**Mechanism of action of SQUID:**

SQUID has high-power magnetic field. Iron interferes with the field and changes in the field are detected [6].

#### **Advantages and disadvantages of SQUID:**

Advantages and disadvantages of SQUID are listed in **Table 1** [6].


#### **Table 1.**

*Beta Thalassemia*

• ICSI/IVF;

**Mutation testing:**

• serum ferritin;

**Technical aspects (Figure 1) [2]:**

1.obtaining embryos (assisted reproductive technology)

2.obtaining biopsy (blastomere or blastocyst biopsy);

4. embryo transfer and cryopreservation of surplus embryos.

3.genetic analysis techniques (PCR, FISH); and

analysis to achieve the highest accuracy rate [4].

• liver biopsy for liver iron content (LIC);

order to avoid serious complications [6].

• MRI T2\* for LIC and cardiac iron.

• echocardiography, MUGA for cardiac iron; and

b.New methods—monitoring of liver iron overload by SQUID:

*1.1.2 Assessment of iron overload in thalassemia*

• controlled ovarian stimulation (COH) to obtain large number of oocytes and

PGD method to diagnose beta thalassemia was initially reported in 1998 and used either denaturing gradient gel electrophoresis or restriction enzyme digestion methods to perform the mutation analysis. The possibility of misdiagnosis due to allele drop out (ADO) and DNA contamination is considered to be the major problems associated with preimplantation genetic diagnosis [3]. Recently, the European Society of Human Reproduction and Technology (ESHRE) recommends doing both direct and indirect mutation testing using short tandem repeat (STR) linkage

a.Well established methods for assessment of iron overload in thalassemia [5]:

• Superconducting QUantum Interference Device (SQUID) has been recently introduced as an integral part of thalassemia care in few centers worldwide. The SQUID allows the thalassemia team to monitor iron concentration in the livers of patients and gives them a reliable tool to help adjust medication in

**16**

**Figure 1.**

*Technical steps of PGD [2].*

*Advantages and disadvantages of SQUID.*

#### **1.2 Updates in treatment**

#### *1.2.1 Gene therapy*

The first obstacle against gene therapy was the extremely complex regulation of the globin genes. The second and equally important obstacle has been the lack of an optimal vector for gene transfer into quiescent hematopoietic stem cells (HSC) [7].

The first successful gene therapy for β-thalassemia major was in 2007. The process is as follow: autologous HSCs are harvested from the patient and then genetically modified with a lentiviral vector expressing a normal globin gene. After the patient has undergone appropriate conditioning therapy to destroy existing defective HSCs, the modified HSCTs are reintroduced to the patient [8].

Two large clinical trials have been recently conducted. The first one was entitled "ß-Thalassemia Major with Autologous CD34+ Hematopoietic Progenitor Cells Transduced with TNS9.3.55 a Lentiviral Vector Encoding the Normal Human ß-Globin Gene." This trial was sponsored by Memorial Sloan Kettering Cancer Center. The second one was entitled "A Study Evaluating the Efficacy and Safety of the LentiGlobin® BB305 Drug Product in Subjects with Transfusion-Dependent β-Thalassemia, who do Not Have a β0/β0 Genotype." It was sponsored by bluebird bio. Expected success awaits these clinical trials (**Figure 2**) [9].

#### *1.2.2 Gene editing*

A newer approach employs genome editing techniques, such as transcription activator-like effectors nucleases (TALEN), zinc finger nucleases (ZFN), or the clustered regularly interspaced short palindromic repeats (CRISPR) with Cas9 nuclease system. They replace the target single-mutation sites with the correct sequence, restoring the functional gene configuration. Producing a sufficiently large number of corrected genes is the major challenge with this new approach (**Figure 3**) [7].

#### *1.2.3 Targeting ineffective erythropoiesis*

A large number of preclinical and early clinical studies investigating erythropoiesis modulators are currently studied. These modulators include

#### **Figure 2.** *Gene therapy of beta thalassemia [9].*

**19**

*Updates in Thalassemia*

*DOI: http://dx.doi.org/10.5772/intechopen.92414*

erythropoiesis and splenomegaly [12].

controls anemia in β-thalassemic mice [13].

*1.2.5 New formulation of iron chelators*

three available iron chelators.

infectious complications [14].

*1.2.5.1 Deferoxamine*

*1.2.5.2 Deferiprone*

*1.2.5.3 Deferasirox*

*1.2.4 Manipulating iron metabolism*

treatment of β-thalassemia and related disorders [10].

TGF-β-like molecules or inhibitors of JAK2, Foxo3 activators, HRI-Eif2ap stimulators, Prx-2 activators, HO-1 inhibitors, Hsp-70 chaperone induction and pyruvate kinase activation. These modulators could soon revolutionize the

Activins, members of TGF-β family signaling, are crucial regulators of hematopoiesis and modulate various cellular responses such as differentiation,

erythropoiesis. Drugs inhibiting JAK2 activity could reverse ineffective

proliferation, migration, and apoptosis. They were observed to ameliorate hematologic parameters and improve hematopoiesis in preclinical and early clinical studies [11]. JAK2 plays an important role in the progression and worsening of ineffective

Hepcidin is the central regulator of iron homeostasis. Hepcidin inhibitors, e.g., ERFE inhibitors are now extensively studied as a possible future treatment of iron overload. Induction of iron restriction by means of transferrin infusions, minihepcidins, or manipulation of the hepcidin pathway prevents iron overload, redistributes iron from parenchymal cells to macrophage stores, and partially

Patients with transfusional iron overload usually require iron chelation therapy

DFO is a hexadentate iron chelator that binds iron in 1:1 complexes. It is given subcutaneously using a pump or intravenously as it is not absorbed orally. The dose ranges from 20 to 50 mg/kg/day. Though it is an effective drug, limited compliance was reported due to the inconvenience of parenteral administration as well as

DFP was the first oral iron chelator to be used for transfusional iron overload. DFP is a bidentate iron chelator that forms 3:1 complexes. The dose ranges from 75 to 100 mg/kg/day divided over three doses. Treatment with DFP was associated with lower myocardial iron deposition compared to deferoxamine. The most common adverse effects of DFP include GIT disturbances, elevated liver enzymes and arthropathy. The most serious adverse event was neutropenia that was

DFX is another iron chelator. It is a tridentate that forms 2:1 complexes. The dose ranges from 20 to 40 mg/kg/day. DFX is a long acting iron chelator. It is given once daily which is convenient to most patients. It lacks the DFP's potentially lifethreatening adverse effect of agranulocytosis. Patient Compliance and adherence to long-term chelation therapy in patients with transfusion-dependent β-thalassemia

recovered after temporary discontinuation of treatment [14].

(ICT) to help decrease the iron burden and to prevent and/or delay long-term complications associated with iron deposition in tissues [14]. There are currently

**Figure 3.** *Gene editing of beta thalassemia [7].*

#### *Updates in Thalassemia DOI: http://dx.doi.org/10.5772/intechopen.92414*

*Beta Thalassemia*

**18**

**Figure 3.**

*Gene editing of beta thalassemia [7].*

**Figure 2.**

*Gene therapy of beta thalassemia [9].*

TGF-β-like molecules or inhibitors of JAK2, Foxo3 activators, HRI-Eif2ap stimulators, Prx-2 activators, HO-1 inhibitors, Hsp-70 chaperone induction and pyruvate kinase activation. These modulators could soon revolutionize the treatment of β-thalassemia and related disorders [10].

Activins, members of TGF-β family signaling, are crucial regulators of hematopoiesis and modulate various cellular responses such as differentiation, proliferation, migration, and apoptosis. They were observed to ameliorate hematologic parameters and improve hematopoiesis in preclinical and early clinical studies [11].

JAK2 plays an important role in the progression and worsening of ineffective erythropoiesis. Drugs inhibiting JAK2 activity could reverse ineffective erythropoiesis and splenomegaly [12].

#### *1.2.4 Manipulating iron metabolism*

Hepcidin is the central regulator of iron homeostasis. Hepcidin inhibitors, e.g., ERFE inhibitors are now extensively studied as a possible future treatment of iron overload. Induction of iron restriction by means of transferrin infusions, minihepcidins, or manipulation of the hepcidin pathway prevents iron overload, redistributes iron from parenchymal cells to macrophage stores, and partially controls anemia in β-thalassemic mice [13].

#### *1.2.5 New formulation of iron chelators*

Patients with transfusional iron overload usually require iron chelation therapy (ICT) to help decrease the iron burden and to prevent and/or delay long-term complications associated with iron deposition in tissues [14]. There are currently three available iron chelators.

#### *1.2.5.1 Deferoxamine*

DFO is a hexadentate iron chelator that binds iron in 1:1 complexes. It is given subcutaneously using a pump or intravenously as it is not absorbed orally. The dose ranges from 20 to 50 mg/kg/day. Though it is an effective drug, limited compliance was reported due to the inconvenience of parenteral administration as well as infectious complications [14].

#### *1.2.5.2 Deferiprone*

DFP was the first oral iron chelator to be used for transfusional iron overload. DFP is a bidentate iron chelator that forms 3:1 complexes. The dose ranges from 75 to 100 mg/kg/day divided over three doses. Treatment with DFP was associated with lower myocardial iron deposition compared to deferoxamine. The most common adverse effects of DFP include GIT disturbances, elevated liver enzymes and arthropathy. The most serious adverse event was neutropenia that was recovered after temporary discontinuation of treatment [14].

#### *1.2.5.3 Deferasirox*

DFX is another iron chelator. It is a tridentate that forms 2:1 complexes. The dose ranges from 20 to 40 mg/kg/day. DFX is a long acting iron chelator. It is given once daily which is convenient to most patients. It lacks the DFP's potentially lifethreatening adverse effect of agranulocytosis. Patient Compliance and adherence to long-term chelation therapy in patients with transfusion-dependent β-thalassemia

#### *Beta Thalassemia*

is challenging and critical to prevent iron overload-related complications. Thanks to oral iron chelators formulations that allow better compliance and improve patients and parents' adherence to the drugs. Once-daily deferasirox dispersible tablets (DT) have proven long-term efficacy and safety in patients ≥2 years old with chronic transfusional iron overload. However, barriers to optimal adherence remain, including palatability, preparation time, and requirements for fasting state. A new film-coated tablet (FCT) formulation was developed, swallowed once daily (whole/ crushed) with/without a light meal [15]. Key differences between deferasirox dispersible and film coated tablets are listed in **Table 2**.

### *1.2.5.4 Combined iron chelators*

Combined DFO and DFP chelation therapy was introduced to manage iron overload in patients with suboptimal chelation with maximum DFP doses. A shuttle mechanism was proposed to explain the synergistic effect of DFP and DFO. DFP enters cells due to its low molecular weight and removes iron, and then passes it on to DFO to be excreted in urine and stool. Other combinations like DFX & DFO and DFX & DFP were used to maximize efficacy, improve compliance and minimize the side effects [14].

#### *1.2.6 Fetal hemoglobin induction*

The main pathophysiological determinant of the severity of β-thalassemia syndromes is the extent of α/non-α globin chain imbalance. Thus in β-thalassemia, pharmacologically induced increase in γ-globin chains would be expected to decrease globin chain imbalance with consequent amelioration of clinical manifestation. Increased production of the fetal γ-globin can bind the excess α-chains to produce fetal hemoglobin and hence improve α/β-globin chain imbalance leading to more effective erythropoiesis. This partly explains the more favorable phenotype in some patients with β-thalassemia intermedia and hemoglobin E/β-thalassemia compared with transfusion-dependent β-thalassemia major [16].

Several pharmacologic compounds including: 5-azacytidine, decitabine, hydroxyurea (HU), butyrate (short-chain fatty acids), erythropoietin and short chain fatty acid derivatives (SCFAD) as fetal hemoglobin-inducing agents had encouraging results in clinical trials [16].

### *1.2.7 Haploidentical hematopoietic stem cell transplantation*

Haploidentical transplant approach have a crucial clinical significance in patients with beta thalassemia major as it provides a graft source to almost all patients who do not have an HLA-matched donors. Haploidentical HSCT is always available and parents of children are highly motivated. Haploidentical means 50% identity and the problem is the subsequent high risk of graft versus host disease (GVHD) caused by donor T cells. Successful haploidentical HSCT depends on effective removal of donor T cells [17].

#### **Methods of T-Cell depletion [17]:**


**21**

**Author details**

**Table 2.**

Tamer Hassan\* and Marwa Zakaria

provided the original work is properly cited.

*Deferasirox formulations: Key differences.*

Department of Pediatric, Faculty of Medicine, Zagazig University, Zagazig, Egypt

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: dr.tamerhassan@yahoo.com

*Updates in Thalassemia*

**Dosage forms and strengths**

*DOI: http://dx.doi.org/10.5772/intechopen.92414*

• White round tablet ◯ 125 mg ◯ 250 mg ◯ 500 mg

≥30 minutes before a meal • Stir to disperse in water, orange

• Tablets should not be chewed or

juice or apple juice ◯ 3.5 oz. liquid for <1 g ◯ 7.0 oz. liquid for ≥1 g • Consume the suspension • Resuspend residue in small amount of liquid and consume

immediately

**Starting dose** • Transfusion-dependent iron

**Titration increments** • Transfusion-dependent iron

**Maximum dose** • Transfusion-dependent iron

swallowed whole

overload: 20 mg/kg/day • NTDT: 10 mg/kg/day

overload: 5–10 mg/kg/day • NTDT: 5–10 mg/kg/day

overload: 40 mg/kg/day • NTDT: 20 mg/kg/day

**Administration** • Once daily on an empty stomach

**Dispersible tablet [EXJADE] Film-coated tablet [JADENU]**

• Film-coated blue oval tablet ◯ 90 mg (light blue) ◯ 180 mg (medium blue) ◯ 360 mg (dark blue)

• Once daily on an empty stomach or with a light meal (<7% fat; ~250

• Swallow whole or crush and mix with soft foods such as yogurt or apple sauce immediately before use

• Transfusion-dependent iron overload:

• Transfusion-dependent iron overload:

• Transfusion-dependent iron overload:

calories)

14 mg/kg/day • NTDT: 7 mg/kg/day

3.5–7 mg/kg/day • NTDT: 3.5–7 mg/kg/day

28 mg/kg/day • NTDT: 14 mg/kg/day


#### **Table 2.**

*Beta Thalassemia*

is challenging and critical to prevent iron overload-related complications. Thanks to oral iron chelators formulations that allow better compliance and improve patients and parents' adherence to the drugs. Once-daily deferasirox dispersible tablets (DT) have proven long-term efficacy and safety in patients ≥2 years old with chronic transfusional iron overload. However, barriers to optimal adherence remain, including palatability, preparation time, and requirements for fasting state. A new film-coated tablet (FCT) formulation was developed, swallowed once daily (whole/ crushed) with/without a light meal [15]. Key differences between deferasirox

Combined DFO and DFP chelation therapy was introduced to manage iron overload in patients with suboptimal chelation with maximum DFP doses. A shuttle mechanism was proposed to explain the synergistic effect of DFP and DFO. DFP enters cells due to its low molecular weight and removes iron, and then passes it on to DFO to be excreted in urine and stool. Other combinations like DFX & DFO and DFX & DFP were used to

maximize efficacy, improve compliance and minimize the side effects [14].

compared with transfusion-dependent β-thalassemia major [16].

*1.2.7 Haploidentical hematopoietic stem cell transplantation*

The main pathophysiological determinant of the severity of β-thalassemia syndromes is the extent of α/non-α globin chain imbalance. Thus in β-thalassemia, pharmacologically induced increase in γ-globin chains would be expected to decrease globin chain imbalance with consequent amelioration of clinical manifestation. Increased production of the fetal γ-globin can bind the excess α-chains to produce fetal hemoglobin and hence improve α/β-globin chain imbalance leading to more effective erythropoiesis. This partly explains the more favorable phenotype in some patients with β-thalassemia intermedia and hemoglobin E/β-thalassemia

Several pharmacologic compounds including: 5-azacytidine, decitabine, hydroxyurea (HU), butyrate (short-chain fatty acids), erythropoietin and short chain fatty acid derivatives (SCFAD) as fetal hemoglobin-inducing agents had

Haploidentical transplant approach have a crucial clinical significance in patients with beta thalassemia major as it provides a graft source to almost all patients who do not have an HLA-matched donors. Haploidentical HSCT is always available and parents of children are highly motivated. Haploidentical means 50% identity and the problem is the subsequent high risk of graft versus host disease (GVHD) caused by donor T cells. Successful haploidentical HSCT depends on

1.CD34+ positive selection with immunomagnetic separation which leads to

2.CD3 depletion with immunomagnetic separation which leads to specific

3.Addition of Alemtuzumab (anti-CD52 antibody) to the transplant bag.

dispersible and film coated tablets are listed in **Table 2**.

*1.2.5.4 Combined iron chelators*

*1.2.6 Fetal hemoglobin induction*

encouraging results in clinical trials [16].

effective removal of donor T cells [17]. **Methods of T-Cell depletion [17]:**

enrichment of stem cells;

reduction of T cells; and

**20**

*Deferasirox formulations: Key differences.*
