Pharmacokinetics of Rac inhibitor EHop-016 in mice by ultra-performance liquid chromatography tandem mass spectrometry

Abstract

The Rho GTPase Rac is an important regulator of cancer cell migration and invasion; processes required for metastatic progression. We previously characterized the small molecule EHop-016 as a novel Rac inhibitor in metastatic breast cancer cells and recently found that EHop-016 was effective at reducing tumor growth in nude mice at 25 mg/kg bodyweight (BW). The purpose of this study was to compare the pharmacokinetics and bioavailability of EHop-016 at different dosages in a single dose input scheme (10, 20 and 40 mg/kg BW) following intraperitoneal (IP) and oral gavage (PO) administration to nude mice. We developed and validated a rapid and sensitive method for the quantitation of EHop-016 in mouse plasma by ultra high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC/MS/MS). Separation was carried out on an Agilent Poroshell 120 EC-C18 column (3.0 mm × 50 mm) using organic and aqueous mobile phases. EHop-016 was identified from its accurate mass and retention time from the acquired full-scan chromatogram and quantified by its peak area. The validated method was linear (R(2)>0.995) over the range of 5-1000 ng/mL (1/x(2) weighting). Pharmacokinetic parameters were obtained by non-compartmental analysis using WinNonlin. The area under the curve (AUC₀-∞) ranged from 328 to 1869 ng h/mL and 133-487 ng h/mL for IP and PO dosing, respectively. The elimination half-life (t₁/₂) ranged from 3.8-5.7 h to 3.4-26.8 h for IP and PO dosing, respectively. For both IP and PO administration, the AUC₀-∞values were proportional to the tested doses demonstrating linear PK profiles. The relative bioavailability of EHop-016 after oral gavage administration ranged from 26% to 40%. These results support further preclinical evaluation of EHop-016 as a new anti-cancer therapy.

Keywords: Cancer; EHop-016; Pharmacokinetics; Rac; UPLC/MS/MS.

Figure 1

(A) Full-scan precursor ion mass spectra of EHop-016 (m/z 100-800). (B) Positive ion mass spectra [M+H]⁺, m/z 179.1, 286.1, 316.2, and 344.2 amu (C,D) Typical total ion chromatograms of EHop-016 obtained after extraction of (C) blank mouse plasma and (D) mouse plasma spiked with EHop-016 (5 ng/mL). (E) Total ion chromatograms of EHop-016 (t_R 1.17) and internal standard EHop-0141 (t_R 1.63), with respective structures.

Figure 2

Blood plasma concentration–time profile of EHop-016 in nude mice following (A) intraperitoneal (IP) injection or (B) per oral (PO) administration. (C) Pharmacokinetic behavior of EHop-016 after intraperitoneal (IP) and oral gavage (PO) administration at specified doses. All data are expressed as the mean ± S.E.M. where n = 5 (IP) and n = 4 (PO).