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Oprozomib, as a second-generation orally bioavailable protease inhibitor (PI), is undergoing clinical evaluation for the treatment of haematological malignancies. In relapsed refractory multiple myeloma (RRMM) patients, oprozomib has shown good efficacy as a single agent or combination therapy. In this experiment, our purpose was to validate a sensitive and rapid method for the determination of oprozomib concentration in rat plasma by ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The samples were treated with acetonitrile as the precipitant and separated by gradient elution using a Waters Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm). Using the selective reaction monitoring (SRM) method, the measurement was finished with the ion transitions of m/z 533.18 ⟶ 199.01 for oprozomib and m/z 493.03 ⟶ 112.03 for tepotinib (internal standard, IS), respectively. Meanwhile, acetonitrile and 0.1% formic acid aqueous solution were used as the mobile phase, and the flow rate was 0.3 mL/min. The lower limit of quantification (LLOQ) of the method was 1.0 ng/mL, and the linear relationship was good in the range of 1.0–100 ng/mL. In addition, the precision of four concentration levels was determined with the values of 3.1–7.3% and the accuracy was from −14.9% to 12.9%. Moreover, the recovery was determined to be from 85.1% to 96.1%, and the values of matrix effect were no more than 110.4%. The optimized UPLC-MS/MS method was also suitable for the pharmacokinetic study of rats after a single oral administration of 21 mg/kg oprozomib.

Angiogenin, a potent angiogenic factor, was cloned and expressed by Escherichia coli and then purified with gel filtration chromatography. Approximately 90% pure angiogenin was obtained to generate a monoclonal antibody. Using western immunoblotting and ELISA, we confirmed that monoclonal antibody C46 secreted from hybridoma cells stably and specifically binds to angiogenin. The fused protein angiogenin-EGF was then expressed in HUVECs, and the subcellular localization of the recombinant protein was determined by confocal microscopy and TEM assay. Recombinant angiogenin was found to mainly concentrate in the pars granulosa of the nucleus, where the protein accumulates to form ribonucleoprotein particles.