Pharmacokinetic studies

Animals: The in-life phase of these studies was performed at Comparative Biosciences, Sunnyvale, California. 7 to 9-wk old Sprague-Dawley rats (males and females) were kept under standard laboratory conditions with free access to food and water. They were allowed to adapt one week before starting the study. The care and use of laboratory animals was in accordance with relevant IACUC-approved animal use protocols.

Administration of encapsulated drug products: A 0.5-mL aliquot of TreXTAM (or TPX6001 alone) in aqueous suspension was prepared by reconstitution of drug products (TPX7001 and/or TPX6001) with distilled water and mixed in appropriate w/v proportions to achieve the targeted dosing. Animals were dosed by oral gavage. Blood samples were collected at fixed times after dosing.

Tissues analysis: 7 to 9-wk old male Sprague-Dawley rats n = 3 per group) were untreated, or treated (oral gavage) with TreXTAM three times per week for four weeks. Four hours after the final dose, gut tissues were taken, frozen at -20 ^oC and stored until used. Tissues were then thawed and homogenized using a glass tube with the pestle insert, in the presence of EDTA-free SIGMAFAST™ Protease Inhibitor Cocktail Tablets (Sigma-Aldrich) used as per manufacturer’s instructions. Levels of TGFβ1 and ATRA in lysates were measured as described above.

Serum analysis: Serum levels of TGFβ1 were measured using an ELISA kit (R&D Systems, Minneapolis, MN; see above) with a slight modification from manufacturer’s instructions. Samples were not acid- activated, minimizing detection of endogenous latent cytokine. For ATRA, a high-performance liquid chromatograph combined with a triple quadrupole mass spectrometer was used as in our previous studies[36].