Why Assuring the Quality of Antimicrobials Is a Global Imperative.

Poor-quality antimicrobial medicines continue to proliferate across supply chains, threatening patients' health and safety, especially in low- and middle-income regions. This article discusses consequences and risks of antimicrobial resistance and other ways in which antimicrobial medicines can be of poor quality and recommends regulatory and policy reforms to help maintain supply chain resilience and quality of antimicrobial medicines.

How Should Regulators and Manufacturers Prevent Avoidable Deaths of Children From Contaminated Cough Syrup?

This commentary responds to a case about diethylene glycol-contaminated glycerin in cough syrup. Glycerin is a commonly used excipient in medicines to improve texture and taste. Excipients are typically pharmacologically inactive ingredients contained in prescription and over-the-counter drugs that play a critical role in the delivery, effectiveness, and stability of active drug substances. The commentary first canvasses how contaminants enter the excipient supply chains. One way is by misleading labeling or intentional adulteration by manufacturers or suppliers. Another way is by human or systemic error. This commentary then discusses quality control testing and suggests the ethical and clinical importance of increased transparency in excipient supply chains.

Which Drugs Should Be on the Essential Medicines List?

The World Health Organization (WHO) published its first Essential Medicines List (EML) in 1977, and it is updated biennially. One might reasonably think drugs on the EML are there because they are critical to effective, evidence-based patient care and intervention. One might not reasonably guess, however, that a particular drug's supply chain vulnerabilities that make it a shortage risk would contribute to a drug's listing on the EML. This commentary on a case first describes why the WHO makes the EML and suggests reasons why it might be important to consider a drug's shortage risk when revising and updating it. This commentary also suggests how distinguishing "essential" drugs from "vulnerable" drugs could bolster supply chain resiliency and mitigate drug shortages' disruptions to patient care.

What Should Dietary Supplement Oversight Look Like in the US?

Most American adults who use dietary supplements (eg, vitamins, minerals, plant and animal extracts, hormones, and amino acids) ingest them orally. The market for these products has grown rapidly and significantly over the last 25 years, but consumer protection regulations have not kept pace. In the United States, supplements' safety is regulated by the US Food and Drug Administration (FDA), but statutory limitations prevent the FDA from effectively regulating these products, exacerbate public health risk, and have generated numerous calls for reform. This article considers key features of reforms likely to strengthen the FDA's capacity to promote safety and consumer protection.

Which Features of Dietary Supplement Industry, Product Trends, and Regulation Deserve Physicians' Attention?

Patients expect that dietary supplements they purchase-and physicians expect that dietary supplements they recommend-are safe, accurately labeled, quality products. Since many dietary supplements, especially vitamins and minerals, are key parts of evidence-based interventions for patients with many conditions, illegal, fraudulent, adulterated, or improperly labeled products should be regarded as sources of clinical and ethical concern. Adverse events (AEs) can occur and, when they do, relevant data should be carefully collected and analyzed. This article considers how many physicians' and patients' confusion about dietary supplement regulation can undermine quality caregiving and responses to AEs. This article also summarizes a recent American Medical Association Council on Science and Public Health report on dietary supplement supply and marketing practices and on physicians' roles in guiding patients when dietary supplement use is clinically indicated.

Seven Points for Athletes to Consider Before Using a Dietary Supplement.

Performance-enhancing drugs (PEDs) have been used by athletes for as long as sporting competitions have existed. To protect the health and safety of athletes and promote fair play, banned substance lists were developed that include several classes of PEDs. Evidence shows that a majority of athletes use dietary supplement products to aid their training and support their health. Evidence also indicates that use of some dietary supplements carries a risk because the products may contain banned PEDs. Consumers and athletes should weigh a number of considerations before purchasing and consuming dietary supplements to protect their health, reputation, and the spirit of fair competition.

Position Paper: Recommendations for the Investigation, Diagnosis, and Certification of Deaths Related to Opioid and Other Drugs.

The National Association of Medical Examiners convened an expert panel to update the association's evidence-based recommendations for investigating and certifying deaths associated with opioids and other misused substances to improve death certificate and mortality data for public health surveillance. The recommendations are as follows:1. Autopsy provides the best information on a decedent's medical condition for optimal interpretation of toxicology results, circumstances surrounding death, medical history, and scene findings. The panel considers autopsy an essential component of investigating apparent overdose deaths.2. Scene investigation includes reconciling prescription information and medication counts. Investigators should note drug paraphernalia or other evidence of using intoxicating substances.3. Retain blood, urine, and vitreous humor whenever available. Blood from the iliofemoral vein is preferable to blood from more central sites.4. A toxicological panel should be comprehensive, including potent depressant, stimulant, and antidepressant medications. Detecting novel substances present in the community may require special testing.5. When death is attributed to a drug or combination of drugs (as cause or contributing factor), the certifier should list the drugs by generic name in the autopsy report and death certificate.6. The best classification for manner of death in an overdose without any apparent intent of self-harm is "accident."

The Opioid Epidemic: Moving Toward an Integrated, Holistic Analytical Response.

In many jurisdictions, public safety and public health entities are working together to enhance the timeliness and accuracy of the analytical characterization and toxicology testing of novel synthetic opioids. The improved sharing and early detection of these analytical data are intended to inform surveillance, interdiction efforts, patient intervention and treatment, all of which are critical to curbing the opioid epidemic. Forensic practitioners working to identify novel synthetic opioids struggle to provide timely results when encountering new or unknown substances, such as the fentanyl analogs. These compounds, which mimic heroin in pharmacologic effect but can be far more potent, are inconsistently present in chemical identification libraries, and are currently largely unavailable as reference materials for analytical comparison. Additionally, federal, state and local governments as well as nongovernmental organizations require potency, toxicity and potential-for-abuse data to evaluate the potential health risks of emerging drug threats. Subsequent scheduling efforts and criminal prosecutions also require these thorough drug characterization studies. Pilot programs have demonstrated that early communication of real-time drug toxicity and analytical data significantly impacts the successful response to emerging opioids. High-quality, real-time, national-level data on chemical composition, toxicological test data, drug toxicity and overdoses, and analysis of seized materials by law enforcement are needed to track drug trends. However, the USA still lacks a national system to coordinate and communicate toxicology, medical and medical examiner and coroner data with the broader medical and law enforcement communities. Opportunities to address these gaps as well as recent advancements collected through interagency efforts and technical workshops in the toxicology and analytical chemistry communities are presented here. Opportunities for partnership, increased communication and expanding best practices to move toward an integrated, holistic analytical response are also explored.

Swift and Certain, Proportionate and Consistent: Key Values of Urine Drug Test Consequences for Probationers.

Traditionally, urine drug testing (UDT) in the correctional population (both prison and community corrections) has been infrequent, is scheduled, and has a high possibility of delayed results. Of practical relevance is that scheduled testing is ineffective for identifying drug misuse. Of ethical relevance is that consequences of positive scheduled tests can be unpredictable-in the form of overly severe punishment or a lack of treatment options-and that the scheduled testing paradigm is a poor way to change behaviors. More innovative programs now use a UDT paradigm with more frequent, random testing providing rapid results and certain, swift consequences and addiction treatment when warranted or requested. Studies have shown these new programs-the foundation of which is frequent, random UDTs-to significantly reduce drug use, criminal recidivism, and incarceration.

Performance-Enhancing Drugs I: Understanding the Basics of Testing for Banned Substances.

Whenever athletes willfully or accidentally ingest performance-enhancing drugs or other banned substances (such as drugs of abuse), markers of those drugs can be detected in biological samples (e.g., biofluids: urine, saliva, blood); in the case of some drugs, that evidence can be apparent for many weeks following the last exposure to the drug. In addition to the willful use of prohibited drugs, athletes can accidentally ingest banned substances in contaminated dietary supplements or foods and inadvertently fail a drug test that could mean the end of an athletic career and the loss of a good reputation. The proliferation of performance-enhancing drugs and methods has required a corresponding increase in the analytical tools and methods required to identify the presence of banned substances in biofluids. Even though extraordinary steps have been taken by organizations such as the World Anti-Doping Agency to limit the use of prohibited substances and methods by athletes willing to cheat, it is apparent that some athletes continue to avoid detection by using alternative doping regimens or taking advantage of the limitations in testing methodologies. This article reviews the testing standards and analytical techniques underlying the procedures used to identify banned substances in biological samples, setting the stage for future summaries of the testing required to establish the use of steroids, stimulants, diuretics, and other prohibited substances.

Mechanism of error caused by isotope-labeled internal standard: accurate method for simultaneous measurement of vitamin D and pre-vitamin D by liquid chromatography/tandem mass spectrometry.

RATIONALE: Bias of up to 25% has been observed for vitamin D3 and D2 samples exposed to heating during sample preparation, even when isotope-labeled internal standards are used. The goals of this study were to identify the mechanism of the positive bias observed in measuring vitamin D3 and D2 by liquid chromatography/tandem mass spectrometry (LC/MS/MS) and determine a way to eliminate the error source. METHODS: Several internal standards with varying locations of labeling were used for comparison in this study. Additionally, different temperatures (25, 37, 55, and 75 °C) and different treatment times were investigated for sample preparation and a LC/MS/MS method capable of simultaneously measuring vitamin D and pre-vitamin D was developed. RESULTS: It was demonstrated that the different conversion behaviors of the analyte and the internal standard were the cause of the positive bias. This bias was eliminated when internal standards with labeling remote from the double-bond area of the molecules were used. Additionally, sample preparation was shortened from overnight saponification at room temperature to 0.5 h at 75 °C. CONCLUSIONS: The use of an internal standard with labeling remote from the conjugated area eliminated the error source and gave accurate correction at all of the temperatures investigated. Heating may be used for rapid sample preparation as an alternative to overnight saponification at room temperature. This work not only describes the mechanism of an inaccurate internal standard correction, but also establishes a rapid LC/MS/MS method for simultaneous measurement of vitamin D and pre-vitamin D.

The androgen receptor and its use in biological assays: looking toward effect-based testing and its applications.

Steroid abuse is a growing problem among amateur and professional athletes. Because of an inundation of newly and illegally synthesized steroids with minor structural modifications and other designer steroid receptor modulators, there is a need to develop new methods of detection which do not require prior knowledge of the abused steroid structure. The number of designer steroids currently being abused is unknown because detection methods in general are only identifying substances with a known structure. The detection of doping is moving away from merely checking for exposure to prohibited substance toward detecting an effect of prohibited substances, as biological assays can do. Cell-based biological assays are the next generation of assays which should be utilized by antidoping laboratories; they can detect androgenic anabolic steroid and other human androgen receptor (hAR) ligand presence without knowledge of their structure and assess the relative biological activity of these compounds. This review summarizes the hAR and its action and discusses its relevance to sports doping and its use in biological assays.

Toxicological determination and in vitro metabolism of the designer drug methylenedioxypyrovalerone (MDPV) by gas chromatography/mass spectrometry and liquid chromatography/quadrupole time-of-flight mass spectrometry.

A method for the toxicological screening of the new designer drug methylenedioxypyrovalerone (MDPV) is described; with an emphasis on its application for anti-doping analysis. The metabolism of MDPV was evaluated in vitro using human liver microsomes and S9 cellular fractions for CYP450 phase I and uridine 5'-diphosphoglucuronosyltransferase (UGT) and sulfotransferase (SULT) phase II metabolism studies. The resulting metabolites were subsequently liquid/liquid extracted and analyzed using gas chromatography/mass spectrometry (GC/MS) as trimethylsilyl (TMS) derivatives. The structures of the metabolites were further confirmed by accurate mass measurement using a liquid chromatography/quadrupole time-of-flight (LC/QTOF) mass spectrometer. The studies demonstrated that the main metabolites of MDPV are catechol and methyl catechol pyrovalerone, which are in turn sulfated and glucuronated. The method for the determination of MDPV in urine has been fully validated by assessing the limits of detection and quantification, linearity, repeatability, and accuracy. This validation demonstrates the suitability for screening of this stimulant substance for anti-doping and forensic toxicology purposes.

The abuse of diuretics as performance-enhancing drugs and masking agents in sport doping: pharmacology, toxicology and analysis.

Diuretics are drugs that increase the rate of urine flow and sodium excretion to adjust the volume and composition of body fluids. There are several major categories of this drug class and the compounds vary greatly in structure, physicochemical properties, effects on urinary composition and renal haemodynamics, and site and mechanism of action. Diuretics are often abused by athletes to excrete water for rapid weight loss and to mask the presence of other banned substances. Because of their abuse by athletes, diuretics have been included on The World Anti-Doping Agency's (WADA) list of prohibited substances; the use of diuretics is banned both in competition and out of competition and diuretics are routinely screened for by anti-doping laboratories. This review provides an overview of the pharmacology and toxicology of diuretics and discusses their application in sports. The most common analytical strategies currently followed by the anti-doping laboratories accredited by the WADA are discussed along with the challenges laboratories face for the analysis of this diverse class of drugs.

Effect of anabolic-androgenic steroids and glucocorticoids on the kinetics of hAR and hGR nucleocytoplasmic translocation.

Although the qualitative nucleocytoplasmic transport of nuclear hormone receptors (NHRs) has been studied, there is little documentation of the cellular kinetics of this transport. Here, translocation studies using the human androgen receptor (hAR) and the human glucocorticoid receptor (hGR) were performed to aid in identifying the mechanism by which anabolic-androgenic steroids (AAS) were activating hAR and potentially interacting with hGR and how glucocorticoid ligands were interacting with the hGR and hAR. The real-time analysis of EGFP-labeled hAR and hGR ligand-induced cytoplasm-to-nucleus translocation was performed using fluorescence microscopy to better understand the action of these NHRs in a physiologically relevant cell-based model. After transient transfection, the hAR and hGR individually translocate as expected (i.e., transport is ligand-induced and dose-dependent) in this model biological system. Testosterone (TEST) had the fastest translocation rate for the hAR of 0.0525 min(-1). The other endogenous steroids, androstenedione (ANE) and dihydrotestosterone (DHT), had considerably lower hAR transport rates. The rates of hAR transport for the exogenous steroids methyltrienelone (MET), nandrolone (NAN), and oxandrolone (OXA) are lower than that of testosterone and similar to those of the endogenous steroids ANE and DHT. The hGR transport rates for cortisol (COR) and dexamethasone (DEX) are also presented. The synthetic GC, DEX, had a more rapid translocation rate (0.1599 min(-1)) at the highest dose of 100 nM compared to the endogenous GC COR (0.0431 min(-1)). The data obtained agrees with the existing qualitative data and adds an important ligand-dependent kinetic component to hAR and hGR transport. These kinetic data can aid our understanding of NHR action and interaction with other regulatory proteins, and can be useful in the development of new therapies.

SHIP-1 increases early oxidative burst and regulates phagosome maturation in macrophages.

Although the inositol phosphatase SHIP-1 is generally thought to inhibit signaling for Fc receptor-mediated phagocytosis, the product of its activity, phosphatidylinositol 3,4 bisphosphate (PI(3,4)P(2)), has been implicated in activation of the NADPH oxidase. This suggests that SHIP-1 positively regulates the generation of reactive oxygen species after phagocytosis. To examine how SHIP-1 activity contributes to Fc receptor-mediated phagocytosis, we measured and compared phospholipid dynamics, membrane trafficking, and the oxidative burst in macrophages from SHIP-1-deficient and wild-type mice. SHIP-1-deficient macrophages showed significantly elevated ratios of PI(3,4,5)P(3) to PI(3,4)P(2) on phagosomal membranes. Imaging reactive oxygen intermediate activities in phagosomes revealed decreased early NADPH oxidase activity in SHIP-1-deficient macrophages. SHIP-1 deficiency also altered later stages of phagosome maturation, as indicated by the persistent elevation of PI(3)P and the early localization of Rab5a to phagosomes. These direct measurements of individual organelles indicate that phagosomal SHIP-1 enhances the early oxidative burst through localized alteration of the membrane 3'-phosphoinositide composition.

Signal sequences for targeting of gene therapy products to subcellular compartments: the role of CRM1 in nucleocytoplasmic shuttling of the protein switch.

PURPOSE: The purpose of this study was to understand the mechanism of nuclear export of the protein switch, used for controlled intracellular delivery of gene products, by studying the involvement of classical export receptor CRM1. METHOD: Transient transfections of protein switch constructs, isolated nuclear export and import signals were carried out. Effect of leptomycin B (inhibitor of export receptor) and geldanamycin (inhibitor of Hsp90) on localization of these constructs was studied using fluorescence microscopy. Putative nuclear export signals in the glucocorticoid and progesterone receptor ligand binding domains were identified and studied. RESULTS: It was observed that treatment with leptomycin B caused nuclear accumulation of the protein switch constructs. However, geldanamycin did not have any pronounced effect on the localization. The isolated nuclear export signal from glucocorticoid receptor localized mostly in the cytoplasm, while its mutated version was present everywhere. CONCLUSION: The localization controlled protein switch constructs are exported out of the nucleus by the classical CRM1 receptors. The ligand binding domain of these protein switch constructs plays an important role in maintaining these constructs in the cytoplasm in the absence of ligand, as well the re-export back to the cytoplasm from the nucleus after ligand washout.

A functional heat shock protein 90 chaperone is essential for efficient flock house virus RNA polymerase synthesis in Drosophila cells.

The molecular chaperone heat shock protein 90 (Hsp90) is involved in multiple cellular processes including protein maturation, complex assembly and disassembly, and intracellular transport. We have recently shown that a disruption of Hsp90 activity in cultured Drosophila melanogaster cells suppresses Flock House virus (FHV) replication and the accumulation of protein A, the FHV RNA-dependent RNA polymerase. In the present study, we investigated whether the defect in FHV RNA polymerase accumulation induced by Hsp90 suppression was secondary to an effect on protein A synthesis, degradation, or intracellular membrane association. Treatment with the Hsp90-specific inhibitor geldanamycin selectively reduced FHV RNA polymerase synthesis by 80% in Drosophila S2 cells stably transfected with an inducible protein A expression plasmid. The suppressive effect of geldanamycin on protein A synthesis was not attenuated by proteasome inhibition, nor was it sensitive to changes in either the mRNA untranslated regions or protein A intracellular membrane localization. Furthermore, geldanamycin did not promote premature protein A degradation, nor did it alter the extremely rapid kinetics of protein A membrane association. These results identify a novel role for Hsp90 in facilitating viral RNA polymerase synthesis in Drosophila cells and suggest that FHV subverts normal cellular pathways to assemble functional replication complexes.