Treatment of Iron Overload With Deferasirox (Exjade) in Hereditary Hemochromatosis and Myelodysplastic Syndrome
Treatment of Iron Overload With Deferasirox (Exjade) in Hereditary Hemochromatosis and Myelodysplastic Syndrome
Hypothesis: Deferasirox can be used as a therapeutic agent to deplete the liver, heart and
 bone marrow of excess iron in patients with iron overload caused by myelodysplastic syndrome
 (MDS) and hemochromatosis (HC.

 Assess the effect of new serum biomarkers (NTBI and hepcidin) and MRI as indicators of iron
 overload and their usefulness to monitor iron depletion treatment.

 Study the effect of iron overload and iron depletion on intracellular signal transduction,
 trace metals concentrations in serum and urine and markers of oxidative stress in blood cells
 and urine.
The two most important causes of iron overload disease in humans are the iron-loading
 disorder hereditary hemochromatosis (HC), and transfusional siderosis in patients with
 chronic hematological diseases like myelodysplastic syndrome (MDS), thalassemia, and
 leukemia.

 Hemochromatosis. In HC the molecular regulation of iron uptake across the intestinal mucosa
 is disturbed, leading to hyperabsorption and accumulation of iron in parenchymal tissues such
 as the liver, pancreas, endocrine organs and heart. Hepcidin, a small peptide hormone
 synthesized in the liver, apparently functions as the master regulator of systemic iron
 homeostasis by its ability to control the efflux of iron from the enterocytes and macrophages
 into blood plasma. This happens by diminishing the iron transfer capacity through the
 basolateral, transmembrane protein, ferroportin. Expression of hepcidin is carefully adjusted
 by the iron status of the hepatocytes through a multimolecular signal transducing pathway
 which acts as a positive feedback mechanism with increased hepcidin synthesis in iron
 overload (and inflammation). Mutation of one of the signal molecules, leads to inadequate
 hepcidin synthesis. The most common are the classic C282Y and H63D point mutations of the
 hemochromatosis protein HFE, which disturbs its interaction with the transferrin receptor 1,
 the first step in the hepcidin signal cascade. Homozygosity for C282Y is the strongest risk
 factor for serious iron overload and disease which develops after a long-lasting,
 asymptomatic period. In Norway the prevalence of C283Y homozygosity is approximately 0.75 in
 both genders. The preclinical, biochemical phenotype of HC is disclosed by blood tests with
 elevated transferrin saturation as a marker of hyperabsorption of iron, and increasing
 ferritin concentration as a surrogate marker of a growing iron overload. As an alternative to
 liver biopsy, magnetic resonance imaging (MRI) seems to be a powerful non-invasive method to
 directly assess the iron content of the liver and heart.

 Myelodysplastic syndrome and transfusional siderosis. Transfusional iron overload is the
 result of multiple blood transfusions supplied over a long time period, each of which adds
 approximately 220 to 250 mg of extra iron to the body. Because iron loading occurs via a
 parenteral route with the macrophages as the primary target cells, the normal homeostatic
 mechanisms are not responding adequately and the plasma hepcidin level can be normal or even
 increased. Even if excess iron is better tolerated in macrophages than in parenchymal cells,
 a life-threatening iron overload develops much faster with transfusional siderosis than
 HFE-associated HC. Myelodysplastic syndrome (MDS) is a heterogeneous group of stem cell
 disorders with inefficient hematopoiesis and increased risk of developing acute myeloid
 leukemia. The MDS International Prognostic Scoring System (IPSS) which is based on numbers of
 cytopenias, cytogenetic features and number of blasts in the bone marrow, enables separation
 of four risk groups with the following median survival years: low-risk patients, 5.7 years;
 intermediate-1, 3.5 years; intermediate-2, 1.2 years; and high-risk 0.4 years. Many patients
 become transfusion-dependent and get serious transfusional iron overload. Iron chelation is
 recommended to low-risk and intermediate-1 patients with probable need of lifelong
 transfusion therapy.

 Consequences of iron overload. If not interrupted, continuous iron loading will at some point
 start to gradually damage vital organs such as liver, heart and endocrine organs, and lead to
 serious disease and a shortened lifetime. At the molecular level persistently increased
 amounts of reactive, small molecular weight iron complexes or the sporadic presence of free
 iron in blood and tissues, are probably the fundamental pathogenetic mechanism in iron
 overload. These substances are highly toxic due to their strong ability to catalyze the
 formation of free radicals such as Reactive Oxygen Species (ROS). The activity of free
 radicals with damage of native molecules (e.g. DNA) and cellular structures is considered to
 be the first step in organ and tissue injury. In blood, reactive iron exists as
 Non-Transferrin Bound Iron (NTBI), mainly in the form of small molecular weight iron-citrate
 complexes. NTBI is rapidly taken up by the liver and other organs where it adds to the
 intracellular, reactive labile iron pool (LIP). The presence of NTBI and LIP has been found
 to correlate with hepatic and cardiac damage in transfusional siderosis and HC. In addition,
 the homeostasis of other trace metals may be abnormal in iron overload. Little is known about
 this issue, which warrants a deeper study.

 Treatment. In HC, the preferred iron removal therapy is venesection, whilst transfusional
 iron overload is treated with iron chelators.

 Clinical aims of study. The major objective is to determine the efficacy of deferasirox to
 remove iron from the liver, heart and bone marrow in HC- and MDS-patients with iron overload.

 Explorative aims. Determine the correlation between the novel biomarkers hepcidin and NTBI,
 and ferritin and transferrin saturation in serum; and liver iron concentration (LIC), heart
 iron concentration (HIC), and bone marrow iron.

 Assess the effect of iron overload and iron depletion with either deferasirox or venesection,
 on the concentration of trace metals in serum and urine in HC and MDS patients.

 Study the influence of iron overload and iron depletion with either deferasirox or
 venesection, on intracellular signal transduction in blood cells isolated from HC and MDS
 patients.

 Study the influence of iron overload and iron depletion with either deferasirox or
 venesection on oxidative damage of DNA (examined in urine), and the antioxidant status in red
 blood cells isolated from HC and MDS patients.

 Establish reference values for the investigational serum and urine tests in healthy control
 subjects.

 Rationale of the study. The major rationale of the study is related to the need to assess the
 potential use of deferasirox to treat HC- and MDS-patients with iron overload. However,
 several "learning objectives" are included: the impact of iron overload and iron depleting
 treatment on NTBI, hepcidin, markers of oxidative stress, trace metals and intracellular
 signal transduction molecules.

 Although venesection is a safe and effective means of depleting iron in HC patients, it may
 suppress hepcidin and thus increase iron absorption, particularly at the end of a long
 treatment period. Also, some HC patients not only feel the cumbersome impact of frequent
 phlebotomies in daily life, but also experience troublesome side effects.

 The subjective perception of venesection side effects was recently communicated in a survey
 study among 210 HC patients across the United States and some european countries. Of these,
 52% of patients undergoing weekly venesections and 37% of the patients in the maintenance
 phase reported side effects "always" or "most of the time", and 16% would "definitely" or
 "probably" refuse phlebotomy if they were offered an alternative treatment. Therefore,
 investigation of the effect of chelator as a potential alternative therapy in HC is
 warranted. Chelation therapy may not only remove excess iron from the body, but may also
 counteract formation of reactive, free radicals.

 In MDS patients transfusion dependency worsens the survival due to development of damaging
 iron overload. With every 500 ng/mL increase of s ferritin above the threshold, the risk of
 death increases by 30%.

 Traditionally, iron depletion therapy has been monitored by s-ferritin, which decreases as
 iron is progressively eliminated. However, s-ferritin is a non-specific parameter which is
 sensitive to acute phase reactions associated with inflammations, chronic diseases and tissue
 injury. Furthermore, s-ferritin does not offer information about the burden of free radical
 production stimulated by reactive iron. For this reason, one aim of this study will be to
 investigate whether new blood markers like NTBI and hepcidin, along with MRI assessment of
 LIC and HIC, will improve the diagnosis of iron overload and more accurately identify those
 patients who are at increased risk of iron induced damage and therefore will benefit from
 iron depletion therapy.

 Iron overload can weaken the cellular antioxidant defence and result in oxidative DNA-damage
 as the first step in carcinogenesis. Examination of the influence of iron overload and iron
 depletion with either deferasirox or venesection on oxidative damage of DNA, and on the
 antioxidant status in red blood cells will be performed.

 Increased knowledge of how intracellular signal transduction is influenced by cellular iron
 and use of deferasirox may provide new insights into the molecular pathogenesis of iron
 overload. This will be examined in single leukocyte cell flowcytometry assays of the
 following intracelluar signal parameters: stress sensor p53, mTOR and NFkB.

 Finally, little is known about how iron overload interacts with the metabolism of other trace
 metals. The concentration of a broad spectrum of trace metals in serum (S) and urine (U) will
 be monitored throughout the study period by Inductively Coupled Plasma Mass Spectrometry
 (ICP-MS) (28) : boron (S,U), barium (S,U), beryllium (S,U), cadmium (S,U), cobalt (S,U),
 cesium (S,U), copper (S,U), mercury (U), lithium (U), manganese (S), molybdenum (S), nickel
 (S,U), lead (S,U), antimony (S,U), selenium (S,U), tin (S,U), strontium (S,U), thallium
 (S,U), tungsten (S), yttrium (S,U), zinc (S,U).

 The tolerability of deferasirox will also be assessed throughout the study, either as sole
 treatment in MDS patients or compared to venesection in HC patients.

 Healthy volunteers are used solely as controls to determine the normal blood concentration of
 the biomarkers and tests which will be performed in the patients. The controls will not be
 treated.

 Previous clinical trials in HC and MDS. Deferasirox has been studied in a variety of diseases
 with transfusional iron overload, particularly in thalassemia. It has a favorable profile
 with respect to efficacy, safety and tolerability. However, controlled clinical studies with
 deferasirox in HC and MDS are limited.

 The study by Phatak et al (2010) was the first clinical trial to demonstrate the safety and
 efficacy of deferasirox in patients with C282Y-homzygot hemochromatosis. They showed that
 serum ferritin levels were reduced similarly in two cohorts given respectively 10 mg/kg/day
 and 15 mg/kg/day of deferasirox. Since the dose of 15 mg/kg/day caused a higher frequency of
 adverse events (AEs), they recommended a staring dose of 10 mg/kg/day to be most appropriate
 in HC- patients. The most important AEs were associated with an increase in serum creatinine
 and liver enzymes. The study was limited by pretreatment of some patients with phlebotomy
 prior to the deferasirox study; in addition, many of the patients had only mild iron
 overload. For this reason, we see no argument to use a higher dose in our study, and
 therefore we choose a starting deferasirox dose of 10 mg/kg/day in HC- patients.

 In a recent study, 24 heavily transfused MDS patients were enrolled to receive 52 weeks of
 deferasirox therapy. Deferasirox was well tolerated and effectively reduced LIC, labile
 plasma iron and s-ferritin in patients completing 24 to 52 weeks of therapy, despite ongoing
 receipt of red blood cell transfusions. In addition to reducing long-term toxicity of iron
 overload, chelation therapy has shown beneficial effects on hematopoiesis in a small
 proportion of MDS patients. One ongoing study comparing deferasirox with placebo was found in
 Cochrane search carried out in June 2010.

 Pharmacokinetics of deferasirox. Deferasirox (Exjade®) is a bivalent iron chelator which is
 taken orally as a tablet, once per day. It is readily absorbed and reaches peak concentration
 in blood after 1 to 2 hours. The half-life is 12 to 17 hours, and effective levels of active
 chelator are maintained in the blood for more than 24 hours. The drug is eliminated via the
 hepatobiliary system. The dose varies from 10 mg/kg/day in patients with mild iron overload
 to 30 mg/kg/day in patients with overt iron toxicity.

 Adverse events (AEs) of deferasirox. The most frequent adverse clinical events are
 gastrointestinal disturbances and skin rash. The most frequent laboratory side effects are
 mild increase in serum creatinine and liver transaminases. The adverse events have for the
 most part been reported in patients with transfusional iron overload. In HC patients the data
 are more scarce due to the very few studies that have been performed in this group. Other
 common AEs include headache, constipation, abdominal distension, dyspepsia, pruritus, and
 proteinuria. Less common AEs include anxiety, sleep disorder, dizziness, early cataract,
 maculopathy, hearing loss, pharyngolaryngeal pain, gastrointestinal hemorrhage, gastric
 ulcer, duodenal ulcer, gastritis, oesophagitis, hepatitis, cholelithiasis, pigmentation
 disorder, renal tubulopathy, glycosuria, pyrexia, edema, and fatigue.

 Adverse reactions reported during post-marketing experience include pancytopenia,
 hypersensitivity reaction, hepatic failure, leukocytoclastic vasculitis, urticaria, erythema
 multiforme, alopecia, acute renal failure, and tubulointerstitial nephritis, gallstones and
 related biliary disorders, elevations of liver transaminases, and hepatic failure. Any such
 adverse events will be recorded in the subject's Case Report Form (CRF).

 Study design.

 The study incorporates 4 arms, which will recruit in parallel:

 Arm 1: (open, randomized, comparative) will consist of 10 patients with confirmed HC, who
 will be randomized to receive standard treatment with venesection (withdrawal of 450 mL blood
 every 8 to 10 days).

 Arm 2: (open, randomized, comparative) will consist of 10 patients with confirmed HC, who
 will be randomized to receive investigational treatment with deferasirox (10 mg/kg orally
 given once daily) for a period of 12 months;

 Arm 3: (open, non-comparative) will consist of 20 patients with confirmed MDS, all of whom
 will receive investigational treatment with deferasirox (initially 10 mg/kg orally given once
 daily, can be increased to max 40 mg/kg) for a period of 12 months;

 Arm 4: will consist of 10 healthy volunteers who will undergo the same screening assessments
 (i.e. with respect to relevant exclusion criteria) and will act as non-treated normal
 controls for comparison of investigational blood tests.

 The study will be non-blinded. Subjects and investigators will be aware of their subject and
 treatment arm.

 Subjects will undergo assessments at screening (within 1-14 days prior to baseline), and for
 patients at baseline Day 0, and at 2,4,6,8 weeks, and thereafter monthly until 12 months, and
 at follow up 4-6 weeks after treatment has been completed. Healthy controls will be examined
 at screening, 2,6 and 12 months. Study-specific assessments will include physical
 examinations/vital signs, sampling of blood, urine, and for patients only: bone marrow and
 MRI to assess the effect of iron overload and iron reduction treatment on the following:

 -  Conventional tests to indicate iron overload: ferritin and transferrin saturation in
 serum; LIC and HIC, bone marrow iron content using microscopy.

 -  Hepcidin concentration in serum;

 -  NTBI concentration in serum;

 -  Intracellular signal transduction parameters: Analysis of the stress sensor p53, NFkB,
 and mTOR in leukocytes from peripheral blood;

 -  Trace metal concentrations in serum and urine;

 -  Antioxidant capability in blood hemolysate: Cu,Zn-SOD;

 -  Free radical damage to DNA and RNA: 8-oxodG concentration in urine;

 -  For MDS patients: number of transfusions;

 -  For HC and MDS patients: number of hospitalizations and overall survival;

 -  Safety and tolerability.

 For patients and controls, samples of peripheral blood will be taken pre-dose, and at 24
 hours after dosing, for assessment of stress sensor p53, NFkB, and mTOR.

 Interim assessments. Routine blood and urine tests will be performed for patients at 2,4,6,8
 weeks, and thereafter monthly until 12 months and at follow up 4-6 weeks after end of
 treatment for monitoring the disease, for safety reasons, and to obtain data for comparison
 traditional biomarkers with the new investigational biomarkers hepcidin and NTBI.

 Volunteers will undergo all safety assessments plus investigational blood and urine tests.
 They will not undergo MRI, bone marrow examinations, registrations of transfusions, or active
 treatment with deferasirox or venesection.

 Patients with HC or MDS will undergo a follow-up assessment 4 to 6 weeks after last dose. At
 this visit, all safety assessments and investigational blood and urine tests, including a
 pregnancy test, will be repeated, but MRI and bone marrow examination will not be performed.
 Volunteer controls will not attend for this visit.

 This is an exploratory study with no sample size calculation and no minimum enrollment
 requirement. It is anticipated that a maximum of 40 patients will be enrolled in order to
 obtain at least 30 patients fulfilling the study criteria; 10 volunteers will also be
 enrolled.
Inclusion Criteria:

 -  Patients with hemochromatosis, aged > 30 years, C282Y- homozygote, with serum-ferritin
 =/> 1000 µg/L

 -  Patients aged > 18 years with verified low-risk or intermediate-1 risk of
 myelodysplastic syndrome, with normal cytogenetics and serum-ferritin > 1500 µg/L, or
 with a transfusion history of =/> red- blood-cell-transfusions.

 Exclusion Criteria:

 -  Previous or current venesection

 -  MDS patients eligible for hematopoietic stem cell transplantation

 -  Subject complies with one or more of the following standard exclusion criteria for MRI
 examination;

 -  If the patient has a pacemaker.

 -  If the patient has a neurostimulator

 -  If the patient has a "aneurismeclips"

 -  If the patient has a foreign object in the eye. If yes, what object.

 -  If the patient has a cochlea-/earimplant.

 -  If the patient has a V/P shunt.

 -  If the patient is claustrophobic.

 -  If the patient has an artificial heart valve.

 -  If the patient has known renal failure, eGFR <30.

 -  If the patient has or will have a liver transplantation.

 -  Other: metal prostheses, metal implant

 -  Presence of inflammation (CRP ≥ 5 mg/L)

 -  Presence of proteinuria or creatinine > 2 x UNL (Upper Normal Limit)

 -  Estimated glomerular filtration rate (GFR) < 60 mL/min

 -  ALAT, ASAT, GT or ALP > 2 x ULN ( Upper Normal Limit)

 -  ALAT > 90 U/L for women, ALAT > 140 U/L for men

 -  ASAT > 70 U/L for women, ASAT > 90 U/L for men

 -  ALP > 210 U/L for women and men

 -  GT > 90 U/L for women ≤ 40 years, GT > 150 U/L for women > 40 years

 -  GT > 160 U/L for men ≤ 40 years, GT > 230 U/L for men > 40 years

 -  Acute or chronic hepatitis

 -  Patients with chronic liver disease Child Pugh Class B and C

 -  Chronic skin disease with rash

 -  Estimated survival < 6 months

 -  Prior or concomitant treatment with other iron chelator therapies within 6 weeks of
 screening

 -  History of non-compliance to medical regimens, or considered potentially unreliable
 and/or not cooperative

 -  Uncontrolled diabetes, defined as glycolated hemoglobin (HbAlc) > 8.5%

 -  Presence of cataracts or hearing loss disease

 -  Presence of a surgical or medical condition which might significantly alter
 absorption, distribution, metabolism or excretion of study drug

 -  Planned in-hospital surgeries during the course of the study

 -  Subjects who are pregnant, breast-feeding, or intending to become pregnant

 -  Hypersensitivity to the active substance or the excipients in drug product

 -  Any other reason why, in the opinion of the investigator, the patient should not
 participate (e.g. serious heart disease, infection, cancer, etc).