Hanspeter Nick

Cardiac Iron Determines Cardiac T2*, T2, and T1 in the Gerbil Model of Iron Cardiomyopathy

Background—Transfusional therapy for thalassemia major and sickle cell disease can lead to iron deposition and damage to the heart, liver, and endocrine organs. Iron causes the MRI parameters T1, T2, and T2* to shorten in these organs, which creates a potential mechanism for iron quantification. However, because of the danger and variability of cardiac biopsy, tissue validation of cardiac iron estimates by MRI has not been performed. In this study, we demonstrate that iron produces similar T1, T2, and T2* changes in the heart and liver using a gerbil iron-overload model. Methods and Results—Twelve gerbils underwent iron dextran loading (200 mg · kg−1 · wk−1) from 2 to 14 weeks; 5 age-matched controls were studied as well. Animals had in vivo assessment of cardiac T2* and hepatic T2 and T2* and postmortem assessment of cardiac and hepatic T1 and T2. Relaxation measurements were performed in a clinical 1.5-T magnet and a 60-MHz nuclear magnetic resonance relaxometer. Cardiac and liver iron concentrations rose linearly with administered dose. Cardiac 1/T2*, 1/T2, and 1/T1 rose linearly with cardiac iron concentration. Liver 1/T2*, 1/T2, and 1/T1 also rose linearly, proportional to hepatic iron concentration. Liver and heart calibrations were similar on a dry-weight basis. Conclusions—MRI measurements of cardiac T2 and T2* can be used to quantify cardiac iron. The similarity of liver and cardiac iron calibration curves in the gerbil suggests that extrapolation of human liver calibration curves to heart may be a rational approximation in humans.

Development of Tridentate Iron Chelators: From Desferrithiocin to ICL670

Successful treatment of beta-thalassemia requires two key elements: blood transfusion and iron chelation. Regular blood transfusions considerably expand the lifespan of patients, however, without the removal of the consequential accumulation of body iron, few patients live beyond their second decade. In 1963, the introduction of desferrioxamine (DFO), a hexadentate chelator, marked a breakthrough in the treatment of beta-thalassemia. DFO significantly reduces body iron burden and iron-related morbidity and mortality. DFO is still the only drug for general use in the treatment of transfusion dependent iron overload. However, its very short plasma half-life and poor oral activity necessitate special modes of application (subcutaneous or intravenous infusion) which are inconvenient, can cause local reactions and are difficult to be accepted by many patients. Over the past four decades, many different laboratories have invested major efforts in the identification of orally active iron chelators from several hundreds of molecules of synthetic, microbial or plant origin. The discovery of ferrithiocin in 1980, followed by the synthesis of the tridentate chelator desferrithiocin and proof of its oral activity raised a lot of hope. However, the compound proved to be toxic in animals. Over a period of about fifteen years many desferrithiocin derivatives and molecules with broader alterations led to the discovery of numerous new compounds some of which were much better tolerated and were more efficacious than desferrithiocin in animals, however, none was safe enough to proceed to the clinical use. The discovery of a new chemical class of iron chelators: The bis-hydroxyphenyltriazoles re-energized the search for a safe tridentate chelator. The basic structure of this completely new chemical class of iron chelators was discovered by a combination of rational design, intuition and experience. More than forty derivatives of the triazole series were synthesized at Novartis. These compounds were evaluated, together with more than 700 chelators from various chemical classes. Using vigorous selection criteria with a focus on tolerability, the tridentate chelator 4-[(3,5-Bis-(2-hydroxyphenyl)-1,2,4)triazol-1-yl]-benzoic acid (ICL670) emerged as an entity which best combined high oral potency and tolerability in animals. ICL670 is presently being evaluated in the clinic.

ICL670A: Preclinical Profile

Man is unable to actively eliminate iron from the body, once it has been acquired. Toxic and eventually lethal levels of iron accumulate as a result of repeated transfusions, e.g. in s-thalassemia major, or due to excessive dietary iron uptake in anemias and hereditary hemochromatosis. Excess iron is deposited in the form of hemosiderins (insoluble “iron cores” of ferritin) mainly in the liver, spleen, many endocrine organs and in the myocardium. The exact mechanism of iron damage to these tissues is unknown, but it is established that organ failure correlates with iron burden in these tissues. Except for infectious diseases, cardiac complications are the major cause of death in s-thalassemia major patients.

Reduction in labile plasma iron during treatment with deferasirox, a once-daily oral iron chelator, in heavily iron-overloaded patients with β-thalassaemia

This subgroup analysis evaluated the effect of once‐daily oral deferasirox on labile plasma iron (LPI) levels in patients from the prospective, 1‐yr, multicentre ESCALATOR study. Mean baseline liver iron concentration and median serum ferritin levels were 28.6 ± 10.3 mg Fe/g dry weight and 6334 ng/mL respectively, indicating high iron burden despite prior chelation therapy. Baseline LPI levels (0.98 ± 0.82 μmol/L) decreased significantly to 0.12 ± 0.16 μmol/L, 2 h after first deferasirox dose (P = 0.0006). Reductions from pre‐ to post‐deferasirox administration were also observed at all other time points. Compared to baseline, there was a significant reduction in preadministration LPI that reached the normal range at week 4 and throughout the remainder of the study (P ≤ 0.02). Pharmacokinetic analysis demonstrated an inverse relationship between preadministration LPI levels and trough deferasirox plasma concentrations. Once‐daily dosing with deferasirox ≥20 mg/kg/d provided sustained reduction in LPI levels in these heavily iron‐overloaded patients, suggesting 24‐h protection from LPI. Deferasirox may therefore reduce unregulated tissue iron loading and prevent further end‐organ damage.

Antiproliferative and apoptotic effects in rat and human hepatoma cell cultures of the orally active iron chelator ICL670 compared to CP20: a possible relationship with polyamine metabolism

Abstract.  Objective: Iron loading has been observed to have a hyperproliferative effect on hepatocytes in vitro and on tumour cells in vivo; removal of this iron being required to induce antitumour activity. Material and Methods: Antiproliferative effects of orally active tridentate iron chelator ICL670 (deferasirox) and bidentate iron chelator CP20 (deferiprone), mediated through the chelation of intracellular iron, were compared in rat hepatoma cell line FAO and human hepatoma cell line HUH7. Results: In FAO cell cultures, we have shown that ICL670 decreased cell viability and DNA replication and induced apoptosis more efficiently than an iron‐binding equivalent concentration of CP20. Moreover, ICL670 decreased significantly the number of the cells in G2‐M phase. In the HUH7 cell cultures, ICL670 and a four‐time higher iron‐binding equivalent concentration of CP20, decreased cell viability and DNA replication in the same range. CP20 increased the number of the cells in G2‐M phase. However, ICL670 inhibited polyamine biosynthesis by decreasing ornithine decarboxylase mRNA level; in contrast, CP20 increased polyamine biosynthesis, particularly putrescine level, by stimulating spermidine‐spermine N1‐acetyl transferase activity that could activate the polyamine retro‐conversion pathway. By mass spectrometry, we observed that ICL670 cellular uptake was six times higher than CP20. Conclusions: These results suggest that ICL670 has a powerful antitumoural effect and blocks cell proliferation in neoplastic cells by a pathway different from that of CP20 and may constitute a potential adjuvant drug for anticancer therapy.

Safety and Efficacy of Combined Chelation Therapy with Deferasirox and Deferoxamine in a Gerbil Model of Iron Overload

Introduction: Combined therapy with deferoxamine (DFO) and deferasirox (DFX) may be performed empirically when DFX monotherapy fails. Given the lack of published data on this therapy, the study goal was to assess the safety and efficacy of combined DFO/DFX therapy in a gerbil model. Methods: Thirty-two female Mongolian gerbils 8–10 weeks old were divided into 4 groups (sham chelated, DFO, DFX, DFO/DFX). Each received 10 weekly injections of 200 mg/kg iron dextran prior to initiation of 12 weeks of chelation. Experimental endpoints were heart and liver weights, iron concentration and histology. Results: In the heart, there was no significant difference among the treatment groups for wet-to-dry ratio, iron concentration and iron content. DFX-treated animals exhibited lower organ weights relative to sham-chelated animals (less iron-mediated hypertrophy). DFO-treated organs did not differ from sham-chelated organs in any aspects. DFX significantly cleared hepatic iron. No additive effects were observed in the organs of DFO/DFX-treated animals. Conclusions: Combined DFO/DFX therapy produced no detectable additive effect above DFX monotherapy in either the liver or heart, suggesting competition with spontaneous iron elimination mechanisms for chelatable iron. Combined therapy was well tolerated, but its efficacy could not be proven due to limitations in the animal model.

Deferasirox reduces iron overload in a murine model of juvenile hemochromatosis.

Mutations in hemojuvelin (HJV) cause severe juvenile hemochromatosis, characterized by iron loading of the heart, liver, and pancreas. Knockout (KO) mice lacking HJV (Hjv−/−) spontaneously load with dietary iron and, therefore, present a model for hereditary hemochromatosis (HH). In HH, iron chelation may be considered in noncandidates for phlebotomy. We examined the effects of deferasirox, an oral chelator, in Hjv−/− mice. Hepatic, cardiac, splenic, and pancreatic iron were determined by measuring elemental iron and scoring histological sections. Heart and liver iron levels were also determined repeatedly by quantitative R2* magnetic resonance imaging (MRI). The time course of iron loading without intervention was followed from Week 8 of age (study start) to Week 20, when once-daily (5×/ week) deferasirox was administered, to Week 28. At 8 weeks, liver iron of KO mice was already markedly elevated versus wild-type mice (P < 0.001) and reached a plateau around Week 14. In contrast, Week 8 cardiac and pancreatic iron levels were similar in both KO and wild-type mice and, compared with the liver, showed a delayed but massive iron loading up to Week 20. Contrary to the liver, heart, and pancreas, the KO mice spleen had lower iron content versus wild-type mice. In Hjv−/− mice, liver and heart iron burden was effectively reduced with deferasirox 100 mg/kg (P < 0.05). Although deferasirox was less efficacious at this dose in the pancreas, over the observed time period, a clear trend toward reduced organ iron load was noted. There was no noticeable effect of deferasirox upon splenic iron in Hjv−/− mice. Quantitative R2* MRI demonstrated the ability to assess iron concentrations in the liver and myocardial muscle accurately and repetitively. Hepatic (R = 0.86; P = 3.2*10− 12) and delayed myocardial (R = 0.81; P = 2.9*10− 10) iron accumulation could be followed noninvasively with high agreement to invasive methods.

Influence of iron chelation on R1 and R2 calibration curves in gerbil liver and heart

MRI is gaining increasing importance for the noninvasive quantification of organ iron burden. Since transverse relaxation rates depend on iron distribution as well as iron concentration, physiologic and pharmacologic processes that alter iron distribution could change MRI calibration curves. This article compares the effect of three iron chelators, deferoxamine, deferiprone, and deferasirox, on R1 and R2 calibration curves according to two iron loading and chelation strategies. Thirty‐three Mongolian gerbils underwent iron loading (iron dextran 500 mg/kg/wk) for 3 weeks followed by 4 weeks of chelation. An additional 56 animals received less aggressive loading (200 mg/kg/week) for 10 weeks, followed by 12 weeks of chelation. R1 and R2 calibration curves were compared to results from 23 iron‐loaded animals that had not received chelation. Acute iron loading and chelation‐biased R1 and R2 from the unchelated reference calibration curves but chelator‐specific changes were not observed, suggesting physiologic rather than pharmacologic differences in iron distribution. Long‐term chelation deferiprone treatment increased liver R1 50% (P < 0.01), while long‐term deferasirox lowered liver R2 30.9% (P < 0.0001). The relationship between R1 and R2 and organ iron concentration may depend on the acuity of iron loading and unloading as well as the iron chelator administered. Magn Reson Med 60:82–89, 2008.

Cloning and sequence analysis of rat hepsin, a cell surface serine proteinase.

A cDNA coding for the rat serine proteinase hepsin was isolated and its nucleotide sequence has been determined. The cDNA was 1739 nucleotides long and contained an open reading frame encoding a protein consisting of 416 amino-acid residues. The deduced amino-acid sequence of the rat enzyme was very similar to the human hepsin sharing an amino-acid sequence identity of 88.7%. Hydropathy plots reveal the presence of a short hydrophobic region close to the N-terminus believed to be a transmembrane domain which anchors the proteinase on the cell surface. The predicted sequence contains the His, Asp and Ser residues which make up the catalytic triad common to all serine proteinases.

Identification of Cardiac Glycoside Molecules as Inhibitors of c-Myc IRES-Mediated Translation

Translation initiation is a fine-tuned process that plays a critical role in tumorigenesis. The use of small molecules that modulate mRNA translation provides tool compounds to explore the mechanism of translational initiation and to further validate protein synthesis as a potential pharmaceutical target for cancer therapeutics. This report describes the development and use of a click beetle, dual luciferase cell-based assay multiplexed with a measure of compound toxicity using resazurin to evaluate the differential effect of natural products on cap-dependent or internal ribosome entry site (IRES)–mediated translation initiation and cell viability. This screen identified a series of cardiac glycosides as inhibitors of IRES-mediated translation using, in particular, the oncogene mRNA c-Myc IRES. Treatment of c-Myc–dependent cancer cells with these compounds showed a decrease in c-Myc protein associated with a significant modulation of cell viability. These findings suggest that inhibition of IRES-mediated translation initiation may be a strategy to inhibit c-Myc–driven tumorigenesis.