Powder suspensions in non-aqueous vehicles for delivery of therapeutic proteins

Protein powder suspensions in non-aqueous vehicles provide an interesting alternative to conventional formulations such as aqueous solutions and lyophilizates and potentially feature a variety of advantages for different applications. Besides powder manufacturing, vehicle selection is key for such preparations. Powders for the use in such formulations can be prepared in several ways including milling of lyophilizates and spray-drying. Chapter III aimed to get a deeper understanding on how milling of model monoclonal antibody (mAb) lyophilizates can be used as a technique to generate protein powders for suspensions in non-aqueous vehicles. It was of interest, if wet media milling can yield protein powder suspensions without damage to the protein. An alternative is dry milling in absence of a suspension vehicle. The chapter aimed to identify critical process parameters and to optimize the milling process. Studied process parameters included the milling ball size, milling frequency, ball to powder mass ratio, milling duration as well as application of cooling. Furthermore, it was tested whether excipients or the protein to stabilizer ratio influence protein stability during milling. With the obtained powder, suspensions were prepared and their viscosities and glide forces were tested. Highly concentrated protein formulations currently play an important role in the pharmaceutical industry, but face different challenges such as decreased protein stability and increased glide forces needed for injection. Protein powder suspensions in non-aqueous vehicles may represent a beneficial alternative and can yield low viscosity formulations depending on the selected suspension vehicle. Traditionally used non-aqueous liquids for injection, such as vegetable oils, are not suitable for high concentration suspension formulations due to their high inherent viscosity. Consequently, low viscosity alternatives are of particular interest. Thus, the goal of Chapter IV was to test various potential vehicles regarding their ability to yield high concentration low viscosity formulations. Promising candidates included semifluorinated alkanes (SFAs), due to their low viscosity, good biocompatibility and inertness towards biopharmaceuticals. In contrary to the previous chapter, spray-drying was used to prepare the protein powder. It was of interest how SFA based suspensions perform with regard to rheology and injection characteristics compared to aqueous solutions. In order to select a suitable syringe design, prediction of glide forces using mathematical models and equations was evaluated. Finally, with commercial manufacturability in mind, the feasibility of the vial filling process of the developed suspensions, using a peristaltic pump, was tested and evaluated. Chapter V provides more detailed insight into long term physical suspension stability of lysozyme, mAb and bevacizumab powder suspensions in different suspension vehicles including SFAs, medium chain triglycerides (MCT) and ethyl oleate (EO). Physical suspension stability includes resuspendability, particle size constancy and injectability. Both excipients and the protein to stabilizer ratio might influence the stability. Furthermore, the application of an additional drying step was tested to improve the physical suspension stability. Suspensions were stored in vials, as well as in prefillable syringes, which provide easier administration. Not only suspension physical stability, but also protein stability has to be maintained over storage time. In the case of protein powder suspensions in non-aqueous vehicles, protein instabilities can have two different causes. First the inherent stability of the powder has to be considered. Secondly, the influence of the suspension vehicle on protein stability might play a role. The focus of Chapter VI lied on the influence of different potential vehicles and powder compositions on protein stability in non-aqueous powder suspensions. For this purpose, stability of a mAb and bevacizumab in SFAs, MCT and EO were studied. Combined with investigations on suspension physical stability this chapter should give valuable information on the applicability of the non-aqueous powder suspension approach for protein formulations. Besides subcutaneous injection, topical ocular protein delivery to the cornea is of interest, especially after the recent approval of a protein drug for the treatment of neurotrophic keratitis. This application route faces poor drug penetration and short residence time as important challenges. In Chapter VII formulations for improved corneal protein delivery based on protein powder suspensions in non-aqueous vehicles, considering the addition of penetration enhancers, were investigated. A main focus lied on the performance of protein powder suspension in SFAs, as previous investigations on topical ocular delivery of small molecule drugs in SFAs showed promising results. The overall goal of the thesis was to study the various opportunities of powder suspensions in non-aqueous vehicles for protein formulation with a focus on SFAs. The various chapters present pathways for successfully developing, characterizing and manufacturing such preparations, which may provide superior suspension and protein stability also at high protein concentration

Gerinnungsphysiologische und genetische diagnostik bei hereditärem protein S-mangel/Coagulation testing and genetic diagnostics in hereditary protein S deficiency

Thrombophilia is the disposition for recurrent idiopathic thromboembolism and is currently seen as a multifactorial disease caused by a disbalance of complex regulated hemostasis. Despite intensive diagnostics the exact defect remains unknown in most cases. An established cause that can be diagnosed in 1–2% of affected individuals is a deficiency of protein S which limits thrombin formation as a cofactor of activated protein C. However, the evaluation of pathogenetic relevance of decreased protein S levels is difficult due to the much more often occurring acquired protein S deficiency under certain physiological or pathological circumstances, for example, intake of hormonal contraceptives or systemic inflammation. By adding aggregated genetic data of 136 patients, this publication gives a new perspective on protein S diagnostics and indicates under which circumstances a real, genetic defined deficiency can be postulated with higher probability.Thrombophilie, die Neigung zu rezidivierenden, idiopathischen Thromboembolien, wird heute als multifaktorielle Erkrankung im Sinne einer Dysbalance des komplex regulierten Hämostasesystems verstanden. Trotz intensiver Abklärung bleibt der genaue Defekt einer erblichen Thromboseneigung oft unbekannt. Eine anerkannte Ursache, welche bei etwa 1 bis 2% der Betroffenen identifiziert werden kann, ist ein Mangel an Protein S, welches indirekt als Kofaktor von aktiviertem Protein C die Thrombinbildung limitiert. Die Einschätzung der pathogenetischen Relevanz verminderter Protein S-Spiegel ist jedoch aufgrund der sehr viel häufiger vorkommenden erworbenen Protein S-Erniedrigung unter bestimmten physiologischen und pathologischen Bedingungen (z.B. Einnahme einer hormonellen Kontrazeption oder systemische Inflammation) schwierig. Diese Arbeit eröffnet durch Hinzufügen aggregierter genetischer Daten von 136 Patienten einen neuen Blick auf die Protein S-Diagnostik im klinischen Alltag und liefert Hinweise, in welcher Konstellation mit größerer Wahrscheinlichkeit von einem echten (genetisch definierten) Mangel ausgegangen werden kann

Development of human antibody fragments using antibody phage display for the detection and diagnosis of Venezuelan equine encephalitis virus (VEEV)

<p>Abstract</p> <p>Background</p> <p>Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus group. Several species of this family are also pathogenic to humans and are recognized as potential agents of biological warfare and terrorism. The objective of this work was the generation of recombinant antibodies for the detection of VEEV after a potential bioterrorism assault or an natural outbreak of VEEV.</p> <p>Results</p> <p>In this work, human anti-VEEV single chain Fragments variable (scFv) were isolated for the first time from a human naïve antibody gene library using optimized selection processes. In total eleven different scFvs were identified and their immunological specificity was assessed. The specific detection of the VEEV strains TC83, H12/93 and 230 by the selected antibody fragments was proved. Active as well as formalin inactivated virus particles were recognized by the selected antibody fragments which could be also used for Western blot analysis of VEEV proteins and immunohistochemistry of VEEV infected cells. The anti-VEEV scFv phage clones did not show any cross-reactivity with Alphavirus species of the Western equine encephalitis virus (WEEV) and Eastern equine encephalitis virus (EEEV) antigenic complex, nor did they react with Chikungunya virus (CHIKV), if they were used as detection reagent.</p> <p>Conclusion</p> <p>For the first time, this study describes the selection of antibodies against a human pathogenic virus from a human naïve scFv antibody gene library using complete, active virus particles as antigen. The broad and sensitive applicability of scFv-presenting phage for the immunological detection and diagnosis of Alphavirus species was demonstrated. The selected antibody fragments will improve the fast identification of VEEV in case of a biological warfare or terroristic attack or a natural outbreak.</p

Extended analysis of a genome-wide association study in primary sclerosing cholangitis detects multiple novel risk loci.

A limited number of genetic risk factors have been reported in primary sclerosing cholangitis (PSC). To discover further genetic susceptibility factors for PSC, we followed up on a second tier of single nucleotide polymorphisms (SNPs) from a genome-wide association study (GWAS). We analyzed 45 SNPs in 1221 PSC cases and 3508 controls. The association results from the replication analysis and the original GWAS (715 PSC cases and 2962 controls) were combined in a meta-analysis comprising 1936 PSC cases and 6470 controls. We performed an analysis of bile microbial community composition in 39 PSC patients by 16S rRNA sequencing. Seventeen SNPs representing 12 distinct genetic loci achieved nominal significance (p(replication) <0.05) in the replication. The most robust novel association was detected at chromosome 1p36 (rs3748816; p(combined)=2.1 × 10(-8)) where the MMEL1 and TNFRSF14 genes represent potential disease genes. Eight additional novel loci showed suggestive evidence of association (p(repl) <0.05). FUT2 at chromosome 19q13 (rs602662; p(comb)=1.9 × 10(-6), rs281377; p(comb)=2.1 × 10(-6) and rs601338; p(comb)=2.7 × 10(-6)) is notable due to its implication in altered susceptibility to infectious agents. We found that FUT2 secretor status and genotype defined by rs601338 significantly influence biliary microbial community composition in PSC patients. We identify multiple new PSC risk loci by extended analysis of a PSC GWAS. FUT2 genotype needs to be taken into account when assessing the influence of microbiota on biliary pathology in PSC.Norwegian PSC Research Center German Ministry of Education and Research (BMBF) through the National Genome Research Network (NGFN) Integrated Research and Treatment Center - Transplantation 01EO0802 PopGen biobank NIH DK 8496

Functional analysis of structural variants in single cells using Strand-seq

Somatic structural variants (SVs) are widespread in cancer, but their impact on disease evolution is understudied due to a lack of methods to directly characterize their functional consequences. We present a computational method, scNOVA, which uses Strand-seq to perform haplotype-aware integration of SV discovery and molecular phenotyping in single cells by using nucleosome occupancy to infer gene expression as a readout. Application to leukemias and cell lines identifies local effects of copy-balanced rearrangements on gene deregulation, and consequences of SVs on aberrant signaling pathways in subclones. We discovered distinct SV subclones with dysregulated Wnt signaling in a chronic lymphocytic leukemia patient. We further uncovered the consequences of subclonal chromothripsis in T cell acute lymphoblastic leukemia, which revealed c-Myb activation, enrichment of a primitive cell state and informed successful targeting of the subclone in cell culture, using a Notch inhibitor. By directly linking SVs to their functional effects, scNOVA enables systematic single-cell multiomic studies of structural variation in heterogeneous cell populations

Hepatocyte specific expression of an oncogenic variant of β-catenin results in cholestatic liver disease

[Background] The Wnt/β-catenin signaling pathway plays a crucial role in embryonic development, tissue homeostasis, wound healing and malignant transformation in different organs including the liver. The consequences of continuous β-catenin signaling in hepatocytes remain elusive. [Results] Livers of Ctnnb1CA hep mice were characterized by disturbed liver architecture, proliferating cholangiocytes and biliary type of fibrosis. Serum ALT and bile acid levels were significantly increased in Ctnnb1CA hep mice. The primary bile acid synthesis enzyme Cyp7a1 was increased whereas Cyp27 and Cyp8b1 were reduced in Ctnnb1CA hep mice. Expression of compensatory bile acid transporters including Abcb1, Abcb4, Abcc2 and Abcc4 were significantly increased in Ctnnb1CA hep mice while Ntcp was reduced. Accompanying changes of bile acid transporters favoring excretion of bile acids were observed in intestine and kidneys of Ctnnb1CA hep mice. Additionally, disturbed bile acid regulation through the FXR-FGF15-FGFR4 pathway was observed in mice with activated β-catenin. [Materials and Methods] Mice with a loxP-flanked exon 3 of the Ctnnb1 gene were crossed to Albumin-Cre mice to obtain mice with hepatocyte-specific expression of a dominant stable form of β-catenin (Ctnnb1CA hep mice). Ctnnb1CA hep mice were analyzed by histology, serum biochemistry and mRNA profiling. [Conclusion] Expression of a dominant stable form of β-catenin in hepatocytes results in severe cholestasis and biliary type fibrosis

The impact of the COVID-19 pandemic on administrative eating disorder prevalence in the outpatient sector and on severity of anorexia nervosa.

The COVID-19 pandemic appears to have had a considerable impact on the mental health of children and adolescents, particularly regarding eating disorders. However, it remains unclear whether the pandemic affected only the frequency or also the severity of eating disorders. We examined potential pandemic-related changes in the administrative prevalence of eating disorders in the outpatient sector compared with other mental disorders using German statutory health insurance data for the age group 10 to 16 years. We also examined disorder severity of anorexia nervosa using data from the multicenter German Registry of Children and Adolescents with Anorexia Nervosa in the same age group. Our results showed a marked increase in the administrative prevalence of eating disorders (based on documented diagnoses) in the outpatient sector among girls but not among boys. A similar pattern was found for internalizing disorders, whereas the administrative prevalences of externalizing disorders decreased. Regarding the severity of anorexia nervosa among inpatients, we found no pandemic-related changes in body mass index standard deviation score at admission, body weight loss before admission, psychiatric comorbidities and psychopharmacological medication. Given the administrative prevalence increase in the outpatient sector, the lack of impact of the pandemic on the inpatient sector may also be partly due to a shift in healthcare utilization towards outpatient services during the pandemic. Thus, the higher number of children and adolescents requiring specialized and timely outpatient care may be a major concern under pandemic conditions