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Genetic models of cleavage-reduced and soluble TREM2 reveal distinct effects on myelination and microglia function in the cuprizone model.
No full textTriggering receptor expressed on myeloid cells 2 (TREM2) is a cell-surface immunoreceptor expressed on microglia, osteoclasts, dendritic cells and macrophages. Heterozygous loss-of-function mutations in TREM2, including mutations enhancing shedding form the cell surface, have been associated with myelin/neuronal loss and neuroinflammation in neurodegenerative diseases, such as Alzheimer`s disease and Frontotemporal Dementia. Using the cuprizone model, we investigated the involvement of soluble and cleavage-reduced TREM2 on central myelination processes in cleavage-reduced (TREM2-IPD), soluble-only (TREM2-sol), knockout (TREM2-KO) and wild-type (WT) mice. The TREM2-sol mouse is a new model with selective elimination of plasma membrane TREM2 and a reduced expression of soluble TREM2. In the acute cuprizone model demyelination and remyelination events were reflected by a T2-weighted signal intensity change in magnetic resonance imaging (MRI), most prominently in the external capsule (EC). In contrast to WT and TREM2-IPD, TREM2-sol and TREM2-KO showed an additional increase in MRI signal during the recovery phase. Histological analyses of TREM2-IPD animals revealed no recovery of neuroinflammation as well as of the lysosomal marker LAMP-1 and displayed enhanced cytokine/chemokine levels in the brain. TREM2-sol and, to a much lesser extent, TREM2-KO, however, despite presenting reduced levels of some cytokines/chemokines, showed persistent microgliosis and astrocytosis during recovery, with both homeostatic (TMEM119) as well as activated (LAMP-1) microglia markers increased. This was accompanied, specifically in the EC, by no myelin recovery, with appearance of myelin debris and axonal pathology, while oligodendrocytes recovered. In the chronic model consisting of 12-week cuprizone administration followed by 3-week recovery TREM2-IPD displayed sustained microgliosis and enhanced remyelination in the recovery phase. Taken together, our data suggest that sustained microglia activation led to increased remyelination, whereas microglia without plasma membrane TREM2 and only soluble TREM2 had reduced phagocytic activity despite efficient lysosomal function, as observed in bone marrow-derived macrophages, leading to a dysfunctional phenotype with improper myelin debris removal, lack of remyelination and axonal pathology following cuprizone intoxication
Relative Bioavailability and Food Effect of Asciminib Pediatric Mini-tablet Formulation Compared to the Reference Tablet Formulation in Healthy Adult Participants
No full textAsciminib, a first-in-class allosteric BCR::ABL1 inhibitor that works by specifically targeting the ABL myristoyl pocket (STAMP), is used in the treatment of chronic myeloid leukemia (CML). This was a randomized, single dose, open-label, four-period cross-over study in healthy adult participants (N=24) which evaluated the relative bioavailability of a single 40 mg dose of asciminib pediatric formulation (1 mg mini-tablets) compared with the reference adult tablet, under fasting conditions. In addition, the effect of food on the bioavailability of the mini-tablet formulation was evaluated. Under fasting conditions, asciminib exposure was similar for both formulations (geometric mean [Gmean] area under the concentration-time curve from time 0 to infinity [AUCinf] 5970 ng·h/mL and 5700 ng·h/mL, respectively). Food decreased the AUCinf and maximum plasma concentration (Cmax) of the asciminib mini-tablets; this effect was more pronounced with a high-fat meal (Gmean ratios [90% confidence interval (CI)]: fasted/low-fat meal, 0.42 [0.38–047], 0.32 [0.28–0.37], respectively; fasted/high-fat meal, 0.30 [0.27–0.34], 0.22 [0.19–0.25], respectively). Both formulations were well tolerated. The mini-tablets were assessed to be easy to ingest with good palatability. Results gained from this study will be utilized in physiologically-based pharmacokinetic (PBPK) modelling to define asciminib starting doses in a pivotal pediatric clinical trial
Remibrutinib inhibits hives effector cells stimulated by serum from chronic urticaria patients independently of FcεR1 expression level and omalizumab clinical response
No full textBackground: Despite advances in the treatment of chronic urticaria, in a significant
percentage of the patients symptoms are not fully controlled with conventional
approaches. New strategies under development include blocking intracellular mediators
of mast cell and basophil activation.
Objective: We aim to investigate the effects of the Bruton's tyrosine kinase (BTK)
inhibitor remibrutinib on human blood basophils and CD34+‐derived mast cells
activation induced by serum obtained from chronic urticaria patients.
Methods: Twenty‐two patients with chronic spontaneous urticaria (mean age
52 years, 27% women) and 22 patients with chronic inducible urticaria (46 years,
27% women) were included in the study together with a sex‐matched control group.
Patients were classified as responders or non‐responders to anti‐IgE therapy on the
basis of their clinical data, FcεR1a expression on blood basophils and total IgE levels.
Changes on CD63 expression—as an activation marker‐, were used to evaluate in
vitro the response of basophils and mast cells to serum exposure and the inhibitory
effects of remibrutinib.
Results: Remibrutinib inhibits degranulation induced by IgE cross‐linking in mast
cells and basophils and also the activation triggered by factors present in the sera of
spontaneous and inducible chronic urticaria patients. Patient's serum induces a
greater degranulation of effector cells than controls. Activation of mast cells and
basophils by patient sera and remibrutinib effects were not related to omalizumab
responsiveness.
Conclusion: Remibrutinib inhibits activation of human basophils and mast cells
induced in vitro by exposure to the serum of chronic urticaria patients independently
of their response to omalizumab
Conformal efficiency as a metric for comparative model assessment befitting federated learning
No full textAs training volume increases predictive model quality, leveraging existing external data sources holds the promise of time- and cost-efficiency. In a drug discovery setting, pharmaceutical companies all own substantial but confidential datasets. The MELLODDY project develops a privacy-preserving federated machine learning solution and deploys it at an unprecedented scale (more than 100,000 tasks across ten major pharmaceutical companies), while ensuring the security and privacy of each partner’s sensitive data. Each partner builds models that benefit from a shared representation, for their own private assays. Established predictive performance metrics such as AUC ROC or AUC PR are constrained to unseen labelled chemical space. However, they cannot gauge performance gains in unlabelled chemical space. Federated learning indirectly extends labelled space, but in a privacy-preserving context, a partner cannot use this label extension for performance assessment. Metrics that estimate uncertainty on a prediction can be calculated even where no label is known. Practically, the chemical space covered with predictions of sufficiently confidence, reflects the applicability domain of a model. After establishing a link to established performance metrics, we propose the efficiency from the conformal prediction framework (‘conformal efficiency’) as a proxy to the applicability domain size. A documented extension of the applicability domain would qualify as a tangible benefit from federated learning. In interim assessments, MELLODDY partners report a median increase in conformal efficiency of the federated over the single-partner model of 5.5% (with increases up to 9.7%). Subject to distributional conditions, that efficiency increase can be directly interpreted as the expected increase in conformal i.e. high confidence predictions. In conclusion, we present the first evidence that privacy-preserving federated machine learning across massive drug-discovery datasets from ten pharma partners indeed extends the applicability domain of property prediction models
Incubation Time Influences Organic Anion Transporter 1 Kinetics In Vitro and Renal Clearance Predictions for Para-Aminohippurate
No full textExamined was the effect of in vitro incubation time on ligand interactions with organic anion transporter 1 (OAT1), and how the time chosen influences renal clearance (CLR) predictions for para-aminohippurate (PAH). Transport studies were performed with Chinese hamster ovary cells expressing OAT1 (CHO-OAT1). The Mechanistic Kidney model (MechKiM) within the Simcyp Simulator (v15, Certera, Inc) was used for pharmacokinetic predictions. Increasing in vitro incubation time led to a decrease in the maximal transport rate and intrinsic uptake clearance (CLint) for PAH, with CLint values ranging 11-fold when using incubation times ranging from 15 sec (CLint,15s) to 45 min (CLint,45min). There was also a significant effect of time on the Michaelis constant. Inhibition potency of five drugs against PAH transport was tested using incubation times of either 15 sec or 10 min. There was no effect of time on inhibition potency for omeprazole or furosemide. Indomethacin was less potent at 10 min, whereas probenecid (~2-fold) and telmisartan (~7-fold) were more potent when using the longer incubation. Pre-treatment of CHO-OAT1 cells with telmisartan for increasing periods of time (30 sec – 30 min) using multiple concentrations (10 nM – 200 nM) showed that its inhibitory effect is time-sensitive. A MechKiM model was developed for PAH using the PAH CLint obtained at 15 sec, in vitro-in vivo extrapolation, and parameter estimation of the OAT1 relative activity factor. Using these parameters, the simulated plasma concentration-time profile, CLR and cumulative urinary excretion-time profile of PAH agreed reasonably well with reported data. Next we used the CLint values obtained from studies using incubation times greater than 15 sec (up to 45 min). The simulated CLR of PAH was sensitive to the time-associated CLint value used, with CLR values 2.7-fold higher when using CLint,15s vs. CLint,45min. The simulated percentages of the PAH dose excreted in the urine after 2 h were 100% (CLint,15sec) vs. 81% (CLint,45min). Simulations predicted a maximum intracellular PAH concentration in tubule cells that was 3.4-fold higher when using CLint,15s vs. CLint,45min. These data show that in vitro incubation time influences transport kinetics and predictions of the involvement of drug transport in pharmacokinetics
Industry Perspective on the Pharmacokinetic and ADME Characterization of Heterobifunctional Protein Degraders
No full textTargeted protein degraders (TPDs), specifically the bifunctional protein degraders discussed in this manuscript, consist of two linked ligands for a protein of interest and an E3 ligase, resulting in molecules which largely violate accepted physicochemical limits (e.g. Lipinski’s Rule-of-5) for oral bioavailability. In 2021, the IQ Consortium Degrader DMPK/ADME Working Group undertook a survey of eighteen IQ member and non-member companies working on degraders to understand whether the characterization and optimization of these molecules were different from any other beyond the rule of 5 (bRo5) compounds. Additionally, the working group sought to identify PK/ADME areas in need of further evaluation and where additional tools could aid in more rapid advancement of TPDs to patients.
The survey revealed that although TPDs reside in a challenging bRo5 physicochemical space, most respondents focus their efforts on oral delivery. Physicochemical properties required for oral bioavailability were generally consistent across the companies surveyed. Many of the member companies used modified assays to address challenging degrader properties (e.g. solubility, non-specific binding), but only half indicated that they modified their drug discovery flow schemes. The survey also suggested the need for further scientific investigation in the areas of CNS penetration, active transport, renal elimination, lymphatic absorption, in silico/machine learning, and human pharmacokinetic prediction. Based on the survey results, the Degrader DMPK/ADME Working Group concludes that TPD evaluation does not fundamentally differ from other bRo5 compounds, but requires some modification compared to traditional small molecules and proposes a generic workflow for PK/ADME evaluation of bifunctional TPDs
Absorption, Distribution, Metabolism, and Excretion of [14C]iptacopan in Healthy Male Volunteers and in In Vivo and In Vitro Studies.
No full textIptacopan (LNP023) is an oral, small-molecule, first-in-class, highly potent proximal complement inhibitor that specifically binds factor B and inhibits the alternative complement pathway. Iptacopan is currently in development as a targeted treatment of paroxysmal nocturnal hemoglobinuria and multiple other complement-mediated diseases. In this study, the absorption, distribution, metabolism, and excretion (ADME) of iptacopan was characterized in six healthy volunteers after a single 100 mg oral dose of [14C]iptacopan. This was supplemented with an in vivo rat ADME study and metabolite exposure comparisons between human, rat, and dog, in addition to in vitro assays, to better understand the clearance pathways and enzymes involved in the metabolism of iptacopan. The fraction of [14C]iptacopan absorbed was estimated to be about 71%, with a time to maximum concentration of 1.5 hours and elimination half-life from plasma of 12.3 hours. Following a single dose of [14C]iptacopan, 71.5% of the radioactivity was recovered in feces and 24.8% in urine. [14C]iptacopan was primarily eliminated by hepatic metabolism. The main biotransformation pathways were oxidative metabolism via CYP2C8, with M2 being the major oxidative metabolite, and acyl glucuronidation via UGT1A1. The two acyl glucuronide metabolites in human plasma, M8 and M9, each accounted for ≤ 10% of the total circulating drug-related material; systemic exposure was also observed in toxicology studies in rat and dog, suggesting a low risk associated with these metabolites. Binding of iptacopan to its target, factor B, in the bloodstream led to a concentration-dependent blood:plasma distribution and plasma protein binding of [14C]iptacopan. SIGNIFICANCE STATEMENT: We characterized the pharmacokinetics, excretion, metabolism and elimination of [14C]iptacopan (an oral, selective small-molecule inhibitor of factor B) in healthy human subjects. [14C]iptacopan was primarily eliminated by metabolism. The primary biotransformation pathways were oxidative metabolism via CYP2C8 and acyl glucuronidation via UGT1A1. Direct secretion of iptacopan into urine and potentially bile represented additional elimination mechanisms. Binding of iptacopan to its target, factor B, in the bloodstream led to a concentration-dependent blood:plasma distribution and plasma protein binding of [14C]iptacopan
Metabolite Bioanalysis in Drug Development: Recommendations from the IQ Consortium Metabolite Bioanalysis Working Group
No full textThe intent of this perspective is to share the recommendations of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ Consortium) Metabolite Bioanalysis Working Group (WG) on the fit-for-purpose metabolite bioanalysis in support of drug development and registration. This report summarizes the considerations for the trigger, timing, and rigor of bioanalysis in the various assessments to address unique challenges due to metabolites, with respect to efficacy and safety, which may arise during drug development from IND enabling studies, and Phase I, Phase II, and Phase III clinical trials to regulatory submission. The recommended approaches ensure that important drug metabolites are identified in a timely manner and properly characterized for efficient drug development
Real-Time Assessment of the Size Changes of Individual Sub-Visible Protein Particles under Buffer Variations: A Microfluidic Study.
No full textProtein particles in biological drugs can significantly impact drug efficacy and carry the risk of adverse effects. Despite advancements, the understanding and control of particle formation in biopharmaceutical manufacturing remain incomplete. Therefore, further investigation into protein particles is warranted, especially considering that novel formats of biological drugs may be more susceptible to aggregation and particle formation than conventional monoclonal antibodies. In this study, we introduce a microfluidic approach for the real-time analysis of individual sub-visible protein particles during buffer exchange. We find that the modulation of intermolecular forces, achieved by changing the buffer pH or urea concentration, leads to the reversible swelling and shrinkage of particles by up to 50%, which is a consequence of altered intermolecular distances. Additionally, we identify a discrepancy in the biophysical behavior of protein particles compared to monomeric protein. This finding highlights the limited predictive power of commonly applied biophysical characterization methods for particle formation in early formulation development. Moreover, the observed particle swelling may be associated with manufacturing deviations, such as filter clogging. These results highlight the importance of studying individual particles to gain a comprehensive insight into particle behavior and the impact of formulation variations in the biopharmaceutical industry
Surfing the biocatalysis wave to new applications Changed to: From nature to industry: Harnessing enzymes for biocatalysis, as per request of Editor
No full textBiocatalysis applies enzymes to make valuable products. Today, this green technology is effectively utilized from bench scale to industrial production. The last decade has seen an explosion in the development of experimental and in silico tools to tailor enzymatic properties, the latter relying on the surge of available bioinformatic data and continuously accelerating computational advances. By harnessing (chemo)-enzymatic synthesis routes or intricate enzyme cascades, completely new targets can be synthesized, ranging from DNA and complex pharmaceuticals to starch made in vitro simply from CO2-derived methanol. In addition, intriguing new chemistries have emerged by combining biocatalysis with transition metal-, photo- and electrocatalysis. Against this exciting backdrop, this review highlights recent key developments, identifies current limitations, and provides a future prospect for this rapidly developing research field