ALBERT

Description: Rationale. The relative effects of monotherapy regimes with oral DFP or sc DFO or combinations of these drugs, on chelation efficiency, iron balance, plasma non-transferrin bound iron (NTBI) and the proportion and speciation of urine iron, have not been compared previously in prospective randomised trials. By examining these variables together in a single study, insights into the effectiveness and mechanisms of action of mono or combination regimens can be gained. Design. A total of 25 patients (pts) with thalassaemia major were randomised into one of the following 3 arms: DFP (LIPOMED AG, Switzerland) was given at a daily dose of 75 mg/kg either in combination with DFO (40–50 mg/kg twice weekly) or as single agent, and pts registered in the DFO control arm received 40–50 mg/kg sc DFO 5 days a week. All pts had been treated with DFO prior to the study. Methodology. Liver iron concentration (LIC) was measured by biopsy at baseline and after 1 year. Total iron excretion (IE)/day was calculated as (iron transfused/year (mg) + (LIC at To - LIC at T1y) x 10.6 x body wt in Kg) /number of days treatment. Chelation efficiency (%) was calculated as [IE (in mg/kg/day)/chelator dose (in mg/kg/day)] x [molecular weight of the respective chelator(s)/56]x n x 100, where 56 is the molecular weight of iron and n is 3 or 1 with DFP and DFO respectively. The average urinary iron excretion (UIE) (weeks 1, 12, 26, 38 and 54) was calculated from atomic absorption measurements. In patients receiving combination, UIE was measured during both days of monotherapy and combination therapy. The % UIE was calculated from mean UIE divided by total IE. In patients receiving DFO with or without DFP, the concentration of feroxamine species were measured in urine, collected into aluminised containers. Plasma NTBI was measured by HPLC at baseline and at weeks 1, 12, 26 and 54 standardised to blood transfusion interval. Results. The results show that DFO sc 5 days a week was the regimen associated with the largest and most efficient IE and stabilised NTBI after 1 year. Single agent DFP showed the lowest IE and efficiency, and a significant increase in NTBI (* p=0.001). The addition of sc DFO two days a week to DFP resulted in significant increase in IE (^ p=0.03) and stabilised NTBI. The proportion of UIE was significantly lower with DFO than with DFP regimens. UIE on the days of combination (0.89± 0.14mg/kg/d) was significantly higher than on days of DFP monotherapy (0.41 ±0.09 mg/kg/d) (p=0.002). Speciation of urinary iron showed that the proportion of DFO bound to iron was higher on days of combination treatment than with DFO monotherapy, consistent with shuttling of iron onto DFO by DFP. Conclusions. The addition of sc DFO twice weekly to DFP at 75mg/kg po is a regimen which appears as effective at producing iron balance as DFO given 5 days a week at standard doses. Both regimens are more effective at stabilising NTBI, and achieving iron balance than DFP at 75mg/kg/day. When DFP is combined with DFO, iron excretion appears to be mainly by the urinary route as feroxamine. Treatment Regimen Iron Excretion mg/kg/d NTBI change μM Efficiency (%) Mean over study Urine Iron (%) Mean over study DFP (n=12) 0.56±0.09 3.00±0.53 * 6.65±0.99 98±14.8 DFO (n=5) 0.77±0.25 0.72±0.45 23.1±10.7 28±10.3 DFP+DFO (n=8) 0.69±0.09 ^ 0.84±0.87 7.34±0.91 84±14.6

Description: Introduction:. Myocardial iron can now be rapidly and reproducibly assessed using a validated cardiovascular magnetic resonance (CMR) T2* technique. However, the calibration of T2* value to absolute myocardial iron concentration is currently unknown in humans. Purpose: To calibrate myocardial T2* values in post-mortem hearts. Methods: Two patients’ hearts have been examined. Each was scanned at 37 Celsius using optimised T2* MR sequences. Iron assays were performed with 100 samples per heart, from 5 short axis slices of the left ventricle. For each slice, 6 segments were cut and a sample taken from the endocardium, mid-wall and epicardium, and 2 from the right ventricular free wall (20 samples per slice). This trial was supported by NIH grant R01 DK66084-01. Results: Heart 1 from Genova Italy, was from a patient with beta-thalassemia major who had been transfused from age 2 to his death at age 47 from a stroke. The patient had heart failure aged 21 years, from which he had recovered. At age 46, the patient had bradycardia and arrhythmia, and his long-term deferoxamine treatment was augmented with deferiprone for 6 months, and then switched to deferiprone alone at 69mg/kg/day for 7 days per week, starting 1 year prior to death. Ex vivo mid-ventricle full thickness septum T2* was 45ms. Heart 2 from London was from a transfusion dependent patient with sideroblastic anemia with poor compliance with deferoxamine. The patient died in heart failure. Further clinical details are awaited. Pre-mortem mid-ventricle septum T2* was 5.2ms, and the T2* value in the same region post-mortem was identical at 5.2ms. The results of the ex-vivo comparison of segmental myocardial T2* and matching segmental absolute iron level are shown in figure 1. Calibration is shown on a log-log scale in accord with previous liver sample evidence showing this allows fitting with a linear function (Anderson LJ. Eur Heart J2001; 22: 2171.9). Conclusion: These are the first human data calibrating human myocardial T2* against absolute iron levels. Myocardial T2* clearly decreases as myocardial iron increases. Of interest was the identical myocardial T2* result between the in-vivo and post-mortem scans in patient 2. Further hearts are now needed to complete the calibration and to determine the consistency of calibration between hearts. Figure Figure

Description: Correction of a disease gene mutation by homologous recombination (HR) with a transfected DNA repair template represents an ideal form of gene therapy for many inherited hematological disorders. Therapeutic gene correction via HR in hematopoietic stem cells is unlikely to succeed, however, without major improvements in its efficiency. One promising approach is to use an engineered zinc finger endonuclease to specifically cleave the target gene and so promote HR between a dsDNA template and its chromosomal target (Urnov et al (2005) Nature 435:646-51). We are exploring an alternative approach based on the expression of Redβ, a single stranded (ss) DNA annealing protein from bacteriophage λ. In bacteria, Redβ promotes HR-mediated gene modification using templates with short regions of homology (e.g. 30–100nt). Such HR is independent of RecA-mediated host cell HR. For double stranded (ds) DNA templates, co-expression of λ exonuclease (the red α gene product) is required, but ssDNA templated modifications are particularly efficient and require only Redβ expression. Such properties could be useful for therapeutic gene correction if reproduced in human cells. Preliminary experiments in mouse embryonic stem cells suggested that Redβ can function in mammalian cells (Zhang Y. et al, (2003) BMC Mol. Biol. 4). To explore this further, we have generated a human cell line in which large amounts of Redβ can be expressed inducibly in the nucleus. In contrast to the over-expression of endogenous HR proteins Rad51 (RecA homologue) or Rad52 (ssDNA annealing protein), sustained Redβ over-expression is well tolerated, with no impairment of cell proliferation or viability. We find that Redβ does indeed promote HR in human cells. Surprisingly, dsDNA and ssDNA templated gene modification are stimulated to similar extents by Redβ. Stimulations are modest, but similar to those achieved by human Rad51 over-expression. The effects of co-expressing Red α are under investigation. We have also shown that ssDNA-templated gene correction is stimulated by transient, RNAi-mediated, depletion of the mismatch repair protein MSH2, and that such stimulation is additive with Redβ-stimulated gene correction.

Description: CD4+CD25+ T regulatory (Treg) cells have been shown to critically regulate self and allograft tolerance in mice. Studies of human Treg cells have been hindered by low numbers present in peripheral blood and difficult purification. We found that cord blood was a superior source for Treg-cell isolation and cell line generation compared with adult blood. Cord blood CD4+CD25+ cells were readily purified and generated cell lines that consistently exhibited potent suppressor activity, with more than 95% suppression of allogeneic mixed lymphocyte reactions (MLRs) (29 of 30 donors). Cultured Treg cells blocked cytokine accumulation in MLRs, with a less robust inhibition of chemokine production. These cell lines uniformly expressed CD25, CD62L, CCR7, CD27, and intracellular cytotoxic T-lymphocyte antigen-4 (CTLA4). FoxP3 protein, but not mRNA, was specifically expressed. Upon restimulation with anti-CD3/CD28 beads, the cultured Treg cells produced minimal cytokines (interleukin-2 [IL-2], interferon-γ [IFN-γ], and IL-10) and preferentially expressed tumor growth factor-β (TGF-β) latency associated protein. Cytokine production, however, was restored to normal levels by restimulation with phorbol myristate acetate (PMA)/ionomycin. Cord blood–derived cultured suppressor cell function was predominantly independent of IL-10 and TGF-β. These results demonstrate cord blood contains a significant number of Treg precursor cells capable of potent suppressor function after culture activation. Banked cord blood specimens may serve as a readily available source of Treg cells for immunotherapy.

Description: CD4+CD25+ regulatory T cells (Tregs) are essential negative regulators of immune responses. Here, we examined the signaling properties of human Tregs, using CD4+CD25+ Treg and CD4+CD25– control (Tcont) cell lines generated from cord blood. Treg cell lines were markedly hyporesponsive to stimulation with dendritic cells and with anti-CD3/CD28–coated beads. Hyporesponsiveness was reversed by exogenous interleukin-2 (IL-2). T-cell receptor (TCR)–CD3/CD28–mediated activation of Rap1 and Akt was retained in Tregs, but activation of Ras, mitogenactivated protein kinase 1/2 (MEK1/2), and extracellular signal-regulated kinase 1/2 (Erk1/2) was impaired. Tregs were blocked from cell cycle progression due to decrease of cyclin E and cyclin A and increase of p27kip1 (p27kip cyclin dependent kinase inhibitor). IL-2 induced sustained increase of cyclin E and cyclin A and prevented up-regulation of p27kip1. Tregs had high susceptibility to apoptosis that was reversed by IL-2, which correlated with activation of Erk1/2, up-regulation of Bcl-xL (B-cell CLL/lymphoma 2-like nuclear gene encoding mitochondrial protein, transcript variant 2), and phosphorylation of Bad (Bcl2 antagonist of cell death) at Ser112. Thus, Tregs share biochemical characteristics of anergy, including abortive activation of Ras-MEK-Erk, increased activation of Rap1, and increased expression of p27kip1. In addition, our results indicate that TCR–CD3/CD28–mediated and IL-2 receptor–mediated signals converge at the level of MEK-Erk kinases to regulate Treg survival and expansion and suggest that manipulation of the MEK-Erk axis may represent a novel strategy for Treg expansion for immunotherapy.

Description: Introduction: Myocardial iron overload is a well established cause of heart failure in a number of haematological disorders and is the leading cause of death in β-thalassaemia major (TM). Once overt heart failure develops prognosis is very poor and it would therefore be desirable to identify patients at risk, prior to the development of symptomatic heart failure. Myocardial iron can now be rapidly and reproducibly assessed using a validated cardiac magnetic resonance (CMR) T2* technique. Left ventricular ejection fraction has been demonstrated to relate to myocardial T2* (normal 〉20ms), and accordingly iron overloaded patients with symptomatic heart failure are likely to have a low T2* but there are no reports of the myocardial T2* level in newly presenting patients with heart failure and the threshold for increased risk is also unknown. Purpose: To establish the distribution of myocardial T2* values in patients presenting with symptomatic heart failure secondary to iron overload. Methods: Database records of CMR T2* assessments over a 5 year period were reviewed to identify iron overloaded patients presenting with heart failure. Results: 28 patients (median age 29y, 11–79) with iron overload and documented heart failure were identified. 22 patients had thalassaemia major, 3 hereditary haemochromatosis, and 3 had miscellaneous transfusion dependent anaemias. The mean myocardial T2* in all groups was 6.8+/− 2.2ms reflecting severe iron loading by clinical criteria. See figure 1. Myocardial T2* values were similar between sub-groups as follows: TM patients 6.7+/− 2.4ms, haemochromatosis 7.7ms (6.7–7.4ms), miscellaneous 6.8ms (4.8–9.1ms). Conclusion: In this database review, the patients developing heart failure secondary to iron overload all had abnormal myocardial T2*, and overall 89% of heart failure patients had values less than 10ms, which reflects severe myocardial iron loading. This data suggests a myocardial T2*

Description: Objective: We examined whether oxidant-stress and inflammation in β-thalassemia can be controlled by deferasirox as effectively as by deferoxamine (DFO) through analysis of body iron burden and biomarkers of lipid peroxidation and inflammation. 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±11.1 yrs) were enrolled in the study. Blood samples were obtained fasting after a 5-day washout of DFO prior to commencing treatment with study drug, and 24 hours post-chelator and antioxidant supplementation at 1, 6, and 12 months on study. Thirty healthy untreated controls matched for age, sex, and race (15 male, 24.5±9 yrs) were also studied. Plasma levels of malondialdehyde (MDA), a marker of lipid peroxidation, were determined by GC-MS; vitamin C by spectrophotometry; α-tocopherol by HPLC; and high-sensitivity C-reactive protein (hsCRP), a marker of inflammation, by nephelometry. In patients with thal, liver iron concentration (LIC) was analyzed by atomic absorption spectrometry and ferritin by immuno-assay. MDA, α-tocopherol, and hsCRP were log-transformed prior to analysis. Back-transformed means are reported. Results: Mean MDA concentrations (nmol/L) at 0, 1, 6, and 12 months were as follows: deferasirox: 78, 77, 46, 48; DFO: 40, 44, 37, 33. Mean MDA concentration in control subjects was 19 nmol/L (range 2–76 nmol/L). MDA was elevated in both treatment groups as compared to controls (p

Description: ICL670 (deferasirox) is an orally active iron chelator, intended as once daily mono-therapy for the treatment of transfusional iron overload, which has undergone extensive multi-centre Phase II and Phase III trials. Data from such studies suggest that 20mg/kg as a single daily dose will achieve iron balance in the majority of transfusionally dependent patients, whereas 30mg/kg/day typically achieves negative iron balance (Cappellini, Abstract 3619, ASH 2004) (Porter, Abstract 3193, ASH 2004). Patients with transfusion dependent anaemia and iron overload who were entered into these multi-centre studies at UCL Hospitals had myocardial T2* CMR performed at the Royal Brompton Hospital, as part of their routine monitoring, in line with previous clinical management at our centre. Study 108, a non-randomized study, included poorly chelated patients with -thalassemia and other iron overload conditions receiving regular blood transfusion: all patients received 10-30mg/kg/day of ICL670. Study 107 consisted exclusively of transfusion dependent -thalassemia patients, randomized to receive either 8–10h sc DFO (30–50mg/kg/day) or ICL670 (10–30mg/kg/day) with doses stratified for baseline liver iron concentration (LIC). We report here changes in cardiac T2*, LIC and serum ferritin, as well as LVEF in all patients on these two studies treated at UCLH for a mean of 13.1± 0.78 months, in whom CMR was obtained. In a total of 23 patients treated with ICL670, mean age 24.6y (range 9–50y), 18 with transfusion dependent -thalassaemia and 6 other iron overload conditions (2 Pyruvate kinase deficiency, 2 sideroblastic anemia, 2 Diamond Blackfan Anemia), myocardial T2* improved significantly from a pre-treatment geometric mean of 18.0ms to 23.1ms (p = 0.013, paired student t test). In the same patients, serum ferritin fell significantly from 3173 ±410 μg/L to 2451 ±242μg/L (p= 0.023, paired student t test) and LIC fell significantly from 18.3 ±2.2 mg/g dry wt to 10.0 ±1.49 (p= 0.0002, paired student t test). There was no significant change in LVEF before or after treatment over the same period. Patients treated in the DFO arm of study 107 (n=8) also showed a small non-significant increase in myocardial T2* from 18.1 ms to 21.1 ms (p= 0.11), These studies suggest that once daily mono-therapy with ICL670 will be effective at improving myocardial T2* and by inference myocardial iron loading in a wide range of patients with transfusional iron overload. Prospective randomised controlled studies in larger patient numbers are now indicated.

Description: Free plasma Hb has been recently implicated in the genesis of pulmonary hypertension in sickle cell disorders (SCD) through a putative NO scavenging mechanism. We have recently reported that free plasma Hb is correlated with increased plasma red cell derived vesicles (RBVes) in sickle cell and thalassemia disorders (Westerman, Blood2004; 104:1671). Since high levels of HbF have been associated with decreased elevation of RBVes in children with SCD (Setty Blood2000;96:1119–1124), we hypothesized that patients treated with hydroxyurea (HU) might show a similar normalisation of RBVes. Given our finding of an association between RBVes and free plasma Hb, such treatment might also be predicted to correct increments in free plasma Hb. In this study we have examined whether sickle cell patients treated with HU show evidence of a decrease in free plasma Hb and RBVes compared to untreated SCD patients. RBVes, free plasma Hb, and markers of activation of thrombolysis were quantitated. 35 adult SCD patients, 27 in steady state, untransfused and not receiving HU and 7 patients on long term HU at a dose of 10–30 mg/kg/day. These parameters were also measured in 27 healthy control subjects without SCD. RBVes were significantly higher in untreated SCD (15.59±3.07 x 103/μL of blood) than in control subjects (2.13 ± 0.5)(p=0.001). In HU-treated SCD patients, vesicles were significantly lower (3.97±0.83)(p=0.0371). than in untreated patients and approached levels found in healthy controls. Free plasma Hb was significaly increased in untreated SCD (16.74±3.87) compared with healthy controls (1.77±0.2)(p=0.001); these levels were also corrected in HU treated SCD patients (13.42±4.48). The proportion of RBVes expressing phosphatadyl serine was also lower in HU-treated SCD (37.25± 9.79) than in untreated SCD (52.52±4.74). This was paralleled by a decrease in red cells expressing PS in HU treated patients (11.83± 3.95) compared with untreated patients (42.71±8.45)(p=0.011). This decrease in both RBVes and RBCs expressing PS in HU-treated patients might in principle decrease activation of coagulation. Indeed, plasma Ddimers, which are increased in untreated SCD (480 ±60.14) compared with healthy controls (117.43±38)(p=0.018). are normalised in HU treated patients (190±28.69). These data provide evidence of a link between RBVes and plasma Hb and suggest that PS expression on RBVes and red cells plays a role in the prothrombotic state seen in SCD. As HU treatment appears to correct plasma Hb, long term treatment may be associated with a decreased scavenging of NO and hence the risk of pulmonary hypertension. The correction of the prothrombotic tendency may also be beneficial in this regard. Prospective trials are indicated to confirm whether HU treated patients have evidence of decreased risk of pulmonary hypertension.

Description: Repeated blood transfusions in Thalassemia Major (TM) may lead to myocardial iron accumulation and death. Left ventricular (LV) function is commonly used to assess for iron overload, however, the reference range in TM for these variables in the absence of myocardial iron loading is not known. We used cardiovascular magnetic resonance (CMR) in 205 TM patients and studied those (N=81) with normal myocardial T2* measurements (T2*〉20ms) and by inference without excess myocardial iron. Resting LV volumes and function normalized to body surface area were compared with 40 age and gender matched healthy controls. All LV parameters were significantly different (p