Hydroxychloroquine (HCQ) is being tested in a number of human clinical trials to determine the role of autophagy in response to standard anticancer therapies. However, preclinical studies in mouse models are equivocal with regard to HCQ exposure (pharmacokinetics) and inhibition of autophagy (pharmacodynamics) in various tissues. Understanding HCQ pharmacokinetics (PK) and pharmacodynamics (PD) in mice allows for a comparison to human PK as reported in recent trials and as such the subsequent PD associated with autophagy inhibition in tissues. In this study, female BALB/c mice were treated with a single intraperitoneal dose of 20, 40, or 80 mg/kg HCQ and tissues and whole blood collected at 3, 6, 12, 24, 48 and 72 hours. Levels of HCQ and desethylhydroxychloroquine (dHCQ), an active metabolite, in whole blood and tissues were determined via a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. Non-compartmental analysis (NCA) measured exposure, as determined by area under the drug concentration versus time curve (AUC0-inf), to both HCQ and dHCQ was dose proportional in whole blood, liver and brain. The maximum concentration (Cmax) of HCQ was 1.3, 3.1 and 4.7 μg/mL and 20.6, 41.7 and 114.7 μg/mL in whole blood and liver, respectively, following 20, 40 and 80 mg/kg. Brain exposure was far less with Cmax achieving 272, 717 and 2220 ng/mL. In general, the time of maximal concentration (Tmax) of HCQ and dHCQ was observed at 3 hr or 6 hr, regardless of dose and location measured. PD assessment for autophagy inhibition was determined by western blotting. The LC3 protein, which is involved in membrane dynamics during autophagy, was visualized and the ratio of LC3-II to tubulin was measured. In the liver, autophagy was inhibited approximately two-fold following all doses at 24 and 48 hr compared to controls. Autophagy was inhibited in both the gut and kidney at 24 and 48 hr at all doses. In the brain, autophagy inhibition was not dose related and may be a result of low HCQ levels. These results demonstrate that a dose of 20 mg/kg is sufficient to inhibit autophagy in most tissues investigated; however, while PK parameters appear to be dose proportional, increased tissue drug levels does not necessarily equate to increased PD effects/autophagy inhibition. In addition, the data establishes a dose of 40 mg/kg in mice results in Cmax and AUC0-inf of HCQ equivalent to that observed in human clinical trials targeting autophagy modulation following a dose of 600 mg daily. Studies are ongoing to assess this relationship in solid tumors.

Citation Format: Kristen M. Jackson, Ryan J. Hansen, Daniel L. Gustafson. Pharmacokinetic and pharmacodynamic assessment of autophagy inhibition following hydroxychloroquine in mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2084.