Elsevier

Biochemical Pharmacology

Volume 65, Issue 3, 1 February 2003, Pages 349-360
Biochemical Pharmacology

Lipophilicity of analogs of pyridoxal isonicotinoyl hydrazone (PIH) determines the efflux of iron complexes and toxicity in K562 cells

https://doi.org/10.1016/S0006-2952(02)01551-4Get rights and content

Abstract

Iron overload secondary to β-thalassemia and other iron-loading anemias is the most serious obstacle to be overcome in the treatment of these diseases, since there is no physiological mechanism for excretion of the excess iron acquired by chronic blood transfusion. Due to the inconvenience and cost of the current iron chelation therapy, the search for an orally available iron chelator is ongoing. Pyridoxal isonicotinoyl hydrazone (PIH) and many of its analogs are effective at mobilizing iron in vivo and in vitro at doses that are not toxic. PIH analogs were approximately equally effective at binding 59Fe within K562 cells; their efficacy depended upon the kinetics of release of the iron–chelator complex from the cell, which was correlated inversely with the lipophilicity of the chelators. Addition of BSA, which has a well-characterized affinity for lipophilic species, to the extracellular medium enhanced iron–chelator efflux, such that all analogs caused 59Fe release from the cells as quickly as it was chelated; this suggests that BSA acts as an extracellular sink for the iron–chelator complexes, many of which are highly lipophilic. The toxicity of the free chelators varied over two orders of magnitude, and was correlated with the amount of intracellular 59Fe–chelator complexes, implicating the complexes in the mechanism of toxicity of the chelators. Understanding the structural features that determine the efficacy and toxicity of iron chelators in biological systems is of value in the selection of PIH analogs for in vivo examination.

Introduction

The importance of iron in biological systems is due mainly to its roles in oxygen distribution and electron transfer. Iron in excess of the capacity of the organism to use or store it is toxic, presumably via formation of reactive oxygen species, including the extremely reactive hydroxyl radical [1], which cause oxidative stress. To prevent uncontrolled redox reactions, and to conserve a poorly bioavailable metal, iron metabolism in mammals is tightly regulated, and involves efficient recycling [2]. Because there is no physiological mechanism of iron excretion, patients receiving chronic blood transfusions develop iron overload, the current treatment for which is desferrioxamine, a drug that must be administered via subcutaneous infusion [3] due to its very short plasma half-time [4].

An orally effective iron chelator is urgently needed as a convenient and inexpensive alternative to desferrioxamine therapy. The efficacy of PIH (Fig. 1) in iron mobilization has been characterized in vitro[5] and in vivo[6]. Screens of PIH analogs using 59Fe-labeled cell culture models have identified several chelators in this series that are more active than PIH [7], [8], [9], including pyridoxal para-methoxybenzoyl hydrazone, pyridoxal meta-chlorobenzoyl hydrazone (m-ClPBH), and pyridoxal meta-fluorobenzoyl hydrazone (m-FPBH). These analogs also mobilized more 59Fe in rats than PIH, whether administered intraperitoneally or orally [10]. A recent study has demonstrated that the property defining the capacity of PIH analogs to mobilize 59Fe from reticulocytes is the rate of efflux of the iron–chelator complexes from the cells [11], presumably via passive diffusion. It is of interest whether these chelators mobilize 59Fe similarly from other cell lines, which, as compared with reticulocytes, may be expected to have different intracellular pathways of iron trafficking.

Several studies have demonstrated the antiproliferative effects of some PIH analogs in cell culture models [8], [12], [13], [14], [15]. It has been demonstrated that 59Fe mobilization and inhibition of DNA synthesis were not correlated [16], suggesting that iron depletion from cells, which is expected to limit the iron available to ribonucleotide reductase, is insufficient to account for the antiproliferative effects of these chelators. Hence, it is of value to examine other mechanisms by which the toxicity of PIH analogs may be mediated.

Section snippets

Synthesis of PIH analogs

Pyridoxal hydrochloride was purchased from Sigma. Salicylaldehyde and 2-hydroxy-1-naphthylaldehyde were purchased from Aldrich. Isonicotinic acid hydrazide and benzoyl hydrazide were purchased from Lancaster. The halogenated acid benzoyl hydrazides used for preparation of the hydrazones were purchased from Transworld Chemicals. PIH and its analogs were synthesized according to standard methods as previously described [17]. All other materials were of the highest quality available.

Preparation of chelator and Fe(chelator)2 solutions

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59Fe mobilization from K562 cells

K562 cells were labeled with 59Fe by incubation for 3 hr with 59Fe2–transferrin as described in Section 2. After this period of incubation, 59Fe is distributed between ferritin, the major iron storage protein, and the poorly characterized LIP [15]. The steps involved in 59Fe mobilization, defined in Fig. 2, were examined separately after the incubation of cells with the chelators by measuring the radioactivity in the incubation medium and in an ethanol-soluble cytoplasmic fraction, which

59Fe mobilization in K562 cells by chelators

PIH analogs were effective in mobilizing 59Fe from K562 cells, many of which were more effective than PIH itself (Fig. 6). At the concentrations used in this study, all chelators bound 59Fe with similar kinetics (Fig. 3, Fig. 4), but the kinetics of 59Fe release from the cells varied markedly. The maximum amount of 59Fe mobilized from K562 cells by 100 μM PIH analogs depended primarily upon the kinetics of release of 59Fe–chelator complexes from the cells (Fig. 3, Fig. 4, Fig. 6), which is

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