Differential effects of inosine monophosphate dehydrogenase (IMPDH/GuaB) inhibition in Acinetobacter baumannii and Escherichia coli.

Inosine 5'-monophosphate dehydrogenase (IMPDH), known as GuaB in bacteria, catalyzes the rate-limiting step in de novo guanine biosynthesis and is conserved from humans to bacteria. We developed a series of potent inhibitors that selectively target GuaB over its human homolog. Here, we show that these GuaB inhibitors are bactericidal, generate phenotypic signatures that are distinct from other antibiotics, and elicit different time-kill kinetics and regulatory responses in two important Gram-negative pathogens: Acinetobacter baumannii and Escherichia coli. Specifically, the GuaB inhibitor G6 rapidly kills A. baumannii but only kills E. coli after 24 h. After exposure to G6, the expression of genes involved in purine biosynthesis and stress responses change in opposite directions while siderophore biosynthesis is downregulated in both species. Our results suggest that different species respond to GuaB inhibition using distinct regulatory programs and possibly explain the different bactericidal kinetics upon GuaB inhibition. The comparison highlights opportunities for developing GuaB inhibitors as novel antibiotics.IMPORTANCEA. baumannii is a priority bacterial pathogen for which development of new antibiotics is urgently needed due to the emergence of multidrug resistance. We recently developed a series of specific inhibitors against GuaB, a bacterial inosine 5'-monophosphate dehydrogenase, and achieved sub-micromolar minimum inhibitory concentrations against A. baumannii. GuaB catalyzes the rate-limiting step of de novo guanine biosynthesis and is highly conserved across bacterial pathogens. This study shows that inhibition of GuaB induced a bacterial morphological profile distinct from that of other classes of antibiotics, highlighting a novel mechanism of action. Moreover, our transcriptomic analysis showed that regulation of de novo purine biosynthesis and stress responses of A. baumannii upon GuaB inhibition differed significantly from that of E. coli.

Human Pharmacokinetic and CYP3A Drug-Drug Interaction Prediction of GDC-2394 Using Physiologically Based Pharmacokinetic Modeling and Biomarker Assessment.

Physiologically based pharmacokinetic (PBPK) modeling was used to predict the human pharmacokinetics and drug-drug interaction (DDI) of GDC-2394. PBPK models were developed using in vitro and in vivo data to reflect the oral and intravenous PK profiles of mouse, rat, dog, and monkey. The learnings from preclinical PBPK models were applied to a human PBPK model for prospective human PK predictions. The prospective human PK predictions were within 3-fold of the clinical data from the first-in-human study, which was used to optimize and validate the PBPK model and subsequently used for DDI prediction. Based on the majority of PBPK modeling scenarios using the in vitro CYP3A induction data (mRNA and activity), GDC-2394 was predicted to have no-to-weak induction potential at 900 mg twice daily (BID). Calibration of the induction mRNA and activity data allowed for the convergence of DDI predictions to a narrower range. The plasma concentrations of the 4ß-hydroxycholesterol (4ß-HC) were measured in the multiple ascending dose study to assess the hepatic CYP3A induction risk. There was no change in plasma 4ß-HC concentrations after 7 days of GDC-2394 at 900 mg BID. A dedicated DDI study found that GDC-2394 has no induction effect on midazolam in humans, which was reflected by the totality of predicted DDI scenarios. This work demonstrates the prospective utilization of PBPK for human PK and DDI prediction in early drug development of GDC-2394. PBPK modeling accompanied with CYP3A biomarkers can serve as a strategy to support clinical pharmacology development plans. SIGNIFICANCE STATEMENT: This work presents the application of physiologically based pharmacokinetic modeling for prospective human pharmacokinetic (PK) and drug-drug interaction (DDI) prediction in early drug development. The strategy taken in this report represents a framework to incorporate various approaches including calibration of in vitro induction data and consideration of CYP3A biomarkers to inform on the overall CYP3A-related DDI risk of GDC-2394.

Matrix effect in bioanalytical assay development using supercritical fluid chromatography-mass spectrometry.

Matrix effect (ME) is commonly caused by coelution of compounds with target analytes, resulting in either suppression or enhancement of analyte ionization. Thus, to achieve the desired accuracy, precision, and sensitivity, ME needs to be evaluated and controlled during bioanalytical method development. As the application of supercritical fluid chromatography-mass spectrometry (SFC-MS) for analysis of biological samples has increased, ME using SFC-MS has also been investigated with a focus on the difference in ME in SFC-MS compared to other chromatographic techniques used for achiral separation in biological samples. Here, we provide a summary of the status of ME evaluation and mitigation in SFC-MS methods. This review presents an overview of the phenomenon of ME and methods for evaluating ME in bioanalysis. Next, the factors that can impact ME in SFC-MS-based bioanalytical methods are discussed in detail with an emphasis on SFC. A literature review of the evaluation of ME in targeted bioanalytical methods using SFC-MS is included at the end. Robust instrumentation, effective sample preparation, and superb separation selectivity are the foundations of reliable analytical methods as well as the ability to mitigate detrimental ME in SFC-MS methods.

Impact assessment of metabolite instability in the development and validation of LC-MS/MS bioanalytical assays for measurement of rosuvastatin in human plasma and urine samples.

During bioanalytical assay development and validation, maintaining the stability of the parent drug and metabolites of interest is critical. While stability of the parent drug has been thoroughly investigated, the stability of unanalyzed metabolites is often overlooked. When an unstable metabolite is known or suspected to interfere with measurement of the parent drug or other metabolites of interest through back-conversion or other routes, additional tests with these unstable metabolites should be conducted. Here, the development and validation of two assays for quantification of rosuvastatin, one in human plasma and one in human urine, was reported. To this end, additional sets of quality control samples were added during assay validation to ensure the reliability of the assays. Acid treatment of samples is shown to be necessary for rosuvastatin quantification. In this regard, stability issues caused by the metabolite, rosuvastatin lactone, may have been overlooked if assay development and validation had only considered the parent drug, rosuvastatin. These assays represent a case study for how to develop and validate assays with unstable metabolites. Taken together, unstable metabolites should be included in all applicable stability tests.

Exploring the potential of dried plasma collection cards for liquid chromatography coupled with tandem mass spectrometry quantitation of giredestrant in human plasma.

Microsampling technology for dried blood-derived samples provides an advantageous alternative to conventional venous blood for drug quantitation. Unlike conventional whole blood microsampling techniques, Noviplex is a novel, card-based technology for rapid dried plasma spot collection that retains the benefits of microsampling during collection and transportation, while avoiding the disadvantages of using whole blood samples. Giredestrant is a promising small-molecule therapeutic agent under development by Genentech to treat patients with estrogen receptor-positive breast cancer. In this study, we investigated the feasibility of using Noviplex cards for pharmacokinetic analysis of giredestrant levels in human plasma, including optimizing extraction recovery, evaluating in-card stability, and assessing batch precision and accuracy. We found that while the Noviplex card demonstrated levels of sensitivity, extraction recovery, and stability at ambient temperature that meet the requirements of pharmacokinetic analysis for clinical studies, further optimization of the filtration layers within the Noviplex card is necessary to improve filtration efficiency and consistency. This study reveals the possibilities as well as the limitations of the Noviplex card and provides a better understanding of the capabilities and risks of using the Noviplex card for drug quantitation in plasma.

A universal surrogate matrix assay for urea measurement in clinical pharmacokinetic studies of respiratory diseases.

In pharmacokinetic studies for respiratory diseases, urea is a commonly used dilution marker for volume normalization of various biological matrices, owing to the fact that urea diffuses freely throughout the body and is minimally affected by disease states. In this study, we developed a convenient liquid chromatography-tandem mass spectrometry (LC-MS/MS) surrogate matrix assay for accurate urea quantitation in plasma, serum and epithelial lining fluid. Different mass spectrometer platforms and ionization modes were compared in parallel. The LC method and mass spectrometer parameters were comprehensively optimized to reduce interferences, to smooth the baseline and to maximize the signal-to-noise ratio. Saline was selected as the surrogate matrix, and its suitability was confirmed by good parallelism and accurate quality control sample measurements. Reliable and robust assay performance was demonstrated by precision and accuracy, dilution integrity, sensitivity, recovery and stability, all of which met bioanalysis requirements to support clinical studies. The assay performance was also verified and better understood by comparing it with a colorimetric assay and to a surrogate analyte assay. The newly developed surrogate matrix assay has the potential to be further expanded for urea quantitation in numerous physiological matrices.

A technical overview of supercritical fluid chromatography-mass spectrometry (SFC-MS) and its recent applications in pharmaceutical research and development.

In this paper, we review the growing development and applications of supercritical fluid chromatography-mass spectrometry (SFC-MS) for the analysis of small molecular analytes and biomarkers in drug discovery. As an alternative chromatographic technique, SFC instrumentation and methodology have dramatically advanced over the last decade. Mass spectrometry (MS) provides the powerful detection capability as it couples with SFC. A growing number of SFC-MS/MS applications were reported over the last decade and the application areas of SFC-MS/MS is rapidly expanding. The first part of this review is devoted to the different aspects of SFC-MS development and recent technological advancements. In the second part of this review, we highlight the recent application areas in pharmaceutical research and development.

Changes in Organic Anion Transporting Polypeptide Uptake in HEK293 Overexpressing Cells in the Presence and Absence of Human Plasma.

Generating accurate in vitro data is crucial for in vitro to in vivo extrapolation and pharmacokinetic predictions. The use of human embryonic kidney (HEK) 293 cells overexpressing organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 in protein-free buffer and 100% human plasma incubations was explored for the uptake of four OATP substrates: pravastatin, rosuvastatin, repaglinide, and pitavastatin. Differences were observed for each parameter [unbound Michaelis constant (K m,u), V max, intrinsic clearance (CLint), and unbound passive diffusion Pdif,u] obtained from the buffer and plasma incubations in both cells, and the fold differences were greatest for the highly protein bound compounds. The fold change in K m,u values ranged from 1.91 to 619, and the fold change in V max values ranged from 1.22 to 97.4. As a result, in both cells, the CLint values generated in the plasma incubations were higher by 0.762- to 31.7-fold than the values generated in the protein-free buffer. The passive diffusion was also higher in the plasma incubations for all four compounds, with a fold difference range of 1.73-23.4. These shifts in the presence and absence of human plasma suggest that plasma proteins may play a role in both the active uptake and passive diffusion processes. The results also support the idea of a transporter-induced protein-binding shift, where high protein binding may not limit the uptake of compounds that have high affinity for transporters. The addition of plasma to incubations leading to higher CLint values for transporter substrates helps mitigate the underprediction commonly noted with in vitro to in vivo extrapolation. SIGNIFICANCE STATEMENT: The current investigation brings a new perspective on how to mitigate the underprediction commonly noted with in vitro to in vivo extrapolation for OATP substrates by using HEK293 cells overexpressing OATP1B1 and OATP1B3. It also supports the idea of a transporter-induced protein-binding shift, where high protein binding may not limit the uptake of compounds that have high affinity for transporters.

A Multiplatform Approach for the Discovery of Novel Drug-Induced Kidney Injury Biomarkers.

Drug-induced kidney injury (DIKI) is a common toxicity observed in pharmaceutical development. We demonstrated the use of label-free liquid chromatography-mass spectrometry (LC-MS) and multiplex liquid chromatography-single reaction monitoring (LC-SRM) as practical extensions of standard immunoassay based safety biomarker assessments for identification of new toxicity marker candidates and for improved mechanistic understanding. Two different anticancer drugs, doxorubicin (DOX) and cisplatin (cis-diamminedichloridoplatinum, CDDP), were chosen as the toxicants due to their different modes of nephrotoxicity. Analyses of urine samples from toxicant treated and untreated rats were compared to identify biochemical analytes that changed in response to toxicant exposure. A discovery (label-free LC-MS) and targeted proteomics (multiplex LC-SRM) approach was used in combination with well established immunoassay experiments for the identification of a panel of urinary protein markers related to drug induced nephrotoxicity in rats. The initial generation of an expanded set of markers was accomplished using the label-free LC-MS discovery screen and ELISA based analysis of six nephrotoxicity biomarker proteins. Diagnostic performance of the expanded analyte set was statistically compared to conventional nephrotoxicity biomarkers. False discovery rate (FDR) analysis revealed 18 and 28 proteins from the CDDP and DOX groups, respectively, exhibiting significant differences between the vehicle and treated groups. Multiplex SRM assays were constructed to more precisely quantify candidate markers selected from the discovery screen and immunoassay experiments. To evaluate the sensitivity and specificity for each of the candidate biomarkers, histopathology severity scores were used as a benchmark for renal injury followed by receiver-operating characteristic (ROC) curve analysis on selected biomarkers. Further examination of the best performing analytes revealed relevant biological significance after consideration of anatomical localization and functional roles. In summary, the inclusion of mass spectrometry together with conventional ELISA based assays resulted in the identification of an expanded set of biomarkers with a realistic potential for providing additional beneficial information in mechanistic investigations of drug induced kidney injury and with similar responsiveness to conventionally applied indicators of renal injury.

Stressful Parental-Bonding Exaggerates the Functional and Emotional Disturbances of Primary Dysmenorrhea.

BACKGROUND: Some evidence suggests that women with primary dysmenorrhea (or painful period) often have traumatic experience with parental attachments, but the exact relationship is still unclear. PURPOSE: This study aims to investigate associations between styles of the parental bonding and the detailed aspects of the disorder in Chinese university-student women. METHODS: From university-student women, we have invited 50 primary dysmenorrhea patients and 111 healthy volunteers, to undergo tests of the Functional and Emotional Measure of Dysmenorrhea (FEMD), the Family Relationship Questionnaire (FRQ), and the visual analogue scale for the pain intensity experienced. RESULTS: Besides the high scores of the FEMD Functional and Emotional scales, the dysmenorrhea patients also scored significantly higher than the healthy controls on the FRQ scales of Paternal Dominance and Maternal Abuse. In patients, the FEMD Emotional scale was negatively predicted by the Paternal Freedom Release scale, and the FEMD Functional scale was positively predicted by the Maternal Dominance scale. CONCLUSIONS: Inappropriate parental bonding or chronic traumatic attachment styles have respective relationships with the functional and emotional disturbances experienced by the primary dysmenorrhea patients.

Metal-induced conformational changes of human metallothionein-2A: a combined theoretical and experimental study of metal-free and partially metalated intermediates.

Electrospray ionization ion mobility mass spectrometry (ESI IM-MS) and molecular dynamics (MD) simulations reveal new insights into metal-induced conformational changes and the mechanism for metalation of human metallothionein-2A (MT), an intrinsically disordered protein. ESI of solutions containing apoMT yields multiple charge states of apoMT; following addition of Cd(2+) to the solution, ESI yields a range of CdiMT (i = 1-7) product ions (see Chen et al. Anal. Chem. 2013, 85, 7826-33). Ion mobility arrival-time distributions (ATDs) for the CdiMT (i = 0-7) ions reveal a diverse population of ion conformations. The ion mobility data clearly show that the conformational diversity for apoMT and partially metalated ions converges toward ordered, compact conformations as the number of bound Cd(2+) ions increase. MD simulations provide additional information on conformation candidates of CdiMT (i = 0-7) that supports the convergence of distinct conformational populations upon metal binding. Integrating the IM-MS and MD data provides a global view that shows stepwise conformational transition of an ensemble as a function of metal ion bound. ApoMT is comprised of a wide range of conformational states that populate between globular-like compact and coil-rich extended conformations. During the initial stepwise metal addition (number of metal ions bound i = 1-3), the metal ions bind to different sites to yield distinct conformations, whereas for i > 4, the conformational changes appear to be domain-specific, attributed to different degrees of disorder of the ß domain; the ß domain becomes more ordered as additional metal ions are added, promoting convergences to the dumbbell-shaped conformation.

Evaluation of Patient-Centric Sample Collection Technologies for Pharmacokinetic Assessment of Large and Small Molecules.

Low-volume sampling devices offer the promise of lower discomfort and greater convenience for patients, potentially reducing patient burden and enabling decentralized clinical trials. In this study, we determined whether low-volume sampling devices produce pharmacokinetic (PK) data comparable to conventional venipuncture for a diverse set of monoclonal antibodies (mAbs) and small molecules. We adopted an open-label, non-randomized, parallel-group, single-site study design, with four cohorts of 10 healthy subjects per arm. The study drugs, doses, and routes of administration included: crenezumab (15 mg/kg, intravenous infusion), etrolizumab (210 mg, subcutaneous), GDC-X (oral), and hydroxychloroquine (HCQ, 200 mg, oral). Samples were collected after administration of a single dose of each drug using conventional venipuncture and three low-volume capillary devices: TassoOne Plus for liquid blood, Tasso-M20 for dry blood, both applied to the arm, and Neoteryx Mitra® for dry blood obtained from fingertips. Serum/plasma concentrations from venipuncture and TassoOne Plus samples overlapped and PK parameters were comparable for all drugs, except HCQ. After applying a baseline hematocrit value, the dry blood concentrations and PK parameters for the two monoclonal antibodies were comparable to those obtained from venipuncture. For the two small molecules, two bridging strategies were evaluated for converting dry blood concentrations to equivalent plasma concentrations. A baseline hematocrit correction and/or linear regression-based correction was effective for GDC-X, but not for HCQ. Additionally, the study evaluated the bioanalytical data quality and comparability from the various collection methods, as well as patient preference for the devices.

Discovery of GuaB inhibitors with efficacy against Acinetobacter baumannii infection.

Guanine nucleotides are required for growth and viability of cells due to their structural role in DNA and RNA, and their regulatory roles in translation, signal transduction, and cell division. The natural antibiotic mycophenolic acid (MPA) targets the rate-limiting step in de novo guanine nucleotide biosynthesis executed by inosine-5´-monophosphate dehydrogenase (IMPDH). MPA is used clinically as an immunosuppressant, but whether in vivo inhibition of bacterial IMPDH (GuaB) is a valid antibacterial strategy is controversial. Here, we describe the discovery of extremely potent small molecule GuaB inhibitors (GuaBi) specific to pathogenic bacteria with a low frequency of on-target spontaneous resistance and bactericidal efficacy in vivo against Acinetobacter baumannii mouse models of infection. The spectrum of GuaBi activity includes multidrug-resistant pathogens that are a critical priority of new antibiotic development. Co-crystal structures of A. baumannii, Staphylococcus aureus, and Escherichia coli GuaB proteins bound to inhibitors show comparable binding modes of GuaBi across species and identifies key binding site residues that are predictive of whole-cell activity across both Gram-positive and Gram-negative clades of Bacteria. The clear in vivo efficacy of these small molecule GuaB inhibitors in a model of A. baumannii infection validates GuaB as an essential antibiotic target. IMPORTANCE: The emergence of multidrug-resistant bacteria worldwide has renewed interest in discovering antibiotics with novel mechanism of action. For the first time ever, we demonstrate that pharmacological inhibition of de novo guanine biosynthesis is bactericidal in a mouse model of Acinetobacter baumannii infection. Structural analyses of novel inhibitors explain differences in biochemical and whole-cell activity across bacterial clades and underscore why this discovery may have broad translational impact on treatment of the most recalcitrant bacterial infections.

Cis-trans isomerizations of proline residues are key to bradykinin conformations.

A recent ion mobility-mass spectrometry (IM-MS) study of the nonapeptide bradykinin (BK, amino acid sequence Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)) found evidence for 10 populations of conformations that depend upon the solution composition [J. Am. Chem. Soc. 2011, 133, 13810]. Here, the role of the three proline residues (Pro(2), Pro(3), and Pro(7)) in establishing these conformations is investigated using a series of seven analogue peptides in which combinations of alanine residues are substituted for prolines. IM-MS distributions of the analogue peptides, when compared to the distribution for BK, indicate the multiple structures are associated with different combinations of cis and trans forms of the three proline residues. These data are used to assign the structures to different peptide populations that are observed under various solution conditions. The assignments also show the connectivity between structures when collisional activation is used to convert one state into another.

An experimental study of the solvent-dependent self-assembly/disassembly and conformer preferences of gramicidin A.

The solvent dependence of self-assembly/disassembly kinetics and conformer preferences of the gramicidin A (GA) dimer is investigated using a combination of techniques, viz., electrospray ionization-ion mobility-mass spectrometry (IM-MS), collision-induced dissociation (CID), and hydrogen/deuterium exchange (HDX)-MS. IM-MS measurements reveal that there are possibly three distinct GA dimeric species, detected as sodium ion adduct ions [2GA + 2Na](2+), and these are assigned as the parallel ß-helix, antiparallel ß-helix, and head-to-head dimer. The monomerization kinetics and equilibrium abundances of the dimer ions depend upon solvent polarity. The antiparallel ß-helix was the thermodynamically preferred species in less polar solvents. HDX measurements and collision-induced dissociation (CID) of the intermediate complex confirm the well-protected dimer geometry with strong intermolecular hydrogen bonds. This combined IM-HDX-CID methodology provides a comprehensive view of GA self-assembly/disassembly in low dielectric solutions, showing its potential utility in solving solution-phase protein self-assembly/disassembly kinetics and providing structural information of the multimers at the same time.

IL-17 neutralization significantly ameliorates hepatic granulomatous inflammation and liver damage in Schistosoma japonicum infected mice.

IL-17 is a signature cytokine of Th17 cells implicated in the induction and progression of chronic inflammatory diseases. Several studies in C57BL/6 mice, immunized with soluble schistosome egg Ags (SEA) in complete Freund's adjuvant (CFA), and subsequently infected with Schistosoma mansoni (S. mansoni) have shown that severe hepatic granulomatous inflammation is correlated with high levels of IL-17. Here, using a Schistosoma japonicum (S. japonicum) larvae infection model in C57BL/6 mice, we analyzed the dynamic expression of IL-17 in infected livers by RT-qPCR and ELISA. Our results showed that IL-17 expression was elevated during the course of infection. The temporal expression of IL-17 and cytokines/chemokines involved in the induction and effector function of Th17 cells was paralleled with hepatic granulomatous inflammation. Treatment of S. japonicum infected mice with IL-17-neutralizing mAb resulted in significant downmodulation of granulomatous inflammation and hepatocyte necrosis. The protection was associated with lower expression of proinflammatory cytokines/chemokines, such as IL-6, IL-1ß, CXCL1, and CXCL2 and a reduced number of infiltrating neutrophils. Anti-IL-17 mAb significantly ameliorated hepatic granulomatous inflammation, partly through the downregulation of proinflammatory cytokines/chemokines and recruitment of neutrophils. Our data indicate a pathogenic role of Th17/IL-17 in hepatic immunopathology in S. japonicum infected mice.

First-in-human phase 1 trial evaluating safety, pharmacokinetics, and pharmacodynamics of NLRP3 inflammasome inhibitor, GDC-2394, in healthy volunteers.

Inappropriate and chronic activation of the cytosolic NOD-, LRR-, and pyrin domain-containing 3 (NLRP3) inflammasome, a key component of innate immunity, likely underlies several inflammatory diseases, including coronary artery disease. This first-in-human phase I trial evaluated safety, pharmacokinetics (PKs), and pharmacodynamics (PDs) of oral, single (150-1800 mg) and multiple (300 or 900 mg twice daily for 7 days) ascending doses (SADs and MADs) of GDC-2394, a small-molecule inhibitor of NLRP3, versus placebo in healthy volunteers. The study also assessed the food effect on GDC-2394 and its CYP3A4 induction potential in food-effect (FE) and drug-drug interaction (DDI) stages, respectively. Although GDC-2394 was adequately tolerated in the SAD, MAD, and FE cohorts, two participants in the DDI stage experienced grade 4 drug-induced liver injury (DILI) deemed related to treatment, but unrelated to a PK drug interaction, leading to halting of the trial. Both participants experiencing severe DILI recovered within 3 months. Oral GDC-2394 was rapidly absorbed; exposure increased in an approximately dose-proportional manner with low-to-moderate intersubject variability. The mean terminal half-life ranged from 4.1 to 8.6 h. Minimal accumulation was observed with multiple dosing. A high-fat meal led to delays in time to maximum concentration and minor decreases in total exposure and maximum plasma concentration. GDC-2394 had minimal CYP3A4 induction potential with the sensitive CYP3A4 substrate, midazolam. Exploratory ex vivo whole-blood stimulation assays showed rapid, reversible, and near-complete inhibition of the selected PD biomarkers, IL-1ß and IL-18, across all tested doses. Despite favorable PK and target engagement PD, the GDC-2394 safety profile precludes its further development.

How alkali metal ion binding alters the conformation preferences of gramicidin A: a molecular dynamics and ion mobility study.

Here, we present a systematic study combing electrospray ionization-ion mobility experiments and an enhanced sampling molecular dynamics, specifically integrated tempering sampling molecular dynamics simulations (ITS-MDS), to explore the conformations of alkali metal ion (Na, K, and Cs) adducts of gramicidin A (GA) in vacuo. Folding simulation is performed to obtain inherent conformational preferences of neutral GA to provide insights about how the binding of metal ions influences the intrinsic conformations of GA. The comparison between conformations of neutral GA and alkali metal ion adducts reveals a high degree of structural similarity, especially between neutral GA and [GA + Na](+); however, the structural similarities decrease as ionic radius of the metal increases. Collision cross section (CCS) profiles for [GA + Na](+) and [GA + Cs](+) ions obtained from by ITS-MDS compare favorably with the experimental CCS, but there are significant differences from CCS profiles for [GA + K](+) ions. Such discrepancies between the calculated and measured CCS profiles for [GA + K](+) are discussed in terms of limitations in the simulation force field as well as possible size-dependent coordination of the [GA + K](+) ion complex.

Case report: Durable response to alectinib in ALK-rearranged lung adenocarcinoma with acquired, crizotinib-resistant ALK C1156F mutation.

Treatment of ALK-rearranged non-small cell lung cancer (NSCLC) with tyrosine kinase inhibitors (TKIs) is challenged by the almost inevitable emergence of therapeutic resistance. Different profiles of resistance mechanisms have been reported for the currently available ALK TKIs. The ALK C1156Y mutation is reported in 2% of patients with acquired resistance to crizotinib. A rare substitution at the same site, C1156F, remains largely unknown. Existing evidence includes identification of C1156F and G1202R in an alectinib-resistant patient and sensitivity to crizotinib and resistance to later-generation 3ALK inhibitors in preclinical models. In this report, we present two cases in which NSCLC patients acquired the ALK C1156F mutation on crizotinib monotherapy. Both patients were men, and one had been heavily treated with chemotherapeutic regimens before identification of ALK rearrangement, whereas the other received crizotinib as first-line treatment. Genomic profiling of blood biopsies after progression on crizotinib suggested emergence of the ALK C1156F variant. Both patients subsequently received and responded favorably to alectinib, achieving respective progression-free survival of 21 and 15 months as of the latest follow-ups. To the best of our knowledge, this work is the first to provide clinical evidence of resistance to crizotinib and sensitivity to alectinib in NSCLC patients harboring acquired ALK C1156F mutation.

Phase I and scintigraphy studies to evaluate safety, tolerability, pharmacokinetics, and lung deposition of inhaled GDC-0214 in healthy volunteers.

Several inflammatory cytokines that promote inflammation and pathogenesis in asthma signal through the Janus kinase 1 (JAK1) pathway. This phase I, randomized, placebo-controlled trial assessed the pharmacokinetics and safety of single and multiple ascending doses up to 15 mg twice daily for 14 days of a JAK1 inhibitor, GDC-0214, in healthy volunteers (HVs; n = 66). Doses were administered with a dry powder, capsule-based inhaler. An accompanying open-label gamma scintigraphy study in HVs examined the lung deposition of a single dose of inhaled Technetium-99m (99m Tc)-radiolabeled GDC-0214. GDC-0214 plasma concentrations were linear and approximately dose-proportional after both single and multiple doses. Peak plasma concentrations occurred at 15-30 min after dosing. The mean apparent elimination half-life ranged from 32 to 56 h across all single and multiple dose cohorts. After single and multiple doses, all adverse events were mild or moderate, and none led to treatment withdrawal. There was no clear evidence of systemic toxicity due to JAK1 inhibition, and systemic exposure was low, with plasma concentrations at least 15-fold less than the plasma protein binding-corrected IC50 of JAK1 at the highest dose. Scintigraphy showed that approximately 50% of the emitted dose of radiolabeled GDC-0214 was deposited in the lungs and was distributed well to the peripheral airways. 99m Tc-radiolabeled GDC-0214 (1 mg) exhibited a mean plasma Cmax similar to that observed in phase I at the same dose level. Overall, inhaled GDC-0214 exhibited pharmacokinetic properties favorable for inhaled administration.