PECAM-1 supports leukocyte diapedesis by tension-dependent dephosphorylation of VE-cadherin.

Leukocyte extravasation is an essential step during the immune response and requires the destabilization of endothelial junctions. We have shown previously that this process depends in vivo on the dephosphorylation of VE-cadherin-Y731. Here, we reveal the underlying mechanism. Leukocyte-induced stimulation of PECAM-1 triggers dissociation of the phosphatase SHP2 which then directly targets VE-cadherin-Y731. The binding site of PECAM-1 for SHP2 is needed for VE-cadherin dephosphorylation and subsequent endocytosis. Importantly, the contribution of PECAM-1 to leukocyte diapedesis in vitro and in vivo was strictly dependent on the presence of Y731 of VE-cadherin. In addition to SHP2, dephosphorylation of Y731 required Ca2+ -signaling, non-muscle myosin II activation, and endothelial cell tension. Since we found that ß-catenin/plakoglobin mask VE-cadherin-Y731 and leukocyte docking to endothelial cells exert force on the VE-cadherin-catenin complex, we propose that leukocytes destabilize junctions by PECAM-1-SHP2-triggered dephosphorylation of VE-cadherin-Y731 which becomes accessible by actomyosin-mediated mechanical force exerted on the VE-cadherin-catenin complex.

Development of Ternary Amorphous Solid Dispersions Manufactured by Hot-Melt Extrusion and Spray-Drying─Comparison of In Vitro and In Vivo Performance.

Producing amorphous solid dispersions (ASDs) by hot-melt extrusion (HME) is favorable from an economic and ecological perspective but also limited to thermostable active pharmaceutical ingredients (APIs). A potential technology shift from spray-drying to hot-melt extrusion at later stages of drug product development is a desirable goal, however bearing the risk of insufficient comparability of the in vitro and in vivo performance of the final dosage form. Hot-melt extrusion was performed using API/polymer/surfactant mixtures with hydroxypropyl methylcellulose acetate succinate (HPMCAS) as the polymer and evaluated regarding the extrudability of binary and ternary amorphous solid dispersions (ASDs). Additionally, spray-dried ASDs were produced, and solid-state properties were compared to the melt-extruded ASDs. Tablets were manufactured of a ternary ASD lead candidate comparing their in vitro dissolution and in vivo performance. The extrudability of HPMCAS was improved by adding a surfactant as plasticizer, thereby lowering the high melt-viscosity. d-α-Tocopheryl polyethylene glycol succinate (TPGS) as surfactant showed the most similar solid-state properties between spray-dried and extruded ASDs compared to those of poloxamer 188 and sodium dodecyl sulfate. The addition of TPGS, however, barely affected API/polymer interactions. The in vitro dissolution experiment and in vivo dog study revealed a higher drug release of tablets manufactured from the spray-dried ASD compared to the melt-extruded ASD; this was attributed to the different particle size. We could further demonstrate that the drug release can be controlled by adjusting the particle size of melt-extruded ASDs leading to a similar release profile compared to tablets containing the spray-dried dispersion, which confirmed the feasibility of a technology shift from spray-drying to HME upon drug product development.

Transport disrupted - Substituting public transport by bike or car under Covid 19.

The Covid 19 pandemic has caused dramatic disruptions in the public transport sector that has seen a stark downturn in many cities across the globe, calling into question previous efforts to reduce air pollution and CO2 emissions by expanding this sector. Especially, the current surge of individual car use is worrying and the question remains which users might be able and willing to substitute public transport by cycling. This effect is interesting to study for the case of Hanover Region, because of the well-developed biking infrastructure that makes biking a viable alternative to individual car use. In this paper, we analyze survey data from June 2020 on the use of transportation modes before and during the pandemic in the Hanover Region. We ask if and how the over 4.000 participants substitute public transport and what characterizes those who chose biking over individual car use. We use multivariate regression models and find evidence that Stadtbahn (local light rail) and bus are substituted by bike, car and working from home, while train use is not significantly replaced by car and seems to be positively related to bike use. The data also shows that women have a higher level of fear of infection than men have during public transport use and therefore reduce public transport use more. Moreover, income displays a positive effect on increased car use while cycling is independent of socio-economic indicators but instead driven by the eco-consciousness of users. Surprisingly, we find that car use was increased in particular by residents of Hanover city, while it was decreased by residents of less densely populated urban areas in the region.

Evaluation of Pharmacobezoar Formation from Suspensions of Spray-Dried Amorphous Solid Dispersions: An MRI Study in Rats.

Spray-dried amorphous solid dispersions of new chemical entities and pH-dependent soluble polymer hydroxypropyl methylcellulose acetate succinate (HPMC-AS) were found to form solid agglomerates in the gastrointestinal tract of rodents after oral administration. These agglomerates, referring to descriptions of intra-gastrointestinal aggregated oral dosage forms termed pharmacobezoars, represent a potential risk for animal welfare. Previously, we introduced an in vitro model to assess the agglomeration potential of amorphous solid dispersions from suspensions and how it can be reduced. In this work, we investigated if the in vitro effective approach of viscosity enhancement of the vehicle used to prepare suspensions of amorphous solid dispersions could reduce the pharmacobezoar formation potential following repeated daily oral dosing to rats as well. The dose level of 2400 mg/kg/day used in the main study was determined in a dose finding study carried out in advance. In the dose finding study, MRI investigations were carried out at short time intervals to gain insights into the process of pharmacobezoar formation. Whereas MRI investigations underlined the importance of the forestomach for the formation of pharmacobezoars, viscosity enhancement of the vehicle reduced the incidence of pharmacobezoars, delayed the onset of pharmacobezoar formation and reduced the overall mass of pharmacobezoars found at necropsy.

Drug Dissolution in Oral Drug Absorption: Workshop Report.

The in-person workshop "Drug Dissolution in Oral Drug Absorption" was held on May 23-24, 2023, in Baltimore, MD, USA. The workshop was organized into lectures and breakout sessions. Three common topics that were re-visited by various lecturers were amorphous solid dispersions (ASDs), dissolution/permeation interplay, and in vitro methods to predict in vivo biopharmaceutics performance and risk. Topics that repeatedly surfaced across breakout sessions were the following: (1) meaning and assessment of "dissolved drug," particularly of poorly water soluble drug in colloidal environments (e.g., fed conditions, ASDs); (2) potential limitations of a test that employs sink conditions for a poorly water soluble drug; (3) non-compendial methods (e.g., two-stage or multi-stage method, dissolution/permeation methods); (4) non-compendial conditions (e.g., apex vessels, non-sink conditions); and (5) potential benefit of having both a quality control method for batch release and a biopredictive/biorelevant method for biowaiver or bridging scenarios. An identified obstacle to non-compendial methods is the uncertainty of global regulatory acceptance of such methods.

An In Vitro Model to Investigate the Potential of Solid Dispersions to Form Pharmacobezoars.

The formation of pharmacobezoars from suspensions of spray-dried amorphous solid dispersions (SD-ASDs) of new chemical entities (NCEs) and hydroxypropyl methylcellulose acetate succinate (HPMC-AS) represents a non-compound related adverse effect in preclinical oral toxicity studies in rodents. Whereas the contribution of the insolubility of the carrier polymer to this process taking place in the acidic environment of the rodent stomach is conclusive, unawareness of the extent of in vivo pharmacobezoar formation is adverse. In order to evaluate the risk of pharmacobezoar formation before in vivo administration, we subsequently introduce an in vitro model to assess the agglomeration potential of solid dispersions. To verify that the pharmacobezoar formation potential can be assessed based on the observed agglomeration potential, we conducted a sequence of experiments with two HPMC-AS-based SD-ASD formulations. In vitro, we found their different in vivo pharmacobezoar formation potential reflected by a significantly increased agglomerated mass of formulation 1 per day compared to formulation 2. In order to find an approach to reduce the agglomeration potential of solid dispersion from suspensions, we further applied the model to investigate the impact of the viscosity of the vehicle used to prepare suspensions on agglomerate formation.

Splicing analyses for variants in MMR genes: best practice recommendations from the European Mismatch Repair Working Group.

Over 20% of the DNA mismatch repair (MMR) germline variants in suspected Lynch syndrome patients are classified as variants of uncertain significance (VUS). Well-established functional assays are pivotal for assessing the biological impact of these variants and provide relevant evidence for clinical classification. In our collaborative European Mismatch Repair Working Group (EMMR-WG) we compared three different experimental approaches for evaluating the effect of seven variants on mRNA splicing in MMR genes: (i) RT-PCR of full-length transcripts (FLT), (ii) RT-PCR of targeted transcript sections (TTS), both from patient biological samples and (iii) minigene splicing assays. An overall good concordance was observed between splicing patterns in TTS, FLT and minigene analyses for all variants. The FLT analysis depicted a higher number of different isoforms and mitigated PCR-bias towards shorter isoforms. TTS analyses may miss aberrant isoforms and minigene assays may under/overestimate the severity of certain splicing defects. The interpretation of the experimental findings must be cautious to adequately discriminate abnormal events from physiological complex alternative splicing patterns. A consensus strategy for investigating the impact of MMR variants on splicing was defined. First, RNA should be obtained from patient's cell cultures (such as fresh lymphocyte cultures) incubated with/without a nonsense-mediated decay inhibitor. Second, FLT RT-PCR analysis is recommended to oversee all generated isoforms. Third, TTS analysis and minigene assays are useful independent approaches for verifying and clarifying FLT results. The use of several methodologies is likely to increase the strength of the experimental evidence which contributes to improve variant interpretation.

Association between neuroendocrinological parameters and learning and memory functions in adolescent anorexia nervosa before and after weight recovery.

A growing body of evidence indicates that hormones play an important role in learning and memory functions as well as in mood modulation. During the acute stage of anorexia nervosa (AN), weight loss has a significant effect on serum levels of estrogen, thyroid hormones, and cortisol. Furthermore deficits in learning and memory functions are evident in patients with eating disorders during emaciation. Hormonal and neuropsychological alterations at least partly remit during weight restoration. We investigated the association between learning and memory functions as well as mood and neuroendocrinological parameters before and after weight gain in adolescent AN. Twenty-eight female subjects with AN, diagnosed according to DSM-IV, were examined before and after weight recovery. Both investigations took place while the patients were receiving hospital treatment, and the results were compared to a control group consisting of 18 age- and IQ-matched normal-weight female adolescents also tested twice within 4 months. Verbal memory and learning were assessed by a German paper-pencil-test (LGT). We performed correlation calculations between neuropsychological functions and depressive symptoms and estrogen, cortisol and free triiodothyronine (fT3) in the plasma at both time points. Compared to normal controls adolescents with AN performed worse in one subtest of the LGT which requires the verbal reproduction of figural material across both time points. Verbal learning was positively correlated with estrogen levels after weight recovery. Depressive symptoms of AN patients significantly decreased during weight rehabilitation and correlated negatively with fT3 at T1. We did not find a relationship between cortisol levels and neuropsychological functions. We observed subtle memory impairments and depressive symptoms in subjects with adolescent AN associated with starvation-induced estrogen and triiodothyronine deficits, respectively. Normalization of body weight and resuming of menses is needed to restore learning and memory functions as well as to alleviate depressive symptoms.

On the usefulness of compendial setups and tiny-TIM system in evaluating the in vivo performance of oral drug products with various release profiles in the fasted state: Case example sodium salt of A6197.

We evaluated the usefulness of quality control dissolution data collected with compendial Apparatus I and II, biorelevant dissolution data collected with compendial apparatus IV, and bioaccessibility data collected with the non-compendial tiny-TIM system in screening modified release formulations during the development of BCS Class I compounds using a Boehringer Ingelheim model experimental compound, A6197. Four products were investigated: an immediate release tablet, an extended release tablet, modified release mini-tablets, and extended release pellets. Data with modified release products collected with the compendial apparatus were evaluated vs. the average intraluminal dissolution estimated after deconvoluting clinical data collected in healthy adults. Data collected with the tiny-TIM system were evaluated vs. the average AUC and Cmax values estimated from the clinical data. Unlike with the quality control data collected with Apparatus I and II, data collected with Apparatus IV data and Level I biorelevant media adequately described the intraluminal dissolution process of the three modified release products. Data deviated less than 10% from the actual average deconvoluted intraluminal dissolution profiles, illustrating the usefulness of Apparatus IV biorelevant data in understanding the intraluminal dissolution process of BCS class I small molecules administered as modified release products in the fasted state. Total bioaccessibility data and maximum bioaccessibility data collected using the tiny-TIM and the immediate release tablet and the three modified release drug products correctly reproduced the ranking of A6197 AUC values (R2 = 0.989) and Cmax values (R2 = 0.962), respectively, illustrating tiny-TIM as a useful system for formulation selection of BCS class I small molecules administered in the fasted state.

Biopharmaceutic IVIVE-Mechanistic Modeling of Single- and Two-Phase In Vitro Experiments to Obtain Drug-Specific Parameters for Incorporation Into PBPK Models.

The physiological relevance of single-phase (aqueous only) and 2-phase (aqueous and organic phase) in vitro dissolution experiments was compared by mechanistic modeling. For orally dosed dipyridamole, stepwise, sequential estimation/confirmation of biopharmaceutical parameters from in vitro solubility-dissolution data was followed, before applying them within a physiologically based pharmacokinetic (PBPK) model. The PBPK model predicted clinical dipyridamole luminal and plasma concentration profiles reasonably well for a range of doses only where the precipitation rate constant was derived from the 2-phase experiment. The population model predicted a distribution of maximal precipitated fractions from 0% to 45% of the 90 mg dose (mean 7.6%). Such population information cannot be obtained directly from a few in vitro experiments; however well they may represent an "average" and several extreme subjects (those with low-high luminal fluid volumes, pH, etc.) because there is no indication of outcome likelihood. For this purpose, direct input of in vitro dissolution/precipitation profiles to a PBPK model is insufficient-mechanistic modeling is required. Biopharmaceutical in vitro-in vivo extrapolation tools can also simulate the effect of key experimental parameters (dissolution volumes, pH, paddle speed, etc.) on dissolution/precipitation behavior, thereby helping to identify critical variables, which may impact the number or design of in vitro experiments.

Full-length transcript amplification and sequencing as universal method to test mRNA integrity and biallelic expression in mismatch repair genes.

In pathogenicity assessment, RNA-based analyses are important for the correct classification of variants, and require gene-specific cut-offs for allelic representation and alternative/aberrant splicing. Beside this, the diagnostic yield of RNA-based techniques capable to detect aberrant splicing or allelic loss due to intronic/regulatory variants has to be elaborated. We established a cDNA analysis for full-length transcripts (FLT) of the four DNA mismatch repair (MMR) genes to investigate the splicing pattern and transcript integrity with active/inhibited nonsense-mediated mRNA-decay (NMD). Validation was based on results from normal controls, samples with premature termination codons (PTC), samples with splice-site defects (SSD), and samples with pathogenic putative missense variants. The method was applied to patients with variants of uncertain significance (VUS) or unexplained immunohistochemical MMR deficiency. We categorized the allelic representation into biallelic (50 ± 10%) or allelic loss (≤10%), and >10% and <40% as unclear. We defined isoforms up to 10% and exon-specific exceptions as alternative splicing, set the cut-off for SSD in cDNA + P to 30-50%, and regard >10% and <30% as unclear. FLT cDNA analyses designated 16% of all putative missense variants and 12% of VUS as SSD, detected MMR-defects in 19% of the unsolved patients, and re-classified >30% of VUS. Our method allows a standardized, systematic cDNA analysis of the MMR FLTs to assess the pathogenicity mechanism of VUS on RNA level, which will gain relevance for precision medicine and gene therapy. Diagnostic accuracy will be enhanced by detecting MMR defects in hitherto unsolved patients. The data generated will help to calibrate a high-throughput NGS-based mRNA-analysis and optimize prediction programs.

In vitro models for the prediction of in vivo performance of oral dosage forms: Recent progress from partnership through the IMI OrBiTo collaboration.

The availability of in vitro tools that are constructed on the basis of a detailed knowledge of key aspects of gastrointestinal (GI) physiology and their impact on formulation performance and subsequent drug release behaviour is fundamental to the success and efficiency of oral drug product development. Over the last six years, the development and optimization of improved, biorelevant in vitro tools has been a cornerstone of the IMI OrBiTo (Oral Biopharmaceutics Tools) project. By bringing together key industry and academic partners, and by linking tool development and optimization to human studies to understand behaviour at the formulation/GI tract interface, the collaboration has enabled innovation, optimization and implementation of the requisite biorelevant in vitro tools. In this paper, we present an overview of the in vitro tools investigated during the collaboration and offer a perspective on their future use in enhancing the development of new oral drug products.

Mechanistic study on hydrodynamics in the mini-scale biphasic dissolution model and its influence on in vitro dissolution and partitioning.

Biphasic dissolution models were proposed to provide good predictive power for in vivo absorption kinetics. However, up to date the impact of hydrodynamics in mini-scale models are not well understood. Consequently, the aim of this work was to investigate different setups of a previously published mini-scale biphasic dissolution model (miBIdi-pH-II) to better understand the relevance of hydrodynamics for evaluating kinetic parameters and to simultaneously increase the robustness of the experimental model. As a first step, the hydrodynamics within the aqueous phase were characterized by in silico simulations of the flow patterns. Different settings, such as higher rotation speeds of the paddles, the implementation of a second propeller into the aqueous phase, and different shapes of aqueous stirrers were investigated. Second, to evaluate the results of the in silico simulations, in vitro experiments with glitter were carried out. Last, the same settings were applied in the miBIdi-pH-II using dipyridamole (DPD) as model compound to estimate kinetic parameters by applying a compartment-based modelling approach. Both in vitro experiments with glitter or DPD demonstrated the adequateness of the previous in silico hydrodynamic simulations. The use of higher rotation speeds and a second aqueous propeller resulted in more homogeneous mixing of the aqueous phase. This resulted in faster distribution of dissolved active pharmaceutical ingredient (API) into the octanol phase. A kinetic model was successfully applied to quantify the influence of hydrodynamics on the partitioning rate of the API into the octanol phase. In conclusion, the combination of in silico and in vitro methods was demonstrated to be powerful for investigating the flow patterns within the miBIdi-pH-II. A comprehensive understanding of the hydrodynamics and the respective influence on the dissolution and apparent partitioning into the octanol phase in the biphasic dissolution model was obtained and completed by using a compartmental kinetic model. This model allowed successful quantification of how the hydrodynamics influence the partitioning of API into the octanol phase.

The effect of prior exposure to imatinib on transplant-related mortality.

BACKGROUND AND OBJECTIVES: Imatinib is an effective treatment for chronic myeloid leukemia (CML) and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). However, relapse is common in patients with advanced or high risk disease. Such patients may be eligible for allogeneic stem cell transplantation (SCT), raising the question whether imatinib therapy may compromise the outcome of subsequent SCT. DESIGN AND METHODS: We retrospectively analyzed 70 patients with CML and 21 with Ph+ ALL who had received imatinib prior to SCT. Data were retrieved by directly contacting centers. Multivariate analysis was used to define factors associated with major outcomes (engraftment, graft-versus-host disease, relapse, non-relapse mortality) in addition to descriptive statistics. For the CML patients major outcomes were compared with those of historical controls drawn from the EBMT registry. RESULTS: At SCT, 44% of CML patients were in accelerated phase or blast crisis and 40% of ALL patients had active disease compared to 84% and 95% prior to imatinib. At 24 months, estimated transplant-related mortality was 44% and estimated relapse mortality 24%. Factors associated with shorter overall and progression-free survival were advanced disease at SCT and a female donor/male recipient pairing. No unusual organ toxicities were observed. Compared to historical controls, prior imatinib treatment did not influence overall survival, progression-free survival or non-relapse mortality, while there was a trend towards higher relapse mortality and significantly less chronic graft-versus-host disease. INTERPRETATION AND CONCLUSIONS: Within the limits of a heterogeneous and relatively small cohort of patients, we found no evidence that imatinib negatively affects major outcomes after SCT, suggesting that imatinib prior to SCT is safe.

A prospective, randomized trial of continuous lateral rotation ("kinetic therapy") in patients with cardiogenic shock.

BACKGROUND: Continuous lateral rotation ["Kinetic therapy" (KT)] has been shown to reduce complications and to shorten hospital stay in trauma patients. Data in non-surgical patients is inconclusive. Retrospective data suggest a beneficial effect of KT in patients with cardiogenic shock (CS) requiring ventilator therapy. KT, however, has not been tested prospectively in those patients. METHODS: A prospective, randomized, open-label trial was performed to compare KT using oscillating beds (TryaDyne Proventa, KCI) with standard care (SC). Patients with cardiogenic shock requiring ventilator therapy for more than 24 h were included. Primary endpoint was the occurrence of hospital-acquired pneumonia. Secondary endpoints were the occurrence of pressure ulcers during the hospital stay and 1-year all-cause mortality. RESULTS: Forty-five patients were randomized to KT, and 44 to SC. All patients required at least one inotropic agent and one vasopressor for circulatory assistance. The groups were comparable in the etiology of heart disease, in the use of revascularization procedures, the use of balloon counterpulsation, and APACHE-II score (33 ± 5 vs. 33 ± 4) and SOFA score (11 ± 1 vs. 11 ± 1) at inclusion; however, more patients in SC were subject to resuscitation before inclusion. Hospital-acquired pneumonia occurred in 10 patients in KT and 28 patients in SC (p < 0.001); pressure ulcers were seen in 10 versus 2 patients (p < 0.001). Hospital mortality tended to be lower in KT, and 1-year all-cause mortality was 41 % in KT and 66 % in SC (p = 0.028). CONCLUSION: The use of KT reduces rates of pneumonia and pressure ulcers as compared to SC. Moreover, in this study, patients with KT had a better outcome. The study suggests that KT should be used in patients with cardiogenic shock requiring ventilator therapy for a prolonged time.