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A cooperative release of mitochondrial DNA from platelets and neutrophils drives an interferon signature in systemic sclerosis.
No full textMitochondria are organelles with a hypomethylated circular genome. Mitochondrial DNA (mtDNA) in the systemic circulation has been implicated in inflammation. This study investigates the role of circulating DNA in systemic sclerosis (SSc) and the cellular mechanisms governing its release.Total DNA was isolated from plasma of healthy individuals and SSc patients. Copy numbers were analyzed for mtDNA (ATP-6) and GAPDH abundance by qPCR. mtDNA was isolated from HC and SSc patients. Neutrophils and platelets were incubated with SSc patients' plasma and mtDNA, and NET formation was assessed by SytoxGreen and immunostainings. Platelets were tested for mtDNA release propensity. DNA oxidation was evaluated by MitoSOX Red staining in vitro and 8-OHdG ELISA of patient plasma. Plasma IFN type 1 and CXCL4 were measured by ELISA. IFN signaling activation capacity was evaluated utilizing THP1 reporter cells and confirmed by a whole blood bulk RNA transcriptomic analysis.Median plasma mtDNA levels were 152-fold higher in SSc patients compared to healthy controls (HC), while nDNA levels were similar. mtDNA from SSc plasma was highly oxidized. SSc-derived mtDNA efficiently promoted its own release by NETosis, most potently in SSc patient neutrophils, and by platelet activation. Oxidized mtDNA from SSc platelets in complex with CXCL4 further stimulated mtDNA release in both neutrophils and platelets. mtDNA plasma concentrations correlated with type I IFN concentrations in SSc patient blood, and SSc blood exhibited elevated interferon-stimulated gene (ISG) expression. SSc plasma-derived mtDNA induced IFN signaling and NET formation via endosomal TLR, cGAS/STING and the JAK/STAT pathway. The type I IFN pathway further promoted NETosis and mtDNA release since IFN receptor (IFNAR) and Janus kinase (JAK) inhibition antagonized the proNETotic effects of IFN.SSc plasma is characterized by highly abundant mtDNA, which drives feedback loops amplifying its own release from both neutrophils and platelets. Thus, mtDNA contributes to inflammation and tissue damage in SSc
The nucleobase guanine at the 3'-terminus of oligonucleotide RGLS4326 drives off-target AMPAR inhibition and CNS toxicity.
No full textDesigning safe and effective oligonucleotide (ON) therapeutics requires thorough understanding of structural-activity relationship (SAR) with the intended on-target(s) as well as the unintended off-target(s). Despite encouraging pharmacodynamic activity in a Phase 1b study, development of the first-generation anti-miR-17 ON RGLS4326 for the treatment of autosomal dominant polycystic kidney disease was discontinued due to dose-limiting central nervous system (CNS)-related toxicity observed in nonclinical chronic toxicity studies. Here, we provide SAR evidence that the nucleobase guanine at the 3'-terminus of RGLS4326 drives an unexpected off-target aptamer-like direct interaction with α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), thereby causing CNS toxicity. By replacing the 3'-terminal guanine with adenine, we discover the next-generation anti-miR-17 RGLS8429 that is devoid of off-target AMPAR interaction and CNS toxicity while preserving the potency against the on-target miR-17. Here, we show a way to avoid off-target CNS effects and, more importantly, data that support the clinical development of RGLS8429
Immunogenicity Risk Assessment for Tailored Mitigation and Monitoring of Biotherapeutics During Development: Recommendations from the European Immunogenicity Platform
No full textBringing safe and effective drugs to patients is of utmost importance for the pharmaceutical industry, with immunogenicity (IG) being a critical factor that influences both aspects. Biotherapeutics can elicit unwanted immune responses, potentially leading to (severe) safety implications, reduced patient benefits, and may result in termination of development. Therefore, understanding IG risks throughout drug development is essential for both drug developers and health agencies (HAs). The Immunogenicity Risk Assessment (IRA) facilitates the identification of IG risk factors and allows the establishment of effective mitigation and monitoring strategies. In this publication, the European Immunogenicity Platform (EIP) presents a comprehensive IRA framework aligned across pharmaceutical industry, emphasizing its significance in product development - from early de-risking to bioanalytical monitoring and mitigation measures during clinical trials. The EIP also provides an updated list of IG risks, offers distinct recommendations for assigning overall IG risk levels prior to the start of clinical development and highlights business considerations within this assessment
InterFace/Off: characterization of competitive adsorption of novel surfactants and proteins at the solid-liquid and oil-liquid interfaces.
No full textInterfacial stress encountered by biopharmaceuticals is often opposed by employing surfactants in their formulations. Surfactants protect proteins from this stress by either shielding the interface or displacing adsorbed proteins. Most previous studies were dedicated to the air-liquid interface, and to characterize monoclonal antibodies or non-pharmaceutically relevant proteins in combination with established surfactants (polysorbates and poloxamer 188). Herein, we employ quartz crystal microbalance with dissipation (QCM-D) and tensiometry to investigate the adsorption behavior of established surfactants as well as the novel surfactants VEDS and VEDG-3.3 at the solid-liquid and silicon oil-liquid interfaces in presence and absence of three model biotherapeutics of different modalities. Our study shows that the individual adsorption behavior is molecule-dependent, as expected. When mixed either simultaneously (co-adsorption) or sequentially (shielding and displacement), both proteins and surfactants were detected to co-adsorb at the interface. Compared to the established surfactants, VEDS and VEDG-3.3 showed a slower adsorption followed by molecular rearrangements that resulted in a denser packing, supporting the mechanistic explanation of their favorable protein stabilization effect previously reported. Collectively, our results support the generation of a unified thermodynamic description of the adsorption of protein-surfactants mixtures in pharmaceutically-relevant conditions
Modular Access to N‑SF5 Azetidines
No full textSynthetic accessible novel scaffolds and chemical functional groups with drug-like properties offer a fresh approach to pharmaceutical molecule design. The introduction of the N-pentafluorosulfanyl (N–SF₅) group may overcome the limited aqueous stability often seen with N-alkyl substitutions—thanks to its larger steric volume, stronger electron-withdrawing effect, and increased lipophilicity. However, the lack of a general, operational simple and efficient synthetic strategy for N–SF₅ compounds has significantly hindered their broader application. To address this, we developed a modular coupling approach using bench-stable, scalable SF₅-transfer reagents, enabling efficient access to N–SF₅-azetidines. This method provides a user-friendly protocol with mild reaction conditions and broad functional group tolerance, making it well-suited for the late-stage modification of commercial drugs and other complex molecules. The resulting scaffolds demonstrate excellent stability and permeability, representing a novel class of potential (bio)isosteres in medicinal chemistry
Exposure-response analysis of asciminib efficacy and safety in patients with chronic myelogenous leukemia in chronic phase.
No full textObjective
This study aimed to evaluate the relationships between asciminib pharmacokinetics with efficacy in first line (1 L) and safety in 1 L and third line (3 L) patients with Philadelphia-positive chronic myeloid leukemia in chronic phase.
Methods
The key primary efficacy endpoint in ASC4FIRST (1 L, phase 3), the week-48 major molecular response (MMR), versus asciminib exposure relationship were evaluated using linear regression model; the same model was applied to assess safety endpoints vs. exposure in ASC4FIRST, ASCEMBL (3 L, phase 3) and first-in-human (3 L, phase 1) trials based on abnormal laboratory data, vital signs and adverse events.
Results
The probability of week-48 MMR in ASC4FIRST versus exposure (averaged daily AUC and Cmin) in 1 L was 70–80% for the second through fourth quartile AUC0−24 h and Cmin where majority of patients had relative dose intensity > 75%; the MMR rate in the first quartile exposure metrics was 45%. Of all safety endpoints in 1 L, the only statistically significant positive relationship with exposure was the worsening event for grade ≥ 3 lipase increase but event probabilities were low (5.8%-11.2%). Line of therapy was investigated as a covariate for all safety endpoints; newly diagnosed patients had a lower probability of all safety events occurring than in pretreated patients.
Conclusion
Treatment compliance optimizes the efficacy of asciminib in newly diagnosed patients. Safety incidences associated with asciminib were low. The 80 mg once-daily regimen provides an optimal benefit-risk profile in newly diagnosed patients. Similar efficacy is expected from the 40 mg twice-daily regimen in the same population
Innovation in Route Design, (Bio)Catalysis and Process Development Applied to the Second Generation Synthesis of Sacubitril.
No full textCommercial manufacturing processes in the pharmaceutical industry need to address numerous goals, ranging from cost effectiveness over process safety to environmental sustainability. While the design of the synthetic route may commonly be considered the centerpiece of chemical process development, only the combination with innovative technologies at scale and in-depth process understanding can unlock the full potential of a synthetic route. The development of a second-generation synthesis of sacubitril, one of the constituent active pharmaceutical ingredients of LCZ696, serves as an example to display how synthesis design can be successfully combined with (bio)catalysis and thorough process development to achieve superior results. In addition, the case at hand highlights the multidisciplinary and team-focused nature of chemical process development
Operating model centralizing open label products used in Clinical Trials
No full textComparator supply decisions are becoming increasingly critical to the success of clinical trials — yet they are often made too late. As study designs become more complex and startup timelines tighten, it’s essential to incorporate comparator considerations early — giving them a seat at the study table from the pre-feasibility phase onwar
Discovery of Selective Low Molecular Weight Interleukin-36 Receptor Antagonists by Encoded Library Technologies
No full textInterleukin-36 receptor (IL-36R), belonging to the IL-1 receptor family, is crucial for host defense and tissue repair. Targeting cytokine receptors with low molecular weight (LMW) compounds remains challenging due to their interaction with the large surface area of cytokine. In this study, two encoded library technologies are used to identify LMW molecules binding to IL-36R’s extracellular domain. The mRNA-based display technique identifies 36R-P138, a macrocyclic peptide blocking IL-36R signaling. Importantly, its optimized analog (36R-P192) also effectively suppresses expression of marker genes induced by IL-36 in human skin biopsies. DNA encoded libraries (DEL) screening delivers 36R-D481, a high affinity LMW IL-36R binder, effectively inhibiting IL-36 signaling. X-ray crystallography analysis reveals that both the cyclic peptide and DEL-compound bind to the IL-36R’s D1 domain, potentially disrupting IL-36 cytokine binding. This study demonstrates that it is possible to target a cytokine receptor within the IL-1 receptor family using a small molecule (< 1000 Da)
Decentralized Clinical Trials in the Era of Real-World Evidence: A Statistical Perspective.
No full textThere has been a growing trend that activities relating to clinical trials take place at locations other than traditional trial sites (hence decentralized clinical trials or DCTs), some of which are at settings of real-world clinical practice. Although there are numerous benefits of DCTs, this also brings some implications on a number of issues relating to the design, conduct, and analysis of DCTs. The Real-World Evidence Scientific Working Group of the American Statistical Association Biopharmaceutical Section has been reviewing the field of DCTs and provides in this paper considerations for decentralized trials from a statistical perspective. This paper first discusses selected critical decentralized elements that may have statistical implications on the trial and then summarizes regulatory guidance, framework, and initiatives on DCTs. More discussions are presented by focusing on the design (including construction of estimand), implementation, statistical analysis plan (including missing data handling), and reporting of safety events. Some additional considerations (e.g., ethical considerations, technology infrastructure, study oversight, data security and privacy, and regulatory compliance) are also briefly discussed. This paper is intended to provide statistical considerations for decentralized trials of medical products to support regulatory decision-making