ABSTRACT Background Effective and safe skin penetration enhancers are highly valuable for improving the efficacy of topical drug and cosmetic formulations. Objective This study aimed to develop and validate novel synergistic combinations of tetrahydropiperine (THP), 2,3‐butanediol (2,3‐BD), and hydrolyzed glycosaminoglycans (hGAGs) as effective skin penetration enhancers. Methods Penetration efficacy was evaluated using a tiered approach: (1) an in vivo zebrafish embryo model with fluorescein sodium (FNa) to screen individual ingredients and their combinations; (2) a zebrafish melanin inhibition assay to measure the enhancement of 4‐butylresorcinol (577) efficacy by lead combinations; and (3) an ex vivo pigskin Franz cell model analyzed by HPLC to quantitatively assess the transdermal delivery of 577. Results Zebrafish embryo FNa penetration assay showed that individual 2% 2,3‐BD had no significant effect on FNa penetration. In contrast, THP (0.01%, 0.03%, 1%) and hGAGs (0.5%, 1.5%, 3%) significantly enhanced FNa penetration by 1.15–1.69‐fold and 5.18–9.14‐fold, respectively (all p < 0.001). Orthogonal experiments identified synergistic combinations: Comb‐F (0.03% THP + 2% 2,3‐BD + 3% hGAGs) and Comb‐G (0.1% THP + 2% 2,3‐BD + 0.5% hGAGs), Comb‐H (0.1% THP + 2% 2,3‐BD + 1.5% hGAGs), Comb‐I (0.1% THP + 2% 2,3‐BD + 3% hGAGs). These combinations achieved FNa penetration enhancements by 18.61, 12.55, 25.56, and 36.83‐fold, respectively, significantly exceeding theoretical additive effects. Control combinations (Comb‐J: 0.1% capsaicin + 2% 2,3‐BD + 0.5% hGAGs; Comb‐K: 0.1% THP + 2% 2,3‐BD + 0.5% hyaluronic acid) lacked synergism, confirming THP/2,3‐BD/hGAGs as a superior SPE trio. Zebrafish melanin assay demonstrated that Comb‐G significantly boosted the melanin inhibition efficacy of 577 by 1.97‐fold. Pigskin‐Franz cell assay confirmed that Comb‐G enhanced 577's delivery by increasing cumulative penetration (4–24 h post‐treatment) by ≥ 22% and improving penetration coefficient from 41.26% (577 alone) to 44.98%. Conclusion We successfully developed and validated a synergistic combination of THP, 2,3‐butanediol, and hGAGs as a potent and effective skin penetration enhancer. This study validates a tiered evaluation pipeline (zebrafish → pigskin) for efficient SPE development and identifies a potent enhancer with direct application potential in topical formulations.
Wang et al. (Mon,) studied this question.