Protein aggregation is a major problem of therapeutic proteins because aggregation decreases their therapeutic activity and shelf life and induces immunogenicity. Stabilization against aggregation is commonly attained by addition of different excipients like sugars, surfactants, buffers, salts, amino acids, polymers, etc. Generally these excipients are required in combination for stabilization. Sugars are required at a higher concentration, and commonly used surfactants like polysorbates have shortcomings due to oxidative degradation. With a view to have a multipurpose excipient to be effective at a lower concentration, we designed antiaggregation agents (AAAs) that would encompass the functionalities of two or more conventional excipients and would curtail the number of excipients to be added for stabilization. Our first designed AAA, trehalose monooleate (TMO), is a sugar-fatty acid derivative. It has been evaluated in silico by docking on aggregation prone regions of model protein bovine serum albumin (BSA), and experimentally its effectiveness has been validated as stabilizer against agitation and thermal stress. TMO has a lower CMC of 6 mg/L, is nonhemolytic, and was found to be nontoxic by sulforhodamine B (SRB) colorimetric assay in Human Hepatoma Cell Line (Hep-G2) using adriamycin as positive contol. Various spectroscopic and separation analytical techniques were employed to monitor the aggregation profile of BSA in presence and absence of TMO. CD spectroscopy showed complete retention of helical structure at concentration as low as 0.05% of TMO, while fluorescence spectroscopy provided vital insights into conformational stability rendered by TMO. Native-PAGE and SEC-HPLC studies demonstrated absence of aggregates. Molecular dynamics study on BSA-TMO docked complex further substantiated the stabilization effect. Overall, it can be said that TMO has good antiaggregation property. The present work is a preliminary attempt toward understanding protein excipient interactions and chemistry to provide rational basis for designing a single excipient for stabilization of protein formulations.

Background:Overactivation of aldose reductase (AR) enzyme has been implicated in the development of various diabetic complications. In the present study, the inhibitory effect of thymol was investigated on AR enzyme and its anti-cataract activity was also examined on isolated goat lens.Materials and Methods:Various concentrations of thymol were incubated with AR enzyme prepared from isolated goat lens. Molecular docking studies were carried out using Schrodinger software to verify the binding of thymol with AR as well as to understand their binding pattern. Further, thymol was evaluated for its anti-cataract activity in high-glucose-induced cataract in isolated goat lens in vitro. Quercetin was maintained as standard (positive control) throughout the study.Results:Thymol showed potent inhibitory activity against goat lens AR enzyme with an IC50 value of 0.65 μg/ml. Docking studies revealed that thymol binds with AR in similar binding pattern as that of quercetin. The high–glucose-induced cataract in isolated goat lens was also improved by thymol treatment. Thymol was also able to significantly (P < 0.001) reduce the oxidative stress associated with cataract.Conclusion:The results suggest that thymol may be a potential therapeutic approach in the prevention of diabetic complications through its AR inhibitory and antioxidant activities.

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