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College of Pharmacy (R.A.Y., K.A.M., T.L.S.), Graduate Center for Toxicology (R.A.Y., A.M.F.), Department of Veterinary Science (C.B.H.), and Division of Laboratory Animal Resources (K.M.D.), University of Kentucky
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Abstract |
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Abstract
Introduction
Results
Discussion
References
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The objectives of the present study were to determine the efficacy and toxicity of repeated oral administration of 3-hydroxypyridin-4-one (HP) chelators in a rabbit model of aluminum (Al) accumulation and toxicity, and the influence of chelator lipophilicity on these effects. Efficacy was assessed as chelator-induced Al mobilization and excretion and reversal of Al accumulation and Al-induced toxicity. Chelator-induced toxicity was assessed by multiple measures. Six HPs were given orally 12 times over 1 month to Al-loaded rabbits, which had significant elevation of Al in most tissues and evidence of Al-induced nephrotoxicity, osteomalacia, and anemia. Intravenous desferrioxamine (DFO), the current chelator of choice for the treatment of Al-overload and toxicity, was included as a positive control.
All six HPs and DFO demonstrated efficacy evidenced by significantly greater urinary and biliary Al elimination after the twelfth dose than seen in saline-treated controls. All of the HPs were more effective than DFO. Chelator-induced urinary Al excretion accounted for 58-98% of total (urinary plus biliary) Al excretion. Chelator-facilitated Al excretion was nearly complete within 12 hr, demonstrating a fairly short duration of action in rabbits with intact renal function.
HP treatments did not consistently affect tissue concentrations of Al or other metals. However, there was a trend toward chelator-induced reduction of Al-induced nephrotoxicity. The influence of HP lipophilicity was limited to a positive correlation between HP · Al lipophilicity and biliary Al output and a negative correlation between HP and HP · Al lipophilicity and reduction of Kupffer cell Al.
Little toxicity was evident after repeated oral HP dosing. Adrenal weight increased after treatment with several HPs. There was a decrease in testes weight after several HPs, which is consistent with an antiproliferative effect. More frequent dosing and/or a longer duration of HP treatment might produce greater reversal of the Al-induced toxicity and perhaps reveal more adverse effects than seen in this study.
There was a lack of profound toxicity during this short-term study. The 1,2-dimethyl (CP20) and 1,2-diethyl (CP94) HPs, which have been the most extensively studied HPs, were the least effective of the HPs examined. These results encourage the further investigation of other HPs as oral alternatives to DFO for the treatment of Al accumulation and toxicity.
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Introduction |
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Abstract
Introduction
Results
Discussion
References
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Al1 is a toxic, nonessential metal. The accumulation of Al is most pronounced in the renally impaired human, but it can occur in those exposed to Al from occupational and numerous iatrogenic sources. Its accumulation can result in a microcytic, hypochromic anemia (1); osteodystrophy (2); encephalopathy (3); joint disease (4); and muscle weakness (5).
DFO is the current drug of choice to treat Al accumulation disorders. However, it is not orally effective; it is expensive; and it can produce ocular toxicities, including cataracts (6), auditory toxicities (7), and neurotoxicities (8). It can also promote infections from iron-dependent organisms (9). These limitations have encouraged the search for a safer, less costly, orally effective alternative.
HPs are under investigation as alternatives to DFO. To be clinically useful, oral Al chelators must reduce Al body burden and have minimal or no toxicity. For the HPs to be effective chelators, they and their metal complexes should be lipophilic enough to cross membranes, yet hydrophilic enough to be excreted by the kidneys (10, 11). The Do/a for these properties was suggested to be between 0.2 and 1.0 (12). Most attention has been devoted to CP20 (also known as L1 or deferiprone). Oral CP20 has been shown to increase urinary Al output in Al-loaded mice and rats (13, 14), and to increase serum and dialysate Al concentration in renal dialysis patients (15). CP20 has also been shown to increase urinary Al output and decrease liver Al burden in Al-loaded uremic rats; however, the CP20 dose studied (188 mg/kg) produced high mortality (16).
HPs with a wide range of lipophilicities (<0.001-2.1) are orally absorbed by the rabbit (17). These same HPs have been shown to increase urinary and biliary Al output in the Al-loaded rabbit after iv injection (18). The present study was conducted to assess the influence of lipophilicity on the ability of repeated oral doses of the HPs to increase Al excretion, to decrease Al body burden and to reverse Al-induced toxicity, and to evaluate HP toxicity.
Materials and Methods
Chelators. The six HPs shown in table 1 were selected for study because they include two with a Do/a < 0.2 and two with a Do/a > 1.0. HPs were synthesized as hydrochloride salts, as described (19). Their purity was >99.5%, confirmed by HPLC, NMR, and elemental analysis. DFO was a gift of Ciba-Geigy (Basel, Switzerland). Solutions of HPs and DFO were prepared in saline immediately before dosing.
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Subjects. Male New Zealand white rabbits, initially weighing 2.4 ± 0.2 kg (mean ± SD), were maintained in a AAALAC-accredited facility. They were weighed 3 times weekly. Food and water were provided ad libitum, except for food deprivation from 24 hr before to 4 hr after chelator or saline treatment. They were fitted with an Elizabethan collar for 24 hr before dosing to prevent coprophagy, further ensuring minimal stomach contents. To achieve Al loading, rabbits (N = 66) were given sterile iv injections of 0.1 mmol Al/kg as the lactate 5 times weekly for 4 weeks. Control rabbits (N = 8) were given sterile Na lactate (0.3 mmol/kg) intravenously on the same schedule.
Treatment Schedules and Sample Collections. Beginning 7 or 10 days after the last Na or Al lactate injection, Na lactate-injected rabbits received 0.9% normal saline and the Al lactate-loaded rabbits received saline, DFO, or one of the HPs by an 8 French pediatric feeding tube, with the exception of DFO, which was given intravenously through a sterilizing (0.22 µm) filter. These treatments were administered 3 times weekly for 4 weeks, in a volume of 1 ml/kg.
An ophthalmoscopic examination was performed weekly to determine the presence of cataracts. Blood samples were drawn for biochemistry assays (multichemical diagnostic panel) and hematological (CBC with differential) evaluation, which were conducted by Roche Bioveterinary Services, (Columbus, Ohio). These samples (4-5 ml) were obtained from an auricular artery before the first Al or Na lactate injection, before the first and seventh treatments, and 24 hr after the twelfth treatment. Blood was collected in a syringe containing 0.2 ml Na4EDTA (100 mg/ml), from which plasma was obtained. At these same time points, with the exception of the last sample that was collected before the twelfth treatment, a 1-ml blood sample was drawn into a syringe containing 0.1 ml heparin (1000 units/ml) for quantitation of Al in plasma. Eight and 2 days before the twelfth treatment, the rabbits' bones were calcein-labeled for histomorphometric analysis. Calcein (30 mg/kg in saline at pH 7.3) was given intravenously over 30 min. One day before the twelfth treatment, a femoral vein and the bile duct were surgically cannulated to enable periodic blood sampling and quantitative bile collection, as described (18). An iv infusion of 2.5% dextrose in 1/2 normal saline (25 ml/hr) was maintained from the time of surgery until euthanasia. Two hours before the twelfth treatment, the rabbit was placed in a Nalgene rabbit restraining cage in which it was housed for 26 hr to enable repeated sample collection. Urine, bile, and blood were collected from 2 hr before to 24 hr after treatment, as previously described (18), with the exception that bile and urine were collected from 4 to 5 and from 5 to 6 hr after treatment. Bile and urine were collected to determine the time course and extent of chelator-facilitated Al excretion. Blood was collected to determine if the HPs chelated vascular or extravascular Al. An aliquot of each bile, urine, and serum sample was stored frozen for Al analysis. Three hours after the twelfth treatment, rabbits were orally administered a slurry containing 10 g homogenized cabbage in 30 ml of bile salt substitute (18, 20) to replace bile salts lost due to bile collection and to encourage bile production. The rabbit was euthanatized 24 hr after the twelfth treatment, ~40 days after the last Al or Na lactate injection. Evaluation of gross pathology was performed immediately. The organs, tissues, and fluids listed in table 2 were harvested. A sample of each tissue other than the tibia was preserved in 10% neutral-buffered formalin for evaluation of histological changes. One tibia was saved in 100% ethanol for histomorphometry. An additional sample of selected tissues (table 2) was stored frozen for multielemental analysis. GFAP was measured in three brain regions; these results will be reported elsewhere.2
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Al and Multielemental Analysis. Al analysis was conducted by ETAAS. Aliquots of serum, plasma, and bile were diluted at least 10-fold (with a solution of 2 mM Mg in 0.2% HNO3) to bring the Al concentration within the range of the Al standards. They were analyzed by ETAAS by comparison with Al standards in the same matrix. Samples of CSF were compared with Al standards prepared in an artificial CSF (21). An aliquot of each urine sample was acid-digested in 0.5 ml HNO3:H2O2 (70:30) solution in a threaded Teflon vial (22), dried, reconstituted with the aforementioned Mg in HNO3 solution, and analyzed compared with aqueous Al standards prepared in the same solution. Approximately 250 mg of each tissue selected for multielemental analysis was dried to constant weight, acid-digested as described for urine, and diluted with distilled/deionized (milli-Q) water to a final volume of 10 ml. An aliquot of the reconstituted tissue samples underwent multielemental (Al, Ca, Cu, Fe, Mg, Ni, and Zn) analysis. This was conducted using an ARL Direct Current Plasma Emission Spectrometer. Ni concentrations in the samples were too low to be quantitated by this method. Al was below the limit of detection in brain and CSF, and occasionally in other tissues, particularly those from Na lactate-injected rabbits. Therefore, a second aliquot of each sample was diluted with the Mg in HNO3 solution and was analyzed for Al by ETAAS by comparison with aqueous standards prepared in the same solution. Standard curves were run before and after every 10 samples. An aliquot of each sample was analyzed twice by ETAAS. The sample was further analyzed when the difference between the two results (absorbance × sec) was >20%. These analyses were conducted on a Perkin-Elmer 4100ZL atomic absorption spectrophotometer.
Histological Assessment of Soft Tissue. Selected tissues were sectioned 5-µm thick and stained with hematoxylin and eosin for evaluation under light microscopy. Grayish-blue-stained material was interpreted as a tissue Al deposit (23). Staining with solachrome azurine (24) and laser microprobe MS (25) were used to verify that the grayish-blue-stained material was indeed Al.
Bone Histomorphometric Analysis. Bone histomorphometric analysis was performed on samples of tibia from the Al-CP20, Al-CP94, Al-DFO, Al lactate-saline, and Na lactate-saline groups. These groups were chosen for initial evaluation, because they have been the most extensively studied HPs (CP20 and CP94), the current chelator of choice (DFO), and the controls. Bone samples were fixed in 100% ethanol, dehydrated, and embedded in methylmethacrylate as previously described (26). Histomorphometry was conducted at a standard site, 1.5 mm below the growth plate. Six serial sections, 4 and 7 µm thick, were cut with a Microm, model HM360 microtome (Carl Zeiss, Thornwood, NY). The 4-µm-thick sections were stained with a modified Masson-Goldner Trichome stain (26). The 7-µm-thick unstained sections were prepared for fluorescent light microscopy for analysis of calcein labeling.
Fifteen static and dynamic parameters of bone structure, bone formation, and resorption in cancellous bone were measured using the Osteoplan system II (Kontron, Munich, Germany, NY). A minimum of 50 optical fields was evaluated at a magnification of 200×. All parameters comply with the nomenclature and were calculated according to the Histomorphometry Nomenclature Committee of the American Society of Bone and Mineral Research (27).Data Analysis. The volume of each bile and urine sample was determined from the weight of the total sample divided by the density of an aliquot. Biliary and urinary Al outputs during each sample collection period were calculated from Al concentration × sample volume. These were divided by the time period of collection to obtain Al output rates, which were then normalized to body weight. Chelator efficiency was calculated as total Al output (cumulative urinary and biliary Al excretion over 24-hr posttreatment) in moles of Al/mol of chelator/kg body weight, as described (28, 29), minus the response to saline treatment of Al-loaded rabbits. The result was multiplied by 100%.
Microscopic evaluation of renal histology revealed microaneurysms, focal sclerosis, and Al deposition in some glomerular tufts. These changes were recorded as absent or present. Interstitial fibrosis/cystic renal tubule was observed in some rabbits and was noted as absent (0), or present and ranked on a scale of 1-3. Al presence in macrophages in stained sections of liver (Kupffer cells), bone marrow, and spleen was ranked as absent (0) or as 1-3 (for extent of deposition).Statistics. Statistically significant chelator-induced differences in bile, urine, and total Al output; organ weights (normalized to body weight); and bone histomorphometric parameters were assessed with one-way ANOVAs. Significance was accepted at the p < 0.05 level for all statistical comparisons in this study, corrected for the number of tests conducted. A p < 0.0055 or 0.0033 was accepted for the comparisons among the 9 organs or the 15 histomorphometric measurements, respectively. Duncan's test was conducted if the ANOVA was significant.
To test for statistically significant effects of Al loading on body weight and on hematology and blood biochemistry measures, two-way mixed ANOVAs were conducted to compare values obtained before, to those obtained after, Na lactate or Al lactate injections. To test for statistically significant effects of the chelators, two-way mixed ANOVAs were conducted to determine differences in body weight and hematology and blood biochemistry measures by comparing values obtained before, to those obtained after, the chelator treatments. To test for statistically significant effects of the chelators on Al and five essential metals, differences in tissue concentrations were examined using mixed ANOVAs across the nine tissues. Significance was accepted at p < 0.0002 for the 25 biochemistry assays and hematological evaluations and <0.0009 for comparisons of the six elements in each of nine tissues. A multiple comparison test was conducted when the ANOVA was significant. Results of the microscopic evaluation that were noted as absent or present were statistically compared by Fisher's exact tests of the Al-saline group vs. the Na lactate-saline group and each of the seven Al-chelator groups. Results of the microscopic evaluation that were ranked from 0 to 3 were statistically compared with sign rank tests. Significance for these tests was accepted at p < 0.017 for the three Fisher's exact tests and p < 0.0125 for the four sign rank tests. Significant relationships between lipophilicity of the HPs, and the HP · Al complexes, and the ability of the HPs to facilitate Al excretion (biliary, urinary, total Al output, and biliary Al output as a percentage of total output) were determined by correlation analyses. Similar analyses were conducted with the extent of microaneurysm, focal sclerosis, and interstitial fibrosis/cystic renal tubule in the kidney and Al in the glomerular tufts and macrophages of the bone marrow, liver, and spleen. Analyses were conducted between the Do/a, log Do/a, Do/a1/2, and Do/a2 vs. mean Al output after each HP, to test for linear and nonlinear relationships. The significance of each correlation was determined by a t test. The accepted p was <0.0015 for Al output vs. lipophilicity and <0.0009 for the morphometric changes and subcellular Al localization. The p values reported are uncorrected. Interpretation of significance is based on the Bonferroni-corrected p values, previously described, when correction for multiple comparisons was appropriate. Due to the conservative nature of this statistical approach, we have also reported values that are not significant by this criteria, but would be in the absence of the Bonferroni correction.| |
Results |
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Abstract
Introduction
Results
Discussion
References
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Al Effects. The Al injections produced Al accumulation that persisted for ~40 days and some toxicity. There was significantly more Al in all tissues [F(72,482) = 10.92, p < 0.0000], except brain and CSF, of Al-loaded rabbits. Tissue Al concentrations are shown in fig. 1. Urinary Al excretion by Al-loaded rabbits averaged 0.8 µmol/kg in the 24 hr after the twelfth saline injection (given ~5 weeks after completion of Al loading), whereas Na lactate-injected rabbits did not excrete measurable amounts of Al in their urine. Cu was significantly decreased [F(5,54) = 9.99, p < 0.0001] in the adrenal, liver, and renal medulla of Al-loaded rabbits. Al loading did not result in any significant changes in Fe levels.
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2.5% of total bone
surface in Al-loaded rabbits, a nonsignificant increase above
non-Al-loaded rabbits.
There were no significant effects of Al loading on plasma biochemistry
values. Hemoglobin was lower in Al-loaded rabbits [F(1,89) = 4.67, p < 0.033].
Chelator Effects. Nine Al-loaded rabbits died during treatment (N = 1 during saline and CP40, N = 2 during CP20, and N = 5 during CP24). Although none of the deaths could be directly attributed to the treatments, some of the CP24-treated rabbits demonstrated poor health, very severe bronchopneumonia and a diffuse subcapsular hepatocytic necrosis involving the entire liver, and hyperactivity that might have contributed to their death. In contrast to all other chelator-treated rabbits, CP24-treated rabbits lost weight during treatment.
Each of the chelators significantly increased total Al elimination, when compared with saline treatment. Urinary [F(7,48) = 12.37, p < 0.0001], biliary [F(8,52) = 7.35, p < 0.0001], and total Al outputs [F(7,47) = 16.39, p < 0.0001] during the 24 hr after each of the treatments are shown in fig. 2. Al output ranged from 237% of Al-saline-treated animals after DFO to 545% after CP52. Calculated efficiencies of the HP chelators ranged from 1.2% for CP20 to 2.5% for CP52, whereas efficiency was 0.8% for DFO. Total Al output was significantly greater after CP40 and CP52 than after DFO, CP20, CP93, and CP94, and was significantly greater after CP24 than DFO, CP20, and CP94. Each chelator significantly increased Al output into urine, compared with Al-saline-treated animals. Urinary Al output was significantly greater after CP52 and CP40 than after the four other HPs and DFO. Only CP24 and CP52 significantly increased biliary Al output, compared with Al-saline-treated animals. The increase with CP24 was significantly greater than with CP52.
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Discussion |
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Abstract
Introduction
Results
Discussion
References
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Al Effects. The presence of lesions in the kidney supports previous reports of Al-induced nephrotoxicity (18, 24, 30). In the present study, the glomerular tuft and renal tubule were the primary sites of renal damage. The location of Al in the Kupffer cells is consistent with the first-pass clearance of Al by the liver (31), and the liver as a site of considerable Al accumulation in this model (fig. 1). The decreases in bone osteoid thickness, osteoid maturation time, and erosion depth are consistent with Al-induced osteomalacia (32). However, the lack of significance of other measures consistent with Al-induced bone disease, the small increase in surface bone Al [2.5% compared with 30% in humans with Al-induced bone disease (33)], and the small increase in bone Al (fig. 1) in Al-loaded rabbits suggests minimal production of Al bone effects. The lack of profound Al-induced bone effects prevented evaluation of the ability of the chelators to influence Al-induced bone disease. The lack of profound Al-induced bone effects was probably due to the relatively short duration of Al loading of these young adult rabbits with normal renal function. The decrease in hemoglobin is consistent with Al-induced anemia.
Chelator Effects. The twelfth treatment in this study was given 5 weeks after completion of Al loading. At that time, baseline Al elimination after saline treatment was 30% of that seen 1 week after Al loading (18), presumably reflecting the lower body burden of Al due to Al clearance over the four additional weeks after Al loading. The Al body burden at the time of the twelfth treatment was estimated to be 4250 µg/kg, based on the total Al content of organs/tissues listed in table 2 in which Al was determined. This was calculated from organ/tissue Al concentration × their weights [assuming bone and muscle represent 3.9 and 49% of body weight, as found for the rat (34)]. Total Al output after CP52, the most effective chelator, minus that after saline, was 2.5% of the estimated Al body burden. All treatments increased total Al output, when calculated as a percentage of saline treatment, as effectively after the twelfth oral dose as after the single iv dose given to rabbits 1 week after the completion of Al loading. Al output after the twelfth chelator treatment ranged from 237% of saline treatment for DFO to 545% for CP52 in the present study, whereas it ranged from 212% of saline treatment for DFO to 456% for CP24 after a single iv dose (18). Therefore, when given orally in doses selected to produce an area under the curve comparable with an iv dose of 450 µmol/kg, the HP chelators were as effective after the twelfth oral dose as the single iv dose.
The present study was conducted in rabbits with intact renal function, which had their bile ducts cannulated before the twelfth treatment. The efficiency of Al chelation and the profile of urinary and biliary Al elimination may be different in renally impaired subjects, due to reduction of urinary clearance of the HPs and the HP · Al complexes; and in non-bile duct-cannulated subjects, if there is significant enterohepatic cycling of the HPs or the HP · Al complexes. The temporal profile of Al elimination (fig. 3) is consistent with the fairly rapid absorption (mean absorption times = 0.5-1.5 hr) and elimination [mean residence times = (0.4-2.6 hr)] of these HPs (17). Changes in serum Al after HP dosing suggest the entrance of these chelators into systemic circulation in the Al-loaded mammal, supporting similar observations in the nonmetal-loaded human (35, 36) and rabbit (17) and in the Al-loaded rabbit after iv dosing (18). The initial increase in serum Al after CP94 and CP20 demonstrates Al mobilization from erythrocytes or extravascular sites. A more pronounced increase in serum Al was seen after the iv dosing of these HPs (18), perhaps due to a greater body burden of Al in those rabbits, previously discussed, and/or due to the higher HP concentrations achieved after iv, rather than oral, dosing. Serum Al decreased below pretreatment concentrations after treatment with each of the chelators. This suggests DFO and HP redistribution of Al out of serum, presumably resulting in Al excretion. Some of the serum Al had to be mobilized from transferrin, which binds >80% of serum Al (37), to account for a >20% reduction of serum Al. The present results do not support the concern that oral Al chelators might produce a net increase in systemic Al due to facilitated Al absorption. Reduction in serum Al argues against chelator-facilitated Al absorption from the gastrointestinal tract as the sole source of the Al eliminated in the bile and urine, because chelator-induced Al absorption would probably increase, and certainly not decrease, serum Al. One extravascular site of Al chelation by the less lipophilic HPs was presumably reticuloendothelial cells, including the Kupffer cell, as shown by the reduction of Kupffer cell Al by CP40, CP20, CP93, and CP52, but not CP94 and CP24. Chelation of this Al presumably resulted in urinary Al excretion, as shown with Fe mobilized from Kupffer cells (38), whereas chelation of Al from hepatocytes and other parenchymal cells by lipophilic HPs presumably resulted in biliary Al excretion (38). The ability of the HPs, except CP94, to decrease bone marrow Al is consistent with chelation of extravascular Al. HP reduction of Al in bone marrow would be expected to improve Al-induced anemia, because bone marrow Al has been shown to be associated with this anemia (39, 40). We also have evidence of HP reduction of Al-induced neurotoxicity.2 The concentration of GFAP, a marker of neurotoxicity, was determined in the frontal cortex, hippocampus, and cerebellum of these rabbits. Frontal cortical GFAP was significantly increased in Al-loaded rabbits, suggesting Al-induced neurotoxicity. Frontal cortical GFAP and Al concentrations positively correlated, supporting this conclusion. GFAP was significantly reduced by CP93, CP52, and CP24, thus suggesting abrogation of the Al-induced toxicity. HPs that decreased GFAP were generally the more lipophilic HPs tested. The nonsignificant reduction of Al in many tissues after 12 treatments with DFO and the HPs may be due to elimination of
2.5% of the total
Al body burden with each treatment. This suggests that treatment would
have to be continued for a much longer period of time, or be given more
frequently, to deplete the substantial Al accumulation significantly.
Clinical studies using DFO to treat Al-induced toxicity were continued
for months to years (reviewed in ref. 41).
The relatively short duration of action of the HPs, and the rapid
elimination of the HP · Al complex, as previously shown in the rat
(42) and shown in this study by the completion of most Al elimination
within 12 hr and return to pretreatment serum Al concentrations,
suggests dosing more than once daily may be beneficial in renally
intact subjects. However, the presence of renal impairment may prolong
the duration of effect and elimination of the HP · Al complex, thus
reducing the potential benefit of more frequent dosing. Further study
of the HPs in renally impaired subjects is needed.
Histology results suggest that chelator-induced reduction in local Al
concentration in the glomerular tuft may have contributed toward the
decreases in Al-induced microaneurysm and sclerosis. Increases in Al
concentration in muscle after CP52 and in lung and liver after CP93 and
the increase in spleen Al content after CP40 and CP94 suggest that Al
may have redistributed after treatment by these chelators. An increase
in spleen Al concentration was also reported after intraperitoneal DFO
and oral CP20 and CP94 treatment of Al-loaded rats (43). The
redistribution of Al to the lung, liver, and spleen may reduce the
toxic potential of the Al to bone and brain, the primary target organs
of Al toxicity. The decrease in Fe in some tissues after HP treatment
is consistent with the greater affinity of the HPs for Fe than for Al
(44). Presumably, the HPs were chelating the Fe in these tissues and causing its excretion as has been reported after CP20 administration (45). Iron excretion during HP therapy to reduce Al accumulation has
the potential to produce Fe deficiency. Although this has been a
concern with DFO treatment of Al accumulation disorders, it has not
proven to be a significant clinical problem because Fe supplements are
often given (46). The lack of significant effect of the HPs on tissue
concentrations of most metals is consistent with the weak association
between the HPs and Ca and Mg, and only moderate association with Zn,
compared with the stronger association with Fe, Cu and Al (44). It is
also consistent with the lack of increased urinary excretion of Cu, Zn,
Mg or Ca after CP20 (45).
The increase in adrenal weight and decrease in testes weight after
several HPs is consistent with results reported for CP20 in
non-metal-loaded rats (47).3 The decrease
in testes weight supports the suggestion that the HPs have an
antiproliferative effect (47). The antiproliferative effect of DFO, and
presumably the HPs, is thought to be due to Fe depletion from
ribonucleotide reductase, consistent with intracellular HP distribution
(10).
Although there are reports that HPs cause a decrease in white cell
counts (48, 49), no HP-induced blood cell toxicity was evident in this
study. This finding is consistent with the unchanged blood cell
profiles seen in Fe-loaded primates after CP20, CP94, or DFO (28) and
unchanged blood cell counts in Al-loaded rats after CP20 and DFO (14).
This lack of blood cell toxicity may be due to the short duration of
treatment. The extent of HP-induced blood cell toxicity is a major
topic of current clinical investigation and warrants investigation in
future long-term animal studies.
Under the short-term oral dosing regimen used, no profound toxicity was
observed that could be directly attributed to the HPs. Overall, the
results from this study reveal the ability of the HPs to increase Al
excretion after repeated oral administration. This demonstrates their
continued effectiveness with repeated dosing, as has been seen with the
ability of CP20 to promote Fe elimination (50). All six HPs studied
were more effective than DFO. CP20 and CP94, the most widely studied
HPs, were the least effective of the six HPs in this study, suggesting
that other HPs be given greater consideration as oral alternatives to
DFO for treatment of Al, and perhaps Fe, accumulation disorders. The more lipophilic HPs more effectively reduced Al-induced neurotoxicity than the hydrophilic HPs (CP40 and CP20). The latter also increased CSF
Al. Lipophilic HPs may be preferable in the treatment of Al accumulation and toxicity in the nervous system. The significant biliary Al excretion following some of the more lipophilic HPs would
favor their use in patients lacking renal function, providing a route
of Al elimination not seen with DFO. However, CP24 may be too toxic,
suggested by its ability to produce seizures after iv, but not oral,
dosing (17, 18) and the higher incidence of death and the weight loss
seen in the present study than with other HPs. A longer term and/or
more aggressive dosing study would better reveal toxic effects of the
HPs. Increased HP lipophilicity did not produce greater total Al
elimination, due to less urinary excretion after lipophilic HPs.
Efficacy of CP40 illustrates the ability of very hydrophilic HPs to
chelate and decorporate Al, suggesting that long-term reduction of Al
body burden can be achieved with quite hydrophilic HPs, perhaps with
less potential for toxicity than lipophilic HPs. The predominance of
urinary Al excretion produced by the hydrophilic HPs suggests their use
in subjects with renal function. The HP · Al complex seems to be
cleared by dialysis (15). Further investigation of HPs as Al chelators should focus on the efficacy/safety profile of individual agents, and
the specific endpoints of Al accumulation and toxicity that are to be
treated, such as reduction of Al and Al-induced toxicity in brain and
bone.
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Acknowledgments |
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Special thanks to Eric Madison, Benjamin Yoo, Charles Xie, and David Ackley for their contributions to this study, and the Center for Applied Energy Research and the Division of Nephrology, Bone and Mineral Metabolism for multielemental analysis and bone histomorphometric analysis, respectively.
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Footnotes |
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Received July 11, 1996; accepted October 31, 1996.
This study was supported by the National Institutes of Health Grant ES RO1 4640 (to R.A.Y.) and by Grant 5 T32 ESO7265-04 (to A.M.F.).
2 R. A. Yokel and J. P. O'Callaghan, submitted for publication.
3 H. P. Schnebli, personal communication.
Send reprint requests to: Dr. Robert A. Yokel, Pharmacy Building, University of Kentucky Medical Center, Lexington, KY 40536-0082.
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Abbreviations |
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Abbreviations used are:
Al, aluminum;
DFO, desferrioxamine;
HP, 3-hydroxypyridin-4-one;
Do/a, partition coefficient;
CP20, 1,2-dimethyl
3-hydroxypyridin-4-one;
iv, intravenous;
Na, sodium;
GFAP, glial
fibrillary acidic protein;
ETAAS, electrothermal atomic absorption
spectroscopy;
CSF, cerebrospinal fluid;
Mg, magnesium;
Ca, calcium;
Cu, copper;
Fe, iron;
Ni, nickel;
Zn, zinc;
CP40, 1-[ethan-1
-ol]-2-methyl-3-hydroxypyridin-4-one;
CP24, 1-n-butyl-2-methyl-3-hydroxypyridin-4-one;
CP52, 1-[3-ethoxypropyl]-2-methyl-3-hydroxypyridin-4-one;
CP93, 1-methyl-2-ethyl-3-hydroxypyridin-4-one;
CP94, 1,2-diethyl-3-hydroxypyridin-4-one.
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References |
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Abstract
Introduction
Results
Discussion
References
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), biliary (
) and total (sum
of the two) Al output in the 24 hr after the twelfth treatment.
