The design, synthesis, and evaluation of organ-specific iron chelators.

A series of iron chelators, three (S)-4,5-dihydro-2-(2-hydroxyphenyl)-4-methyl-4-thiazolecarboxylic acid (DADFT) and three (S)-4,5-dihydro-2-(2-hydroxyphenyl)-4-thiazolecarboxylic acid (DADMDFT) analogues are synthesized and assessed for their lipophilicity (log Papp), iron-clearing efficiency (ICE) in rodents and iron-loaded primates (Cebus apella), toxicity in rodents, and organ distribution in rodents. The results lead to a number of generalizations useful in chelator design strategies. In rodents, while log Papp is a good predictor of a chelator's ICE, chelator liver concentration is a better tool. In primates, log Papp is a good predictor of ICE, but only when comparing structurally very similar chelators. There is a profound difference in toxicity between the DADMDFT and DADFT series: DADMDFTs are less toxic. Within the DADFT family of ligands, the more lipophilic ligands are generally more toxic. Lipophilicity can have a profound effect on ligand organ distribution, and ligands can thus be targeted to organs compromised in iron overload disease, for example, the heart.

Antioxidant and lysosomotropic properties of acute D-propranolol underlies its cardioprotection of postischemic hearts from moderate iron-overloaded rats.

The benefits of acute D-propranolol (D-Pro, non-beta-adrenergic receptor blocker) pretreatment against enhanced ischemia/reperfusion (I/R) injury of hearts from moderate iron-overloaded rats were examined. Perfused hearts from iron-dextran-treated rats (450 mg/kg/week for 3 weeks, intraperitoneal administration) exhibited normal control function, despite iron treatment that elevated plasma iron and conjugated diene levels by 8.1-and 2.5-fold, respectively. However, these hearts were more susceptible to 25 mins of global I/R stress compared with non-loaded hearts; the coronary flow rate, aortic output, cardiac work, left ventricular systolic pressure, positive differential left ventricular pressure (dP/dt), and left ventricular developed pressure displayed 38%, 60%, 55%, 13%, 41%, and 15% lower recoveries, respectively, and a 6.5-fold increase in left ventricular end-diastolic pressure. Postischemic hearts from iron-loaded rats also exhibited 5.6-, 3.48-, 2.43-, and 3.45-fold increases in total effluent iron content, conjugated diene levels, lactate dehydrogenase (LDH) activity, and lysosomal N-acetyl-beta-glucosaminidase (NAGA) activity, respectively, compared with similarly stressed non-loaded hearts. A comparison of detection time profiles during reperfusion suggests that most of the oxidative injury (conjugated diene) in hearts from iron-loaded rats occurred at later times of reperfusion (8.5-15 mins), and this corresponded with heightened tissue iron and NAGA release. D-Pro (2 microM infused for 30 mins) pretreatment before ischemia protected all parameters compared with the untreated iron-loaded group; pressure indices improved 1.2- to 1.6-fold, flow parameters improved 1.70- to 2.96-fold, cardiac work improved 2.87-fold, and end-diastolic pressure was reduced 56%. D-Pro lowered total release of tissue iron, conjugated diene content, LDH activity, and NAGA activity 4.59-, 2.55-, 3.04-, and 4.14-fold, respectively, in the effluent of I/R hearts from the iron-loaded group. These findings suggest that the enhanced postischemic dysfunction and tissue injury of hearts from iron-loaded rats was caused by excessive iron-catalyzed free radical stress, and that the membrane antioxidant properties of D-Pro and its stabilization of sequestered lysosomal iron by D-Pro may contribute to the cardioprotective actions of D-Pro.

Effects of iron dextran injections on the incidence of abomasal bloat, clinical pathology and growth rates in lambs.

A preliminary study revealed significantly lower serum iron concentrations in lambs that developed abomasal bloat about one week later, than in lambs that did not develop bloat. In a subsequent trial, with 754 naturally reared twin lambs from five flocks, iron dextran injections were found to have a preventive effect on the development of abomasal bloat. Clinical signs of abomasal bloat were observed in the placebo-treated lamb of 16 couples and in the iron-treated lamb of six couples (P<0.05). In three further couples, both lambs developed bloat. The iron-treated group had significantly (P<0.001) better average weight gains, both from birth to summer, and from birth to autumn of approximately 0.5 and 1 kg, respectively. There was a reduction in red blood cell values and iron saturation in the placebo group 14 days after treatment but not in the iron-treated lambs. In one of the flocks, there was a decrease in the cumulative incidence of abomasal bloat from 37 per cent to 3 per cent during the period of four years after measures such as later lambing, earlier turnout and iron injections were introduced.

Iron-dextran as a magnetic susceptibility contrast agent: flow-related contrast effects in the T2-weighted spin-echo MRI of normal rat and cat brain.

Iron-dextran (1 mmol Fe/kg) was used as an intravascular, paramagnetic contrast agent in rat and cat brain in conventional spin-echo T2-weighted (TR 2800/TE 100) 1H magnetic resonance imaging. The resulting images displayed differential decreases (30-50%) in intensity whose pattern was similar to that obtained with the superparamagnetic particulate iron oxide AMI-25 (0.18 mmol Fe/kg). Postcontrast images displayed improved anatomic detail, and contrast effects were observed to be greater in cortical and subcortical gray matter than in adjacent white matter. Intravenous injection of acetazolamide after administration of iron-dextran caused a small additional decrease in image intensity. Measurement of whole blood and plasma at 5 min postinjection of either contrast agent revealed significant increases in their volume magnetic susceptibilities. The contrast effect appears to be related to magnetic susceptibility changes brought about by the iron-dextran; it has both blood volume and blood flow components. The static model of magnetic susceptibility effects in brain capillaries is modified to include bolus flow of erythrocytes, providing a mechanism for the observed flow effects.

Dextran magnetite as a liver contrast agent.

The superparamagnetic particle dextran magnetite was studied as a liver tumor contrast agent for magnetic resonance imaging (MRI). The effects of dextran magnetite on the longitudinal (T1) and transverse (T2) relaxation times in liver, spleen, and an implanted rat liver tumor were measured at 0.47 T (IBM/Bruker PC-20 relaxometer) over the dose range of 23 to 69 mumol Fe/kg. Dextran magnetite substantially reduced the T2 of the liver and spleen, but not of the tumor, thereby providing a basis for improved tumor imaging. The T1 of the tumor was not affected following injection of dextran magnetite in the dose range studied, while the spleen T1 was reduced substantially more than the T1 of the liver. Histological studies using the iron reaction for Prussian blue clearly showed dextran magnetite in the liver and spleen, but not in the tumor. While dextran magnetite was sequestered in macrophages in both liver and spleen, the distribution in the liver was more diffuse (70 microns average particle separation) than that in the spleen (25 microns separation). The lack of a T1 effect in the liver is consistent with the fact that a majority of the water in the tissue cannot diffuse to the relaxational centers on the time scale of the liver's intrinsic T1 (280 ms). In the spleen, however, the dextran magnetite is more densely packed in the red pulp allowing a significant fraction of the water to be relaxed by a T1 mechanism. Spin-echo images of the implanted tumor (mammary adenocarcinoma. R3230AC) in the livers of Fischer 344 rats were obtained at 0.50 T (Siemens Magnetom). The tumor-to-liver contrast was improved for both T1 and T2-weighted spin-echo images after intravenous injection of the dextran magnetite contrast agent. The contrast determined from these images agreed with that predicted by the measured T1 and the T2 (Hahn spin-echo) values. In addition, gradient-echo T2-weighted images with good contrast were obtained in a much shorter imaging time than was needed for T2-weighted spin-echo images. These results demonstrate that the MRI contrast enhancement observed with dextran magnetite is based on its selective uptake and distribution in the macrophages in the liver and spleen and that this agent has substantial potential as a superparamagnetic MR contrast agent.

Colorimetry and constant-potential coulometry determinations of transferrin-bound iron, total iron-binding capacity, and total iron in serum containing iron-dextran, with use of sodium dithionite and alumina columns.

After the parenteral administration of iron-dextran (imferon), the increased total iron concentrations in serum can be determined by atomic absorption spectroscopy and by colorimetric methods involving sodium dithionite, which reductively dissociates iron from the dextran complex. We report that constant-potential coulometry detects only about 55-70% of dextran-bound iron before dithionite reduction and variable amounts after reaction with the reducing agent. In addition, we have developed a procedure for determining transferrin-bound iron, total iron-binding capacity (TIBC), total iron, and dextran-bound iron with the Kodak Ektachem colorimetric system. In determining total serum iron, the sample is first mixed with sodium dithionite, which rapidly dissociates all dextran-bound iron, but does not remove iron from either transferrin or hemoglobin. After the mixture is applied to an Ektachem slide, transferrin-bound iron is released at pH 4 and is detected together with the iron previously bound to dextran. TIBC is determined by mixing serum with ferric citrate in moderate excess and filtering through a small alumina (Al2O3) column, which binds excess free iron and iron-dextran; the iron in the column eluate represents the TIBC. Transferrin-bound iron is determined by applying diluted serum without added ferric citrate to an alumina column and measuring the iron in the column eluate. Dextran-bound iron is equivalent to the difference between total and transferrin-bound iron. Using this method, we found that transferrin iron-binding sites are saturated in vitro by excess iron-dextran less efficiently than by ferric citrate.

Iron and total iron-binding capacity in serum of patients receiving iron-dextran: Kodak Ektachem methodologies, spectrophotometry, and atomic-absorption spectrometry compared.

We compared the analytical performance of the Kodak Ektachem XR700 assays of iron (Fe) and total iron-binding capacity (TIBC) with that of a conventional ferrozine assay (performed with a Cobas-Bio) and occasionally with that of atomic-absorption spectrometry (AAS). The correlation was modest between Kodak and Cobas-Bio concentrations of Fe in serum (r = 0.79). Multiple outliers were noticed in samples from hemodialysis patients, with Cobas values exceeding those of Kodak by as much as 26 mumol/L. These intermethod differences were not dissipated by dialysis, but were invariably accompanied by an even higher total Fe content of the serum as judged by AAS (20 to 80 mumol/L higher). TIBC values by Kodak and Cobas-Bio were highly correlated (r = 0.99); again, the Cobas-Bio results exceeded those by Kodak in some hemodialysis patients, but always for samples with higher Fe concentrations by the Cobas-Bio. By AAS, the TIBC values of these patients also exceeded those by Kodak, to about the same extent as observed for serum Fe. These intermethod differences in Fe and TIBC were seen only in patients who had received an intravenous Fe-dextran (Imferon) injection two to three days before blood sampling but could be generated in vitro by adding Imferon to serum from normal controls. Less than 6% of dextran-bound Fe is measured as Fe by Kodak, as opposed to 20-30% by Cobas-Bio and 89-120% by AAS. We conclude that the Kodak Fe slides are superior to liquid reagents, by exclusively measuring protein-bound circulating Fe pools.

Vascular to alveolar leak of iron dextran (120 kD) in the immature ventilated rabbit lung.

Rabbit fetuses were delivered by hysterotomy on day 27 or 28 of gestation. Immediately after birth, the animals were tracheotomized and received by intravenous injection 0.2 mu Ci radiolabeled albumin and 11 mg iron dextran in 0.2 ml saline. The newborn rabbits then were ventilated artificially with a tidal vol of 12 ml/kg for 5-20 min. One group of nonventilated animals served as controls. At the end of the experiment, one lung was lavaged via the airways and the other was fixed for histologic examination. The recovery of labeled albumin and iron dextran in the lavage fluid was quantified. Iron dextran complexes were easily identified in the lung sections by staining with Prussian blue. Iron dextran accumulated in the airspaces of animals delivered on day 27 (about 4% of the injected dose during 10-20 min of ventilation). The albumin leakage was slightly higher than that of the dextran, a result consistent with different mol wt of the markers. The vol density of leaking alveoli in histologic sections increased with time, from 0 at birth to a mean value of 0.36 after 20 min of ventilation. The leakage starts as a focal event, gradually involving more and more terminal airspaces. In the histologic sections, there was no indication of a significant leakage at the bronchiolar level, although the epithelium of terminal and preterminal airways was clearly injured in all ventilated animals.

The measurement of serum unsaturated iron-binding capacity in the presence of iron-dextran.

The standard colorimetric methods most often used for the measurement of serum unsaturated iron binding capacity (UIBC) are subject to gross interference by iron dextran. This paper describes a brief evaluation of an alternative radiometric assay for serum UIBC, based on a commercially available kit method, but incorporating a modification to the manufacturer's protocol. The effects of iron dextran on the assay were determined.

[Toxicity, tolerance and blood concentrations of iron and tylosin with the use of preparation FV-82]. / Toksichnost, ponosimost i kruvni kontsentratsii na zheliazoto i tilozina pri prilagane na preparata FV-82.

A pharmacologic evaluation was made of a technologic model of a liquid drug form (code name phi B-82), having the following composition: tylosine tartrate 3500000 UI, cyanocobalamine 0.008 g, pyridoxin hydrochloride 0.500 g, tartaric acid 0.100 g, and feridextran (dextrofer-100) up to 100 cm3; pH from 5.5 to 6.5, and Fe3+ 100 mg/cm3. It was found that phi B-82 at i/m application to rabbits, subcutaneous injection to albino mice, and intra-abdominal introduction to albino rats and mice at rates that were equal to ED100 and 3 to 5 times higher than those used with pigs did not lead to local and total lack of tolerance. The acute toxicity (LD50) of phi B-82 at intra-abdominal application to 18-20 g albino mice was 29.2 cm3/kg. The single muscular application to guinea pigs at 2 cm3 per kg of body mass showed good absorption of the preparation - it did not differ essentially from those of dextrofer-100 and aquaous solution of tylosine tartrate used in equivalent amounts. The bacteriostatic concentrations of tylosine were maintained for 24 hours. It was shown that the optimal effect would be produced by a combined preparation having the qualities of the feridextrane complexes with a rapid absorption and those of the erythropoietic vitamins of the B12 group and B6 along with the participation of tylosine as an antibiotic.

Interference of imferon in colorimetric assays for iron.

Imferon (Merrell-National), an iron-dextran complex, is widely used in patients with iron deficiency. It is present in the circulation in appreciable amounts for two to three weeks after administration and interferes with all tested colorimetric iron assays, both with and without deproteinization. The amount of the plasma Imferon iron interference depends primarily on the choice of reductant. With dithionite it is essentially 100%. In the presence of ascorbic acid and hydroxylamine, the interference depends also on assay conditions, especially temperature, but also incubation time and pH. The minimum interference in a homogeneous assay was about 3%. The relative amount of interference from hemoglobin iron under the various assay conditions is different from that of Imferon iron. In the presence of a reducing agent, the dextran-iron complex decomposes--instantaneously with dithionite, and gradually with sulfite, ascorbic acid, and hydroxylamine. The freed iron becomes dialyzable, can react with bathophenanthroline, and elutes on a Sephadex G-50 or G-15 column in the same fractions as an ammonium ferrous sulfate.

The assay of iron-poly (sorbitol-gluconic acid) complex (Ferastral) and its separation from transferrin in serum.

A new iron-poly (sorbitol-gluconic acid) complex (Ferastral) has been studied. A method of assay is described. The iron complex may be separated from serum transferrin using a Sephadex DEAE A50 column. This binds the iron complex and elutes iron-transferrin which can then be assayed. It is shown that the assay of serum transferrin unsaturated binding capacity using excess 59FeCl2 and MgCO3 adsorption, is valid in the presence of Ferastral. Serum unsaturated iron binding capacity may therefore be used to follow the binding of Ferastral iron by transferrin. These methods may be used to follow the distribution of iron in plasma after an intramuscular injection of Ferastral.

Transfer of iron-dextran across the placenta.

In pregnant mice. 55Fe-labeled iron-dextran (Imferon) is transferred across the placenta. It was detected in the bone marrow, liver, spleen and peripheral blood of the pregnant animal, as well as in the embryonic liver erythroid precursors and peripheral blood. Uptake by liver and peripheral blood cells of pregnant anemic mice and by liver erythroid precursors of anemic embryos was significantly higher than in normal control animals. Electron-microscopic examination revealed that the iron deposits in the embryonic liver erythroid precursors had the same structure as the injected Imferon.

Utilization of iron dextran.

The utilization of iron dextran was investigated in normal subjects, in patients with iron-deficiency anaemia, and in anaemias associated with rheumatoid arthritis, reticulosis, and uraemia. Utilization of iron for haemoglobin formation at 14 days was found to be depressed in patients with rheumatoid arthritis, reticulosis, and uraemia, but when a concomitant iron-deficiency anaemia was present utilization was significantly increased. When iron dextran is used to treat anaemias in such conditions an optimum therapeutic response will be obtained only when bone marrow iron stores are absent.