Determination of a new oral iron chelator, ICL670, and its iron complex in plasma by high-performance liquid chromatography and ultraviolet detection

Abstract

ICL670 is a representative of a new class of orally active tridentate selective iron chelators. Two molecules of ICL670 are required to form a complete hexacoordinate chelate Fe–[ICL670]₂ with one ferric iron. A simple and rapid HPLC–UV method for the separate determination of ICL670 and Fe–[ICL670]₂ in the plasma of iron-overloaded patients is described. Plasma samples were prepared as rapidly as possible, the tubes being kept at 4°C. Plasma proteins were precipitated with methanol. The supernatant was diluted with water and placed on the refrigerated sample rack of an autosampler before injection. The chromatographic separations were achieved on an Alltima C₁₈ column using 0.05 M Na₂HPO₄ and 0.01 M tetrabutylammonium hydrogen sulfate–acetonitrile–methanol (41:9:50, v/v/v) as mobile phase. The analytes were detected at 295 nm. Calibration and quality control samples were prepared in normal human plasma. The mean accuracy (n=6) over the entire investigated concentration range 0.25–20 μg/ml ranged from 91 to 109% with a coefficient of variation (C.V.) from 4 to 8% for ICL670, and from 95 to 105% with a C.V. from 2 to 20% for the iron complex. The dissociation of the complex during analysis was shown to be marginal. The iron removal from plasma of iron-overloaded patients by free ICL670 during analysis was low. The in vitro iron transfer from the iron pools of iron-overloaded plasma onto ICL670 was shown to be a slow process.

Introduction

Iron accumulates in the body as a result of repeated transfusions, e.g. in β-thalassemia major, or due to excessive dietary iron uptake in anemias and hereditary hemochromatosis. Man is unable to actively eliminate iron from the body once it has been acquired. Iron chelation therapy has been shown to reduce iron-related morbidity and to improve quality of life in patients with β-thalassemia. Desferal^® (desferrioxamine) is currently the only widely adopted drug available to mobilize iron deposits. But the poor oral bioavailability and the short plasma half-life of desferrioxamine necessitate its application as slow subcutaneous or intravenous infusions, thereby limiting the acceptance of long-term therapy by patients. Difficulties in separating the pharmacological effect from toxic effects have hampered for a long time the development of iron chelators which can be given orally. Currently, deferiprone (L1; a bidentate ligand) is the orally active iron chelator with the broadest clinical experience. Treatment with deferiprone may involve some complications and requires close monitoring [1]. ICL670 is a representative of a new class of orally active tridentate selective iron chelators. Two molecules of ICL670 are required to form a complete hexacoordinate chelate Fe–[ICL670]₂ with one ferric iron (Fig. 1).

In order to establish relationships between the pharmacological effect of an iron chelator and its plasma kinetics, the separate determination of the ligand and its iron complex in plasma is required. Many attempts to determine separately the free ligand and the iron complex in plasma have been made with desferrioxamine. Most of the described methods involve high-pressure liquid chromatography (HPLC). One of the major difficulties encountered was due to the high chelating property of desferrioxamine which binds labile iron from the HPLC devices. In order to avoid conversion of the ligand to the iron complex during chromatography, the proposed methods involve either the use of iron-free HPLC devices [2], or addition of a chelating agent to the mobile phase such as nitrilotriacetic acid [3] or ethylenediaminetetraacetic acid (EDTA) [4], [5]. The addition of radioactive iron has been used in another HPLC method [6] for conversion of unbound drug and metabolites to radio-iron bound species in order to overcome stability problems in frozen samples. Dual detection (UV–Vis absorption and radioactive substance measurement) was applied.

With deferiprone, the encountered difficulties in setting up an HPLC assay were peak tailing, conversion of the ligand to the complex and dissociation of the complex during chromatography. Goddard et al. [7] proposed an HPLC method based on protein precipitation and reversed-phase ion-pair chromatography at pH 2.2. Under these conditions, total deferiprone (sum of the ligand and the complex) is determined since the iron complex is dissociated and determined as deferiprone. Epemolu et al. [8] proposed another HPLC method involving liquid–liquid extraction into dichloromethane, chromatography of the ligand on a PGC Hypercarb column using a pH 3 mobile phase containing EDTA, and chromatography of the complex in a separate run at pH 7.

The chromatographic behavior of an iron chelator and its chelate is dependent on its binding capacity. ICL670 has a lower binding affinity for iron than desferrioxamine but binds about one thousand-fold more tightly than deferiprone. The pM-value at pH 7.4 for desferrioxamine, ICL670 and deferiprone is 26.6, 22.5 and 19.5, respectively. Considering these differences in iron-binding affinity and the chromatographic behavior previously observed for desferrioxamine and deferiprone, an HPLC assay has been developed for the determination of ICL670 and Fe–[ICL670]₂ in plasma. This method is described herein.