Deciphering in vitro-in vivo correlation of single-dose injectable sustained-release tofacitinib-loaded microspheres for long-acting anti-inflammatory therapy of rheumatoid arthritis.

Tofacitinib is a potent Janus kinase inhibitor for the treatment of rheumatoid arthritis (RA); however, its clinical application is limited by inherent side effects, a short half-life, and the requirement for frequent dosing. In this study, poly(lactic-co-glycolic acid) (PLGA)-based injectable sustained-release tofacitinib microspheres with distinct in vitro release profiles (High, Medium, and Low) were fabricated via a microreactor-assisted technique. The microspheres were systematically characterized regarding their morphology, particle size, drug loading, encapsulation efficiency, and in vitro drug release. Cytotoxicity and anti-inflammatory activity were evaluated in RAW264.7 macrophage cells. Pharmacokinetic (PK) studies were performed in rats following intramuscular administration, and a preliminary in vitro-in vivo correlation (IVIVC) was established. The therapeutic efficacy of the microspheres was assessed using an adjuvant-induced arthritis (AIA) rat model. The prepared microspheres exhibited a uniform morphology with a mean particle size range of approximately 30-40 μm, encapsulation efficiency exceeding 80%, and sustained in vitro drug release lasting for more than 28 days. In vitro experiments demonstrated that the sustained-release formulations significantly reduced the secretion of interleukin-1β, interleukin-6, and tumor necrosis factor-α in RAW264.7 cells. Notably, the Low-release tofacitinib microspheres (Tofacitinib-Microspheres-Low) showed a more pronounced inhibitory effect at 72 h compared to 24 h (p < 0.001). PK studies revealed that the microspheres significantly prolonged systemic exposure to tofacitinib, with a markedly extended apparent half-life and increased area under the concentration-time curve relative to the conventional tofacitinib suspension. The IVIVC analysis has preliminarily established the predictability of in vivo drug behavior, with Level A IVIVC achieved for both the High- and Low-release formulations. In the AIA rat model, treatment with tofacitinib-loaded microspheres significantly reduced clinical arthritis scores, paw swelling, and inflammatory progression compared to the model group (p < 0.05), while maintaining normal hematological and biochemical parameters. In conclusion, high-molecular-weight 85/15 PLGA (90 kDa) is the optimal polymer grade for lab-scale single-dose injectable tofacitinib microspheres, enabling 42-day long-term anti-RA efficacy with mitigated acute peak drug-related toxicities. This microreactor-produced sustained-release injectable depot provides a mechanistic strategy to reduce administration frequency and improve safety of tofacitinib for RA management.