1. Art and Diseño

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Hydroxyurea Increases Hemoglobin F Levels and Improves the Effectiveness
of Erythropoiesis in P-ThalassemialHemoglobin E Disease
By Suthat Fucharoen, Noppadol Siritanaratkul, Pranee Winichagoon, Jew Chowthaworn, Wipapun Siriboon,
Wanna Muangsup, Suchet Chaicharoen, Nalinee Poolsup, Busaba Chindavijak, Pensri Pootrakul,
Anong Piankijagum, Alan N. Schechter, and Griffin P. Rodgers
* 8% ( P
Hydroxyurea (HU) is one of several agents that have been
shown t o enhance hemoglobin (Hb) F levels in patients with
sickle cell disease and may be useful as a therapy for /3globinopathies. However, limited information exists on the
effects of HU in patients with thalassemia. Accordingly, we
examined the hematologic effects of orally administered HU
in 13 patients with /3-thalassemialHb E, including four patients who had been splenectomized.These patients were
treated with escalating doses (final range, 10 t o 20 mg/kg/
d) for 5 months and were observed in the outpatient hematology clinic every 2 t o 4 weeks. Complete blood counts
including reticulocyte counts, amounts of Hb E and Hb F,
'y:*y and a:non-a globin biosynthetic ratios were evaluated
before and during treatment. Almost all patients responded
with an average increase
of 33% in Hb F levels, from a mean
(*SDI of 4
2% 11% t o 56% 2 8% ( P < .0001), and areciprocal decline in thepercentage of Hb E from 59% k 9% t o &%
.001). Reticulocytosiswas decreasedfrom a mean
(+SDI of 18.0% 15.6% t o 11.7% k 9.1% ( P < .05); there
was also a slight (10%) but statistically significant increase
in hemoglobin levels and an improved balance in a:non-a
globin chains ratios. The side effectswere minimal in most
patients, although these patients tended t o tolerate a lower
dose of HU before significant myelosuppression than has
been ourprevious experience in sickle cell disease. Onesplenectomized patient died of sepsis during the trial. We conclude that increased HbF production in fi-thalassemia/Hb E
patients, with an improvement in thea:non-a globin ratios
and, probably, the effectiveness of erythropoiesis, can be
achieved using HU. Longer trials of HU in this population,
including at other dosesand in combination with other
agents, appear warranted.
This is a US government work. Thereare no restrictions on
its use.
T
in patients with @-thalassemiaand sickle cell anemia, which
could be a therapeutic approach for these patients.1°12
Among these agents, hydroxyurea (HU), acell cycle-specific
agent that blocks DNA synthesis, has been shown to enhance
Hb F levels in both nonhuman primates and in patients with
myeloproliferative and sickle cell
Although early
results in thalassemia have been variable," recently it has
been reported that virtually all of 14 patients with sickle cell/
@-thalassemiatreated with HU have had an increase in Hb
F levels and an apparent decrease in crisis freq~encies.'~
In
this study, we examined the hematologic effects of orally
administered HU in 13 patients with P-thalassemialHb E.
*
HALASSEMIA is a heterogeneous group of genetic defects that results in defective globin synthesis. It is the
most common genetic disorder among the people living in
Southeast Asia; a-thalassemia, @-thalassemia, hemoglobin
(Hb) E, and Hb Constant Springs (CS) are prevalent. The
gene frequencies of a-thalassemia reach 20% to 30% in
Northern Thailand and Laos. Frequencies of @-thalassemia
gene vary between 1% and 9%. Hb E is the hallmark of
Southeast Asia, attaining a gene frequency of 50% to 60%
at the junction of Thailand, Laos, and Cambodia. Hb CS
frequencies vary between 1% and 8%.'z2 These abnormal
genes in different combinations lead to over 60 thalassemia
syndromes. Interaction between @-thalassemia and Hb E
genes leads to homozygous @-thalassemia and P-thalassemia/Hb E diseases that are major @-thalassemiasyndromes
in this region. P-thalassemialHbE can be as severe as homozygous @-thalassemia. Indeed, hemoglobin levels in 802
cases of @-thalassemia/HbE disease at steady state varied
from 2.6 to 13.3 g/dL, with an average of 7.7 g/dL.3-5
Many factors may contribute to this heterogeneity, such
as concomitant inheritance of a-thalassemia, which can ameliorate the severity of @-thalassemia.6Recently, we found
that the inheritance of a @-thalassemiachromosome with the
Xmn I cleavage site at position - 158 of the 'y-globin gene
that was linked to the haplotype -+ - ++ was associated
witha milder anemia, as in the case of homozygous pthalassemia.' Increased expression of the 'y-globin gene and
higher production of hemoglobin F, which could reduce the
overall globin chain imbalance, were also associated with
homozygosity for the Xmn I cleavage site and, thus, with
less severe anemia.
The finding that higher production of Hb F paralleled
higher hemoglobin levels in P-thalassemia/Hb E patients
was similar to findings in other @-thalassemiadisease and
sickle cell
During the last 10 years, there have
been several studies attempting to enhance Hb F production
Blood, Vol 87, No 3 (February l), 1996: pp 887-892
*
PATIENTSELECTION AND METHODS
Patients were considered eligible for the study if they exhibited
signs of moderate to severe thalassemia, including evidence of osteoFrom the Thalassemia Center, Division of Hematology, Department of Medicine, Faculty of Medicine, Siriraj Hospital, Bangkok,
Thailand; the Institute of Sciences and Technology for Research
and Development, Bangkok, Thailand; the Department of Pharmacy,
Faculty of Pharmacy, Mahidol University9Bangkok, Thailand; and
the Laboratory of Chemical Biology, National Institute of Diabetes,
Digestive and Kidney Diseases, Bethesda, MD.
Submitted September 26, 1994; accepted August 30, 1995.
Supported in part by the Commission of the European Communities, Science and Technology for the Development Program, Contract No. TS3*-CT92-0081; and the Prajadhipok-Rambhai Bami
Foundation.
Address reprint requests to Gri@n P. Rodgers, MD, Chief; Molecular Hematology Section, Laboratory of Chemical Biology, Bldg 10,
Room 9N-318,IO Center Dr MSC-1822, Bethesda, MD 20895-1822.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
This is a US govemment work. There are m restrictions on its use.
00oS-4971/96/8703-0$0.00/0
887
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FUCHAROEN ET AL
888
porotic bone disease, intermittent transfusion-dependency, and hemochromatosis. Thirteen patients with p-thalassemialHb E-nine
nonsplenectomized and four splenectomized cases-were recruited
in this study. The patients, six menand seven women,rangedin
age from 18 to 55 years (average age, 34 years). All of them were
treated at the outpatient hematology clinic of Siriraj Hospital (Bangkok, Thailand) with regular followup every 2 to 4 weeks. An exemption for treatment with an investigational new drug was obtained
from the Food and Drug Administration. The treatment protocol was
approved separately by clinical research subpanels at Siriraj Hospital, Mahidol University (Bangkok, Thailand) and the National Heart,
Lung and Blood Institute (Bethesda, MD). After informed consent
was obtained, baseline and serial hematologic tests and measurements were performed. Hematologic data, Hb analysis, and reticulocyte count were determined by conventional methods. Percentages
of Hb E and Hb F were obtained by elution after cellulose acetate
electrophoresis. Determination ofthe
ratio was obtained from
the globin chains separated by Triton X-100 acid urea polyacrylamide gel electrophoresis.16In vitro globin chain synthesis was determined from the reticulocytes of heparinized bloodas previously
described," and theglobin chains were separated using a Fast Protein
Liquid Chromatography (FPLC) system (Pharmacia Biotech, Piscataway, NJ). Complete blood counts including reticulocyte count, the
amounts of Hb E and Hb F, serum tests indicating the level of renal
and hepatic function, and ' y : * y and ct:non-ct ratios were evaluated
before and serially during treatment. Restriction endonuclease analysis of genomic leukocyte DNA was performed to assess the alphaglobin genotype'"'' and the haplotype of the beta-globin gene cluster.'
The patients were initially treated with a dose of HU of approximately 10 mgikg (to nearest 250-mg capsule). This dose was maintained for approximately 10 weeks, at which point, in the absence
of hematologic toxicity, the dose was increased to 20 mgikg and
continued for an additional 10 weeks. Therapy was discontinued at
the end of the 20-week trial. Evidence of hematologic suppression
was defined as an absolute reticulocyte count less than 50,00O/pL,
a white blood cell count less than 3,OOO/pL (or neutrophil count less
than l,OOO/pL), or a platelet count less than 100,00O/pL.
Statistical methods to detect the significance of the differences in
the individual pairs of data included the paired Student's f test for
data with a Gaussian distribution and Mann-Whitney U test for data
with a non-Gaussian distribution. Repeated measures-analysis of
variance (ANOVA) with Bonferroni's correction-was also used to
analyze multiple measurements on the same individual. Univariate
and multivariate analyses were used to describe the strength of relationships between variables.'')
RESULTS
Fetal hemoglobin responses. Overall, patients responded with a 32.5% increase in fetal hemoglobin from a
mean (?SD) of 42% ? 11% to 56% -c 8% (P < .0001). As
indicated in Table 1, while there was a spectrum in the Hb
F responses, only 1 of the 13 patients (patient 8) did not
show an increase in Hb F expressed in grams per deciliter
and, thus, would be considered a nonresponder. Hb E levels
declined from 59% t 9% to 49% t 8% (P < .001). A
sample of the reciprocal changes in the levels of Hb E and
Hb F in one patient is shown in Fig 1, whilethemean
changes for the entire group of 13 patients are depicted in
Fig 2. The increase in Hb F due to increased y-globin chain
synthesis led to improvement in the a:non-cu ratios, and there
were no significant changes in the G y : A y ratio with this
enhanced production of fetal hemoglobin (Table 2). We were
unable to detect an absolute relationship between the initial
percent Hb F, Gy:Ay
ratio, @-globinhaplotype, a-globin genotype, or basal level of renal or hepatic function with the
magnitude of the Hb F response. Fetal hemoglobin was detectedin all of the red cells (F cells) before and during
therapy by the modified Kleihauer acid-elution technique."
Thus, we estimate that with treatment the proportion of Hb
F per red cell increased by about one third.
Hematologicresponses.
There was a small but statistically significant increase in the level of hemoglobin, from
6.6 ? 0.6 g/dL to7.3 t- 0.7 g/dL (Student's t-test, P <
.02). When a more conservative method of analysis (repeated
measures-ANOVA with Bonferonni's correction) was applied, taking into account the multiple measurements of hemoglobin before andat entry, this change inhemoglobin
remained statistically significant (F = 6.95, P = .15; with
Bonferroni correction, P < .05). There was also a moderate
decrease in bilirubin (4.9 ? 1.6 mg/dL to 3.5 ? 1.9 mg/dL,
P < ,001) and the reticulocyte count (18.0% ? 15.6%to
1 1.7% ? 9.1 %, P < .05). As noted in previous clinical trials,
HU therapy was associated with a marked increase in the
meanred
cell volume (MCV) andhemoglobin
content
(MCH), while there was no net change in the meancorpuscular hemoglobin concentration, which iscalculated from these
two indices. Changes in these parameters correlated with the
increments in fetal hemoglobin augmentation ( r = .61, P =
.03; Table 1). A summary of all patients' hematologic profiles is shown in Table 2. Subset analysis indicated that there
were no statistically significant differences before or after
HU treatment in any of these listed parameters between the
splenectomized and nonsplenectomized patients.
During this 20-week trial, therewas indication ofmild
hematopoietic toxicity in the majority of our patients. The
maximal average tolerable dose in these patients was 15 mg/
kg/d (range, 11.1 to 17.9 mg/kg/d). There was a significant
decline in the white blood cell (WBC) count (average decrease, approximately 50%) in 6 of 13 patients, in the platelet
count (average decrease, approximately 30%) in 3 of 13
patients, and in the reticulocyte count in 1 of 13 patients. It
should be notedthat the nadir of these counts in most patients
was at a level above what wasconsidered significant hematologic suppression (see Patient Selection and Methods), and
only three patients required a dose adjustment due to WBC
count less than 3,00O/pL. These declines in blood cell counts
occurred precipitously within the 2 weeksbetween clinic
visits and are to be distinguished from the moregradual
decline in the WBC and reticulocyte counts occurring over
the 5-month treatment period (Table 2). Thus, it may be
concluded that requisite doses of HU tostimulate fetal hemoglobin levels in patients with P-thalassemiab E are at or
near the threshold for myelosuppression.
Clinical effects. In addition to the modest increase in the
hemoglobin, most patients described an increase in exercise
tolerance and sense of well-being in comparison with their
symptoms before treatment. Because this wasnot a controlled trial, we cannot formally exclude a placebo effect.
Side effects were minimal in most patients, except for the
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HU
IN P-THALASSEMINHbE DISEASE
889
Table 1. Changes in the Level of Hemoglobin and Absolute Amount of Hb F in 13 Patients With pThalassemialHbE
During 5 Months of HU Treatment
HbF (g/dL)
Hemoglobin (g/dL)
Patient
6.6
30/F
7.6
6.0
1. P.N.' 6.9
2. P.P.* 6.1
3. CS.' 6.9
4. T.S. 7.2
7.6
5.B.T.
6. V.N. 6.2
7. S.I.' 6.1
8. RN.' 4.8
9. I.J. 7.5
10.6.6M.I.'
11. B.C.
12.
7.1L.S.
13.
6.0 S S .
Mean 2 SD
Age
(yr)/Sex
18/M
26/M
29/M
36/M
42/M
48/M
23/F
26/F
27/F
28/F
0-Day
Pre
7.2
6.6
6.8
6.0
7.4
5.9
6.4
6.0
7.1
8.0
7.1
7.4
6.7
8.0
6.2
7.2
6.0
7.3
7.7
7.8
4.2
8.1
7.4
8.1
7.7
5 mo 0-Day
2 mo
Pre
2.2
1.9
2.8
3.0
2.5 NAt
2.7
2.2
2.0 5.9*
4.0
2.2
1.9
2.6
2.5
2.4
2.6
1.8
2.7
3.4
3.2
3.4
2.0
4.0
3.0
3.2
3.5
3.6
3.4
2.4$
4.1
2 mo
5 mo
3.2
2.6
3.4
3.3
3.6
4.4
3.2
1.9
4.4
NAt
2.5
34/F
42/F
6.51 2 0.59
6.56
2 0.83
6.91
2 1.07
7.25 2 0.63
2.60
2 0.57
2.85
2 0.75
3.51
2 0.73
3.67
2 0.65
The pretreatment value of hemoglobin is the average of three consecutive baseline values, while the 0-day value is the average of two
determinations obtained within 2 weeks of initiation of HU.
Abbreviations: Pre, pretreatment value; NA, not applicable.
Splenectomized patient.
t HU was stopped because the patient could not come for regular follow-up.
Patient had upper respiratory tract infection, and 1 U of blood was given at the end of the third month of HU.
§ The last hematologic data were obtained 2 weeks before patient died.
*
anticipated changes in hematologic parameters, as described
above. One splenectomized patient died in the last month of
the trial because of septicemia after diarrhea. She had been
on a stable dose of HU (14 mg/kg/d) for the preceding 3.5
months, which she tolerated well. Her W C count was
12,OOO/pL with 85% neutrophils when she was brought to
the hospital in a semicomatose condition, and she died within
a few hours after her arrival at the hospital. Hemoculture
revealed Streptococcal pneumococci in all three blood samples. This specific complication should beviewed in the
context of an underlying significantly increased incidence
of fatal pneumococal bacteremia among splenectomized pthalassemia patients in Southeast Asia, which we have previously reported."
DISCUSSION
Numerous studies have demonstrated that HU can enhance
Hb F production in patients with sickle cell anemia. However, there has been limited and mainly negative information
on the effect of HU in thalassemia." Two recent reports
of HU therapy of P-thalassemia intermedia suggest modest
beneficial effect^:^.'^ while we have observed several patients with P-thalassemia with the IVS-654 mutation who
have responded to HU with increased &globin ~ynthesis.'~
Recently, HU has been shown to be of potential benefit
in sicklelp-thalassemia double heterozygotes, although no
effect on hemoglobin levels was achieved.I5The two major
hemoglobin components in patients with p-thalassemia/Hb
E disease are Hb E and Hb F, and the levels of Hb F can
vary from 30% to 70%.' Recent studies have demonstrated
that there is a good correlation of hemoglobin levels with
the amount of Hb F production in certain groups of patients
with @'-thalassemia/Hb
E.5.7
Our results demonstrated that increased Hb F production
in patients with P-thalassemiakIb E associated with an improvement in the adnon-a globin production can be achieved
using HU. Red cell MCV and MCH were also increased in
all subjects (Table 2). Although there were no dramatic
changes in the hemoglobin levels, the percentage and the
absolute amount of reticulocytes were decreased, which reflects the improvement in bone marrow stress due to tissue
anoxia. Alternatively, this decrease in reticulocyte count may
reflect marrow toxicity. However, the fact that the reticulocytes decreased after the hemoglobin increased and that the
patients exhibited a decrease in serum bilirubin and lactic
dehydrogenase (LDH; data not shown) would support the
improved erythropoiesis hypothesis. The temporal relationship between the increase in the percent HbF (and the reciprocal decrease in percent HbE) and the increase in total
hemoglobin (Fig 2) is also consistent with this view. Bone
marrow failure due to aplastic crisis (and presumably secondary to myelotoxicity) in the severe &globin disorders
may be accompanied by a transient decrease in the reticulocyte count and bilirubin and LDH levels, and a transient
increase in Hb F levels.26However, such effects are most
often accompanied by more profound declines in the reticulocyte count (often to zero) and a concomitant decrease in
hemoglobin levels, while the mean (red) cell volume remains
unchangedZ6-findings in marked contrast with our observations. Finally, we observed that the hemoglobin levels and
red cell indices in these patients returned to baseline levels
within 8 weeks of cessation of HU (data not shown), again
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890
FUCHAROEN ET AL
IA
was not possible to study F-reticulocyte levels because of
technical limitations in our method. The enumeration of F
cells by acid-elution shows that all red cells in patients with
,@"thalassemia/Hb E give a positive reaction with heterochromatic staining for Hb F. Ideally, it would be necessary
to use immunofluorescence staining and flow cytometry to
distinguish increases in the F-cell production and/or in the
quantity of Hb F per F cell.
--I--Hemoglobin (@dl)
6
.
5
'
m
'
'
z
.
1
5
-100
0
100
2
Treatment Day
W
16
'O
U.
.
n
I
z
7
z
50
-
A
I I 1 1 1 1 1 1 1 , 1 ,
6 8 10 12
14
16 18 20 22 24
"
'
.
.
~
.
.
.
.
~
8 6.5
5
-100
5 J I
0 2 4
. 5 "
40
W
40
.
I
I
0
100
30
200
0
m
1
5.5
Treatment Day
Fig 1. Effect of HU onhematologicvalues in a patient with pthalassemia/Hb E. (A) Sequential change in hemoglobin values. (B)
Reciprocal changes in the proportion of Hb F.
reflecting that the changes in hematologic parameters were
coincident with HU therapy.
It is noteworthy that in these patients, the fetal hemoglobin
and total hemoglobin levels continued to increase during
treatment, and thus, the absolute improvement in the level of
ineffective erythropoiesis and the effects on the characteristic
peripheral hemolysis of these patients achievable with HU
cannot be stated definitively. Indeed, long-term HU treatment of patients with sickle cell anemia indicates that a
continued improvement in Hb F levels and in the hemoglobin
values can bedemonstrated for up to a year while ona stable
dose ofHU (G.P.R., unpublished observations, December
1 994).
Fetal hemoglobin levels in patients with beta-globin disorders are determined by three factors: F-cell production, the
amount of Hb F per F cell, and the preferential survival of
F cells.27We had previously shown that during short-term
trials of HU in patients with sickle cell anemia, F-cell production, as estimated by the F-reticulocyte levels, accounted
for 70% of the increase in Hb F 1e~els.I~
Unfortunately, it
~
1
50
2
n
I
~
"-a"% HbE
WW
~
4.5
Week on Hydroxyurea
Fig 2. Effect of HU onhematologicvaluesin
13 patients with
p-thalassemialHb E.Theaveragesandstandarddeviationsinthe
percentages of (A) Hb F, (B) Hb E, and (C1 total hemoglobin in this
group of patients duringHU treatment is shown.
~
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HU THERAPY IN P-THALASSEMINHbEDISEASE
891
Table 2. HematologicEffects of HU in 13 Patients
With B-ThalassemialHbE
Test
Hb (g/dL)
MCV (fL)
MCH (pg)
WBC (//L)
Reticulocytes (%)
nRBC (X103/pL)
= y A y ratio
a:Non-a globin
Baseline Value
(mean 2 SE)
After 5 Months HU
(mean 2 SE)
P Value
7.3 2 0.6
6.6 2 0.6
<.05
64.8 t 7.1
74.2 2 6.2
< .05
19.4 t 1.4
23.3 t 2.0
<.05
13,900t 9,500
9,500t 6,300<.05
18.0 f 15.6
<.05
11.7 t 9.1
2.26 t 3.24
3.04 2 4.52
.l5
1.8 t- 0.7
.53 0.6 1.4
2.6 t- 0.5
<.02
0.5 2.1
Baseline values were obtained within 1 month before the initiation
of H U treatment; P value determined by two-tailed Student's t-test.
Abbreviation: nRBC, nucleated red blood cell count.
Because of the short period of this clinical trial, it is very
difficult to draw conclusions on the clinical response to HU
treatment. Most of the patients reported feeling better; side
effects generally have been minimal. In this group of patients
who exhibit a @-thalassemiaintermedia phenotype, even a
modest increase in red cell production with coincident suppression of bone marrow turn-over might be highly beneficial; the need for transfusion support might be delayed or
eliminated, Further studies will be required to evaluate the
long-term toxicity and benefit to the patients. It is notable
that these patients with P-thalassemialHbE seem to tolerate
a lower dose of HU than has been reported in patients with
sickle cell anemia'3314before hematopoietic toxicity is evident. Nonetheless, we have treated two Chinese patients with
@-thalassemiaintermedia for periods of 1 and 2 years both
on a dose of HU of 8 mgkgld, which has led to a 50% and
100% increase, respectively, in their baseline hemoglobin
values.25Based on these results expanded studies of HU in
patients with P-thalassemia seem warranted, especially for
longer periods of time and at other doses. Moreover, clinical
trials of other agents such as butyric acidz8and its derivatives29.30 alone and in combination with HU3' or erythropoietin3' should be considered to identify optimal regimens in
this patient population.
ACKNOWLEDGMENT
We acknowledge Bristol-Myers Squibb (Princeton, NJ) for the
contribution of HU. We also thank Drs Marvin Podgor and Craig
Stotts for advice with statistical methods.
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effectiveness of erythropoiesis in beta-thalassemia/hemoglobin E
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S Fucharoen, N Siritanaratkul, P Winichagoon, J Chowthaworn, W Siriboon, W Muangsup, S
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Rodgers
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