The enantioselective binding of three proton pump inhibitors (PPIs)—omeprazole, rabeprazole, and lansoprazole—to two key plasma proteins, α1-acid glycoprotein (AGP) and human serum albumin (HSA), was characterized. The interactions between PPI enantiomers and proteins were investigated using a multifaceted analytical approach, including high-performance liquid chromatography (HPLC), fluorescence and UV spectroscopy, as well as in silico molecular docking. HPLC analysis demonstrated that all three PPIs exhibited enantioseparation on an AGP-based chiral stationary phase, suggesting stereoselective binding to AGP, while only lansoprazole showed enantioselective binding on the HSA-based column. Quantitatively, the S-enantiomers of omeprazole and rabeprazole showed higher binding affinity to AGP, while the R-enantiomer of lansoprazole displayed greater affinity for AGP, with a reversal in the elution order observed between the two protein-based columns. Protein binding percentages, calculated via HPLC, were greater than 88% for each enantiomer across both transport proteins, with all enantiomers displaying higher affinity for AGP compared to HSA. Thermodynamic analysis indicated that on the HSA, the more common, enthalpy-controlled enantioseparation was found, while in contrast, on the AGP, entropy-controlled enantioseparation was observed. The study also identified limitations in using fluorescence titration due to the high native fluorescence of the compounds, whereas UV titration was effective for both proteins. The determined logK values were in the range of 4.47–4.83 for AGP and 4.02–4.66 for HSA. Molecular docking supported the experimental findings by revealing the atomic interactions driving the binding process, with the predicted enantiomer elution orders aligning with experimental data. The comprehensive use of these analytical methods provides detailed insights into the enantioselective binding properties of PPIs, contributing to the understanding of their pharmacokinetic differences and aiding in the development of more effective therapeutic strategies.

The interactions between human α₁-acid glycoprotein (AGP) and the enantiomers of apremilast (APR) were investigated by using a combination of HPLC, isothermal titration calorimetry (ITC), ultracentrifugation, and molecular docking. HPLC analyses on a chiral AGP column revealed enantioselective binding, with S-APR showing slightly higher stability for AGP compared with R-APR. However, the stability difference is sufficient for the baseline separation of APR enantiomers. ITC experiments confirmed the spontaneous and exothermic nature of the binding, with similarly high binding constants (log K > 5) for both enantiomers but a more exothermic enthalpy for the S-form. Ultracentrifugation studies further supported the high stability of the AGP–APR complexes, with the S-enantiomer again demonstrating marginally stronger binding. Molecular docking revealed that S-APR formed more direct interactions, including additional hydrogen bonds and π–π interactions with AGP compared to R-APR, corroborating the experimental findings. This enhanced interaction also contributes to the chiral separation. Comparison of the protein binding data suggests that AGP plays a significant role in APR transport. Altogether, the multianalytical approach provided detailed insight into the enantioselective binding of APR to AGP, which may contribute to understanding its pharmacokinetic behavior and therapeutic action.

Curcumin and natural curcuminoids possess numerous beneficial properties, including antioxidant, antitumor, and anti-inflammatory effects. However, their application is challenging because of instability and rapid metabolism. Cyclic C₅-curcuminoids, a subgroup of curcuminoids, are chemically more stable and less susceptible to hydrolysis or metabolism. In our study, seven synthetic compounds—five cyclic C₅-curcuminoids (compounds 8, 9, 11, 12, and 13) and two cyclic chalcones (compounds 4 and 5)—were examined using an in vitro 6-hydroxydopamine-induced neuronal damage model established with all-trans retinoic acid-differentiated SH-SY5Y and BV-2 microglial cells. The antioxidant, anti-apoptotic (including ferroptosis markers (iron, GSH/GSSG, and malondialdehyde)), apoptotic (PARP, cytochrome c, caspase 9, active caspase-3, and oligonucleosome release), and anti-inflammatory effects of these compounds were also investigated. Compounds 5, 9, 12, and 13 significantly decreased reactive oxygen species production (percentage changes: 5: 28.9%; 9: 32%; 12: 29.8%; 13: 49.2%) and increased antioxidant capacity (percentage changes: 5: 63.4%; 9: 85.8%; 12: 31.2%; 13: 85.9%) and antioxidant enzyme activity (catalase, superoxide dismutase, and glutathione peroxidase) in SH-SY5Y cells. Compounds 5, 9, and 12 decreased microglial activation by downregulating Iba1 expression (fold changes: 5: 0.73; 9: 0.39; 12: 0.33) and decreasing glutamate concentration (percentage changes: 5: 26.5%; 9: 41.2%; 12: 27.3%). Together with compound 13, they reduced pro-inflammatory cytokine (IL-6, TNFα) secretion and induced anti-inflammatory cytokine (IL-10) release in SH-SY5Y and BV-2 cells. Based on these results, the blood–brain barrier penetrations of compounds 5, 9, 12, and 13 were also investigated. The highest blood–brain barrier penetration was observed for compound 12 (P_app value 13.6 × 10⁻⁶ cm/s). Synthetic cyclic C₅-curcuminoids derived from curcumin overcome the limitations of stability and bioavailability and exhibit strong antioxidant, anti-inflammatory, and anti-apoptotic effects, making them promising candidates for treating neurodegenerative diseases.