Pharmacokinetics and pharmacodynamics of AZD6244 (ARRY-142886) in tumor-bearing nude mice

Abstract

Purpose: AZD6244 (ARRY-142886) (AstraZeneca, Macclesfield, UK) is a novel small molecule MEK1/2 inhibitor that is currently being tested in Phase II trials. With the recent publication of human pharmacokinetic data from clinical studies, we now know the achievable levels and range of AZD6244 exposure in humans. This study aimed to describe the pharmacokinetic profile of AZD6244 in mice in order to design preclinical studies that recapitulate exposure levels in humans.

Methods: Male athymic, nude mice received subcutaneous inoculation of A375 human melanoma cells. Once tumors reached 400-700 mm(3), mice were given a single dose of either 5 or 10 mg/kg AZD6244 via oral gavage. Additionally, a subset of mice was dosed once daily for 1 week (10 mg/kg). Mice were killed and plasma and tissues were collected at various time points after the last dose. Samples were analyzed by LC/MS/MS for AZD6244 concentration. Additionally, pharmacodynamic endpoints such as tumor proliferation and ERK phosphorylation were analyzed at various time points after the last dose.

Results: After either a single dose or at steady state, at clinically equivalent exposures, AZD6244 effectively inhibits ERK phosphorylation and suppresses proliferation. Furthermore, we describe a hysteretic relationship between the pharmacokinetics and the pharmacodynamics of AZD6244 and both target and pharmacologic responses.

Conclusions: The information presented herein will drive the rational design of pre-clinical studies that are not only relevant to the clinical setting, but also pave the way to understand the biological response to AZD6244 treatment.

Fig. 1

AZD6244 chemical structure and single dose plasma pharmacokinetics. a AZD6244 structure. b, c Plasma concentration versus time curves of AZD6244 and the metabolite, AZD6244-N-desmethyl. Tumor-bearing mice were administered a single oral dose of AZD6244 at either b 5 or c 10 mg/kg (mean, SD, n = 4)

Fig. 2

Single dose tissue pharmacokinetics. AZD6244 tissue concentration versus time in a liver, b gut, c lung, d muscle and e skin. Tumor-bearing mice were administered a single oral dose of AZD6244 at either 5 or 10 mg/kg (mean, SD, n = 4)

Fig. 3

Daily dose pharmacokinetics and tissue distribution. AZD6244 and AZD6244-N-desmetnyl concentration versus time in a plasma, b liver, c gut, d muscle. Tumor-bearing mice were administered a once daily oral dose of 10 mg/kg AZD6244 daily for 7 days. Samples were taken at the indicated time after the last dose (mean, SD, n = 4)

Fig. 4

Comparison of the plasma concentration versus time curves of AZD6244 and AZD6244-N-desmethyl for the 10 mg/kg single dose and 10 mg/kg daily dose regimens. SD single dose, SS steady state (mean, SD, n = 4)

Fig. 5

Time profile comparison of inhibition of tumor ERK phosphorylation and tumor AZD6244 concentration. Mice were given a single dose of a 5 mg/kg, b 10 mg/kg, or c 10 mg/kg daily for 7 days. Tumor samples were taken at the indicated time after the last dose. Tumors were lysed and equal amounts of total protein were resolved by SDS-PAGE. Membranes were probed with ERK and phospho-ERK antibodies. Ratios of the pixel intensity of pERK:ERK were analyzed for % inhibition = 100 × [1 – (treated/control)] (mean, SD, n = 4). One-way ANOVA p values comparing each time point to control tumor as follows: *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 6

Time profile comparison of tumor proliferation as measured by BrdU incorporation and tumor AZD6244 concentration. Mice were given a a single dose of 5 mg/kg, b a single dose of 10 mg/kg or c 10 mg/kg daily for 7 days. Tumor samples were taken at the indicated time after the last dose, fixed in formalin and mounted on glass slides. Immunohistochemistry for BrdU was performed and blinded slides were scored (mean, SD, n = 3, One-way ANOVA p values comparing each time point to control tumor as follows: *p < 0.05; **p < 0.01; ***p < 0.001). d Representative images of tumor sections from vehicle- and AZD6244-treated mice

Fig. 7

AZD6244 shows hysteretic PK/PD relationships. a % inhibition of tumor ERK phosphorylation versus plasma (AZD6244) ng/mL; b % inhibition of tumor ERK phosphorylation versus tumor (AZD6244) ng/mL; c % inhibition of tumor proliferation versus tumor (AZD6244) ng/g; and d schematic representation of AZD6244 PK/PD relationships to cell signaling and proliferation (mean, SEM, n = 3)