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Project report (for Faculty of Pharmacy): Structural characterization of proteins in solid pharmaceutical forms
No full textWe started the projected studies by preparing samples, i.e. lyophilizates. We decide to use two different monoclonal antibodies and several different excipients, among them two sugars as stabilizing agents. We employed different lyophilization cycles, one for mAb1 and two for mAb2. Some of the samples of monclonal antibody mAb2 were later subjected to stability studies at 40ºC for three and nine months. For samples of mAb1 we used also different stresses, such as UV light and temperature. Samples were analyzed by spectroscopic methods (Infrared, UV-Vis, Fluorescence and Raman spectroscopy, and solid-state NMR), Circular dichroism, Dynamic differential calorimetry, X-ray diffraction analysis and others. Samples with monoclonal antibody mAb1 have shown to be relatively stable, except when exposed to higher temperatures. Within samples of mAb2 we observed less secondary and tertiary structure for samples that were subjected to stability studies. Later we decide also to analyze sample for aggregates formation, which is already a part of the projected studies implementation within Lek d.d.. As an important part of this project we also take care of the dissemination by participating on several lectures, conferences and other activities. We are in the final process of publishing a review that covers the literature of the projected studies
Assessing the Relevance of Solution Phase Stress Testing of Solid Dosage Form Drug Products: A Cross-Industry Benchmarking Study
No full textStress testing (also known as forced degradation) of pharmaceutical products has long been recognized as a critical part of the drug development process, providing foundational information related to intrinsic stability characteristics and to the development of stability-indicating analytical methods. A benchmarking study was undertaken by nine pharmaceutical companies and the Brazilian Health Regulatory Agency (Agência Nacional de Vigilância Sanitária, or ANVISA) with a goal of understanding the utility of various stress testing conditions for producing pharmaceutically-relevant chemical degradation of drugs. Special consideration was given to determining whether solution phase stress testing of solid drug products produced degradation products that were both unique when compared to other stress conditions and relevant to the formal drug product stability data. The results from studies of 62 solid dosage form drug products were compiled. A total of 387 degradation products were reported as being observed in stress testing studies, along with 173 degradation products observed in accelerated and/or long-term stability studies for the 62 drug products. Among these, 25 of the stress testing degradation products were unique to the solution phase stress testing of the drug products; however, none of these unique degradation products were relevant to the formal stability data. The relevant degradation products were sufficiently accounted for by stress testing studies that included only drug substance stressing (in solution and in the solid state) and drug product stressing (in the solid state). Based on these results, it is the opinion of the authors that for solid dosage form drug products, well-designed stress testing studies need not include solution phase stress testing of the drug product in order to be comprehensive
Re-programming and optimization of a L-proline cis-4-hydroxylase for the cis-3-halogenation of its native substrate
No full textFreestanding non-heme iron/α-ketoglutarate dependent halogenases enable the regio- and stereoselective halogenation of inactivated C(sp3)-H bonds. Yet, with only a handful of these halogenases characterized, the biosynthetic potential of enzymatic radical halogenation remains limited. Herein, we describe the remodeling of L-proline cis-4-hydroxylase from Sinorhizobium meliloti into a halogenase by introduction of a single point mutation into the enzyme’s active site (D108G). The re-programmed halogenase displays a striking regio-divergent reaction chemistry: While halogenation of L-proline exclusively occurs at the C3-position, the retained hydroxylation activity leads to derivatization at the C-4 position, corresponding to the regioselectivity of the wildtype enzyme. By employing several rounds of directed evolution, an optimized halogenase variant with 98-fold improved apparent kcat / Km for chlorination of L-proline compared to the parental enzyme SmP4H (D108G) was identified. The development and optimization of this novel halogenation biocatalyst highlights the possibility to rationally harness the chemical versatility of non-heme Fe/αKG dependent dioxygenases for C-H functionalization
Process analytical technology as a tool to optimize and accelerate pharmaceutical process development
No full textOver the last decades process analytical technology (PAT) has been widely used in the pharmaceutical development to support process understanding and optimization. Among the different technologies, near infrared spectroscopy (NIRS) has maintained its status as a most versatile and valuable PAT tool for probing the manufacturing of solid dosage forms. Two industrial examples of PAT NIRS applications are presented in this paper. The first case tackles the challenges of process development in an international environment, with different locations for manufacture and traditional analytics. The study shows how it was possible to troubleshoot segregation by means of in-line monitoring of the blending process combined to an at-line content uniformity determination on high number of tablets. The second case describes in-line monitoring by NIRS for assay determination in a wet met media milling process of a nanosuspension
Visible Light mediated micellar one-pot Triazole formation
No full textThe amphiphilic Cu nanoparticles (NPs) stabilized with proline linker of the amphiphile is reported. The NPs was characterized using various spectroscopic techniques like XAS, XPS, cryoTEM and HRTEM analysis. DFT calculations confirm the tertiary amide core of the amphilphile as an active binding site for the Cu NPs. The highly active Cu NPs is employed in visible light mediated photo-initiator free cycloaddition reaction and C-H aminations using parts per million level of catalyst loading. The Cu NPs is easy to synthesize and ound to be highly stable and recyclable.
Long-Term Stability Predictions of Therapeutic Monoclonal Antibodies in Solution Using Arrhenius Based Kinetics
No full textLong-term stability of monoclonal antibodies is the key aspect in their development for use as (bio)pharmaceutical products; therefore, possible prediction of long-term stability from accelerated stability studies is of major interest, despite currently regarded as not sufficiently robust. In this work, using combination of accelerated stability studies (up to 6 months) and first order degradation kinetic modelling, we are able to predict long-term stability (up to 3 years) including temperature dependence of changes of multiple quality attributes and for multiple monoclonal antibody formulations. More specifically, we can robustly predict the long-term stability behavior of a protein at the intended storage condition (5°C), based on up to six months data obtained from different temperatures, usually from intended (5°C), accelerated (25°C) and stress conditions (40°C). We have performed stability studies and evaluated the stability data of several mAbs including IgG1, IgG2, IgG4 and fusion proteins and validated our model by overlaying the 95% prediction interval and experimental stability data from up to 36 months. We demonstrated improved robustness, speed and accuracy of kinetic long-term stability prediction as compared to classical linear extrapolation used today, justifying long-term stability prediction and shelf life extrapolation for some biological products such as monoclonal antibodies. More generally, this work aims to contribute towards further development and refinement of the regulatory landscape that will allow extrapolation for biological products during the developmental phase, clinical phase and also in marketing authorization applications, as already established today for small molecules
TNF leads to mtDNA release and cGAS/STING-dependent interferon responses that support inflammatory arthritis.
No full textTumor necrosis factor (TNF) is a key driver of several inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel disease, and psoriasis, in which affected tissues show an interferon-stimulated gene signature. Here, we demonstrate that TNF triggers a type-I interferon response that is dependent on the cyclic guanosine monophosphate-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. We show that TNF inhibits PINK1-mediated mitophagy and leads to altered mitochondrial function and to an increase in cytosolic mtDNA levels. Using cGAS-chromatin immunoprecipitation (ChIP), we demonstrate that cytosolic mtDNA binds to cGAS after TNF treatment. Furthermore, TNF induces a cGAS-STING-dependent transcriptional response that mimics that of macrophages from rheumatoid arthritis patients. Finally, in an inflammatory arthritis mouse model, cGAS deficiency blocked interferon responses and reduced inflammatory cell infiltration and joint swelling. These findings elucidate a molecular mechanism linking TNF to type-I interferon signaling and suggest a potential benefit for therapeutic targeting of cGAS/STING in TNF-driven diseases
SWnet: a deep learning model for drug response prediction from cancer genomic signatures and compound chemical structures.
No full textOne of the major challenges in precision medicine is accurate prediction of individual patient's response to drugs. A great number of computational methods have been developed to predict compounds activity using genomic profiles or chemical structures, but more exploration is yet to be done to combine genetic mutation, gene expression, and cheminformatics in one machine learning model
Discovery of a Covalent FEM1B Recruiter for Targeted Protein Degradation Applications
No full textProteolysis Targeting Chimeras (PROTACs), heterobifunctional compounds that consist of protein-targeting ligands linked to an E3 ligase recruiter, have arisen as a powerful therapeutic modality for targeted protein degradation (TPD). Despite the popularity of TPD approaches in drug discovery, only a small number E3 ligase recruiters are available for the >600 E3 ligases that exist in human cells. Here, we have discovered a cysteine-reactive covalent ligand EN106 that targets FEM1B, an E3 ligase recently discovered to be critical in cellular reductive stress response. EN106, through targeting cysteine C186, disrupts FEM1B substrate recognition of FNIP1. We further establish that EN106 can be used as a covalent recruiter for FEM1B in TPD applications, in which we demonstrate that a PROTAC linking EN106 to the BET Bromodomain inhibitor JQ1 leads to the selective NEDDylation, proteasome, and FEM1B-dependent degradation of BRD4 in cells. Our study showcases a covalent ligand that targets a substrate recognition site within the E3 ligase FEM1B involved in reductive stress response and highlights the utility of covalent ligand screening in expanding the arsenal of E3 ligase recruiters that can be deployed for TPD applications
Molecular targets and approaches to restore autophagy and lysosomal capacity in neurodegenerative disorders.
No full textAutophagy is a catabolic process that promotes cellular fitness by clearing aggregated protein species, pathogens and damaged organelles through lysosomal degradation. The autophagic process is particularly important in the nervous system where post-mitotic neurons rely heavily on protein and organelle quality control in order to maintain cellular health throughout the lifetime of the organism. Alterations of autophagy and lysosomal function are hallmarks of various neurodegenerative disorders. In this review, we conceptualize some of the mechanistic and genetic evidence pointing towards autophagy and lysosomal dysfunction as a causal driver of neurodegeneration. Furthermore, we discuss rate-limiting pathway nodes and potential approaches to restore pathway activity, from autophagy initiation, cargo sequestration to lysosomal capacity