Magneto-optical Q-switching using magnetic garnet film with micromagnetic domains.

High-power giant pulses can be used applied in various applications with Q-switched micro-lasers. This method can shorten the pulse duration; however, active control is currently impossible in micro-lasers. To achieve precise pulse control while maintaining compactness and simplicity, we exploit the magneto-optical effect in magnetic garnet films with micromagnetic domains that can be actively controlled by a pulsed magnetic field. Our Q-switching technique enhances the output power by a factor of 4 × 103. Moreover, the device itself is smaller than other Q-switching devices. This novel type of active Q-switch can be combined with a micro-laser to obtain megawatt-order pulses.

Synthesis and determination of absolute configuration of a non-peptidic αvß6 integrin antagonist for the treatment of idiopathic pulmonary fibrosis.

A diastereoselective synthesis of (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-((R)-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)butanoic acid (1), a potential therapeutic agent for the treatment of Idiopathic Pulmonary Fibrosis, which is currently undergoing Phase I clinical trials is reported. The key steps in the synthesis involved alkylation of 2-methylnaphthyridine with (R)-N-Boc-3-(iodomethyl)-pyrrolidine, and an asymmetric Rh-catalysed addition of an arylboronic acid to a 4-(N-pyrrolidinyl)crotonate ester. The overall yield of the seven linear step synthesis was 8% and the product was obtained in >99.5% ee proceeding with 80% de. The absolute configuration of 1 was established by an alternative asymmetric synthesis involving alkylation of an arylacetic acid using Evans oxazolidinone chemistry, acylation using the resulting 2-arylsuccinic acid, and reduction. The absolute configuration of the benzylic asymmetric centre was established as (S).

Activating p53 abolishes self-renewal of quiescent leukaemic stem cells in residual CML disease.

Whilst it is recognised that targeting self-renewal is an effective way to functionally impair the quiescent leukaemic stem cells (LSC) that persist as residual disease in chronic myeloid leukaemia (CML), developing therapeutic strategies to achieve this have proved challenging. We demonstrate that the regulatory programmes of quiescent LSC in chronic phase CML are similar to that of embryonic stem cells, pointing to a role for wild type p53 in LSC self-renewal. In support of this, increasing p53 activity in primitive CML cells using an MDM2 inhibitor in combination with a tyrosine kinase inhibitor resulted in reduced CFC outputs and engraftment potential, followed by loss of multilineage priming potential and LSC exhaustion when combination treatment was discontinued. Our work provides evidence that targeting LSC self-renewal is exploitable in the clinic to irreversibly impair quiescent LSC function in CML residual disease - with the potential to enable more CML patients to discontinue therapy and remain in therapy-free remission.

Discovery and Development of Highly Potent and Orally Bioavailable Nonpeptidic αvß6 Integrin Inhibitors.

A series of 3-aryl((S)-3-fluoropyrrolidin-1-yl)butanoic acids were developed as potent orally bioavailable αvß6 integrin inhibitors. Starting from a zwitterionic peptidomimetic series optimized for inhaled administration, the balancing of potency and passive permeability to achieve suitable oral agents through modification and exploration of aryl substituents and pKa of the central cyclic amine is described. (S)-4-((S)-3-Fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoic acid was found to have highly desirable oral pharmacokinetic profiles in rat, dog, and minipig, with low to moderate clearance (26%, 7%, and 18% liver blood flow, respectively), moderate volumes of distribution (3.6, 1.4, and 0.9 L/kg, respectively), high to complete oral bioavailabilities, high αvß6 integrin potency of pIC50 of 8.0, and high solubility in physiological media (>2 mg/mL). Equating to the estimated human dose range of 10-75 mg b.i.d. to achieve 90% αvß6 target engagement at Cmin, it was selected for further investigation as a potential therapeutic agent for the treatment of idiopathic pulmonary fibrosis.

Regulation of renal organic anion transporter 3 (SLC22A8) expression and function by the integrity of lipid raft domains and their associated cytoskeleton.

BACKGROUND/AIMS: In humans and rodents, organic anion transporter 3 (Oat3) is highly expressed on the basolateral membrane of renal proximal tubules and mediates the secretion of exogenous and endogenous anions. Regulation of Oat3 expression and function has been observed in both expression system and intact renal epithelia. However, information on the local membrane environment of Oat3 and its role is limited. Lipid raft domains (LRD; cholesterol-rich domains of the plasma membrane) play important roles in membrane protein expression, function and targeting. In the present study, we have examined the role of LRD-rich membranes and their associated cytoskeletal proteins on Oat3 expression and function. METHODS: LRD-rich membranes were isolated from rat renal cortical tissues and from HEK-293 cells stably expressing human OAT3 (hOAT3) by differential centrifugation with triton X-100 extraction. Western blots were subsequently analyzed to determine protein expression. In addition, the effect of disruption of LRD-rich membranes was examined on functional Oat3 mediated estrone sulfate (ES) transport in rat renal cortical slices. Cytoskeleton disruptors were investigated in both hOAT3 expressing HEK-293 cells and rat renal cortical slices. RESULTS: Lipid-enriched membranes from rat renal cortical tissues and hOAT3-expressing HEK-293 cells showed co-expression of rOat3/hOAT3 and several lipid raft-associated proteins, specifically caveolin 1 (Cav1), ß-actin and myosin. Moreover, immunohistochemistry in hOAT3-expressing HEK-293 cells demonstrated that these LRD-rich proteins co-localized with hOAT3. Potassium iodide (KI), an inhibitor of protein-cytoskeletal interaction, effectively detached cytoskeleton proteins and hOAT3 from plasma membrane, leading to redistribution of hOAT3 into non-LRD-rich compartments. In addition, inhibition of cytoskeleton integrity and membrane trafficking processes significantly reduced ES uptake mediated by both human and rat Oat3. Cholesterol depletion by methyl-ß-cyclodextrin (MßCD) also led to a dose dependent reduction Oat3 expression and ES transport by rat renal cortical slices. Moreover, the up-regulation of rOat3-mediated transport seen following insulin stimulation was completely prevented by MßCD. CONCLUSION: We have demonstrated that renal Oat3 resides in LRD-rich membranes in proximity to cytoskeletal and signaling proteins. Disruption of LRD-rich membranes by cholesterol-binding agents or protein trafficking inhibitors altered Oat3 expression and regulation. These findings indicate that the integrity of LRD-rich membranes and their associated proteins are essential for Oat3 expression and function.

TGM6, a helminth secretory product, mimics TGF-ß binding to TßRII to antagonize TGF-ß signaling in fibroblasts.

The murine helminth parasite Heligmosomoides polygyrus expresses a family of proteins structurally related to TGF-ß Mimic 1 (TGM1), a secreted five domain protein that activates the TGF-ß pathway and converts naïve T lymphocytes to immunosuppressive Tregs. TGM1 signals through the TGF-ß type I and type II receptors, TßRI and TßRII, with domains 1-2 and 3 binding TßRI and TßRII, respectively, and domains 4-5 binding CD44, a co-receptor abundant on T cells. TGM6 is a homologue of TGM1 that is co-expressed with TGM1, but lacks domains 1 and 2. Herein, we show that TGM6 binds TßRII through domain 3, but does not bind TßRI, or other type I or type II receptors of the TGF-ß family. In TGF-ß reporter assays in fibroblasts, TGM6, but not truncated TGM6 lacking domains 4 and 5, potently inhibits TGF-ß- and TGM1-induced signaling, consistent with its ability to bind TßRII but not TßRI or other receptors of the TGF-ß family. However, TGM6 does not bind CD44 and is unable to inhibit TGF-ß and TGM1 signaling in T cells. To understand how TGM6 binds TßRII, the X-ray crystal structure of the TGM6 domain 3 bound to TßRII was determined at 1.4 Å. This showed that TGM6 domain 3 binds TßRII through an interface remarkably similar to the TGF-ß:TßRII interface. These results suggest that TGM6 has adapted its domain structure and sequence to mimic TGF-ß binding to TßRII and function as a potent TGF-ß and TGM1 antagonist in fibroblasts. The coexpression of TGM6, along with the immunosuppressive TGMs that activate the TGF-ß pathway, may prevent tissue damage caused by the parasite as it progresses through its life cycle from the intestinal lumen to submucosal tissues and back again.

4-Phenyl-7-azaindoles as potent, selective and bioavailable IKK2 inhibitors demonstrating good in vivo efficacy.

The lead optimization of a series of potent azaindole IKK2 inhibitors is described. Optimization of the human whole blood activity and selectivity over IKK1 in parallel led to the discovery of 16, a potent and selective IKK2 inhibitor showing good efficacy in a rat model of neutrophil activation.

Smart dry powder inhalers and intelligent adherence management.

Adherence to inhaled treatments is a complex challenge for patients with chronic obstructive pulmonary disease (COPD) and asthma, it not only involves following the prescribed treatment plans but also administering the medications correctly. When using a dry powder inhaler (DPI), the inhalation flow is particularly critical. Patients frequently fail to use a rapid enough onset and fast enough inhalation when using DPIs. At the same time, there is increasing pressure on physicians to switch patients to DPIs, to minimise the environmental impact of pMDI propellants. This makes it critical to understand whether a patient will maintain or improve disease control by using their new inhaler correctly. However, it is challenging for health care professionals to understand how a patient behaves away from the clinic. Therefore, it would be beneficial to obtain real-world data through the use of monitoring tools, i.e., "smart inhalers". This paper reviews the technologies used to monitor DPIs, how effective they have been in a clinical setting, and how well these have been adopted by patients and health care providers.

Pilot trial of a technology assisted treatment for trichotillomania.

The present study examined the usability, acceptability, feasibility, and preliminary efficacy of a prototype wrist-worn motion detection device and accompanying mobile app, developed by HabitAware®, as a system for delivering self-administered Habit Reversal Training (HRT). As an exploratory aim, the effect of the device and HRT app combination was compared to a reminder bracelet. The pilot trial included 15 adults with trichotillomania who interacted with the device and app system (n = 10) or reminder bracelet (n = 5) for 4 weeks. Participants in the device and app condition reported high usability, acceptability, and perceived efficacy of the system. The device and HRT app combination reduced hair pulling severity. Individuals in the reminder bracelet condition also showed a significant improvement in hair pulling. A future efficacy study with a larger sample size, longer timeframe, and improved gesture detection algorithm is warranted.

Lead optimization using matched molecular pairs: inclusion of contextual information for enhanced prediction of HERG inhibition, solubility, and lipophilicity.

Previous studies of the analysis of molecular matched pairs (MMPs) have often assumed that the effect of a substructural transformation on a molecular property is independent of the context (i.e., the local structural environment in which that transformation occurs). Experiments with large sets of hERG, solubility, and lipophilicity data demonstrate that the inclusion of contextual information can enhance the predictive power of MMP analyses, with significant trends (both positive and negative) being identified that are not apparent when using conventional, context-independent approaches.

The Climate is Changing for Metered-Dose Inhalers and Action is Needed.

Increases in global temperature are already having a significant impact on our climate. The hydrofluorocarbon (HFC) propellants used today in pressurized metered-dose inhalers (pMDIs) have global warming potential (GWP) many times that of carbon dioxide. Their use, together with all other emissive uses of HFCs, is being phased down under the Montreal protocol. This has prompted calls to switch patients to dry powder inhalers (DPIs). This paper presents a new analysis of the top 15 respiratory drug markets by drug class. It shows that a switch to DPIs would be economically feasible for most countries and most drugs. However, a wholesale switch of reliever medications, notably short-acting  ß-agonists, would lead to significant increases in the cost of these life-saving medications. Reviewing the evidence, whilst most patients are capable of using DPIs, the very young, very old and those undergoing an acute exacerbation still require a pMDI. Thus, there is a clinical and economic need to have both pMDIs and DPIs available. At the same time, it is projected that the reduction in non-medical uses of propellants is likely to give rise to a 5-fold increase in their cost for pMDI uses and is likely to hit the Western world in 2025. This may lead to a price increase in reliever medication that will make it unaffordable for the poorer communities in some markets. At the same time, opportunities to save money by developing new formulations using propellants with lower GWP, such as HFC 152a or HFO 1234ze(E), are described. Two companies have made this commitment, but neither currently have a strong presence in reliever medication. For them, or other companies, now is the time to act; 2025 is not far away in terms of product development timescales and the climate cannot wait.

Monitoring of urothelial cancer disease status after treatment by digital droplet PCR liquid biopsy assays.

OBJECTIVES: Real-time monitoring of disease status would be beneficial for timely decision making in the treatment of urothelial cancer (UC), and may accelerate the evaluation of clinical trials. Use of cell free tumor DNA (cftDNA) as a biomarker in liquid biopsy is minimally invasive and its successful use has been reported in various cancer types, including UC. The objective of this study was to evaluate the use of digital droplet PCR (ddPCR)-based assays to monitor UC after treatment. METHOD AND MATERIALS: Blood, urine and matching formalin fixed, paraffin embedded diagnostic specimens were collected from 20 patients diagnosed with stage T1 (n = 2) and T2/T3 (n = 18) disease. SNaPshot assays, Sanger sequencing and whole exome sequencing were used to identify tumor-specific mutations, and somatic mutation status was confirmed using patient-matched DNAs extracted from buffy coats and peripheral blood mononucleocytes. The ddPCR assays of the tumor-specific mutations were used to detect the fractional abundance of cftDNA in plasma and urine. RESULTS: SNaPshot and Sanger sequencing identified point mutations in 70% of the patients that were assayable by ddPCR. Cases of remission and relapse monitored by assays for PIK3CA E542K and TP53 Y163C mutations in plasma and urine concurred with clinical observations up to 48 months from the start of chemotherapy. A new ddPCR assay for the telomerase reverse transcriptase (TERT) promoter (-124) mutation was developed. The TERT assay was able to detect mutations in cases below the limit of detection by SNaPshot. Whole exome sequencing identified a novel mutation, CNTNAP4 G727*. A ddPCR assay designed to detect this mutation was able to distinguish mutant from wild-type alleles. CONCLUSIONS: The study demonstrated that ddPCR assays could be used to detect cftDNA in liquid biopsy monitoring of the post-therapy disease status in patients with UC. Overall, 70% of the patients in our study harbored mutations that were assayable by ddPCR.

A Turing Test for Molecular Generators.

Machine learning approaches promise to accelerate and improve success rates in medicinal chemistry programs by more effectively leveraging available data to guide a molecular design. A key step of an automated computational design algorithm is molecule generation, where the machine is required to design high-quality, drug-like molecules within the appropriate chemical space. Many algorithms have been proposed for molecular generation; however, a challenge is how to assess the validity of the resulting molecules. Here, we report three Turing-inspired tests designed to evaluate the performance of molecular generators. Profound differences were observed between the performance of molecule generators in these tests, highlighting the importance of selection of the appropriate design algorithms for specific circumstances. One molecule generator, based on match molecular pairs, performed excellently against all tests and thus provides a valuable component for machine-driven medicinal chemistry design workflows.

4-Phenyl-7-azaindoles as potent and selective IKK2 inhibitors.

The synthesis and SAR of a novel series of IKK2 inhibitors are described. Modification around the hinge binding region of the 7-azaindole led to a series of potent and selective inhibitors with good cellular activity.

A randomised, open-label, cross-over clinical study to evaluate the pharmacokinetic profiles of cigarettes and e-cigarettes with nicotine salt formulations in US adult smokers.

E-cigarettes containing 'nicotine salts' aim to increase smoker's satisfaction by improving blood nicotine delivery and other sensory properties. Here, we evaluated the pharmacokinetic profiles and subjective effects of nicotine from two e-cigarette device platforms with varying concentrations of nicotine lactate (nicotine salt) e-liquid relative to conventional cigarettes. A randomised, open-label, cross-over clinical study was conducted in 15 healthy US adult smokers. Five different e-cigarette products were evaluated consecutively on different days after use of own brand conventional cigarette. Plasma nicotine pharmacokinetics, subjective effects, and tolerability were assessed following controlled use of the products. The rate of nicotine absorption into the bloodstream was comparable from all e-cigarettes tested and was as rapid as that for conventional cigarette. However, in all cases, nicotine delivery did not exceed that of the conventional cigarette. The pharmacokinetic profiles of nicotine salt emissions were also dependent upon the properties of the e-cigarette device. Subjective scores were numerically highest after smoking a conventional cigarette followed by the myblu 40-mg nicotine salt formulation. The rise in nicotine blood levels following use of all the tested e-cigarettes was quantified as 'a little' to 'modestly' satisfying at relieving the desire to smoke. All products were well tolerated with no notable adverse events reported. These results demonstrate that, while delivering less nicotine than a conventional cigarette, the use of nicotine salts in e-cigarettes enables cigarette-like pulmonary delivery of nicotine that reduces desire to smoke.

Discovery of an Orally Bioavailable Pan αv Integrin Inhibitor for Idiopathic Pulmonary Fibrosis.

The heterodimeric transmembrane αv integrin receptors have recently emerged as potential targets for the treatment of idiopathic pulmonary fibrosis. Herein, we describe how subtle modifications of the central aromatic ring of a series of phenylbutyrate-based antagonists of the vitronectin receptors αvß3 and αvß5 significantly change the biological activities against αvß6 and αvß8. This resulted in the discovery of a pan αv antagonist (compound 39, 4-40 nM for the integrin receptors named above) possessing excellent oral pharmacokinetic properties in rats (with a clearance of 7.6 mL/(min kg) and a bioavailability of 97%).

Profile of a Highly Selective Quaternized Pyrrolidine Betaine αvß6 Integrin Inhibitor-(3S)-3-(3-(3,5-Dimethyl-1H-pyrazol-1-yl)phenyl)-4-((1S and 1R,3R)-1-methyl-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-ium-1-yl)butanoate Synthesized by Stereoselective Methylation.

A quaternary ammonium betaine 7 is described which shows exceptional potency and selectivity (1.4 to >3 logs) for the αvß6 integrin receptor over the other αv integrins as determined in cell adhesion assays. 7 is prepared by remarkably stereoselective methylation, the origins of which are discussed. The chemical, biological, physicochemical, and pharmacokinetic properties of 7 and its docking into αvß6 are described along with related analogues.

Localization of P-gp (Abcb1) and Mrp2 (Abcc2) in freshly isolated rat hepatocytes.

Freshly isolated hepatocytes are widely accepted as the "gold standard" for providing reliable data on drug uptake across the sinusoidal (basolateral) membrane. However, the suitability of freshly isolated hepatocytes in suspension to assess efflux by canalicular (apical) proteins or predict biliary excretion in the intact organ is unclear. After collagenase digestion, hepatocytes rapidly lose polarity, but localization of canalicular transport proteins in the first few hours after isolation has not been well characterized. In this study, immunostaining and confocal microscopy have provided, for the first time, a detailed examination of canalicular transport protein localization in freshly isolated rat hepatocytes fixed within 1 h of isolation and in cells cultured for 1 h. Organic anion transporting polypeptide 1a1 (Oatp1a1) was expressed in all hepatocytes and distributed evenly across the basolateral membrane; there was no evidence for colocalization of Oatp1a1 with P-glycoprotein (P-gp) or multidrug resistance-associated protein 2 (Mrp2). In contrast, P-gp and Mrp2 expression was lower than Oatp1a1 and confined to junctions between adjacent cells, intracellular compartments, and "legacy" network structures at or near the cell surface. P-gp and Mrp2 staining was more predominant in regions adjacent to former canalicular spaces, identified by zonula occludens-1 staining. Functional analysis of rat hepatocytes cultured for 1 h demonstrated that the fluorescent anion and Mrp2 substrate, 5-(and-6)-carboxy-2',7'-dichlorofluorescein (CDF), accumulated in cellular compartments; compartmental accumulation of CDF was sensitive to (E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-[[3-dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid (MK571, Mrp inhibitor) and was not observed in hepatocytes isolated from Mrp2-deficient rats. Drug efflux from freshly isolated hepatocytes as an estimate of apical efflux/biliary excretion would give an inaccurate assessment of true apical elimination and, as such, should not be used to make in vivo extrapolations.

Delay Between Shaking and Actuation of a Hydrofluoroalkane Fluticasone Pressurized Metered-Dose Inhaler.

BACKGROUND: Inhaled corticosteroids are used to treat pediatric asthma. The shaking of a pressurized metered-dose inhaler (pMDI) is required to ensure consistency of emitted dose. Delays between shaking and actuating the pMDI are frequent during administration of aerosols to children where a valved holding chamber is used. METHODS: In a recent clinical trial, we used a monitoring device to record shaking and actuation of the pMDI and the inhalation profiles of children with asthma while they were inhaling fluticasone hydrofluoroalkane from a valved holding chamber onto an external filter. During the procedure, in vitro and transport samples were generated without a delay between shaking and actuating the pMDI. Emitted dose, expressed as percentage of ex-actuator nominal dose, obtained from the second actuation following a recorded shake-actuation interval for subjects and from in vitro/transport samples (no delay) were compared. RESULTS: The mean emitted dose was 158.6% (95% CI 150.1-167.2%) (subjects) and 106.8% (95% CI 104.7-108.9%) (in vitro + transport) of the ex-actuator nominal dose (P < .001). The mean delay between shaking and actuating the pMDI was 12.9 s (95% CI 11.9-13.9 s) for the subject samples. A strong correlation was observed between shake and actuation delay and the emitted dose of the second actuation following the delay (Spearman correlation coefficient = 0.61). A 10-, 20-, and 30-s delay resulted in an emitted dose of the second actuation following the delay of 147, 187, and 227% of the ex-actuator nominal dose, respectively. CONCLUSIONS: Delays between shaking and actuating a corticosteroid suspension pMDI resulted in an increase in the emitted dose of the second actuation following the delay. This can be a common occurrence when doses are administered by a caregiver to a patient via a holding chamber. This should be addressed by practitioners educating patients and parents on proper inhaler use. (ClinicalTrials.gov registration NCT01714063.).

Mesh nebulizers have become the first choice for new nebulized pharmaceutical drug developments.

In the 24 years since first being marketed, the mesh nebulizer has been developed by five main manufacturers into a viable solution for the delivery of high-value nebulized drugs. Mesh nebulizers provide increased portability, convenience and energy efficiency along with similar lung deposition and increased ease of use compared with jet nebulizers. An analysis of EU and US clinical trial databases has shown that mesh nebulizers are now preferred over jet nebulizers for clinical trials sponsored by pharmaceutical companies. The results show a strong preference for the use of mesh nebulizers in trials involving high cost and niche therapy areas. Built-in capability to optimize the way patients use their mesh nebulizer and manage their disease will further increase uptake. [Formula: see text].