Rescuing a Troubled Tolcapone with PEGylated PLGA Nanoparticles: Design, Characterization, and Hepatotoxicity Evaluation.

Tolcapone is an orally active catechol-O-methyltransferase (COMT) inhibitor used as adjuvant therapy in Parkinson's disease. However, it has a highly hepatotoxic profile, as recognized by the U.S. Food and Drug Administration. As a possible solution, nanoscience brought us several tools in the development of new functional nanomaterials with tunable physicochemical properties, which can be part of a solution to solve several drawbacks, including drug's short half-life and toxicity. This work aims to use PEGylated poly(lactic-co-glycolic acid) (PLGA) nanoparticles as a stable carrier with lower hydrodynamic size and polydispersity to encapsulate tolcapone in order to overcome its therapeutic drawbacks. Using the nanoprecipitation method, tolcapone-loaded nanoparticles with a DLC% of 5.7% were obtained (EE% of 47.0%) and subjected to a lyophilization optimization process to obtain a final shelf-stable formulation. Six different cryoprotectants in concentrations up to 10% (w/v) were tested. A formulation of PLGA nanoparticles with 3% hydroxypropyl-ß-cyclodextrin (HPßCD) as a cryoprotectant (PLGA-HP@Tolc), presenting sub-200 nm sizes and low polydispersity (PdI < 0.200) was selected. Cytotoxicity assays, namely, MTT and SRB, were used to study the metabolic activity and cell density of tolcapone and PLGA-HP@Tolc-treated cells. In both assays, a hepatocarcinoma cell line (HepG2) growing in glucose or glucose-free media (galactose-supplemented medium) was used. The results demonstrated that the treatment with the PLGA-HP@Tolc formulation led to a decrease in cytotoxicity in comparison to free tolcapone-treated cells in both media tested. Moreover, the elected formulation also counteracted ATP-depletion and excessive ROS production induced by tolcapone. The results suggest that HPßCD might have a dual function in the formulation: cryoprotectant and anticytotoxic agent, protecting cells from tolcapone-induced damage. Using an in vitro COMT inhibition assay, the PLGA-HP@Tolc formulation demonstrated to inhibit COMT as efficiently as free tolcapone. Overall, the results suggest that tolcapone-loaded PLGA NPs could be an interesting alternative to free tolcapone, demonstrating the same in vitro efficacy in inhibiting COMT but with a safer cytotoxic profile.

Tolcapone, a potent aggregation inhibitor for the treatment of familial leptomeningeal amyloidosis.

Hereditary transthyretin amyloidosis (ATTR) is a disease characterized by the extracellular deposition of transthyretin (TTR) amyloid fibrils. Highly destabilizing TTR mutations cause leptomeningeal amyloidosis, a rare, but fatal, disorder in which TTR aggregates in the brain. The disease remains intractable, since liver transplantation, the reference therapy for systemic ATTR, does not stop mutant TTR production in the brain. In addition, despite current pharmacological strategies have shown to be effective against in vivo TTR aggregation by stabilizing the tetramer native structure and precluding its dissociation, they display low brain permeability. Recently, we have repurposed tolcapone as a molecule to treat systemic ATTR. Crystal structures and biophysical analysis converge to demonstrate that tolcapone binds with high affinity and specificity to three unstable leptomeningeal TTR variants, stabilizing them and, consequently, inhibiting their aggregation. Because tolcapone is an FDA-approved drug that crosses the blood-brain barrier, our results suggest that it can translate into a first disease-modifying therapy for leptomeningeal amyloidosis. DATABASES: PDB codes for A25T-TTR, V30G-TTR, and Y114C-TTR bound to tolcapone are 6TXV, 6TXW, and 6XTK, respectively.

Repurposing Benzbromarone for Familial Amyloid Polyneuropathy: A New Transthyretin Tetramer Stabilizer.

Transthyretin (TTR) is a homotetrameric protein involved in human amyloidosis, including familial amyloid polyneuropathy (FAP). Discovering small-molecule stabilizers of the TTR tetramer is a therapeutic strategy for these diseases. Tafamidis, the only approved drug for FAP treatment, is not effective for all patients. Herein, we discovered that benzbromarone (BBM), a uricosuric drug, is an effective TTR stabilizer and inhibitor against TTR amyloid fibril formation. BBM rendered TTR more resistant to urea denaturation, similarly to iododiflunisal (IDIF), a very potent TTR stabilizer. BBM competes with thyroxine for binding in the TTR central channel, with an IC50 similar to IDIF and tafamidis. Results obtained by isothermal titration calorimetry (ITC) demonstrated that BBM binds TTR with an affinity similar to IDIF, tolcapone and tafamidis, confirming BBM as a potent binder of TTR. The crystal structure of the BBM-TTR complex shows two molecules binding deeply in the thyroxine binding channel, forming strong intermonomer hydrogen bonds and increasing the stability of the TTR tetramer. Finally, kinetic analysis of the ability of BBM to inhibit TTR fibrillogenesis at acidic pH and comparison with other stabilizers revealed that benzbromarone is a potent inhibitor of TTR amyloidogenesis, adding a new interesting scaffold for drug design of TTR stabilizers.