Twelve-month efficacy and safety of omidenepag isopropyl, a selective EP2 agonist, in open-angle glaucoma and ocular hypertension: the RENGE study.

PURPOSE: To assess the long-term safety and efficacy of omidenepag isopropyl (OMDI) 0.002% (a first-in-class, selective, non-prostaglandin, prostanoid EP2 receptor agonist), alone or administered concomitantly with timolol 0.5%, in patients with open-angle glaucoma (OAG, including normal-tension and exfoliation glaucoma) or ocular hypertension (OHT). STUDY DESIGN: Open-label, multicenter, Phase 3 study (NCT02822729). METHODS: Patients aged ≥ 20 years, with OAG or OHT, and a baseline diurnal intraocular pressure (IOP) ≥ 16- < 22 mmHg (Group 1) or ≥ 22- ≤ 34 mmHg (Groups 2 and 3) were enrolled. All patients (N = 125) received OMDI 0.002% once daily. Group 3 also received timolol 0.5% twice daily. IOP was measured at baseline and at Weeks 2, 4, 8, 12, 26, 40, and 52. RESULTS: Significant reductions in mean diurnal IOP from baseline occurred at every visit (P < 0.0001). Mean ± SE diurnal IOP reduction at Week 52 was -3.7 ± 0.3 mmHg (Group 1), -5.6 ± 0.5 mmHg (Group 2), and -8.4 ± 0.6 mmHg (Group 3). Most adverse events (AEs) were mild, and no serious treatment-related AEs were reported. Conjunctival hyperemia (incidence: monotherapy [Groups 1 and 2], 18.8%; concomitant [Group 3], 45.0%) and macular edema (ME)/cystoid macular edema (CME) (incidence: monotherapy, 11.8%; concomitant, 15.0%) occurred most frequently. All treatment-related ME/CME cases occurred in pseudophakic eyes and responded to standard-of-care treatment and study drug discontinuation. CONCLUSIONS: In this study, OMDI 0.002%, alone or administered concomitantly with timolol 0.5%, resulted in sustained IOP reduction over 52 weeks in patients with OAG or OHT. Concomitant treatment resulted in increased efficacy and increased incidence of conjunctival hyperemia.

Effects of the Selective EP2 Receptor Agonist Omidenepag on Adipocyte Differentiation in 3T3-L1 Cells.

Purpose: We aimed at comparing the effects of omidenepag (OMD) with those of prostaglandin F (FP) receptor agonists (FP agonists) on adipogenesis in mouse 3T3-L1 cells. Methods: To evaluate the agonistic activities of OMD against the mouse EP2 (mEP2) receptor, we determined cAMP contents in mEP2 receptor-expressing CHO cells by using radioimmunoassays. Overall, 3T3-L1 cells were cultured in differentiation medium for 10 days and adipocyte differentiation was assessed according to Oil Red O-stained cell areas. Changes in expression levels of the adipogenic transcription factors Pparg, Cebpa, and Cebpb were determined by using real-time polymerase chain reaction (PCR). OMD at 0.1, 1, 10, and 40 µmol/L, latanoprost free acid (LAT-A) at 0.1 µmol/L, or prostaglandin F2α (PGF2α), at 0.1 µmol/L were added to cell culture media during adipogenesis. Oil Red O-stained areas and expression patterns of transcription factor targets of OMD or FP agonists were compared with those of untreated controls. Results: The 50% effective concentration (EC50) of OMD against the mEP2 receptor was 3.9 nmol/L. Accumulations of Oil Red O-stained lipid droplets were observed inside control cells on day 10. LAT-A and PGF2α significantly inhibited the accumulation of lipid droplets; however, OMD had no effect on this process even at concentrations up to 40 µmol/L. LAT-A and PGF2α significantly suppressed Pparg, Cebpa, and Cebpb gene expression levels during adipocyte differentiation. Conversely, OMD had no obvious effects on the expression levels of these genes. Conclusions: A selective EP2 receptor agonist, OMD, did not affect the adipocyte differentiation in 3T3-L1 cells, whereas FP agonists significantly inhibited this process.

ARG098, a novel anti-human Fas antibody, suppresses synovial hyperplasia and prevents cartilage destruction in a severe combined immunodeficient-HuRAg mouse model.

BACKGROUND: The anti-human Fas/APO-1/CD95 (Fas) mouse/human chimeric monoclonal IgM antibody ARG098 (ARG098) targets the human Fas molecule. The cytotoxic effects of ARG098 on cells isolated from RA patients, on normal cells in vitro, and on RA synovial tissue and cartilage in vivo using implanted rheumatoid tissues in an SCID mouse model (SCID-HuRAg) were investigated to examine the potential of ARG098 as a therapy for RA. METHODS: ARG098 binding to each cell was analyzed by cytometry. The effects of ARG098 on several cells were assessed by a cell viability assay in vitro. Effects on the RA synovium, lymphocytes, and cartilage were assessed in vivo using the SCID-HuRAg mouse model. RESULTS: ARG098 bound to cell surface Fas molecules, and induced apoptosis in Fas-expressing RA synoviocytes and infiltrating lymphocytes in the RA synovium in a dose-dependent manner. However, ARG098 did not affect the cell viability of peripheral blood mononuclear cells of RA patients or normal chondrocytes. ARG098 also induced apoptosis in RA synoviocytes and infiltrating lymphocytes in the RA synovium in vivo. The destruction of cartilage due to synovial invasion was inhibited by ARG098 injection in the modified SCID-HuRAg mouse model. CONCLUSIONS: ARG098 treatment suppressed RA synovial hyperplasia through the induction of apoptosis and prevented cartilage destruction in vivo. These results suggest that ARG098 might become a new therapy for RA.

Cytosolic targeting of macromolecules using a pH-dependent fusogenic peptide in combination with cationic liposomes.

pH-Sensitive peptides and polymers have been employed as additives to enhance the cytosolic delivery of drugs and genes by facilitating their endosomal escape. However, little attention has been paid to the intracellular fate of these peptides and polymers. In this study, we explored the possibility of utilizing GALA, a pH-sensitive fusogenic peptide, as a cytosol-targeting vehicle. In combination with cationic liposomes, Lipofectamine 2000 (LF2000), the feasibility of this approach for the cytosolic targeting of proteins and nanoparticles was exemplified through the delivery of avidin (68 kDa) and streptavidin-coated quantum dots (15-20 nm) in serum-containing medium. The use of cationic liposomes is critical to enhance the cell-surface adhesion of the GALA conjugates and eventual endosomal uptake. Circular dichroism studies suggest that the GALA can be liberated from cationic liposomes at a reducing pH to form a helical structure and this may eventually lead to disruption of the endosomal membrane to achieve an efficient leakage of the GALA conjugates into the cytosol.

Interaction of arginine-rich peptides with membrane-associated proteoglycans is crucial for induction of actin organization and macropinocytosis.

Arginine-rich peptides, including octaarginine (R8), HIV-1 Tat, and branched-chain arginine-rich peptides, belong to one of the major classes of cell-permeable peptides which deliver various proteins and macromolecules to cells. The importance of the endocytic pathways has recently been demonstrated in the cellular uptake of these peptides. We have previously shown that macropinocytosis is one of the major pathways for cellular uptake and that organization of the F-actin accompanies this process. In this study, using proteoglycan-deficient CHO cells, we have demonstrated that the membrane-associated proteoglycans are indispensable for the induction of the actin organization and the macropinocytic uptake of the arginine-rich peptides. We have also demonstrated that the cellular uptake of the Tat peptide is highly dependent on heparan sulfate proteoglycan (HSPG), whereas the R8 peptide uptake is less dependent on HSPG. This suggests that the structure of the peptides may determine the specificity for HSPG, and that HSPG is not the sole receptor for macropinocytosis. Comparison of the HSPG specificity of the branched-chain arginine-rich peptides in cellular uptake has suggested that the charge density of the peptides may determine the specificity. The activation of the Rac protein and organization of the actin were observed within a few minutes after the peptide treatment. These data strongly suggest the possibility that the interaction of the arginine-rich peptides with the membrane-associated proteoglycans quickly activates the intracellular signals and induces actin organization and macropinocytotis.

Cellular uptake of arginine-rich peptides: roles for macropinocytosis and actin rearrangement.

The use of membrane-permeable peptides as carrier vectors for the intracellular delivery of various proteins and macromolecules for modifying cellular function is well documented. Arginine-rich peptides, including those derived from human immunodeficiency virus 1 Tat protein, are among the representative classes of these vectors. The internalization mechanism of these vector peptides and their protein conjugates was previously regarded as separate from endocytosis, but more recent reevaluations have concluded that endocytosis is involved in their internalization. In this report, we show that the uptake of octa-arginine (R8) peptide by HeLa cells was significantly suppressed by the macropinocytosis inhibitor ethylisopropylamiloride (EIPA) and the F-actin polymerization inhibitor cytochalasin D, suggesting a role for macropinocytosis in the uptake of the peptide. In agreement with this we observed that treatment of the cells with R8 peptide induced significant rearrangement of the actin cytoskeleton. The internalization efficiency and contribution of macropinocytosis were also observed to have a dependency on the chain length of the oligoarginine peptides. Uptake of penetratin, another representative peptide carrier, was less sensitive to EIPA and penetratin did not have such distinct effects on actin localization. The above observations suggest that penetratin and R8 peptides have distinct internalization mechanisms.