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.
Iron is involved in essential biochemical reactions ranging from respiration to DNA synthesis. Consequently, iron deprivation has been proposed as a strategy for inhibition of tumor cell growth. We recently described a novel iron chelator, tachypyridine [N,N',N"-tris(2-pyridylmethyl)-cis,cis-1,3,5-triaminocyclohexane], and demonstrated that it not only inhibited growth of cultured tumor cells, but was actively cytotoxic. Here we explore the mechanisms underlying tachpyridine cytotoxicity. Using several criteria, including time-lapse video microscopy, DNA staining and TUNEL assays, tachpyridine was shown to specifically induce apoptotic cell death. Further, unlike numerous cytotoxic chemotherapeutic drugs which induce apoptosis by activating p53-dependent pathways, tachpyridine-mediated cell death did not require p53 activation. Although immunoblotting revealed rapid accumulation of p53 following treatment with tachpyridine, p21(WAF1) was not induced. Further, neither cytotoxicity nor apoptosis required p53. p53 null human lung cancer H1299 cells transfected with an ecdysone-inducible p53 exhibited equivalent sensitivity to tachpyridine in the presence and absence of p53, demonstrating the lack of requirement for p53 in an isogenic cell system. Further, time-lapse video microscopy and TUNEL assays demonstrated that both p53 null and p53 wild-type cells underwent apoptotic cell death in response to tachpyridine. In addition, in 55 human cancer cell lines the mean GI(50) of tachpyridine in cells with mutant p53 was virtually identical to the GI(50) in cells with wild-type p53. These results demonstrate that tachpyridine initiates an apoptotic mode of cell death that does not require functional p53. Since over 50% of human tumors contain a functionally defective p53 that reduces sensitivity to commonly used chemotherapeutic agents, such as etoposide and cisplatin, the ability of tachpyridine to induce apoptosis independently of p53 may offer an advantage in anti-tumor therapy.
In order to define a predictive animal model for the effects of hydroxypyridinone (HPO) iron chelators in humans, we have compared the 28 d oral efficacy and toxicology of the HPO, 1,2-diethyl-3-hydroxypyridin-4-one (CP94) in rats and guinea-pigs and related the results to the contrasting metabolism of this compound in the two species. CP94 was highly effective at mobilizing liver iron in rats but showed toxicity at higher doses, whereas in the guinea-pig the compound lacked toxicity but was ineffective at mobilizing liver iron. These differences can be explained by the contrasting metabolism of the drug between the two species. In rats, at the top dose of 300 mg/kg intragastrically, all animals died before the end of the study, with no deaths or weight loss at lower doses. At 100 mg/kg, rat liver non-haem iron concentrations were reduced by 53% and 44% in females and males respectively (P < 0.001). At this dose, adrenal medullary cell vacuolation, increased mammary secretory activity, vacuolation of corpora luteal cells and single cell hepatocyte necrosis were seen. There were no reductions in the white cell count. At 50 mg/kg rat liver non-haem iron concentrations were decreased by 50% and 34% in females and males respectively (P < 0.02). In female rats this was associated with increased mammary secretory activity. In iron-overloaded rats given 100 mg/kg by gavage for 28 d, liver non-haem iron concentration was reduced by 39% (P < 0.01) and serum ferritin by 71% (P < 0.001). Ovarian and mammary changes were not influenced by iron loading. In guinea-pigs, CP94 was evaluated at 50 mg/kg, 100 mg/kg or 200 mg/kg by oral insufflation for 28 d. No reduction in liver iron was seen and no systematic dose related histological, biochemical or haematological effects were observed. Whereas in guinea-pigs 99% of urinary recovery following an oral dose of CP94 (100 mg/kg) was as the inactive glucuronide metabolite, in the rat only 23% of the dose was excreted in the urine as the glucuronide with remainder as the free drug or an iron binding metabolite. The lack of both efficacy and toxicity in the guinea-pig may therefore be explained by the rapid inactivation of CP94 by glucuronidation. This metabolism of CP94 in the guinea-pig is closer to humans than the rat, suggesting that both the efficacy and toxicity of this compound in humans may also be limited by glucuronidation.
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.
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