Engineered Hydrophobin as a Crystallization Inhibitor for Flufenamic Acid

Hydrophobins are multifunctional, highly surface-active proteins produced in filamentous fungi. Due to their surface-active properties, resistance to degradation, and potential immunological inertness, hydrophobins have been used in many applications such as protein purification, increasing implant biocompatibility, increasing water solubility of insoluble drugs, and foam stabilizers for food products. To further explore surface-active and self-assembly properties of hydrophobins, we evaluated an engineered, recombinant hydrophobin (class II type 1, HFB1) as a potential crystallization inhibitor for maintaining drug supersaturation for an amorphous drug delivery system. A supersaturation-precipitation method was employed utilizing an ultraviolet (UV) fiber optic system for tracking precipitation kinetics of a model drug, flufenamic acid (FA), that was selected due to its low aqueous solubility in its crystalline form. The effectiveness of HFB1 as a crystallization inhibitor was compared with commonly used pharmaceutical grade polymeric crystallization inhibitors. The following polymers were selected to compare with HFB1: methocel (A4C grade), methocel (K15M grade), Kollidon vinylpyrrolidone-vinyl acetate (VA64), and hydroxypropyl methylcellulose acetate succinate (HPMCAS) (MF grade). The supersaturation-precipitation experiments concluded that HFB1 outperformed all polymers tested in this study and can potentially be used as a crystallization inhibitor at significantly lower concentrations in amorphous drug delivery systems. Dynamic light scattering (DLS) and circular dichroism (CD) results suggest a crystallization inhibition mechanism in which HFB1 functions differently depending on whether flufenamic acid is molecularly dispersed but supersaturated relative to its crystalline solubility or it has exceeded its amorphous solubility limit and exists as a phase-separated drug-rich colloid.