Revisiting the Landscape of Potential Small and Drug Substance Related Nitrosamines in Pharmaceuticals.

N-Nitrosamines are a class of indirect acting mutagens, as their metabolic degradation leads to the formation of the DNA-alkylating diazonium ion. Following up on the in-silico identification of thousands of nitrosamines that can potentially be derived from small molecule drugs and their known impurities described in a previous publication, we have now re-analyzed this dataset to apply EMA's Carcinogenic Potency Categorization Approach (CPCA) introduced with the 16th revision of their Q&A document for Marketing Authorization Holders. We find that the majority of potential nitrosamines from secondary amine precursors belongs to potency categories 4 and 5, corresponding to an acceptable daily intake of 1500 ng, whereas nitrosamines from tertiary amine precursors distribute more evenly among all categories, resulting in a substantial number of structures that are assigned the more challenging acceptable intakes of 18 ng/day and 100 ng/day for potency categories 1 and 2, respectively. However, the nitrosative dealkylation pathway for tertiary amine is generally far slower than the direct nitrosation on secondary amines, with a direct nitrosation mechanism suspected only for structures featuring electron-rich (hetero)aromatic substituents. This allows for greater focus towards those structures that require further review, and we demonstrate that their number is not substantial. In addition, we reflect on the nitrosamine risk posed by secondary amine API impurities and demonstrate that based on the ICH Q3A/B identification threshold unknown impurities may exist that could be transformed to relevant amounts of NA. We also demonstrate that the analytical sensitivity required for the quantification of high potency nitrosamines can be problematic especially for high dose APIs. In summary, the regulatory framework rolled out with the latest Q&A document represents a substantial improvement compared with the previous situation, but further refinement through interaction between manufacturers, regulators, not-for-profit and academic institutions will be required to ensure patient access to vital medicines without compromising safety.

The Landscape of Potential Small and Drug Substance Related Nitrosamines in Pharmaceuticals.

This article reports the outcome of an in silico analysis of more than 12,000 small molecule drugs and drug impurities, identifying the nitrosatable structures, assessing their potential to form nitrosamines under relevant conditions and the challenges to determine compound-specific AIs based on data available or read-across approaches for these nitrosamines and their acceptance by health authorities. Our data indicate that the presence of nitrosamines in pharmaceuticals is likely more prevalent than originally expected. In total, 40.4 % of the analyzed APIs and 29.6 % of the API impurities are potential nitrosamine precursors. Most structures identified through our workflow could form complex API-related nitrosamines, so-called nitrosamine drug substance related impurities (NDSRIs), although we also found structures that could release the well-known small and potent nitrosamines NDMA, NDEA, and others. Due to common structural motifs including secondary or tertiary amine moieties, whole essential drug classes such as beta blockers and ACE inhibitors are at risk. To avoid the risk of drug shortages or even the complete loss of therapeutic options, it will be essential that the well-established ICH M7 principles remain applicable for nitrosamines and that that the industry and regulatory authorities keep an open communication not only about the science but also to make sure there is a good balance between risk and benefit to patients.

Use of Lhasa Limited Products for the In Silico Prediction of Drug Toxicity.

Lhasa Limited have had a role in the in silico prediction of drug and other chemical toxicity for over 30 years. This role has always been multifaceted, both as a provider of predictive software such as Derek Nexus, and as an honest broker for the sharing of proprietary chemical and toxicity data. A changing regulatory environment and the drive for the Replacement, Reduction and Refinement (the 3Rs) of animal testing have led both to increased acceptance of in silico predictions and a desire for the sharing of data to reduce duplicate testing. The combination of these factors has led to Lhasa Limited providing a suite of products and coordinating numerous data-sharing consortia that do indeed facilitate a significant reduction in the testing burden that companies would otherwise be laboring under. Many of these products and consortia can be organized into workflows for specific regulatory use cases, and it is these that will be used to frame the narrative in this chapter.

Predicting moisture content during maize nixtamalization using machine learning with NIR spectroscopy.

KEY MESSAGE: Moisture content during nixtamalization can be accurately predicted from NIR spectroscopy when coupled with a support vector machine (SVM) model, is strongly modulated by the environment, and has a complex genetic architecture. Lack of high-throughput phenotyping systems for determining moisture content during the maize nixtamalization cooking process has led to difficulty in breeding for this trait. This study provides a high-throughput, quantitative measure of kernel moisture content during nixtamalization based on NIR scanning of uncooked maize kernels. Machine learning was utilized to develop models based on the combination of NIR spectra and moisture content determined from a scaled-down benchtop cook method. A linear support vector machine (SVM) model with a Spearman's rank correlation coefficient of 0.852 between wet laboratory and predicted values was developed from 100 diverse temperate genotypes grown in replicate across two environments. This model was applied to NIR spectra data from 501 diverse temperate genotypes grown in replicate in five environments. Analysis of variance revealed environment explained the highest percent of the variation (51.5%), followed by genotype (15.6%) and genotype-by-environment interaction (11.2%). A genome-wide association study identified 26 significant loci across five environments that explained between 5.04% and 16.01% (average = 10.41%). However, genome-wide markers explained 10.54% to 45.99% (average = 31.68%) of the variation, indicating the genetic architecture of this trait is likely complex and controlled by many loci of small effect. This study provides a high-throughput method to evaluate moisture content during nixtamalization that is feasible at the scale of a breeding program and provides important information about the factors contributing to variation of this trait for breeders and food companies to make future strategies to improve this important processing trait.

Mitochondrial respiration controls the Prox1-Vegfr3 feedback loop during lymphatic endothelial cell fate specification and maintenance.

Recent findings indicate that mitochondrial respiration regulates blood endothelial cell proliferation; however, its role in differentiating lymphatic endothelial cells (LECs) is unknown. We hypothesized that mitochondria could work as a sensor of LECs' metabolic specific needs by determining their functional requirements according to their differentiation status and local tissue microenvironment. Accordingly, we conditionally deleted the QPC subunit of mitochondrial complex III in differentiating LECs of mouse embryos. Unexpectedly, mutant mice were devoid of a lymphatic vasculature by mid-gestation, a consequence of the specific down-regulation of main LEC fate regulators, particularly Vegfr3, leading to the loss of LEC fate. Mechanistically, this is a result of reduced H3K4me3 and H3K27ac in the genomic locus of key LEC fate controllers (e.g., Vegfr3 and Prox1). Our findings indicate that by sensing the LEC differentiation status and microenvironmental metabolic conditions, mitochondrial complex III regulates the critical Prox1-Vegfr3 feedback loop and, therefore, LEC fate specification and maintenance.

Steric and electronic effects on the 1 H hyperpolarisation of substituted pyridazines by signal amplification by reversible exchange.

Utility of the pyridazine motif is growing in popularity as pharmaceutical and agrochemical agents. The detection and structural characterisation of such materials is therefore imperative for the successful development of new products. Signal amplification by reversible exchange (SABRE) offers a route to dramatically improve the sensitivity of magnetic resonance methods, and we apply it here to the rapid and cost-effective hyperpolarisation of substituted pyridazines. The 33 substrates investigated cover a range of steric and electronic properties and their capacity to perform highly effective SABRE is assessed. We find the method to be tolerant to a broad range of electron donating and withdrawing groups; however, good sensitivity is evident when steric bulk is added to the 3- and 6-positions of the pyridazine ring. We optimise the method by reference to a disubstituted ester that yields signal gains of >9000-fold at 9.4 T (>28% spin polarisation).

Optical confinement in the nanocoax: coupling to the fundamental TEM-like mode.

The nanoscale coaxial cable (nanocoax) has demonstrated optical confinement in the visible and the near infrared. We report on a novel nanofabrication process which yields optically addressable, sub-µm diameter, and high aspect ratio metal-insulator-metal nanocoaxes made by atomic layer deposition of Pt and Al2O3. We observe sub-diffraction-limited optical transmission via the fundamental, TEM-like mode by excitation with a radially polarized optical vortex beam. Our experimental results are based on interrogation with a polarimetric imager. Finite element method numerical simulations support these results, and their uniaxial symmetry was exploited to model taper geometries with both an electrically large volume, (15λ)3, and a nanoscopic exit aperture, (λ/200)2.

A simple and cost-efficient technique to generate hyperpolarized long-lived 15N-15N nuclear spin order in a diazine by signal amplification by reversible exchange.

Signal Amplification by Reversible Exchange (SABRE) is an inexpensive and simple hyperpolarization technique that is capable of boosting nuclear magnetic resonance sensitivity by several orders of magnitude. It utilizes the reversible binding of para-hydrogen, as hydride ligands, and a substrate of interest to a metal catalyst to allow for polarization transfer from para-hydrogen into substrate nuclear spins. While the resulting nuclear spin populations can be dramatically larger than those normally created, their lifetime sets a strict upper limit on the experimental timeframe. Consequently, short nuclear spin lifetimes are a challenge for hyperpolarized metabolic imaging. In this report, we demonstrate how both hyperpolarization and long nuclear spin lifetime can be simultaneously achieved in nitrogen-15 containing derivatives of pyridazine and phthalazine by SABRE. These substrates were chosen to reflect two distinct classes of 15N2-coupled species that differ according to their chemical symmetry and thereby achieve different nuclear spin lifetimes. The pyridazine derivative proves to exhibit a signal lifetime of ∼2.5 min and can be produced with a signal enhancement of ∼2700. In contrast, while the phthalazine derivative yields a superior 15 000-fold 15N signal enhancement at 11.7 T, it has a much shorter signal lifetime.

Facile fabrication and formation mechanism of aluminum nanowire arrays.

Anodized alumina membranes (AAMs) have proven effective at making vertically-oriented and well-ordered metal nanowire arrays, which are useful in plasmonics and electrochemistry. Here, we produced Al nanowires via directed AAM pore nucleation: a patterned oxide mask on a flat Al surface directed where pores did and did not form, the pores acting to oxidize Al around the sites without pores. This left Al nanowires embedded in the AAM, and produced freestanding Al nanowires after etching the AAM. The nanowire tops had two distinct contours, smooth bowls and flat rough surfaces-suggesting that nanowires with bowl tops result from slow pore development relative to pattern-nucleated pores, not pore blockage as prior literature suggests. The observed low porosity of ∼2%, as opposed to the more typical 10%, suggests pore nucleation in the electrolyte employed may need greater local variations in electric field or pH, possibly explaining the electrolyte's peculiar ability to make Al nanowires. Finally, a soft nano-imprint lithography process was developed here to pattern the mask without damaging the stamp, avoiding a stamp degradation problem in previous work that utilized hard nano-imprint lithography.

Pertussis: The Identify, Isolate, Inform Tool Applied to a Re-emerging Respiratory Illness.

Pertussis, commonly referred to as "whooping cough," is a highly contagious acute respiratory infection that has exhibited cyclical outbreaks throughout the last century. Although vaccines have provided some immunity, many populations, including infants and pregnant women, remain at risk for serious illness. Through the use of the novel "Identify, Isolate, Inform" (3I) tool, emergency department (ED) providers can readily recognize key symptoms of the disease and risk factors for exposure, thus curbing its transmission through early initiation of antimicrobial therapy and post-exposure prophylaxis. The three classic stages of pertussis include an initial catarrhal stage, characterized by nonspecific upper respiratory infection symptoms, which may advance to the paroxysmal stage, revealing the distinctive "whooping cough." This cough can persist for weeks to months leading into the convalescent stage. Household contacts of patients with suspected pertussis or other asymptomatic, high-risk populations (infants, pregnant women in their third trimester, and childcare workers) may benefit from post-exposure prophylactic therapy. The Pertussis 3I tool can also alert healthcare professionals to the proper respiratory droplet precautions during contact with a symptomatic patient, as well as isolation practices until antimicrobial treatment is in progress. ED personnel should then inform local public health departments of any suspected cases. All of these actions will ultimately aid public health in controlling the incidence of pertussis cases, thus ensuring the protection of the general public from this re-emerging respiratory illness.

Using 2 H labelling to improve the NMR detectability of pyridine and its derivatives by SABRE.

By introducing a range of 2 H labels into pyridine and the para-substituted agents, methyl isonicotinate and isonicotinamide, we significantly improve their NMR detectability in conjunction with the signal amplification by reversible exchange process. We describe how the rates of T1 relaxation for the remaining 1 H nuclei are increased and show how this leads to a concomitant increase in the level of 1 H and 13 C hyperpolarization that can ultimately be detected.

Achieving Biocompatible SABRE: An in†vitro Cytotoxicity Study.

Production of a biocompatible hyperpolarized bolus for signal amplification by reversible exchange (SABRE) could open the door to simple clinical diagnosis via magnetic resonance imaging. Essential to successful progression to preclinical/clinical applications is the determination of the toxicology profile of the SABRE reaction mixture. Herein, we exemplify the cytotoxicity of the SABRE approach using in vitro cell assays. We conclude that the main cause of the observed toxicity is due to the SABRE catalyst. We therefore illustrate two catalyst removal methods: one involving deactivation and ion-exchange chromatography, and the second using biphasic catalysis. These routes produce a bolus suitable for future in vivo study.

Hepatitis A Virus: Essential Knowledge and a Novel Identify-Isolate-Inform Tool for Frontline Healthcare Providers.

Infection with hepatitis A virus (HAV) causes a highly contagious illness that can lead to serious morbidity and occasional mortality. Although the overall incidence of HAV has been declining since the introduction of the HAV vaccine, there have been an increasing number of outbreaks within the United States and elsewhere between 2016 and 2017. These outbreaks have had far-reaching consequences, with a large number of patients requiring hospitalization and several deaths. Accordingly, HAV is proving to present a renewed public health challenge. Through use of the "Identify-Isolate-Inform" tool as adapted for HAV, emergency physicians can become more familiar with the identification and management of patients presenting to the emergency department (ED) with exposure, infection, or risk of contracting disease. While it can be asymptomatic, HAV typically presents with a prodrome of fever, nausea/vomiting, and abdominal pain followed by jaundice. Healthcare providers should maintain strict standard precautions for all patients suspected of having HAV infection as well as contact precautions in special cases. Hand hygiene with soap and warm water should be emphasized, and affected patients should be counseled to avoid food preparation and close contact with vulnerable populations. Additionally, ED providers should offer post-exposure prophylaxis to exposed contacts and encourage vaccination as well as other preventive measures for at-risk individuals. ED personnel should inform local public health departments of any suspected case.

Delivering strong 1H nuclear hyperpolarization levels and long magnetic lifetimes through signal amplification by reversible exchange.

Hyperpolarization turns typically weak NMR and MRI responses into strong signals so that ordinarily impractical measurements become possible. The potential to revolutionize analytical NMR and clinical diagnosis through this approach reflect this area's most compelling outcomes. Methods to optimize the low-cost parahydrogen-based approach signal amplification by reversible exchange with studies on a series of biologically relevant nicotinamides and methyl nicotinates are detailed. These procedures involve specific 2H labeling in both the agent and catalyst and achieve polarization lifetimes of ca 2 min with 50% polarization in the case of methyl-4,6-d2 -nicotinate. Because a 1.5-T hospital scanner has an effective 1H polarization level of just 0.0005% this strategy should result in compressed detection times for chemically discerning measurements that probe disease. To demonstrate this technique's generality, we exemplify further studies on a range of pyridazine, pyrimidine, pyrazine, and isonicotinamide analogs that feature as building blocks in biochemistry and many disease-treating drugs.

A Case of Erythema Nodosum with Coccidioidomycosis.

Erythema nodosum (EN) is associated with many systemic diseases and infections. This case report provides an image of erythematous nodules, an overview of the various causes of EN, and the laboratory tests and imaging that can be done in the emergency department to narrow its broad differential diagnosis.

Opioid Dependent Malingerer with Self-Induced Sepsis.

A 21-year-old woman was admitted to the emergency department (ED) with severe sepsis. Both the mechanism of infection and organisms discovered were unusual.

Mumps Virus: Modification of the Identify-Isolate-Inform Tool for Frontline Healthcare Providers.

Mumps is a highly contagious viral infection that became rare in most industrialized countries following the introduction of measles-mumps-rubella (MMR) vaccine in 1967. The disease, however, has been re-emerging with several outbreaks over the past decade. Many clinicians have never seen a case of mumps. To assist frontline healthcare providers with detecting potential cases and initiating critical actions, investigators modified the "Identify-Isolate-Inform" tool for mumps infection. The tool is applicable to regions with rare incidences or local outbreaks, especially seen in college students, as well as globally in areas where vaccination is less common. Mumps begins with a prodrome of low-grade fever, myalgias and malaise/anorexia, followed by development of nonsuppurative parotitis, which is the pathognomonic finding associated with acute mumps infection. Orchitis and meningitis are the two most common serious complications, with hearing loss and infertility occurring rarely. Providers should consider mumps in patients with exposure to a known case or international travel to endemic regions who present with consistent signs and symptoms. If mumps is suspected, healthcare providers must immediately implement standard and droplet precautions and notify the local health department and hospital infection control personnel.

Wireless communication system via nanoscale plasmonic antennas.

Present on-chip optical communication technology uses near-infrared light, but visible wavelengths would allow system miniaturization and higher energy confinement. Towards this end, we report a nanoscale wireless communication system that operates at visible wavelengths via in-plane information transmission. Here, plasmonic antenna radiation mediates a three-step conversion process (surface plasmon → photon → surface plasmon) with in-plane efficiency (plasmon → plasmon) of 38% for antenna separation 4λ0 (with λ0 the free-space excitation wavelength). Information transmission is demonstrated at bandwidths in the Hz and MHz ranges. This work opens the possibility of optical conveyance of information using plasmonic antennas for on-chip communication technology.

Shielded Coaxial Optrode Arrays for Neurophysiology.

Recent progress in the study of the brain has been greatly facilitated by the development of new tools capable of minimally-invasive, robust coupling to neuronal assemblies. Two prominent examples are the microelectrode array (MEA), which enables electrical signals from large numbers of neurons to be detected and spatiotemporally correlated, and optogenetics, which enables the electrical activity of cells to be controlled with light. In the former case, high spatial density is desirable but, as electrode arrays evolve toward higher density and thus smaller pitch, electrical crosstalk increases. In the latter, finer control over light input is desirable, to enable improved studies of neuroelectronic pathways emanating from specific cell stimulation. Here, we introduce a coaxial electrode architecture that is uniquely suited to address these issues, as it can simultaneously be utilized as an optical waveguide and a shielded electrode in dense arrays. Using optogenetically-transfected cells on a coaxial MEA, we demonstrate the utility of the architecture by recording cellular currents evoked from optical stimulation. We also show the capability for network recording by radiating an area of seven individually-addressed coaxial electrode regions with cultured cells covering a section of the extent.

Identify-Isolate-Inform: A Tool for Initial Detection and Management of Zika Virus Patients in the Emergency Department.

First isolated in 1947 from a monkey in the Zika forest in Uganda, and from mosquitoes in the same forest the following year, Zika virus has gained international attention due to concerns for infection in pregnant women potentially causing fetal microcephaly. More than one million people have been infected since the appearance of the virus in Brazil in 2015. Approximately 80% of infected patients are asymptomatic. An association with microcephaly and other birth defects as well as Guillain-Barre Syndrome has led to a World Health Organization declaration of Zika virus as a Public Health Emergency of International Concern in February 2016. Zika virus is a vector-borne disease transmitted primarily by the Aedes aegypti mosquito. Male to female sexual transmission has been reported and there is potential for transmission via blood transfusions. After an incubation period of 2-7 days, symptomatic patients develop rapid onset fever, maculopapular rash, arthralgia, and conjunctivitis, often associated with headache and myalgias. Emergency department (ED) personnel must be prepared to address concerns from patients presenting with symptoms consistent with acute Zika virus infection, especially those who are pregnant or planning travel to Zika-endemic regions, as well as those women planning to become pregnant and their partners. The identify-isolate-inform (3I) tool, originally conceived for initial detection and management of Ebola virus disease patients in the ED, and later adjusted for measles and Middle East Respiratory Syndrome, can be adapted for real-time use for any emerging infectious disease. This paper reports a modification of the 3I tool for initial detection and management of patients under investigation for Zika virus. Following an assessment of epidemiologic risk, including travel to countries with mosquitoes that transmit Zika virus, patients are further investigated if clinically indicated. If after a rapid evaluation, Zika or other arthropod-borne diseases are the only concern, isolation (contact, droplet, airborne) is unnecessary. Zika is a reportable disease and thus appropriate health authorities must be notified. The modified 3I tool will facilitate rapid analysis and triggering of appropriate actions for patients presenting to the ED at risk for Zika.