Zudong Liu

Chelation and determination of labile iron in primary hepatocytes by pyridinone fluorescent probes

A series of fluorescent iron chelators has been synthesized such that a fluorescent function is covalently linked to a 3-hydroxypyridin-4-one. In the present study, the fluorescent iron chelators were loaded into isolated rat hepatocytes. The intracellular fluorescence was not only quenched by an addition of a highly lipophilic 8-hydroxyquinoline-iron(III) complex but also was dequenched by the addition of an excess of the membrane-permeable iron chelator CP94 (1,2-diethyl-3-hydroxypyridin-4-one). The time course of uptake of iron and iron chelation in single, intact cells was recorded on-line by using digital fluorescence microscopy. Intracellular concentrations of various fluorescent iron chelators were determined by using a spectrofluorophotometer subsequent to lysis of probe-loaded cells and were found to depend on their partition coefficients; the more hydrophobic the compound, the higher the intracellular concentration. An ex situ calibration method was used to determine the chelatable iron pool of cultured rat hepatocytes. CP655 (7-diethylamino-N-[(5-hydroxy-6-methyl-4-oxo-1,4-dihydropyridin-3-yl)methyl]-N-methyl-2-oxo-2H-chromen-3-carboxamide), which is a moderately lipophilic fluorescent chelator, was found to be the most sensitive probe for monitoring chelatable iron, as determined by the intracellular fluorescence increase induced by the addition of CP94. The concentration of the intracellular chelatable iron pool in hepatocytes was determined by this probe to be 5.4+/-1.3 microM.

The Treatment of Malaria with Iron Chelators

New routes to chemotherapeutic attack on malaria are urgently required as drug resistant mutants to the antifolate pyrimethamine and the quinine family are becoming widespread. Without new drugs, falciparum malaria could become an untreatable disease in some regions within the next decade. The idea that iron chelation might have antiplasmodial effect emerged from the awareness that many microbial pathogens depend on host-derived iron for virulence. In 1982 it was demonstrated by Pollack and co-workers that desfemoxm i n e possessed an antiplasmodial effect in-vitro (RaventosSuarez et a1 1982). Some years later this was followed by a clinical trial of iron-chelation therapy in children with cerebral malaria (Gordeuk et a1 1992a). Desfenioxamine quickly reduced the symptoms of parasitaemia. Thus it is possible that iron chelation offers a novel approach to controlling malaria infections.

Determination of the Labile Iron Pool of Human Lymphocytes using the Fluorescent Probe, CP655

Background: We have recently developed a new technique for quantitatively measuring protein-bound 3-nitrotyrosine (3-NT), a footprint of nitrosative stress, utilizing high-performance liquid chromatography with an electrochemical detection (HPLC-ECD) system. Using this system, we showed that 3-NT formation was upregulated in the sputum of both COPD and asthmatic patients. However, in order to improve the accuracy of the measurement system, We have to resolve some problems which were the influence of free amino acid form of 3-NT and of salivary contamination. Objectives: We initially investigated the amount of the free amino acid form of 3-NT in induced sputum and compared with that of protein-bound 3-NT. Next, we evaluated the concentration of protein-bound 3-NT in saliva and compared with that in induced sputum by means of HPLC-ECD. Methods: Five male COPD patients were enrolled. Induced sputum and saliva were obtained from the patients. The free amino acid form of 3-NT in sputum and saliva was measured by HPLC-ECD, and the protein-bound 3-NT and tyrosine in sputum and saliva were enzymatically hydrolyzed by Streptomyces griseus Pronase and measured for the protein hydrolysate by HPLC-ECD. Results: The mean value of the amount of protein-bound 3-NT was 65.0 fmol (31.2 to 106.4 fmol). On the other hand, the amount of the free amino acid form of 3-NT was under the detection limit (<10 fmol). The levels of both 3-NT (sputum: 0.55 ± 0.15 pmol/ml, saliva: 0.02 ± 0.01 pmol/ml, p < 0.01) and tyrosine (sputum: 0.81 ± 0.43 μmol/ml, saliva: 0.07 ± 0.04 μmol/ml, p < 0.01) in saliva were significantly lower than in sputum. The percentage of 3-NT in saliva to that in sputum was about 3.1%, and that of tyrosine was about 9.0%. Conclusion: The free amino acid form of 3-NT does not affect the measurement of protein-bound 3-NT. Furthermore, the influence of salivary contamination on the measurement of protein-bound 3-NT in induced sputum by means of HPLC-ECD was very small and could be negligible.A protein digestion system using immobilized enzymes for protein identification and glycochain analyses has been developed, and a vibration reaction unit for micro-scale sample convection on an enzyme-immobilized solid surface was constructed. BSA as a model substrate was digested by this unit, and was successfully identified by mass spectrometry (MS) analyses. Compared to the conventional liquid-phase digestion, the reaction unit increased the number of matched peptides from 9 to 26, protein score from 455 to 1247, and sequence coverage from 21% to 48%. Glycopeptidase F (NGF), an enzyme that cleaves N-glycans from glycoproteins, was also immobilized and used to remove the glycochains from human immunoglobulin G (IgG). Trypsin and NGF were immobilized on the same solid surface and used to remove glycochains from IgG in single-step. Glycochains were labeled with fluorescent reagent and analyzed by HPLC. Several peaks corresponding to the glycochains of IgG were detected. These results suggested that the single-step digestion system, by immobilized multiple enzymes (trypsin and NGF) would be effective for the rapid structural analysis of glycoproteins.This research shows a novel method for hazard identification of a chemical and UV light on a single cell level with a laser probe beam. The laser probe beam was passed through interface of cell membrane/culture medium of a cultured human hepatoblastoma cell line HepG2. Deflection of the laser probe beam, which was induced by changes in concentration gradients due to the active materials movement across the cell membrane, was monitored. When a toxic hazard existed, a living cell was expected to be killed or injured, or cellular behaviors to be changed greatly. Then, the changing deflection signal from the living cell would become unchanged or altered in a different way. This was successfully demonstrated with cytotoxity of UV light and H2O2. Most of the cultured HepG2 cells showed changing deflection signals after 10 min illumination of UV-visible light longer than 370 nm, while almost all HepG2 cells showed unchanged deflection signal after 10 min illumination of UV-visible light with wavelength longer than 330 nm. The results suggested that UV light between 330–370 nm could kill the cells. Additions of H2O2 solution with different concentrations to the cell cultures caused the changing deflection signal from a living cell either unchanged or changed in different trend, suggesting toxicity of H2O2 to the cells. The results from the beam deflection detection agreed well with those obtained by the conventional trypane blue method.Escherichia coli as a plasmid recipient cell was dispersed in a chrysotile colloidal solution, containing chrysotile adsorbed to plasmid DNA (chrysotile-plasmid cell mixture). Following this, the chrysotile-plasmid cell mixture was dropped onto the surface of an elastic body, such as agarose, and treated physically by sliding a polystyrene streak bar over the elastic body to create friction. Plasmid DNA was easily incorporated into E. coli, and antibiotic resistance was conferred by transformation. The transformation efficiency of E. coli cultured in solid medium was greater than that of E. coli cultured in broth. To obtain greater transformation efficiency, we attempted to determine optimal transformation conditions. The following conditions resulted in the greatest transformation efficiency: the recipient cell concentration within the chrysotile-plasmid cell mixture had an optical density greater than or equal to 2 at 550 nm, the vertical reaction force applied to the streak bar was greater than or equal to 40 g, and the rotation speed of the elastic body was greater than or equal to 34 rpm. Under these conditions, we observed a transformation efficiency of 107 per μg plasmid DNA. The advantage of achieving bacterial transformation using the elastic body exposure method is that competent cell preparation of the recipient cell is not required. In addition to E. coli, other Gram negative bacteria are able to acquire plasmid DNA using the elastic body exposure method.We have determined and quantified spectrophotometrically the capacity of producing reactive oxygen species (ROS) as 1O2 during the photolysis with UV-A light of 5 new synthesized naphthyl ester derivates of well-known quinolone antibacterials (nalidixic acid (1), cinoxacin (2), norfloxacin (3), ciprofloxacin (4) and enoxacin (5)). The ability of the naphthyl ester derivatives (6–10) to generate singlet oxygen were detecting and for the first time quantified by the histidine assay, a sensitive, fast and inexpensive method. The following tendency of generation of singlet oxygen was observed: compounds 7 > 10 > 6 > 8 > 9 >> parent drugs 1–5.High-performance liquid chromatography (HPLC) analysis was used for identification of two problematic ureides, asparagine and citrulline. We report here a technique that takes advantage of the predictable delay in retention time of the co-asparagine/citrulline peak to enable both qualitative and quantitative analysis of asparagine and citrulline using the Platinum EPS reverse-phase C18 column (Alltech Associates). Asparagine alone is eluted earlier than citrulline alone, but when both of them are present in biological samples they may co-elute. HPLC retention times for asparagine and citrulline were influenced by other ureides in the mixture. We found that at various asparagines and citrulline ratios [= 3:1, 1:1, and 1:3; corresponding to 75:25, 50:50, and 25:75 (μMol ml−1/μMol ml−1)], the resulting peak exhibited different retention times. Adjustment of ureide ratios as internal standards enables peak identification and quantification. Both chemicals were quantified in xylem sap samples of pecan [Carya illinoinensis (Wangenh.) K. Koch] trees. Analysis revealed that tree nickel nutrition status affects relative concentrations of Urea Cycle intermediates, asparagine and citrulline, present in sap. Consequently, we concluded that the HPLC methods are presented to enable qualitative and quantitative analysis of these metabolically important ureides.Microalbuminuria is associated with hypertension and is a strong risk factor for subsequent chronic disease, both renal and coronary heart disease (CHD), Presently there are several methods available for measurement of microalbuminuria. The aim of this study was to evaluate if the three different methods gave similar information or if one of the assays were superior to the others. Blood pressure, inflammatory markers and cardiovascular mortality and morbidity were correlated with urine albumin analysed with a point-of-care testing (POCT) instrument, nephelometric determination of albumin and albumin/creatinine ratio in elderly males. The study population consisted of 103 diabetic and 603 nondiabetic males (age 77 years) in a cross-sectional study. We analyzed urine albumin with a HemoCue® Urine Albumin POCT instrument and a ProSpec® nephelometer and albumin/creatinine ratio. There were strong correlations between both systolic and diastolic blood pressure and all three urine albumin methods (p < 0.0001). There were also significant correlations between the different urine albumin measurements and serum amyloid A component, high-sensitivity C-reactive protein and interleukin-6. The three different urine albumin methods studied provided similar information in relation to cardiovascular disease. There was a strong correlation between systolic and diastolic blood pressure and microalbuminuria in both the whole study population and in nondiabetic males emphasizing the role of hypertension in glomerular damage. The good correlation between the studied urine albumin measurements show that all three methods can be used for monitoring urine albumin excretion.Chromium is an important constituent widely used in different industrial processes for production of various synthetic materials. For evaluation of workers’ exposure to trace toxic metal of Cr (III), environmental and biological monitoring are essential processes, in which, preparation of samples is one of the most time-consuming and error-prone aspects prior to analysis. The use of solid-phase extraction (SPE) has grown and is a fertile technique of sample preparation as it provides better results than those produced by liquid-liquid extraction (LLE). SPE using mini columns filled with XAD-4 resin was optimized regarding to sample pH, ligand concentration, loading flow rate, elution solvent, sample volume, elution volume, amount of resins, and sample matrix interferences. Chromium was retained on solid sorbent and was eluted with 2 M HNO3 followed by simple determination of analytes by using flame atomic absorption spectrometery. Obtained recoveries of metal ion were more than 92%. The optimized procedure was also validated with three different pools of spiked urine samples and showed a good reproducibility over six consecutive days as well as six within-day experiments. Through this study, suitable results were obtained for relative standard deviation, therefore, it is concluded that, this optimized method can be considered to be successful in simplifying sample preparation for trace residue analysis of Cr in different matrices for evaluation of occupational and environmental exposures. To evaluate occupational exposure to chromium, 16 urine samples were taken, prepared, and analyzed based on optimized procedure.Mistletoe Extracts (ME) are of growing interest to pharmacological research because of their apoptosis-inducing/cytostatic and immunomodulatory effects. The standardization of the three different groups of Mistletoe Isolectins (ML-I, II and III) is often rendered more difficult since the primary structures are nearly identical. Their classification is based on their Galactose- and N-acetyl-D-galactosamine (GalNAc)-specificity which was measured by various inhibitory assays. The aim of the present study was to improve the characterization of the direct binding activity of the isolectins from ME to immobilized lactose, GalNAc and to the oligosaccharide asialofetuin. After careful ultrafiltration of fresh ME, affinity chromatography was carried out using lactose- agarose, GalNAc—agarose and asialofetuin—affigel 15 columns. MLs were further purified by Sephadex G-100 or by cation exchange chromatography which was adapted to a Fast Protein Liquid Chromatography (FPLC) system. Proteins from both fresh plants and commercial ME were able to bind immobilized lactose to a considerable extent. The majority of this lectin has a B-chain with a Molecular Weight (MW) of 34kD and an A-chain with a MW of 29 kD (ML-I). Only a minor part of the lactose-binding proteins has a lower MW, namely 32kD and 27kD (MLII). However, neither MLs which were eluted from lactose columns, nor the proteins from fresh plant or ME showed a direct binding to the immobilized GalNAc. In spite of this deficiency, GalNAc was able to induce a considerable (25% and 32%) inhibitory effect on their binding to immobilized asialofetuin indicating a discrepancy between the lectin binding and inhibiting effects of GalNAC. Consequently, for an improved standardization of ME more specific sugar molecules are necessary.Pentavalent technetium-99m dimercaptosuccinic acid (99mTc-(V)DMSA) is a tumor-seeking agent which was introduced to evaluate, image, and manage many types of cancers. In this review, the beginning of, and the most recent applications of using this agent was appraised. The relation with tumor cell detection and proliferation was reported and several mechanisms of uptake of 99mTc-(V)DMSA in tumor cells are described.We studied the near UV absorption spectrum of canine plasminogen. There are 19 tryptophans, 19 phenylalanines and 34 tyrosines in the protein. 4th derivative spectra optimized for either tryptophan or tyrosine give a measure of the polarity of the environments of these two aromatic amino acids. Plasminogen at temperatures between 0 °C and 37 °C exists as a mixture of four conformations: closed-relaxed, open-relaxed, closed-compact, and open-compact. The closed to open transition is driven by addition of ligand to a site on the protein. The relaxed to compact transition is driven by increasing temperature from 0 °C to above 15–20 °C. When the conformation of plasminogen is mainly closed-relaxed, the 4th derivative spectra suggest that the average tryptophan environment is similar to a solution of 20% methanol at the same temperature. Under the same conditions, 4th derivative spectra suggest that the average tyrosine environment is similar to water. These apparent polarities change as the plasminogen is forced to assume the other conformations. We try to rationalize the information based on the known portions of the plasminogen structure.Chirasil-β-Dex containing an undecamethylene spacer (C11-Chirasil-Dex) was synthesized and used as chiral stationary phase (CSP) in enantioselective gas chromatography (GC). The versatility of the new stationary phase in the simultaneous enantiomeric separation of a set of N-alkylated barbiturates is demonstrated.We have developed a two-dimensional (2D-) gel system of zymography and reverse zymography for the detection and characterization of proteases and protease inhibitors. Isoelectric focusing (IEF) agarose gels with pH gradients were employed for separation in the first-dimension and sodium dodecyl sulfate (SDS)-polyacrylamide gel copolymerized with gelatin used for the second dimension. Proteases and protease inhibitors separated by IEF gel were applied on the second gel without trichloroacetic acid (TCA) fixation. Protease activity in the 2D-gel was visualized as transparent spots where gelatin substrate was digested after commassie brilliant blue (CBB) staining. Some of the transparent spots from the skin mucus extract of rainbow trout were determined to be a cysteine protease through use of E-64 or CA-074. In the reverse zymography technique, the gel was incubated with papain solution at 37 degrees C for 18 h. Cysteine protease inhibitors from broad bean seeds were detected as clear blue spots after CBB staining. The amino (N-) terminal sequences of four papain inhibitor spots thus detected were demonstrated to be identical to that of favin beta chain, a broad bean lectin. Taken together, our system can be considered to be an efficient technique for discovering and characterizing new proteases and protease inhibitors in biological samples. This is the first report describing a 2D-gel system of zymography and reverse zymography.The present study introduces a method for determining the labile iron pool (LIP) in human lymphocytes. It is measured using the probe CP655, the fluorescence of which is stoichiometrically quenched by the addition of iron. The intracellular CP655 fluorescence in lymphocytes was quenched by increasing intracellular iron concentrations using the highly lipophilic 8-hydroxyquinoline iron complex. Intracellular fluorescence quenching, mediated by the physiological intracellular labile iron, can be recovered on the addition of excess membrane-permeable iron chelator, CP94. The intracellular probe concentration was measured using laser scanning microscopy. An ex situ calibration was performed in a “cytosolic” medium based on the determined intracellular CP655 concentration and probe fluorescence quenching in the presence of iron. The concentration of the LIP of healthy human lymphocytes was determined to be 0.57 ± 0.27 μM. The use of the fluorescent probe CP655 renders it possible to record the time course of iron uptake and iron chelation by CP94 in single intact lymphocytes.The aim of this study is to adopt the approach of metabolic fingerprinting through the use of Fourier Transform Infrared (FTIR) technique to understand changes in the chemical structure in Padina tetrastromatica (Hauck). The marine brown alga under study was grown in two different environmental conditions; in natural seawater (P. tetrastromatica (c)) and in seawater suplemented with 50 ppm of cadmium (P. tetrastromatica (t)) for a three-week period in the laboratory. The second derivative, IR specrum in the mid-infrared region (4000–400 cm−1) was used for discriminating and identifying various functional groups present in P. tetrastromatica (c). On exposure to Cd, P. tetrastromatica (t) accumulated 412 ppm of Cd and showed perturbation in the band structure in the mid-IR absorption region. Variation in spectral features of the IR bands of P. tetrastromatica (untreated and treated) suggests that cadmium ions bind to hydroxyl, amino, carbonyl and phosphoryl functionalities. This was attributable to the presence of the following specific bands. A band at 3666 cm−1 in untreated P. tetrastromatica (c) while a band at 3560 cm−1 in Cd-treated P. tetrastromatica (t) due to non bonded and bonded O-H respectively. Similarly, non bonded N-H for P. tetrastromatica (c) showed two bands at 3500 cm−1 and 3450 cm−1 due to the N-H stretching vibrations and a band at 1577 cm−1 due to N-H bending vibrations, while an intense band at 3350 cm−1 due to bonded N-H stretching vibrations and at 1571 cm−1 due to bending vibrations was observed for Cd-treated P. tetrastromatica (t). Involvement of ester carbonyl group is characterized by the presence of a band at 1764 cm−1 in untreated P. tetrastromatica (c) while the Cd-treated P. tetrastromatica (t) showed the band at 1760 cm−1. The intensity of the band at 1710 cm−1 in the control samples decreased drastically after cadmium treatment indicating carbonyl of COOH to be involved in metal chelation. A band at 1224 cm−1 for untreated P. tetrastromatica (c) and at 1220 cm−1 for Cd-treated P. tetrastromatica (t) is indicative of the involvement of phosphoryl group in metal binding. Several other such changes were also evident and discussed in this paper. Based on our observation, FTIR technique proves to be an efficient tool for detecting structural changes and probable binding sites induced by the presence of a metal pollutant, cadmium, in the marine environment.A high-performance liquid chromatographic (HPLC) method has been developed for the separation and determination of S- and R-enantiomers of betaxolol in tablets and ophthalmic preparations. Baseline resolution was achieved by using teicoplanin macrocyclic antibiotic chiral stationary phase (CSP) known as Chirobiotic T with fluorescence detection at excitation/emission wavelengths 275/305 nm. The polar ionic mobile phase (PIM) consists of methanol-glacial acetic acid-triethylamine, (100:0.020:0.025, v/v/v) has been used at a flow rate of 1.5 ml/min. All analytes with S-(–)-atenolol as internal standard were conducted at ambient temperature. The method is highly specific where another coformulated compounds did not interfere. The stability of betaxolol enantiomers under different degree of temperature also studied. The results showed that it is stable for at least 7 days at 70°C. The method validated for its linearity, accuracy, precision and robustness. Experimental design was used during validation to evaluate method robustness. Using the chromatographic conditions described, S- and R-betaxolol were well resolved with mean retention times of 11.3 and 12.6 min, respectively. Linear response (r > 0.997) was observed over the range of 10–500 ng/ml of betaxolol enantiomers, with detection limit of 5 ng/ml. The recoveries of S- and R-betaxolol from tablets and ophthalmic preparation ranged from 97.4 to 101.4% and 98.0 to 102.0%, respectively. The mean relative standard deviation (R.S.D.%) for both enantiomers were 1.1–1.4% and 1.3–1.7% in tablets and ophthalmic solution, respectively.Protein kinases catalyze the transfer of the γ-phosphoryl group of adenosine triphosphate (ATP) to the hydroxyl groups of protein side chains, and they play critical roles in regulating cellular signal transduction and other biochemical processes. They are attractive targets for today’s drug discovery and development, and many pharmaceutical companies are intensively developing various kinds of protein kinase inhibitors. A good example is the recent success with the Bcr-Abl tyrosine kinase inhibitor imatinib mesylate (Gleevec™) in the treatment of chronic myeloid leukemia. Though imatinib has dramatically improved the treatment of Bcr-Abl-positive chronic myeloid leukemia, resistance is often found in patients with advanced-stage disease. Several mechanisms have been proposed to explain this resistance, including point mutations within the Abl kinase domain, amplification of the bcr-abl gene, overexpression of the corresponding mRNA, increased drug efflux mediated by P-glycoprotein, and activation of the Src-family kinase (SFK) Lyn. We set out to develop a novel drug whose affinity for Abl is higher than that of imatinib and whose specificity in inhibiting Lyn is higher than that of SFK/Abl inhibitors such as dasatinib (Sprycel™) or bosutinib (SKI-606). Our work has led to the development of NS-187 (INNO-406), a novel Abl/Lyn dual tyrosine kinase inhibitor with clinical prospects. To provide an overview of how a selective kinase inhibitor has been developed, this review presents chemical-modification studies carried out with the guidance of molecular modeling, the structural basis for the high potency and selectivity of NS-187 based on the X-ray structure of the NS-187/Abl complex, and the biological profiling of NS-187, including site-directed mutagenesis experiments.

In-vivo metabolism of 1-(3′-hydroxypropyl)-2-methyl-3-hydroxypyridin-4-one (CP41) and 1-(2′-hydroxyethyl)-2-ethyl-3-hydroxypyridin-4-one (CP102) by rat

Both 1 -(3 ’-hydroxypropyl)-2-methyl-3 -hydroxy-pyridin4-one (CP4 1) and 1 -(2’-hydroxyethyl)-2-ethyl-3hydroxypyridin-4-one (CP 102) are orally effective iron chelators, which are able to enhance the excretion of iron in vivo (Zaninelli et al. 1997). Although the pharmacokinetics of CP102 and its metabolic profile in urine has been previously reported (Singh et al. 1996), its biliary metabolic profile remains unclear. In addition, a clear understanding of the metabolism of CP41 is needed in order to study its pharmacokinetic characteristics. The rat bile duct cannulation model was utilised in the present investigation. After the animals were orally administered with a single dose of CP102 or CP41 (450pmol/kg body weight), the bile samples were collected hourly up to a period of 10 hours. The urine samples were also collected using separate animals without bile duct cannulation. The bile and urine samples were analysed using a HPLC method and the amounts of iron excreted in bile and urine were determined utilising a colorimetric method. In order to determine the conjugated metabolites of CP41 and CP102, samples were treated separately in the presence of glucuronidase or sulfatase before being applied for HPLC analysis. The elimination of CP102 from rat urine showed a similar pattern to that of a previous report (Zaninelli et al. 1997), i.e. the unchanged CP102 was the dominant form excreted in urine. Minor amounts of glucuronide and sulfate metabolites of CP102 were also found in urine. In the bile, the unchanged CP102 was only found to be a major elimination form, however, glucuronide and sulfate metabolites were excreted in approximately equal amounts to that of CP 102. Unlike CP102, 1 -(3 ’-hydroxy) group oxidation of CP41 was demonstrated to be the main metabolic pathway. The resulting oxidative metabolite (CP38), was excreted at high levels (-30%) in the urine. Other CP41 elimination forms in urine included the unchanged CP4 1 (-45%), glucuronide (-10%) and sulfate (-15%) of CP41. The biliary metabolic profile of CP41 was again found to be different to its urinary metabolic profile. CP41 is rapidly oxidised to CP38 in the liver and the latter is excreted in bile forming over 90% of the total recovery of all elimination forms of CP41. Thus unchanged CP41 was excreted in bile to a much lesser extent than in urine. Small amounts of conjugated metabolites of CP4 1 were also detected in the bile. In the normal rat, CP102 is found to be more efficient than CP41 at removing iron from the liver. This is surprising, as when CP102 is administered, over 60% of pyridinone secreted in the bile is conjugated (and therefore unable to chelate iron) whereas with CP41 over 90% of the pyridinone secreted is nonconjugated. This may indicate that the negatively charged CP38 can not access the major labile iron pool as efficiently as CP41. Lysosomal compartmentalisation could account for this difference, a factor currently under investigation.

MALDI mass spectrometric determination of dendritic iron chelation stoichiometries and conditional affinity constants

The iron chelation stoichiometries of a dendritic iron(III) chelator with N(1), N(3), N(5)-trimethylbenzene-1,3,5-tricarboxamide at its core, and containing 3 identical hexadentate tris-hydroxypyridinone branches D was studied by MALDI mass spectrometry. At pH 7.2, the speciation of the system included FeD, Fe(2)D and Fe(3)D species with the respective conditional stability constants of 26.74, 26.03 and 25.36. The differences in the stepwise affinity constants arise from the statistical distribution of iron(III), and there was no evidence for cooperativity between the iron-binding sites.

Characterization of two isomeric β‐D‐glucosiduronic acids derived from 1,2‐diethyl‐3‐hydroxypyridin‐4‐one (CP94) in rat liver homogenate incubates

1,2‐Diethyl‐3‐hydroxypyridin‐4‐one (CP94) is an orally active iron chelator with potential for use in photodynamic therapy. This investigation reports the formation and characterization of two isomeric glucuronides of CP94 in rat liver homogenate incubates. To assign the glucuronidation sites in the CP94 molecule, two O‐methylated derivatives of CP94 have been synthesized. By comparing the spectral characteristics of the CP94 3‐O‐ and 4‐O‐methyl derivatives with CP94 and the CP94 glucuronides formed during incubation, evidence was obtained which enabled the assignment of these two isomeric glucuronides to the 3‐O‐glucuronide and 4‐O‐glucuronide of CP94. It was found that the 3‐O‐glucuronide was the dominant CP94 metabolite under in‐vitro conditions. In an attempt to understand the potential influence of structural variation on the glucuronidation of CP94 analogues, the 1‐and 2‐monoethyl derivatives of CP94 were investigated. The 2‐monoethyl derivative of CP94 yielded only the 3‐O‐glucuronide in rat liver homogenate incubate, while no glucuronide was formed from the 1‐monoethyl derivative. In addition, no glucuronide from the 3‐O‐methyl or 4‐O‐methyl derivatives of CP94 could be detected. The relevance of these findings to the development of new 3‐hydroxypyridin‐4‐one iron chelators is discussed.

3-Hydroxpyridin-4-one prodrugs as novel antimalarials

Unfortunately, many of the compounds which show good antimalarial activity are also toxic to mammalian cells due to low selectivity as a result of their distribution coefficients (D7.4). To date, the one exception to be tested is CP38 (Rl= CH~CHZCO~H, R2= CH3) [Hershko et al, 19921. This compound has an extremely low D7.4 (<O.OOO 1 ), and consequently penetrates host cellular membranes only slowly (therefore minimising toxicity). It is predicted that CP38 enters cells infected with malaria by another mechanism, possibly a carrier mediated transport system that is induced/ activated in the parasitised erythrocyte. This property also leads to poor bioavailability, associated with its relatively low oral activity. The prodrug concept has been utilised to overcome the problem of poor oral absorption and to facilitate the transport of CP38 to the blood supply. A number of esters have been synthesised. (Tablel). Table 1. Distribution coefficients of CP38 ester prodrugs.

Design of 3-hydroxypyridin-4-one chelators with high pFe3+ values

Regular blood transfusion can lead to iron overload and associated toxicity. Desferrioxamine (DFO) has been used to scavenge excess iron, however because of the inconvenient administration, we have attempted to identify an orally active, nontoxic and selective iron chelator. 3-Hydroxypyridin-4-one (HPO) chelators have currently been developed as an alternative to DFO (Tilbrook and Hider, 1998). CP20 (1,2-dimethy1-3-hydroxypyridin-4-one), is currently in clinical trials but has been found to be (Brittenham, 1992). improvement.

Design of ester prodrugs of 3-hydroxypyridin-4-one chelators with clinical potential

3-Hydroxypyridin-4-ones (HPOs) are currently one of the main candidates for the development of orally active iron chelators (Hider et al. 1996). The simple lY2-dialkyl derivatives are highly effective at removing iron from iron overloaded animals including man but are associated with two disadvantages; (i) they penetrate cells easily and therefore gain ready access to the bone marrow and the brain and (ii) they are rapidly conjugated with glucuronic acid, thereby loosing their iron binding properties (Singh et al. 1992). Designing more hydrophilic hydroxypyridinones, for instance hydroxyalkyl hydroxypyridinone can effectively decrease penetration of membranes. Some of 1 hydroxyalkyl derivatives of HPOs such as CP102 and CP41 (Table) are not extensively metabolised via phase I1 reactions and therefore their chelating action is more prolonged (Singh et al. 1996). Not surprisingly the increased hydrophilicity of such compounds is associated with relatively poor oral absorption and insufficient extraction by the liver, which is the major iron storage organ. A strategy to improve chelation efficacy and hence to minimize drug-induced toxicity can be achieved by the selective delivery of the drug to target organs such as the liver. The development of hydrophobic ester prodrugs of 1 -hydroxyalkyl HPO derivatives is one route, which has been considered to improve both drug absorption and hepatic extraction.

In-vivo metabolism of 1, 2-diethyl-3-hydroxypyridin-4-one (CP94) by rat

1,2-DiethyI-3-hydroxypyridin-4-one (CP94, I) is an extensively studied orally effective iron chelator. The metabolic profiles of this compound in the urine and blood of rats have been previously investigated (Epemolu et al. 1994; Singh et al. 1992; Epemolu et al. 1992). However, the biliary metabolic profile of CP94 and its relationship with iron excretion have not been investigated. As bile is the main pathway for iron secretion in rat, the establishment of the biliary metabolic profile of CP94 was considered to be critically important. The present study compares the biliary and urinary metabolic profiles of CP94 in the normal rat. The rat bile duct cannulation model was used to collect bile samples up to a period of 10 hours after oral administration of a single dose of CP94 (450 pmole/kg). The urine samples were collected using separate animals without bile duct cannulation. The bile and urine samples were analysed using a HPLC method (Singh et al. 1992) and the amount of iron excreted in the bile and urine was determined using a colorimetric method (Gosriwatana and Hider, 1997). In order to determine the potential conjugated metabolites of CP94, the bile and urine samples were treated with either P-glucuronidase or sufatase before application for HPLC analysis. The results confirmed the CP94 urinary metabolic profile reported by Singh (1992). Only low levels of unchanged CP94 were detected in the urine. However, two glucuronidated metabolites of CP94 were found to be major elimination forms in the urine. One is the 3-glucuronide (11), whilst the other is likely to be the 4-glucuronide (111). The precise assignment of these two peaks requires further investigation. In addition, the 2-( 1 -hydroxy) metabolite of CP94 (IV) was found to be the dominant metabolite in urine. Unlike the urinary profile, the parent CP94 and its conjugated metabolites (I1 and III) were all found to be major excreted forms in the bile. Metabolite IV was also detected in the bile, but at low level. In addition, an unstable and potentially toxic metabolite, possibly the 6-hydroxy metabolite (V), was detected in the bile. When the bile was collected and processed under nitrogen, metabolite V was detected in much larger amounts, indeed it is clearly one of the major metabolites of CP94. This metabolite is destroyed when the bile is incubated in the presence of oxygen. It is likely that metabolite V is oxidised to a reactive semi-quinone or quinone and that this metabolite could therefore account for some of the toxicity associated with CP94. The excretion of iron in bile showed a parallel pattern to the excretion of the parent CP94 but in the molar ratio range of 0.9-1.2. This range of values implies that the active metabolites of CP94, e.g. IV and V, may also play an important role in iron secretion as the iron will be excreted as a 3:l ligand-metal complex. ,e ,&lucumnic acid