Quantitative study of two indirubin derivatives: 6-Bromoindirubin-3′-oxime (6BIO) and 6-Bromoindirubin-3′-[O-(2-piperazine-1-ylethyl) oxime] (6BIO-pip) in mice plasma by LC-MS/MS. Application in a pharmacokinetic study

J Tchoumtchoua; M Halabalaki; E Gikas; L Liu; R Jove; S Nam; AL Skaltsounis

doi:10.1055/s-0034-1394554

Planta Medica, Table of Contents

Quantitative study of two indirubin derivatives: 6-Bromoindirubin-3′-oxime (6BIO) and 6-Bromoindirubin-3′-[O-(2-piperazine-1-ylethyl) oxime] (6BIO-pip) in mice plasma by LC-MS/MS. Application in a pharmacokinetic study

J Tchoumtchoua ¹, M Halabalaki ¹, E Gikas ², L Liu ³, R Jove ⁴, S Nam ³, AL Skaltsounis ¹

¹Division of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, University of Athens, 15771, Athens, Greece
²Division of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Athens, 15771, Athens, Greece
³Department of Molecular Medicine, Beckman Research Institute, City of Hope Comprehensive Cancer Center, Duarte, CA, USA
⁴Vaccine and Gene Therapy Institute of Florida, Port St. Lucie, FL, USA

Abstract

Indirubins represent a group of natural and synthetic products with bio-activities against numerous human cancer cell lines by inhibiting protein kinases. The natural sources of indirubins are plants of Isatis sp., Indigofera sp., and Polygonum sp., recombinant bacteria, mammalian urine and some marine mollusks [1]. Specifically, the halogenated derivative 6-bromo indirubin-3′-oxime (6BIO) possesses an increased selectivity against GSK-3 and 6-Bromoindirubin-3′-[O-(2-piperazine-1-ylethyl) oxime] (6BIO-pip) demonstrates dual JAK/Src inhibitory activity and suppresses tumor cell growth both in vitro and in vivo [2,3]. However, to our knowledge, no analytical method to determine 6BIO and 6BIO-pip in biological fluids has been developed till now. Therefore, rapid, sensitive and high throughput UHPLC-MS/MS methods were developed and validated to evaluate the concentrations of 6BIO and 6BIO-pip in mice plasma. Plasma samples were pre-treated by protein precipation using cold mixture of methanol: acetonitrile (9: 1, v/v) and separations were carried out on a Hypersil Gold C18 column (50 × 2.1 mm i.d.; 1.9 µm p.s.) using 0.1% acetic acid and methanol as mobile phase at a flow rate of 500 mL/min in a gradient mode. For quantitation, a hybrid LTQ-Orbitrap MS equipped with an electro-spray ionization source was used applying a selected reaction monitoring (SRM) option. The monitored transitions were m/z 354→324 in negative mode for 6BIO and m/z 468→356 in positive mode for 6BIO-pip. Following the guidelines of EMA, the assay methods were fully validated in terms of selectivity, linearity, recovery, matrix effect, accuracy, precision and stability. The validated methods were successfully applied to the pharmacokinetic studies of 6BIO and 6BIO-pip following their oral administration to mice at the dose of 50 mg/kg. The results indicated that the higher solubility of 6BIO-pip provided it a higher plasma bioavailability and stability as compared to 6BIO.

Keywords: Indirubins; LC-MS/MS; Pharmacokinetic study; Quantitation.

References:

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