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F Reticulocyte Response in Sickle Cell Anemia Treated With Recombinant
Human Erythropoietin: A Double-Blind Study
By Ronald L. Nagel, Elliot Vichinsky, Maya Shah, R. Johnson, E. Spadacino, Mary E. Fabry,
L. Mangahas, Robert Abel, and G. Stamatoyannopoulos
Studies on baboons and preliminary observations in three
patients with sickle cell anemia (SS) suggested that high
doses of pulse administered recombinant human erythropoietin (rHuEPO) stimulate F-reticulocyte production. We
now report on the administration of rHuEPO in a doubleblind format t o ascertain frequency of response and potential precipitation of side effects. Ten patients were enrolled,
but one was discontinued due t o the indication of a blood
transfusion. Of the other nine, five received rHuEPO in escalating doses (from 400 to 1,500 U per kg twice daily
[BID] per week), alternating with a placebo, in blinded
fashion. The second group, consisting of four patients, followed an identical protocol (except starting dose was 1,000
U/Kg, BID per week) and were iron supplemented during
treatment. The criterion of response was a transient doubling (as a minimum) of the steady-state F-reticulocytelevel.
We found that none of the five patients in the first group
respondedt o rHuEPO, and t w o of them became iron deficient, as judged by a significant decrease in ferritin. Of the
second group, four patients responded with F-reticulocyte
increases. In three patients, open label administration of
rHuEPO confirmed the effect. We observed seven painful
episodes during this study, t w o during the EPO administration and five during the placebo arm. Three patients were
phlebotomized because the hemoglobin level increased 1.5
g/dL more than steady-state levels. Of the six patients followed-up by percent dense cell determinations, one exhibited increased levels during periods of the treatment,
whereas the other five showed no change. No anti-rHuEPO
antibodies were detected. We conclude that rHuEPO can
stimulate F-reticulocyte response in some patients with
sickle cell anemia, without apparent negative clinical side
effects. The state of iron stores may be critical. Whether
higher doses of rHuEPO and/or a different regimen might
induce sustained F cells and fetal hemoglobin increases remains t o be determined.
0 1993 by The American Society of Hematology.
A
( I ) homozygous SS for sickle cell anemia, as evidenced by two methods of Hb electrophoreses; (2) monitored clinical status of at least 3
months; (3) aged between 18 and 45 years; (4) clinically stable as
otherwise evidenced by medical history, complete physical examination (including vital signs and weight), and 12-lead electrocardiogram, each performed within 2 weeks of the start of the study; ( 5 )
no clinically significant, acute abnormalities on chest x-ray (within
6 months before study participation); (6) no clinically significant abnormal laboratory values for the following tests, except for those
values out of the normal range due to the disease process; in addition,
all medications and dosages must have been stable for 2 weeks before
study entry, and were continued throughout the duration of the study;
(7) all females were either postmenopausal for at least 1 year, had a
hysterectomy or tubal ligation, or had taken oral contraceptives for
at least 1 month before study entry, or agreed to use spermicide and
bamer methods of protection (The patients continued with the same
SIGNIFICANT increase in the intraerythrocytic concentration of fetal hemoglobin (HbF) is very likely to
inhibit polymerization of sickle cell Hb (HbS)’-3and, consequently, the sickling phenomena: Several efforts have been
recently made to pharmacologically induce this effect. Cytotoxic agents such as Ara-C and hydroxyurea have exhibited
some promise’s6 and the use ofthe latter agent is being pursued
actively.‘ Nevertheless, concerns about potential long-term
toxicity and the need to elicit the highest possible increase
in HbF expression have led other groups to continue the
search for HbF-inducing agents that can be administered
alone or in combination with cytotoxic agents.
The observation that bleeding in baboons induced an Freticulocyte response similar to cytotoxic agents (vinblastine
and Am-C) prompted Stamatoyannopoulos et a18to postulate
that erythropoietin (EPO) could be useful in the induction
of HbF. Further studies in baboonsg with pulse doses gave
credence to this approach because HbF reticulocyte response
was observed. Also, preliminary, uncontrolled administration
of recombinant human EPO (rHuEPO) to three sickle cell
anemia patients suggested that F-reticulocyte response could
be elicited.”
We report here, using a double-blind format, on a study
of the administration of rHuEPO to ascertain frequency of
response (defined as a doubling of the F reticulocytes) and
potential side effects in a group of 10 sickle cell anemia patients. Of note is that this protocol did not result in the observation of, nor is it expected to lead to increased levels of
HbF cells or percent HbF because neither effect was obtained
with high pulse doses in the baboon.
MATERIALS AND METHODS
Patient Selection
The study population consisted of 10 sickle cell anemia patients
who met the following inclusion criteria at the prestudy examination:
Blood, Vol81, No 1 (January 1). 1993: pp 9-14
From the Albert Einstein College of Medicine/MonteJiore Medical
Center, New York, NY; Children’sHospital Medical Center, Oakland,
CA; R. W. Johnson Pharmaceutical Research Institute, Philadelphia.
PA; and the University of Washington, Seattle, WA.
Submitted May 15, 1992; accepted August 25, 1992.
Supported by National Institutes of Health Grant Nos. HL20899
and HL21016.
An abbreviated report of these Jindings was published: Vichinsky
E, et al: The Stimulation of Fetal Hemoglobin by hrErythropoietin
in Sickle Cell Anemia: A Double Blind Study in Regulation of Hemoglobin Switching. Stamatoyannopoulos G, Nienhuis A W (eds); p
394-402, 1991.23
Address reprint requests to Ronald L. Nagel, MD, Head, Division
of Hematology, Albert Einstein College of Medicine, I300 Morris
Park Ave, Ullmann 921, Bronx, NY 10461.
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 I734 solely to
indicate this fact.
0 1993 by The American Society of Hematology.
0006-4971/93/8101-0013$3.00/0
9
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10
NAGEL ET AL
method for the duration of the study.); (8) all females of childbearing
potential had a negative serum pregnancy test (human chorionic gonadotropin /3 radioimmunoassay [HCGB RIA] test) and history of a
normal menstrual flow within 1 month before study entry; and (9)
an informed consent was signed after the nature of the study has
been fully explained.
The criteria for exclusion were: (1) presence of significanthepatic,
cardiovascular, pulmonary, malignant, hematologic, neurologic, infectious, or inflammatory diseases unrelated to sickle cell anemia;
(2) sickle cell crisis within 3 weeks of study admission; (3) uncontrolled
hypertension (ie, diastolic blood pressure > 100 mmHg) or history
of seizures; (4) presence of asthma or severe atopic disease;(5) presence.
of current (last 3 months) ischemic heart disease; (6) presence of
thrombocytopenia (<100,000 cells/pL); (7) presence of neutropenia
( ~ 2 , 0 0 0cellslpl); (8) history of Coombs-positive hemolytic anemia;
(9) presence of apparent iron-deficiency anemia; ( 10) corticosteroid
therapy or immunosuppressant therapy within 1 month before study
entry; (1 1) history of substance abuse; (12) exposure to an investigational drug within 1 month of the start of the study; (1 3) acute
illness within 7 days of the start of the study; and (14) plasma creatinine greater than 1.5 mg/dL.
The procedures for screeningwere performed within 2 weeks before
dosing, except where otherwise stated, and included the following:
(1) medical history, including sickle cell crisis history; (2) physical
examination, including weight and vital signs; (3) 12-lead electrocardiogram;(4) chest x-ray (within 6 months); (5) the following routine
laboratory tests: (A) CBC plus differential, and platelet count; (B)
serum chemistry and serum ferritin; and (C) urinalysis, including
pH, specific gravity, and microscopic; (6) the following special laboratory measurements: (A) percent and total reticulocyte count; (B)
F reticulocytes; (C) F cells; and (D) HbF, by alkali denaturation; (7)
pregnancy test, RIA, if applicable; (8) rHuEPO antibody; (9) naturally
occurringrHuEPO levels; and (IO) signed consent form in the presence
of witnesses.
Sickle cell anemia patients were defined by two methods of Hb
electrophoreses (cellulose acetate at pH 8.6 and agar electrophoresis,
pH 6.4) as having exclusively HbS, HbF, and HbA2in their red blood
cells (RBCs). S//3 thalassemia combinations were excluded by family
studies, normal mean corpuscular volume (MCV), and, in some cases,
Mst I1 digestion of their DNA.
Table I depicts steady-state hematologic characteristics of the patients that completed the study.
Study Protocol
Group A patients. Five patients not supplemented with iron received either rHuEPO or placebo once a week for 12 weeks after a
blinded randomization schedule in which rHuEPO alternated with
a placebo. The weekly administration was divided in two doses separated by 12 hours. Doses were escalated weekly from 400 to 1,500
U/kg weight, except for one patient who was initially treated with
1,000 U/kg twice daily (BID) per week and escalated to 1,500 U/kg
BID per week.
Group B patients. Five patients were enrolled in the study, but
one patient did not complete the study because he required exchange
transfusion therapy because of acute chest syndrome in the initial
part of the protocol. Patients in this group received a supplement of
325 mg of ferrous sulphate three times a day throughout the study,
except for the two patients who had very high steady-state ferritin
levels (710,000 U). The escalating doses of rHuEPO began at 1,000
and escalated to 1,500 U/kg week.
Because the increase of total Hb is considered an unwelcome event
in the absence of antisickling therapy, the increase in 1.5 g/dL over
steady-state during the protocol was an indication for phlebotomy.
If the patient doubled the F-reticulocyte percentage, the patient
was eligible to an open label phase at the doses at which the effect
was seen. In this follow-up protocol, rHuEPO was administered
weekly and was not alternated with placebo.
Laboratory Procedures
Patients were monitored three times a week in group A and four
times a week in group B. Complete blood counts with differential
white blood cell counts were determined with a Coulter Counter
(Coulter, Hialeah, FL). Reticulocyte counts were performed in 2,000
cells using the brilliant cresyl blue method. For the determination of
red RBCs cells that contained HbF (F cells), the cells were washed
and resuspended in fetal calf serum. Smears made with these cells
were dried and fixed in a mixture of acetone, methanol, and ethanol
and reacted with murine anti-y-globin and a goat antimouse yG(ab'),
fragment conjugated to fluorescein isothiocyanate." F reticulocytes
were counted as previously described by Papayannopoulou et
HbF levels were determined by alkaline denaturation and RBC densities as previously described." Density gradients were performed as
described by Fahry et a1.I4Liver and renal function test and ferritins
were performed weekly. Baseline serum EPO was determined by ra-
Table 1. Hematologic Parameters of the Sample
Total
Retic
Patient
Cells
HbF
(%)
F
Retic
(%)
F
(%)
Iron
EPO'
(%)
Bili
Gg/dL)
(mU/d)
Creat
(mg/dL)
LDH
(U/L)
SUPPI
Group A
KM
AL
AJ
HL
KW
35/M
26/M
39/M
27/M
29/M
9.8
10.2
9.1
8.3
9.6
28.1
30.7
27.2
22.3
27.9
106.9
85.2
106.9
86.3
96.4
29.9
13.7
13.5
22.7
14.1
12.0
11.0
11.8
7.0
11.8
48.2
40.8
34.3
16.3
27.3
10.2
6.3
5.5
2.07
4.9
2.9
1.7
1.7
2.4
3.0
No
No
No
No
No
237
80
157
300
79
1.0
1.0
0.8
0.9
0.8
390
316
352
363
331
Group B
CW
IP
EP
JP
25/M
25/M
24/F
20/F
8.7
9.6
8.7
8.7
26.2
28.1
26.3
28.0
75.4
101.2
93.2
92.7
12.8
25.9
8.1
10.8
0.9
10.8
5.5
10.6
3.5
38.9
38.5
16.3
2.5
7.0
6.3
2.4
6.0
2.3
1.5
1.1
Yes
10
Yes
IO
185
92
63
54
0.9
0.8
0.4
0.5
493
385
232
237
Ferritin
Before
Ferritin
After
EPO
EPO
507
51
800
153
163
671
700
1,080
42.4
147
73
13,480
503
21,240
133
15,910
436
12,340
Abbreviations: Hct, hematocrit; Retic, reticulocytes; Bili, total bilirubin; Creat, creatine; LDH, lactic dehydrogenase levels; 10. iron overload
Pretrial plasma EPO levels.
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11
ERYTHROPOIETIN AND SS DISEASE
dioimmune assay (Smithmine Bioscience Laboratories, Evansville,
IN) and serum anti-EPO antibodies were determined by Ortho Pharmaceutical Corp (Raritan, NJ).
Criteria of Response
The doubling of the F-reticulocyte level after the administration
of rHuEP0 (particularly when appropriately delayed by 1 or 2 days)
was used as a criterion of response. This protocol was not designed
to result in an increase in HbF, nor were significant increases in HbF
levels expected to be seen. Its objective was limited to qualitatively
reproduce the baboon response to EPO in humans, that is, the increase
in F-reticulocytes secondary to EPO administration.
RESULTS
Group A Patients
None of the five patients in this group had a frank response
to rHuEPO administration, according to our established criteria, except that one patient reached the exact doubling of
F reticulocytes on one occasion. In most of the patients, there
were smaller but consistent increases in the F reticulocytes
in association with the administration of rHuEPO, but not
with placebo. In this group of patients that were not supplemented with iron before or during treatment, interesting
changes in femtin levels were observed. Serum femtins decreased in all patients and two patients developed frank iron
deficiency; coincidentally, with hypofemtinemia, they exhibited microcytosis and hypochromia (Table 1).
Group B Patients
Complete results were obtained in four of the five patients
recruited. Three were supplemented with 325 mg of ferrous
sulphate three times daily (TID) and the other two had iron
overload (due to previous chronic transfusion protocols) with
ferritin levels greater than 10,000. No significant decrease of
femtin was observed in these four patients (Table I). The
fifth patient was discontinued early in the protocol due to
medical indication for transfusion (acute chest syndrome).
All four patients had an F-reticulocyte response according
to our criteria. In three patients (examples are presented in
Figs 1 through 3), the increase in F reticulocytes was consistent
and was confirmed in an open label format in which the
administration of rHuEPO was made weekly, and not alternated with placebo. The fourth patient's (JP) F reticulocytes
also reached doubling (Fig 4), and an open label study was
attempted, but due to a cluster of painful crises, no reliable
data were collected. The F-reticulocyte response, as in baboons, was transient and occurred 1 to 3 days after rHuEPO
administration.
All four patients in group B had increased total reticulocytes
after rHuEPO administration, which slightly preceded the
maximal F-reticulocyte response.
Of the three patients that responded with a confirmed Freticulocyte increase, one (CW) had a significant increase in
F cells during the period of treatment (from 3.5% to 7.6%).
The others showed no change.
Dense cells were determined for five S S patients, one from
group A and four from group B. Only one patient (CW) exhibited an increase of dense cells, not accompanied with any
clinical symptoms. A decrease to steady-state levels was observed before the open label phase was finished and before
the last two doses of rHuEPO. The increase in dense cells
preceded painful crises, particularly on day 132 (see Discussion).
Of the three responders, one of them (IP) had six episodes
in which total Hb exceeded I .5 g/dL. In each of these episodes,
1 U (500 mL) was removed by phlebotomy. This patient had
a significant iron overload with a femtin level greater than
10,000 (Table 1).
-0
B
5
n
I
f
40
2 0
d
100
15
750
Fig 1. Double-blind phase of patient CW. In this
graph, F reticulocytes, total reticulocytes, HbF, F
cells, and dense cell determinations are depicted.
In the lower part of the diagram, the period of a
painful crises is depicted by a horizontal line, as well
as the days the patient received placebo (Pia) or
€PO.
25
0
i
p
0"
z
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12
NAGEL ET AL
30
25
20
--
I
0
15 ?
f5
9
X
IO
5
2
0
100
132
(10o0UlkgbLdD"kl
EPO
Crlsls
4
4 4 4 4 4
Fig 2. Open label phase of patient CW. Same
parameters as in Fig 1.
4 4
H
All patients in group A responded maximally with the lower
doses administered ( 1,000 U/kg BID per week). The size of
the response was comparable with each dose when measured
from the nadir of the placebo week.
Serial physical and laboratory examinations, and, in some
cases, patient diaries, gave no indication of clinical complications, except for the instances of iron deficiency in group
A. Seven painful crises were observed during the treatment
of the nine patients, two after rHuEPO administration and
five after placebo. Anti-rHuEPO antibodies were not detected
in any of the patients.
DISCUSSION
There are two important reasons to pursue alternatives to
hydroxyurea treatment aimed at enhancing HbF synthesis
in sickle cell anemia: (1) the concerns that exist that this
cytotoxic agent could represent long-term health hazard^'^;
and (2) the possibility of finding other agents that, in conjunction with a lower dose of hydroxyurea, could induce a
higher HbF response.
The results presented here show that the administration
of rHuEPO in high pulse doses can induce a transient increase
10
Days
Fig 3. Double-blind phase of patient EP. Same
parameters as in Fig 1.
t
w
t
EFU
t
w
t
EFU
t
w
t
EFU
-10
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ERYTHROPOIETIN AND SS DISEASE
Fig 4. Double-blind phase of patient JP. Same
parameters as in Fig 1.
in F reticulocytes in sickle cell anemia patients, similar to,
although less intense than, those previously found using a
similar protocol in the baboon? This is significant, because
experiments in baboons have a good record in predicting
human responses (if not quantitatively, at least qualitatively).
The F-reticulocyte response was commonly preceded by a
burst of all reticulocytes, as expected. These results also confirm the preliminary study of AI-Khatti et a1.I'
The results are at variance, nevertheless, with the report
of Goldberg et a1.I6 The lack of iron supplementation and
the infrequent monitoring of F reticulocytes might be responsible for their apparent negative findings.
The fact that the F-reticulocyte response was observed almost exclusively among patients supplemented with iron (or
in iron overload) raises the possibility that iron for erythropoiesis might be a limiting factor in rHuEPO response. While
iron deficiency is more common in sickle cell anemia disease
than previously tho~ght,~',~''
the diagnosisis difficult because
femtin levels are not a good predictor." In addition, the experience of Peterson et alZ0during the cyanate trials shows
that iron can be a limiting factor in erythropoietic response
in sickle cell disease.
No clinical complications were observed with the administration of rHuEPO in sickle cell anemia, and the doubleblind design of the study helped to put to rest the notion that
administration of high doses of rHuEPO could induce painful
crises, because more were observed associated with the use
of a placebo than the use of rHuEPO.
The only laboratory abnormality was the significant increase in dense cells observed in one patient (unaccompanied
by clinical symptoms), which disappeared before rHuEPO
was discontinued. The interpretation of this phenomena is
not clear, but it could be related to impending painful crises.
Ballas and Smithe2' have detected an increase in dense cells
preceding the appearance of crises, and have confirmed the
decrease in dense cells observed during painful crises, as re-
13
t
EPO
wt
EPO
t
p€PO
w
t
ported by Fabry and Nagel.'* Nevertheless, this observation
should be kept in mind and all patients be monitored for
dense cells in future studies.
We conclude that the administration of rHuEPO at high
doses and in a pulse regimen is capable of producing a transient F-reticulocyte response in sickle cell anemia, apparently
without any negative effects. The effect observed here in sickle
cell anemia is qualitatively similar to the response to pulse
doses of EPO in baboons: including the lack of HbF response.
The challenge is to find the dosage and protocol to obtain an
F-cell- and HbF-sustained response by rHuEPO alone or in
combination with other drugs, such as hydroxyurea.
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1993 81: 9-14
F reticulocyte response in sickle cell anemia treated with recombinant
human erythropoietin: a double-blind study
RL Nagel, E Vichinsky, M Shah, R Johnson, E Spadacino, ME Fabry, L Mangahas, R Abel and G
Stamatoyannopoulos
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