Off-Target In Vitro Profiling Demonstrates that Remdesivir Is a Highly Selective Antiviral Agent.

Remdesivir (RDV, GS-5734), the first FDA-approved antiviral for the treatment of COVID-19, is a single diastereomer monophosphoramidate prodrug of an adenosine analogue. It is intracellularly metabolized into the active triphosphate form, which in turn acts as a potent and selective inhibitor of multiple viral RNA polymerases. RDV has broad-spectrum activity against members of the coronavirus family, such as SARS-CoV-2, SARS-CoV, and MERS-CoV, as well as filoviruses and paramyxoviruses. To assess the potential for off-target toxicity, RDV was evaluated in a set of cellular and biochemical assays. Cytotoxicity was evaluated in a set of relevant human cell lines and primary cells. In addition, RDV was evaluated for mitochondrial toxicity under aerobic and anaerobic metabolic conditions, and for the effects on mitochondrial DNA content, mitochondrial protein synthesis, cellular respiration, and induction of reactive oxygen species. Last, the active 5'-triphosphate metabolite of RDV, GS-443902, was evaluated for potential interaction with human DNA and RNA polymerases. Among all of the human cells tested under 5 to 14 days of continuous exposure, the 50% cytotoxic concentration (CC50) values of RDV ranged from 1.7 to >20 µM, resulting in selectivity indices (SI, CC50/EC50) from >170 to 20,000, with respect to RDV anti-SARS-CoV-2 activity (50% effective concentration [EC50] of 9.9 nM in human airway epithelial cells). Overall, the cellular and biochemical assays demonstrated a low potential for RDV to elicit off-target toxicity, including mitochondria-specific toxicity, consistent with the reported clinical safety profile.

Key Metabolic Enzymes Involved in Remdesivir Activation in Human Lung Cells.

Remdesivir (RDV; GS-5734, Veklury), the first FDA-approved antiviral to treat COVID-19, is a single-diastereomer monophosphoramidate prodrug of an adenosine analogue. RDV is taken up in the target cells and metabolized in multiple steps to form the active nucleoside triphosphate (TP) (GS-443902), which, in turn, acts as a potent and selective inhibitor of multiple viral RNA polymerases. In this report, we profiled the key enzymes involved in the RDV metabolic pathway with multiple parallel approaches: (i) bioinformatic analysis of nucleoside/nucleotide metabolic enzyme mRNA expression using public human tissue and lung single-cell bulk mRNA sequence (RNA-seq) data sets, (ii) protein and mRNA quantification of enzymes in human lung tissue and primary lung cells, (iii) biochemical studies on the catalytic rate of key enzymes, (iv) effects of specific enzyme inhibitors on the GS-443902 formation, and (v) the effects of these inhibitors on RDV antiviral activity against SARS-CoV-2 in cell culture. Our data collectively demonstrated that carboxylesterase 1 (CES1) and cathepsin A (CatA) are enzymes involved in hydrolyzing RDV to its alanine intermediate MetX, which is further hydrolyzed to the monophosphate form by histidine triad nucleotide-binding protein 1 (HINT1). The monophosphate is then consecutively phosphorylated to diphosphate and triphosphate by cellular phosphotransferases. Our data support the hypothesis that the unique properties of RDV prodrug not only allow lung-specific accumulation critical for the treatment of respiratory viral infection such as COVID-19 but also enable efficient intracellular metabolism of RDV and its MetX to monophosphate and successive phosphorylation to form the active TP in disease-relevant cells.

Inflammatory signature in lung tissues in patients with combined pulmonary fibrosis and emphysema.

Background: The aetiology and inflammatory profile of combined pulmonary fibrosis and emphysema (CPFE) remain uncertain currently. Objective: We aimed to examine the levels of inflammatory proteins in lung tissue in a cohort of patients with emphysema, interstitial pulmonary fibrosis (IPF), and CPFE. Materials and methods: Explanted lungs were obtained from subjects with emphysema, IPF, CPFE, (or normal subjects), and tissue extracts were prepared. Thirty-four inflammatory proteins were measured in each tissue section. Results: The levels of all 34 proteins were virtually indistinguishable in IPF compared with CPFE tissues, and collectively, the inflammatory profile in the emphysematous tissues were distinct from IPF and CPFE. Moreover, inflammatory protein levels were independent of the severity of the level of diseased tissue. Conclusions: We find that emphysematous lung tissues have a distinct inflammatory profile compared with either IPF or CPFE. However, the inflammatory profile in CPFE lungs is essentially identical to lungs from patients with IPF. These data suggest that distinct inflammatory processes collectively contribute to the disease processes in patients with emphysema, when compared to IPF and CPFE.

Zonisamide Reduces Withdrawal Symptoms But Does Not Enhance Varenicline-Induced Smoking Cessation.

INTRODUCTION: Varenicline (Chantix) is a first-line treatment for smoking cessation but does not produce cessation in many individuals. It may be possible to improve abstinence by co-administering varenicline with other medications. Zonisamide (Zonegran) has a similar pharmacologic profile to topiramate, which has been shown to reduce smoking, but is better tolerated. This study evaluated whether combined zonisamide and varenicline reduced tobacco withdrawal and increased abstinence among smokers trying to quit, relative to varenicline and placebo. METHODS: This was a double-blind, randomized, placebo-controlled pilot trial of zonisamide + varenicline versus placebo + varenicline for smoking cessation. Smokers received brief counseling and study medications, and completed weekly assessments for 10 consecutive weeks. The primary outcome was continuous abstinence rates (biochemically verified) during the final 4 weeks of treatment. RESULTS: Results are presented as intent-to-treat and completer analyses. Seventy-four individuals were enrolled; 45 completed the study. Overall, 14.9% (intent-to-treat) and 25.0% (completer) of participants maintained sustained abstinence during the final 4 weeks of treatment. There were no differences between groups for biochemically-verified smoking, but zonisamide + varenicline reduced self-reported smoking, nicotine withdrawal, and craving compared to placebo + varenicline. CONCLUSIONS: Zonisamide decreased nicotine withdrawal and craving, though not of sufficient magnitude to modify smoking behavior. The sample size was small and low rates of abstinence across groups suggest the study population was difficult to treat. Additional evaluation of zonisamide or other medications that increase GABA or decrease glutamate in larger or more diverse populations may yield positive clinical benefit for nicotine/tobacco cessation. IMPLICATIONS: This study provides support for layering novel medications with varenicline for smoking cessation, for investigating medications that target the GABA and glutamate system, and for assessing the contribution that reductions in nicotine withdrawal have on ultimate cessation outcomes.

Multiplanar MDCT measurement of esophageal hiatus surface area: association with hiatal hernia and GERD.

BACKGROUND: Accurate measurement of esophageal hiatus size is clinically important, especially when antireflux surgery is planned. We present a novel method for in vivo measurement of esophageal hiatal surface area using MDCT multiplanar reconstruction. We aimed to determine whether large hiatal area is associated with hiatal hernia and gastroesophageal reflux disease. METHODS: We retrospectively analyzed subjects prospectively enrolled in the COPDGene(®) project. We created two test groups, one with hiatal hernia on chest CT and one with GERD on medical treatment identified by history without hernia. Matched control groups were formed. We performed CT postprocessing to define the double-oblique plane of the esophageal hiatus, on which the hiatal surface area is manually traced. RESULTS: Subjects with hernia (n = 48) had larger mean hiatus areas than matched controls (6.9 vs. 2.5 cm(2), p < 0.0001), and were more likely to have GERD (42 vs. 10 %, p < 0.0005). Subjects with mixed (type III) hernias had larger hiatuses compared to subjects with sliding (type I) hernias, who, in turn, had larger hiatuses than subjects without hernia (p < 0.0001). Hernia-negative subjects with GERD (n = 55) did not have significantly larger mean hiatal areas compared to matched controls (3.0 vs. 2.5 cm(2), p = 0.12). Twenty measurements obtained by two radiologists showed correlation of 0.93, with mean difference of 0.5 cm(2) (p = 0.20). CONCLUSIONS: We devised a method to measure in vivo esophageal hiatal surface area using MDCT reconstruction and established the normal size range for the first time. This methodology has the potential to guide decision-making in antireflux surgery technique preoperatively, and assess surgical result postoperatively. The presence of hernia correlated with large hiatuses and GERD. However, hiatal area failed to identify those with GERD in the absence of hiatal hernia.

Integrating Palliative Care into Critical Care: A Quality Improvement Study.

BACKGROUND: Many terminally ill patients experience an increasing intensity of medical care, an escalation frequently not consistent with their preferences. In 2009, formal palliative care consultation (PCC) was integrated into our medical intensive care unit (ICU). We hypothesized that significant differences in clinical and economic outcomes exist between ICU patients who received PCC and those who did not. METHODS: We reviewed ICU admissions between July and October 2010, identified 41 patients who received PCC, and randomly selected 80 patients who did not. We measured clinical outcomes and economic variables associated with patients' ICU courses. RESULTS: Patients in the PCC group were older (average 64 years, standard deviation [SD] 19.2 vs 55.6 years, SD 14.5; P = .021) and sicker (median Acute Physiology and Chronic Health Evaluation IV score 85.5, interquartile range [IQR] 60.5-107.5 vs 60, IQR 39.2-74.75; P < .001) than the non-PCC controls. PCC patients received significantly more total days of ICU care on average (8 days, IQR 4-15 vs 4 days, IQR 2-7; P < .001), had more ICU admissions, and were more likely to die during their ICU stay (64.3% vs 12.5%, P < .001). Median total hospital charges per patient attributable to ICU care were higher in the PCC group than in the controls (US$315,493, IQR US$156,470-US$486,740 vs US$116,934, IQR US$54,750-US$288,660; P < .001). After we adjusted for ICU length of stay, we found that median ICU charges per day per patient did not differ significantly between the groups (US$37,463, IQR US$27,429-US$56,230 vs US$41,332, IQR US$30,149-US$63,288; P = .884). Median time to PCC during the ICU stay was 7 days (IQR 2-14.5 days). CONCLUSIONS: Patients who received PCC had higher disease acuity, longer ICU lengths of stay, and higher ICU mortality than controls. "Trigger" programs in the ICU may improve utilization of PCC services, improve patient comfort, and reduce invasive, often futile end-of-life care.

Designing Effective Mentorship for Underrepresented Faculty in Academic Medicine.

BACKGROUND AND OBJECTIVES: A dearth of training and resources exists for mentors to address the unique needs of faculty from racial/ethnic groups that are underrepresented in medicine (URiM). Mentoring Underrepresented Faculty for Academic Excellence (MUFAE) was a multi-institutional mentoring program designed to provide mentors where there were none. METHODS: In 2020, 25 early career URiM faculty mentees each were paired with advanced faculty, and pairs met individually for monthly calls for 1 year. Mentees completed pre- and postassessment surveys regarding their experience in the program. Mentees and mentors also participated in virtual group check-ins where they gave feedback on their experience to program leaders while also networking with fellow participants. RESULTS: Twenty-two of the 25 mentor-mentee pairs (88%) completed the program, and 17 of the 22 (77%) mentees completed the pre- and postsurveys. Survey responses showed significant increases in mentees reports of feeling they received mentorship focused on their needs as URiM faculty members, feeling equipped to advance in their careers, and feeling supported in their efforts to complete antiracism/health-equity programs. Feedback at the check-ins indicated that URiM mentors appreciated the opportunities to talk about their own frustrations and that White mentors appreciated having an increased understanding of challenges that their URIM colleagues faced. CONCLUSIONS: MUFAE is a model for academic societies to address the lack of mentors for URiM faculty. Mentees and mentors found the experience a meaningful one that fills a need in academic mentoring.

Inhaled remdesivir reduces viral burden in a nonhuman primate model of SARS-CoV-2 infection.

Remdesivir (RDV) is a nucleotide analog prodrug with demonstrated clinical benefit in patients with coronavirus disease 2019 (COVID-19). In October 2020, the US FDA approved intravenous (IV) RDV as the first treatment for hospitalized COVID-19 patients. Furthermore, RDV has been approved or authorized for emergency use in more than 50 countries. To make RDV more convenient for non-hospitalized patients earlier in disease, alternative routes of administration are being evaluated. Here, we investigated the pharmacokinetics and efficacy of RDV administered by head dome inhalation in African green monkeys (AGM). Relative to an IV administration of RDV at 10 mg/kg, an approximately 20-fold lower dose administered by inhalation produced comparable concentrations of the pharmacologically active triphosphate in lower respiratory tract tissues. Distribution of the active triphosphate into the upper respiratory tract was also observed following inhaled RDV exposure. Inhalation RDV dosing resulted in lower systemic exposures to RDV and its metabolites as compared with IV RDV dosing. An efficacy study with repeated dosing of inhaled RDV in an AGM model of SARS-CoV-2 infection demonstrated reductions in viral replication in bronchoalveolar lavage fluid and respiratory tract tissues compared with placebo. Efficacy was observed with inhaled RDV administered once daily at a pulmonary deposited dose of 0.35 mg/kg beginning approximately 8 hours post-infection. Moreover, the efficacy of inhaled RDV was similar to that of IV RDV administered once at 10 mg/kg followed by 5 mg/kg daily in the same study. Together, these findings support further clinical development of inhalation RDV.

Prodrugs of a 1'-CN-4-Aza-7,9-dideazaadenosine C-Nucleoside Leading to the Discovery of Remdesivir (GS-5734) as a Potent Inhibitor of Respiratory Syncytial Virus with Efficacy in the African Green Monkey Model of RSV.

A discovery program targeting respiratory syncytial virus (RSV) identified C-nucleoside 4 (RSV A2 EC50 = 530 nM) as a phenotypic screening lead targeting the RSV RNA-dependent RNA polymerase (RdRp). Prodrug exploration resulted in the discovery of remdesivir (1, GS-5734) that is >30-fold more potent than 4 against RSV in HEp-2 and NHBE cells. Metabolism studies in vitro confirmed the rapid formation of the active triphosphate metabolite, 1-NTP, and in vivo studies in cynomolgus and African Green monkeys demonstrated a >10-fold higher lung tissue concentration of 1-NTP following molar normalized IV dosing of 1 compared to that of 4. A once daily 10 mg/kg IV administration of 1 in an African Green monkey RSV model demonstrated a >2-log10 reduction in the peak lung viral load. These early data following the discovery of 1 supported its potential as a novel treatment for RSV prior to its development for Ebola and approval for COVID-19 treatment.

Species differences in liver accumulation and metabolism of nucleotide prodrug sofosbuvir.

Sofosbuvir (SOF) is a nucleotide prodrug which has been used as a backbone for the clinical treatment of hepatitis C viral infection. Because sofosbuvir undergoes complex first pass metabolism, including metabolic activation to form its pharmacologically active triphosphate (GS-331007-TP) to inhibit the viral RNA polymerase in the liver, it is difficult to project the human dose for clinical evaluation based on preclinical data. Selecting an appropriate animal model for drug exposure in the target tissue is challenging due to differences in absorption, stability, hepatic uptake, and intracellular activation across species. Efficient liver delivery has been established in human liver following administration in a clinical trial of patients receiving sofosbuvir prior to liver transplantation. Using the clinical liver exposure as a benchmark, we assessed and compared the pharmacokinetic profile in mouse, rat, hamster, dog and monkey. Liver accumulation was also assessed in the PXB mouse model in which the liver is mostly populated with human hepatocytes. At human equivalent dose, the hepatic concentrations of GS-331007-TP in dog and PXB mouse were comparable to those observed in the human livers. In these species, high and sustained levels of GS-331007-TP were observed in both primary hepatocytes in vitro and the liver in vivo.

Pocket Reference Card Improves Pediatric Resident Comfort in Caring for Children at End of Life.

BACKGROUND: Studies have shown that pediatricians in all stages of training are uncomfortable managing patients at end of life. Our goal was to create and test a portable reference card to improve pediatric resident education in comprehensive care for children nearing end of life. METHODS: We evaluated the impact of the Pediatric End-of-Life Care Management Reference Card on residents' perceived comfort and knowledge through pre- and post-intervention surveys. The preintervention questionnaires and pocket cards were distributed to all first- and second-year residents, and then a follow-up survey was provided six months later. Based on Likert scales, questions focused on self-reported understanding of palliative care principles and knowledge regarding and comfort in performing end-of-life symptom management. RESULTS: Twenty-six pediatric residents completed pre- and post-intervention surveys. Following receipt of the reference card, no significant changes were noted consistently across all groups of residents. The majority of improvements were noted when comparing second to third year residents, including knowledge and comfort related to pain management, comfort in managing secretions and nausea, and documentation following death. The first to second year residents demonstrated improvement in knowing what language to use to tell a family that their child has died. CONCLUSION: This study demonstrates that a portable reference card may be a convenient, simple, and useful component of education for pediatric residents in end-of-life care management. This reference card is a foundation from which to develop a standardized educational tool. Additional research is required to assess the impact of this type of intervention in pediatric palliative care education.

Laser acupuncture reduces pain in pediatric kidney biopsies: a randomized controlled trial.

Evaluate laser acupuncture (LA) as an adjuvant therapy in pain management during percutaneous kidney biopsy procedure in children and adolescents. This prospective, double-blinded, randomized controlled trial enrolled patients aged 7 to 26 years admitted to a children's hospital for percutaneous kidney biopsy. Patients received LA to treatment points (acupuncture group) or sham points (control group) before the procedure. The laser delivered a dose of 42 J/cm over 10 acupoints. Patients and parents rated the pain during and after the biopsy, and change in pain scores were calculated for each patient. Anxiety, vital signs, sedation medication, and patient's biopsy experience were secondary outcomes. Sixty-nine treatments (33 in the acupuncture group and 36 in the control group) were eligible for analysis. Patients in the acupuncture group reported a significantly improved change in the pain score after the biopsy compared with the controls (0.8 vs -0.5, P = 0.044). Patients in the acupuncture group had a statistically significant decrease in procedure vital signs including heart rate (-1.8 vs 5.6, P = 0.043) and respiratory rate (-2.4 vs 0.4, P = 0.045) when compared with controls. Parents also perceived a correspondingly greater improvement in their child's pain for those in the acupuncture group compared with the controls (2.3 vs 0.3, P = 0.04). Adjunctive LA significantly improved pain after pediatric percutaneous kidney biopsies.

Hiatal Hernia on Chest High-Resolution Computed Tomography and Exacerbation Rates in COPD Individuals.

Background: Gastroesophageal reflux disease (GERD) is associated with frequent chronic obstructive pulmonary disease (COPD) exacerbations. Hiatal hernia (HH) contributes to GERD pathogenesis and is identifiable on chest high-resolution computed tomography (HRCT). We hypothesize that the presence of an HH on HRCT identifies those at increased risk for acute exacerbation of COPD. Methods: We retrospectively reviewed a prospectively enrolled cohort of smokers with and without airflow obstruction. HHs were identified visually on inspiratory HRCT. Individuals' demographic and clinical information was compared with secondary analysis performed using a propensity score generated matched cohort. Results: There were 523 COPD individuals and 607 unobstructed smokers. COPD individuals had more HHs than unobstructed smokers, (11.6% versus 6.1%, p < 0.001). COPD individuals with hernias were older, female, overweight and GERD positive as compared to those without hernia. There was no difference in self-reported exacerbation rates or hospitalizations per year, but similar severity of obstruction, smoking rates and long-term oxygen use. Analysis with the matched cohort revealed no significant difference in exacerbation rates. Conclusions: Presence of HHs on inspiratory HRCT scan did not predict worse symptoms or exacerbation rate in COPD individuals. Those with HHs were older, more obese, and predominantly female compared to those without HHs.

Revealing neuronal circuitry using stem cell-derived neurons.

Mouse embryonic stem cell (mESC)-derived neurons are a renewable cell source for investigation of neuronal circuits. Engineering circuit-tracing components into stem cells facilitates studies on mechanisms of synaptic coupling and circuitogenesis. This unit details methods for the generation of mESC-derived neurons harboring trans-synaptic viral tracing elements, which are used for investigation of synaptic connections within circuits in vitro, ex vivo, and in vivo. The first protocol describes procedures for feeder-free passaging of mESCs, modified to carry reporter and rabies virus tracing elements. The second protocol describes in vitro generation of neurons from these ESCs. The last protocols describe the use of ESC-derived neurons as "source cells" for rabies virus circuit-tracing to identify inputs onto synaptically connected neurons. Given the broad applicability, these protocols can be applied to investigate the ability of in vitro-derived neurons to establish/maintain synaptic connections in disease models, and/or with human-induced pluripotent stem cells.

The microbiology of postobstructive pneumonia in lung cancer patients.

Recurrent pneumonias often occur in the setting of an airway obstruction and can be the presenting symptom of an undiagnosed malignancy. Little is known regarding the microbiology of these pneumonias making antibiotic therapy difficult to direct; however, the few studies available show these pneumonias to be polymicrobial. Examining the colonization patterns of at-risk populations such as patients with chronic obstructive pulmonary disease and using techniques such as ultrasound and computed tomography-guided biopsies may help in the treatment of these pneumonias. The following review is presented to highlight the current medical knowledge as well as suggest areas for future evaluation.

Genetic strategies to investigate neuronal circuit properties using stem cell-derived neurons.

The mammalian brain is anatomically and functionally complex, and prone to diverse forms of injury and neuropathology. Scientists have long strived to develop cell replacement therapies to repair damaged and diseased nervous tissue. However, this goal has remained unrealized for various reasons, including nascent knowledge of neuronal development, the inability to track and manipulate transplanted cells within complex neuronal networks, and host graft rejection. Recent advances in embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) technology, alongside novel genetic strategies to mark and manipulate stem cell-derived neurons, now provide unprecedented opportunities to investigate complex neuronal circuits in both healthy and diseased brains. Here, we review current technologies aimed at generating and manipulating neurons derived from ESCs and iPSCs toward investigation and manipulation of complex neuronal circuits, ultimately leading to the design and development of novel cell-based therapeutic approaches.