Medicinal Chemistry of Fetal Hemoglobin Inducers for Treatment of -Thalassemia Roberto Gambari

Current Medicinal Chemistry, 2007, 14, 199-212
199
Medicinal Chemistry of Fetal Hemoglobin Inducers for Treatment of
-Thalassemia
Roberto Gambari*,1,2 and Eitan Fibach3
1ER-GenTech,
Department of Biochemistry and Molecular Biology, Section of Molecular Biology, University of Ferrara,
Ferrara, Italy
2GenTech-for-Thal,
Laboratory for the Development of Pharmacological and Pharmacogenomic Therapy of Thalassaemia,
Biotechnology Centre, Ferrara, Italy
3Department
of Haematology, Hadassah - Hebrew University Medical Centre, Jerusalem, Israel
Abstract: In this review we summarize the achievements of medicinal chemistry in the field of
pharmacological approaches to the therapy of β-thalassemia using molecules able to stimulate the production
of fetal hemoglobin (HbF). We first describe the molecular basis of the pathology and the biochemical rational
of using HbF inducers for therapy; we then outlined the in vitro and in vivo experimental systems suitable for
screening of such potential drugs, and finally we describe the different classes of compounds with emphasis
on their advantages and disadvantages in the treatment. The results of these reviewed studies indicate that: (a)
HbF inducers can be grouped in several classes based on their chemical structure and mechanism of action; (b)
clinical trials with some of these inducers demonstrate that they are effective in ameliorating the symptoms of
β-thalassemia; (c) a good correlation was found between HbF stimulation in vivo and in vitro indicating that in
vitro testing might be predictive of the in vivo response; (d) combined use of different inducers might
maximize the effect, both in vitro and in vivo. However, (e) the response to HbF inducers, evaluated in vitro and
in vivo, is variable, and some patients might be refractory to HbF induction by certain inducers; in addition, (f)
several considerations call for caution, including the fact that most of the inducers exhibit in vitro
cytotoxicity, predicting side effects in vivo following prolonged treatments.
Keywords: Fetal hemoglobin; β-thalassemia; Histone deacethylases; Hydroxyurea; DNA-binding drugs; Rapamycin.
This paper is dedicated to the memory of Professor Panos Ioannou.
INTRODUCTION
The objective of this review is to summarize the
achievements of medicinal chemistry in the field of
pharmacological approaches to the therapy of β-thalassemia.
We will shortly describe the molecular basis of the
pathology and the experimental systems suitable for the
screening of potential therapeutic compounds. Major
emphasis will be dedicated to the description of the different
classes of molecules employed in in vitro and in vivo
preclinical studies, as well as recently performed clinical
trials.
1. HEMOGLOBINS AND THEIR SWITCH DURING
ONTOGENY
Hemoglobin (Hb) is a tetramer of two α-like and two βlike globin polypeptide chains. In human, the genes for αglobins are clustered on chromosome 16, which contains one
gene for ζ and two genes for α (α 1 and α 2, the proteins of
which are identical). The genes for β-like globins are
clustered on chromosome 11, that contains genes for ε, β
and δ, one gene for each, and two slightly different genes for
γ (Gγ and Aγ , the proteins of which differ in one amino
*Address correspondence to this author at the Department of Biochemistry
and Molecular Biology, Via L.Borsari n.46, 44100 Ferrara, Italy; Tel: +39532-424443; Fax: +39-532-424500; E-mail: gam@unife.it
0929-8673/07 $50.00+.00
acid). In addition, these clusters contain various sites that are
responsible for the regulation of the expression of each gene
[1].
The expression of the globin genes is regulated during
ontogeny. In humans, globin production is characterized by
two major "switches" [2]. Production of embryonic Hbs
switches after the first two months of gestation into fetal Hb
(HbF) (α 2γ 2), and then again, before and immediately after
birth, into adult Hb (HbA) (α 2β2). Since both HbA and HbF
contain α chains, the switch from the former to the latter
represents a decrease in the expression of the γ -globin genes,
associated with an increase of β-globin gene expression. The
prevalence of HbF during embryonic life is explained by its
high affinity to oxygen, a property that allows it to remove
oxygen from HbA in the maternal red blood cells (RBCs)
through the placenta.
Immediately after birth the newborn has 85-98% HbF,
which gradually decreases to < 5% at the age of one year. In
adult life HbA is the major Hb, a small < 5% is HbA2
(α 2δ2) and the rest (<5%) is HbF which is concentrated in a
few RBC [3-5].
2. -THALASSEMIAS
In β-thalassemias, mutations affecting the β-globin gene
or its regulatory regions cause absence (β0) or reduced (β+ )
synthesis of β-globin chains [1-4]. This is associated with a
corresponding excess of the complementary α-globin. The
© 2007 Bentham Science Publishers Ltd.
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Current Medicinal Chemistry, 2007, Vol. 14, No. 2
outcome of this unbalanced globin production is the
destruction by apoptosis of erythroid precursors in the bone
marrow and at extramedullary sites (ineffective
erythropoiesis) and short survival of RBCs in the peripheral
blood [5-10]. The disease is associated with morbidity and
mortality due to severe chronic anemia and treatment-related
complications (such as multi-organ failure due to iron
overload which is caused by increased absorption and
therapeutic blood transfusion).
More than 200 mutations have been found to cause βthalassemia [10]. In contrast to α-thalassemia, where
deletions in the α-globin gene cluster account for the
disease, the molecular defects causing β-thalassemia are
usually point mutations involving only one or few
nucleotide(s) [10,11]. For instance, β°39-thalassemia is
caused by a stop codon mutation that leads to premature
termination of β-globin chain synthesis [12,13]; the β°IVSI1 mutation suppresses correct maturation of the β-globin
RNA precursor [14], while in thalassemia with the β+ IVSI110 mutation normal and abnormal spliced β-globin RNA
precursor coexist [15].
3. FETAL HEMOGLOBIN AMELIORATES THE
CLINICAL SYMPTOMS OF -THALASSEMIA
The proportion of HbF in postnatal life is influenced by
various physiological [6] and genetic [7,8] factors. An
increase in HbF may be acquired, e.g., in juvenile myelomonocytic leukemia [9] or during acute erythropoietic stress
[10] and is frequently observed in β-hemoglobinopathies
[11,12].
Epidemiological findings have shown that increased HbF
in β-thalassemia ameliorates the clinical symptoms [16,17].
The most convincing finding was found in individuals with
mutations associated with hereditary persistence of HbF
(HPFH) in adults [22-25]. Coexistence of homozygous βthalassemia with HPFH is asymptomatic. It seems that HbF
can functionally compensate for the absence of β-globin
chains [16-21]. These findings have generated conside-rable
interest in identifying molecular and pharmacological ways
to increase the production of HbF [26-36]. Indeed, several
groups of compounds were found to reactivate the γ -globin
genes in post-natal erythroid cells. However, many of these
drugs have low efficacy and specificity, while some are
potentially toxic or carcinogenic. There is therefore an urgent
need for new agents that can induce HbF production with
greater efficiency and lower toxicity.
4. IN VITRO MODEL SYSTEMS FOR SCREENING
OF POTENTIAL INDUCERS OF FETAL HEMOGLOBIN
Cells Expressing Reporter Genes Under the Control of
the -Globin Promoter
In order to screen for HbF inducers, several groups have
used cells transfected with reporter genes that are under the
transcriptional control of the Gγ -globin gene promoter. The
main advantage of these systems is that they can potentially
be automated and thus serve for high-throughput screening
of HbF inducers. For instance, Skarpidi et al. [37] used
Gambari and Fibach
recombinant DNA constructs in which the coding sequences
of two different luciferase reporter genes, firefly and renilla,
were substituted for those of human Gγ -globin and β-globin
genes, respectively. When HbF inducers were added to
cultures of cells stably transfected with these constructs, the
activity of their human globin genes could be determined by
a sensitive enzymatic assay of the two luciferases in the cell
lysates. The specificity of the inducers was determined by
their ability to increase the activity γ -globin/firefly luciferase
gene more than that of the β-globin/renilla luciferase.
Other groups introduced reporter genes within an intact
β-globin gene locus. Thus, Vadolas et al. [38] developed a
stable cellular genomic reporter assay based on a variant of
the green fluorescence protein (EGFP) gene under the Gγ globin promoter in the intact human β-globin locus. They
demonstrated that K562 cells stably transfected with this
construct maintain a uniform basal level of EGFP expression
over long periods of continuous culture and that induction of
EGFP expression is associated with the induction of the
endogenous γ -globin genes. Furthermore, they compared the
GFP-induction potency of several agents, demonstrating that
this system might help in identifying novel HbF inducers
[38,39].
Fig. (1) reports results obtained using cells stably
transfected with a Gγ -Aγ -EGFP β-globin locus construct
[38] (Fig. 1A) or with reporter genes (coding green EGFP
and far-red fluorescent protein, red-FP) under the control of
the human γ -globin or β-globin promoters, respectively
(Breveglieri, G.; Salvatori, F.; Feriotto, G.; Gambari, R.,
manuscript in preparation) (Fig. 1B).
Human Erythroid-Like Cell Lines
Human erythroid-like cell lines, such as K562 [40], HEL
[41] and UT-7 [42], were derived from cells explanted from
patients with various forms of myeloid leukemia. The cells
grow in suspension cultures as single, undifferentiated cells,
with low production of Hbs. When stimulated by various
agents, they respond within few days with a significant
increase in the production of Hbs and the expression of other
erythroid-specific differentiation markers. Among these
erythroid-like cell lines, K562 cells, isolated and
characterized by Lozzio & Lozzio [40] from a patient with
chronic myelogenous leukemia in blast crisis, have been
extensively employed as a useful in vitro model to study the
molecular mechanism(s) regulating the expression of
embryonic and fetal human globin genes [43-46], as well as
for screening of new differentiation-inducing compounds
[47-55]. Rutherford et al. first reported in 1979 [47] that
hemin induces a reversible accumulation of embryonic and
fetal Hbs without affecting the rate of cell growth; the cells
can be indefinitely subcultured in the presence of the
inducer. Thus, this cell line appears to be committed along
the embryo-fetal erythroid differentiation pathway and, when
induced by hemin, produces Hb independently of terminal
differentiation. Since then, many inducers of erythroid
differentiation were described, such as cytosine arabinoside
(ara-C) [54], butyrates [56], 5-azacytidine [57], chromomycin
and mithramycin [51], tallimustine [49,58], cisplatin and
cisplatin analogs [50]. Unlike hemin, most of these inducers
inhibit cell growth and activate terminal cell division. When
K562 cells committed to differentiation by these inducers
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Current Medicinal Chemistry, 2007 Vol. 14, No. 2
201
Fig. (1). Screening of HbF-inducers – The use of cells expressing reporter genes under the control of the γ-globin promoter.
A. The green fluorescence protein (EGFP) gene is under the transcriptional control of the Gγ-globin promoter in the intact human βglobin locus [38]. B. A construct was generated, cloning the EGFP and red-FP under the control of γ-globin and β-globin promoters,
respectively. The constructs in (A) and (B) were stably transfected into K562 cells, which were treated with HbF-inducers. C. The effect
of hydroxyurea (a), mithramycin (b), cisplatin (c), carboplatin (d), butyrate (e) and trichostatin (f) are shown as percent increase in
EGFP expression (data from Vadolas et al. [38]). D. The selective γ-globin inducing effect of angelicin is indicated by the increase in
EGFP (which is under the control of the γ-globin promoter), but not of red-FP (which is under the control of the β-globin promoter).
Cells were examined and photographs with a fluorescence microscope.
were cloned in semi-solid culture, they generated small
colonies of Hb-containing cells that could not be subcultured [55].
Fig. (2) demonstrates the induction of Hb synthesis by
cytosine arabinoside in K562 cells. Quantitative methods to
follow erythroid induction in K562 and other cells include
(a) benzidine-staining to measure the proportion of Hbcontaining cells (Fig. 2, C-E), high-performance liquid
chromatography (HPLC) (b) cellulose-acetate gel
electrophoresis to characterize the produced embryonic Hbs
usually Hb Portland, ζ2γ 2, and Hb Gower 1, ζ2ε 2, and (c)
Northern blotting (Fig. 2, F and G) and RT-PCR analyses
to determine the expression of globin genes [55].
Although these cell lines serve as convenient
experimental models, because of their leukemic origin and
long history in culture, they do not recapitulate all aspects of
erythroid cell development. For example, K562 cells do not
respond to erythropoietin (EPO), the physiological erythroid
hormone and they do not produce adult Hbs. Therefore, in
the absence of expression of β-globin, this model does not
recapitulate certain aspects of the pathology of β-thalassemia
cells (e.g., excess of α-chains). Finally, although many
agents have been shown to increase Hb production in such
cell lines, not all are effective on primary cultures of
erythroid cells, and, vice versa, other agents (e.g.,
cytokines), that affect primary cultures, fail to stimulate
these cell lines.
Cultures of Human Erythroid Progenitors
It is possible to obtain large cultures of relatively pure
and synchronized erythroid cell population and compounds
can be added on different days when the culture consists of
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Gambari and Fibach
Fig. (2). Screening of HbF-inducers – The use of human erythroid cells.
A-G. K562 cells were treated with 250 nM cytosine arabinoside for 7 days. The pellets of un-treated (A) and treated (B) cells. The red
color of the pellet in (B) indicates hemoglobin production. C-D. Uninduced (C) and induced (D) cells stained with dihydrobenzidine
(representative hemoglobin-containing blue cells are arrowed). E. Kinetics of the increase of benzidine-positive, hemoglobincontaining cells treated with 50 (¡) and 100 (l) nM rapamycin. n = control uniduced cells. F-G. Northern blotting analysis of
uninduced (left side) and erythroid induced (right side) cells. Hybridization was carried out with a γ-globin (F) or a β-actin (G) probe.
The increase of signal in erythroid induced cells (F) indicates an increase of production of γ-globin mRNA. No major changes are
observed studying β-actin mRNA (G). H-M. Cultures of human erythroid progenitors. Peripheral blood progenitors derived from
normal donors were cultured according to the two-phase liquid culture protocol [59,60]. (H) Large aggregates of erythroid precursors
on day 6 of phase II. Unstained cells were photographed in situ using an inverted microscope. I. A red pellet of erythroid precursor
cells harvested from day-12 phase II cultures. (L) Benzidine staining of cells from day 8 phase II cultures. Cells were smeared on a
glass slide using a cytocentrifuge, and stained with 3,3'-dimethoxybenzidine. Mature erythroid hemoglobin-containing cells, which
are benzidine positive, (representative cells arrowed) and early erythroid precursors, which are benzidine negative, are seen. M. HPLC
chromatogram of hemoglobins produced by cultured erythroid cells. Control (upper chromatogram) and rapamycin-treated (lower
chromatogram) cells were harvested on day 12, washed and lysed. The hemoglobins in the lysate were separated on cation-exchange
HPLC. The peaks are labeled with the corresponding hemoglobin type. Increase in the proportion of HbF is detectable in lysates from
treated cells.
cells at specific stages of maturation. In a procedure
developed by Fibach et al. [59], the procedure is divided
into two phases: an EPO-independent phase, in which
peripheral blood cells are first cultured in the presence of a
combination of growth factors, but in the absence of EPO.
In the second phase, the culture, supplemented with EPO,
generates orthochromatic normoblasts and enucleated
erythrocytes (Fig. 2, H-L) [65]. During phase I, erythroid,
myeloid and megakaryocytic progenitors proliferate and
differentiate. The early erythroid-committed progenitors,
BFUe, proliferate and differentiate into CFUe-like
progenitors. At this stage, the cultures contain some
adherent cells (mainly macrophages) and non-adherent cells
(mainly lymphocytes). After one week, the non-adherent
cells are harvested, washed and re-cultured in fresh medium
supplemented with EPO. In the absence of necessary
cytokines to support their proliferation and differentiation,
non-erythroid progenitors cease their development. The
CFUe proliferate and differentiate into erythroid precursors.
Proerythroblasts are discernible by inverted microscopy on
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Current Medicinal Chemistry, 2007 Vol. 14, No. 2
days 4-5 of phase II as large, round and smooth cells. At
this stage they may be purified from remaining lymphocytes
and erythrocytes on Percoll gradient and re-cultured in the
same medium. As the proerythroblasts continue to multiply,
they form clusters and then large aggregates which, when
undisturbed, can reach hundreds of cells (Fig. 2H). As these
cells differentiate, they decrease in size and accumulate Hb
(Fig. 2L, arrowed cells), and the aggregates assume a
reddish color (Fig. 2I).
Peripheral blood cells are employed in this procedure for
the following reasons: (a) the availability of blood from
normal individuals and patients; (b) the homogeneity of the
peripheral blood erythroid progenitors, namely early BFUe,
as opposed to the bone marrow which contains progenitors
at various developmental stages. Good results can be
obtained with cells derived from other sources, including
CD34+ cells purified by immuno-magnetic bead
technologies, but, in these cases, some modifications of the
procedure are required.
This system recapitulates many aspects of in vivo
erythropoesis including globin RNA metabolism, cell cycle
kinetics, expression of cell surface antigens, iron and ferritin
metabolism and transcription factors [60-62].
Several research groups have used this system to study
the effects of hundreds of compounds, including butyrate
derivatives [63,19], hemin [65], EPO [64]. For studying
their potential to enhance HbF production, compounds can
be added to phase I, phase II or both. Non-toxic drugs, such
as cytokines and hemin, may be added to the cultures at any
time. With cytotoxic drugs, such as hydroxyurea (HU) and
5-azacytidine, because of their cytotoxic/cytostatic effects,
they are usually added on day 4-8 of phase II.
Since the erythroid cells in phase II are grown in
suspension, samples of cells can be withdrawn at any time
without disturbing the cultures and assayed for morphology,
size, number, cell viability and apoptosis, cell cycle or
expression of surface antigens. Hemoglobinization can be
easily followed by staining the cells with benzidine.
The Hb content of the developing erythroid cells can be
measured by a variety of techniques, such as alkaline
denaturation, benzidine staining, cation-exchange HPLC for
hemoglobins (Fig. 2M) and reverse-phase HPLC for globin
chains. Using the HPLC techniques, Hb is measurable in
culture as early as 5 days in phase II. On day 12, one ml
culture is sufficient for multiple measurements. The mean
cellular Hb or HbF concentrations of erythroid cells are
calculated from the values of the HPLC determinations
divided by the number of benzidine-positive cells. The
distribution of the erythroid cell population with respect to
intracellular content of HbF can be analyzed by flow
cytometry using monoclonal antibodies directed specifically
against HbF [60]. Dual/triple staining with the
corresponding antibodies can be used for simultaneous
analysis of both HbA and HbF, or Hb and another marker of
interest such as glycophorin A or CD34 surface antigens
[60].
5. MOUSE MODELS FOR -THALASSEMIA
Murine models mimicking β°-thalassemia are very
difficult to be obtained [66]. This is due to the fact that the
mouse β-globin locus contains four functional β-globin
genes: βh1 and ε y, which are transcribed only during the
embryonic development and become silenced in 14-15 dayold embryos, and the b1 (βmajor ) and b2 (βminor) genes,
Table 1. In Vivo Experimental Model Systems for Studying the Effects of HbF Inducers
Genotype
Phenotype
Reference
HbbTh1/Th1 (a)
Mild anemia
Skow et al., 1983 [68]
HbbTh2/Th2 (b)
Severe anemia
Sheehee et al., 1993 [70]
HbbTh3/- (c)
Thalassemia intermedia
Yang et al., 1995 [71]
HbbTh3/Th3(c)
Death in utero
Yang et al., 1995 [71]
HbbTh3/Th3 fetal liver cells
Adult thalassemia major
Rivella et al., 2005 [73]
HbbTh3/- carrying human globin locus
Recapitulates β-IVS-110
with a β-IVSI-110 globin gene
splicing mutation
Vadolas et al., 2006 [74]
HbbTh3/- carrying human globin locus
Recapitulates β°39
Jamsai et al., 2005 [75]
with a β°39 globin gene
stop mutation
HbbTh3/- carrying human globin locus
Recapitulates HbE
with a β-thal/HbE globin gene
phenotype
Irradiated mouse transplanted with
(a) Deletion including the b1 gene.
(b)Targeted deletions of b1 gene with the insertion of a non-globin promoter.
(c) Deletion of both b1 and b2 genes.
203
Jamsai et al., 2006 [76]
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Current Medicinal Chemistry, 2007, Vol. 14, No. 2
Gambari and Fibach
Fig. (3). Strategies for screening and developing of HbF-inducers for treatment of β-thalassemia.
which are activated around 11 days after conception and in
adults produce 80% and 20%, respectively, of the total (1315 g/dl) Hb [66]. Notably, unlike humans, mice lack γ -like
globin genes, and the embryonic to adult Hb switch occurs
before birth, rather than during the first 6 months after birth.
Accordingly, no naturally occurring mutations that
completely inhibit expression of the b genes have ever been
observed, as mice homozygous for such β° mutations are
expected to die prenatally of severe anemia [66].
However, a few adult mouse models of β-thalassemia
intermedia have been described (Table 1). In the th1 model,
a spontaneous DNA deletion includes the b1 gene and its
adjacent upstream sequence, including the promoter [67].
Mice that are homozygous (Hbbth1/th1) have reduced Hb
levels but only a mild anemia, possibly due to a
translational compensatory mechanism that increases b2
(βminor) globin synthesis [66,68]. Another mouse model
(th2) was generated by targeted deletion of the b1 gene,
introducing a non-globin promoter into the disrupted b1
locus. Homozygous Hbbth2/th2 mice are severely anemic and
do not survive more than a few hours after birth, while
heterozygous mice show a very mild phenotype [69]. The
th3 model was generated by deletion of both the βmajor and
βminor genes [70,71]. Mice homozygous for this deletion die
late in gestation, as expected by the absence of the b1 and b2
gene products. Heterozygotes (Hbbth3/+ ), however, are viable
but exhibit severe anemia (7 to 9 g/dL of Hb), abnormal
RBC morphology, splenomegaly and hepatic iron
deposition similar to that found in patients with βthalassemia intermedia.
Recently, an adult mouse model of β0-thalassemia was
generated by engrafting wild-type mice, after myelo-ablation,
with β-globin-null fetal liver cells harvested from 14.5- day
Hbbth3/th3 embryos which lack both the βmajor and βminor
genes. After 6-7 weeks, these mice exhibited a severe anemia
(2-4 g Hb/dL), low RBC and reticulocyte counts and
hematocrit values. The profound anemia settled in after 50
days, consistent with the clearance rate of the recipient’s
normal RBCs, and the mice succumbed to ineffective
erythropoiesis within 60 days [72]. Post mortem
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Current Medicinal Chemistry, 2007 Vol. 14, No. 2
examination demonstrated severe body mass reduction with
massive splenomegaly due to erythroid hyperplasia as well
as extensive hepatic extra-medullary hematopoiesis and iron
overload [66,72].
Since the work by Tariq et al. [73], showing the crucial
role of the human Locus Control Region (LCR) in
reproducing the correct switch from γ -globin to β-globin
gene expression, β-thalassemic mouse models were generated
which, in addition to deletion of the mouse β-like globin
genes, carry a mutated human β-globin gene cluster. For
instance, Vadolas et al. [74] generated a "humanized" mouse
model carrying the common thalassemic β+ -IVSI-110
splicing mutation on a bacterial artificial chromosome that
contains the human β-globin locus. They examined
heterozygous murine β-globin knock-out mice carrying either
the IVSI-110 or the normal human β-globin locus. A 90%
decrease in human β-globin chain synthesis was observed in
mice with the IVSI-110 mutation compared to mice with
normal human β-globin cluster. This difference has been
attributed to aberrant splicing; therefore, the humanized
IVSI-110 mice recapitulate the splicing defect found in
comparable β-thalassemia patients. This model can therefore
serve as a platform for testing strategies for restoration of
normal splicing. Other examples of “humanized” transgenic
β-thalassemia mice are listed in Table (1) [75,76].
6. INDUCERS OF FETAL HEMOGLOBIN
HbF inducers can be grouped in several classes according
to their chemical structure and mechanism of action (Table
2) [77-115]. Fig. (3) shows a flow-chart of the necessary
Table 2.
steps for screening, characterization and developing of a
useful clinical HbF inducer.
Hypomethylating Agents
Decreased HbF production after birth is associated with
DNA methylation at the γ -globin gene promoter by DNA
methyltransferases [77-79]. These enzymes are inhibited by
the cytosine analogs, 5-azacytidine and 5-aza-2'deoxycytidine (decitabine) [80]. In early studies, 5azacytidine significantly increased HbF in thalassemia
patients, but clinical development of these analogs was
halted after a poorly controlled animal study that suggested
that 5-azacytidine might be carcinogenic. However, the
majority of the preclinical studies with decitabine have
suggested a chemopreventive rather than carcinogenic effect
[81]. Furthermore, decitabine, unlike 5-azacytidine, is not
incorporated into RNA and is a more directed DNAhypomethylating agent [82]. Accordingly, this class of HbF
inducers should be carefully reexamined for their therapeutic
potential.
Hydroxyurea
One of the first studies showing a clear effect of
hydroxyurea [HU] was published in 1993 by Fibach et al.
[63] using the two-phase liquid culture in which, as
previously described, human peripheral blood-derived
progenitor cells undergo proliferation and differentiation. HU
was found to have multiple effects on these cultured cells:
(a) an increase in the proportion of HbF produced; (b) a
decrease in cell number due to inhibition of cell
Inducers of Fetal Hemoglobin in Erythroid Precursor Cells from Human Donors
Inducer
Mechanism of action
References (a)
Hydroxyurea
Inhibition of DNA synthesis
Fibach et al., 2003 [63]
5-azacytidine
Hypomethylation of DNA
De Simone, 2004 [82]
Citarabine
Hypomethylation of DNA
Sauthararajah et al, 2003 [80]
Butyrates
HDAC inhibitors
Cao et al., 2004 [86]
Trichostatin
HDAC inhibitor
Marianna et al., 2001[84]
Apicidin
HDAC inhibitors
Witt et al., 2003 [88]
Scriptaid
HDAC inhibitors
Johnson et al., 2005 [90]
Mithramycin
DNA-binding drug
Fibach et al., 2003 [108]
Cisplatin and analogues
DNA-binding drugs
Bianchi et al., 2000 [50]
Tallimustine and analogues
DNA-binding drugs
Bianchi et al., 2001 [49]
Angelicin
DNA-binding drug
Lampronti et al., 2003 [107]
Rapamycin
FRAP-mTOR signal transduction
Mischiati et al., 2003 [111]
Fibach et al., 2006 [113]
Triple-helix oligodeoxynucleotides
Peptiole nucleic acids (PNAs)
(a) In some cases, only a representative reference in given.
205
Activation of γ-globin
gene promoter
Xu et al., 2000 [118]
Artificial promoters
Wang et al., 1999 [122]
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Current Medicinal Chemistry, 2007, Vol. 14, No. 2
proliferation; (c) an increase in Hb content per cell (mean
corpuscular hemoglobin, MCH); and (d) an increase in cell
size (mean corpuscular volume). The extent of these effects
was related to the HU dose and time of addition. When
added to cell cultures from normal individuals 4 days
following their exposure to EPO, HU caused a 1.3- to 3.5fold increase in the proportion of HbF, from 0.4% to 5.2%
(mean 1.6%) in untreated to 1.5% to 8.2% (mean 3.1%) in
HU-treated cultures. Addition of HU to cells isolated from
four patients with β-thalassemia showed a 1.3- to 6.2-fold
increase.
When HU was administered to thalassemic mice it
improved the β-thalassemic phenotype. Sauvage te al.
reported [93] that the hematocrit rose from 29 ± 3% at day 0
to 37 ± 4% at day 30, despite myelo-suppression and
decreased reticulocyte counts. The βminor/α ratio of globin
chain synthesis increased from 0.78 at day 0 to 0.97 at day
30. Membrane defects improved: the proportion of bound α
chains decreased, the proportion of spectrin and ankyrin
increased as did the deformability of RBC.
Several reports confirmed the HbF augmenting effect of
HU both in vitro and in vivo [94-104]. For example,
Yavarian et al. [105] reported the treatment with HU of 133
patients with transfusion-dependent β-thalassemia. After one
year of treatment these patients were classified into: good
responders (61%) who shifted from monthly blood
transfusion dependency to a stable transfusion-free condition;
moderate responers (23%) who remained transfusion
dependent but at longer intervals (6 months or more), and
non responders, who remained at the same level of
transfusion dependency.
Table (3) summarizes the clinical studies with HU in βthalassemia patients. The results indicate that a significant
proportion of the patients became transfusion-free after
treatment.
Inhibitors of Histone Deacetylases
Several findings suggest that inhibition of the activity of
histone deacetylases (HDACs) is associated with an
Table 3.
Gambari and Fibach
increased expression of the γ -globin genes [83-92]. HDAC
inhibition leads to hyperacetylation of ε-amino groups of
lysine residues in histones [92]. This in turn causes a
decreased association of basic core histone proteins with the
DNA, rendering certain genes more accessible to the
transcriptional machinery. Among HDAC inhibitors,
trichostatin was found to possess high HbF-inducing
activity in human and mouse erythroleukemia cells. In a
recent report, Witt et al. [64] have showed that, among
several specific HDAC inhibitors tested, apicidin was by far
the most efficient HbF-inducer (at nM to µM concentrations)
in K562 cells [88], and that its effect involved, in addition
to HDAC inhibition, p38 mitogen-activated protein (MAP)
kinase signaling [64]. Scriptaid, a novel HDAC inhibitor,
was shown to induce γ -globin in K562 cells and human
erythroid progenitors in vitro. Treatment with scriptaid of βYAC transgenic mice, where the γ -globin gene is
completely silenced, caused reticulocytosis and synthesis of
human γ -globin mRNA [90]. In a recent report, Johnson et
al. [90] demonstrated that scriptaid induces γ -globin
expression via the p38 MAPK signaling; the p38-selective
inhibitor SB203580 completely reversed the ability to
induce HbF.
Further HDAC inhibitors were recently characterized for
their effect on human γ -globin gene expression in transgenic
mice. Among the hydroxamic acid derivatives of short-chain
fatty acids studied, butyryl and propionyl hydroxamate were
most effective, increasing the human γ /murine α-globin
mRNA ratios by 33.9% and 71%, respectively. This was
associated with an increase in reticulocytes hematocrit, and
the in vivo levels of BFU-E [91].
DNA-Binding Drugs
Figs. (4 and 5) show the molecular structures and
representative results of two DNA-binding drugs (DBDs)
(mithramycin and angelicin) found to increase HbF in
erythroid precursor cells. Interestingly, several DBDs are or
have been used in therapy. Chromomycin and mithramycin
were used in cancer and in hypercalcemia; tallimustine and
Clinical Trials Employing Hydroxyurea as In Vivo Inducer of Fetal Hemoglobin
Number of patients treated(a)
Genotype/phenotype(b)
Number of patients responding to
the treatment(c)
5
β-thalassemia intermedia
3 (60%)
Hoppe et al., 1999 [123]
7
β-thalassemia intermedia
3 (43%)
de Paula et al., 2003 [124]
References
4
β-thalassemia major
1 (25%)
de Paula et al., 2003 [124]
2
β-thalassemia intermedia
2 (100%)
Bradai et al., 2003 [125]
5
β-thalassemia major
5 (100%)
Bradai et al., 2003 [125]
36
β-thalassemia major
25 (69%)
Alebouyeh et al., 2004 [126]
133
β-thalassemia major
81 (61%)
Yavarian et al., 2004 [105]
37
H-β-thalassemia intermedia
26 (70%)
Dixit et al., 2005 [127]
166
β-thalassemia intermedia
83 (50%)
Karimi et al., 2005 [128]
42
HbE/β°-thalassemia
50%
Singer et al., 2005 [132]
(a) Dosages were between 5-20 mg/kg/day.
(b)Only transfusion-dependent patients are included.
(c) Transfusion independence or reduction of the transfusion frequency.
Fetal Hemoglobin Inducers and -Thalassemia
tallimustine analogues are anticancer and antiviral agents;
angelicin and the analogue bergaptene are used in PUVA
(psoralen plus UVA) therapy. Many reports demonstrated
that some DBDs display DNA sequence selectivity, and that
even similar DBDs differ with respect to stability of their
complexes with DNA. In any case, DBDs interacting with
the major groove of DNA are expected to inhibit complex
formation between transcription factors and target DNA
elements [106].
Our group has recently demonstrated that tallimustine
[49-58] and some cisplatin analogues [50,107] as well as the
GC-rich binders chromomycin and mithramycin [51] are
powerful inducers of differentiation of K562 cells,
suggesting that the expression of crucial genes involved in
erythroid differentiation of these cells are influenced by
DBDs. Several DBDs, such as tallimustine, mithramycin,
cisplatin and angelicin, increase HbF production in erythroid
precursor cells from normal human subjects. The extent of
induction was found to be higher than that of HU.
Current Medicinal Chemistry, 2007 Vol. 14, No. 2
207
Since, among the DBDs studied, mithramycin displayed
the lowest cytotoxicity, we compared it to HU on HbF
production by thalassemic erythroid precursors [108]. The
results demonstrated that in cultures derived from 12
patients, mithramycin increased HbF production in all cases,
while HU was not effective in two cases and was toxic in
one. In the majority of cases the activity of mithramycin was
higher than HU. In all cases, HU strongly inhibited cell
proliferation, while, at concentrations able to induce HbF
production, mithramycin had minimal effect on cell growth.
Rapamycin
Rapamycin (Fig. 6) is a lipophilic macrolide also called
sirolimus, isolated from a strain of Streptomyces
hygroscopicus found in a soil from Easter Island (known by
the inhabitants as Rapa Nui) [109,110]. We recently
demonstrated
that
rapamycin
induces
erythroid
differentiation of K562 cells and increases HbF production
Fig. (4). HbF-inducers – the DNA-binding drug mithramycin.
A. Molecular structure of mithramycin. B. Structure of molecular interaction between mithramycin and 5'-AAGGCCTT-3' DNA (PDP
code: 1D83, Image Library of Biological Macromolecules, www.imb.jena.de/IMAGE.html). C. Effects of mithramycin on the
interaction between Sp1 and its target DNA, evaluated by gel-shift analysis. A 32 P-labelled Sp1 mer was incubated with purified Sp1
in the presence of the indicated concentrations of mithramycin. After binding, gel electrophoresis and autoradiography were
performed. D. Effects of mithramycin on HbF production by erythroid precursor cells from three β-thalassemia patients [108]. Open
boxes represent control cells, grey boxes mithramycin-treated cells.
208
Current Medicinal Chemistry, 2007, Vol. 14, No. 2
Gambari and Fibach
Fig. (5). HbF-inducers – the DNA-binding drugs bergapten and angelicin.
A. Molecular structure of the compounds. B. C. Effects of angelicin on HbF production (C) and γ-globin mRNA expression (B). HbF
was analyzed by HPLC, γ-globin mRNA accumulation by real-time quantitative RT-PCR. Open boxes represent control cells, black
boxes hydroxyurea-treated cells, grey boxes angelicin-treated cells [107].
in erythroid cultures derived from normal donors [111] as
well as from β-thalassaemia patients who differ widely with
respect to their HbF levels, ranging from 4.6% to 93.7% of
the total Hb. The results indicated that: (a) rapamycin
increases HbF in cultures with different basal HbF levels; (b)
rapamycin increases the overall Hb content/cell; (c)
rapamycin
selectively
induces
γ -globin
mRNA
accumulation, with only a minor effect on β-globin and no
effect on α-globin mRNAs; (d) there is a strong correlation
between the increase by rapamycin of HbF and the increase
in γ -globin mRNA content [113].
As for the mechanism of action of this and related
compounds, we found that rapamycin-mediated erythroid
induction is associated with a hypophosphorylation of α-pS6 ribosomal protein and with hyper-phosphorylation of 4EBP-1 [113]. Furthermore, we found that inactivation of both
4E-BP1 and p70-S6K are sufficient steps, since we induced
differentiation when these molecular events were
simultaneously produced by a mixture of drugs involved in
downstream alterations of the FRAP-mTOR signal
transduction pathway [113].
The interest in rapamycin as an HbF-inducer is related to
the fact that its effect is not associated with cytotoxicity and
cell growth inhibition, in contrast to other inducers [111].
Interestingly, Rapamycin (as Sirolimus or Rapamune) was
approved by the U.S. Food and Drug Administration for
prevention of acute rejection in renal transplant recipients.
Moreover, the drug levels in the blood of these patients are
very similar to those effective in HbF stimulation in vitro
[112]. We, therefore, suggest that this, and structurally
related molecules, warrant careful evaluation as potential
drugs for stimulation of γ -globin gene expression and
increased HbF in patients with β-thalassaemia and sickle cell
anemia [111,113] .
Oligonucleotides and Peptide Nucleic Acids
Several strategies have been developed for increasing γ globin gene expression using oligonucleotides (ODN) or
their analogs [116-122]. One approach, described by Xu et
al., is based on the use of triple-helix forming
oligonucleotides (TFOs) [118]. Using a psoralen-conjugated
Fetal Hemoglobin Inducers and -Thalassemia
Current Medicinal Chemistry, 2007 Vol. 14, No. 2
209
Fig. (6). HbF-inducers – the DNA-binding drug rapamycin.
A. Molecular structure of rapamycin. B. Effects of rapamycin on HbF production in erythroid precursor cells from four β-thalassemic
patients with different starting levels of HbF. HbF was analyzed by HPLC [113]. Open boxes represent control cells, grey boxes
rapamycin-treated cells.
TFO designed to bind to a site overlapping with an Oct-1
binding site at the -280 region of the γ -globin gene resulted
in targeted mutagenesis with a frequency of 20%. In vitro
protein binding assays indicated that these mutations
reduced Oct-1 binding to its target site. In vivo gene
expression assays in mouse erythroleukemia cells
demonstrated activation of γ -globin gene expression by
these mutations. These findings suggest that this site
negatively regulates γ -globin gene expression, and that
mutations at the site can activate the γ -globin gene.
We attempted to apply a transcription factor decoy
strategy for targeting putative repressor factors. One decoy
oligonucleotide was able to induce erythroid differentiation
in K562 cells, and increased γ -globin mRNA expression and
HbF production in erythroid precursor cells from normal
donors [119].
Activation of γ -globin genes was recently proposed with
the aid of “artificial promoters”, as recently reviewed
[120,121]. Using peptide-nucleic acids (PNAs) designed to
bind to the 5' flanking region of the γ -globin gene,
induction of expression of a reporter gene construct was
demonstrated both in vitro and in vivo. In this case, PNAs
are designed for a stable triple helix targeting the sense
strand of DNA, allowing RNA polymerase to start
transcription [122]. Interestingly, PNA-mediated induction
of endogenous γ -globin gene expression was also
demonstrated in K562 cells.
In summary, these molecular technologies provide novel
approaches for induction of γ -globin gene expression and
treatment of β-thalassemia.
Erythropoietin
The rationale for in vivo treatment with recombinant
human EPO in thalassemia comes from studies in baboons,
thalassemic mice and in erythroid cultures of β-thalassemia
patients. The results demonstrated an increase in γ -globin
synthesis and consequently in HbF, resulting in
improvement in erythropoietic parameters. Studies with
EPO, mainly in patients with β-thalassemia intermedia,
showed a significant, dose-related increase in thalassemia
erythropoiesis without changes in the proportion of HbF, in
MCV and in MCH, and no major side effects during 9
months of continuous treatment. Accordingly, the use of
EPO in combination with true HbF inducers is expected to
cause both increased erythropoiesis and preferential induction
of HbF. Indeed, EPO was reported to be required to
maximize HbF induction by inducers such as HU and
HDAC inhibitors [114,115].
7. COMBINED USE OF VARIOUS INDUCERS OF
FETAL HEMOGLOBIN
Several recent reports show that a combined use of HbF
inducers might maximize the effect on HbF with lower
210
Current Medicinal Chemistry, 2007, Vol. 14, No. 2
toxicity than each individual inducer. For example,
Marianna et al. [131] investigated the effects of the butyrate
anaolg valproic acid and the HDAC inhibitor trichostatin in
combination with hemin. They showed that the combination
had a significant higher γ -globin enhancing capacity with
lower toxicity in human erythroid liquid cultures. These
findings suggest that combination of these drugs (all FDAapproved) might be helpful for the treatment of
hemoglobinopathies. This and similar studies are very
important, in consideration of the concerns about the doselimiting myelotoxicity and potential carcinogenicity of HbF
inducing agents.
Gambari and Fibach
9. CONCLUSIONS AND FUTURE PERSPECTIVES
Several conclusions should be drawn following the
comparative analysis of the data found in the literature on
HbF inducers as potential drugs for pharmacological
treatment of β-thalassemia.
1
The approach is reasonable, on the basis of the
clinical parameters exhibited by HPFH patients.
2
Clinical trials (even if still limited) employing HbF
inducers were effective in ameliorating in vivo clinical
parameters of β-thalassemia patients.
3
Good correlation of in vivo and in vitro results of
HbF synthesis and globin mRNA accumulation
indicates that in vitro testing might be predictive of
in vivo responses.
4
A combined use of HbF inducers might be useful to
maximize HbF induction, both in vitro and in vivo.
8. CLINICAL TRIALS
Clinical trials aimed at increasing HbF synthesis in βthalassemia have included administration of cell-cycle
specific agents (e.g., HU), hematopoietic growth factors
(e.g., EPO) and short-chain fatty acids (e.g., butyrate and its
analogues), all of which stimulate γ -globin synthesis by
different mechanisms. HU is the most studied drug and it is
currently the only HbF-inducing drug in routine use [105,
125-134] (Table 3).
For example, Dixit et al. studied the response to HU of
thirty-seven patients with β-thalassemia intermedia [127].
Major response was defined as transfusion independence or
Hb rise of more than 20g/l, and minor response as rise in Hb
of 10-20 g/l or reduction in transfusion frequency by 50%.
Twenty-six patients (70.2%) showed response to HU
therapy. Seventeen patients (45.9%) were major responders,
and nine patients (24.3%) showed minor response. Mean
HbF levels rose on HU therapy. Older age, low baseline F
cell percent, and low baseline HbF levels (below 10%) were
predictors of poor response. Response was evident within 1
month of starting HU therapy in the majority of responders.
Thus, a short trial of HU therapy can predict durable
response [127].
Singer et al. [132] reported a multicenter trial of 42
patients treated with HU for two years; almost half the
patients demonstrated a significant increase in steady-state
Hb level. Combined treatment of HU with EPO benefited
selected patients, but the addition of sodium phenyl butyrate
was ineffective. After 5 years of follow-up, a subset of
patients remained off transfusions.
As for demethylatying agents, in phase I/II studies,
decitabine, at DNA hypomethylating but noncytotoxic
doses, was well tolerated and effective in increasing HbF and
total Hb levels both in patients who had and had not
responded to prior HU therapy [81,82]. Therapy with
butyrates has been also reported [135,136].
As for the possibility to predict in vivo response, the data
suggest a correlation between the in vitro results on
erythroid precursor cells isolated from β-thalassemia patients
and the patients' response to treatment (e.g., [134]). If this
correlation is confirmed, it will be possible by testing
cultures of cells derived from the patient's peripheral blood
to design the optimal treatment for each patient. This will
prevent both expensive and potentially risky treatment from
patients who do no respond to treatment and suggest an
alternative treatment with other agents.
However, several considerations introduce cautions.
1
Most of the HbF inducers exhibit in vitro
cytotoxicity, predicting side effects in vivo during
prolonged treatment (as expected for the therapy of
the β-thalassemia).
2
The response to HbF inducers, evaluated in vitro and
in vivo, is variable, and some β-thalassemia patients
(and the erythroid cells derived from them) might be
refractory to induction; the reasons for this
phenomenon are largely unknown.
Accordingly, several approaches need to be followed,
including gene expression profiling and proteomic studies to
fully characterize the pharmacogenomic effects of HbF
inducers. In addition, medicinal chemistry will continue to
play a pivotal role in the development of novel HbF
inducers, with the aim to identify agents acting through
different mechanisms and exhibiting low toxic effects.
Finally, a strict collaboration between clinicians and
researchers is required to bring the most interesting HbF
inducers from the laboratory to the clinic.
ACKNOWLEDGEMENTS
R.G. is granted by AIRC, Fondazione Cassa di
Risparmio di Padova e Rovigo, Cofin-2005, by UE
obiettivo 2, by UE ITHANET Project and by the STAMINA
Project of Ferrara University. This research is also supported
by Regione Emilia-Romagna (Spinner Project) and by
Associazione Veneta per la Lotta alla Talassemia, Rovigo
(AVLT).
ABBREVIATIONS
Hb
=
Hemoglobin
HbF
=
Fetal hemoglobin
HPFH =
High persistence of fetal hemoglobin
EPO
=
Erythropoietin
GFP
=
Green fluorescence protein
RFP
=
Red fluorescence protein
Fetal Hemoglobin Inducers and -Thalassemia
HDAC =
Histone deacethylase
HU
Hydroxyurea
=
HPLC =
High performance liquid chromatography
CML
Chronic myelogenous leukemia
=
DNMT =
DNA methyltransferases
RBC
Red blood cell
=
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