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When cross-linked by heating or by gamma-irradiation and entrapped in cellulose, whey proteins can generate insoluble biofilms with good mechanical properties and high resistance to attack by proteolytic enzymes. Interchain cross-linking of proteins generated an increase in the puncture strength, and a decrease in water vapor permeability. Gelatin was added in film formulation as a stabilizer to improve the puncture strength and film appearance. The structure of the biofilms was also analyzed. SDS-PAGE revealed that heating and gamma-irradiation produce an increase of the molecular weight of the cross-linked protein. Size exclusion chromatography showed a molecular mass of 40 kDa for un-cross-linked whey proteins, whereas for the soluble fractions of the cross-linked proteins, molecular distributions were between 600 and 3800 kDa for the heated proteins and between 1000 and 2000 kDa for gamma-irradiated proteins. No major alteration of the structural conformation of the proteins was observed by FTIR for biofilms obtained after heat treatment, whereas gamma-irradiation induced some modifications in the protein structure. X-ray diffraction analysis suggests that cross-linking by gamma-irradiation seems to modify the conformation of proteins, which became more ordered and more stable.

The kinase-activating mutation G2019S in leucine-rich repeat kinase 2 (LRRK2) is one of the most common genetic causes of Parkinson’s disease (PD) and has spurred development of LRRK2 inhibitors. Preclinical studies have raised concerns about the safety of LRRK2 inhibitors due to histopathological changes in the lungs of nonhuman primates treated with two of these compounds. Here, we investigated whether these lung effects represented on-target pharmacology and whether they were reversible after drug withdrawal in macaques. We also examined whether treatment was associated with pulmonary function deficits. We conducted a 2-week repeat-dose toxicology study in macaques comparing three different LRRK2 inhibitors: GNE-7915 (30 mg/kg, twice daily as a positive control), MLi-2 (15 and 50 mg/kg, once daily), and PFE-360 (3 and 6 mg/kg, once daily). Subsets of animals dosed with GNE-7915 or MLi-2 were evaluated 2 weeks after drug withdrawal for lung function. All compounds induced mild cytoplasmic vacuolation of type II lung pneumocytes without signs of lung degeneration, implicating on-target pharmacology. At low doses of PFE-360 or MLi-2, there was ~50 or 100% LRRK2 inhibition in brain tissue, respectively, but histopathological lung changes were either absent or minimal. The lung effect was reversible after dosing ceased. Lung function tests demonstrated that the histological changes in lung tissue induced by MLi-2 and GNE-7915 did not result in pulmonary deficits. Our results suggest that the observed lung effects in nonhuman primates in response to LRRK2 inhibitors should not preclude clinical testing of these compounds for PD.

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