Dual-Drug Loaded Separable Microneedles for Efficient Rheumatoid Arthritis Therapy.

Although the inhibitors of the interleukin-6 receptor (IL-6R) and tumor necrosis factor-α (TNF-α) have achieved a certain success in the clinical treatment of rheumatoid arthritis (RA), great effort should be made to overcome side effects and to improve patient compliance. The present research aimed to address these problems by the co-delivery of tocilizumab (TCZ)-an inhibitor of IL-6R-and an aptamer Apt1-67, which specifically inhibits TNF receptor 1 via separable microneedles (MN). MN were featured with a sustained release of TCZ from needle tips and a rapid release of Apt1-67 from needle bodies by using methacrylate groups grafted hyaluronic acid as the fillings of needle tips and polyvinyl alcohol/polyvinyl pyrrolidone as the fillings of needle bodies. Our results demonstrated that TCZ and Apt1-67 were distributed in MN as expected, and they could be released to the surroundings in the skin. In vivo studies revealed that combined medication via MN (TCZ/Apt1-67@MN) was superior to MN loaded with a single drug. Compared with subcutaneous injection, TCZ/Apt1-67@MN was of great advantage in inhibiting bone erosion and alleviating symptoms of CIA mice. This study not only provides a novel approach for combined medication with different release properties but also supplies a strategy for improving drug efficacy.

Novel DEK-Targeting Aptamer Delivered by a Hydrogel Microneedle Attenuates Collagen-Induced Arthritis.

DEK protein is critical to the formation of neutrophil extracellular traps (NETs) in rheumatoid arthritis (RA). Blocking DEK using the aptamer DTA via articular injection has been shown to have robust anti-inflammatory efficacy in a previous study. However, DTA is prone to nuclease degradation and renal clearance in vivo. RA is a systemic disease that involves multiple joints, and local injection is impractical in clinical settings. In this study, DTA was modified with methoxy groups on all deoxyribose sugar units and inverted deoxythymidine on the 3' end (DTA4) to enhance its stability against nuclease. DTA4 is stable for 72 h in 90% mouse serum and maintains a high binding affinity to DEK. DTA4 effectively inhibits the formation of NETs and the migration of HUVECs in vitro. DTA4 was then modified with cholesterol on its 5' end to form DTA6. DTA6 dramatically reduces DEK expression in inflammatory RAW264.7 cells. A hydrogel microneedle (hMN) was then fabricated for the transdermal delivery of DTA6. The hMN maintains morphological integrity after absorbing the aptamer solution, effectively pierces the skin, and rapidly releases DTA6 into the dermis. The DTA6-loaded hMN significantly attenuates inflammation and protects joints from cartilage/bone erosion in collagen-induced arthritis (CIA) mice.

Simultaneous determination of 24 free amino acids in MGC803 cells by hydrophilic interaction liquid chromatography with tandem mass spectrometry.

Amino acids play key roles in cellular protein biosynthesis and energy metabolism pathways. In this study, a simple, rapid and sensitive method was developed for the simultaneous determination of 24 free amino acids in cell samples using hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry (HILIC-MS/MS). Cell samples were deproteinized with methanol/H2O (80:20, v/v) without intricate derivatization process. The analytes were separated on a Waters BEH Amide column (2.1 mm × 100 mm, 1.7 µm), and accomplished within 5 min at a flow rate of 0.2 mL/min. The good linearity was obtained for all analytes (r2 > 0.99) with the limits of quantification from 0.1 to 25 ng/mL. The intra- and inter-day precision ranged from 0.35 to 10.36% and from 2.22 to 9.93%, respectively. The recoveries of most analytes were between 80% and 120% with RSD less than 10.0%. The developed method was then applied to the direct analysis of 24 underivatized amino acids in human gastric cancer cell line MGC803 treated with the antitumor candidate drug J3, and significant differences in the concentration levels of amino acids were also assessed.

Simultaneous determination of the novel anti-tumor candidate drug MDH-7 and 5-fluorouracil in rat plasma by LC-MS/MS: Application to pharmacokinetic interactions.

MDH-7 (2,3,9-tri-O-acetyl-5,6-dideoxy-1,10-di-[N4'-pentoxycarbonyl-5'-fluoro cytosine]-4-ulose 1,4: 7,10-difuranose-4,8-pyranose) is a novel anti-tumor drug candidate. To study the pharmacokinetic interaction between MDH-7 and 5-fluorouracil (5-FU), a sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to simultaneously determine the concentrations of MDH-7 and 5-fluorouracil (5-FU) in rat plasma. Plasma samples were prepared by simple liquid-liquid extraction with ethyl acetate. Chromatographic separation was performed on a Waters XBridge™ C18 column (5 µm, 2.1 mm × 150 mm) with the mobile phase of methanol and H2O (80:20, v/v). The ESI positive and negative ion switch was operated in the multiple reactions monitoring (MRM) mode. The calibration curves showed good linearity (r2 > 0.99) over the ranges of 50-8000 ng/mL for MDH-7 and 10-2000 ng/mL for 5-FU, respectively. The lower limit of quantitations (LLOQs) was 50 ng/mL (MDH-7) and 10 ng/mL (5-FU) with relative standard deviation (RSD) < 13.0%. The proposed method was successfully applied to simultaneous assessment of pharmacokinetic drug-drug interaction between MDH-7 and 5-FU in rats.