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CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
From the Departments of Pediatric Oncology/Hematology,
General Pediatrics, Neonatology, Clinical Chemistry/Biochemistry, and
Pharmacy, Charité, Humboldt University, Berlin,
Germany.
The butyrate derivative isobutyramide (IBT) increases fetal
hemoglobin (HbF) in patients with The term Several observations have indicated that a number of short-chain fatty
acids, and butyrate compounds in particular, are able to influence the
developmental program of globin synthesis.3-6 Subsequently, butyrate compounds were found to increase HbF levels in
animal models and in erythroid cells of patients with
Sodium phenylbutyrate is an alternative compound that is absorbed
enterally and has been reported to increase HbF levels in patients with
sickle cell disease15 and to increase F reticulocytes in
patients with We report here a long-term clinical trial to examine the hematologic
effects of orally administered IBT, particularly regarding changes in
the transfusion requirement and subsequent changes in iron burden in
patients with transfusion-dependent homozygous After written informed consent was obtained from the patients and
their parents where applicable, 5 male and 3 female patients with
homozygous Before entry into the study, the patients were transfused with 10 to 20 mL/kg of body weight of packed RBCs with a hematocrit of 67% ± 3%
(mean ± SD) at 4-week intervals. The baseline Hb level was 100 g/L or greater, and the post-transfusion Hb level was about 130 g/L,
resulting in a mean Hb level of about 120 g/L. On entry into the study,
the baseline Hb level was therefore reduced to 85 g/L and the mean
post-transfusion level to 115 g/L, which was expected to activate
endogenous RBC production. The regular 4-week blood transfusion regimen
was discontinued during the study period. Instead, patients were
transfused when the Hb declined to a level of 85 g/L or less,
regardless of the time interval between 2 blood transfusions. However,
when transfusions were necessary, the number of units of packed RBCs
remained unchanged. The total duration of the study was 14 months,
beginning with a prestudy phase consisting of 3 transfusion cycles (3 months), a treatment phase of 8 months, and a follow-up period of 3 months. An option to prolong treatment in patients obtaining
clinical benefits by IBT was included in the study protocol.
IBT was prepared as a suspension immediately before use. The solid IBT
was diluted 1:10 with water at room temperature. To cover the bitter
taste of the substance, the carrier was natural grapefruit juice
additionally flavored with grapefruit oil. IBT was administered
starting at a dose of 250 mg/kg per day in 2 divided doses. The
starting dose was maintained for approximately 8 weeks, at which point,
in the absence of hepatic or renal toxicity, the dose was increased to
350 mg/kg per day and continued for an additional 24 weeks.
Safety parameters were monitored by physical examination, vital signs,
and clinical laboratory evaluation (blood chemistry, blood counts, and
differential counts). Efficacy was evaluated by changes in the
transfusion interval, iron load, Hb, mean corpuscular volume (MCV),
mean corpuscular hemoglobin (MCH), lactate dehydrogenase (LDH), and
plasma-free Hb. Assuming that 1 mL of packed RBCs contains 1 mg of
iron, the transfusion-related daily iron loading was calculated from
the volume of packed RBCs transfused and the actual transfusion interval. To obtain the exact volume, RBC bags were weighed before and
after transfusion each time, and the hematocrit was determined. The
measured weight was then divided by the specific weight (1.055 g/mL) and multiplied by the hematocrit, providing the volume transfused.
The reticulocyte count was obtained manually after staining with cresyl
blue brilliant. The number and percentage of nucleated RBCs (nRBCs) was
also determined in peripheral blood smears. The percentage of HbF was
obtained by elution after cellulose acetate electrophoresis. The
absolute HbF concentration was calculated as the product of the
percentage of HbF and total Hb. Circulating erythropoietin (EPO) levels
were measured by a commercially available radioimmunoassay.20 Mutations of the The post-transfusion Hb level was determined 1 hour after the end of
transfusion treatment. The decline of Hb level thereafter was first
assessed in 2- to 3-week intervals. Upon approaching the baseline level
of 85 g/L, the intervals between Hb measurements became increasingly
shorter (1-3 times a week) until the pretransfusion Hb level of 85 g/L
was reached. Each automated Hb measurement was paralleled by a manual
count of nRBCs to control for possible artificial elevations in
automated Hb measurements caused by the presence of nRBCs in the
peripheral blood. MCV, MCH, LDH, free Hb, HbF, and reticulocyte counts
were obtained 1 day before and 1 hour after completion of blood
transfusion as well as once every 2 to 3 weeks within the transfusion
interval. EPO levels were determined 1 day before transfusion.
For the purpose of this study, a clinical response was defined as a
decrease in the daily body iron load of at least 20%, achieved by a
prolongation of the transfusion interval during IBT therapy, based on
an intention-to-treat analysis.
Because a normal distribution could not be assumed, all numeric data
are presented as median and interquartile ranges unless stated
otherwise. For the calculation of the median of Hb, HbF (% and
absolute), MCV, MCH, free Hb, LDH, nRBCs, reticulocyte counts, and EPO,
pretransfusion values were used. Groups were compared using the
Mann-Whitney test.
Pretransfusion and post-transfusion hemoglobin
Transfusion interval and changes in iron load Patients were grouped into responders and nonresponders depending on whether the transfusion interval could be prolonged and the average daily iron load decreased by at least 20% (Table 2). By this definition, 2 patients (nos. 2 and 5) responded by a reduction of the daily iron burden by 21% and 54%, respectively. In patient no. 5, the transfusion intervals were extended from 4 weeks before the start of therapy to 8 and 9 weeks, respectively, during treatment (Figure 1). The prolonged transfusion intervals were interrupted only once when he developed an acute episode of catheter sepsis with
hemolysis, during which time IBT was stopped. This was followed by an
immediate relapse, with 2 transfusions being required at a 4-week
interval. However, an extension of the transfusion intervals to 8 and 9 weeks, respectively, was possible after IBT treatment could be resumed.
In this patient, the average daily iron load decreased from 455 µg/kg
per day (range 451-459 µg/kg per day) before the start of therapy to
211 µg/kg per day (range 203-286 µg/kg per day) during the
treatment period of 12 months.
In patient no. 2, the effect of IBT was more protracted (Figure 1). During the pretreatment phase, this patient needed blood transfusions every 4 weeks. This did not change during the first 8 weeks after the initiation of IBT treatment, but transfusion intervals could be extended to 5 and 6 weeks thereafter. After a subsequent return to 4-week intervals for 3 cycles, a consistent extension to 8-week intervals ensued. In this patient, the average iron load declined from 683 µg/kg per day (range 618-748 µg/kg per day) prestudy to 542 µg/kg per day (range 340-596 µg/kg per day) during 13 months of IBT treatment. In contrast to the 2 responders, in all of the 6 other patients the decline of post-transfusion Hb levels to the transfusion threshold of 85 g/L occurred within 4-week intervals irrespective of IBT treatment. These patients were therefore considered to represent nonresponders (Figure 1). Their daily iron load during the 3-month post-IBT phase was 449 µg/kg per day (range 480-586 µg/kg per day) and did not differ from iron load before or during IBT (Table 2). Fetal hemoglobin production Patient no. 5 showed a progressive increase in the HbF level over 300 days on IBT therapy (Figure 1). This was interrupted only onceby
an acute episode of anemia related to septicemia from days 144 to 172, during which time IBT therapy was discontinued. This was paralleled by
a rapid decrease of the HbF level. When IBT therapy was resumed, the
HbF level increased again and peaked at 22% (22 g/L) on day 308. Patient no. 2 displayed a less marked response to IBT therapy than
patient no. 5 (Figure 1). The HbF level increased to a maximum of 18%
(17 g/L) on day 365.
It is notable that increases in HbF levels were not only seen in clinical responders but also in nonresponders (Figure 1; Table 2). Based on an intention-to-treat analysis, all 8 patients showed an increase in HbF production after oral IBT therapy was initiated. The median pretransfusion percentage of HbF increased from 3.1% (range 1.9%-4.8%) before therapy to 6.0% (range 3.3%-8.7%) (P = .0017) during oral IBT treatment. The median pretransfusion absolute HbF level rose from 2.7 g/L (range 1.6-4.1 g/L) to 5.1 g/L (range 2.7-6.6 g/L) (P = .0013). Upon discontinuation of IBT, the HbF level decreased rapidly and did not differ from pretreatment values; the absolute HbF concentration was 2.8 g/dL (range 0.83-4 g/L), and percent HbF was 2.4% (range 1.7%-4.5%); P > 0.1. Interestingly, HbF concentrations were higher in clinical responders not only during but also before IBT treatment: Although at comparable baseline Hb levels, responders no. 2 and 5 displayed an HbF level of 5% (range 4.7%-5.7%) and 5.3% (range 4.9%-6.1%), respectively, before start of therapy, whereas the nonresponders' HbF was either not detectable or did not exceed 4.3%. Parental HbF levels of the responders were also higher than those of the nonresponders (Table 1), suggesting a genetic factor that influences the capacity to respond to IBT treatment. Hematologic responses and indicators of hemolysis During IBT therapy, all patients experienced an increase of pretransfusion MCV from 79 fL (range 77-81 fL) to 86 fL (range 82.6-88 fL) (P < .0001) and of pretransfusion MCH from 27.6 pg (range 27-28 pg) to 28.9 pg (range 28.3-29.7) (P < .0001) (Table 2). After cessation of IBT therapy, both MCV and MCH returned to pretreatment values within the 3-month follow-up period (data not shown). In addition, all 8 patients showed evidence of reduced hemolysis/ineffective erythropoiesis during IBT therapy. Median values for free Hb (pretransfusion) decreased from 0.48 g/L (range 0.39-0.81 g/L) to 0.19 g/L (range 0.16-0.24 g/L) (P < .0001) (Table 2). Upon discontinuation of IBT, free Hb returned to pretreatment levels within the 3-month follow-up period (data not shown). In neither of the 8 patients were significant prestudy, study, or poststudy changes observed for the following parameters: LDH 232 IU/L (range 207-347 IU/L) prestudy, 218 IU/L (range 195-322 IU/L) during the study, and 241 IU/L (range 188-362 IU/L) poststudy P > .1; reticulocyte counts 5% (range 3%-8.8%), 4%
(range 2%-8%), and 3.5% (range 1%-7.8%),
respectivelyP > .1; nRBCs 0.29/nL (range 0.066-1.46/nL), 0.18/nL (range 0.0-0.58/nL), and 0.42/nL (range 0.19-9.0/nL), respectively P > 0.1.
Circulating EPO concentrations were markedly higher in clinical responders than nonresponders (Table 2). Of the 6 patients with EPO levels below 150 IU/L, none responded to therapy, but the 2 responders displayed EPO levels above 150 IU/L. It is notable that EPO levels in the responders were significantly higher than in the nonresponders right from the start of IBT therapy. However, there were no further increases during the trial period thereafter (data not shown). The  86 C  G mutation. The other
responder (no. 2) was homozygous for the common nonsense mutation of
codon 39, which results in the complete inactivation of the affected gene. In the 6 other patients who were classified as nonresponders, severe  -globin mutations that are common in the Mediterranean area
could be identified (Table 1).
Compliance with IBT therapy Four-week supplies of IBT were provided to the patients. Compliance was recorded by counting empty vials. Reported compliance with medication was 94% ± 3% (mean ± SD). Therapy in patient no. 6 was discontinued at day 126 after he had developed DFO-induced liver toxicity (see "Adverse events"). After elective cessation of therapy, patient no. 1 had no post-treatment phase because he could no longer visit the outpatient clinic. The 2 responders (nos. 2 and 5) continue to take IBT medication.Adverse events The most common adverse effect reported by all patients was a bitter taste and a full feeling immediately after the drug was taken,. However, this adverse effect was not severe enough to cause discontinuation of the treatment in any of the patients. Patients were allowed to vary the aqueous vehicle and, in the course of the trial, 6 of the 8 patients switched to cherry juice, which was felt to cover the bitter taste best. Only 1 patient continued taking grapefruit juice as the flavoring agent; 3 patients complained about recurrent epigastric discomfort, especially after the morning dose of the drug. Patient no. 6 complained of repeated episodes of nausea after taking IBT. By the 28th week of treatment, patient no. 3 developed bilateral numbness of the fingers and toes and weakness of the legs, with difficulties walking. She already had an episode of similar symptoms 1 year before the initiation of IBT. The symptoms were interpreted to represent peripheral neuropathy, which was supported by a decreased nerve conduction velocity. However, these findings persisted after the withdrawal of IBT. The patient then discontinued chelation therapy, and neuropathy slowly improved despite reintroduction of IBT after 4 weeks. After approximately 12 weeks of IBT, patient no. 5 developed septicemia caused by Staphylococcus cohnii in the implanted venous access port, which was treated with antibiotics. IBT therapy was discontinued for 4 weeks and resumed thereafter. Patient no. 6 developed jaundice and an increase in serum LDH levels causing discontinuation of IBT on day 126. However, these signs of liver toxicity only improved after the patient was taken off DFO. Several attempts to restart chelation therapy at a much lower dose were followed by a prompt rise in serum bilirubin levels, which returned to normal after cessation of DFO. Subject no. 7 developed a follicular pruritic rash covering the arms and extending to the trunk and thighs after 4 weeks of treatment. Within several days, the rash subsided spontaneously.
The present study aimed to determine the safety and efficacy of a
prolonged administration of oral IBT in a group of patients with
homozygous A transfusion regimen with a baseline Hb of at least 100 g/L results in
the down-regulation of endogenous EPO production and subsequently in
the suppression of marrow activity.22 Assuming that a drug
that activates endogenous HbF production cannot display its full effect
in the presence of erythroid marrow inhibition, we lowered the
pretransfusion Hb to 85 g/L during the study. Serial reticulocyte and
nRBC counts assessed before, during, and after IBT treatment were
performed to distinguish F-cell selection from a genuine increase of
HbF synthesis. After initiation of IBT treatment, 2 of 8 patients
demonstrated an improvement in the effectiveness of erythropoiesis as
measured by a slower decrease of post-transfusion Hb to baseline
levels. These effects were associated with an increased HbF production.
The slower decline of Hb resulted in an extension of the transfusion
intervals, with a corresponding reduction of the average daily iron
loading by 21% to 54%. Such a reduction in excess body iron is
expected to be beneficial because the mortality of patients with
A correlation between the Both dosage and duration of IBT therapy appear to be critical for sustained rises of HbF. In a previous short-term pilot study in patients with thalassemia intermedia, modest increases of HbF were attained by IBT in dosages not exceeding 150 mg/kg of body weight.19 The data of the study presented here suggest that dosages of 350 mg/kg or more and a longer treatment period may be needed to achieve functionally significant increases of Hb concentrations. Although in all patients an increase of the HbF level was demonstrated
during IBT treatment, a clinical benefit was achieved in only 2 patients. It is notable that the parental HbF level was increased in
those 2 patients, suggesting a genetic factor responsible for a
particular propensity to activate HbF synthesis under conditions of
erythroid stress. Similarly, raised parental HbF levels have previously
been associated with an increase of A second feature that becomes apparent in the current study is the
positive correlation between serum EPO levels and the likelihood to
respond to IBT therapy. Although all patients were transfused at
similar baseline Hb levels during the 3-month prestudy phase, circulating EPO levels were markedly higher in responders right from
the start of therapy. This observation correlates with data obtained
with oral sodium phenylbutyrate in In summary, the data presented here demonstrate that IBT monotherapy is
capable of achieving a reduction of transfusion requirements in
selected patients who were characterized by raised pretreatment and
parental HbF levels and elevated endogenous EPO concentrations. Future
trials will have to show whether these predicting parameters are valid
in a larger number of patients and whether an IBT-EPO combination
represents an option for patients with a low baseline level of
We are very grateful to Annette Bode, Ute Brueckner, and Karin Roth-Ostermann for technical assistance and the management of the patients. Special thanks go to the pediatric intensive care unit of the Charité for patient and helpful support at any time.
Submitted April 19, 1999; accepted June 19, 2000.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Andreas E. Kulozik, Department of General Pediatrics, Charité, Humboldt University, Augustenburger Platz 1, D-13353 Berlin, Germany; e-mail: andreas.kulozik{at}charite.de.
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© 2000 by The American Society of Hematology.
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  S. P. Perrine Fetal Globin Induction--Can It Cure {beta} Thalassemia? Hematology, January 1, 2005; 2005(1): 38 - 44. [Abstract] [Full Text] [PDF]
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O. Witt, S. Monkemeyer, G. Ronndahl, B. Erdlenbruch, D. Reinhardt, K. Kanbach, and A. Pekrun Induction of fetal hemoglobin expression by the histone deacetylase inhibitor apicidin Blood, March 1, 2003; 101(5): 2001 - 2007. [Abstract] [Full Text] [PDF]
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-thalassemia
Top
Abstract
Introduction
-globin chains
precipitates within the red cell, which causes ineffective
erythropoiesis and severe anemia.
-globin
chain and fetal hemoglobin (HbF) synthesis is capable of compensating
for the imbalance between 