Ding Yong Liu

Oral ferrous sulphate leads to a marked increase in pro‐oxidant nontransferrin‐bound iron

Sir, Serum nontransferrin-bound iron (NTBI) is a potential catalyst for the production of reactive oxygen species, contributing to tissue damage. Nontransferrin-bound iron not only occurs in iron overload but also during intravenous iron infusion, possibly owing to the rapid saturation of transferrin, as a result of a rapid influx of iron [1]. More recently, NTBI was found to be positively correlated with lipid peroxidation in Beta-Thalassaemia and haemodialysis patients [2,3], although the relationship between NTBI and oxidative damage in vivo is contentious. Nevertheless, the presence of NTBI may explain published reports of lipid peroxidation after oral FeSO 4 [4,5], and as this is the usual treatment for IDA the occurrence of pro-oxidant NTBI during FeSO 4 treatment could have serious clinical implications. Therefore, we aimed to determine whether oral FeSO 4 results in the formation of serum NTBI in anaemic women. Seven anaemic women (mean haemoglobin 111 ± 5 g L − 1 ; serum ferritin 11·4 ± 2 μ g L − 1 ) who were otherwise healthy and of childbearing age completed this study. All subjects were recruited from the student and staff population at Kings College London and were not taking any medications nor had any history of chronic illness including gastrointestinal disease, although the exact causes of anaemia were not investigated. After an overnight fast, subjects were administered a tablet containing 200 mg of FeSO 4 (65 mg of elemental iron; Alpharma, Barnstaple, UK), with two slices of toasted white bread, margarine, honey and dilute orange cordial (visit 1), or 200 mg of FeSO 4 with the same drink but without food (visit 2). The meal (including drink) was low in inhibitors of iron absorption and contained only a trace of vitamin C. Serial blood samples were taken through an indwelling venous catheter before and after oral iron for 4 h on both occasions. Blood was drawn into vacutainers without additives to determine total serum iron, total iron binding capacity (TIBC) and serum NTBI. Serum ferritin, full blood count, total serum iron and TIBC were measured using routine laboratory methods, and serum was prepared for NTBI analysis using a method described elsewhere [6] and analyzed using an iron-free high-performance liquid chromatography system. Transferrin saturation was calculated (serum iron × 100/TIBC), and the rate of iron absorption was estimated using a recently reported equation [7]. We used a repeated measures  to test the increase in total serum iron, NTBI and transferrin saturation following FeSO 4 , and a simple linear fit to evaluate the correlation between NTBI and iron absorption, and NTBI and transferrin saturation. Neither the increase in total serum iron nor NTBI differed significantly when FeSO 4 was taken with or without food (both P < 0·001; Fig. 1), probably because the meal was low in compounds that inhibit iron absorption and vitamin C levels were similarly low on both occasions. On both occasions, serum transferrin concentration was in the normal range and was similar at baseline and 210 min after 200 mg of FeSO 4 (visit 1: 2·65 ± 0·10 g L − 1 at 0 min vs. 2·67 ± 0·08 g L − 1 at 210 min; and visit 2: 2·71 ± 0·09 g L − 1 vs. 2·82 ± 0·08 g L − 1 ). However, transferrin saturation increased from baseline to 210 min post-FeSO 4 , in conjunction with an increase in serum NTBI (Fig. 2). The mean ( ± SE) peak increase in serum NTBI was 4·6 ± 0·5 μ mol L − 1 and 3·9 ± 1·1 μ mol L − 1 , following 200 mg of All authors contributed to the study concept, while C. Hutchinson designed the study and prepared the manuscript with contributions from C.A. Geissler, R.C. Hider and J.J. Powell. W. Al-Ashgar carried out the study under the supervision of C. Hutchinson, and D.Y. Liu analyzed serum samples for NTBI. All authors approved and contributed academically to the final manuscript.

Synthesis, physicochemical properties and biological evaluation of ester prodrugs of 3-hydroxypyridin-4-ones: design of orally active chelators with clinical potential

Abstract The synthesis of a range of hydrophobic ester prodrugs of 3-hydroxypyridin-4-ones with potential for oral administration is described. The distribution coefficient values of a range of these ester prodrugs and the corresponding alcohols in 1-octanol and MOPS buffer pH 7.4 are presented together with their rates of hydrolysis at pH 2, pH 7.4, in rat blood and liver homogenate. In vivo iron mobilisation efficacy of the pivaloyl and benzoyl prodrugs has been compared with their parent drugs using a 59 Fe-ferritin loaded rat model. Both classes of prodrug enhanced the ability of the parent hydroxypyridinone to facilitate the excretion of 59 Fe. The influence of the pivaloyl function was more marked than that of the benzoyl function. The optimal effect was observed with 1-[2′-(pivaloyloxy)ethyl]-2-methyl-3-hydroxy-4(1 H )-pyridinone 25 . However, not all the prodrugs provide increased efficacy which suggests that lipophilicity is not the only factor which influences the drug efficacy. The metabolism of the compound may have a dominating influence on the overall efficacy.

Synthesis, physicochemical properties and biological evaluation of aromatic ester prodrugs of 1-(2'-hydroxyethyl)-2-ethyl-3-hydroxypyridin-4-one (CP102): orally active iron chelators with clinical potential.

The synthesis of seven aromatic ester derivatives of 1‐(2′‐hydroxyethyl)‐2‐ethyl‐3‐hydro‐xypyridin‐4‐one is described. These ester prodrugs have been designed to target iron chelators to the liver, the major iron storage organ. In principle this should improve chelation efficacy and minimize toxicity.

Iron requirements based upon iron absorption tests are poorly predicted by haematological indices in patients with inactive inflammatory bowel disease

Fe deficiency and Fe-deficiency anaemia are common in patients with inflammatory bowel disease (IBD). Traditional clinical markers of Fe status can be skewed in the presence of inflammation, meaning that a patients Fe status can be misinterpreted. Additionally, Fe absorption is known to be down-regulated in patients with active IBD. However, whether this is the case for quiescent or mildly active disease has not been formally assessed. The present study aimed to investigate the relationship between Fe absorption, Fe requirements and standard haematological indices in IBD patients without active disease. A group of twenty-nine patients with quiescent or mildly active IBD and twenty-eight control subjects undertook an Fe absorption test that measured sequential rises in serum Fe over 4 h following ingestion of 200 mg ferrous sulphate. At baseline, serum Fe, transferrin saturation, non-transferrin-bound Fe (NTBI), ferritin and soluble transferrin receptor were all measured. Thereafter (30-240 min), only serum Fe and NTBI were measured. Fe absorption did not differ between the two groups (P = 0·9; repeated-measures ANOVA). In control subjects, baseline haematological parameters predicted Fe absorption (i.e. Fe requirements), but this was not the case for patients with IBD. Fe absorption is normal in quiescent or mildly active IBD patients but standard haematological parameters do not accurately predict Fe requirements.

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.

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.

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