Effective in vitro evaluation of the risk of histamine release related to valemetostat tosylate using MRGPRX2-expressing cells.

Mas-related G-protein-coupled receptor X2 (MRGPRX2), expressed on mast cells, is associated with drug-induced pseudo-allergic reactions. Although it is well known that there are differences of sensitivity between species in the pseudo-allergic reactions, no platform for evaluating a human risk of the pseudo-allergic reactions observed in nonclinical studies has been established. Valemetostat tosylate, developed as an anti-cancer drug, induced histamine release in a nonclinical study with dogs. The purpose of the current study was to identify the mechanism and assess the human risk of valemetostat-tosylate-induced histamine release using dog and human MRGPRX2-expressing cells. In an experiment with human or dog MRGPRX2-expressing cells, valemetostat tosylate caused activation of human and dog MRGPRX2. Importantly, the EC50 for dog MRGPRX2 was consistent with the Cmax value at which histamine release was observed in dogs. Furthermore, the EC50 for human MRGPRX2 was ca. 27-fold higher than that for dog MRGPRX2, indicating a species difference in histamine-releasing activity. In a clinical trial, histamine release was not observed in patients receiving valemetostat tosylate. In conclusion, an in vitro assay using human and animal MRGPRX2-expressing cells would be an effective platform to investigate the mechanism and predict the human risk of histamine release observed in nonclinical studies.

Determination of key residues in MRGPRX2 to enhance pseudo-allergic reactions induced by fluoroquinolones.

MAS-related G protein-coupled receptor X2 (MRGPRX2), expressed in human mast cells, is associated with drug-induced pseudo-allergic reactions. Dogs are highly sensitive to the anaphylactoid reactions induced by certain drugs including fluoroquinolones. Recently, dog MRGPRX2 was identified as a functional ortholog of human MRGPRX2, with dog MRGPRX2 being particularly sensitive to fluoroquinolones. The aim of this study was to determine key residues responsible for the enhanced activity of fluoroquinolone-induced histamine release associated with MRGPRX2. Firstly, a structure model of human and dog MRGPRX2 was built by homology modeling, and docking simulations with fluoroquinolones were conducted. This model indicated that E164 and D184, conserved between human and dog, are essential for the binding to fluoroquinolones. In contrast, F78 (dog: Y) and M109 (dog: W) are unconserved residues, to which the species difference in fluoroquinolone sensitivity is attributable. Intracellular calcium mobilisation assay with human MRGPRX2 mutants, in which residues at positions 78 and 109 were substituted to those of dog MRGPRX2, revealed that M109 and F78 of human MRGPRX2 are crucial residues for enhancing the fluoroquinolone-induced histamine release. In conclusion, these key residues have important clinical implications for revealing the mechanisms and predicting the risks of fluoroquinolone-mediated pseudo-allergic reactions in humans.

Identification of the dog orthologue of human MAS-related G protein coupled receptor X2 (MRGPRX2) essential for drug-induced pseudo-allergic reactions.

MAS-related G protein coupled receptor-X2 (MRGPRX2), expressed in human mast cells, is associated with drug-induced pseudo-allergic reactions. Dogs are highly susceptible to drug-induced anaphylactoid reactions caused by various drugs; however, the distribution and physiological function of canine MRGPR family genes, including MRGPRX2, remain largely unknown. In the present study, we clarified the distribution of dog MRGPR family genes by real-time quantitative PCR and in situ hybridisation. We also investigated the stimulatory effects of various histamine-releasing agents, including fluoroquinolones, on HEK293 cells transiently transfected with dog MRGPR family genes to identify their physiological function. Dog MRGPRX2 and MRGPRG were distributed in a limited number of tissues, including the skin (from the eyelid, abdomen, and cheek), whereas MRGPRD and MRGPRF were extensively expressed in almost all tissues examined. Histochemical and in situ hybridisation analyses revealed that MRGPRX2 was expressed in dog connective tissue-type mast cells in the skin. Intracellular Ca2+ mobilisation assay revealed that HEK293 cells, expressing dog MRGPRX2 or human MRGPRX2, but not dog MRGPRD, MRGPRF, and MRGPRG, responded to histamine-releasing agents. Our results suggest that dog MRGPRX2 is the functional orthologue of human MRGPRX2 and plays an essential role in drug-induced anaphylactoid reactions in dogs.

Host cell proteins induce inflammation and immunogenicity as adjuvants in an integrated analysis of in vivo and in vitro assay systems.

INTRODUCTION: Host cell proteins (HCPs) are contaminated proteins remaining after purification of biopharmaceuticals. Recent reports revealed clinical implications of HCPs in anti-drug antibody (ADA) development in patients without any inflammatory effects. Therefore, we evaluated the inflammatory effects and immunogenicity of HCPs in an in vivo study by intravitreal administration to rabbits and an in vitro THP-1 cells assay. METHODS: Escherichia coli-derived HCPs at 200 ng/eye with or without ranibizumab at 0.25 mg/eye were administrated intravitreally to rabbits. For in vitro examination, differentiated THP-1 cells were stimulated with HCPs at 0.17 to 10.88 µg/mL with or without ranibizumab at 0.2 mg/mL. RESULTS: Co-administration of HCPs with ranibizumab, but not HCPs alone, induced ocular inflammation. Presence of ADA (anti-ranibizumab) was detected in the vitreous fluid of rabbits in which HCPs and ranibizumab were co-administered. HCPs increased cytokine release and upregulated cell surface markers involved in the antigen presentation in the THP-1 cell assay, which was enhanced by co-stimulation with ranibizumab. DISCUSSION: These finding suggests that HCPs may induce inflammation and immunogenicity as an adjuvant. Furthermore, integrated analyses by an in vivo rabbit model and in vitro assay system using THP-1 cells would be useful to evaluate the immunological risk of HCPs.

Immunostimulatory effects on THP-1 cells by peptide or protein pharmaceuticals associated with injection site reactions.

Injection site reaction (ISR) is a common side-effect associated with the use of peptide or protein pharmaceuticals. These types of pharmaceuticals-induced activation of antigen-presenting cells is assumed to be a key step in the pathogenesis of immune-mediated ISR. The present study was designed to evaluate the immunostimulatory properties of peptide or protein pharmaceuticals using human monocytic THP-1 cells. Here, THP-1 cells, with or without phorbol-12-myristate-13-acetate (PMA) pretreatment, were exposed to enfuvirtide and glatiramer acetate (positive controls) or evolocumab (negative control) for 6 or 24 h. PMA treatment differentiated non-adherent monocytic THP-1 (nTHP-1) cells into adherent macrophagic THP-1 (pTHP-1) cells that highly express CD11b and CD36. Enfuvirtide increased the release of cytokines, e.g. TNFα, MIP-1ß, and MCP-1, and expression of CD86 and CD54 on nTHP-1 cells at 24 h. Similar immunostimulatory properties of glatiramer acetate were observed both in the nTHP-1 and pTHP-1 cells at 6 h, but the responses were very weak in the pTHP-1 cells. Evolocumab did not affect cytokine secretion or cell surface marker expression in either cell type. Taken together, these in vitro THP-1 cell assays revealed the immunostimulatory properties of enfuvirtide and glatiramer acetate. This assay platform thus could serve as a powerful tool in evaluating potential immune-related ISR risks of peptide or protein pharmaceuticals in humans.