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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
Discovery of ligands for TRIM58, a novel tissue selective E3 ligase
No full textRedirecting E3 ligases to neo-substrates leading to their proteasomal disassembly, known as targeted protein degradation (TPD), has emerged as a promising alternative to traditional, occupancy driven pharmacology. Although the field has expanded tremendously over the last years, the choice of E3 ligases remains limited, with an almost exclusive focus on CRBN and VHL. Here, we report the discovery of novel ligands to the PRY-SPRY domain of TRIM58, a RING ligase that is specifically expressed in erythroid precursor cells. A DSF screen, followed by validation using additional biophysical methods, led to the identification of the TRIM58 ligand TRIM-473. A basic SAR around the chemotype was established by utilizing a competitive binding assay employing a short FP pep-tide probe derived from an endogenous TRIM58 substrate. The X-ray co-crystal structure of TRIM58 in complex with TRIM-473 gave insights to the binding mode and potential exit vectors for bifunctional degrader design
Design of a Super-Soft Topical JAK Inhibitor, which Is Efficacious in Human Skin but Rapidly Deactivated in Blood
No full textWe describe the discovery and characterization of the super-soft topical JAK inhibitor 3(R), which is potent in biochemical and cellular assays as well as in human skin models. In blood, the neutral ester 3(R) is rapidly hydrolyzed (t1/2~6 min) to the corresponding charged carboxylic acid 4 exhibiting >30-fold reduced permeability. Consequently, acid 4 does not reach the intracellular JAK kinases and is inactive in cellular assays and in blood. Thus, hydrolysis by blood esterases leads to the rapid deactivation of the topically active ester 3(R) at a rate beyond the maximal hepatic clearance
Circulating CD200 is increased in the secretory phase of women with endometriosis as is endometrial mRNA, and endometrial stromal cell CD200R1 is increased in spite of reduced mRNA.
No full textPROBLEM
Estrogen-dependent extrauterine implantation and growth of menstrual endometrial tissue affects roughly 10% of reproductive age women and depends on suppression of local innate immune defenses to prevent ectopic tissue rejection. Immunohistochemistry has shown the immune check-point inhibitor CD200 which can suppress rejection is expressed in eutopic endometrium and in ectopic deposits. Soluble CD200 accumulated in venules draining eutopic and ectopic endometrium of endometriosis cases in the secretory phase but not proliferative phase of the menstrual cycle, and should be increased in the circulation.
METHOD OF STUDY
Sera from endometriosis and non-endometriosis controls were tested by ELISA for CD200. Endometrial CD200, CD200R1 and CD200R2 mRNA in eutopic was quantified by RT-PCR and localized by in situ hybridization. CD200R1 protein was quantified by immunohistochemistry.
RESULTS
Secretory phase serum CD200 was elevated in women with endometriosis compared to controls. Serum CD200 correlate
IQ Therapeutic Protein Drug-Drug Interaction White Paper
No full textTypically, therapeutic proteins (TPs) do not elicit clinically meaningful drug interactions (DIs). However, there are select instances where TP drug interactions (TP-DIs) of clinical concern can occur. This white paper discusses the various types of TP-DIs involving mechanisms such as changes in disease state, target-mediated drug disposition (TMDD), FcRN, or anti-drug antibodies (ADAs). The nature of drug interaction being investigated should determine whether the study is conducted in healthy subjects, in patients, or assessed via population pharmacokinetic (PopPK) analysis. DIs involving antibody-drug conjugates are discussed briefly but the primary focus will be DIs involving cytokine modulation. Cytokine modulation can occur directly by certain TPs, or indirectly due to moderate-to-severe inflammation, infection, or injury. Disease states that have been shown to result in indirect disease-DIs that are clinically meaningful (i.e., typically a 2-fold change in the systemic exposure of a co-administered sensitive CYP substrate drug) have been listed. Type of disease and severity of inflammation should be the primary drivers for risk assessment for disease-DIs. While more clinical inflammatory marker data needs to be collected, the use of two or more clinical inflammatory markers (such as c-reactive protein, albumin, or interleukin-6) may help broadly categorize whether the predicted magnitude of inflammatory disease-drug interaction risk is negligible, weak, or moderate-to-strong. Based on current knowledge, clinical DI studies are not necessary for all TPs, and in particular, should no longer be conducted in psoriasis trials as psoriasis patients have insufficient systemic inflammation to cause a meaningful indirect disease-DI
Neurofilament Light Chain and Dorsal Root Ganglia Injury After Adeno-Associated Virus 9 Gene Therapy in Nonhuman Primates
No full textIn nonhuman primates (NHPs), adeno-associated virus serotype 9 (AAV9) vectorized gene therapy can cause asymptomatic microscopic injury to dorsal root (DRG) and trigeminal ganglia (TG) somatosensory neurons, causing neurofilament light chain (NfL) to diffuse into cerebrospinal fluid (CSF) and blood. Data from 260 cynomolgus macaques administered vehicle or AAV9 vectors (intrathecally or intravenously) were analyzed to investigate NfL as a soluble biomarker for monitoring DRG/TG microscopic findings. The incidence of key DRG/TG findings with AAV9 vectors was 78% (maximum histopathology severity, moderate) at 2–12 weeks post-dose. When examined up to 52 weeks post-dose, the incidence was 42% (maximum severity, minimal). Terminal NfL concentrations in plasma, serum, and CSF correlated with microscopic severity. After 52 weeks, NfL returned to pre-dose baseline concentrations, correlating with microscopic findings of lesser incidence and/or severity compared with interim time points. Blood and CSF NfL concentrations correlated with asymptomatic DRG/TG injury, suggesting that monitoring serum and plasma concentrations is as useful for assessment as more invasive CSF sampling. Longitudinal assessment of NfL concentrations related to microscopic findings associated with AAV9 administration in NHPs indicates NfL could be a useful biomarker in nonclinical toxicity testing. Caution should be applied for any translation to humans
Discovery and Characterization of the Topical Soft JAK Inhibitor CEE321 for Atopic Dermatitis.
No full textThe JAK kinases JAK1, JAK2, JAK3, and TYK2 play key roles in cytokine signaling. Activation of the JAK/STAT pathways is linked to many diseases involving the immune system, including atopic dermatitis. As systemic JAK inhibitor pharmacology is associated with side effects, topical administration to the skin has been considered to locally restrict the site of action. Several orally bioavailable JAK inhibitors repurposed for topical use have been recently approved or are in clinical development. Here, we disclose our clinical candidate CEE321, which is a potent pan JAK inhibitor in enzyme and cellular assays. In contrast to repurposed oral drugs, CEE321 does not display high potency in blood and has a high clearance in vivo. Therefore, we consider CEE321 to be a "soft drug". When applied topically to human skin that was stimulated with the cytokines IL4 and IL13 ex vivo, CEE321 potently inhibited biomarkers relevant to atopic dermatitis
Meeting report of the third European Biotransformation Workshop
No full textChallenges, strategies and new technologies in the field of biotransformation were presented and discussed at the 3rd European Biotransformation Workshop which was held in collaboration with the DMDG on October 5-6, 2022 in Amsterdam. In this meeting report we summarise the presentations and discussions from this workshop.
The topics covered are listed below:
• Accelerator Mass Spectrometry for the support of Microtracer Studies
• Biotransformation of the novel myeloperoxidase inhibitor AZD4831 in preclinical species and humans
• AMS in biotransformation studies: Unusual case studies
• Discussion on new FDA draft guidance and AMS
• Multimodal molecular imaging and ion mobility applications in drug discovery and development
• Metabolites in Safety Testing (MIST) considerations for large molecule