Site-Selective Electrochemical Arene C-H Amination.

Here we present the discovery and development of a highly selective aromatic C-H amination reaction. This electrochemical strategy involves a cathodic reduction process that generates highly electrophilic dicationic N-centered radicals that can efficiently engage in aromatic C-H functionalization and channel the regioselectivity of the aromatic substitution. The nitrogen-radical cation-pi interaction with arenes used throughout nature leads to a charge transfer mechanism, with subsequent aromatic C-N bond formation. This electrochemical process generates aryl DABCOnium salts in excellent yields and regioselectivities (single regioisomer in most cases). The scope of the reaction on arene is broad where various functionalities such as aryl halides (bromides, chlorides, fluorides), carbonyls (ketones, esters, imides), sulfonamides, and heteroarenes (pyridines, bipyridines, and terpyridines) are well tolerated. Moreover, we disclose the synthetic utility of the aryl DABCOnium salt adducts leading to the direct access of diverse aryl piperazines and the chemoselective cleavage of the exocyclic aryl C(sp2)-N bond over electrophilic C(sp3)-N+ bonds via photoredox catalysis to afford synthetically useful aryl radicals that can engage in aryl C-C and C-P bond formation.

Unmasking the Silent Threat: Periodontal Health's Impact on COPD Severity and Hospitalization.

OBJECTIVE: This study investigated the relationship between chronic obstructive pulmonary disease (COPD) and periodontitis, focusing on how periodontal health impacts COPD airflow limitation, exacerbations, and hospitalization. BACKGROUND: Periodontitis, a multifactorial inflammatory disease, is characterized by destruction of tooth-supporting structures, while COPD is a global pulmonary disorder with high mortality. METHODS: A total of 199 COPD patients aged over 40 years underwent lung function tests (spirometry), 6 min walk test, and St George's Respiratory Questionnaire-COPD (SGRQ-C) to assess lung health. Periodontal indices such as probing depth (PD), clinical attachment loss (CAL), and plaque index (PI) were assessed. RESULTS: We found a significant negative correlation between periodontal disease severity and lung function (lower FEV1, FVC, and FEV1/FVC ratio) after adjusting for smoking. Likewise, periodontal parameters (PPD, PI, and CAL) exhibited negative correlations with lung function. These periodontal indices were independently associated with airflow limitation severity, exacerbations frequency, and prior-year hospitalization. Linear regression indicated that each unit increase in PPD, PI, and CAL corresponded to estimated increases in GOLD airflow limitation grading (0.288, 0.718, and 0.193, respectively) and number of exacerbations (0.115, 0.041, and 0.109, respectively). In logistic regression, PPD, PI, and CAL adjusted odds ratios (ORs) were estimated to increase by 1.29 (95%CI: 1.03-1.62), 3.04 (95%CI: 1.28-7.2), and 1.26 (95%CI: 1.06-1.49), respectively, for hospitalization in previous year. CONCLUSION: Periodontitis is associated with COPD airflow limitation, exacerbation, and hospitalization, with PI being the most clinically relevant periodontal factor. Dentists and physicians should monitor and increase awareness among COPD patients to maintain oral hygiene for prevention of periodontal diseases and mitigate its effect on COPD progression.

The Validity of the ROX Index and APACHE II in Predicting Early, Late, and Non-Responses to Non-Invasive Ventilation in Patients with COVID-19 in a Low-Resource Setting.

The use of the Ratio of Oxygen Saturation (ROX) index to predict the success of high-flow nasal oxygenation (HFNO) is well established. The ROX can also predict the need for intubation, mortality, and is easier to calculate compared with APACHE II. In this prospective study, the primary aim is to compare the ROX (easily administered in resource limited setting) to APACHE II for clinically relevant outcomes such as mortality and the need for intubation. Our secondary aim was to identify thresholds for the ROX index in predicting outcomes such as the length of ICU stay and failure of non-invasive respiratory support therapies and to assess the effectiveness of using the ROX (day 1 at admission, day 2, and day 3) versus Acute physiology and chronic health evaluation (APACHE) II scores (at admission) in patients with Coronavirus Disease 2019 (COVID-19) pneumonia and Acute Respiratory Distress Syndrome (ARDS) to predict early, late, and non-responders. After screening 208 intensive care unit patients, a total of 118 COVID-19 patients were enrolled, who were categorized into early (n = 38), late (n = 34), and non-responders (n = 46). Multinomial logistic regression, receiver operating characteristic (ROC), Multivariate Cox regression, and Kaplan-Meier analysis were conducted. Multinomial logistic regressions between late and early responders and between non- and early responders were associated with reduced risk of treatment failures. ROC analysis for early vs. late responders showed that APACHE II on admission had the largest area under the curve (0.847), followed by the ROX index on admission (0.843). For responders vs. non-responders, we found that the ROX index on admission had a slightly better AUC than APACHE II on admission (0.759 vs. 0.751). A higher ROX index on admission [HR (95% CI): 0.29 (0.13-0.52)] and on day 2 [HR (95% CI): 0.55 (0.34-0.89)] were associated with a reduced risk of treatment failure. The ROX index can be used as an independent predictor of early response and mortality outcomes to HFNO and NIV in COVID-19 pneumonia, especially in low-resource settings, and is non-inferior to APACHE II.

Cochlear implantation in unilateral hearing loss: impact of short- to medium-term auditory deprivation.

Introduction: Single sided deafness (SSD) results in profound cortical reorganization that presents clinically with a significant impact on sound localization and speech comprehension. Cochlear implantation (CI) has been approved for two manufacturers' devices in the United States to restore bilateral function in SSD patients with up to 10 years of auditory deprivation. However, there is great variability in auditory performance and it remains unclear how auditory deprivation affects CI benefits within this 10-year window. This prospective study explores how measured auditory performance relates to real-world experience and device use in a cohort of SSD-CI subjects who have between 0 and 10 years of auditory deprivation. Methods: Subjects were assessed before implantation and 3-, 6-, and 12-months post-CI activation via Consonant-Nucleus-Consonant (CNC) word recognition and Arizona Biomedical Institute (AzBio) sentence recognition in varying spatial speech and noise presentations that simulate head shadow, squelch, and summation effects (S0N0, SSSDNNH, SNHNSSD; 0 = front, SSD = impacted ear, NH = normal hearing ear). Patient-centered assessments were performed using Tinnitus Handicap Inventory (THI), Spatial Hearing Questionnaire (SHQ), and Health Utility Index Mark 3 (HUI3). Device use data was acquired from manufacturer software. Further subgroup analysis was performed on data stratified by <5 years and 5-10 years duration of deafness. Results: In the SSD ear, median (IQR) CNC word scores pre-implant and at 3-, 6-, and 12-months post-implant were 0% (0-0%), 24% (8-44%), 28% (4-44%), and 18% (7-33%), respectively. At 6 months post-activation, AzBio scores in S0N0 and SSSDNNH configurations (n = 25) demonstrated statistically significant increases in performance by 5% (p = 0.03) and 20% (p = 0.005), respectively. The median HUI3 score was 0.56 pre-implant, lower than scores for common conditions such as anxiety (0.68) and diabetes (0.77), and comparable to stroke (0.58). Scores improved to 0.83 (0.71-0.91) by 3 months post-activation. These audiologic and subjective benefits were observed even in patients with longer durations of deafness. Discussion: By merging CI-associated changes in objective and patient-centered measures of auditory function, our findings implicate central mechanisms of auditory compensation and adaptation critical in auditory performance after SSD-CI and quantify the extent to which they affect the real-world experience reported by individuals.

The Use of High-Flow Nasal Cannula and Non-Invasive Mechanical Ventilation in the Management of COVID-19 Patients: A Prospective Study.

High-flow nasal cannula (HFNC) and ventilator-delivered non-invasive mechanical ventilation (NIV) were used to treat acute respiratory distress syndrome (ARDS) due to COVID-19 pneumonia, especially in low- and middle-income countries (LMICs), due to lack of ventilators and manpower resources despite the paucity of data regarding their efficacy. This prospective study aimed to analyse the efficacy of HFNC versus NIV in the management of COVID-19 ARDS. A total of 88 RT-PCR-confirmed COVID-19 patients with moderate ARDS were recruited. Linear regression and generalized estimating equations (GEEs) were used for trends in vital parameters over time. A total of 37 patients were on HFNC, and 51 were on NIV. Patients in the HFNC group stayed slightly but not significantly longer in the ICU as compared to their NIV counterparts (HFNC vs. NIV: 8.00 (4.0-12.0) days vs. 7.00 (2.0-12.0) days; p = 0.055). Intubation rates, complications, and mortality were similar in both groups. The switch to HFNC from NIV was 5.8%, while 37.8% required a switch to NIV from HFNC. The resolution of respiratory alkalosis was better with NIV. We conclude that in patients with COVID-19 pneumonia with moderate ARDS, the duration of treatment in the ICU, intubation rate, and mortality did not differ significantly with the use of HFNC or NIV for respiratory support.

Circulatory Serum Krebs von Den Lungen-6 and Surfactant Protein-D Concentrations Predict Interstitial Lung Disease Progression and Mortality.

There is a need for biomarkers to predict outcomes, including mortality, in interstitial lung disease (ILD). Krebs von den Lungen-6 (KL-6) and surfactant protein D (SP-D) are associated with lung damage and fibrosis in all ILDs and are related to important clinical outcomes. Though these two biomarkers have been associated with ILD outcomes, there are no studies that have evaluated their predictive potential in combination. This study aims to determine whether KL-6 and SP-D are linked to poor disease outcomes and mortality. Additionally, we plan to examine whether changes in KL-6 and SP-D concentrations correspond with changes in lung function and whether serial measurements improve their predictive potential to identify disease progression and mortality. Forty-four patients with ILD participated in a prospective 6-month longitudinal observational study. ILD patients who succumbed had the highest KL-6 levels (3990.4 U/mL (3490.0-4467.6)) and highest SP-D levels (256.1 ng/mL (217.9-260.0)), followed by those who deteriorated: KL-6 levels 1357.0 U/mL (822.6-1543.4) and SP-D levels 191.2 ng/mL (152.8-210.5). The generalized linear model (GLM) analysis demonstrated that changes in forced vital capacity (FVC), diffusing capacity of lungs for carbon monoxide (DLCO), forced expiratory volume in 1 s (FEV1), and partial pressure of arterial oxygen (PaO2) were correlated to changes in KL6 (p = 0.016, 0.014, 0.027, 0.047) and SP-D (p = 0.008, 0.012, 0.046, 0.020), respectively. KL-6 (odds ratio (OR): 2.87 (1.06-7.79)) and SPD (OR: 1.76 (1.05-2.97)) were independent predictors of disease progression, and KL-6 (hazard ratio (HR): 3.70 (1.46-9.41)) and SPD (HR: 2.58 (1.01-6.59)) were independent predictors of death by Cox regression analysis. Combined biomarkers (KL6 + SPD + CT + FVC) had the strongest ability to predict disease progression (AUC: 0.797) and death (AUC: 0.961), on ROC analysis. Elevated KL-6 and SPD levels are vital biomarkers for predicting the severity, progression, and outcomes of ILD. High baseline levels or an increase in levels over a six-month follow-up despite treatment indicate a poor prognosis. Combining KL6 and SPD with conventional measures yields a more potent prognostic indicator. Clinical studies are needed to test additional interventions, and future research will determine if this combined biomarker benefits different ethnicities globally.

Allergic sensitization to foods in India and other Low-Middle-income countries.

Food allergy is an important cause of morbidity, significantly affecting the quality of life of the sufferer. Most food allergy research has been undertaken in high-income countries. Here, we summarize literature regarding food allergy in India and other low-middle-income countries (LMIC). We provide summaries of self-reported adverse food reactions and food sensitization in these regions by reviewing published community-based studies of prevalence, burden, and risk factors. We identified 2 community-based studies of food allergy prevalence in Karnataka, India, which estimate that food allergy affects just 0.14% of children and 1.2% of adults. The overall prevalence of allergic sensitization to 'any' food was 26.5% in adults and 19.1% in children by serum-specific IgE; but only 4.48% in children by skin prick test. We identified a further 28 studies in other LMICs, mainly from China but also Turkey, South Africa, Ghana, Mexico, Brazil, Thailand, Philippines, and Korea. The overall prevalence of allergic sensitization to 'any' food ranged from 0.11% to 16.8% in children using serum-specific IgE and 0.14% to 9.6% in children by skin prick test. The questionnaires and skin prick testing materials used and number of allergens tested varied significantly between studies. Other than Karnataka, there is no information on prevalence of food sensitization and probable food allergy in the community in India. Similar lack of information is noted among the majority of the 136 LMICs. Where community-based studies have been undertaken, there is wide variation in the prevalence and patterns of food sensitization across different LMICs, at least partly due to variations in study methodology. International collaboration is required in order to formally assess food allergy prevalence and burden across representative samples from multiple LMICs.

Association between ADAM33 Single-Nucleotide Polymorphisms and Treatment Response to Inhaled Corticosteroids and a Long-Acting Beta-Agonist in Asthma.

ADAM33 has been linked to airway structural changes in patients with asthma, leading to airway hyperresponsiveness, narrowing, and ultimately poor treatment responsiveness. This study aimed to evaluate the genetic association of ADAM33 SNPs with asthma, disease severity, and treatment responsiveness to ICS+LABA in the South Indian population. In this case-control study (486 controls and 503 cases), we performed genotyping using MassArray for six SNPs of ADAM33, namely rs2280091, rs2787094, rs3918396, rs67044, rs2853209, and rs3918392. We studied the association with asthma and treatment responsiveness to ICS+LABA, using genotype, allele frequency distribution, and haplotype analysis. A significant clinical finding of the study was that certain patients in the disease severity group (moderate and mild) showed poor or no improvement after a three-month follow-up of regular ICS+LABA therapy. Of the studied ADAM33 SNPs, rs2853209 showed an association with asthma. The further analysis of asthma patients according to disease severity suggested an association between moderate disease and the minor allele "T" for rs2853209. The homozygous minor allele of SNP rs2787094 was found to be associated with poorer lung function and the least lung-function improvement after three months of ICS+LABA therapy. The haplotype analysis of six SNPs showed a significant association between the rs2853209 and rs3918396 blocks and asthma. ADAM33 gene polymorphism has clinical relevance in terms of disease association and response to treatment. SNP rs2853209 seemed most relevant to asthma, and SNP rs2787094 could be a genetic marker for predicting response to ICS+LABA therapy in the study population.

The Assessment of Serum Fibronectin Levels as a Potential Biomarker for the Severity of Drug-Sensitive Pulmonary Tuberculosis: A Pilot Study.

BACKGROUND: Tuberculosis (TB) is a global health burden caused by Mycobacterium tuberculosis (Mtb) infection. Fibronectin (Fn) facilitates Mtb attachment to host cells. We studied the Fn levels in smear-positive TB patients to assess its correlation with disease severity based on sputum smears and chest X-rays. METHODS: Newly detected consecutive sputum AFB-positive pulmonary TB patients (n = 78) and healthy control subjects (n = 11) were included. The mycobacterial load in the sputum smear was assessed by IUATLD classification, ranging from 0 to 3. The severity of pulmonary involvement was assessed radiologically in terms of both the number of zones involved (0-6) and as localized (up to 2 zones), moderate (3-4 zones), or extensive (5-6 zones). The serum human fibronectin levels were measured by using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Catalogue No: CK-bio-11486, Shanghai Coon Koon Biotech Co., Ltd., Shanghai, China). RESULTS: The PTB patients showed lower Fn levels (102.4 ± 26.7) compared with the controls (108.8 ± 6.8), but they were not statistically significant. Higher AFB smear grades had lower Fn levels. The chest X-ray zones involved were inversely correlated with Fn levels. The Fn levels, adjusted for age and gender, decreased with increased mycobacterial load and the number of chest radiograph zones affected. A Fn level <109.39 g/mL predicted greater TB severity (sensitivity of 67.57% and specificity of 90.38%), while a level <99.32 pg/mL predicted severity based on the chest radiology (sensitivity of 84.21% and specificity of 100%). CONCLUSIONS: The Fn levels are lower in tuberculosis patients and are negatively correlated with severity based on sputum mycobacterial load and chest radiographs. The Fn levels may serve as a potential biomarker for assessing TB severity, which could have implications for early diagnosis and treatment monitoring.

Impact of Acute Exacerbation and Its Phenotypes on the Clinical Outcomes of Chronic Obstructive Pulmonary Disease in Hospitalized Patients: A Cross-Sectional Study.

Acute exacerbations of COPD (AECOPD) are clinically significant events having therapeutic and prognostic consequences. However, there is a lot of variation in its clinical manifestations described by phenotypes. The phenotypes of AECOPD were categorized in this study based on pathology and exposure. In our cross-sectional study, conducted between 1 January 2016 to 31 December 2020, the patients were categorized into six groups based on pathology: non-bacterial and non-eosinophilic; bacterial; eosinophilic; bacterial infection with eosinophilia; pneumonia; and bronchiectasis. Further, four groups were classified based on exposure to tobacco smoke (TS), biomass smoke (BMS), both, or no exposure. Cox proportional-hazards regression analyses were performed to assess hazard ratios, and Kaplan-Meier analysis was performed to assess survival, which was then compared using the log-rank test. The odds ratio (OR) and independent predictors of ward admission type and length of hospital stay were assessed using binomial logistic regression analyses. Of the 2236 subjects, 2194 were selected. The median age of the cohort was 67.0 (60.0 to 74.0) and 75.2% were males. Mortality rates were higher in females than in males (6.2% vs. 2.3%). AECOPD-B (bacterial infection) subjects [HR 95% CI 6.42 (3.06-13.46)], followed by AECOPD-P (pneumonia) subjects [HR (95% CI: 4.33 (2.01-9.30)], were at higher mortality risk and had a more extended hospital stay (6.0 (4.0 to 9.5) days; 6.0 (4.0 to 10.0). Subjects with TS and BMS-AECOPD [HR 95% CI 7.24 (1.53-34.29)], followed by BMS-AECOPD [HR 95% CI 5.28 (2.46-11.35)], had higher mortality risk. Different phenotypes have different impacts on AECOPD clinical outcomes. A better understanding of AECOPD phenotypes could contribute to developing an algorithm for the precise management of different phenotypes.

Trends in the Bacterial Prevalence and Antibiotic Resistance Patterns in the Acute Exacerbation of Chronic Obstructive Pulmonary Disease in Hospitalized Patients in South India.

Exacerbation due to antimicrobial-drug-resistant bacteria among chronic obstructive pulmonary disease (AECOPD) patients contributes to mortality and morbidity. We examined the prevalence of the bacterial organisms and trends in drug resistance in AECOPD. In this retrospective study, between January 2016 to December 2020, among 3027 AECOPD patients, 432 (14.3%) had bacteria isolated. The regression and generalized estimating equations (GEE) were used for trends in the resistance patterns over five years, adjusting for age, gender, and comorbidities. Klebsiella pneumoniae (32.4%), Pseudomonas aeruginosa (17.8%), Acinetobacter baumannii (14.4%), Escherichia coli (10.4%), and Staphylococcus aureus (2.5%) were common. We observed high levels of drug resistance in AECOPD patients admitted to ICU (87.8%) and non-ICU (86.5%). A Cox proportional hazard analysis, observed infection with Acinetobacter baumannii and female sex as independent predictors of mortality. Acinetobacter baumannii had 2.64 (95% confidence interval (CI): 1.08−6.43) higher odds of death, compared to Klebsiella pneumoniae. Females had 2.89 (95% CI: 1.47−5.70) higher odds of death, compared to males. A high proportion of bacterial AECOPD was due to drug-resistant bacteria. An increasing trend in drug resistance was observed among females.

TMEM106B deficiency impairs cerebellar myelination and synaptic integrity with Purkinje cell loss.

TMEM106B, a type II lysosomal transmembrane protein, has recently been associated with brain aging, hypomyelinating leukodystrophy, frontotemporal lobar degeneration (FTLD) and several other brain disorders. TMEM106B is critical for proper lysosomal function and TMEM106B deficiency leads to myelination defects, FTLD related pathology, and motor coordination deficits in mice. However, the physiological and pathological functions of TMEM106B in the brain are still not well understood. In this study, we investigate the role of TMEM106B in the cerebellum, dysfunction of which has been associated with FTLD and other brain disorders. We found that TMEM106B is ubiquitously expressed in neurons in the cerebellum, with the highest levels in the Purkinje neurons. Aged TMEM106B-deficient mice show significant loss of Purkinje neurons specifically in the anterior lobe of the cerebellum. Increased microglia and astrocyte activation, as well as an accumulation of ubiquitinated proteins, p62 and TDP-43 were also detected in the cerebellum of aged TMEM106B deficient mice. In the young mice, myelination defects and a significant loss of synapses between Purkinje and deep cerebellar nuclei neurons were observed. Interestingly, TMEM106B deficiency causes distinct lysosomal phenotypes in different types of neurons and glia in the cerebellum and frontal cortex. In humans, TMEM106B rs1990622 risk allele (T/T) is associated with increased Purkinje neuron loss. Taken together, our studies support that TMEM106B regulates lysosomal function in a cell-type-specific manner and TMEM106B is critical for maintaining synaptic integrity and neural functions in the cerebellum.

Addressing Today's Absorption, Distribution, Metabolism, and Excretion (ADME) Challenges in the Translation of In Vitro ADME Characteristics to Humans: A Case Study of the SMN2 mRNA Splicing Modifier Risdiplam.

Small molecules that present complex absorption, distribution, metabolism, and elimination (ADME) properties can be challenging to investigate as potential therapeutics. Acquiring data through standard methods can yield results that are insufficient to describe the in vivo situation, which can affect downstream development decisions. Implementing in vitro-in vivo-in silico strategies throughout the drug development process is effective in identifying and mitigating risks while speeding up their development. Risdiplam (Evrysdi)-an orally bioavailable, small molecule approved by the US Food and Drug Administration and more recently by the European Medicines Agency for the treatment of patients ≥2 months of age with spinal muscular atrophy-is presented here as a case study. Risdiplam is a low-turnover compound whose metabolism is mediated through a non-cytochrome P450 enzymatic pathway. Four main challenges of risdiplam are discussed: predicting in vivo hepatic clearance, determining in vitro metabolites with regard to metabolites in safety testing guidelines, elucidating enzymes responsible for clearance, and estimating potential drug-drug interactions. A combination of in vitro and in vivo results was successfully extrapolated and used to develop a robust physiologically based pharmacokinetic model of risdiplam. These results were verified through early clinical studies, further strengthening the understanding of the ADME properties of risdiplam in humans. These approaches can be applied to other compounds with similar ADME profiles, which may be difficult to investigate using standard methods. SIGNIFICANCE STATEMENT: Risdiplam is the first approved, small-molecule, survival of motor neuron 2 mRNA splicing modifier for the treatment of spinal muscular atrophy. The approach taken to characterize the absorption, distribution, metabolism, and excretion (ADME) properties of risdiplam during clinical development incorporated in vitro-in vivo-in silico techniques, which may be applicable to other small molecules with challenging ADME. These strategies may be useful in improving the speed at which future drug molecules can be developed.

In vitro and clinical investigations to determine the drug-drug interaction potential of entrectinib, a small molecule inhibitor of neurotrophic tyrosine receptor kinase (NTRK).

Background Entrectinib is a CNS-active, potent inhibitor of tyrosine receptor kinases A/B/C, ROS1 and anaplastic lymphoma kinase approved for use in patients with solid tumors. We describe the in vitro and clinical studies investigating potential entrectinib drug-drug interactions. Methods In vitro studies with human biomaterials assessed the enzymes involved in entrectinib metabolism, and whether entrectinib modulates the activity of the major cytochrome P450 (CYP) enzymes or drug transporter P-glycoprotein. Clinical studies investigated the effect of a strong CYP3A4 inhibitor (itraconazole) and inducer (rifampin) on single-dose entrectinib pharmacokinetics. The effect of entrectinib on sensitive probe substrates for CYP3A4 (midazolam) and P-glycoprotein (digoxin) were also investigated. Results Entrectinib is primarily metabolized by CYP3A4. In vitro, entrectinib is a CYP3A4/5 inhibitor (IC50 2 µM) and a weak CYP3A4 inducer. Entrectinib inhibited P-glycoprotein (IC50 1.33 µM) but is a poor substrate. In healthy subjects, itraconazole increased entrectinib Cmax and AUC by 73% and 504%, respectively, and rifampin decreased entrectinib Cmax and AUC by 56% and 77%, respectively. Single dose entrectinib did not affect midazolam AUC, although Cmax decreased by 34%. Multiple dose entrectinib increased midazolam AUC by 50% and decreased Cmax by 21%. Single dose entrectinib increased digoxin AUC and Cmax by 18% and 28%, respectively, but did not affect digoxin renal clearance. Conclusions Entrectinib is a CYP3A4 substrate and is sensitive to the effects of coadministered moderate/strong CYP3A4 inhibitors and strong inducers, and requires dose adjustment. Entrectinib is a weak inhibitor of CYP3A4 and P-glycoprotein and no dose adjustments are required with CYP3A4/P- glycoprotein substrates.Registration Number (Study 2) NCT03330990 (first posted online November 6, 2017) As studies 1 and 3 are phase 1 trials in healthy subjects, they are not required to be registered.

Calculation of an Apical Efflux Ratio from P-Glycoprotein (P-gp) In Vitro Transport Experiments Shows an Improved Correlation with In Vivo Cerebrospinal Fluid Measurements in Rats: Impact on P-gp Screening and Compound Optimization.

P-glycoprotein (P-gp) is a major blood-brain barrier (BBB) efflux transporter. In vitro approaches, including bidirectional efflux ratio (ER), are used to measure P-gp-mediated transport, but findings can be inconsistent across models. We propose a novel, more physiologically relevant, in vitro model: unidirectional apical efflux ratio (AP-ER)-a ratio of permeability rates at the apical side of the BBB with and without P-gp inhibitor. To test our approach, ER and AP-ER were calculated for 3227 structurally diverse compounds in porcine kidney epithelial cells (LLC-PK1) overexpressing human or mouse P-gp and classified based on their passive transcellular P-gp permeability or charged properties. In vivo rat infusion studies were performed for selected compounds with high ER but low AP-ER. One-third of the 3227 compounds had bidirectional ER that was much higher than AP-ER; very few had AP-ER higher than ER. Compounds with a large difference between AP-ER and ER were typically basic compounds with low-to-medium passive permeability and high lipophilicity and/or amphiphilicity, leading to strong membrane binding. Outcomes in the human model were similar to those in mice, suggesting AP-ER/ER ratios may be conserved for at least two species. AP-ER predicted measured cerebrospinal fluid (CSF) concentration better than ER for the five compounds tested in our in vivo rat infusion studies. We report superior estimations of the CSF concentrations of the compounds when based on less resource-intensive AP-ER versus classic ER. Better understanding of the properties leading to high P-gp-mediated efflux in vivo could support more efficient brain-penetrant compound screening and optimization. SIGNIFICANCE STATEMENT: To address inconsistencies associated with the historical, bidirectional efflux ratio (ER) calculation of P-glycoprotein-mediated transport, we propose to use the novel, more physiologically relevant, unidirectional apical efflux ratio (AP-ER) model. In vitro experiments suggested that compounds with strong membrane binding showed the largest difference between AP-ER and ER, and in vivo infusion studies showed that AP-ER predicted cerebrospinal fluid concentrations of compounds better than ER; outcomes in the human model were similar to those in mice.

Application of the Extended Clearance Classification System (ECCS) in Drug Discovery and Development: Selection of Appropriate In Vitro Tools and Clearance Prediction.

In vitro to in vivo extrapolation (IVIVE) to predict human hepatic clearance, including metabolism and transport, requires extensive experimental resources. In addition, there may be technical challenges to measure low clearance values. Therefore, prospective identification of rate-determining step(s) in hepatic clearance through application of the Extended Clearance Classification System (ECCS) could be beneficial for optimal compound characterization. IVIVE for hepatic intrinsic clearance (CLint,h) prediction is conducted for a set of 36 marketed drugs with low-to-high in vivo clearance, which are substrates of metabolic enzymes and active uptake transporters in the liver. The compounds were assigned to the ECCS classes, and CLint,h, estimated with HepatoPac (a micropatterned hepatocyte coculture system), was compared with values calculated based on suspended hepatocyte incubates. An apparent permeability threshold (apical to basal) of 50 nm/s in LLC-PK1 cells proved optimal for ECCS classification. A reasonable performance of the IVIVE for compounds across multiple classes using HepatoPac was achieved (with 2-3-fold error), except for substrates of uptake transporters (class 3b), for which scaling of uptake clearance using plated hepatocytes is more appropriate. Irrespective of the ECCS assignment, metabolic clearance can be estimated well using HepatoPac. The validation and approach elaborated in the present study can result in proposed decision trees for the selection of the optimal in vitro assays guided by ECCS class assignment, to support compound optimization and candidate selection. SIGNIFICANCE STATEMENT: Characterization of the rate-determining step(s) in hepatic elimination could be on the critical path of compound optimization during drug discovery. This study demonstrated that HepatoPac and plated hepatocytes are suitable tools for the estimation of metabolic and active uptake clearance, respectively, for a larger set of marketed drugs, supporting a comprehensive strategy to select optimal in vitro tools and to achieve Extended Clearance Classification System-dependent in vitro to in vivo extrapolation for human clearance prediction.

Loss of TMEM106B and PGRN leads to severe lysosomal abnormalities and neurodegeneration in mice.

Haploinsufficiency of progranulin (PGRN) is a leading cause of frontotemporal lobar degeneration (FTLD). Loss of PGRN leads to lysosome dysfunction during aging. TMEM106B, a gene encoding a lysosomal membrane protein, is the main risk factor for FTLD with PGRN haploinsufficiency. But how TMEM106B affects FTLD disease progression remains to be determined. Here, we report that TMEM106B deficiency in mice leads to accumulation of lysosome vacuoles at the distal end of the axon initial segment in motor neurons and the development of FTLD-related pathology during aging. Ablation of both PGRN and TMEM106B in mice results in severe neuronal loss and glial activation in the spinal cord, retina, and brain. Enlarged lysosomes are frequently found in both microglia and astrocytes. Loss of both PGRN and TMEM106B results in an increased accumulation of lysosomal vacuoles in the axon initial segment of motor neurons and enhances the manifestation of FTLD phenotypes with a much earlier onset. These results provide novel insights into the role of TMEM106B in the lysosome, in brain aging, and in FTLD pathogenesis.

A role of the frontotemporal lobar degeneration risk factor TMEM106B in myelination.

TMEM106B encodes a lysosomal membrane protein and was initially identified as a risk factor for frontotemporal lobar degeneration. Recently, a dominant D252N mutation in TMEM106B was shown to cause hypomyelinating leukodystrophy. However, how TMEM106B regulates myelination is still unclear. Here we show that TMEM106B is expressed and localized to the lysosome compartment in oligodendrocytes. TMEM106B deficiency in mice results in myelination defects with a significant reduction of protein levels of proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG), the membrane proteins found in the myelin sheath. The levels of many lysosome proteins are significantly decreased in the TMEM106B-deficient Oli-neu oligodendroglial precursor cell line. TMEM106B physically interacts with the lysosomal protease cathepsin D and is required to maintain proper cathepsin D levels in oligodendrocytes. Furthermore, we found that TMEM106B deficiency results in lysosome clustering in the perinuclear region and a decrease in lysosome exocytosis and cell surface PLP levels. Moreover, we found that the D252N mutation abolished lysosome enlargement and lysosome acidification induced by wild-type TMEM106B overexpression. Instead, it stimulates lysosome clustering near the nucleus as seen in TMEM106B-deficient cells. Our results support that TMEM106B regulates myelination through modulation of lysosome function in oligodendrocytes.

Entrectinib, a TRK/ROS1 inhibitor with anti-CNS tumor activity: differentiation from other inhibitors in its class due to weak interaction with P-glycoprotein.

BACKGROUND: Studies evaluating the CNS penetration of a novel tyrosine kinase inhibitor, entrectinib, proved challenging, particularly due to discrepancies across earlier experiments regarding P-glycoprotein (P-gp) interaction and brain distribution. To address this question, we used a novel "apical efflux ratio" (AP-ER) model to assess P-gp interaction with entrectinib, crizotinib, and larotrectinib, and compared their brain-penetration properties. METHODS: AP-ER was designed to calculate P-gp interaction with the 3 drugs in vitro using P-gp-overexpressing cells. Brain penetration was studied in rat plasma, brain, and cerebrospinal fluid (CSF) samples after intravenous drug infusion. Unbound brain concentrations were estimated through kinetic lipid membrane binding assays and ex vivo experiments, while the antitumor activity of entrectinib was evaluated in a clinically relevant setting using an intracranial tumor mouse model. RESULTS: Entrectinib showed lower AP-ER (1.1-1.15) than crizotinib and larotrectinib (≥2.8). Despite not reaching steady-state brain exposures in rats after 6 hours, entrectinib presented a more favorable CSF-to-unbound concentration in plasma (CSF/Cu,p) ratio (>0.2) than crizotinib and larotrectinib at steady state (both: CSF/Cu,p ~0.03). In vivo experiments validated the AP-ER approach. Entrectinib treatment resulted in strong tumor inhibition and full survival benefit in the intracranial tumor model at clinically relevant systemic exposures. CONCLUSIONS: Entrectinib, unlike crizotinib and larotrectinib, is a weak P-gp substrate that can sustain CNS exposure based on our novel in vitro and in vivo experiments. This is consistent with the observed preclinical and clinical efficacy of entrectinib in neurotrophic tropomyosin receptor kinase (NTRK) and ROS1 fusion-positive CNS tumors and secondary CNS metastases.