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Industry Perspective of International Consortium for Innovation through Quality in Pharmaceutical Development: Complementary LBA and LC-MS Strategies for Large Molecule Protein Bioanalysis and Biotransformation
No full textIncreasingly diverse large molecule modalities have driven the need for complex bioanalysis involving both traditional ligand binding assays (LBA) and more recent hybrid immunoaffinity liquid
chromatography-mass spectrometry (LC-MS) platforms. Given the scientific expertise in LBA and LCMS typically resides in different functions within the industry, this has presented operational challenges for an integrated approach for bioanalysis. Encouragingly, over time, the industry has recognized the complementary value. This has not been an easy transition as organizational structures vary widely within the industry. However, there are tremendous benefits in adopting fully integrated strategies for biopharma. This paper highlights the technical and operational challenges in current large molecule bioanalysis, value of collaborations across LBA and LC-MS platforms, and scientific expertise for fully integrated strategies
p53 dynamics in single cells are temperature-sensitive.
No full textCells need to preserve genome integrity despite varying cellular and physical states. p53, the guardian of the genome, plays a crucial role in the cellular response to DNA damage by triggering cell cycle arrest, apoptosis or senescence. Mutations in p53 or alterations in its regulatory network are major driving forces in tumorigenesis. As multiple studies indicate beneficial effects for hyperthermic treatments during radiation- or chemotherapy of human cancers, we aimed to understand how p53 dynamics after genotoxic stress are modulated by changes in temperature across a physiological relevant range. To this end, we employed a combination of time-resolved live-cell microscopy and computational analysis techniques to characterise the p53 response in thousands of individual cells. Our results demonstrate that p53 dynamics upon ionizing radiation are temperature dependent. In the range of 33 °C to 39 °C, pulsatile p53 dynamics are modulated in their frequency. Above 40 °C, which corresponds to mild hyperthermia in a clinical setting, we observed a reversible phase transition towards sustained hyperaccumulation of p53 disrupting its canonical response to DNA double strand breaks. Moreover, we provide evidence that mild hyperthermia alone is sufficient to induce a p53 response in the absence of genotoxic stress. These insights highlight how the p53-mediated DNA damage response is affected by alterations in the physical state of a cell and how this can be exploited by appropriate timing of combination therapies to increase the efficiency of cancer treatments
Causal prophylactic efficacy of ganaplacide (KAF156) in a controlled human malaria infection model
No full textAbstract
BACKGROUND
KAF156 is a novel antimalarial drug that is active against both liver- and blood- stage Plasmodium parasites, including drug-resistant strains. Here, we investigated the causal prophylactic efficacy of KAF156 in a controlled human malaria infection (CHMI) model.
METHODS
In Part 1, healthy, malaria-naïve participants received 800 mg KAF156 or placebo three hours before CHMI with P. falciparum-infected mosquitoes. In Part 2, KAF156 was administered as single doses of 800, 300, 100, 50, or 20 mg 21 hours post-CHMI. All participants received atovaquone/proguanil treatment if blood-stage infection was detected or on day 29. For each cohort, 7-14 subjects were enrolled to KAF156 treatment and up to four subjects to placebo.
RESULTS
KAF156 at all dose levels was safe and well tolerated. Two serious adverse events were reported - both resolved without sequelae and neither was considered related to KAF156. In Part 1, all participants treated with KAF156 and none of those randomized to placebo were protected against malaria infection. In Part 2, all participants treated with placebo or 20 mg KAF156 developed malaria infection. In contrast, 50 mg KAF156 protected 3/14 participants from infection, and doses of 800, 300, and 100 mg KAF156 protected all subjects against infection. An exposure-response analysis suggested that a 24-hour post-dose concentration of KAF156 of 21.5 ng/mL (90% CI 17.66 to 25.32 ng/mL) would ensure a 95% chance of protection from malaria parasite infection.
CONCLUSIONS
KAF156 was safe and well tolerated and demonstrated high levels of pre- and post-CHMI protective efficacy. (Funded by Novartis
Increased lysosomal biomass is responsible for the resistance of triple-negative breast cancers to CDK4/6 inhibition
No full textBreast cancer is the most common type of cancer in women and the second leading cause of cancer death among women. While significant progress has been made in the treatment of hormone receptor (HR) positive, HER2 negative and HER2-amplified breast cancer in the last decades, Triple negative breast cancer (TNBC) is lacking behind. Still today, chemotherapy is the only treatment option, but response rates are low and only a small fraction of patients can successfully be treated. Here we show, that CDK4/6 inhibitors which are FDA-approved for the treatment of HR+/HER2negative breast cancer and significantly improved treatment outcome in this type of breast cancer, also represent a promising therapy option for TNBC in contrast to the current view. We show, that a subset of TNBCs despite being resistant to chemical inhibition of CDK4/6 strictly depend on CDK4/6 kinases for proliferation. We identified lysosomal sequestration of the drug and therefore limited bioavailability at its target site responsible for the discrepancy. We show that increased number of lysosomes correlates with resistance and inhibition of the lysosome renders these cells fully sensitive to CDK4/6 inhibitors. We provide strategies how to overcome resistance by means of FDA-approved lysosomotropic agents that raise lysosomal pH, co-inhibition of CDK2 or the use of structurally altered CDK4/6 inhibitors with decreased basic characteristics. We also provide a biomarker to stratify patients for successful CDK4/6 inhibitor therapy. Moreover, we show that this mechanism of resistance also underlies cases of acquired resistance in HR+/HER2negative breast cancer and importantly we provide evidence that increased lysosomal sequestration operates in patients presenting resistant to treatment with the CDK4/6 inhibitor palbociclib. Our study therefore suggests that CDK4/6 inhibitor therapy could be a treatment option for a subset of TNBCs where new targeted therapeutic intervention is so urgently needed and furthermore, offers strategies how to overcome resistance in HR+/HER2-negative breast cancer where CDK4/6 inhibition is already successfully applied. In summary, our study provides ways on how to improve therapeutic use of this promising new class of anti-cancer agents
Understanding metabolism related differences in ocular efficacy of MGV354
No full textMGV354 was being developed as a novel ocular therapy for lowering of intraocular pressure, a key modifiable risk factor for glaucoma. MGV354 is an activator of soluble guanylate cyclase, an enzyme known to be involved in the regulation of IOP. MGV354 has been shown to robustly lower IOP over 24 h after a single topical ocular drop in rabbit and monkey pharmacology models. However, MGV354 failed to produce similar results in patients with ocular hypertension or open-angle glaucoma. With an objective of explaining the lack of efficacy in the clinic, we attempted to study whether human metabolism was significantly different from animal metabolism. The present study documents the investigation of metabolism of MGV354 in an effort to understand potential differences in biotransformation pathways of MGV354 in rabbits, monkeys, and humans. Overall twenty-six metabolites, formed via oxidative and conjugative pathways, were identified in vitro and in vivo. In vitro hepatic metabolism was qualitatively similar across species, with minor but distinct differences. There were no observable interspecies differences in the hepatic and ocular metabolism of MGV354. Although ocular metabolism was not as extensive as hepatic, the results do not explain the lack of efficacy of MGV354 in clinical studies
Evaluation of a novel blood microsampling device for clinical trial sample collection and protein biomarker analysis
No full textAim: Evaluation of a novel microsampling device for its use in clinical sample collection and biomarker analysis. Methodology: Matching samples were collected from 16 healthy donors (ten females, six males; age 42 ± 20) via K2EDTA touch activated phlebotomy (TAP) device and phlebotomy. The protein profile differences between sampling groups was evaluated using aptamer-based proteomic assay SomaScan and selected ELISA. Conclusion: Somascan signal concordance between phlebotomy- and TAP-generated samples was studied and comparability of protein abundances between these blood sample collection methods was demonstrated. Statistically significant correlation in selected ELISA assays also confirmed the TAP device applicability to the quantitative analysis of protein biomarkers in clinical trials
Reaction Calorimetry in continuous flow mode. A new approach for the thermal characterization of high energetic and fast reactions
No full textA new method for the calorimetric characterization of high-energetic, fast reactions in flow mode has been developed. The use of an engineered flow reactor in combination with a process modelling software allowed the deconvolution of the reaction enthalpy from space-resolved temperature profiles. The new procedure was verified in a comparison with a conventional batch calorimeter and subsequently implemented for the thermal characterization of an organolithium flow process. The information collected for this reaction successfully supported a scale-up to the pilot plant. Overall, the new approach resulted to be superior when compared with established procedures, enabling the generation of precise calorimetric data in an accurate scale-down flow device
Toward a Scalable Synthesis and Process for EMA401. Part II: Development and Scale-up of a Pyridine- and Piperidine-free Knoevenagel-Doebner Condensation
No full textDuring the route scouting of EMA401 (1), a angiotensin II type 2 antagonist, we identified the synthesis of key amino acid intermediate 2 via its cinnamic acid derivative 3 as a streamlined option. In general, cinnamic acids can be synthesized from the corresponding aldehyde in a Knoevenagel-Doebner condensation in pyridine with piperidine as an organocatalyst. We aimed at replacing both of these reagents and found novel conditions in toluene as the solvent and morpholine as the organocatalyst. Scale-up of the process allowed production of 25 kg of the cinnamic acid 3, which was of required quality for process development on the subsequent phenylalanine ammonia lyase-catalyzed step. The modified conditions were found to be widely applicable to alternative aldehydes, and so of relevance to practitioners of chemical scale-up
The Current Status and Future of Two- and Multidimensional Liquid Chromatography in Pharmaceutical R&D and QC
No full textTwo-dimensional liquid chromatography (2D-LC) has recently seen major developments and attracted significant interest. The limited resolving power and insufficient chromatographic selectivity of conventional one-dimensional liquid chromatography (1D-LC) for more complex pharmaceutical products, alongside the introduction of modern commercial 2D-LC instruments means the technique is no longer considered a purely academic or specialist research tool. 2D-LC is now viewed as a powerful technique in the “analytical toolbox” and is being widely adopted in the analysis of both small molecules and more complex molecules, specifically biopharmaceuticals. This article will discuss the benefits of 2D-LC and multiple application areas in (bio)pharmaceutical analysis, and will highlight the challenges and future outlook
Repolarization Studies using Human Stem Cell-Derived Cardiomyocytes: Validation Studies and Best Practice Recommendations
No full textHuman stem-cell derived cardiomyocytes (hSC-CMs) hold great promise as in vitro models to study the electrophysiological effects of novel drug candidates on human ventricular repolarization. Two recent large validation studies have demonstrated the ability of hSC-CMs to detect drug-induced delayed repolarization and “cellrhythmias” (interrupted repolarization or irregular spontaneous beating of myocytes) linked to Torsade-de-Pointes proarrhythmic risk. These (and other) studies have also revealed variability of electrophysiological responses attributable to differences in experimental approaches, protocols, technology platforms used, and pharmacologic sensitivity of different human-derived models. Thus, when evaluating drug-induced repolarization effects there is a need to consider 1) the advantages and disadvantages of different approaches, 2) the need for robust functional characterization of hSC-CMs preparations to define “fit for purpose” applications, and 3) adopting standardized best practices to guide future studies with evolving hSC-CM preparations. Examples provided and suggested best practices are instructional in defining consistent, reproducible, and interpretable “fit for purpose” hSC-CM based applications. Implementation of best practices should enhance the clinical translation of hSC-CM-based cell and tissue preparations in drug safety evaluations and support their growing role in regulatory filings