Real-World Experience on the Use of Mepolizumab from the Severe Asthma Registry of the German Asthma Net (MepoGAN-Study).

Purpose: The German Asthma Net (GAN) operates a Severe Asthma Registry that provides an overview of the clinical presentation and management of patients with severe asthma. Based upon data from the GAN registry, the MepoGAN study aimed to describe clinical profiles and treatment outcomes of patients who were treated with the anti-IL-5 monoclonal antibody mepolizumab (NucalaTM) in routine practice in Germany. Patients and Methods: The MepoGAN study is a descriptive retrospective non-interventional cohort study. Mepolizumab patients enrolled in the GAN registry were evaluated with results being described in two different data sets: Cohort 1 (n=131) started on mepolizumab when the patients entered the registry. Results were reported after 4 months of therapy. Patients in Cohort 2 (n=220) were on treatment with mepolizumab at the time of enrollment and follow-up data were collected after a further year of treatment. Outcome measures included asthma control, lung function, disease symptoms, OCS use, and exacerbations. Results: Patients enrolled in the registry who started on mepolizumab in Cohort 1 had a mean age of 55 years, were former smokers in 51% of the cases, had a mean blood eosinophil count of 500 cells/µL, and frequently had maintenance OCS use (55%). In this real-world setting, mepolizumab therapy was associated with a clinically relevant reduction in blood eosinophils (-445.7 cells/µL), OCS use (-30%), and improvement in asthma control. Fifty-five percent (vs 10% at baseline) of the patients reported controlled or partially controlled asthma 4 months after starting therapy. In patients who were already treated with mepolizumab at registry enrollment (Cohort 2), asthma control and lung function remained stable after a further year of observation. Conclusion: The GAN registry data confirm the effectiveness of mepolizumab in a real-world setting. Treatment benefits are maintained over time. While the asthma of patients treated in routine practice was more severe, the results observed with mepolizumab are broadly consistent with RCTs.

Is IQWiG's 15% Threshold Universally Applicable in Assessing the Clinical Relevance of Patient-Reported Outcomes Changes? An ISPOR Special Interest Group Report.

This article discusses a recent methodological change to assess the additional benefit of drug intervention by the German Federal Joint Committee (Gemeinsamer Bundesausschuss), a key stakeholder in EUnetHTA21 (European Network for Health Technology Assessment joint consortium for future EU HTA regulation), methodological workstream. The German Federal Joint Committee (Gemeinsamer Bundesausschuss) set a universal individual response threshold at ≥ 15% of the scale range of the measurement instrument, for all patient-reported outcomes, to achieve an additional benefit rating for a given pharmaceutical intervention. This approach is originally based on a corresponding recommendation from the Institute for Quality and Efficiency in Health Care. The merits of this approach are reviewed from various perspectives, including the evidence basis, statistical and psychometric considerations, and regulatory perspectives by the ISPOR Clinical Outcomes Assessment Special Interest Group's multistakeholder group of authors (academia, contract research organizations, and industry). Particular focus is placed on the patient perspective within the Institute for Quality and Efficiency in Health Care approach. The article development incorporated feedback from ISPOR members during well-attended ISPOR US and European conference presentations and 2 formal rounds of written review. The authors concluded that the ≥ 15% response threshold is incongruent with previously defined and scientifically established thresholds and is not well-suited for universal implementation. Further scientific evidence and discussion among all stakeholders are needed, especially should this universal rule be considered in the context of future joint clinical assessments of health technologies in the European Union scheduled from 2025 onward.

Cryo-EM structures of a LptDE transporter in complex with Pro-macrobodies offer insight into lipopolysaccharide translocation.

Lipopolysaccharides are major constituents of the extracellular leaflet in the bacterial outer membrane and form an effective physical barrier for environmental threats and for antibiotics in Gram-negative bacteria. The last step of LPS insertion via the Lpt pathway is mediated by the LptD/E protein complex. Detailed insights into the architecture of LptDE transporter complexes have been derived from X-ray crystallography. However, no structure of a laterally open LptD transporter, a transient state that occurs during LPS release, is available to date. Here, we report a cryo-EM structure of a partially opened LptDE transporter in complex with rigid chaperones derived from nanobodies, at 3.4 Å resolution. In addition, a subset of particles allows to model a structure of a laterally fully opened LptDE complex. Our work offers insights into the mechanism of LPS insertion, provides a structural framework for the development of antibiotics targeting LptD and describes a highly rigid chaperone scaffold to enable structural biology of challenging protein targets.

Acoustic levitation and rotation of thin films and their application for room temperature protein crystallography.

Acoustic levitation has attracted attention in terms of chemical and biochemical analysis in combination with various analytical methods because of its unique container-less environment for samples that is not reliant on specific material characteristics. However, loading samples with very high viscosity is difficult. To expand the scope, we propose the use of polymer thin films as sample holders, whereby the sample is dispensed on a film that is subsequently loaded onto an acoustic levitator. When applied for protein crystallography experiments, rotation controllability and positional stability are important prerequisites. We therefore study the acoustic levitation and rotation of thin films with an aspect ratio (the diameter-to-thickness ratio) of 80-240, which is an order of magnitude larger than those reported previously. For films with empirically optimized shapes, we find that it is possible to control the rotation speed in the range of 1-4 rotations per second while maintaining a positional stability of 12 ± 5 µm. The acoustic radiation force acting on the films is found to be a factor of 26-30 higher than that for same-volume water droplets. We propose use cases of the developed films for protein crystallography experiments and demonstrate data collections for large single crystal samples at room temperature.

Apo and ligand-bound high resolution Cryo-EM structures of the human Kv3.1 channel reveal a novel binding site for positive modulators.

Kv3 ion-channels constitute a class of functionally distinct voltage-gated ion channels characterized by their ability to fire at a high frequency. Several disease relevant mutants, together with biological data, suggest the importance of this class of ion channels as drug targets for CNS disorders, and several drug discovery efforts have been reported. Despite the increasing interest for this class of ion channels, no structure of a Kv3 channel has been reported yet. We have determined the cryo-EM structure of Kv3.1 at 2.6 Å resolution using full-length wild type protein. When compared to known structures for potassium channels from other classes, a novel domain organization is observed with the cytoplasmic T1 domain, containing a well-resolved Zinc site and displaying a rotation by 35°. This suggests a distinct cytoplasmic regulation mechanism for the Kv3.1 channel. A high resolution structure was obtained for Kv3.1 in complex with a novel positive modulator Lu AG00563. The structure reveals a novel ligand binding site for the Kv class of ion channels located between the voltage sensory domain and the channel pore, a region which constitutes a hotspot for disease causing mutations. The discovery of a novel binding site for a positive modulator of a voltage-gated potassium channel could shed light on the mechanism of action for these small molecule potentiators. This finding could enable structure-based drug design on these targets with high therapeutic potential for the treatment of multiple CNS disorders.

Fate of weathered multi-walled carbon nanotubes in an aquatic sediment system.

The widespread application of carbon nanotubes (CNT) in various consumer products leads to their inevitable release into aquatic systems. But only little is known about their distribution among aquatic compartments. In this study, we investigated the partitioning of radiolabeled, weathered multi-walled CNT (14C-wMWCNT) in an aquatic sediment system over a period of 180 days (d). The applied nanomaterial concentration in water phase was 100 µg L-1. Over time, the wMWCNT disappeared exponentially from the water phase and simultaneously accumulated in the sediment phase. After 2 h incubation just 77%, after seven days 30% and after 180 d only 0.03% of applied radioactivity (AR) remained in the water phase. The respective values for the disappearance times DT50 and DT90 were 3.2 d and 10.7 d. Further, minor mineralization of 14C-wMWCNT to 14CO2 was observed with values below 0.06% of AR. In addition, a study was carried out to estimate the deposition of wMWCNT in the water phase with and without sediment in the test system for 28 d. We found no influence of a sediment phase on the sedimentation behavior of wMWCNT in the water phase: After 6.5 d and 7.3 d 50% of the applied wMWCNT subsided in the presence and absence of sediment, respectively. The slow removal of wMWCNT from the water body by deposition into sediment implies that in addition to sediment-dwelling organisms, pelagic organisms are also at risk of exposure to nanomaterials and prone for their take-up.

A trophic transfer study: accumulation of multi-walled carbon nanotubes associated to green algae in water flea Daphnia magna.

Carbon nanotubes (CNT) are promising nanomaterials in modern nanotechnology and their use in many different applications leads to an inevitable release into the aquatic environment. In this study, we quantified trophic transfer of weathered multi-walled carbon nanotubes (wMWCNT) from green algae to primary consumer Daphnia magna in a concentration of 100 µg L-1 using radioactive labeling of the carbon backbone (14C-wMWCNT). Trophic transfer of wMWCNT was compared to the uptake by daphnids exposed to nanomaterials in the water phase without algae. Due to the rather long observed CNT sedimentation times (DT) from the water phase (DT50: 3.9 days (d), DT90: 12.8 d) wMWCNT interact with aquatic organisms and associated to the green algae Chlamydomonas reinhardtii and Raphidocelis subcapitata. After the exposition of algae, the nanotubes accumulated to a maximum of 1.6 ± 0.4 µg 14C-wMWCNT mg-1 dry weight-1 (dw-1) and 0.7 ± 0.3 µg 14C-wMWCNT mg-1 dw-1 after 24 h and 48 h, respectively. To study trophic transfer, R. subcapitata was loaded with 14C-wMWCNT and subsequently fed to D. magna. A maximum body burden of 0.07 ± 0.01 µg 14C-wMWCNT mg-1 dw-1 and 7.1 ± 1.5 µg 14C-wMWCNT mg-1 dw-1 for D. magna after trophic transfer and waterborne exposure was measured, respectively, indicating no CNT accumulation after short-term exposure via trophic transfer. Additionally, the animals eliminated nanomaterials from their guts, while feeding algae facilitated their excretion. Further, accumulation of 14C-wMWCNT in a growing population of D. magna revealed a maximum uptake of 0.7 ± 0.2 µg mg-1 dw-1. Therefore, the calculated bioaccumulation factor (BAF) after 28 d of 6700 ± 2900 L kg-1 is above the limit that indicates a chemical is bioaccumulative in the European Union Regulation REACH. Although wMWCNT did not bioaccumulate in neonate D. magna after trophic transfer, wMWCNT enriched in a 28 d growing D. magna population regardless of daily feeding, which increases the risk of CNT accumulation along the aquatic food chain.

Advances in long-wavelength native phasing at X-ray free-electron lasers.

Long-wavelength pulses from the Swiss X-ray free-electron laser (XFEL) have been used for de novo protein structure determination by native single-wavelength anomalous diffraction (native-SAD) phasing of serial femtosecond crystallography (SFX) data. In this work, sensitive anomalous data-quality indicators and model proteins were used to quantify improvements in native-SAD at XFELs such as utilization of longer wavelengths, careful experimental geometry optimization, and better post-refinement and partiality correction. Compared with studies using shorter wavelengths at other XFELs and older software versions, up to one order of magnitude reduction in the required number of indexed images for native-SAD was achieved, hence lowering sample consumption and beam-time requirements significantly. Improved data quality and higher anomalous signal facilitate so-far underutilized de novo structure determination of challenging proteins at XFELs. Improvements presented in this work can be used in other types of SFX experiments that require accurate measurements of weak signals, for example time-resolved studies.

Persistence and adherence to dutasteride/tamsulosin fixed-dose versus free-combination alpha blocker/5ARI therapy in patients with benign prostate hyperplasia in Germany
.

OBJECTIVE: To evaluate real-world persistence and adherence in patients with benign prostate hyperplasia (BPH) receiving a fixed-dose combination of dutasteride plus tamsulosin (DUT-TAM FDC) versus α-blocker plus 5-α reductase inhibitor (AB/5ARI) free-combination therapy. MATERIALS AND METHODS: This retrospective, observational cohort study utilized the German IMS LRx (IQVIA) database. Patients ≥ 45 years old with BPH receiving DUT-TAM FDC or AB/5ARI free-combination therapy from July 1, 2011 to November 30, 2017 were included. Data were analyzed for 48 months from index date (date of first prescription). Persistence, measured as time to discontinuation (defined as a 90-day gap in therapy), was evaluated using Kaplan-Meier curves (log-rank tests). Adherence, measured as medication possession ratio (MPR), was based on a comparison of mean prescribing duration and expected treatment duration. RESULTS: A total of 141,667 patients were included (DUT-TAM FDC, n = 86,057; free AB/5ARI: n = 55,610). Small differences in persistence were observed between treatment arms. At month 12, 41.8% of DUT-TAM FDC-treated and 41.0% of AB/5ARI free-combination therapy-treated patients were persistent; at month 24, 28.2% and 27.1% were persistent, respectively. A higher proportion of DUT-TAM FDC-treated patients had MPR ≥ 0.80, ≥ 0.75 and ≥ 0.70 compared with AB/5ARI free-combination therapy (p < 0.0001). CONCLUSION: Small differences observed in persistence between treatment arms may not translate to meaningful clinical relevance. Adherence was significantly better in the FDC arm, which may be clinically relevant as improved adherence is associated with better outcomes. Persistence and adherence to BPH therapy in Germany is low; further studies exploring the reasons behind this are required.

In situ crystallography as an emerging method for structure solution of membrane proteins: the case of CCR2A.

The in meso in situ serial X-ray crystallization method (Huang et al., (2015) Acta Crystallogr D Biol Crystallogr 71, 1238) combines lipid cubic phase crystallization, direct freezing of the crystallization droplet without handling of the crystals, and data collection in situ. Recently, this method was used to overcome the mechanical fragility of crystals which enabled the X-ray structure determination of chemokine receptor 2A (Apel et al., (2019) Structure 27, 427) at 2.7 Å resolution. The CCR2 structure provides the structural basis for ligand selectivity of CCR2 against chemokine receptor 5 and provides insights into the residence time of MK-0812 analogs based on molecular dynamics simulations. These findings offer new opportunities for drug discovery targeting chemokine receptors.

Crystal Structure of CC Chemokine Receptor 2A in Complex with an Orthosteric Antagonist Provides Insights for the Design of Selective Antagonists.

We determined two crystal structures of the chemokine receptor CCR2A in complex with the orthosteric antagonist MK-0812. Full-length CCR2A, stabilized by rubredoxin and a series of five mutations were resolved at 3.3 Å. An N- and C-terminally truncated CCR2A construct was crystallized in an alternate crystal form, which yielded a 2.7 Å resolution structure using serial synchrotron crystallography. Our structures provide a clear structural explanation for the observed key role of residue E2917.39 in high-affinity binding of several orthosteric CCR2 antagonists. By combining all the structural information collected, we generated models of co-structures for the structurally diverse pyrimidine amide class of CCR2 antagonists. Even though the representative Ex15 overlays well with MK-0812, it also interacts with the non-conserved H1213.33, resulting in a significant selectivity over CCR5. Insights derived from this work will facilitate drug discovery efforts directed toward highly selective CCR2 antagonists with potentially superior efficacy.

Ligand channel in pharmacologically stabilized rhodopsin.

In the degenerative eye disease retinitis pigmentosa (RP), protein misfolding leads to fatal consequences for cell metabolism and rod and cone cell survival. To stop disease progression, a therapeutic approach focuses on stabilizing inherited protein mutants of the G protein-coupled receptor (GPCR) rhodopsin using pharmacological chaperones (PC) that improve receptor folding and trafficking. In this study, we discovered stabilizing nonretinal small molecules by virtual and thermofluor screening and determined the crystal structure of pharmacologically stabilized opsin at 2.4 Å resolution using one of the stabilizing hits (S-RS1). Chemical modification of S-RS1 and further structural analysis revealed the core binding motif of this class of rhodopsin stabilizers bound at the orthosteric binding site. Furthermore, previously unobserved conformational changes are visible at the intradiscal side of the seven-transmembrane helix bundle. A hallmark of this conformation is an open channel connecting the ligand binding site with the membrane and the intradiscal lumen of rod outer segments. Sufficient in size, the passage permits the exchange of hydrophobic ligands such as retinal. The results broaden our understanding of rhodopsin's conformational flexibility and enable therapeutic drug intervention against rhodopsin-related retinitis pigmentosa.

X-ray free electron laser: opportunities for drug discovery.

Past decades have shown the impact of structural information derived from complexes of drug candidates with their protein targets to facilitate the discovery of safe and effective medicines. Despite recent developments in single particle cryo-electron microscopy, X-ray crystallography has been the main method to derive structural information. The unique properties of X-ray free electron laser (XFEL) with unmet peak brilliance and beam focus allow X-ray diffraction data recording and successful structure determination from smaller and weaker diffracting crystals shortening timelines in crystal optimization. To further capitalize on the XFEL advantage, innovations in crystal sample delivery for the X-ray experiment, data collection and processing methods are required. This development was a key contributor to serial crystallography allowing structure determination at room temperature yielding physiologically more relevant structures. Adding the time resolution provided by the femtosecond X-ray pulse will enable monitoring and capturing of dynamic processes of ligand binding and associated conformational changes with great impact to the design of candidate drug compounds.

Serial millisecond crystallography for routine room-temperature structure determination at synchrotrons.

Historically, room-temperature structure determination was succeeded by cryo-crystallography to mitigate radiation damage. Here, we demonstrate that serial millisecond crystallography at a synchrotron beamline equipped with high-viscosity injector and high frame-rate detector allows typical crystallographic experiments to be performed at room-temperature. Using a crystal scanning approach, we determine the high-resolution structure of the radiation sensitive molybdenum storage protein, demonstrate soaking of the drug colchicine into tubulin and native sulfur phasing of the human G protein-coupled adenosine receptor. Serial crystallographic data for molecular replacement already converges in 1,000-10,000 diffraction patterns, which we collected in 3 to maximally 82 minutes. Compared with serial data we collected at a free-electron laser, the synchrotron data are of slightly lower resolution, however fewer diffraction patterns are needed for de novo phasing. Overall, the data we collected by room-temperature serial crystallography are of comparable quality to cryo-crystallographic data and can be routinely collected at synchrotrons.Serial crystallography was developed for protein crystal data collection with X-ray free-electron lasers. Here the authors present several examples which show that serial crystallography using high-viscosity injectors can also be routinely employed for room-temperature data collection at synchrotrons.

SPR-based fragment screening with neurotensin receptor 1 generates novel small molecule ligands.

The neurotensin receptor 1 represents an important drug target involved in various diseases of the central nervous system. So far, the full exploitation of potential therapeutic activities has been compromised by the lack of compounds with favorable physicochemical and pharmacokinetic properties which efficiently penetrate the blood-brain barrier. Recent progress in the generation of stabilized variants of solubilized neurotensin receptor 1 and its subsequent purification and successful structure determination presents a solid starting point to apply the approach of fragment-based screening to extend the chemical space of known neurotensin receptor 1 ligands. In this report, surface plasmon resonance was used as primary method to screen 6369 compounds. Thereby 44 hits were identified and confirmed in competition as well as dose-response experiments. Furthermore, 4 out of 8 selected hits were validated using nuclear magnetic resonance spectroscopy as orthogonal biophysical method. Computational analysis of the compound structures, taking the known crystal structure of the endogenous peptide agonist into consideration, gave insight into the potential fragment-binding location and interactions and inspires chemistry efforts for further exploration of the fragments.

Current practice and perspectives in CRO oversight based on a survey performed among members of the German Association of Research-Based Pharmaceutical Companies (vfa).

In recent years, the number and scope of outsourced activities in the pharmaceutical industry have increased heavily. In addition, also the type of outsourcing has changed significantly in that time. This raises the question of whether and how sponsors retain the capability to select and to control the contract research organizations (CROs) involved and what expertise still has to be present in the development department as well as other relevant departments to ensure adequate oversight, also in line with the expectations of regulators and health authorities. In order to answer these questions, a survey was conducted among the German vfa member companies. The survey describes the latest developments and experiences in outsourcing by 18 German vfa member companies. It concentrates on measures how to implement Quality Assurance (QA) when performing outsourced clinical studies. This study shows that the majority of companies apply a full-outsourcing, preferred-provider model of clinical trial services, with the clinical research department playing the major role in this process. A large amount of guiding documents, processes and tools are used to ensure an adequate oversight of the services performed by the CRO(s). Finally the guiding principles for all oversight processes should be transparent communication, a clearly established expectation for quality, a precise definition of accountability and responsibility while avoiding silo mentality, and a comprehensive documentation of the oversight's evidence. For globally acting and outsourcing sponsors, oversight processes need to be aligned with regards to local and global perspectives. This survey shows that the current implementation of oversight processes in the participating companies covers all relevant areas to ensure highest quality and integrity of the data produced by the outsourced clinical trial.

Effectiveness of alitretinoin in severe chronic hand eczema: PASSION, a real-world observational study.

PURPOSE: Severe chronic hand eczema (CHE) has a debilitating effect on quality of life (QoL). PASSION evaluated the effectiveness of oral alitretinoin on QoL and work productivity in patients with severe CHE following prescribing guidelines. METHODS: A non-interventional, open-label, observational, multicentre study conducted in Germany in fulfilment of German guidelines. Patients (n = 631) were treated with once-daily alitretinoin for ≤24 weeks under standard daily practise conditions. Effectiveness was assessed by Physician Global Assessment (PGA), QoL Assessment (EQ-5D) and work impairment. Tolerability and safety were assessed by adverse event (AE) monitoring. RESULTS: In total, 279 (44.2%) patients dropped out before Week 24. Of the 631 patients enrolled, 29.8% achieved a PGA rating of clear/almost clear at Week 24. Mean (standard deviation) EQ-5D utility and EQ-5D visual analogue scale scores at baseline were 0.76 (0.25) and 53.6 (23.55), respectively, and increased to 0.94 (0.12) and 80.8 (19.23) at Week 24, indicating improved QoL. At baseline, 49.4%/29.1% of patients reported strong/very strong workplace impairment, respectively, and decreased to 8.5%/1.4%, respectively, at Week 24. AEs were reported in 116 (18.4%) patients. No new safety signals were observed. CONCLUSIONS: Alitretinoin produced marked improvement in the QoL and work productivity of patients with severe CHE.

Biophysics in drug discovery: impact, challenges and opportunities.

Over the past 25 years, biophysical technologies such as X-ray crystallography, nuclear magnetic resonance spectroscopy, surface plasmon resonance spectroscopy and isothermal titration calorimetry have become key components of drug discovery platforms in many pharmaceutical companies and academic laboratories. There have been great improvements in the speed, sensitivity and range of possible measurements, providing high-resolution mechanistic, kinetic, thermodynamic and structural information on compound-target interactions. This Review provides a framework to understand this evolution by describing the key biophysical methods, the information they can provide and the ways in which they can be applied at different stages of the drug discovery process. We also discuss the challenges for current technologies and future opportunities to use biophysical methods to solve drug discovery problems.

Release of (14)C-labelled carbon nanotubes from polycarbonate composites.

Waste disposal of carbon nanotube (CNT) containing products is expected to be the most important pathway for release of CNTs into the environment. In the present work, the use of radiolabelled CNTs ((14)C-CNT) for polycarbonate polymer nanocomposites with 1 wt% (14)C-CNT content allowed for the first time to quantify and differentiate the CNT release according to the type of impact along the materials' ageing history. After an initial exposure of the nanocomposite by solar-like irradiation, further environmental impacts were applied to composite material. They aimed at mimicking disposal site conditions that may induce further ageing effects and CNT release. This study included shaking in water, rapid temperature changes, soaking in humic acid solution as well as waste water effluent, and, finally, gentle mechanical abrasion. All ageing impacts were applied sequentially, both on pristine (control) and on solar-irradiated nanocomposites. All experiments were accompanied by absolute quantification of radioactive release as well as chemical and morphological analyses of the nanocomposite surfaces using infra-red (IR) spectroscopy, X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). The morphological analysis showed that spectral irradiation can uncover CNT networks on the outer nanocomposite surface layers by polymer degradation. After having subjected the solar-irradiated nanocomposite to all studied disposal site effect, the total radioactive release was quantified to amount to 64 mg CNT/m(2), whereas only 0.8 mg CNT/m(2) were found for the un-irradiated control sample. Solar degradation of polymers was thus found to significantly increase the propensity of the studied polymer nanocomposites to release CNTs during ageing effects at the product's end-of-life typical for disposal sites.