Robert W. Grady

Effectiveness and safety of ICL670 in iron-loaded patients with thalassaemia: a randomised, double-blind, placebo-controlled, dose-escalation trial

BACKGROUND Transfusional iron overload is a potentially fatal complication of the treatment of thalassaemia. We aimed to investigate short-term efficacy, pharmacokinetic/pharma- codynamic (PK/PD) relations, and safety of ICL670, a novel, tridentate, orally active iron chelator. METHODS We enrolled 24 patients and divided them into three cohorts consisting of a minimum of seven individuals. Patients were admitted to a metabolic unit and consumed a diet with a defined content of iron. Two patients in each cohort were randomly allocated placebo. Five or more patients received one daily dose of ICL670 at 10, 20, or 40 mg x kg(-1) x day(-1), from day 1 to 12. Net iron excretion (NIE) was measured between days 1 and 12. Primary objectives included assessment of safety and tolerability (measured by adverse events and clinical laboratory monitoring), pharmacokinetics (measured as drug and drug-iron complex), and cumulative net iron excretion (measured by faecal and urine output minus food input). Analysis was for efficacy. FINDINGS ICL670 was absorbed promptly and was detectable in the blood for 24 h. Exposure (area under the curve of plasma concentration) to ICL670 at pharmacokinetic steady state was proportional to dose. All three doses resulted in positive NIE. The NIE achieved at 20mg x kg(-1) day(-1) would prevent net iron accumulation in most patients transfused with 12-15 mL packed red-blood-cells kg(-1) month(-1), equivalent to 0.3-0.5 mg iron kg(-1) x day(-1). A linear relation (PK/PD) was recorded between exposure to ICL670 and total iron excretion, by contrast with placebo (r2=0.54, p<0.0001). Skin rashes were noted in four patients treated at 20 and 40 mg x kg(-1) x day(-1), and one patient also developed grade 2 transaminitis. INTERPRETATION ICL670 given once daily at 20 mg/kg seems to be an effective orally active iron chelator and is reasonably well tolerated. Long-term studies are now necessary to establish the practical contribution of this drug.

Hepcidin as a therapeutic tool to limit iron overload and improve anemia in β-thalassemic mice

Excessive iron absorption is one of the main features of β-thalassemia and can lead to severe morbidity and mortality. Serial analyses of β-thalassemic mice indicate that while hemoglobin levels decrease over time, the concentration of iron in the liver, spleen, and kidneys markedly increases. Iron overload is associated with low levels of hepcidin, a peptide that regulates iron metabolism by triggering degradation of ferroportin, an iron-transport protein localized on absorptive enterocytes as well as hepatocytes and macrophages. Patients with β-thalassemia also have low hepcidin levels. These observations led us to hypothesize that more iron is absorbed in β-thalassemia than is required for erythropoiesis and that increasing the concentration of hepcidin in the body of such patients might be therapeutic, limiting iron overload. Here we demonstrate that a moderate increase in expression of hepcidin in β-thalassemic mice limits iron overload, decreases formation of insoluble membrane-bound globins and reactive oxygen species, and improves anemia. Mice with increased hepcidin expression also demonstrated an increase in the lifespan of their red cells, reversal of ineffective erythropoiesis and splenomegaly, and an increase in total hemoglobin levels. These data led us to suggest that therapeutics that could increase hepcidin levels or act as hepcidin agonists might help treat the abnormal iron absorption in individuals with β-thalassemia and related disorders.

Bone loss caused by iron overload in a murine model: importance of oxidative stress.

Osteoporosis is a frequent problem in disorders characterized by iron overload, such as the thalassemias and hereditary hemochromatosis. The exact role of iron in the development of osteoporosis in these disorders is not established. To define the effect of iron excess in bone, we generated an iron-overloaded mouse by injecting iron dextran at 2 doses into C57/BL6 mice for 2 months. Compared with the placebo group, iron-overloaded mice exhibited dose-dependent increased tissue iron content, changes in bone composition, and trabecular and cortical thinning of bone accompanied by increased bone resorption. Iron-overloaded mice had increased reactive oxygen species and elevated serum tumor necrosis factor-α and interleukin-6 concentrations that correlated with severity of iron overload. Treatment of iron-overloaded mice with the antioxidant N-acetyl-L-cysteine prevented the development of trabecular but not cortical bone abnormalities. This is the first study to demonstrate that iron overload in mice results in increased bone resorption and oxidative stress, leading to changes in bone microarchitecture and material properties and thus bone loss.

Bone Disease in Thalassemia: A Frequent and Still Unresolved Problem

Adults with β thalassemia major frequently have low BMD, fractures, and bone pain. The purpose of this study was to determine the prevalence of low BMD, fractures, and bone pain in all thalassemia syndromes in childhood, adolescence, and adulthood, associations of BMD with fractures and bone pain, and etiology of bone disease in thalassemia. Patients of all thalassemia syndromes in the Thalassemia Clinical Research Network, ≥6 yr of age, with no preexisting medical condition affecting bone mass or requiring steroids, participated. We measured spine and femur BMD and whole body BMC by DXA and assessed vertebral abnormalities by morphometric X‐ray absorptiometry (MXA). Medical history by interview and review of medical records, physical examinations, and blood and urine collections were performed. Three hundred sixty‐one subjects, 49% male, with a mean age of 23.2 yr (range, 6.1–75 yr), were studied. Spine and femur BMD Z‐scores < −2 occurred in 46% and 25% of participants, respectively. Greater age, lower weight, hypogonadism, and increased bone turnover were strong independent predictors of low bone mass regardless of thalassemia syndrome. Peak bone mass was suboptimal. Thirty‐six percent of patients had a history of fractures, and 34% reported bone pain. BMD was negatively associated with fractures but not with bone pain. Nine percent of participants had uniformly decreased height of several vertebrae by MXA, which was associated with the use of iron chelator deferoxamine before 6 yr of age. In patients with thalassemia, low BMD and fractures occur frequently and independently of the particular syndrome. Peak bone mass is suboptimal. Low BMD is associated with hypogonadism, increased bone turnover, and an increased risk for fractures.

The influence of heme-binding proteins in heme-catalyzed oxidations.

We show here that heme-binding proteins may enhance, decrease, or completely inhibit heme-catalyzed oxidations and that in doing so the proteins themselves may be oxidized depending upon their relative affinities for heme and the nature of their interactions with this metalloporphyrin. That release of iron from heme was not responsible for the catalytic effect is indicated by the observation that heme induced more peroxidation of rat liver microsomal lipid in the presence of H2O2 than iron and that iron release is very low under the conditions employed. Hemopexin, which binds heme with high affinity, completely inhibited heme-catalyzed lipid peroxidation at concentrations slightly higher than that of heme, suggesting a unique role for this acute phase protein in antioxidant defense mechanisms. The protein itself was not oxidized, presumably because the putative bis-histidyl heme-hemopexin complex cannot interact with H2O2. Rat and human albumin and rat glutathione S-transferases (GST), proteins with moderate affinities for heme, decreased heme-catalyzed lipid peroxidation in a dose-dependent manner but were subject to oxidation. The GST were crosslinked forming a nondisulfide covalently linked subunit dimer as well as products of higher molecular weight whereas the oxidation products of the albumins had molecular weights only slightly higher than those of the native proteins. The changes in the electrophoretic patterns of GST and albumin were accompanied by a decrease in their tryptophan fluorescence and the formation of bityrosine-like products. Proteins with lower affinities for heme, such as bovine albumin and rat liver fatty acid-binding protein (L-FABP), enhanced lipid peroxidation at all concentrations tested. While bovine albumin was modified, L-FABP was not crosslinked nor were its tyrosine residues oxidized. Thus, the susceptibility of a protein to heme-mediated oxidative damage would appear to be determined by factors such as its affinity for heme, the nature of the amino acids in the vicinity of the bound catalyst and the availability of a free coordination site on the iron.

Decreased differentiation of erythroid cells exacerbates ineffective erythropoiesis in β-thalassemia

In beta-thalassemia, the mechanism driving ineffective erythropoiesis (IE) is insufficiently understood. We analyzed mice affected by beta-thalassemia and observed, unexpectedly, a relatively small increase in apoptosis of their erythroid cells compared with healthy mice. Therefore, we sought to determine whether IE could also be characterized by limited erythroid cell differentiation. In thalassemic mice, we observed that a greater than normal percentage of erythroid cells was in S-phase, exhibiting an erythroblast-like morphology. Thalassemic cells were associated with expression of cell cycle-promoting genes such as EpoR, Jak2, Cyclin-A, Cdk2, and Ki-67 and the antiapoptotic protein Bcl-X(L). The cells also differentiated less than normal erythroid ones in vitro. To investigate whether Jak2 could be responsible for the limited cell differentiation, we administered a Jak2 inhibitor, TG101209, to healthy and thalassemic mice. Exposure to TG101209 dramatically decreased the spleen size but also affected anemia. Although our data do not exclude a role for apoptosis in IE, we propose that expansion of the erythroid pool followed by limited cell differentiation exacerbates IE in thalassemia. In addition, these results suggest that use of Jak2 inhibitors has the potential to profoundly change the management of this disorder.

Mitochondrial development in Trypanosoma brucei brucei transitional bloodstream forms

Intermediate and short stumpy bloodstream forms of Trypanosoma brucei brucei are transitional stages in the differentiation of mammal-infective long slender bloodstream forms into the procyclic forms found in the midgut of the tsetse vector. Although the mitochondria of the proliferative long slender forms do not accumulate rhodamine 123, the mitochondria of the transitional forms attain this ability thus revealing the development of an electromotive force (EMF) across the inner mitochondrial membrane. The EMF is inhibited by 2,4-dinitrophenol, rotenone and salicylhydroxamic acid but not by antimycin A or cyanide. Consequently, NADH dehydrogenase, site I of oxidative phosphorylation, is the source of the EMF and the plant-like trypanosome alternative oxidase (TAO) supports the electron flow serving as the terminal oxidase of the chain. Although the TAO is present in the long slender forms as well, it serves only as the terminal oxidase for electrons from glycerol-3-phosphate dehydrogenase. The data presented here, combined with older data, lead to the conclusion that the mitochondria of transitional intermediate and short stumpy forms likely produce ATP. This putative production is either by F1F0 ATPase driven by the complex I proton pump or by mitochondrial substrate level phosphorylation, or most likely by both. These conclusions contrast with the previously held dogma that all bloodstream form mitochondria are incapable of ATP production.

Differences in the prevalence of growth, endocrine and vitamin D abnormalities among the various thalassaemia syndromes in North America.

This study aimed to determine differences in the rates of growth, endocrine‐ and calcium‐related abnormalities in the various thalassemia syndromes in North America treated with current therapies. Medical history, physical examinations and blood and urine collections were obtained from patients with all thalassemia syndromes age 6 years and older in the Thalassemia Clinical Research Network. 361 subjects, 49% male, mean age 23·2 years (range 6·1–75 years) were studied. Approximately 25% of children and adults, regardless of the thalassemia syndrome, had short stature. Overall growth in children was mildly affected. Final height was close to midparental height (z = −0·73 ± 1·24). Patients with beta thalassemia major (TM) had higher rates of hypogonadism, multiple endocrinopathies, worse hyperglycaemia, subclinical hypoparathyroidism and hypercalciuria. Hypogonadism remained the most frequent endocrinopathy and was frequently under‐treated. 12·8% of the subjects had 25 vitamin D concentrations less than 27 nmol/l and 82% less than 75 nmol/l, regardless of the thalassemia syndrome. Adolescents had lower 25 vitamin D levels than children and adults. Compared to patients with other thalassemia syndromes, those with beta TM suffered from higher rates of multiple endocrinopathies, abnormal calcium metabolism and hypercalciuria. Vitamin D abnormalities were high among adolescents.

Serum Ferritin Iron, a New Test, Measures Human Body Iron Stores Unconfounded by Inflammation

Serum ferritin protein is an acute phase reactant. We hypothesized that serum ferritin protein generated in response to an inflammatory process would have much less iron (Fe) in it than would “normal” ferritin protein, and therefore measuring serum ferritin iron would assess human body iron status unconfounded by inflammation.

Sodium Cyanate Induced Polyneuropathy in Patients with Sickle-Cell Disease

Abstract Two patients with sickle-cell disease who were receiving sodium cyanate for 440 and 600 days, respectively, in doses of up to 44 and 41 mg/kg body weight · day, respectively, had polyneuro...