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This exploratory study assessed apoptosis in peripheral blood leucocytes (PBL) from β-thalassaemia patients receiving chronic transfusions and chelation therapy (deferasirox or deferoxamine) at baseline, 1, 6, and 12 months. At baseline, thalassaemic PBLs presented 50% greater levels of Bax (BAX), 75% higher caspase-3/7, 48% higher caspase-8 and 88% higher caspase-9 activities and 428% more nucleosomal DNA fragmentation than control subjects. Only neutrophils correlated significantly with apoptotic markers. Previously, we showed that over the treatment year, hepatic iron declined; we now show that the ratio of Bax/Bcl-2 (BCL2), (-27·3%/year), and caspase-9 activity (-13·3%/year) declined in both treatment groups, suggesting that chelation decreases body iron and indicators of PBL apoptosis.

Patients with β-thalassemia require iron chelation therapy to protect against progressive iron overload and non-transferrin-bound iron. Some patients fail to respond adequately to deferoxamine and deferasirox monotherapy while others have side effects which limit their use of these drugs. Since combining deferiprone and deferoxamine has an additive effect, placing all patients into net negative iron balance, we investigated the possibility that combining deferasirox and deferoxamine would lead to similar results. We conducted 34-day metabolic iron balance studies in six patients in whom the relative effectiveness of deferasirox (30 mg/kg/day) and deferoxamine (40 mg/kg/day) was compared, alone and in combination. Patients consumed fixed low-iron diets; daily urinary and stool iron excretion were determined by atomic absorption. Red blood cell transfusions were given prior to each drug treatment to minimize the effects of ineffective erythropoiesis. Serial safety measures, hematologic parameters, serum chemistries, ferritin levels and urinalyses were determined. All patients were in negative iron balance when treated with deferoxamine alone while four of six patients remained in positive balance when deferasirox monotherapy was evaluated. Daily use of both drugs had a synergistic effect in two patients and an additive effect in three others. Five of six patients would be in negative iron balance if they used the combination of drugs just 3 days a week. No significant or drug-related changes were observed in the blood work-ups or urinalyses performed. We conclude that supplementing the daily use of deferasirox with 2 - 3 days of deferoxamine therapy would place all patients into net negative iron balance thereby providing a convenient way to tailor chelation therapy to the individual needs of each patient.

β-Thalassemia is a genetic disorder caused by mutations in the β-globin gene and characterized by chronic anemia caused by ineffective erythropoiesis, and accompanied by a variety of serious secondary complications such as extramedullary hematopoiesis, splenomegaly, and iron overload. In the past few years, numerous studies have shown that such secondary disease conditions have a genetic basis caused by the abnormal expression of genes with a role in controlling erythropoiesis and iron metabolism. In this article, the most recent discoveries related to the mechanism(s) responsible for anemia/ineffective erythropoiesis and iron overload are discussed in detail. Particular attention is paid to the pathway(s) controlling the expression of hepcidin, which is the main regulator of iron metabolism, and the Epo/EpoR/Jak2/Stat5 signaling pathway, which regulates erythropoiesis. Better understanding of how these pathways function and are altered in β-thalassemia has revealed several possibilities for development of new therapeutic approaches to treat of the complications of this disease.

Patients with thalassemia major accumulate body iron over time as a consequence of continuous red blood cell transfusions which cause hepatic, endocrine, and cardiac complications. Despite the availability of three iron chelators, some patients fail to respond adequately to monotherapy with any of them. Combination therapy, consisting in the use of two chelators on the same day, has been introduced to increase the efficacy and to induce negative iron balance in patients with severe iron overload. Extensive long-term experience has shown that combined chelation with deferiprone and deferoxamine (DFO) rapidly reduces liver iron, serum ferritin, and myocardial siderosis, improves cardiac function, reverses and prevents endocrine complications, reduces cardiac mortality, and improves survival. Side effects, though significant, are manageable if properly monitored. Preliminary promising results have been obtained using combined chelation with deferasirox and DFO. As more drug combination regimes are evaluated, it should be possible to better tailor iron chelation to the needs of the patients, minimizing toxicity and maximizing efficacy throughout life.

Summary 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.

Adults with beta 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, > or =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 most widely used drug for iron chelation is deferoxamine (DFO) mesylate. While effective in promoting iron excretion, it requires prolonged daily infusions, often resulting in poor compliance. A clinical trial was conducted using starch-conjugated DFO (S-DFO; 40SD02), a high-molecular-weight iron chelator possessing prolonged vascular retention. Single doses of S-DFO were infused intravenously into groups of four transfusion-dependent patients with beta-thalassaemia at doses of 150, 300, 600 and 900 mg/kg. Urinary iron excretion and various pharmacologic parameters were evaluated for 1 week and safety for 3 weeks. No drug-related effects were observed on clinical chemistries, haematological and coagulation parameters, urinalyses, vital signs or electrocardiograms. Drug-related adverse events were limited to four urticarial reactions, none requiring termination of the infusion. The drug stimulated clinically significant urinary iron excretion, with the highest dose (900 mg/kg) inducing excretion of 1.31 mg of iron/kg (range 0.79-1.90 mg/kg) over 1 week, with residual iron-binding capacity present in the plasma for over 6 d. In summary, treatment with S-DFO, administered weekly, has the potential to achieve iron balance in the poorly compliant patient.

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.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.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, p0.0001). 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.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.

The effectiveness of deferoxamine (DFO), deferiprone (DFP), or deferasirox (DFX) in thalassemia major was assessed. Outcomes were reported as means±SD, mean differences with 95% CI, or standardized mean differences. Statistical heterogeneity was tested using χ2 (Q) and I2. Sources of bias and Grading of Recommendations Assessment, Development and Evaluation system (GRADE) were considered. Overall, 1520 patients were included. Only 7.4% of trials were free of bias. Overall measurements suggest low trial quality (GRADE). The meta-analysis suggests lower final liver iron concentrations during associated versus monotherapy treatment (p

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.

Myocardial iron overload is the leading cause of death in patients with β-thalassemia major. An intensification monotherapy with deferoxamine (DFO) as well as a combination therapy with DFO and deferiprone (DFP) reduces myocardial iron and improves cardiac function. However, the prognosis for thalassemia major patients with established cardiac disease switched from DFO monotherapy to combined DFP/DFO chelation is unknown. Twenty-eight thalassemia major patients with cardiac disease were enrolled in a prospective study lasting 42 ± 6 months. Fifteen (9 high-ferritin and 6 low-ferritin) were placed on DFP/DFO (DFP, 75 mg/kg t.i.d.; DFO, 40–50 mg/kg over 8–12 h at night 5–7 days/week), while 13 (5 high- and 8 low-ferritin) received DFO alone. No cardiac events were observed among high-ferritin patients on combination therapy, whereas 4 cardiac events ( p = 0.0049), including three deaths, occurred in high-ferritin patients on DFO monotherapy. These findings demonstrate that in thalassemia major patients with well-established cardiac disease combined iron-chelation therapy with DFP/DFO is superior to DFO monotherapy.

Osteoporosis and fractures occur frequently in patients with beta-thalassemias, a group of congenital hemolytic anemias characterized by decreased synthesis of the beta chain of hemoglobin. In this study, we determined the bone abnormalities of the th3 thalassemia mouse, generated by deletion of the mouse beta-chain genes. The heterozygous th3/+ mouse has moderate anemia and serves as a model of beta-thalassemia intermedia, which represents the mild thalassemia phenotype. The th3/th3 mouse has lethal anemia and is a model of beta-thalassemia major, which is characterized by life-threatening anemia requiring regular transfusions to sustain life. Compared to controls, (1) microCT of trabecular bone showed decreased bone volume fraction, number of trabeculae, and trabecular thickness in both th3/+ and th3/th3 (P0.05); (2) cortical bone analysis showed thinner cortices and increased marrow area in th3/+ (P0.05); (3) microCT abnormalities in th3/+ mice were present by 2 months and did not worsen with age; (4) histomorphometry was significant for decreased bone formation and resorption in both th3/+ and th3/th3, and expression of cathepsin K and osteocalcin from bone of both th3/+ and th3/th3 animals was reduced (P0.05); (5) biomechanics showed reduced maximum load, maximum moment, and structural stiffness in both th3/+ and th3/th3 (P0.01). In conclusion, the th3 mouse model of thalassemia manifests bone changes reminiscent of those in humans and can be used for further bone studies in thalassemia. Bone changes are associated with decreased bone turnover and develop early during the period of bone accrual.

Iron chelation therapy with desferrioxamine (DFO) has dramatically improved the outlook in beta-thalassemia. Parenteral DFO reduces tissue iron stores, prevents iron-induced organ damage, and reduces morbidity and mortality, with little serious toxicity. However, the burden of prolonged subcutaneous portable pump infusions, high cost, and patient noncompliance have prompted the development of new methods of administration and new formulations of DFO as well as oral iron chelators. Deferiprone (L1), the only oral iron chelator studied in large long-term clinical trials, is less effective and more toxic than DFO and may not adequately control iron overload; however, compliance and quality of life are improved. Combinations of two iron chelators (such as parenteral DFO plus oral L1, or 2,3-DHB; or oral L1 plus HBED) have been shown to produce additive and synergistic effects, explained by the shuttle hypothesis. Iron bound to a "shuttle"--an oral agent that mobilizes tissue iron--is exchanged in the bloodstream with a "sink"--such as parenteral DFO--and excreted via the kidneys, while the shuttle is reutilized. Combination therapy may produce enhanced iron excretion, target specific iron compartments, minimize side effects, increase treatment options, improve compliance, and facilitate individualization of therapy. Better understanding of the kinetics of iron metabolism, iron overload, and chelation should improve therapeutic strategies.

In a rat model of Pneumocystis carinii pneumonia, a 3-week infusion of deferoxamine producing concentrations in plasma of > or = 1.5 micrograms m-1 eliminated the trophozoite life cycle stage. Since this concentration is well below that routinely achieved in patients treated for iron overload, deferoxamine has promise as a therapy for AIDS-associated P.carinii pneumonia.

The iron-chelating drug deferoxamine (DFO) has been shown to be active in animal models of Pneumocystis carinii pneumonia (PCP), with effective daily intraperitoneal bolus dosages being 400 and 1,000 mg of DFO mesylate kg of body weight-1 in mouse and rat models, respectively. Continuous infusion produced a moderately improved response in a rat model. The data reported here demonstrate that the response achieved by continuous infusion of 195 and 335 mg of DFO mesylate kg-1 day-1 in the rat model is associated with mean concentrations in plasma of 1.3 and 2.5 micrograms of DFO ml-1 and mean concentrations in lung tissue of 4.9 and 6.0 micrograms of DFO g of lung tissue-1, respectively. Since current clinical use of DFO mesylate for the treatment of iron overload produces higher concentrations in the plasma of patients, DFO may prove to be a useful anti-PCP treatment. The 2.4- to 3.8-fold higher DFO concentration observed in lung tissue compared with that observed in plasma may be important in the response of PCP to DFO.

The iron-chelating drug deferoxamine mesylate (DFO) is active against Pneumocystis carinii in vitro and in rat and mouse models of P. carinii pneumonia. Because DFO has a short half-life, daily divided or continuous dosage was expected to improve the dose response, as is the case with DFO treatment of malaria. Therefore, results of single daily intraperitoneal injections were compared with results of an evenly divided four-times-daily dosage and the efficacy of delivery with implanted infusion pumps. The highest bolus dosage (1,000 mg kg-1 of body weight day-1) was as effective as the standard combination of trimethoprim with sulfamethoxazole. Unexpectedly, very little improvement was observed with the divided or continuous dosage, and several mechanisms that could account for this are discussed.

Background Understanding patients’ views about medication is crucial to maximize adherence. Thalassemia is a congenital blood disorder requiring chronic blood transfusions and daily iron chelation therapy. Methods The Beliefs in Medicine Questionnaire (BMQ) was used to assess beliefs in chelation in thalassemia patients from North America and London in the Thalassemia Longitudinal Cohort (TLC) of the Thalassemia Clinical Research Network (TCRN). Chelation adherence was based on patient report of doses administered out of those prescribed in the last four weeks. Results Of 371 patients (ages 5-58y, mean 24y), 93% were transfused and 92% receiving chelation (26% deferoxamine (DFO; a slow subcutaneous infusion via portable pump), 63% oral, 11% combination). Patients expressed high “necessity” for transfusion (96%), DFO chelation (92%) and oral chelation (89%), with lower “concern” about treatment (48%, 39%, 19% respectively). Concern about oral chelation was significantly lower than that of DFO (p Conclusions Despite their requirement for multimodal therapy, thalassemia patients have positive views about medicine, more so than in other disease populations. Patients may benefit from education about the tolerability of chelation and strategies to effectively cope with side effects, both of which might be beneficial in lowering body iron burden. Clinicaltrials.gov identifier NCT00661804

The experience of the past dozen years clearly indicates that iron plays a significant role in the pathogenesis of a wide variety of disease states apart from hemochromatosis. 1 In cases of rheumatoid arthritis and reperfusion injury, iron catalyzes the formation of free radicals which destroy membranes and thereby lead to tissue damage. Sequestration of this iron would obviate much of this damage. In lymphopro-liferative disorders and parasitic diseases such as malaria, iron serves as an essential nutrient for dividing cells. Withholding iron would inhibit cell division and hence slow the progression of such diseases. While unlikely to affect cures, chelation of iron would undoubtedly be of benefit as adjunctive therapy in these and related disorders.

Abstract 3187 ß-Thalassemia and sickle cell disease (SCD) are the most common genetic red blood cell (RBC) disorders characterized respectively by limited production of aberrant ß-globin chains. In both cases, chronic transfusions and iron chelation are required to treat the anemia and/or formation of abnormal RBC. In ß-thalassemia, anemia stimulates erythropoietin (Epo) synthesis, which in turn leads to increased erythropoiesis and development of hepatosplenomegaly, often resulting in the need for splenectomy. Recently, we demonstrated that erythroid cells from ß-thalassemic mice have a hyper-activation of Jak2, a kinase that mediates the signaling triggered by the binding of Epo to the Epo receptor. This led us to hypothesize that Jak2 inhibitors could be utilized to minimize erythroid expansion in this disorder, limiting splenomegaly. A Jak2 inhibitor (Tg101209 or Tg) was first tested in mice affected by ß-thalassemia intermedia (th3/+). Two doses of Tg (150 and 100 mg/Kg/day) were given orally for 10 days. Tg administration induced a mild decrease of hemoglobin levels (8.8±0.2, 8±0.2 and 7.8±0.2g/dL for placebo, Tg 100 mg/Kg and Tg 150 mg/Kg treated mice, respectively. p We then evaluated the outcome of combining Tg with blood transfusion, a common therapy in b-thalassemia and SCD. In b-thalassemia, massively enlarged spleens are believed to sequester a significant proportion of circulating RBC, thereby limiting their lifespan and the efficacy of transfusion regimens. We hypothesize that decreasing splenomegaly by administration of Jak2 inhibitors could increase the efficacy of transfusion. This was first tested in th3/+ animals. In this case, transfusion alone was sufficient to increase the hemoglobin (Hb) levels approximately 3 g/dL and reduce the spleen size to 65% of that seen in non-transfused controls. In this model, the combined effect of transfusion and administration of Tg was more effective, the spleen size been 50% of non-transfused controls (p Lastly, we tested combination therapy in a mouse model of SCD. Mice treated with Tg and transfusion exhibited slightly lower levels of Hb than transfused controls (Hb=9.7±0.2g/dL versus Hb=10.9±0.2g/dL). However, compared to the control, mice receiving combination therapy exhibited a larger percentage of donor RBCs, while endogenous erythropoiesis was markedly suppressed along with the production of sickle RBCs (1.3±0.3×106 RBC/ul compared to transfused-controls exhibiting 2.7±0.3×106 RBC/ul). In summary, administration of Jak2 inhibitors might reduce the production of pathological cells that, together with preservation of the splenic architecture, could minimize the propensity of patients to thrombotic events. Furthermore, suppression of endogenous erythropoiesis and reduction of the transfusion regimen would be expected to also reduce iron accumulation, making it easier to prevent its toxic effects through chelation therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 5163 Introduction: Deferasirox (Exjade, ICL670) is an orally effective iron chelating agent approved for use in patients 2 years of age and older with transfusional iron overload. While it is effective as a single agent, there are patients who do not attain net negative iron balance despite being at the upper limit of approved dosing (40 mg/kg/day). Deferiprone (Ferriprox, L1) is another orally effective iron chelator. Through a series of metabolic iron balance studies we were able to demonstrate that various regimens involving the combined use of deferiprone and deferoxamine (Desferal, DFO) were capable of placing all patients into net negative iron balance with the potential to adjust dosing schedules and the ratio of drugs to maximize effectiveness while minimizing toxicity. A variety of such regimens are now in widespread clinical use. We have taken the same approach in order to optimize the use of deferasirox. Methods: Six patients with thalassemia major were enrolled in a 34-day metabolic iron balance study wherein deferasirox and deferoxamine were evaluated alone and in combination, each patient serving as his/her own control. Deferoxamine (40 mg/kg) was administered on days 5 – 10 as an 8-hour subcutaneous infusion during the night. On days 15 – 20, deferasirox (30 mg/kg) was given orally, 30 minutes prior to breakfast. Both drugs were given on days 25 – 30, the same doses and dosing schedules being employed. Non drug days allowed for washout of stool iron induced by the previous treatment. The patients consumed a fixed low-iron diet consisting of four individualized meal plans. Daily collections of urine and stool were made and their iron content determined by atomic absorption, a correction being made for all uneaten food. Results: As in previous studies, there was significant patient to patient variability in terms of the amount of drug-induced iron excretion. Combination therapy placed all patients into iron balance exceeding 200% (206% - 270%, mean 251%). The combination was synergistic in two patients (35% and 57%), additive in three patients, and less than additive in one patient. Where iron excretion was more than additive, all of the excess appeared in the urine (a 2.1- and 3.4-fold increase). Deferasirox proved to be less effective than deferoxamine in all six patients (relative iron excretion: 23% - 59%, mean 42%), stool iron excretion in response to the respective drugs being 94% -100% (mean 98%) and 49% - 74% (mean 59%). Net negative iron balance was achieved in 2/6 patients when on deferasirox (iron balance 28% - 129%, mean 72%) and 6/6 patients when infusing deferoxamine (iron balance 125% - 219%, mean 167%). Discussion: These results suggest that chelation therapy can be tailored to the individual needs of each patient, selectively removing iron from different pools while minimizing any side effects. Deferasirox appears to shuttle iron to deferoxamine for excretion in the urine, not unlike the situation when deferiprone and deferoxamine are combined, although the source of the iron may be somewhat different. It is hoped that these results will provide patients with further options to optimize their chelation regimens. Disclosures: Grady: Novartis Pharmaceuticals: Research Funding. Off Label Use: Deferasirox and Deferoxamine are both iron chelating agents. Their use in combination is not indicated on their labeling. Galanello:Novartis Pharmaceuticals: Speakers Bureau. Paley:Novartis Pharmaceuticals: Employment. Giardina:Novartis Pharmaceuticals: Research Funding.

Abstract 2020 Poster Board I-1042 β-thalassemia intermedia (TI) and major (TM) are characterized by Ineffective Erythropoiesis (IE). We hypothesized that the kinase Jak2 plays a major role in IE and splenomegaly. To test this hypothesis we administered a Jak2 inhibitor (TG101209) to mice affected by TI, showing that this treatment was associated with a marked decrease in IE, and a moderate decrease in hemoglobin (Hb) levels (∼1 g/dL). This last observation indicates that the use of a Jak2 inhibitor might exacerbate anemia in thalassemia. However, we hypothesized that using standard transfusion to treat TM mice would also be adequate to prevent any further anemia caused by Jak2 inhibition while still allowing for decreased splenomegaly. Therefore, we analyzed the erythropoiesis and iron metabolism in TM animals treated with a Jak2 inhibitor and transfused. Use of TG101209 in TM mice not only reduced the spleen size dramatically (0.42±0.15 g and 0.19±0.10 g respectively in transfused+placebo (N=4) vs transfused+TG101209 (N=8), P= 0.007), but also allowed the mice to maintain higher Hb levels (respectively 7.3±1.1 g/dl vs 9.3±1.2 g/dl, P=0.019). This was likely due to reduced spleen size and limited red cell sequestration. Contrary to TM mice treated with transfusion+placebo, no foci of extra-medullary hematopoiesis were detectable in the parenchema of mice treated with TG101209. Hamp1 expression inversely correlated with the spleen weight, possibly indicating that suppression of IE (due both to blood transfusion and TG101209 administration) had a positive effect on Hamp1 expression. Hb levels also directly correlated with Hamp1 expression in the same animals. In this case, however, only transfusion played a role in increasing Hamp1 expression, although TG101209 undoubtedly had a positive effect by reducing the spleen size and thereby indirectly increasing the Hb levels. The suppression of erythropoiesis by blood transfusion limits the extent of our interpretations as it may mask the effect of the Jak2 inhibitor. Therefore we hypothesized that the administration of a tailored and reduced dose of the drug could be effective in reducing the splenomegaly in non-transfused TI mice, without affecting the Hb levels. We also hypothesized that the suppression of erythropoiesis would also lead to increased Hamp1 expression in the presence of iron overload. Compared to mice treated with placebo (N=5), analysis of TI mice treated with a tailored dose of 100mg/kg/day of the drug (N=11) showed a significant decrease in spleen size (0.18±0.05 g and 0.27±0.05 g, P=0.006 for drug treated mice and placebo treated mice respectively). Of note no significant difference of Hb levels was detectable between the 2 groups. In the drug treated mice we observed a significant decrease of the immature erythroid cell population (P=0.012) and amelioration of the architecture of the spleen, with the reappearance of white pulp foci and a significant restoration of the splenic lymphocitic populations. Drug treated mice showed increased levels of Hamp1 mRNA that inversely correlated with the spleen weight, suggesting a direct feedback between erythropoietic rate and expression of Hamp1. To determine if the use of Jak2 inhibitors could be beneficial in a mouse model mimicking a human form of hereditary ellyptocytosis, we treated mice KO for the 4.1R protein isoforms with TG101209, in presence or absence of blood transfusions. These mice exhibit moderate splenomegaly and anemia and the drug treatment was effective in reducing the spleen weight and the associated IE. We also plan to analyze Sickle Cell mice that we are treating with a Jak2 inhibitor. In conclusion our data show that the administration of Jak2 inhibitors is efficient in decreasing the spleen size and ameliorating the pathologic iron metabolism in thalassemia, both in the presence or absence of blood transfusions. Moreover we show that Jak2 inhibitors could transform the therapeutic approach for other forms of anemias. Disclosures: No relevant conflicts of interest to declare.

Abstract 2023 Poster Board I-1045 Macrophages represent an important link between erythropoiesis and iron metabolism. They support maturation and differentiation of erythroblasts and clear senescent red cells from circulation, thus recycling iron within the hematopoietic system. However, little is known about the role of macrophages in conditions of ineffective erythropoiesis like beta-thalassemia intermedia (β-TI) or sickle cell anemia (SCD). Our goal was to study the role of macrophages under such conditions, where there is a large expansion of the erythron, the lifespan of the red cells is decreased, and both iron absorption and organ iron content are increased. Clodronate liposomes have been shown to efficiently eliminate macrophages from spleen, liver and bone marrow when administered by intravenous injection. Therefore, we utilized this technique to eliminate macrophages from the spleen, liver and bone marrow of β-TI and SCD mice. Three different treatment schedules were used; a) acute high dose; b) medium term split dose; c) chronic low dose. Animals were analyzed for erythropoietic and pathological parameters. All treatments effectively depleted splenic and bone marrow macrophages as shown by flow citometric and immunohistochemical analyses. At doses that led to only a small decrease of hemoglobin (Hb) levels in normal mice, clodronate treatment induced high level of mortality in β-TI and SCD mice (70% and 75%, respectively). In contrast, administration of clodronate to mice affected by beta-thalassemia major, mice that do not make any adult or fetal Hb/mature red cells, caused no mortality suggesting that circulating abnormal red cells might be responsible for the mortality observed in β-TI and SCD mice. Pathological analysis of β-TI mice revealed the formation of thrombi in the lungs and heart to be the likely cause of death. Analysis of SCD mice is in progress. β-TI and SCD have been described as hypercoagulable states with a high risk of thrombosis. Damaged red cell membranes and ROS have been implicated as factors that increase the risk of thrombosis in β-TI and SCD patients. We hypothesize that clodronate treatment enhances this process. To test this hypothesis, we administered an anti-coagulant (heparin) and an anti-oxidant (NAC) to β-TI mice. Both these treatments resulted in increased survival from 31% to 65% compared to clodronate alone. Analysis of SCD animals is in progress. β-TI mice that survived clodronate treatment exhibited a milder thalassemic phenotype, characterized by increased Hb (∼1.5g/dL), HCT (∼7%) and RBC count (∼2e6 cells/ul), partially associated with a decreased RBC clearance. Ineffective erythropoiesis and splenomegaly were also ameliorated (∼40% decrease in spleen size). Animals depleted of macrophages also showed a marked decrease in serum iron parameters, but no difference in their organ iron concentrations. Analysis of hepcidin expression is in progress. To further corroborate these observations, β-TI mice are being crossed with mice expressing the human diphtheria toxin (CD11b-DTR) which allows for conditional depletion of macrophages. Our data suggests that macrophages play a major role in conditions of abnormal red cell production and ineffective erythropoiesis. In the former case, they have an important protective function related to the high risk of thrombosis associated with β-TI and SCD. In the latter, macrophages may act as negative modulators of erythropoietic development. The amelioration of splenomegaly and ineffective erythropoiesis observed suggests that erythroid cells might proliferate less and/or differentiate more in the absence of macrophages. One hypothesis is that macrophages sense erythroid maturation and/or differentiation and act as modulators of these processes to ensure the release of high quality red cells into the circulation. Second, macrophages, under conditions of iron overload, might produce inflammatory cytokines that further impair erythropoiesis in thalassemia. Analysis of RNA extracted from macrophages and of inflammatory cytokines in β-TI mice before and after clodronate treatment is in progress. Disclosures: No relevant conflicts of interest to declare.

Abstract 4070 Poster Board III-1005 Introduction A worldwide multicenter clinical trial of deferoxamine (DFO) in comparison to deferasirox (DFX/Exjade) established optimal therapeutic doses of DFX for use in b-thalassemia patients (pts) with moderate to severe iron burdens as determined by serum ferritin levels greater than 1000 ng/mL and liver iron concentrations (LICs) greater than 3 mg/g-dry weight (dw) resulting from regular red blood cell (RBC) transfusion regimens designed to maintain pre-tx hemoglobin (Hgb) levels greater than 9 to 10 gm/dL. To date, the use of DFX in less heavily iron overloaded transfused patients is not well documented. Therefore, we studied the safety and efficacy of DFX at a dose of approximately 20 mg/kg/day in a group of regularly transfused thalassemia major pts who had previously been well chelated with subcutaneous (sc) DFO as evidenced by serum ferritin levels less than 1000 ng/mL and LICs less than 3 mg/gm-dw. Patients The pt group consisted of eight transfusion dependent thalassemia major pts (2M/6F) who ranged in age from 13 to 49 yrs (mean ± SEM 28.3 ± 13.2 yrs). Six of the pts were splenectomized (2M/4F) and maintained on moderate to high transfusion regimens (2 to 4 RBC units per month) so as to maintain a pre-tx Hgb level of 9 to 10 gm/dL. Assessments/Methods Clinical parameters, including pre-tx Hgb levels, RBC units transfused, serum ferritins, liver function tests (ALT/AST), and renal function studies (BUN/creatinine) were determined at baseline and monitored monthly thereafter in all patients for one year. Hearing tests, eye exams and determinations of LIC by T2* magnetic resonance imaging (MRI) were done at baseline and 12 months. Annual tx requirements were calculated and expressed as mL of RBCs/kg/yr. All pts previously infused DFO sc 5 days each week at doses of 40 to 60 mg/kg/day. During the study, they ingested DFX daily 30 minutes prior to breakfast at a dose of 20 to 30 mg/kg as a suspension in water, apple or orange juice. Dose adjustments were made depending upon trends in serum ferritin levels, liver function tests or renal studies. Results Each pt received 2 to 4 leukocyte depleted washed RBC units per month (mean 119 ± 8.2 mL/kg/yr). Their mean pre-tx Hgb levels were 9.7 ± 0.2 gm/dL. Average monthly chemistry parameters were as follows: serum ferritin = 550 ± 9.4 ng/mL; AST = 36 ± 5; ALT = 36 ± 7; BUN = 14.8 ± 1.0; creatinine = 0.7 ± 0.1. There were no significant changes in the parameters related to liver or renal function, or in LICs during the 12-month observation period, the mean LICs were 2.0 ± 0.4 mg/gm-dw at baseline vs. 1.6 ± 0.3 mg/gm-dw at 12 months. Serum ferritin levels also remained stable during the course of therapy (577 ± 71 ng/mL at baseline vs. 480 ± 106 ng/mL at 12 months). The starting dose of DFX ranged from 20 to 30 mg/kg/day. Over the course of the study, the dose was increased to 25 and 30 mg/kg/day in pts 2 and 7, respectively, owing to a progressive increase in serum ferritin levels and decreased to 10 mg/kg in pt 3 and 18 mg/kg in pt 8 due to declining ferritin levels. On average, a dose of 20.5 ± 2 mg/kg/day was needed to maintain iron balance. No serious adverse events occurred during the 12-month course of DFX and no significant side effects (e.g. rash or gastrointestinal symptoms) were reported. Conclusion Moderate doses of DFX, slightly more than 20 mg/kg/day, were safe and effective in maintaining iron balance as measured by LICs (MRI) and serum ferritin levels in thalassemia major patients with low iron burdens (LICs Disclosures: No relevant conflicts of interest to declare.

β-Thalassemia intermedia and major are characterized by ineffective erythropoiesis (IE), requiring sporadic or chronic blood transfusions, respectively. Some of the major consequences of IE are extra-medullary hematopoiesis (EMH), splenomegaly and systemic iron overload mediated by transfusion therapy and down-regulation of hepcidin. Using mouse models of β-thalassemia intermedia (th3/+) and major (th3/th3), and human specimens we investigated IE in this disorder. Th3/+ and th3/th3 erythroid cells were analyzed with respect to rates of apoptosis and degrees of cell proliferation and differentiation. We found that there was both a relative and absolute expansion of the immature erythroid progenitor cell fraction in thalassemic mice compared to cells in the final stages of differentiation. Further investigation of the thalassemic erythroid cells in vivo and in vitro indicated that a larger number of the thalassemic cells are associated with the phosphorylated form of the Jak2 protein kinase than in normal mice. In fact, their proliferation was prevented by TG101209, a Jak2 inhibitor. Similar compounds are currently utilized or being considered for use in the treatment of myeloproliferative diseases such as polycythemia vera. In order to assess the potential of Jak2 inhibitors in limiting IE in β-thalassemia, we administered TG101209 to th3/+ mice. We found that both 10 and 18 days of treatment were sufficient to dramatically reduce the spleen size and the percentage of immature erythroid progenitors therein compared with administration of a placebo. However, these changes were associated with decreasing hemoglobin levels (Blood, 2008, 112:875–85). We speculated that this problem could be overcome by administration of blood transfusions during treatment, an extension of current management in thalassemia as noted above. To test this hypothesis, we administered TG101209 to th3/th3 mice in conjunction with regular blood transfusions. Administration of TG101209 to transfused th3/+ mice is in progress. Our preliminary data on th3/th3 mice indicates that simultaneous administration of TG101209 and transfused blood not only reverses splenomegaly, but also results in higher Hb levels (N ≥ 3). The increased hemoglobin levels observed, compared to those in mice treated with transfusion therapy alone, suggest that the use of Jak2 inhibitors may reduce the amount of blood per transfusion and/or the rate of transfusion required in thalassemia. Since the increased iron absorption in thalassemic mice is a direct consequence of IE, treatment with TG101209 could also be beneficial in ameliorating this process. According to several observations, suppression of erythropoiesis should lead to increased Hamp1 expression in the presence of iron overload. Therefore, under conditions of Jak2 inhibition, Hamp1 transcription is expected to increase. To test this hypothesis we are currently analyzing organ iron levels and the expression of iron-related genes in drug-treated mice. In conclusion, although our study does not exclude a role for apoptosis in the IE of β-thalassemia, we have demonstrated that increased cell proliferation and limited cell differentiation play a significant role in this process. Moreover, we show for the first time that a Jak2 inhibitor is effective in decreasing the spleen size of thalassemic mice. This could represent a completely new approach to the treatment of splenomegaly in β-thalassemia patients, perhaps coupled with blood transfusion. That administration of a Jak2 inhibitor reverses splenomegaly and also ameliorates the degree of iron overload could provide an opportunity to gain new insight into the dynamic processes of iron absorption and erythropoiesis in this pathological condition.

Neonatal hemochromatosis (NH), an uncommon and generally fatal disorder of infancy, is defined by hepatic disease of antenatal onset, generally manifest at birth, and by stainable iron in a tissue distribution like that seen in heritable adult-onset hemochromatosis (HH). It is not known if parents and sibs of infants with NH are at risk of iron storage disease in later life. We provide 30-year follow-up for iron overload of a family in which 6 of 9 children died in utero or early in the neonatal period. Four of the 6 came to autopsy, where severe liver disease was found; in 3 of the 4, autopsy material could be reviewed. These 3 children had NH. Studies 30 years later did not identify HH or other iron storage disease in the parents or surviving sibs. These findings suggest that first-degree relatives of persons with NH are not necessarily at increased risk of iron storage disease in later life.

The iron chelator deferoxamine and the polyamine biosynthesis inhibitor eflornithine (DL-alpha-difluoromethylornithine) were examined for anti-Pneumocystis carinii activity in the rat model of P. carinii pneumonia. The activity of deferoxamine at 250, 500, and 1,000 mg/kg given intraperitoneally provides evidence that iron chelation is a promising novel approach to P. carinii chemotherapy. Results with eflornithine at 2, 3, and 4% in drinking water confirm and extend previously reported activity in the rat model.

Starch-conjugated deferoxamine (S-DFO) is a long-acting, polymeric iron chelator designed to achieve iron balance in patients requiring frequent transfusions. Results from an ascending single dose study in 16 patients with beta-thalassemia showed that S-DFO is capable of achieving up to 7 days of iron balance (Br. J. Haematol. 138, 374–381, 2007). The multiple dose toxicology study described here was conducted to support extended clinical studies in patients with thalassemia and other iron overload disorders. The study was designed to evaluate physiological effects of S-DFO in iron-loaded animals;to seek evidence of cardiovascular side-effects;to find evidence of drug accumulation in plasma and other tissues; andto determine the No Observable Adverse Effect Level (NOAEL) following biweekly intravenous infusion for 4 weeks. Iron loading was achieved by multiple biweekly infusions of iron-dextran so as to give a cumulative dose of 500 mg of iron/kg body weight. Equilibration of iron was then allowed to proceed for 10 weeks before initiation of treatment. The study included four groups of 10 iron-loaded animals (5M/5F): saline controls; 210 and 420 mg/kg of deferoxamine (DFO) equivalents as S-DFO; 105 mg/kg of non-conjugated DFO; and two groups of 10 normoferremic animals (5M/5F): saline controls; and 210 mg/kg of DFO equivalents as S-DFO. All animals received 8 doses of either S-DFO, DFO or saline over 28 days. Six animals in each group (3M/3F) were euthanized on Day 29 for histological examination, the remaining 4 animals in each group on Day 43. Safety parameters for cardiovascular pharmacology, hematology and clinical chemistry showed no changes attributable to S-DFO treatment. The polymeric chelator is cleaved/metabolized by serum amylase(s). While trough concentrations of S-DFO fragments prior to the final drug infusion on Day 25 showed some accumulation, there was no evidence of resulting pathological changes. Thus, the NOAEL is at least 420 mg/kg of DFO equivalents. The hepatic iron content of the iron-loaded animals was approximately 20 times greater than that of the normoferremic controls. Treatment with 420 mg/kg of DFO equivalents led to a 13% reduction in the liver iron content. Neither iron loading nor treatment with S-DFO had a significant effect upon the cardiac iron status. The lack of toxicity in normoferremic animals suggests that S-DFO may have applications in clinical situations where increased iron stores are not characteristic of the disease. Furthermore, the ability to safely infuse up to 420 mg/kg of DFO equivalents without concern for hypotension indicates that S-DFO may also be well suited for treatment of acute iron poisoning. Finally, these results support initiation of multidose studies of S-DFO in patients with thalassemia and other iron overload disorders. Supported by NIDDK Contract Number N01-DK-32624.

Iron overload has been shown to increase mitochondrial dysfunction and apoptosis and may be implicated in leukocyte apoptosis. We assessed whether markers of leukocyte apoptosis and mitochondrial dysfunction are higher in iron-overloaded thalassemia patients compared with control subjects and whether improvement in the control of iron overload with deferasirox or deferoxamine (DFO) is associated with a change in the level of apoptotic markers. Methods: Thalassemia Clinical Research Network patients participating in the Novartis CICL670A0107 trial (a randomized comparison of deferasirox, an oral iron chelator, vs. DFO) were eligible and 44 (25 male, 21.8 yrs) were enrolled in the study. Fasting blood samples were obtainedafter a 5-day washout of DFO prior to commencing treatment with study drug, and24 hours post-chelator at 1, 6, and 12 months on study. Thirty healthy controls matched for age, sex, race and antioxidant usage (15 male, 24.5 yrs) also supplied a blood sample at baseline. After blood collection, plasma, serum and cells were separated by centrifugation. The pro-apoptotic marker Bax (an inducer of mitochondrial dysfunction) and the anti-apoptotic marker Bcl-2 were determined by ELISA. A high ratio of Bax/Bcl-2 indicates decreased stability of the mitochondrial outer membrane and increased potential for mitochondrial dysfunction and apoptosis. Activity of caspase–3 and −9 (both pro-apoptotic) were determined by a luminescent enzyme activity assay and reported as the average relative light units (RLU)/μg protein. Apoptotic markers were log-transformed prior to analysis and means and % change are reported. Results: At baseline, thalassemia patients had elevated Bax compared to the controls (17.4 vs. 11.6 pg/μg protein, p = 0.006). Similarly the activity of caspase-3 and −9 was high relative to the controls (for caspase-3, 1823 vs. 1041 RLU/μg protein, p = 0.01; and for caspase-9, 2482 vs. 1322 RLU/μg protein, p = 0.001). In longitudinal analysis, liver iron concentration and ferritin declined in both treatment groups (p ≤ 0.02 for both). This paralleled a decline in the ratio of Bax/Bcl-2 (–27.3% /yr, average decline, p = 0.033) and ALT (–7.3% /yr, average decline, p = 0.040). There was no significant difference between chelators in the rate of change of these markers over time. Conclusions: These findings demonstrate that markers of leukocyte apoptosis and mitochondrial dysfunction are high in thalassemia compared to controls and that the Bax/Bcl-2 ratio (a marker of mitochondrial dysfunction in apoptosis) decreased with effective iron chelation. DFO and deferasirox showed equal effectiveness in decreasing iron burden, ALT and the ratio of Bax/Bcl-2. Thus, with effective chelation, it may be possible to improve leukocyte and mitochondrial function and the levels of apoptosis.

Deferiprone (L1, Ferriprox) and deferasirox (ICL670, Exjade), two orally effective iron-chelating agents, have revolutionized the management of iron overload. Nonetheless, neither drug is effective in all patients, deferoxamine (DFO) still being the only drug capable of placing all affected individuals in net negative iron balance. Deferitrin (4,5-dihydro-2-(2,4-dihydroxyphenyl)-4-methylthiazole-4(S)-carboxylic acid, GT56-252), is a tridentate ligand with a demonstrated efficacy and an acceptable toxicity profile in preclinical evaluations in primates. In Phase 1 studies, it was well absorbed and no safety issues were identified. Thus, given the need for additional oral chelation options, we explored the efficacy of deferitrin in iron-overloaded patients with β-thalassemia major. Total iron balance studies were carried out wherein the effectiveness of single daily ascending doses of deferitrin (4.5, 6.75, 11 and 17 mg/kg/day) was compared with that of a standard DFO regimen (40 mg/kg infused subcutaneously over 8 hours). Twenty patients were admitted to our clinical research center for 28 days and placed on fixed individualized low-iron diets. On days 5 – 10 they were infused nightly with DFO and on days 15 – 24 given a dose of deferitrin with breakfast. Groups of 4 patients were studied at the three lowest doses of deferitrin, only 2 patients being given 17 mg/kg. Drug-free days allowed for washout of stool iron due to previous treatments, a stool marker being given at the beginning and end of each period of drug evaluation. Safety was assessed by hematology (CBC and coagulation parameters), chemistry (electrolytes, BUN, creatinine, liver function tests), urinalysis, and urinary β-2-microglobulin as well as by EKG, physical examination and monitoring of adverse events. Iron balance due to DFO ranged from 52% – 325% (mean 157%) with 40% – 77% (mean 61%) of total iron excretion appearing in the stool. Only 4 patients failed to achieve net negative iron balance. The response to deferitrin was highly variable at each dose studied, there being patients who responded poorly and others in whom there was a good response. Overall, iron balance ranged from 7% – 42%, nearly all of the iron excreted (0.04 – 0.14 mg/kg/day) appearing in the stool. Of note, total iron excretion appeared to reach a plateau at a deferitrin dose of 11 mg/kg/day. As animal studies suggested that more iron might be excreted upon giving the drug in divided doses, we interrupted our evaluation of 17 mg/kg/day and studied an additional 6 patients at 25 mg/kg/day, the drug being divided t.i.d. with breakfast, dinner and a bedtime snack. Iron balance in these patients ranged from 28% – 62% (mean 43%), stool iron excretion (0.14 – 0.29 mg/kg/day) accounting for 99% of the total. Their DFO-induced iron balance was similar to that of the patients previously studied, ranging from 123% – 233% (mean 173%). At all doses, no significant changes were noted in the EKGs or any hematological, biochemical or urinary parameters. There were no serious adverse events. These results suggest that deferitrin was orally effective and, while less effective than DFO, it was of sufficient efficacy to warrant further evaluation in a longer-term study as an alternative to DFO.

We conducted a 4-yr prospective trial to evaluate the long-term effects of combined deferiprone (DFP)/deferoxamine (DFO) on reversal of cardiac complications in thalassemia major compared to those of DFO alone. Twenty-eight patients (pts) with cardiac disease requiring medication were stratified according to their risk for cardiac death. Fourteen pts were high risk, serum ferritin (SF) > 2500 ug/L on two-thirds of occasions since the onset of cardiac disease. Of those with a SF < 2500 ug/L (low risk), six had progressive decrements of left ventricular ejection fraction (LVEF). Nine high-risk pts and six low-risk pts were placed on DFP/DFO (DFP, 75 mg/kg/d divided t.i.d.; DFO, 40 – 50 mg/kg over 8 – 12 h at night 5 – 7 d/wk. The others infused DFO alone. If SF fell below 500 ug/L, DFO infusions were reduced to 2 d/wk. Cardiac follow-up (including blood work and ECG) was done at 4-m intervals. M-mode and two-dimensional echocardiograms were done at 4- to 6-m intervals. Cardiac T2* was not available at the beginning of the study. All but eight patients (3 death, 1 refusal, 2 claustrophobic, 2 pacemaker) subsequently had at least one T2* assessment. Routine lab tests were done at 1- to 6-m intervals. Blood counts were done at 7- to 10-d intervals for those taking DFP. Mean follow-up was approximately 40 m. Compliance with DFO was significantly better among low-risk pts in both treatment groups (DFP/DFO, 82% vs 61%; DFO alone, 83% vs 52%) as was that with DFP (94% vs 76%). At baseline, no statistically significant differences were observed between the SF levels, LVEFs or left ventricular shortening fractions (LVSFs) of pts on DFP/DFO or DFO alone in either risk group except for the LVEFs of the low-risk group (DFP/DFO, 56.5% +/− 5.5%; DFO alone, 65.4% +/− 5.0%; p = 0.032). In the high-risk group, four cardiac events (3 deaths, 1 worsening of CHF) occurred in the group getting DFO alone vs none in the DFP/DFO-treated group. The latter pts showed a decrease in SF and an increase in both LVEF and LVSF at the end of study (EOS). The three pts who died (at 17 to 35 m) had increased SFs. These pts were not rescued by IV DFO (98 +/− 12 mg/kg/d). The two DFO-treated pts who survived had marginally improved T2*s (1.5 to 3.0 ms and 7.6 to 8.8 ms) over the year prior to EOS. Only one of the seven evaluable pts on DFP/DFO had a T2* < 10 ms, the others averaging 19.4 +/− 6.7 ms. Among the low-risk pts, those on DFP/DFO showed a reduction in SF and an improvement in both LVEF and LVSF. Those on DFO alone had increased SF but essentially no change in LVEFs or LVSFs. Five pts on DFP/DFO had T2* evaluations. In two pts, T2* rose from 9.0 to 37 ms (38 m) and from 9.3 to 11.8 ms (17 m). The remaining three had T2* values > 20 ms at EOS. Similar results were seen in low-risk pts on DFO alone. These finding clearly support the notion that DFP/DFO has a beneficial effect upon the heart, even in well established disease. Moreover, our finding of low T2* values associated with low SF levels indicates the importance of tailoring treatment to each individual.

DEFERIPRONE, BUT NOT DEFEROXAMINE, INHIBITS BIOSYNTHESIS OF FIBRILLAR PROCOLLAGENS IN IRON-OVERLOADED THALASSEMIA PATIENTS. † 943

Summary. 2,3-Dihydroxybenzoic acid was evaluated as a potentially useful, orally effective iron-chelating drug by performing iron balance studies in patients with β-thalassaemia major. The administration of this substance at 25 mg/kg/d to five patients for 8 d caused an average increase in iron excretion of 4.5 mg/d. When the drug was administered at 25 mg/kg q.i.d. to eight patients for 21 d, iron excretion increased to 6.5 mg/d. Chelation was highly specific for iron with changes in magnesium and calcium excretion being insignificant. The drug was well tolerated with side effects limited to gastrointestinal complaints which ameliorated when the drug was taken with food. These studies provide a rationale for further evaluation of 2,3-dihydroxybenzoic acid in patients with iron overload.

The relative proportions of T-cell (OKT3-positive, OKT4-positive and OKT8-positive) and B-cell (SIg-positive) populations in peripheral blood obtained from 29 chronically transfused patients with beta-thalassaemia major were compared with those of 17 healthy controls. Changes attributable to blood transfusion and/or splenectomy are described. The percentage of OKT8-positive (T-suppressor) cells found in the thalassaemic patients increased linearly (P less than 0.001) with the number of units transfused, irrespective of splenectomy. The percentage of OKT4-positive (T-helper) cells varied inversely with increasing transfusion in nonsplenectomized patients while in those who were splenectomized no significant correlation was apparent. Thus, in both groups of patients the T4/T8 ratio declined in a transfusion-related manner. The splenectomized patients experienced a marked and persistent lymphocytosis due to an increase in the number of both T- and B-cells. When the results were expressed as percentages, the greatest increase occurred in the number of B-cells, this increase being unrelated to the number of transfusions received. None of the serum parameters usually associated with iron overload or abnormal liver function correlated with the observed increases in T-suppressor and SIg-positive cells. These findings corroborate reports that transfusion of blood products may lead to decreased T4/T8 ratios. However, none of the patients studied manifested clinical signs of acquired immune deficiency syndrome (AIDS). Accordingly, studies which define transfusion related AIDS on the basis of analyses with monoclonal antibodies must be viewed with caution.

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...

The search for better medication preliminary to anesthesia continues. The authors believe this ataractic drug has certain assets which may offer advantages in premedication.

Abstract 81 We investigated the contribution of macrophages to physiological and pathological conditions in which erythropoietic activity is enhanced. We utilized mouse models of a) anemia by phlebotomy-induced stress erythropoiesis (SE); b) increased erythropoiesis by erythropoietin (Epo) administration; c) Polycythemia Vera (Jak2V617F/+ or PV) and d) beta-thalassemia intermedia (Hbbth3/+ or BTI) in which macrophages were chemically depleted by injection of liposome-clodronate (LC). While chronic injection (up to 3 months) of LC in normal mice had little effect on steady state erythropoiesis, depletion of macrophages severely impaired recovery from anemia following phlebotomy and significantly limited the increase in hematocrit (Htc) in animals treated with Epo. To exclude that this effect was mediated by decreased serum iron parameters, we used mice iron overloaded by dietary means or affected by hemochromatosis (Hfe-KO and Hamp-KO). In these mice, recovery from anemia was still impaired following macrophage depletion, even though serum iron and transferrin saturation levels were elevated and unaffected by LC administration. In vitro studies using both mouse and human primary erythroblasts (EBs) indicated that EBs in S-phase were twice as many compared to EBs cultured in absence of macrophages. The numbers of terminally mature erythroid cells were up to six fold higher in co-culture conditions. Experiments using transwells indicate that direct contact between EBs and macrophages was required to generate this effect. Since our data highlighted an important role of macrophages in enhancing erythropoiesis, we investigated two disorders in which the pool of erythroid progenitor cells is expanded, such as PV and BTI. Chronic administration of LC in PV mice completely reversed splenomegaly and the Htc (P Our observations indicate that macrophages directly modulate stress- and pathological erythropoiesis. Several adhesion molecules participate in the formation of interactions within the erythroblastic islands, including integrins. Interestingly, βeta1integrin and its associated protein, focal adhesion kinase-1 (Fak1), were reported to be necessary for the compensatory response to anemia, suggesting that this pathway might be involved in the macrophage-EB cross-talk. More EBs co-cultured with macrophages retained cell surface expression of βeta1integrin molecule during the last stage of cell differentiation compared to EBs cultured alone, even though other differentiation markers did not shown any variation. Fak1 phosphorylation in EBs was induced by co-culturing them with splenic macrophages, suggesting that Fak1 signaling is one of the pathways activated in EBs through contact with macrophages. Administration of a FAK1 inhibitor (FAK1i) decreased proliferation of EB co-cultured with macrophages, while delayed recovery from anemia and decreased the spleen size in phlebotomized animals (40% decrease compared to phlebotomized control animals at day 4; P=0.032). Finally, short-term administration of FAK1i to BTI animals rapidly reverted splenomegaly with a concurrent reduction of erythroid expansion in both BM and spleen and led to amelioration of anemia, supported by increased RBCs count. Our data indicate that, while macrophages allow proper erythroid response under conditions of induced anemia or increased erythropoiesis in wt mice, they contribute to the pathological progression of PV and BTI. Activation of Fak1 promotes erythroid proliferation and pathological development, while its inhibition limits ineffective erythropoiesis and splenomegaly in BTI. In conclusion, we identified a new mechanism contributing to the pathophysiology of these disorders, which we believe will have critical scientific and therapeutic implications in the near future. Disclosures: Levine: Agios Pharmaceuticals: Research Funding. Rivella:Novartis Pharmaceuticals: Consultancy; Biomarin: Consultancy; Merganser Biotech: Consultancy, Equity Ownership, Research Funding; Isis Pharma: Consultancy, Research Funding.