–278.7 ng/mL]). The mean maximum concentrations in the 0.5 mg/kg and 1 mg/kg groups were 35.8 ± 49.0 ng/mL and 63.3 ± 121.1 ng/mL, respectively. OND concentrations were below the lower limit of quantification (LLOQ) in 50% (18/36) of samples in the 0.5 mg/kg groups and 39% (14/36) of samples in the 1 mg/kg groups. Six dogs (6/24, 25%) did not have OND detected at any time. The mean nausea scores at baseline were similar amongst all groups and decreased over time. The bioavailability of oral OND appears to be poor. Despite low plasma OND concentrations, nausea scores improved over time. [ABSTRACT FROM AUTHOR]

This document is a part of the Pharmacotherapy Self-Assessment Program (PSAP) and provides information on the acute management of prescription and nonprescription drug overdoses. It emphasizes the importance of supportive care, exposure history, and understanding toxidromes in guiding treatment decisions. The article discusses the treatment paradigm in toxicology, including decontamination methods, enhanced elimination techniques, and focused antidotes. It recommends consulting toxicologists or poison centers for guidance in managing drug overdoses and highlights the value of involving poison centers and toxicologists in patient care. The document also provides information on the management and treatment of specific drug overdoses, such as Type 1 antiarrhythmics, diphenhydramine, bupropion, digoxin, and various toxic substances. It covers symptoms, treatment options, and the importance of early toxicologist involvement. Additionally, the document presents clinical cases to guide healthcare professionals in making appropriate treatment decisions. [Extracted from the article]

Delayed hypersensitivity reactions (DHRs) have been reported in association with COVID-19 vaccines, particularly those that are mRNA-based. Classic DHRs result in induration, erythema, tenderness, and urticaria. However, soft tissue mass is an uncommon complication of a COVID-19 vaccination-associated DHR and is rarely reported in the literature. We present a case of a 49-year-old male who recognized a mildly painful, firm soft tissue mass within the biceps mimicking neoplasm six months after receiving the booster dose of the Moderna vaccine. Non-operative conservative treatment modalities, including heating pads, ice packs, acetaminophen, and ibuprofen, failed to improve the patient's mass. The mass, which proved histologically to be an inflammatory pseudo-tumor, did not recur after complete excision. While there have been many reported cases of DHRs following COVID-19 vaccinations, we present this case to raise awareness of the development of pseudo-tumors as a possible, yet rare, clinical manifestation of DHRs following vaccination. [ABSTRACT FROM AUTHOR]

Charged aerosol detection, increasingly recognized for quantifying pharmaceutical compounds with weak ultraviolet absorption, is a universal detection technique for high‐performance liquid chromatography (HPLC). Charged aerosol detection shows a non‐linear response with increasing analyte concentration over a wide range, limiting its versatility in various analytical applications. In this work, a co‐optimization strategy for power function value (PFV) and power laws was proposed and applied to broaden the linear range of the standard curve of saccharides in Qishen Yiqi dripping pills using the HPLC‐charged aerosol detection (HPLC‐CAD) method. Power function values for all analytes were optimized based on empirical models. Subsequently, the optimum power laws were investigated based on a preferred PFV. Additionally, various regression equations were evaluated to ensure the accuracy and precision of the results. With the optimized PFV and power law, the ordinary least squares model demonstrated a satisfactory fit. The optimal PFVs and power laws expanded the standard curve's linear range by 2.7 times compared to default settings, reducing model uncertainty. This paper presents a vital method for developing a multi‐component quantitative HPLC‐CAD approach without external data transformation outside the provided software, especially suitable for analytical applications of traditional Chinese medicine with significant quality differences. [ABSTRACT FROM AUTHOR]

Toxicology testing is performed in clinical settings, forensic settings, and for controlling doping. Drug screening is a toxicology test to determine if drugs are present in biological samples. The most common specimen type for drug testing is urine, as drugs and/or their metabolites are often more concentrated in the urine, extending the detection window of drugs. The dilute-and-shoot method is a simple procedure used in toxicology testing, where a sample is diluted before being directly injected into the liquid chromatography-mass spectrometry (LC-MS) system. This method is easy, quick, and cost-saving, and can be used for protein-poor liquid specimens such as urine. Thus, it is reasonable and attractive for busy toxicology laboratories to combine the dilute-and-shoot method with high-resolution hyphenated-MS for urine drug screening. This method has several disadvantages, including a suboptimal detection capability for certain analytes, as well as interference from co-eluting matrix components called matrix effects, in which co-eluting matrix molecules alter the ionization efficiency of the analyte molecules at the ionization source in LC-MS, altering (mostly reducing) the analyte detection capability. The matrix effect testing is essential for the validation of LC-MS-based assays. A reasonable approach to addressing these undesirable effects would be to minimize these components. The most straightforward approach is to reduce the amounts of matrix components by using a higher dilution of the specimen and a lower volume for specimen injection. Optimization of the chromatographic separation is another reasonable approach for reducing co-eluting matrix components with the analyte. [ABSTRACT FROM AUTHOR]

A facile and sensitive electrochemical sensor for determining imatinib (IMA) was constructed by modifying a glassy carbon electrode (GCE) with a nanocarbon material, acetylene black (AB). The electrochemical behavior of IMA on the prepared GCE/AB was studied using electrochemical techniques, namely, differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy. The direct determination of IMA by the GCE/AB sensor was accomplished using DPV under optimized conditions. The method verification showed that the oxidation peak current was proportional to the concentrations of IMA in the linear ranges of 0.01–0.5 and 0.5–4 μM, with correlation coefficients of 0.9856 and 0.9946, respectively. The limit of detection of the GCE/AB sensor was 0.15 nM. Moreover, the GCE/AB sensor showed good precision and accuracy. Finally, the GCE/AB sensor was successfully applied to determine IMA in human serum samples, and the recoveries were satisfactory. [ABSTRACT FROM AUTHOR]

Simple Summary: ARID1A is frequently mutated in cancers, especially in gynecologic ones, but very little attention has been paid to determine ARID1A levels in tumor specimen. Therefore, the main aim of this work was to develop an enzyme-linked immunosorbent assay (ELISA) to measure ARID1A protein levels in an accurate and precise manner. In addition, we performed western blot analysis to validate the ELISA results. As we can show the high specificity, accuracy, and precision of our assay, further research can now focus on whether ARID1A protein levels are of predictive value, as most current methods analyze its gene ARID1A only. In terms of functionality, ARID1A is a member of the SWI/SNF complex, but it is not clear how mutations in members of this chromatin remodeling complex lead to tumorigenesis. We hope to facilitate the quantitative analysis of proteins involved in tumorigenesis with this detailed ELISA protocol for the nuclear protein ARID1A. ARID1A is a subunit of the mammalian SWI/SNF complex, which is thought to regulate gene expression through restructuring chromatin structures. Its gene ARID1A is frequently mutated and ARID1A levels are lowered in several human cancers, especially gynecologic ones. A functional ARID1A loss may have prognostic or predictive value in terms of therapeutic strategies but has not been proposed based on a quantitative method. Hardly any literature is available on ARID1A levels in tumor samples. We developed an indirect enzyme-linked immunosorbent assay (ELISA) for ARID1A based on the current EMA and FDA criteria. We demonstrated that our ELISA provides the objective, accurate, and precise quantification of ARID1A concentrations in recombinant protein solutions, cell culture standards, and tissue lysates of tumors. A standard curve analysis yielded a 'goodness of fit' of R2 = 0.99. Standards measured on several plates and days achieved an inter-assay accuracy of 90.26% and an inter-assay precision with a coefficient of variation of 4.53%. When tumor lysates were prepared and measured multiple times, our method had an inter-assay precision with a coefficient of variation of 11.78%. We believe that our suggested method ensures a high reproducibility and can be used for a high sample throughput to determine the ARID1A concentration in different tumor entities. The application of our ELISA on various tumor and control tissues will allow us to explore whether quantitative ARID1A measurements in tumor samples are of predictive value. [ABSTRACT FROM AUTHOR]

Immunogenicity testing and characterization is an important part of understanding the immune response to administration of a protein therapeutic. Neutralizing antibody (NAb) assays are used to characterize a positive anti-drug antibody (ADA) response. Harmonization of reporting of NAb assay performance and results enables efficient communication and expedient review by industry and health authorities. Herein, a cross-industry group of NAb assay experts have harmonized NAb assay reporting recommendations and provided a bioanalytical report (BAR) submission editable template developed to facilitate agency filings. This document addresses key bioanalytical reporting gaps and provides a report structure for documenting clinical NAb assay performance and results. This publication focuses on the content and presentation of the NAb sample analysis report including essential elements such as the method, critical reagents and equipment, data analysis, study samples, and results. The interpretation of immunogenicity data, including the evaluation of the impact of NAb on safety, exposure, and efficacy, is out of scope of this publication., (© 2024. The Author(s).)

We previously identified a lipopeptide, EK1C4, by linking cholesterol to EK1, a pan-CoV fusion inhibitory peptide via a polyethylene glycol (PEG) linker, which showed potent pan-CoV fusion inhibitory activity. However, PEG can elicit antibodies to PEG in vivo, which will attenuate its antiviral activity. Therefore, we designed and synthesized a dePEGylated lipopeptide, EKL1C, by replacing the PEG linker in EK1C4 with a short peptide. Similar to EK1C4, EKL1C displayed potent inhibitory activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other coronaviruses. In this study, we found that EKL1C also exhibited broad-spectrum fusion inhibitory activity against human immunodeficiency virus type 1 (HIV-1) infection by interacting with the N-terminal heptad repeat 1 (HR1) of viral gp41 to block six-helix bundle (6-HB) formation. These results suggest that HR1 is a common target for the development of broad-spectrum viral fusion inhibitors and EKL1C has potential clinical application as a candidate therapeutic or preventive agent against infection by coronavirus, HIV-1, and possibly other class I enveloped viruses. [ABSTRACT FROM AUTHOR]

Survivors of acute radiation exposure are likely to experience delayed effects that manifest as injury in late-responding organs such as the heart. Non-invasive indicators of radiation-induced cardiac dysfunction are important in the prediction and diagnosis of this disease. In this study, we aimed to identify urinary metabolites indicative of radiation-induced cardiac damage by analyzing previously collected urine samples from a published study. The samples were collected from male and female wild-type (C57BL/6N) and transgenic mice constitutively expressing activated protein C (APCHi), a circulating protein with potential cardiac protective properties, who were exposed to 9.5 Gy of γ-rays. We utilized LC-MS-based metabolomics and lipidomics for the analysis of urine samples collected at 24 h, 1 week, 1 month, 3 months, and 6 months post-irradiation. Radiation caused perturbations in the TCA cycle, glycosphingolipid metabolism, fatty acid oxidation, purine catabolism, and amino acid metabolites, which were more prominent in the wild-type (WT) mice compared to the APCHi mice, suggesting a differential response between the two genotypes. After combining the genotypes and sexes, we identified a multi-analyte urinary panel at early post-irradiation time points that predicted heart dysfunction using a logistic regression model with a discovery validation study design. These studies demonstrate the utility of a molecular phenotyping approach to develop a urinary biomarker panel predictive of the delayed effects of ionizing radia-tion. It is important to note that no live mice were used or assessed in this study; instead, we focused solely on analyzing previously collected urine samples. [ABSTRACT FROM AUTHOR]

The main objective of this study was the preparation and evaluation of the primary reagents for the liquid phase human chorionic gonadotropin-radioimmunoassay (HCG-RIA) kit. Polyclonal antibody is specific and valid; monoclonal antibodies are more specific. In HCG-RIA technique, polyclonal antibodies for βHCG subunits with high binding and displacement % were used. 125I-HCG radioactive tracer was prepared with high yield 71.58 ± 0.92%, purity 99.2 ± 0.05%, and specific activity 170.42 ± 1.65 µCi/µg using chloramine T method, βHCG polyclonal antisera and HCG standards in assay buffer matrix were locally prepared. In-house HCG-RIA was developed with high sensitivity 0.5 mIU/ml, specificity (99%), precision (CV% < 6.4 and < 8.3 for intra-and inter-assay, respectively), and accuracy (recovery range 96.4–104.3%) could be used for quantitative estimation of HCG in human sera for monitoring pregnancy and diagnosis of gestational trophoblastic diseases. [ABSTRACT FROM AUTHOR]

The bioanalysis of drugs that undergo acyl glucuronidation presents an analytical challenge due to poor stability of acyl glucuronide metabolites in biological matrices. The objective of this study was to investigate the impact of back conversion of acyl glucuronide metabolites on drug concentration measurement in bioequivalence (BE) studies submitted to Abbreviated New Drug Applications (ANDAs). The prevalence of several treatments for preventing the back conversion of acyl glucuronide metabolites and the results of incurred sample reanalysis (ISR) were analyzed. In total, 322 ANDAs for 26 drugs known to generate acyl glucuronide metabolites were surveyed. Many studies have applied multiple preventive treatments during the clinical and bioanalytical phases. More than two-thirds (67.2%) of the studies utilized procedures of lowering the temperature for sample collection during clinical phase. Fewer studies have utilized procedures for lowering the pH of plasma samples (12.3%) or adding enzyme inhibitors (4.4%) in the clinical phase. A small fraction (16.9%) validated the pre-study method in the presence of the acyl glucuronide metabolites. The majority (62.2%) of the studies employed the procedure of lowering the pH during the sample extraction process in the bioanalytical phase. Among the studies that had significantly higher (p-value < 0.01 by sign test) ISR results than the corresponding original concentration values, 41 BE studies did not carry out any preventive treatments during the bioanalysis phase, suggesting that back conversion of acyl glucuronide metabolites to parent drugs may be present in these studies. The awareness of appropriate treatments of study samples for possible back-conversions of acyl glucuronide metabolites is expected to assist generic drug applicants in improving the quality of their future applications. [ABSTRACT FROM AUTHOR]

Molecular imprinted polymers (MIPs) as extraordinary compounds with unique features have presented a wide range of applications and benefits to researchers. In particular when used as a sorbent in sample preparation methods for the analysis of biological samples and complex matrices. Its application in the extraction of medicinal species has attracted much attention and a growing interest. This review focus on articles and research that deals with the application of MIPs in the analysis of components such as biomarkers, drugs, hormones, blockers and inhibitors, especially in biological matrices. The studies based on MIP applications in bioanalysis and the deployment of MIPs in high-throughput settings and optimization of extraction methods are presented. A review of more than 200 articles and research works clearly shows that the superiority of MIP techniques lies in high accuracy, reproducibility, sensitivity, speed and cost effectiveness which make them suitable for clinical usage. Furthermore, this review present MIP-based extraction techniques and MIP-biosensors which are categorized on their classes based on common properties of target components. Extraction methods, studied sample matrices, target analytes, analytical techniques and their results for each study are described. Investigations indicate satisfactory results using MIP-based bioanalysis. According to the increasing number of studies on method development over the last decade, the use of MIPs in bioanalysis is growing and will further expand the scope of MIP applications for less studied samples and analytes. [ABSTRACT FROM AUTHOR]

Vaccination is an essential public health measure that helps to reduce the burden of infectious diseases in children. Although vaccines have an excellent safety record and the association of severe allergic reactions is rare, public concerns about vaccine safety can lead to incomplete vaccination coverage in children with or without allergies. Therefore, it is important to understand the mechanisms and implications of allergic reactions to vaccines and define strategies to manage them to provide the safest care for vaccine recipients. In this review, we provide an overview on the types of allergic reactions that can occur after vaccination, including those caused by various vaccine constituents. We also discuss the mechanisms underlying these allergic reactions and the recommended diagnosis and management strategies for children with a history of suspected allergic reactions to vaccines. An improved understanding of allergic reactions to vaccines can aid in the enhancement of the safety and effectiveness of vaccination. [ABSTRACT FROM AUTHOR]

Unlike many dose-response curves used in clinical chemistry, the immunoassay curve used to quantitate measurands is often sigmoidal rather than linear. Consequently, a more complex curve fitting model is required. Various models are available, but they can introduce bias, and there can be little awareness of why this error can be introduced. These curve-fitting models include those based on the law of mass-action, empirical models such as splines or linearization models such as the log/logit function. All these models involve assumptions, which can introduce bias as the dose-response curve is 'forced' to fit or minimize the distance between the standard concentration points to the theoretical curve. The most common curve fitting model is the four or five parameter model, which uses four or five parameters to fit a sigmoidal curve to a set of standard points. Measurement of cardiac troponin is an important element in establishing a diagnosis of acute myocardial infarction. We use troponin, a cardiac biomarker, to demonstrate the potential effect of the bias that the curve fit could introduce. Troponin is used for both rule-in and rule-out decisions at different concentrations and at either end of the dose-response curve. The curve fitting process can cause lot-to-lot reagent (and calibrator) variation in immunoassay. However, laboratory staff need to be aware of this potential source of error and why it occurs. Understanding how the error occurs leads to a greater awareness of the importance of validating new reagent/calibrator assessment using patient samples with concentrations at crucial decision points. [ABSTRACT FROM AUTHOR]

In this study, for the first time an electrochemiluminescence sensor for ultra‐trace monitoring of lamotrigine is reported. The sensing probe was fabricated using silver prussian blue analogue as a new effective co‐reaction accelerator and carboxylated sulfur‐doped graphitic carbon nitride nanocomposite as a new green luminophore. Also potassium peroxydisulfate was utilized as a strong co‐reactant. The proposed ECL sensor exhibited excellent wide linear range (3.45×10−15 to 2.98×10−8 M), low detection limit (4.89×10−16 M), the relative standard deviation of 1.07 %, outstanding reproducibility and superior long‐term stability. The prominent advantages of the sensor showed that it can be used to determine LMT in clinical samples. [ABSTRACT FROM AUTHOR]

Folic acid (FA) and its other forms known as folates are small molecules vital for humans. The high demand for increasingly sensitive methods of measuring folate concentrations is due to the fact that abnormal levels of FA cause severe health disorders. Besides, folates are used as recognition molecules in targeted drug delivery. The majority of FA measuring techniques are rather expensive, laborious, sometimes not sufficiently sensitive and specific, and often employ consumables that are too costly to be single-use for routine medical diagnostics. Here, we present a procedure for transformation of a simple microscope cover glass slip without deposition of any metal or dielectric films into a cost-efficient chemosensor chip interrogated by spectral correlation interferometry for highly sensitive measurements of the concentration of small molecules, as well as a feasibility study of long-term monitoring of such molecules in a flow mode. The obtained chips were tested for folate detection. The highly specific and sensitive measurements can be performed in real-time in a wide dynamic range of 0.9–220,000 pM. The developed method and single-use consumables are promising for concentration measurements of low molecular weight substances in pharmaceuticals and in vitro diagnostics. [ABSTRACT FROM AUTHOR]

Prussian blue nanoparticles (PBNPs), also called nanozymes, are very attractive as an alternative to horseradish peroxidase in immunoassay development due to their simple and low-cost synthesis, stability and high catalytic activity. Today, there is a method for highly effective PBNP synthesis based on the reduction of an FeCl 3 /K 3 [Fe(CN) 6 ] mixture by hydrogen peroxide. However, there is a lack of research showcasing the use of these highly effective PBNPs for specific target detection in clinical settings, as well as a lack of comprehensive comparisons with conventional methods. To address this gap, we prepared diagnostic reagents based on highly effective PBNPs by modifying them using gelatin and attaching anti-C-reactive protein (CRP) monoclonal antibodies through cross-linking with glutaraldehyde. As a result, a solid-phase colorimetric immunoassay in a sandwich format (nanozyme-linked immunosorbent assay [NLISA]) using highly effective PBNPs as a label for CRP detection has been demonstrated for the first time. The assay demonstrated a detection limit of 21.8 pg/mL, along with acceptable selectivity, precision (CV < 25%) and accuracy (the recovery index was within acceptable limits (75-125%) for LLOQ /ULOQ range. The analytical performance of this method is on par with sensitive assays developed in the last 5 years. Notably, the results obtained from NLISA align with those from an immunofluorescence assay conducted by a certified clinical laboratory. Furthermore, this study underscores the technological challenges involved in constructing an analysis that necessitate further exploration., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Medical providers are increasingly confronted with clinical decision-making that involves (meth)amphetamines. And clinical laboratories need a sensitive, efficient assay for routine assessment of D- and L-isomers to determine the probable source of these potentially illicit analytes. This paper presents a validated method of D- and L-isomer detection in human oral fluid from an extract used for determination of a large oral fluid assay (63 analytes) on an older AB SCIEX 4000 instrument. Taken from the positive extract, D- and L-analytes were added. The method for extraction included addition of internal standard and a 2-step liquid-liquid extraction and dry-down step to concentrate and clean the samples. The samples were suspended in 50% MeOH in water, diluted with mobile phase, with separation and detection accomplished using LC-MS/MS to determine analyte concentration. Once samples were confirmed positive for (meth)amphetamine from the large oral fluid assay, they were further examined for the enantiomeric forms with 50 μl aliquots of the standards and samples of interest combined with 450 μl of D- and L-assay mobile phase, then analyzed using chiral column separation, and LC-MS/MS detection with standard curve spanning the range from 2.5 to 1000 ng/mL. The result is a sensitive and accurate detection of D- and L-isomers of amphetamine and methamphetamine in human oral fluid performed on an older model mass spectrometer (AB SCIEX 4000). The novelty of this assay is twofold (a) the 2-step liquid-liquid extraction and dry-down step to concentrate and clean the samples, and (b) its adoption characteristics as a reflex test from a large ODT panel without the need to invest in newer or expensive LC-MS/MS instruments. Finally, this assay also has potential to add a valuable option to high-throughput laboratories seeking a D- and L-testing alternative to urine drug testing methods. [ABSTRACT FROM AUTHOR]