Nanolithography Based on the Formation and Manipulation of Nanometer-Size Organic Liquid Menisci

In this work we form and manipulate nanometer-size liquid bridges of nonpolar organic solvents such as octane and 1-octene with a dynamic force microscope. These menisci have been used to confine chemical reactions that gave rise to the fabrication of nanometer-size structures.Peer reviewe

5qSMA: standardised retrospective natural history assessment in 268 patients with four copies of SMN2

Newborn screening for 5qSMA offers the potential for early, ideally pre-symptomatic, therapeutic intervention. However, limited data exist on the outcomes of individuals with 4 copies of SMN2 , and there is no consensus within the SMA treatment community regarding early treatment initiation in this subgroup. To provide evidence-based insights into disease progression, we performed a retrospective analysis of 268 patients with 4 copies of SMN2 from the SMArtCARE registry in Germany, Austria and Switzerland. Inclusion criteria required comprehensive baseline data and diagnosis outside of newborn screening. Only data prior to initiation of disease-modifying treatment were included. The median age at disease onset was 3.0 years, with a mean of 6.4 years. Significantly, 55% of patients experienced symptoms before the age of 36 months. 3% never learned to sit unaided, a further 13% never gained the ability to walk independently and 33% of ambulatory patients lost this ability during the course of the disease. 43% developed scoliosis, 6.3% required non-invasive ventilation and 1.1% required tube feeding. In conclusion, our study, in line with previous observations, highlights the substantial phenotypic heterogeneity in SMA. Importantly, this study provides novel insights: the median age of disease onset in patients with 4 SMN2 copies typically occurs before school age, and in half of the patients even before the age of three years. These findings support a proactive approach, particularly early treatment initiation, in this subset of SMA patients diagnosed pre-symptomatically. However, it is important to recognize that the register will not include asymptomatic individuals.Open Access funding enabled and organized by Projekt DEAL.Biogenhttp://dx.doi.org/10.13039/100005614Roche Deutschlandhttp://dx.doi.org/10.13039/100020957Novartis Pharmahttp://dx.doi.org/10.13039/100008792Universitätsklinik München (6933

Long-term efficacy and safety of nusinersen in adults with 5q spinal muscular atrophy: a prospective European multinational observational study

Background Evidence for the efficacy of nusinersen in adults with 5q-associated spinal muscular atrophy (SMA) has been demonstrated up to a period of 16 months in relatively large cohorts but whereas patients reach a plateau over time is still to be demonstrated. We investigated the efficacy and safety of nusinersen in adults with SMA over 38 months, the longest time period to date in a large cohort of patients from multiple clinical sites. Methods Our prospective, observational study included adult patients with SMA from Germany, Switzerland, and Austria (July 2017 to May 2022). All participants had genetically-confirmed, 5q-associated SMA and were treated with nusinersen according to the label. The total Hammersmith Functional Motor Scale Expanded (HFMSE) and Revised Upper Limb Module (RULM) scores, and 6-min walk test (6 MWT; metres), were recorded at baseline and 14, 26, and 38 months after treatment initiation, and pre and post values were compared. Adverse events were also recorded. Findings Overall, 389 patients were screened for eligibility and 237 were included. There were significant increases in all outcome measures compared with baseline, including mean HFMSE scores at 14 months (mean difference 1.72 [95% CI 1.19–2.25]), 26 months (1.20 [95% CI 0.48–1.91]), and 38 months (1.52 [95% CI 0.74–2.30]); mean RULM scores at 14 months (mean difference 0.75 [95% CI 0.43–1.07]), 26 months (mean difference 0.65 [95% CI 0.27–1.03]), and 38 months (mean difference 0.72 [95% CI 0.25–1.18]), and 6 MWT at 14 months (mean difference 30.86 m [95% CI 18.34–43.38]), 26 months (mean difference 29.26 m [95% CI 14.87–43.65]), and 38 months (mean difference 32.20 m [95% CI 10.32–54.09]). No new safety signals were identified. Interpretation Our prospective, observational, long-term (38 months) data provides further real-world evidence for the continuous efficacy and safety of nusinersen in a large proportion of adult patients with SMA. Funding Financial support for the registry from Biogen, Novartis and Roche

Untersuchungen zum Freisetzungs- und Verteilungsverhalten von Wirkstoffen nach intramuskulärer Applikation

In der Arzneimitteltherapie nehmen parenteral zu applizierende Arzneimittel einen immer größer werdenden Stellenwert ein. Diese Entwicklung beruht vor allem auf der steigenden Anzahl von Peptiden und Proteinen als Wirkstoffe, deren perorale Applikation aufgrund unzureichender Resorptionsraten aus dem Gastrointestinaltrakt unmöglich ist, aber auch auf der Möglichkeit Depotformulierungen anzuwenden, die Wirkstoffe nach subkutaner oder intramuskulärer Injektion über einen langen Zeitraum freisetzen können. Dennoch sind Arzneimitteltransportwege nach subkutaner oder intramuskulärer Applikation im Detail weitestgehend unerforscht. In der vorliegenden Arbeit wurde der Fokus auf die intramuskuläre Applikation gelegt. Im ersten Teil der Arbeit wurde eine tierexperimentelle Studie an Ratten durchgeführt, die es zum Ziel hatte, ausgewählte physiologische Einflussparameter auf die Wirkstoffresorption nach intramuskulärer Injektion zu untersuchen. Die Tiere erhielten sowohl wässrige Lösungen als auch ölige Suspensionen von Paracetamol, Prednisolon und Diclofenac-Natrium in die rechte Oberschenkelmuskulatur und das jeweilige Placebo in den Muskel des linken Oberschenkels. Anschließend wurde das intramuskuläre Arzneistoffdepot erstmals mittels Magnetresonanztomographie (MRT) als bildgebendes Verfahren zeitabhängig untersucht und parallel die Anflutungskinetik der Wirkstoffe ins Blut bestimmt. Die Volumina und Oberflächen der Depots sowie die Zeit bis zu deren Abtransport aus dem Muskelgewebe wurden mit den Blutspiegelkurven verglichen. Allen Formulierungen war gemein, dass der Wirkstoff deutlich schneller resorbiert wurde als das Depot. Daraus lässt sich schließen, dass die Wirkstoffaufnahme nicht über die Resorption des Depots erfolgte. Ein schnelleres Verschwinden der Depots führte nicht zu höheren Blutspiegelmaxima oder zu einer beschleunigten systemischen Wirkstoffresorption. Es wurde keine Korrelation zwischen dem Volumen und der Oberfläche der Depots und den Blutspiegelkurven gefunden. Ein weiterer Aspekt, der während der durchgeführten Studie näher untersucht wurde, ist die Möglichkeit, mit Hilfe der MRT lokale Reaktionen in Folge der Injektion zu detektieren. Bei Diclofenac-Natrium wurde in allen Fällen eine Ansammlung interstitieller Flüssigkeit am Ort der Injektion beobachtet. Histopathologische Untersuchungen des Muskelgewebes konnten einen Zusammenhang zwischen der Größe des visualisierten Ödems und dem Ausmaß der Entzündung belegen. Im zweiten Teil dieser Arbeit galt es, mit den gewonnenen In vivo-Daten möglichst biorelevante Methoden für Freisetzungstests intramuskulärer Arzneiformen zu entwickeln. Dabei sollte sowohl die Simulation der Durchblutung des Muskelgewebes Berücksichtigung finden als auch die Simulation des Muskelgewebes oder der Depots im Muskelgewebe. Es wurden verschiedene Versuchsaufbauten entwickelt, adaptiert und modifiziert. Darunter zählen Gel-Schaum-Blöcke in der Durchflusszelle, die Keramikmembran und der Membranadapter in der Durchflusszelle. Während die Simulation der Durchblutung und der injizierten Depots in allen Testmethoden möglich war, konnte das Muskelgewebe nicht befriedigend nachgebildet werden. Die Anwendung verschiedener Freisetzungstest-Methoden hatte auf die Verteilung der Wirkstoffe aus den wässrigen Lösungen keinen nennenswerten Einfluss. Der größte Einfluss auf die Verteilungsgeschwindigkeit wurde bei der öligen Suspension von Prednisolon beobachtet. Im Rahmen der vorliegenden Arbeit konnte nicht abschließend geklärt werden, welche Parameter der Situation in vivo entscheidend für das Anflutungsverhalten der Wirkstoffe nach intramuskulärer Injektion sind. Es konnte jedoch gezeigt werden, dass die Kombination aus pharmakokinetischen Untersuchungen mit MRT-Bildgebung eine vielversprechende Möglichkeit darstellt, Einblicke in die Biopharmazie intramuskulärer Depots zu erhalten. Außerdem konnte gezeigt werden, dass die Verwendung der MRT zur Feststellung der lokalen Verträglichkeit, vor allem neu entwickelter Arzneiformen, eine sehr gut geeignete nicht-invasive Methode darstellt. Die Ergebnisse der In vitro-Untersuchungen verdeutlichen, dass die Freisetzungstestmethode vor allem bei langsam freisetzenden Arzneiformen einen erheblichen Einfluss ausüben kann. Eine universelle Testmethode, mit der zuverlässig das In vivo-Freisetzungsverhalten nach intramuskulärer Injektion vorhergesagt werden kann, ist derzeit nicht verfügbar. Die entwickelten Modelle stellen einen ersten Schritt zur Entwicklung biorelevanter Freisetzungstestmethoden für intramuskulär applizierte Darreichungsformen dar. Zur Verbesserung dieser Modelle sind ein umfassenderes Verständnis der Arzneimittelverteilungs- und -Transportmechanismen notwendig und weitere Studien, die bildgebende Verfahren mit der pharmakokinetischen Analyse kombinieren, wünschenswert.Parenteral dosage forms are of rising importance in pharmacotherapy. This trend is mainly due to the increasing number of peptides and proteins as active ingredients, which cannot be applied orally since their absorption from the gastrointestinal tract is insufficient. Furthermore a controlled release over a time period up to several months is conceivable via subcutaneous or intramuscular application. Despite the considerable increase in the number of extravascular parenterally applied drugs, drug transport following subcutaneous or intramuscular injection has not been investigated in detail. This work was focused on intramuscular application. The first part of this work describes the conduction of a study in rats, which had the aim to investigate certain physiological parameters influencing drug absorption following intramuscular injection. Aqueous solutions and oily suspensions of prednisolone, paracetamol and diclofenac sodium were injected into the muscle tissue of the right thigh. At the same time placebos of these formulations were applied into the muscle tissue of the contralateral thigh. Afterwards the fate of the intramuscular depots was investigated for the first time by using magnetic resonance imaging (MRI) as imaging method over a certain time period combined with pharmacokinetic investigations. Volumes and surface areas of the depots as well as the time that was needed for removal of the depot from muscle tissue were compared to blood level curves. In all applied formulations the drug was absorbed much faster than the depot. Consequently drug absorption did not depend on absorption of the depot. A faster absorption of the depot did not lead to higher blood concentrations or an accelerated systemic drug absorption. No correlation between volume and surface areas of the depots and blood level curves were obtained. A further aspect that was investigated in more detail during the study was the possibility of detecting local reactions in consequence of the injection using MRI. An accumulation of interstitial fluid at the local site of injection was observed in all animals that received an injection of diclofenac sodium. Histopathological investigations of the muscle tissue showed a correlation between the size of the visualized edema and the extent of inflammation. In the second part of this work it was the aim to develop preferably biorelevant test methods for in vitro dissolution of intramuscular dosage forms based on the obtained in vivo data. If possible, simulation of the muscle tissue or of the depots within the muscle tissue as well as the process of injection should be considered. Different dissolution test setups were developed, adapted and modified. Amongst them was the gel-foam-block in the flow through cell, the ceramic membrane and the membrane adapter in the flow through cell. While simulation of blood perfusion and of the injected depots was possible in all test setups, imitation of muscle tissue within the used dissolution test methods was not satisfactorily achieved. Applying different dissolution test setups did not have any appreciable influence on distribution of the drugs from aqueous solutions. The highest influence on velocity of distribution was observed in the oily suspensions of prednisolone. In the presented work it could not be finally clarified, which in vivo parameters are crucial for the absorption behavior of drugs following intramuscular injection. However, it was shown, that the combination of pharmacokinetic investigations with magnetic resonance imaging is a promising method to gain insights into the biopharmacy of intramuscular depots. Furthermore it was shown, that MRI is an excellent non-invasive method in detecting local tolerability, which may be extremely beneficial during dosage form development. The results of the in vitro experiments illustrate, that the chosen dissolution test method can have major influence on the test outcome, especially in long acting dosage forms. Up to date there is no universal test method that allows a reliable prediction of the in vivo dissolution behavior following intramuscular injection. The applied models represent a first step in the development of biorelevant dissolution test methods for intramuscularly applied dosage forms. In order to improve these models a comprehensive understanding of drug distribution and transport mechanisms is necessary. Therefore the conduction of further studies that combine imaging methods with pharmacokinetic analysis is desirable

Partitioning, duality, and linkage disequilibria in the Moran model with recombination

Esser M, Probst S, Baake E. Partitioning, duality, and linkage disequilibria in the Moran model with recombination. JOURNAL OF MATHEMATICAL BIOLOGY. 2016;73(1):161-197.The multilocus Moran model with recombination is considered, which describes the evolution of the genetic composition of a population under recombination and resampling. We investigate a marginal ancestral recombination process, where each site is sampled only in one individual and we do not make any scaling assumptions in the first place. Following the ancestry of these loci backward in time yields a partition-valued Markov process, which experiences splitting and coalescence. In the diffusion limit, this process turns into a marginalised version of the multilocus ancestral recombination graph. With the help of an inclusion-exclusion principle and so-called recombinators we show that the type distribution corresponding to a given partition may be represented in a systematic way by a sampling function. The same is true of correlation functions (known as linkage disequilibria in genetics) of all orders. We prove that the partitioning process (backward in time) is dual to the Moran population process (forward in time), where the sampling function plays the role of the duality function. This sheds new light on the work of Bobrowski et al. (J Math Biol 61:455-473, 2010). The result also leads to a closed system of ordinary differential equations for the expectations of the sampling functions, which can be translated into expected type distributions and expected linkage disequilibria

In vitro simulation of distribution processes following intramuscular injection

There is an urgent need for in vitro dissolution test setups for intramuscularly applied dosage forms. Especially biorelevant methods are needed to predict the in vivo behavior of newly developed dosage forms in a realistic way. There is a lack of knowledge regarding critical in vivo parameters influencing the release and absorption behavior of an intramuscularly applied drug. In the presented work the focus was set on the simulation of blood perfusion and muscle tissue. A solid agarose gel, being incorporated in an open-pored foam, was used to mimic the gel phase of muscle tissue and implemented in a flow through cell. An aqueous solution of fluorescein sodium was injected. Compared to recently obtained in vivo results the distribution of the model substance was very slow. Furthermore an agarose gel of lower viscosity an open-pored foam and phosphate buffer saline pH 7.4 were implemented in a multi-channel-ceramic membrane serving as a holder for the muscle imitating material. Blood simulating release medium was perfused through the ceramic membrane including filling materials. Transport of the dissolved fluorescein sodium was, in case of the gel, not only determined by diffusion but also by convective transport processes. The more realistic the muscle simulating materials were constituted the less reproducible results were obtained with the designed test setups

Automated Detection and Counting of Wild Boar in Camera Trap Images

Camera traps are becoming widely used for wildlife monitoring and management. However, manual analysis of the resulting image sets is labor-intensive, time-consuming and costly. This study shows that automated computer vision techniques can be extremely helpful in this regard, as they can rapidly and automatically extract valuable information from the images. Specific training with a set of 1600 images obtained from a study where wild animals approaching wild boar carcasses were monitored enabled the model to detect five different classes of animals automatically in their natural environment with a mean average precision of 98.11%, namely ‘wild boar’, ‘fox’, ‘raccoon dog’, ‘deer’ and ‘bird’. In addition, sequences of images were automatically analyzed and the number of wild boar visits and respective group sizes were determined. This study may help to improve and speed up the monitoring of the potential spread of African swine fever virus in areas where wild boar are affected