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Introduction: Drug-induced liver injury (DILI) covers a spectrum of rare but potentially severe adverse effects. DILI diagnosis is an exclusion process of other possible causes, with the occasional misdiagnosis. DILI accounts for high attrition rates in drug development and drug withdrawal from the market. The development of an early (preclinical) DILI predictive model is therefore of high importance. Cholestatic DILI is a result of (drug-induced) cholestasis, which is an alteration in bile acid homeostasis and bile flow. The mechanisms involved in both drug-induced cholestasis and liver injury are complex and not fully cleared up yet. However, several pathways have shown a correlation with cholestatic liver injury, one being the malfunctioning of the bile salt export pump (BSEP). BSEP is the main hepatic efflux transporter for conjugated bile acids. Two genetic diseases (PFIC-2 and BRIC-2) are caused by mutations in the gene coding for BSEP and clinical presentation includes cholestasis. The correlation between BSEP malfunction and cholestasis has led to the hypothesis that the inhibition of BSEP by drugs is a mechanism involved in cholestatic DILI. The development of an in vitro BSEP inhibition assay is therefore seen as a promising tool for the preclinical prediction of the DILI potential of drugs. Materials and methods: Inside-out BSEP membrane vesicles were used as in vitro model system to determine BSEP-mediated substrate uptake and the influence of inhibitors. Fluorescent substrates used were tauro-nor-THCA-24-DBD and cholyl-L-lysyl-fluorescein (CLF), while glycocholic acid (GCA) was used as the endogenous substrate. Bioanalysis was performed by fluorescence spectrometry for fluorescent substrates and LC-MS/MS for GCA. Net uptake was calculated by subtracting uptake in BSEP membrane vesicles by uptake in MOCK membrane vesicles. Results: Inhibition experiments with tauro-nor-THCA-24-DBD showed high interexperimental variability. Adsorption of the substrate to the glass-fiber filter plate was seen to highly influence substrate uptake measurements. This problem could not be overcome by the use of filter plates lacking a glass-fiber layer, as uptake rates were even lower. Additionally, the dynamic range was not sufficient. CLF was used as alternative probe substrate in an exploratory experiment and highest uptake was seen at 15 min incubation time and 20 µM substrate concentration. However, the dynamic range was not increased compared to tauro-nor-THCA-24-DBD. GCA showed increasing uptake with prolonged incubation times and in function of increasing substrate concentration. The dynamic range was comparable to tauro-nor-THCA-DBD and CLF, but could still be increased by assay optimization. Conclusion: Tauro-nor-THCA-24-DBD nor CLF were found suitable substrates for membrane vesicle-based assessment of BSEP activity and evaluation of BSEP inhibitors. Dynamic ranges were too narrow, primarily due to non-specific binding to the in vitro assay setup. However, the endogenous bile acid GCA was found to be remarkably suitable for the purpose of this BSEP assay. Despite the disadvantage that LC-MS/MS-based bioanalysis is required, this assay is currently being optimized to increase its sensitivity and robustness.

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The implementation of international guidelines on qualification/comparability protocols has the potential to benefit the company and regulators by reducing the review time and the number of submissions needed for specific cases of periodic and predictable changes. The study aimed to establish an overview of recently submitted qualification/comparability protocols for reference standard and starting material, and developing a strategy within GlaxoSmithKline (GSK), to enable the update or construction of regulatory affairs Chemistry, Manufacturing and Controls (CMC) templates for these protocols. This strategy and the templates will be implemented for all future GSK submissions for these changes. Information on recently submitted protocols was collected by a general and a company internal literature study. Recent submission packages from different GSK vaccines were identified and studied for their content and structure. Information on the strategy of the company was gathered and established by the Global Regulatory Affairs (GRA) Regional Team. The gathered information enabled the implementation and optimization of a clear structure to show the required qualification/comparability protocol with the content necessary to be aligned with regulatory guidelines. The strategy of the company focuses on gaining acceptance of an EU-like model in other countries or regions by local advocacy. Countries and regions are classified into A-and B-countries to determine further submissions and advocacy efforts.

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The pharmaceutical industry is known for the many regulations, guidelines and laws imposed by the authorities. The Regulatory Affairs department forms therefore the bridge between the health authorities and the company in order to correctly obey the regulations. The Regulatory Affairs department is involved throughout the whole lifecycle management of the medicinal products with the main task to register medicinal products and to maintain the registration dossiers. Besides obeying the health authorities, the local Regulatory Affairs team needs to report to the Regulatory Affairs department on global level. The 'Safety Labelling Changes' procedure includes guidelines and rules imposed by these two parties. A safety change in the product information must be implemented within 6 months of approval imposed by health authorities. In addition to this, Sandoz Global applies some internal deadlines in order to succeed in this 6-months deadline. These deadlines have changed twice during the past few months. At first, Global expected that the local team would succeed to finish the whole process in 70 days, starting from the approval date of the variation until the date of sending the artwork to the manufacturer. This in contrast with the previous situation, where a deadline of 90 days was followed. In order to succeed within this deadline, a reformation of the process and applying local milestones were necessary to track in Excel files possible bottlenecks or the causes of delay. In the most recent procedure, the end-process deadline is softened to align with the production schedule, grace period, national regulations, etc. Along with the notification of the production site within 30 days of the health approval date including information about the impact on the leaflet or labelling. The results of the reformed procedure showed no impact on the amount of delayed artworks sent to manufacturer. There is even an improvement in comparison with 2019. The late deliveries are often attributable to non-local responsibilities or to processes that are intentionally put on hold and less taking into account the strict end-process deadline. For the most recent procedure little information is available but based on the analysis of the previous procedure, it should lead to less late deliveries to the manufacturer. An interesting project, for which a pilot study is currently ongoing, is the electronic product information. Thanks to this project, the patient should always have access to the most up-to-date leaflet and simplify and speed-up the local regulatory processes.

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Haemostasis is a delicate balance between blood coagulation and fibrinolysis. The fibrinolytic system is the body’s innate system for dissolving blood clots, which is crucial for maintaining blood fluidity. The central fibrinolytic enzyme, plasmin, is generated upon cleavage of plasminogen by tissue plasminogen activator (tPA) or urokinase (uPA). Plasmin is a highly potent enzyme that cleaves the fibrin network of the thrombus, resulting in thrombus dissolution. It is well established that platelets are integral to haemostasis and thrombus formation, yet, they harbour numerous fibrinolytic proteins, many of which are stored and released from secretory α-granules upon platelet activation. Earlier studies from our laboratory have demonstrated that platelets act as a site for local plasmin generation, highlighting novel profibrinolytic functions of platelets. Currently, there is no tool available that is sensitive enough and permits to study real-time plasmin generation on the platelet surface and is viable to study plasmin dynamics of plasmin in thrombi formed under flow. Therefore, a stable and reproducible plasmin-selective bioreceptor (PlnS-Ab) was constructed using bioconjugation, and consisting of an anti-human CD41 antibody, a dibenzocyclooctyne (DBCO) and a plasmin-selective peptide (PlnS-P). The PlnS-P carries a plasmin-releasable quencher that upon plasmin mediated cleavage a signal releases at 516 nm. Rapid cleavage of the PlnS-P and the PlnS-Ab (Km of 28.18 µM; Kcat of 4.5 s-1) by purified plasmin (10nM) was quantified. Moreover, a linear relationship between a changing plasmin (0-10 nM) concentration and the corresponding maximum fluorescence release of the PlnS-P was measured. Use of platelet rich plasma (PRP) preincubated with the anti-CD41 antibody (10 µg/ml), resulted in no significant loss of platelet functions as measured as collagen-induced platelet aggregation and PRP clot lysis. Furthermore, endogenous plasmin activity on the platelet surface was monitored using the PlnS-P and the PlnS-Ab on both unstimulated platelets with significant augmentation upon stimulation with thrombin/convulxin (Th/CVX) and addition of tPA or uPA. Whereas plasmin activity was inhibited by inclusion of Aprotinin (1 mM). The PlnS-Ab showed selectivity for plasmin with a negligible affinity for thrombin, an not further identified protease and no cleavage by tPA or uPA. Finally, flow cytometry analysis of isolated platelets stained with the PlnS-Ab and a phycoerythrin (PE) labelled CD42b antibody as a second platelet marker confirmed PlnS-Ab-platelet affinity. The developed bioreceptor, targeted directly to platelets while carrying a plasmin-releasable quencher, will be used in flow-based and in vivo models, generating a plasmin-selective biosensor. Subsequently, use of the biosensor will permit characterisation of novel profibrinolytic platelet functions in real-time within the dynamic thrombus environment. 

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In 2020, a new cholesterol-lowering medicine will receive market authorization from the European medicines agency and in 2019, the European Society of Cardiology (ESC) guidelines were updated. A training program is needed to train the internal staff on this new medicine. The purpose of this training program is to provide an overview of the current medicines against hyperlipidemia, the new ESC guidelines and to discuss the efficacy, safety and positioning of the new medicine bempedoic acid. The development of atherosclerosis is mainly due to hypercholesterolemia. The causal relationship between low density lipoprotein (LDL) and atherosclerosis has been extensively proven and concluded that the lower the LDL value, the lower the risk of cardiovascular events. This was one of the reasons, why the guidelines for the treatment of hyperlipidemia have been updated. The new guidelines have lowered their LDL target values. This will have consequences for the prescribing behaviour of doctors because they will have to evolve from monotherapy to more combination therapy. Statins remain the first line therapy but will be combined with ezetimibe and in some cases with proprotein convertase subtilisin-kexin type 9 (PCSK-9) inhibitors. The use of PCSK-9 inhibitors is therefore more recommended by the new ESC guidelines. This will have a major impact on the healthcare budget and health expenditure of patients, because the price of these medicines is much higher compared to the price of statins and ezetimibe (both generic). The new medicine bempedoic acid, acts on a different enzyme of the same pathway as statins. It can provide added value for patients, who do not reach the new LDL targets and for patients who cannot tolerate daily use of statins. The efficacy and safety of bempedoic acid was extensively tested in these patient populations by several phase 3 studies. CLEAR Tranquility and CLEAR Serenity investigated efficacy and safety in patients, who were statin intolerant. CLEAR Wisdom and CLEAR Harmony investigated efficacy and safety in patients, who were statin tolerant and had a high cardiovascular risk. A fixed dose combination of ezetimibe and bempedoic acid was also investigated. These studies all concluded that the LDL value was reduced by 15.1-36.2% and bempedoic acid was well tolerated. Bempedoic acid has a major advantage over statins because patients report fewer muscle related side effects, what could be a major benefit for statin intolerant patients. The target population for bempedoic acid and fixed dose combination, will be (very) high-risk patients who cannot reach the LDL goals with optimized maximally tolerated statins and ezetimibe. The anticipated place for bempedoic acid in the treatment regimen is between a maximum tolerated doses of statins/ezetimibe and the PCSK-9 inhibitors.