A Perfused In Vitro Human iPSC-Derived Blood-Brain Barrier Faithfully Mimics Transferrin Receptor-Mediated Transcytosis of Therapeutic Antibodies.

Delivering biologics to elicit a therapeutic response in the central nervous system (CNS) remains challenging due to the presence of the blood-brain barrier (BBB). Receptor-mediated transcytosis is a strategy to improve brain exposure after systemic drug administration. The availability of a clinically relevant in vitro BBB model is crucial to investigate transcytosis pathways and to predict the penetration of biologics into the CNS. We created a perfused human in vitro BBB model made of induced pluripotent stem cells (iPSC)-derived brain microvascular endothelial cells (BMEC) for studying transferrin receptor-mediated transcytosis. iPSC-derived BMEC were seeded in the top channel of a three-lane microfluidic device (OrganoPlate®). After 2 days in culture, the established cell model exhibited relevant BBB features, including physiological transendothelial electrical resistance in a transwell setting (1500 Ω*cm2), reduced apparent permeability (Papp) to the fluorescence tracer Lucifer yellow (20-fold less than cell-free chips), expression of key BBB markers such as tight junctions proteins, transporters, receptors and functional P-gp efflux pump. Moreover, the model exhibited functional transferrin receptor-mediated uptake and transcytosis. To assess selective transferrin receptor-mediated transcytosis, a mixture of anti-human transferrin receptor (MEM-189) and control (sheep IgG anti-bovine serum albumin) antibodies was perfused in the top channel for 2 h. The Papp of MEM-189 was 11-fold higher than that of the control antibody, demonstrating facilitated receptor-mediated transcytosis. Compared to published work reporting a 2-fold ratio, this result is remarkable and establishes the suitability of our model for exploring receptor-mediated transcytosis and screening of antibodies for putative brain shuttle application. A perfused in vitro human model made of iPSC-derived BMEC with the chief characteristics (barrier tightness, functionality) of the human BBB can be applied to study transferrin receptor (TfR)-mediated transcytosis of therapeutic antibodies. This may bring critical advances in drug shuttle technology. Graphical abstract generated with biorender.com.

Transapical Delivery of a Sapien Valve for Transcatheter Aortic Valve Replacement in an 11-Year-Old Patient with Truncus Arteriosus.

Complex congenital heart defects may necessitate repeated surgical interventions throughout a patient's lifetime. Each subsequent procedure exposes patients to a greater cumulative risk, thus adding to the potential morbidity and mortality of the surgery. Transcatheter interventions can help mitigate the surgical risk for many defects and can delay or mitigate the need for surgery. This case report describes the rare use of a transapically delivered transcatheter aortic valve replacement (TAVR) therapy in a high-risk pediatric patient to postpone the need for surgery and potentially reduce the number of lifelong surgical interventions. The case highlights how transcatheter aortic valve therapies can be considered for non-standard, higher risk pediatric patients to postpone the need for surgical valve replacement and may serve as a paradigm shift in the care of complex patients with aortic valve pathology.

Absorption, Distribution, Metabolism, and Excretion of Therapeutic Proteins: Current Industry Practices and Future Perspectives.

Therapeutic proteins (TPs) comprise a variety of modalities, including antibody-based drugs, coagulation factors, recombinant cytokines, enzymes, growth factors, and hormones. TPs usually cannot traverse cellular barriers and exert their pharmacological activity by interacting with targets on the exterior membrane of cells or with soluble ligands in the tissue interstitial fluid/blood. Due to their large size, lack of cellular permeability, variation in metabolic fate, and distinct physicochemical characteristics, TPs are subject to different absorption, distribution, metabolism, and excretion (ADME) processes as compared with small molecules. Limited regulatory guidance makes it challenging to determine the most relevant ADME data required for regulatory submissions. The TP ADME working group was sponsored by the Translational and ADME Sciences Leadership Group within the Innovation and Quality (IQ) consortium with objectives to: (1) better understand the current practices of ADME data generated for TPs across IQ member companies, (2) learn about their regulatory strategies and interaction experiences, and (3) provide recommendations on best practices for conducting ADME studies for TPs. To understand current ADME practices and regulatory strategies, an industry-wide survey was conducted within IQ member companies. In addition, ADME data submitted to the U.S. Food and Drug Administration was also collated by reviewing regulatory submission packages of TPs approved between 2011 and 2020. This article summarizes the key learnings from the survey and an overview of ADME data presented in biologics license applications along with future perspectives and recommendations for conducting ADME studies for internal decision-making as well as regulatory submissions for TPs. SIGNIFICANCE STATEMENT: This article provides comprehensive assessment of the current practices of absorption, distribution, metabolism, and excretion (ADME) data generated for therapeutic proteins (TPs) across the Innovation and Quality participating companies and the utility of the data in discovery, development, and regulatory submissions. The TP ADME working group also recommends the best practices for condu-cting ADME studies for internal decision-making and regulatory submissions.

Meeting report: 1st workshop of the peptide ADME discussion group.

1. Challenges and opportunities within peptide ADME (absorption, distribution, metabolism and elimination) were presented and discussed at the 1st peptide workshop of the Peptide ADME Discussion Group in Gothenburg, Sweden (15th of October 2018). This article summarises the presentations and discussions from this 1st workshop. The following topics were covered: Background science presentation on peptidases Presentation of various peptide ADME packages Peptide drug-drug interactions (DDI).

A long-lived IL-2 mutein that selectively activates and expands regulatory T cells as a therapy for autoimmune disease.

Susceptibility to multiple autoimmune diseases is associated with common gene polymorphisms influencing IL-2 signaling and Treg function, making Treg-specific expansion by IL-2 a compelling therapeutic approach to treatment. As an in vivo IL-2 half-life enhancer we used a non-targeted, effector-function-silent human IgG1 as a fusion protein. An IL-2 mutein (N88D) with reduced binding to the intermediate affinity IL-2Rßγ receptor was engineered with a stoichiometry of two IL-2N88D molecules per IgG, i.e. IgG-(IL-2N88D)2. The reduced affinity of IgG-(IL-2N88D)2 for the IL-2Rßγ receptor resulted in a Treg-selective molecule in human whole blood pSTAT5 assays. Treatment of cynomolgus monkeys with single low doses of IgG-(IL-2N88D)2 induced sustained preferential activation of Tregs accompanied by a corresponding 10-14-fold increase in CD4+ and CD8+ CD25+FOXP3+ Tregs; conditions that had no effect on CD4+ or CD8+ memory effector T cells. The expanded cynomolgus Tregs had demethylated FOXP3 and CTLA4 epigenetic signatures characteristic of functionally suppressive cells. Humanized mice had similar selective in vivo responses; IgG-(IL-2N88D)2 increased Tregs while wild-type IgG-IL-2 increased NK cells in addition to Tregs. The expanded human Tregs had demethylated FOXP3 and CTLA4 signatures and were immunosuppressive. These results describe a next-generation immunotherapy using a long-lived and Treg-selective IL-2 that activates and expands functional Tregsin vivo. Patients should benefit from restored immune homeostasis in a personalized fashion to the extent that their autoimmune disease condition dictates opening up the possibility for remissions and cures.

Implementation of the National Breast and Cervical Cancer Early Detection Program: the beginning.

In 1990, Congress passed the Breast and Cervical Cancer Mortality Prevention Act because of increases in the number of low-income and uninsured women being diagnosed with breast cancer. This act authorized the Centers for Disease Control and Prevention (CDC) to establish the National Breast and Cervical Cancer Early Detection Program (NBCCEDP) to provide high-quality and timely breast and cervical cancer screening and diagnostic services to low-income, uninsured women. The program started in 1991, and, in 1993, Congress amended the act to allow the CDC to fund American Indian and Alaska Native tribes and tribal organizations. By 1996, the program was providing cancer screening across the United States. To ensure appropriate delivery and monitoring of services, the program adopted detailed policies on program management, evidence-based guidelines for clinical services, a systematized clinical data system to track service quality, and key partnerships that expand the program's reach. The NBCCEDP currently funds 67 programs, including all 50 states, the District of Columbia, 5 US territories, and 11 tribes or tribal organizations.

An Ex-Vivo Study Comparing the Accuracy of the E-Connect S+ and Morita Tri Auto ZX2+ Endodontic Handpieces in Root Canal Length Determination.

INTRODUCTION: This ex vivo study evaluated the accuracy of the Electronic Apex Locator (EAL) and Automatic Apical Stop (AAS) functions of the E-Connect S+ and Morita Tri Auto ZX2+ cordless apex locators in determining patency length. METHODS: Sixty-four human teeth with a single root were randomly allocated into E-connect or Morita groups (n = 32). The canals were accessed and preflared, after which a size 15 K-file was inserted into the canal to the major foramen and recorded as the actual length (AL). Matched measurements were taken using the AAS and EAL functions and visually confirmed with confocal microscopy. The variance between canal length (mm), the persons correlation (ρ) between function and AL, and the accuracy (%) of the canal length relative to the AL (Δmm) between devices and functions were assessed. RESULTS: Regardless of device or function, all measurements were within 1±Δmm and correlated strongly (ρ > 0.97) with the AL. When considering a more stringent clinically acceptable range of 0.5±Δmm from the AL, all devices and functions demonstrated similar accuracy levels (84%-94%). However, at lower tolerance ranges, the E-connect device with the EAL function exhibited the highest accuracy. On average, all devices and functions stopped short of the AL (mean Δmm>0). CONCLUSION: The E-Connect S+ and Morita Tri Auto ZX2+ apex locators provided reliable accuracy in determining the position of the major foramen. These findings demonstrate a high level of reproducibility in canal length measurements using both cordless endodontic handpieces, regardless of whether the EAL or AAS functions were employed.

Safe, long-term hepatic expression of anti-HCV shRNA in a nonhuman primate model.

The hepatitis C virus (HCV) chronically infects 2% of the world population and effective treatment is limited by long duration and significant side-effects. Here, we describe a novel drug, intended as a "single-shot " therapy, which expresses three short hairpin RNAs (shRNAs) that simultaneously target multiple conserved regions of the HCV genome as confirmed in vitro by knockdown of an HCV replicon system. Using a recombinant adeno-associated virus (AAV) serotype 8 vector for delivery, comprehensive transduction of hepatocytes was achieved in vivo in a nonhuman primate (NHP) model following a single intravenous injection. However, dose ranging studies performed in 13 NHP resulted in high-expression levels of shRNA from wild-type (wt) Pol III promoters and dose-dependent hepatocellular toxicity, the first demonstration of shRNA-related toxicity in primates, establishing that the hepatotoxicity arises from highly conserved features of the RNA interference (RNAi) pathway. In the second generation drug, each promoter was re-engineered to reduce shRNA transcription to levels that circumvent toxicity but still inhibit replicon activity. In vivo testing of this modified construct in 18 NHPs showed conservation of hepatocyte transduction but complete elimination of hepatotoxicity, even with sustained shRNA expression for 50 days. These data support progression to a clinical study for treatment of HCV infection.

Virtual medical scribes: making electronic medical records work for you.

There is increasing buzz around the term "medical scribe" in healthcare today. Medical scribes help meet the growing electronic medical record (EMR) data entry challenge healthcare providers face. Medical scribes reduce providers' paperwork burden, increase a medical practice's net margins, and reduce stress levels for doctors and their staff. They do this by charting patient encounters in real-time during patient examinations, thus reducing significantly the data entry workload that EMRs place on providers. Medical scribes can work onsite or offsite from a HIPAA-secure location, the latter being known as "virtual medical scribes." This article explores the uses and benefits of scribes to give you the background to employ them effectively in your clinic or hospital.

Delivery of the Brainshuttle™ amyloid-beta antibody fusion trontinemab to non-human primate brain and projected efficacious dose regimens in humans.

There are few treatments that slow neurodegeneration in Alzheimer's disease (AD), and while therapeutic antibodies are being investigated in clinical trials for AD treatment, their access to the central nervous system is restricted by the blood-brain barrier. This study investigates a bispecific modular fusion protein composed of gantenerumab, a fully human monoclonal anti- amyloid-beta (Aß) antibody under investigation for AD treatment, with a human transferrin receptor 1-directed Brainshuttle™ module (trontinemab; RG6102, INN trontinemab). In vitro, trontinemab showed a similar binding affinity to fibrillar Aß40 and Aß plaques in human AD brain sections to gantenerumab. A single intravenous administration of trontinemab (10 mg/kg) or gantenerumab (20 mg/kg) to non-human primates (NHPs, Macaca fascicularis), was well tolerated in both groups. Immunohistochemistry indicated increased trontinemab uptake into the brain endothelial cell layer and parenchyma, and more homogeneous distribution, compared with gantenerumab. Brain and plasma pharmacokinetic (PK) parameters for trontinemab were estimated by nonlinear mixed-effects modeling with correction for tissue residual blood, indicating a 4-18-fold increase in brain exposure. A previously developed clinical PK/pharmacodynamic model of gantenerumab was adapted to include a brain compartment as a driver of plaque removal and linked to the allometrically scaled above model from NHP. The new brain exposure-based model was used to predict trontinemab dosing regimens for effective amyloid reduction. Simulations from these models were used to inform dosing of trontinemab in the first-in-human clinical trial.

In vitro characterization of the activity of PF-05095808, a novel biological agent for hepatitis C virus therapy.

PF-05095808 is a novel biological agent for chronic hepatitis C virus (HCV) therapy. It comprises a recombinant adeno-associated virus (AAV) DNA vector packaged into an AAV serotype 8 capsid. The vector directs expression of three short hairpin RNAs (shRNAs) targeted to conserved regions of the HCV genome. These shRNAs are processed by the host cell into the small interfering RNAs which mediate sequence-specific cleavage of target regions. For small-molecule inhibitors the key screens needed to assess in vitro activity are well defined; we developed new assays to assess this RNA interference agent and so to understand its therapeutic potential. Following administration of PF-05095808 or corresponding synthetic shRNAs, sequence-specific antiviral activity was observed in HCV replicon and infectious virus systems. To quantify the numbers of shRNA molecules required for antiviral activity in vitro and potentially also in vivo, a universal quantitative PCR (qPCR) assay was developed. The number of shRNA molecules needed to drive antiviral activity proved to be independent of the vector delivery system used for PF-05095808 administration. The emergence of resistant variants at the target site of one shRNA was characterized. A novel RNA cleavage assay was developed to confirm the spectrum of activity of PF-05095808 against common HCV clinical isolates. In summary, our data both support antiviral activity consistent with an RNA interference mechanism and demonstrate the potential of PF-05095808 as a therapeutic agent for chronic HCV infection.

Kappa agonist CovX-Bodies.

Small peptidic kappa agonists were covalently linked to the reactive lysine of the CovX antibody to create compounds having potent activity at the kappa receptor with greatly extended half-life when compared to the parent peptide as exemplified by compound 20.

Uptake, efficacy, and systemic distribution of naked, inhaled short interfering RNA (siRNA) and locked nucleic acid (LNA) antisense.

Antisense oligonucleotides (ASOs) and small interfering RNA (siRNA) promise specific correction of disease-causing gene expression. Therapeutic implementation, however, has been forestalled by poor delivery to the appropriate tissue, cell type, and subcellular compartment. Topical administration is considered to circumvent these issues. The availability of inhalation devices and unmet medical need in lung disease has focused efforts in this tissue. We report the development of a novel cell sorting method for quantitative, cell type-specific analysis of siRNA, and locked nucleic acid (LNA) ASO uptake and efficacy after intratracheal (i.t.) administration in mice. Through fluorescent dye labeling, we compare the utility of this approach to whole animal and whole tissue analysis, and examine the extent of tissue distribution. We detail rapid systemic access and renal clearance for both therapeutic classes and lack of efficacy at the protein level in lung macrophages, epithelia, or other cell types. We nevertheless observe efficient redirection of i.t. administered phosphorothioate (PS) LNA ASO to the liver and kidney leading to targeted gene knockdown. These data suggest delivery remains a key obstacle to topically administered, naked oligonucleotide efficacy in the lung and introduce inhalation as a potentially viable alternative to injection for antisense administration to the liver and kidneys.

A Rare Case of Large-Vessel Vasculitis following Checkpoint Inhibitor Therapy and Pegfilgrastim.

Checkpoint inhibitors (CPIs) and pegfilgrastim, a long-acting growth factor agent, are vital components of current cancer treatments. Immune-related adverse events (irAEs) such as colitis and pneumonitis are well-established toxicities associated with CPI therapy. However, large-vessel vasculitis secondary to CPI utilization is reported only in rare case reports and case series. Interestingly, large-vessel vasculitis has also been reported as a rare complication of pegfilgrastim use. We present a 59-year-old female with left stage IIA (cT2N0M0) triple-negative breast cancer receiving neoadjuvant decitabine and pembrolizumab prior to neoadjuvant chemotherapy (NAC). NAC included standard-of-care dose dense doxorubicin and cyclophosphamide (ddAC) supported with pegfilgrastim use followed by weekly carboplatin and paclitaxel. After receiving her second cycle of ddAC with pegfilgrastim, the patient reported five days of left shoulder and arm pain. Subsequent CT imaging demonstrated wall thickening and inflammatory changes surrounding the left subclavian artery, aortic arch, left carotid artery, proximal innominate arteries, and the mid internal carotid arteries and its branching vessels. These findings were extremely concerning for large-vessel vasculitis. Excluding CPI therapy and pegfilgrastim use, no additional inciting event or medication that the patient was exposed to was noted to be associated with large-vessel vasculitis. We present this case to report on this rare but severe complication from commonly utilized agents in cancer treatment. We also extend the possibility of large-vessel vasculitis development in relation to the COVID-19 vaccine due to shared ingredients found in both the vaccine and pegfilgrastim. It is important to outline the treatment used for such a complication as no standardized treatment has been established for large-vessel vasculitis caused by CPI therapy or pegfilgrastim use.

Uniformed Services and the Field Hospital Experience During Coronovirus Disease 2019 (SARS-CoV-2) Pandemic: Open to Closure in 30 Days With 1,100 Patients: The Javits New York Medical Station.

INTRODUCTION: The surge of SARS-CoV-2-virus infected (COVID-19) patients presenting to New York City (NYC) hospitals quickly overwhelmed and outnumbered the available acute care and intensive care resources in NYC in early March 2020. Upon the arrival of military medical assets to the Javits Convention Center in NYC, the planned mission to care for non-SARS-CoV-2 patients was immediately changed to manage patients with (SARS-CoV-2)COVID-19 and their comorbid conditions.Healthcare professionals from every branch of the uniformed services, augmented by state and local resources, staffed the Javits New York Medical Station (JNYMS) from April 2020. METHODS: The data review reported aggregated summary statistics and participant observations collected by N.Y. State and U.S. military officials. RESULTS: During the 28 days of patient intake at the JNYMS, 1,095 SARS-CoV-2-positive patients were transferred from NYC hospitals to the JNYMS. At its peak, the JNYMS accepted 119 patients in a single day, had a maximum census of 453, and had a peak intensive care unit census of 35. The median length of stay was 4.6 days (interquartile range: 3.1-6.9 days). A total of 103 patients were transferred back to local hospitals, and there were 6 deaths, with an overall mortality rate of 0.6% (95% CI, 0.3-1.2). DISCUSSION AND CONCLUSIONS: This is the first report of the care provided at the JNYMS. Within 2 weeks, this multi-agency effort was able to mobilize to care for over 1,000 SARS-CoV-2 patients with varying degrees of illness in a 1-month period. This was the largest field hospital mobilization in the U.S. medical history in response to a non-wartime pandemic. Its success with huge patient throughput including disposition and low mortality relieved critical overcrowding and supply deficiencies throughout NYC hospitals. The downstream impact likely saved additional hundreds of lives and reduced stress on the system during this healthcare crisis.

The proximity of the N- and C- termini of bovine knob domains enable engineering of target specificity into polypeptide chains.

Cysteine-rich knob domains can be isolated from the ultralong heavy-chain complementarity-determining region (CDR) 3, which are unique to a subset of bovine antibodies, to create antibody fragments of ~4 kDa. Advantageously, the N- and C- termini of these small binding domains are in close proximity, and we propose that this may offer a practical route to engineer extrinsic binding specificity into proteins. To test this, we transplanted knob domains into various loops of rat serum albumin, targeting sites that were distal to the interface with the neonatal Fc receptor. Using knob domains raised against the clinically validated drug target complement component C5, we produced potent inhibitors, which exhibit an extended plasma half-life in vivo via attenuated renal clearance and neonatal Fc receptor-mediated avoidance of lysosomal catabolism. The same approach was also used to modify a Camelid VHH, targeting a framework loop situated at the opposing end of the domain to the CDRs, to produce a small, single-chain bispecific antibody and a dual inhibitor of Complement C3 and C5. This study presents new protein inhibitors of the complement cascade and demonstrates a broadly applicable method to engineer target specificity within polypeptide chains, using bovine knob domains.

Selection, optimization, and pharmacokinetic properties of a novel, potent antiviral locked nucleic acid-based antisense oligomer targeting hepatitis C virus internal ribosome entry site.

We have screened 47 locked nucleic acid (LNA) antisense oligonucleotides (ASOs) targeting conserved (>95% homology) sequences in the hepatitis C virus (HCV) genome using the subgenomic HCV replicon assay and generated both antiviral (50% effective concentration [EC(50)]) and cytotoxic (50% cytotoxic concentration [CC(50)]) dose-response curves to allow measurement of the selectivity index (SI). This comprehensive approach has identified an LNA ASO with potent antiviral activity (EC(50) = 4 nM) and low cytotoxicity (CC(50) >880 nM) targeting the 25- to 40-nucleotide region (nt) of the HCV internal ribosome entry site (IRES) containing the distal and proximal miR-122 binding sites. LNA ASOs targeting previously known accessible regions of the IRES, namely, loop III and the initiation codon in loop IV, had poor SI values. We optimized the LNA ASO sequence by performing a 1-nucleotide walk through the 25- to 40-nt region and show that the boundaries for antiviral efficacy are extremely precise. Furthermore, we have optimized the format for the LNA ASO using different gapmer and mixomer patterns and show that RNase H is required for antiviral activity. We demonstrate that RNase H-refractory ASOs targeting the 25- to 40-nt region have no antiviral effect, revealing important regulatory features of the 25- to 40-nt region and suggesting that RNase H-refractory LNA ASOs can act as potential surrogates for proviral functions of miR-122. We confirm the antisense mechanism of action using mismatched LNA ASOs. Finally, we have performed pharmacokinetic experiments to demonstrate that the LNA ASOs have a very long half-life (>5 days) and attain hepatic maximum concentrations >100 times the concentration required for in vitro antiviral activity.

Implementing a Registry Federation for Materials Science Data Discovery.

As a result of a number of national initiatives, we are seeing rapid growth in the data important to materials science that are available over the web. Consequently, it is becoming increasingly difficult for researchers to learn what data are available and how to access them. To address this problem, the Research Data Alliance (RDA) Working Group for International Materials Science Registries (IMRR) was established to bring together materials science and information technology experts to develop an international federation of registries that can be used for global discovery of data resources for materials science. A resource registry collects high-level metadata descriptions of resources such as data repositories, archives, websites, and services that are useful for data-driven research. By making the collection searchable, it aids scientists in industry, universities, and government laboratories to discover data relevant to their research and work interests. We present the results of our successful piloting of a registry federation for materials science data discovery. In particular, we out a blueprint for creating such a federation that is capable of amassing a global view of all available materials science data, and we enumerate the requirements for the standards that make the registries interoperable within the federation. These standards include a protocol for exchanging resource descriptions and a standard metadata schema for encoding those descriptions. We summarize how we leveraged an existing standard (OAI-PMH) for metadata exchange. Finally, we review the registry software developed to realize the federation and describe the user experience.

Incidental Finding of a Large Right Atrial Thrombus in a Patient With Cerebral Lymphoma.

Right atrial (RA) masses are rare, challenging to diagnose, and potentially life-threatening with high mortality if untreated. We present a patient presenting with diffuse large B-cell lymphoma in the brain that was incidentally found to have a large RA mass. For a better definition of the RA mass, extensive workup using multimodality imaging including chest computed tomography, transthoracic echocardiography, transesophageal echocardiography, cardiac magnetic resonance imaging, and left heart catheterization was warranted. The imaging demonstrated a large RA mass extending through the tricuspid valve into the right ventricle and superior and inferior vena cava without a mobile component. The mass was then successfully resected, and further histology examination was performed to rule out lymphoma and rare subtypes of diffuse large B-cell lymphoma. The comprehensive workup proved the RA mass to be a calcified thrombus rather than a direct metastatic spread of lymphoma.