The relationship between lipophilicity of hydroxypyrid-4-one iron chelators and cellular iron mobilization, using an hepatocyte culture model

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The N-alkyl-3-hydroxypyridones are a group of compounds with clinical potential as oral iron chelators (Hider et al., 1982). In order to determine the physicochemical properties of iron chelators which are most important for optimizing cellular iron mobilization, an hepatocyte culture system has been used which allows the simultaneous comparison of cellular iron release and toxicity. In this communication, the ability of chelators to mobilize hepatocellular iron has been related to their partition coefficients between aqueous and lipid phases, in both the iron-free and complexed forms. Hepatocytes were isolated by collagenase perfusion of livers from adult male Wistar rats (Berry & Friend, 1969). After differential centrifugation hepatocytes of greater than 95% purity were plated on to collagen-coated sterile dishes by a modification of previously described methods (Page et al . , 1983). After overnight culture at 37C, the non-adherent dead cells were washed free leaving an hepatocyte monolayer of greater than 95% viability. These were pulsed for a variable time with 59 Fe-transferrin before further washes and incubation with the test chelator. Release of 59Fe was then measured over a known time period. The N-alkyl-3-hydroxypyrid-4-one iron chelators coordinate Fe'+ in a 3 : 1 ratio and with a stability constant of lo (Hider et al., 1982), which is five orders of magnitude greater than that of desferrioxamine (Keberle, 1964). By increasing the alkyl function substitution on the ring N atom, a variet of compounds with increasing lipophilicity but similar Fey. stability constants are produced (Table 1). The percentage increase release of 59 Fe above control at 2 h with compounds CP20-CP25 is presented (Table 1). The iron release from hepatocyte monolayers is significantly less with the more hydrophilic compounds CP20 and CP21 than with the more lipophilic compound CP22 ( P < 0.008, Student's t test). This compound which has partition coefficients near to 1 in both the iron-free and complexed forms, appears to be the most active of these chelators in this system. CP23, which has a slightly higher lipophilicity in the free form, thus facilitating its diffusion into the cell, has a relatively low lipid solubility in the iron-complexed form. This may explain the lower iron release seen compared with CP22. Compounds with higher solubility in the lipid than aqueous phase, CP24 and CP24, were less active than CP22, presumably because they partitioned preferentially within the cell membrane. Furthermore, CP25 was toxic at 500 ~ L M as measured by increased lactate dehydrogenase release into incubation medium. CP22 consistently caused more 5'Fe release than desferrioxamine at equimolar concentrations ( P < 0.007) whereas the more hydrophilic CP20 was less active than desferrioxamine. This suggests that both the affinity constant for ferric iron and the partition coefficients of free and complexed forms of chelators may determine net iron mobilization from parenchymal cells. As the majority of radioiron in this model will be in the form of ferritin 15 min after pulsing of the cells (Octave et al . , I983), and as studies in vitro suggest that the hydroxypyrid-Cones mobilize ferritin iron at a greater rate than desferrioxamine (Hider et al., 1982), ferritin iron may represent an important source of chelatable iron for the hydroxypyrid-4-ones which is less available to desferrioxamine. It is concluded that chelators which have approximately equal lipid and aqueous solubilities in both free and complexed forms are likely to be the most effective mobilizers of parenchymal cellular iron, and that the most active hydroxypyrid-Cones mobilize hepatocellular iron at a greater rate than desferioxamine in this system.