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A series of bidentate hydroxypyridinone iron chelators that have therapeutic potential as oral iron chelators, have been studied systematically to determine which properties are the most critical for the mobilization of hepatocyte iron. The relationship between lipid solubility of the free and complexed forms of each chelator and hepatocyte iron release has been investigated as well as the contribution of the binding constant for iron (III). Hydroxypyridin-4- ones that were approximately equally soluble in lipid and aqueous phases were the most active compounds, the partition coefficient of the free chelator appearing to be more critical in determining iron release than that of the iron-complexed form. Highly hydrophilic chelators did not mobilize intracellular iron pools, whereas highly lipophilic compounds were toxic to hepatocytes. The contribution of the binding constant for iron (III) to cellular iron release was assessed by comparing hydroxypyridin-4-ones (log beta 3 = 36) and hydroxypyridin-2- ones (log beta 3 = 32), which possess similar partition coefficients. The results show that the binding for iron (III) is particularly important at low concentrations of chelator (less than 100 mumol/L) and that at higher concentrations (greater than 500 mumol/L) iron mobilization is limited by the available chelatable pool. Measurement of iron release with other chelators confirms the importance of both the lipid solubilities and iron (III)-binding constants to iron mobilization. The most active hydroxypyridin-4-ones released more hepatocyte iron than did deferoxamine when compared at equimolar concentrations. The results suggest that the ability of an iron chelator to enter the cell is crucial for effective iron mobilization and that once within the cell the binding constant of the chelator for iron (III) becomes a dominant factor.

The rapidity and duration of the response of non-transferrin-bound iron (NTBPI) to chelation therapy are largely unknown and have important implications for the design of optimal chelation regimens. Methodology was developed to measure simultaneously NTBPI, deferoxamine (DFO), and its major metabolite. NTBPI was present in all but 2 of 28 thalassaemia major (TM) patients who had received conventional subcutaneous DFO the previous night, suggesting a short duration of NTBPI clearance by DFO. The detailed kinetics of NTBPI were therefore studied in response to intravenous DFO at 50 mg/kg/27 h for 48 hours and compared in 17 regularly transfused TM and 8 untransfused thalassaemia intermedia (TI) patients to determine the influence of hypertransfusion and iron overload on NTBPI response. Before DFO infusion, NTBPI was present in all patients and was significantly higher in TI (4.52 +/- 0.53 mumol/L) than TM (2.92 +/- 0.03 mumol/L; P = .03). NTBPI values in TM correlated with transferrin saturation (r = .6, P = .03) but not with serum ferritin. Removal of NTBPI by intravenous DFO is in a biphasic manner. The initial rapid rate constant (alpha) was similar in TI (1.5 hour-1) and TM (1.6 hour-1), but the subsequent beta phase was slower (0.04 hour-1) in TI when compared with TM (0.4 hour-1, P = .002). Detectable NTBPI persisted during the beta phase, particularly in TI, despite an excess of plasma DFO also being present (steady state 8 mumol/L). On cessation of DFO infusion, NTBPI reappearance was rapid; the kinetics also being biphasic. The rapid initial rate constant (alpha = 2.5 hour- 1) lasted less than 30 minutes and was approximately equal to the summation of the initial rate constant for removal of DFO (1.8 hour-1) and its major metabolite (0.6 hour-1). This was followed by a slower return to pretreatment levels, usually between 6 and 12 hours, which was faster in TI than in TM. This marked NTBPI lability supports the use of continuous rather than intermittent DFO in high risk patients.

Five orally effective iron chelators of the 3-hydroxypyridin-4-one series have been administered intraperitoneally to iron-overloaded and nonoverloaded male mice at a dose of 200 mg/kg/24 h for a total of 60 days to investigate the effect on iron loading and toxicity. There was a significant reduction in hepatic iron at the end of the study in the iron-overloaded mice with all compounds studied using chemical iron quantitation (P less than .001) and with Perls' stain (P less than .01). Liver iron removal with the hydroxypyridinones ranged from 37% with CP20 to 63% with CP51, compared with 46% removal for desferrioxamine (DFO). There was no significant reduction in splenic or cardiac iron with any chelator. There were no deaths in iron-overloaded animals receiving any of the hydroxypyridin-4-ones, but significantly more deaths in the nonoverloaded groups as a whole (P less than .03). No weight loss was observed with any chelator. Significant reductions in hemoglobin and white cell count were observed with CP20(L1). No histologic abnormalities of kidney, spleen, bone marrow, or stifle joints were observed. Intracytoplasmic inclusion bodies were observed in the centrilobular hepatocytes of animals administered each of the hydroxypyridin-4-ones, while the DFO-treated and control groups showed no such changes.

The relationship between the oral efficacy and the acute toxicity of hydroxypyridin-4-one iron chelators has been investigated to clarify structure-function relationships of these compounds in vivo and to identify compounds with the maximum therapeutic safety margin. By comparing 59Fe excretion following oral or intraperitoneal administration of increasing doses of each chelator to iron-overloaded mice, the most effective compounds have been identified. These have partition coefficients (Kpart) above 0.3 in the iron-free form with a trend of increasing oral efficacy with increasing Kpart values (r = .6). However, this is achieved at a cost of increasing acute toxicity, as shown by a linear correlation between 59Fe excretion increase per unit dose and 1/LD50 (r = .83). A sharp increase in the LD50 values is observed for compounds with Kpart values above 1.0, suggesting that such compounds are unlikely to possess a sufficient therapeutic safety margin. Below a Kpart of 1.0, acute toxicity is relatively independent of lipid solubility. All the compounds are less toxic by the oral route than by the intraperitoneal route, although iron excretion is not significantly different by these two routes. At least five compounds (CP51, CP94, CP93, CP96, and CP21) are more effective orally than the same dose of intraperitoneal desferrioxamine (DFO) (P less than or equal to .02) or orally administered L1(CP20) (P less than or equal to .02).

Currently, the clinical use of 5-aminolaevulinic acid (ALA)-induced protoporphyrin IX (PPIX) for photodynamic therapy (PDT) is limited by the maximum tolerated oral ALA dose (60 mg kg(-1)). This study investigates whether hydroxypyridinone iron-chelating agents can be used to enhance the tissue levels of PPIX, without increasing the administered dose of ALA. Quantitative charge-coupled device (CCD) fluorescence microscopy was employed to study PPIX fluorescence pharmacokinetics in the colon of normal Wistar rats. The iron chelator, CP94, when administered with ALA was found to produce double the PPIX fluorescence in the colonic mucosa, compared with the same dose of ALA given alone and to be more effective than the other iron chelator studied, CP20. Microspectrofluorimetric studies demonstrated that PPIX was the predominant porphyrin species present. PDT studies conducted on the colonic mucosa showed that the simultaneous administration of 100 mg kg(-1) CP94 i.v. and 50 mg kg(-1) ALA i.v. produced an area of necrosis three times larger than similar parameters without the iron-chelating agent with the same light dose. It is possible, therefore, to increase the amount of necrosis produced by ALA-induced PDT substantially, without increasing the administered dose of ALA, through the simultaneous administration of the iron-chelating agent, CP94.