Molecular factors affecting the complex formation between deferiprone (L1) and Cu(II). Possible implications on efficacy and toxicity.

Deferiprone (1,2-dimethyl-3-hydroxypyrid-4-one, L1, CAS 30652-11-0) is a new chelating drug used worldwide for the treatment of iron overloading conditions. Spectrophotometric and potentiometric measurements were carried out to investigate the interaction of L1 with Cu(II) ions under different conditions. The complexation of Cu(II) ions with L1 in aqueous solution leads predominantly to the formation of the Cu(L1)2 species at a pH range of 4-9. The experimental results indicate that L1 has high affinity for Cu(II) with stability constants log beta 11 = 10.3 +/- 0.9 and log beta 12 = 19.2 +/- 0.6. The effect of Cu(II) ions on the affinity of L1 for Fe(III) ions by competition reactions in vitro indicate displacement of Fe(III) in a concentration dependent manner by Cu(II). Similarly, the presence of different buffers at various pH values resulted in the formation of different stoichiometry L1 complexes with Cu(II) and of mixed complexes with buffer anions. The strong interaction of L1 with Cu(II) may have implications on the therapeutic and toxicological properties of this chelating drug. In particular, L1 may be used in the treatment of copper overloading conditions, such as Wilson's disease or other conditions where copper toxicity is implicated.     

The design and development of deferiprone (L1) and other iron chelators for clinical use: targeting methods and application prospects

Iron is essential for all human cells as well as neoplastic cells and invading microbes. Natural and synthetic iron chelators could affect biological processes involving iron and other metal ions in health and disease states. Iron overload is the most common metal toxicity condition worldwide. There are currently two iron chelating drugs, which are mostly used for the treatment of thalassaemia and other conditions of transfusional iron overload. Deferoxamine was until recently the only approved iron chelating drug, which is effective but very expensive and administered parenterally resulting in low compliance. Deferiprone (L1 or 1,2-dimethyl-3-hydroxypyrid-4-one) is the world's first and only orally active iron chelating drug, which is effective and inexpensive to synthesise thus increasing the prospects of making it available to most thalassaemia patients in third world countries who are not currently receiving any form of chelation therapy. Deferiprone has equivalent iron removal efficacy and comparable toxicity to deferoxamine. There are at least four other known iron chelators, which are currently being developed. Even if successful, these are not expected to become available for clinical use in the next five years and to be as inexpensive as deferiprone. The variation in the chemical, biological, pharmacological, toxicological and other properties of the chelating drugs and experimental chelators provide evidence of the difference in the mode of action of chelators and the need to identify and select molecular structures and substituents based on structure/activity correlations for specific pharmacological activity. Such information may increase the prospects of designing new chelating drugs, which could be targeted and act on different tissues, organs, proteins and iron pools that play important role not only in the treatment of iron overload but also in other diseases of iron and other metal imbalace and toxicity including free radical damage. Chelating drugs could also be designed, which could modify the enzymatic activity of iron and other metal containing enzymes, some of which play a key role in many diseases such as cancer, inflammation and atherosclerosis. Other applications of iron chelating drugs could involve the detoxification of toxic metals with similar metabolic pathways to iron such as Al, Cu, Ga, In, U and Pu.     

Pharmaceutical analysis of the oral iron chelator deferiprone (DMHP, L1)

The oral iron chelator deferiprone (1,2-dimethyl-3-hydroxypyrid-4-one, DMHP, L1 or CP20) can be a useful drug in patients with transfusional hemosiderosis. From 1987 about 1000 patients in 16 countries have taken this drug on the base of clinical trials or compassionate use. Since this compound is only available as a raw substance, it is important to ascertain its purity before bringing the drug into a pharmaceutical formulation. Because deferiprone is administered chronically and in high doses, intake of potential toxic impurities can be substantial.In this article a proposal for the quality control of deferiprone is presented in the form of a pharmaceutical monograph. This includes the analytical methods required for identification, purity checking and assay. Furthermore the way we synthesized the drug to get hold of it in a pure form is described. This synthesis is also used in manufacturing the drug commercially. The monograph can be used as a guideline for standardization of the quality of deferiprone to be used for further study and treatment.     

Clinical use,therapeutic aspects and future potential of deferiprone in thalassemia and other conditions of iron and other metal toxicity

The therapeutic aspects and future prospects of the new iron chelating drug deferiprone are reviewed, with an emphasis on its clinical use in thalassemia and other conditions of iron overload, imbalance and toxicity, as well as its possible use in other metal toxicity conditions. Orally administered deferiprone appears to be as effective as subcutaneous deferoxamine in the removal of iron in transfused iron loaded patients, with an equivalent therapeutic index profile in both animals and humans. Only about 10% of patients requiring iron chelation therapy worldwide receive deferoxamine mainly because of its high cost, toxicity and low compliance with subcutaneous administration. Deferiprone has been used by over 6000 patients in 40 countries worldwide, in some cases daily for more than 10 years, with very promising results. Doses of 50-120 mg/kg/day are effective in bringing patients to negative iron balance. Deferiprone increases urinary iron excretion, decreases serum ferritin levels and reduces liver iron in the majority of chronically transfused iron loaded patients. All of the toxic side effects of deferiprone are considered reversible and manageable, and include agranulocytosis, musculoskeletal and joint pains, gastrointestinal complaints and zinc deficiency. In general, the incidence of toxic side effects could be reduced by using lower doses or combination therapy with deferoxamine. The suggestion that deferiprone therapy may cause liver fibrosis has not been confirmed. New therapeutic protocols for maximizing the efficacy and minimizing the toxicity of deferiprone are being considered based on new findings in relation to its metal chelation, pharmacological, toxicological and metabolic properties. (c) 2001 Prous Science. All rights reserved.     

Efficacies of cyclodextrin-complexed and liposome-encapsulated clarithromycin against Mycobacterium avium complex infection in human macrophages

Cyclodextrins and liposomes have been used in recent years as drug delivery vehicles, improving the bioavailability and therapeutic efficacy of many poorly water-soluble drugs. In this study, we used two approaches to enhance the availability of the poorly water-soluble antibiotic, clarithromycin, by inclusion complex formation and by liposome-encapsulation. We examined the efficacies of these formulations against Mycobacterium avium complex (MAC) in human peripheral blood monocyte-derived macrophages. The water solubility of clarithromycin was enhanced by about 700-fold by complexation with cyclodextrin. The use of a rapid radiometric (BACTEC) method for the detection of MAC growth and susceptibility showed identical MICs against MAC for both the free and complexed drug. The anti-MAC efficacy of the cyclodextrin complex of clarithromycin in macrophages was slightly lower than the free drug, probably due to the high stability of the inclusion complex. At higher drug concentrations, Liposome-encapsulated clarithromycin was slightly more effective against intracellular MAC growth than the free drug.     

Advances in iron overload therapies. prospects for effective use of deferiprone (L1), deferoxamine, the new experimental chelators ICL670, GT56-252, L1NA11 and their combinations

Effective new therapies and mechanisms have been developed for the targeting and prevention of iron overload and toxicity in thalassaemia and idiopathic haemochromatosis patients. A new era in the development of chelating drugs began with the introduction of deferiprone or L1, which as a monotherapy or in combination with deferoxamine can be used universally for effective chelation treatments, rapid iron removal, maintenance of low iron stores and prevention of heart and other organ damage caused by iron overload. Several experimental iron chelators such as deferasirox (4-[3,5-bis (2-hydroxyphenyl)-1,2,4-triazol-1-yl]-benzoic acid) or ICL670, deferitrin (4,5-dihydro-2- (2,4-dihydroxyphenyl)-4-methylthiazole-4 (S)-carboxylic acid) or GT56-252, 1-allyl-2-methyl-3-hydroxypyrid-4-one or L1NAll and starch deferoxamine polymers have reached different stages of clinical development. The lipophilic ICL670, which can only be administered once daily is generally ineffective in causing negative iron balance but is effective in reducing liver iron. It is suspected that it may increase iron absorption and the redistribution of iron from the liver to the heart and other organs. The experimental iron chelators do not appear to have significant advantages in efficacy and toxicity by comparison to deferiprone, deferoxamine or their combination. However, the prospect of combination therapies using deferiprone, deferoxamine and new chelators will provide new mechanisms of chelator interactions, which may lead to higher efficacy and lower toxicity by comparison to monotherapies. A major disadvantage of the experimental chelators is that even if they are approved for clinical use, they are unlikely to be as inexpensive as deferiprone and become available to the vast majority of thalassaemia patients, who live in developing countries.     

UGT1A6 genotype-related pharmacokinetics of deferiprone (L1) in healthy volunteers

AIMS

To examine the effects of UGT1A6 polymorphisms on the pharmacokinetics of deferiprone in healthy volunteers.

METHODS

Twenty-two healthy volunteers were enrolled and grouped according to UGT1A6 genotype. After an overnight fast, the subjects received a single oral dose of 25 mg kg⁻¹ deferiprone. Blood samples were collected at 0, 15, 30, 45, 60, 90, 120, 180, 240, 300 and 360 min after dosing. Urine output was collected at 0, 0–2, 2–4, 4–8, 8–12 and 12–24 h. Deferiprone (L1) and deferiprone-glucuronide (L1G) concentrations in serum and urine were determined using a validated high-performance liquid chromatography method. UGT1A6 genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism analysis.

RESULTS

No statistically significant differences in any pharmacokinetic parameters of either deferiprone or deferiprone-glucuronide among the genotype groups were noted. Likewise, there were no statistically significant differences in 24-h urinary deferiprone and deferiprone-glucuronide excretion among the genotype groups. Significant differences between men and women were found in AUC_0–∞, V_d/F, and CL/F of deferiprone. Gender differences in 24-h urinary deferiprone and its metabolite excretion, however, failed to reach statistical significance. The V_d/F of deferiprone was found to correlate significantly with serum ferritin (r_s = 0.665; P = 0.001).

CONCLUSION

The studied single nucleotide polymorphisms in UGT1A6 do not appear to exert statistically significant effects on the single-dose pharmacokinetics of deferiprone. Gender appears to influence the serum pharmacokinetics of deferiprone, but not urinary excretion of deferiprone and its metabolite. Body iron stores may have an influence on the extent of extravascular deferiprone distribution.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT

• UGT1A6 has been proposed as the predominant isoform responsible for the glucuronidation of deferiprone.
• UGT1A6*2 allele has been associated with the altered enzyme activity.

WHAT THIS STUDY ADDS

• There is no statistically significant effect of UGT1A6 genotype on the single-dose pharmacokinetics of deferiprone in healthy volunteers.
• Gender influences serum pharmacokinetics of deferiprone.
• Body iron stores reflected by serum ferritin levels may have an influence on the extent of extravascular deferiprone distribution.

     

Serotype-specific modulation of human monocyte functions by glycopeptidolipid (GPL) isolated from Mycobacterium avium complex

Immunomodulating activity of glycopeptidolipids (GPL), separated from different serovars of Mycobacterium avium complex (MAC), on macrophage functions was compared. When peripheral blood mononuclear cells (PBMC), from healthy donors showing strongly positive reactions to mycobacterial purified protein derivatives (PPD), were incubated with heat-killed Staphylococcus aureus coated with GPL serovar 4, phagocytosis of monocytes increased in dose-dependent manner. However, coating with GPL serovar 9 did not show any effects. After phagocytosis of heat-killed S. aureus, the phagosome-lysosome (P-L) fusion in monocytes was inhibited dose-dependently by coating of S. aureus with GPL serovar 4, but not serovar 9. These results indicate that GPL serovar 4 facilitates invasion of MAC into monocytes and renders resistance to bactericidal reactions due to the inhibition of P-L fusion. Regarding accessory function of macrophages in proliferative responses of T cells, the addition of GPL serovar 4 to cultures resulted in significant inhibition of anti-CD3 monoclonal antibody (mAb)-induced proliferation, whereas both serovar GPLs did not cause reduction of cell viability. Furthermore, the PPD-specific T cell proliferative response was downregulated markedly by GPL serovar 4, but weakly suppressed by GPL serovar 9. These results indicated that the immunomodulating activity of GPL on macrophage functions is serovar-dependent.     

Effect of poloxamer CRL-1072 on drug uptake and nitric-oxide-mediated killing of Mycobacterium avium by macrophages

Mycobacterium avium-intracellulare complex (MAI) are common pathogens of opportunistic infections that are naturally resistant to most antibiotics and develop acquired resistance rapidly. An experimental drug, poloxamer CRL-1072, was found to have two unusual properties: it synergistically enhanced the activity of several antibiotics against MAI even though it had little activity as a single agent and it had greater activity against MAI in macrophage culture or in mice than in broth culture. Studies were undertaken to investigate the mechanisms of these effects. CRL-1072 was taken up by MAI and enhanced the uptake of fluorescent-labeled streptomycin and erythromycin in broth culture. The labeled antibiotics had reduced activity so the relevance for naive antibiotics must be inferred. In culture with human U937 monocytoid cells, CRL-1072 became localized in phagosomes and promoted uptake of streptomycin. Finally, CRL-1072 was found to induce production of mRNA for inducible nitric oxide synthase (iNOS) and nitric oxide (NO) by U937 cells. The antimycobacterial effect in macrophages was reversed by the iNOS inhibitor N-monomethyl L-arginine (NMMA), suggesting that CRL-1072 promotes killing of MAI by inducing NO. These effects were induced by noncytotoxic concentrations of CRL-1072. These data suggest that the antimycobacterial mechanisms of CRL-1072 include enhancing the delivery of antibiotic to targets within MAI and enhancement of the ability of macrophages to kill ingested organisms.     

红花对活化巨噬细胞产生一氧化氮的影响作用

目的 水提醇沉法对红花进行提取,研究红花提取物对脂多糖(LPS)活化小鼠巨噬细胞RAW264.7产生一氧化氮(NO)的影响作用,同时测定提取物中的总黄酮含量.方法 ATP-Lite法测定红花对RAW264.7细胞毒性的影响,姜黄素为阳性对照.采用Griess法测定由LPS活化RAW264.7细胞产生NO的含量,阿司匹林为阳性对照.以山奈酚为标准品测定红花提取物中总黄酮的含量.结果 姜黄素对RAW264.7的半数致死浓度为(29.9±4.3)μg/ml.红花提取物在低于125μg/ml时,对RAW264.7细胞的致死率低于5％.在12.5～100 μg/ml的浓度范围内,红花表现出对LPS活化RAW264.7细胞产生NO的抑制作用.以山奈酚计,1 g生药红花中含1.14 mg总黄酮.结论 红花对LPS活化巨噬细胞产生NO具有抑制作用,其作用可能与其所含黄酮类成分有关.     

Benefits and risks of deferiprone in iron overload in Thalassaemia and other conditions: comparison of epidemiological and therapeutic aspects with deferoxamine.

Deferiprone is the only orally active iron-chelating drug to be used therapeutically in conditions of transfusional iron overload. It is an orphan drug designed and developed primarily by academic initiatives for the treatment of iron overload in thalassaemia, which is endemic in the Mediterranean, Middle East and South East Asia and is considered an orphan disease in the European Union and North America. Deferiprone has been used in several other iron or other metal imbalance conditions and has prospects of wider clinical applications. Deferiprone has high affinity for iron and interacts with almost all the iron pools at the molecular, cellular, tissue and organ levels. Doses of 50-120 mg/kg/day appear to be effective in bringing patients to negative iron balance. It increases urinary iron excretion, which mainly depends on the iron load of patients and the dose of the drug. It decreases serum ferritin levels and reduces the liver and heart iron content in the majority of chronically transfused iron loaded patients at doses >80 mg/kg/day. It is metabolised to a glucuronide conjugate and cleared through the urine in the metabolised and a non-metabolised form, usually of a 3 deferiprone: 1 iron complex, which gives the characteristic red colour urine. Peak serum levels of deferiprone are observed within 1 hour of its oral administration and clearance from blood is within 6 hours. There is variation among patients in iron excretion, the metabolism and pharmacokinetics of deferiprone. Deferiprone has been used in more than 7500 patients aged from 2-85 years in >50 countries, in some cases daily for >14 years. All the adverse effects of deferiprone are considered reversible, controllable and manageable. These include agranulocytosis with frequency of about 0.6%, neutropenia 6%, musculoskeletal and joint pains 15%, gastrointestinal complains 6% and zinc deficiency 1%. Discontinuation of the drug is recommended for patients developing agranulocytosis. Deferiprone is of similar therapeutic index to subcutaneous deferoxamine but is more effective in iron removal from the heart, which is the target organ of iron toxicity and mortality in iron-loaded thalassaemia patients. Deferiprone is much less expensive to produce than deferoxamine. Combination therapy of deferoxamine and deferiprone has been used in patients not complying with subcutaneous deferoxamine or experiencing toxicity or not excreting sufficient amounts of iron with use of either drug alone. New oral iron-chelating drugs are being developed, but even if successful these are likely to be more expensive than deferiprone and are not likely to become available in the next 5-8 years. About 25% of treated thalassaemia patients in Europe and more than 50% in India are using deferiprone. For most thalassaemia patients worldwide who are not at present receiving any form of chelation therapy the choice is between deferiprone and fatal iron toxicity.     

Disposition, accumulation and toxicity of iron fed as iron (II) sulfate or as sodium iron EDTA in rats.

A study was performed to provide data on the disposition, accumulation and toxicity of sodium iron EDTA in comparison with iron (II) sulfate in rats on administration via the diet for 31 and 61 days. Clinical signs, body weights, food consumption, food conversion efficiency, hematology, clinical chemistry and pathology of selected organs were used as criteria for disclosing possible harmful effects. Determination of iron and total iron binding capacity in blood plasma and non-heme iron analysis in liver, spleen and kidneys were used to assess the disposition and accumulation of iron originating from sodium iron EDTA or iron (II) sulfate. It was concluded that, under the conditions of the present study, iron is accumulated from the diet in liver, spleen and kidneys in a dose-dependent manner, and iron derived from FeEDTA is taken up and/or accumulated less efficiently in liver and spleen than iron from FeSO(4). Moreover, feeding iron up to 11.5 and 11.2 mg/kg body weight/day, derived from FeSO(4) and FeEDTA, respectively, did not result in tissue iron excess nor in any other toxicologically significant effects.     

Growth inhibition of Mycobacterium bovis, Mycobacterium tuberculosis and Mycobacterium avium in vitro: effect of 1-beta-D-2'-arabinofuranosyl and 1-(2'-deoxy-2'-fluoro-beta-D-2'-ribofuranosyl) pyrimidine nucleoside analogs

The resurgence of tuberculosis and the emergence of multiple-drug-resistant strains of Mycobacteria necessitate the search for new classes of antimycobacterial agents. We synthesized a series of 1-beta-D-2'-arabinofuranosyl and 1-(2'-deoxy-2'-fluoro-beta-D-ribofuranosyl) pyrimidine nucleosides possessing diverse sets of alkynyl, alkenyl, alkyl, and halo substituents at the C-5 position of the uracil and investigated their effect on activity against M. tuberculosis, M. bovis, and M. avium. Among these molecules, 5-alkynyl-substituted derivatives emerged as potent inhibitors of M. bovis, M. tuberculosis, and M. avium. Nucleosides 1-beta-D-2'-arabinofuranosyl-5-dodecynyluracil (5), 1-(2'-deoxy-2'-fluoro-beta-D-ribofuranosyl)-5-dodecynyluracil (24), and 1-(2'-deoxy-2'-fluoro-beta-D-ribofuranosyl)-5-tetradecynyluracil (25) showed the highest antimycobacterial potency against M. bovis and M. tuberculosis. The MIC90 exhibited by compounds 5, 24, and 25 was similar or close to that of the reference drug rifampicin. The most active compounds 5, 24, and 25 were also found to retain sensitivity against a rifampicin-resistant strain of M. tuberculosis H37Rv at similar concentrations. Some of these analogs also revealed in vitro antimicrobial effect against several other gram-positive pathogens.     

Protective effect of metallothionein against the toxicity of cadmium and other metals(1)

In the present study, we have investigated the effects of extracellular redox status and metal/thiol interactions on glutathione distribution in HeLa cell cultures. No effects were seen on glutathione distribution after the addition of different thiols, whereas the pro-oxidant copper ions affected glutathione distribution in several ways. The addition of dithiothreitol (DTT) but not the other thiols potentiated the effects of mercury ions on glutathione distribution and cell toxicity. In the presence of DTT, increased intra- and extracellular glutathione concentrations were noted already at 0.05 micromol/l, which was below the previously reported toxicity threshold for mercury ions in blood. Likewise DTT potentiated the effects of copper ions on glutathione distribution and cell toxicity, whereas the addition of DTT to cell cultures with a non-metal thiol reactive agent (hydroquinone) or an oxidative agent (hydrogen peroxide) did not affect glutathione distribution or cell toxicity. Thus, it seems as the synergistic effects between DTT and thiol reactive agents only apply to metal ions.     

Possible role of glutathione in chromium(VI) metabolism and toxicity in rats

The effect of Cr(VI) on liver, kidney, and lung glutathione (GSH) levels and the effect of GSH depletion on Cr(VI)-induced nephrotoxicity were studied in male Sprague-Dawley rats (150-200 g). GSH levels, measured as nonprotein sulfhydryls, were determined between 0.5 and 26 hr after intraperitoneal injection of the maximum non-toxic dose of sodium dichromate (10 mg/kg). While Cr(VI) at this dose did not significantly change hepatic, renal, or pulmonary GSH levels, there appeared to be an initial decrease of hepatic GSH followed by an increase to approximately 120% of control between 5 and 12.5 hr after Cr(VI) treatment. The increase in hepatic GSH levels was significant 5 hr after treatment with 20 mg/kg sodium dichromate, was manifested as an increase in both non-protein sulfhydryls and total glutathione, and was prevented by L-buthionine sulfoximine (BSO) pretreatment. In rats pretreated with 4.0 mmol/kg BSO to deplete GSH, subsequent treatment with Cr(VI) further reduced hepatic GSH levels 2 hr after Cr(VI) treatment and inhibited weight gain in the first 24 hr after treatment. Intraperitoneal injection of Cr(VI) did not inhibit hepatic glutathione reductase activity, even at toxic doses. Depletion of renal GSH to approximately 25% of control with BSO potentiated the acute nephrotoxicity of 30 mg/kg sodium dichromate as measured by serum urea nitrogen levels and relative kidney weight. However, GSH depletion with BSO did not appear to affect the incidence of glucosuria, haematuria, or lysozymuria over a range of Cr(VI) doses, nor did it affect renal uptake of Cr. Taken together, these data show that GSH protects against the acute nephrotoxicity of Cr(VI), although it is not clear whether GSH is directly involved in the intracellular metabolism of Cr(VI) at non-toxic doses.     

Effects of selenium on toxicity and ultrastructural localization of silver in cultured macrophages

The effects of selenium on cellular toxicity and histochemical distribution of silver were examined in a cell culture system of mouse peritoneal macrophages. Selenium caused a significant delay in the appearance of coagulation necrosis induced by high silver concentrations and reduced the cytostatic effect of lower doses of silver when long-term toxicity was examined. Furthermore, selenium increased the amount of silver that could be visualized by autometallography. The additional silver made available for this histochemical demonstration was located in the cytosol as well as in lysosomes, the sole localization of silver when selenium was not administered.     

Synergistic inhibition of human immunodeficiency virus type 1 (HIV-1) replication in vitro by 1-[(2-hydroxyethoxy)methyl]-6-phenylthiothymine (HEPT) and recombinant alpha interferon.

1-[(2-Hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) has recently proved to be a potent and selective inhibitor of human immunodeficiency virus type 1 (HIV-1) in vitro. Combinations of HEPT and recombinant alpha interferon (IFN-alpha) synergistically inhibit the replication of HIV-1 in MT-4 cells at non-toxic concentrations. Synergistic inhibition of HIV-1 replication has also been observed in peripheral blood lymphocytes. These results indicate that the combination of HEPT with IFN-alpha should be further pursued in the treatment of retrovirus infections [i.e. acquired immune deficiency syndrome (AIDS)].