Therapeutic potential of crystal-engineered berberine cocrystals ameliorates PCOS phenotypes via AMPK-IRS1/PI3K/Akt...

Polycystic ovary syndrome (PCOS) is a complex endocrine-metabolic disorder characterized by hyperandrogenism, chronic anovulation, and insulin resistance, with hyperinsulinemia playing a pivotal role in exacerbating ovarian dysfunction. Although berberine (BBR) exhibits insulin-sensitizing properties, its therapeutic potential is limited by poor oral bioavailability. Our previous study developed BBR cocrystals with gallic and gentisic acid, achieving a 1.8-fold increase in C_max to overcome bioavailability issues. The present study evaluated berberine-gallic acid (BBR-GAL) and berberine-gentisic acid (BBR-GEN) using an integrated in silico and in vivo approach in a letrozole-induced PCOS mouse model. Molecular docking simulations suggested stronger predicted binding of BBR relative to gallic acid and gentisic acid across all receptor systems, supporting its role as the dominant binder. Further, the coformers exhibited weaker but spatially proximal interactions, indicating potential indirect effects on the local binding environment. In vivo, letrozole administration induced hallmark PCOS features, including weight gain, impaired glucose tolerance, hyperandrogenism, ovarian inflammation, and disruption of hepatic insulin signaling. Treatment with BBR-GAL and BBR-GEN significantly improved metabolic parameters and restored insulin responsiveness. Notably, treatment with cocrystals was associated with increased adenosine monophosphate-activated protein kinase activation and reduced insulin receptor substrate-1 Ser307 phosphorylation, leading to enhanced phosphatidylinositol 3-kinase/protein kinase B signaling. Furthermore, treatment attenuated ovarian inflammation by downregulating interleukin 1-beta, interleukin 6, and tumor necrosis factor-alpha, and normalized luteinizing hormone receptor and androgen receptor expression. Collectively, these findings support the further investigation of BBR-GAL and BBR-GEN as multitarget agents that integrate insulin sensitization, antiinflammatory activity, and hormonal regulation for the management of PCOS. SIGNIFICANCE STATEMENT: This study demonstrates that crystal-engineered BBR cocrystals show improved efficacy against metabolic and reproductive dysfunction in an experimental model of PCOS. By restoring hepatic insulin signaling through modulation of the adenosine monophosphate-activated protein kinase-insulin receptor substrate-1/phosphatidylinositol 3-kinase/protein kinase B axis and attenuating ovarian inflammation, these formulations provide mechanistic validation for improved therapeutic efficacy. These findings indicate that crystal-engineered BBR could be a useful approach in improving the endocrine-metabolic phenotypes of preclinical PCOS.