1. No category

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1995 85: 43-49
Oral sodium phenylbutyrate therapy in homozygous beta
thalassemia: a clinical trial
AF Collins, HA Pearson, P Giardina, KT McDonagh, SW Brusilow and GJ Dover
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Oral Sodium Phenylbutyrate Therapy in Homozygous p Thalassemia:
A Clinical Trial
By Anne F. Collins, Howard A. Pearson, Patricia Giardina, Kevin T. McDonagh, Saul W. Brusilow,
and George J. Dover
Butyrate analogues have beenshown t o increase fetal hemoglobin (HbF) production in vitroand in vivo. Sodium phenylbutyrate (SPB), an oral agent used to treat individuals
with urea-cycla disorders, has beenshown t o increase HbF
in nonanemic individuals and in individuals with sickle cell
disease. We have treated eleven patients with homozygous
/3 thalassemia (three transfusion dependent) and one sickle&thalassemia patient with 20 g/d (forty 500-mg tablets!of
SPB for 41 t o 460 days. All patients showed an increase in
the percent of F reticulocytes associatedwith treatment, but
only four patients responded by increasing their Hb levels
by greater than 1 g/dL (mean increase, 2.1 g/dL; range, 1.2
t o 2.8 g/dL). None of the transfusion-dependent thalassemia
subjects reapondod. Increase in Hb was associated with an
increase in red blood cell number (mean increase, 0.62 x
10’z/L), and mean corpuscular volume (mean increase, 6 fL).
Changes in percent HbF, absolute HbF levels, or Q- to non-
a-globin ratios as measured by levels of mRNA and globin
protein in peripheral blood did not correlate with response
to treatment. Response to treatment was not associated
with the
type of p-globin mutation, but baselineerythropoietin levels of greater than 120 mU/mL was seen in all responders and onlytwo of eight nonresponderst o SPB. Compliance
with treatment was greater than 90% as measured by pill
counts. Side effects of the drug included weight gain and/
oredemacaused by increase salt load in 2/12, transient
epigastric discomfort in 7/12, and abnormal body odor in 3/
12 subjects. Two splenectomized patients who were not on
prophylactic antibiotics developedsepsis while on treatment. We conclude that SPB increases Hbin some patients
with thalassemia, but the precise mechanism of action is
unknown.
6 1995 by The American Society of Hematology.
OMOZYGOUS 0 THALASSEMIA, a disease in which
there is inadequate production of 0 globin leading to
severe anemia, affects thousands of individuals worldwide.
Current management of this condition includes the use of
regular red blood cell (RBC)transfusions and iron chelation
therapy. The development of an effective therapy to increase
hemoglobin (Hb) levels in homozygous fJ thalassemia without the use of RBC transfusions could allow normal growth
and development, whereas decreasing or eliminating transfusional iron overload, which remains the major cause of death,
reduced life expectancy and morbidity in individuals with
this disease.’ Although bone marrow transplantation can
achieve these aims: it is not a therapeutic option for the
majority of patients.
For some years, there has been interest in increasing y globin transcription and fetal Hb (HbF) production in patients with ,B hemogl~binopathies.~.~
For patients with homozygous fJ thalassemia, increased y-globin production and a
reduction in the ratio of a- to non-a-globin could reasonably
be expected to ameliorate the seventy of the anemia. To this
end, trials of chemotherapeutic agents including 5-azacytidine3,5-7 and hydroxy~rea’,~.~
have been conducted, but myelotoxicity, fears of long-term carcinogenesis, and only modest responses to treatment have limited the clinical usefulness
of these agents. Erythropoietin has also been used, but responses to this therapy have been variable.L’*L1
There is considerable evidence that butyrate analogues
induce erythroid differentiati~n””~
and stimulate HbF production in human erythroid progenitors in v i t r ~ . In
~ ~vivo,
”~
these agents have also been shown to reactivate embryonic
globin production in an avian model,” delay the switch from
fetal to adult globin in ovine fetuses,” and to increase HbF
production in adult primates.17.20-22
In humans, several fatty acids including a amino-butyric
acid?’ arginine
isob~tyramide,~~~”
sodium phen y l b ~ t y r a t e ,propionic
~ ~ . ~ ~ acid:’ and 2-propylpentanoic (dipropylacetic) acid (unpublished data) have now been shown
to stimulate HbF production, suggesting that they may play
a role in the treatment of the p-globin disorders. However,
previous clinical trials of these agents in &thalassemia have
been limited to relatively short-term trials of the intravenous
agent, arginine b~tyrate,”.~~.~’
and oral i ~ o b u t y r a m i d e . ~ . ~ ~
Sodium phenylbutyrate is an orally administered agent
originally developed to promote waste nitrogen excretion in
the treatment of urea-cycle disorders3’ and is currently used
for this purpose in an Federal Drug Administration-approved
phase I11 trial. Over 100 patient-years experience with this
drug has now accumulated with no untoward effects being
found. The finding of increased HbF levels in these patients2*
stimulated clinical trials of sodium phenylbutyrate in patients
with P-hemoglobinopathies.
We report here the first long-term clinical trial of an orally
administered fatty acid, sodium phenylbutyrate, in patients
with homozygous p thalassemia. This represents the largest
clinical trial of any Hb switching agent used in thalassemic
patients to date.
H
Blood, Vol 85, No 1 (January l), 1995: pp 43-49
PATIENTS AND METHODS
This study was approved by the Joint Committee on Clinical
Investigation of the Johns Hopkins Medical Institutions and written
informed consent was obtained from all patients. Eleven patients
From The Johns Hopkins University School of Medicine, Baltimore MD; Yale University School of Medicine, New Haven CT; New
York Hospital-Cornell Medical Center NY; and the National Heart,
h n g , and Blood Institute, Bethesda MD.
Submitted June 28, 1994; accepted September 8,1994.
Supported in part by National Institute of Health Grants No.
HL 28028 (to G.J.D.), HD 11134, HD 26358 (to S.W.B.); Clinical
Research Center Grants No. RR-0035 and RR-00722; The Cooley’s
Anemia Foundation Inc; The Cooley ’S Anemia Foundation of Maryland Inc; and The Connecticut Campaign Against Cooley’s Anemia.
Address reprint requests to George J. Dover, MD, Ross Research
Bldg, Room 1125, 720 Rutland Ave, Baltimore, MD 21205.
The publication costs of this article were defrayed in parr 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 f a r .
8 1995 by The American Society of Hematology.
0006-4971/95/8501-0024$3.00/0
43
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COLLINS ET AL
44
Table 1. Clinical Details of Patients Studied
Prestudy Hb
Patient No.
Age lyrs)
l
32.7
26.5
20.5
32.2
36.7
20.6
42.3
30.5
24.8
26.9
25.1
35.7
2
3
4
5
6
7
8’
9
10
11
12
All patients had normal
0-Globin Mutations
Sex
M
F
F
F
F
M
F
F
M
F
M
F
LY
Codon 39P?
Homozyg IVS1, nt 110
Homozyg IVSl, nt 110
Codon 3911VS1, nt 1
Codon 39/IVSl, nt 1
Codon 39/IVSl, nt 6
IVSl, nt 6/??
Codon 39/FR 5/6
Homozyg IVSl, nt 110
Homozyg IVSl, nt 110
IVS1, nt 1/IVS2, nt 745
p8/IVS2, nt 745
(g/dL)
7.8t
6.0
6.9
7.4
6.5
8.1
4.5
5.5
Transfused
Transfused
Transfused
8.2
genes.
* Pretreated with 5-azacytidine.
t Transfused 6 and 3 weeks before study.
with homozygous p thalassemia and one patient with sickle p plus
thalassemia were studied. Of the 11 patients with homozygous p
thalassemia, 3 were receiving regular RBC transfusions and 8 were
not. Of these 8, 4 had never received regular transfusions, 3 had
discontinued regular transfusions because of severe RBC alloimmunization and one had discontinued transfusions to hasten the treatment of severe iron overload. All patients had previously undergone
splenectomy. Details of each patient’s age, p-globin mutations, a
gene number and prestudy Hb are shown in Table 1.
Patients 1 through 7 and 9 through 12 commenced sodium phenylbutyrate therapy during a 21-day inpatient stay in the Johns Hopkins
Hospital Clinical Research Unit. Patients 2 and 9 underwent the
protocolontwo occasions, recommencing sodium phenylbutyrate
after S9 and 440 days off treatment. All received 20 g/d of sodium
phenylbutyrate (10.6 to 13.8 g/m2/d) given as forty 500-mg tablets
in three divided doses: 12 tablets were given with breakfast and 14
tablets with each of lunch and dinner. All were discharged home on
medication and were followed from between 31 and 500 days as
outpatients. Five of these patients continue on medication.
Patient 8, unable to be transfused because of severe alloimmunization, was receiving intravenous infusions of 5-azacytidine every 3
to 4 weeks’ before starting sodium phenylbutyrate therapy. Her therapy was initiated at a lower dose of 12 g/d (9.6 g/m2/d) during a
21-day inpatient stay at the National Institutes of Health, Bethesda,
MD. The infusions of S-azacytidine were subsequently discontinued
and she continues on sodium phenylbutyrate alone.
All patients were documented to have normal RBC folate levels
(and serum folate in the case of regularly transfused patients) before
the commencement of therapy and received folic acid 1 mg/d orally
while on the protocol.
Blood counts were determined using Coulter STK-S or STK-R
counters (Coulter Immunology, Hialeah, FL) with manual spun Hb
measurements being performed to overcome any artifactual elevation
in automated Hb measurements caused by the presence of nucleated
RBCs in the peripheral blood.” A Technicon H-l counter was used
for the determination ofRBCmean corpuscular volume (MCV).
Reticulocyte counts were measured with a Sysmex R-1000.34Percentages of HbF, F cells, and F reticulocytes were measured as
previously de~cribed.’~
Globin-chain synthesis was measured by incubating peripheral blood (PB) with tritiated leucine using standard
methods36with globin-chain separation being achieved by high-performance liquid chromatography. PB ratios of a, p- and y-globin
mRNA were measured using an RNAase protection assay (RPA I1
kt; Ambion, Inc, Austin, TX). 32P-labeledRNA probes were made
complimentary to a 130-bp segment of exon 1 of a globin, a 205-
bp segment of exon 2 of p globin and a 170-bp segment of exon 2
of y globin. The protected fragments were separated by electrophoresis on an 8 mol/L urea6%polyacrylamide gel, located by autoradiography and quantitated by counting the radioactivity of each isolated
band in a scintillation counter. Levels of sodium phenylbutyrate,
phenylacetate, and phenylacetylglutamine were measured in plasma
and urine by previously described method^.^'
Data are expressed asmean ? standard deviation (range) and
were compared using the paired Student’s r-test. Differences between
observed and expected frequencies were compared using a chisquare test.
RESULTS
Changes in Hb. We divided the patients into two broad
categories, responders and nonresponders, based on changes
in Hb while on sodium phenylbutyrate therapy (Table 2). A
response was defined as a sustained rise in Hb of greater
than 1 g/dL. A sustained increase in Hb of 2.1 t 0.7 g/dL
(1.2 to 2.8g/dL) was seen in 4 of the 8 (50%) patients
with homozygous thalassemia noton regular transfusion
programs (Fig I). Patient 4 showed a progressive increase
in Hb over 350 days on therapy, with an acute episode of
anemia related to septicemia at day 200 during which she
was not transfused. Her Hb peaked at 10.2 g/dL on day 351
and then fell to around 9 g/dL when her dose of sodium
phenylbutyrate was reduced by 25% to 15 gld. Interestingly,
her sibling, patient 5, also responded to sodium phenylbutyrate therapy with an increase in Hb of 1.8 g/dL over the first
140 days on therapy. Patient 8, who was receiving intravenous infusions of 5-azacytidine at the time sodium phenylbutyrate therapy was commenced, has shown a progressive
increase in Hb over more than 450 days, interrupted by an
acute episode of anemia related to a major gastrointestinal
hemorrhage at day 220. She received two units of blood at
this time. Her Hb has continued to increase allowing the 5azacytidine infusions to be discontinued at day 280. Patient
3 showed an increase in Hb of 1.2
g/dL while on sodium
phenylbutyrate, andher Hb had returned to pretreatment
levels within 40 days after stopping therapy. Although this
increase in Hb is only modest, in the other patients responding to therapy, Hb continued to increase for 500 days
or more, suggesting that a trial of only 100 days of sodium
phenylbutyrate therapy mayhave been inadequate to achieve
the maximum possible response. Four of the eight nontransfused patients and all three previously regularly transfused
patients showed no response to therapy (Table 2, Fig 2).
F reticulocyteresponse.
All 12 patients showedanincrease in the percentage of F reticulocytes after the commencement of sodium phenylbutyrate therapy, with levels
at day 21 being 70% It 74% (mean +- 1SD; range, 6% to
248%) above baseline levels (P = .001). In all patients, the
increased level of F reticulocytes persisted as long as therapy
was continued. In five of eight patients who discontinued
sodium phenylbutyrate therapy, F reticulocytes decreased
rapidly, but in three, nos. 1, 6, and 11, the F reticulocytes
remained at the higher level for 4 weeks or more after the
cessation of therapy. Two patients, 2 and 9, again showed an
increase in F reticulocytes when therapy was recommenced.
These changes suggest that the increase in F reticulocytes
was directly related to the sodium phenylbutyrate therapy
(Fig 3 ) .
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THERAPY
IN 4 THALASSEMIA
PHENYLBUTYRATE
45
Table 2. Hb, RBC Count, and MCV Pro- and Post-Sodium Phenylbutyrate Therapy
Hb WdL)
Days
Treated
Pre
Patient No.
Post
Pre
Pre Post
Post
Erythropoietin
(mUlrnLl
MCV (fL)
RBC XlO''/L
Pre
LDH 1 U/L
Indirect Bilirubin
(rng/dL)
Pre
Day 21
Pre
Day 21
334
729
237
666
4.8
4.1
3.5
3.1
Nonresponders
'1
2
6
7
'
9
10'
11'
12
74
323
7.8
91
6.0
89
5.7
95
8.1
97
4.5
39t
9.1
31t
9.0
86t
9.4
71
8.4
68 3.62
443.748.1 8.2
5.1 2.872.95
5.8
2.42
6.1
2.27
3.00
6.1
5.0
2.55
7.9
3.10
8.8
3.15
6.8
3.25
5.0
2.90
85
2.43
2.38
2.77
2.5075
2.80a5
3.10
ND 90
1.71
88
87
80
73
86
3.9773
3.36
3.37
2.4997
66
86
76
93
96
185
81
85
90
80
86
88
84
88
96
ND
ND
ND
ND
ND
18
104
56
44
25
240
41
601
340
385
148
ND
ND
214
144
302
308
251
210
371
346
3.7
4.6
3.9
2.5
1.3
1 .o
3.0
1.7
1.7
2.5
0.9
1.4
0.9
166
3,882
207
682
3.54185.6
440
247
531
289
259
251
422
3.3
2.6
4.3
1.7
1.6
4.6
1.1
Responders
3
4*
5*
8*
100
490
137
468
6.9
7.4
6.5
5.5
8.1
10.2
8.3
8.3
3.63
2.58
2.61
1.91
94
83
Abbreviation: ND, not done.
Patient was transfused before commencing sodium phenylbutyrate.
t Patient was transfusedwhile on sodium phenylbutyrate therapy.
Patient continues ontherapy.
*
nor was there any correlation between the change in globin
mRNA ratios and peripheral Hb levels. Similarly, when repeated in three patients after more than 270 days on therapy,
no correlation between ratios of globin mRNA and change
in Hb could be shown. In addition to mRNA studies, in three
patients, the production of a- and non-cy-globin chains in
PB reticulocytes was measured at day 0 and at day 21. Again,
in these patients, neither ratios of globin mFWA nor globinchain synthesis seemed related to changes in Hb on therapy.
Patient 12, with sickle p plus thalassemia, did not experience an increase in Hb and, therefore, was called a nonresponder. However, over the first 21 days of therapy, she did
show an increase in F reticulocytes (14% to 34%) and F
cells (13% to 20%) associated with an increase inHbF%
Hemoglobin F response. Table 3 outlines the HbF response to sodium phenylbutyrate therapy in each patient, as
measured by percentage of HbF, absolute HbF g/&, and cy/
non-a ratios of both globin mRNA and globin-chain synthesis. In those patients responding to therapy, increase in total
Hb was not solely explained by increased HbF.In those
patients previously on regular transfusion programs, at termination of therapy both HbF% and absolute HbF levels were
higher than at baseline, probably reflecting the reduced suppression of erythropoiesis related to fall in Hb and not being
necessarily directly attributable to the sodium phenylbutyrate
therapy. Ratios of a-to non-a-globin mRNA, measured before therapy and again at day 21 in all patients, showed no
uniformity of response to sodium phenylbutyrate therapy,
11
Fig 1. Hb in the four patient. responding to aodium phenylbutyrate therapy: patient
4 (upper left),
patient 5 (upper right), patient 8 (lower left), and
patiant 3 (lower right). IN), sodium phenylbutyrate
thwapy, with changoa in the size of the bar coneaponding to &angm in dose. (*), intravenous infusions of C-azacytidine.
1
#B
3
-200
-lw
0
100
m
DAYS ON SPB
m
m
5w
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COLLINS ET AL
46
4.51
0
10
20
30
40
50
60
70
80
DAYS ON SPB
Fig 2. Hb in two patients who did not respond to therapy: patient
2 l*), who is not regularly transfused and patient 11 (01, who is
transfusion-dependent.
(3.1% to 5.0%). This response to therapy is very similar to
those previously seenin patients with homozygous sickle
cell di~ease.’~
In sickle thalassemia, an increase in the percentage of HbF is probably a more desirable outcome than
increase inHb. Interestingly, while on therapy, her MCV
increased from 67 fL to 74 fL and returned to its pretreatment
value when sodium phenylbutyrate therapy was discontinued.
Indicators of hemolysis. The nine patients not on regular
transfusion programs showed indirect evidence of a reduction in hemolysis, with significantly lower levels of serum
lactate dehydrogenase ( P < .03) and indirect bilirubin ( P =
.OOO5)when pretreatment levels were compared with those
at day 21 (Table 2). This was not observed in the transfusiondependent patients, possibly related to the mean fall in Hb
of 2.0 g/dL, which occurred in these patients during the 21day inpatient study period. In contrast, no significant difference was seen in Hb between baseline measurements and
day 21 in those patients who were not regularly transfused.
Predictors of increased Hb in response to sodium phenylbutyrate therapy. Response to sodium phenylbutyrate therapy, as defined by a sustained increase in total Hb of greater
than 1 g/dL above baseline, did not appear to be predicted
by P-globin mutation; baseline percentage of HbF, absolute
HbF, or F-reticulocyte levels; or baseline Hb or baseline ato non-a-globin ratios. Similarly, significant falls in lactate
dehydrogenase and indirect bilirubin, traditional measures
of hemolysis, could be shown in all nontransfused patients
with no differences being observed between responders and
nonresponders. Interestingly, those patients with baseline
erythropoietin levels greater than 120 mU/mL were significantly ( P < .05) more likely to experience an increase in
Hb (4/6) than those whose baseline erythropoietin level was
below 120 mU/mL (0/6) (Table 2). A similar trend existed
between baseline HbF percentage in those patients not receiving regular RBC transfusions and response to sodium
phenylbutyrate therapy, although this did not reach statistical
significance. Of the four patients with baseline HbF less than
40%, none responded to therapy. In contrast, four of the five
patients with baseline HbF greater than 40% did respond (P
= ,075).
Compliancewith sodium phenylbutyratetherapy.
Sodium phenylbutyrate tablets wereprovidedtothe
patients
with a 25-day supply each time; a further supply was provided only when the patient specifically requested more tablets. In this way, compliance was calculated for each patient
by comparing the number of tablets dispensed to that prescribed. Compliance with therapy wasa problem in only one
patient, no. 3, who abruptly discontinued therapy after 100
days, having been 95% compliant up until that time. For the
patients as a group, compliance with medication was 97%
? 3%.
PhenyZbuQrate pharmucokinetics. Peak daytime levels
of phenylbutyrate, phenylacetate, and phenylacetylglutamine
ranged between 0.60 and 1.70 mmol/L, 0.50 and 1.SO mmol/
L, and 0.56 and 2.67 mmol/L, respectively. Serial-fasting
morning plasma levels of phenylacetate, measured in all patients, were less than l .O mmoln and showed no progressive
accumulation. Serial 24-hour urine collections performed in
9 of the 12 patients studied showed a mean excretion of
76% 2 13% (53% to 97%) of the molar amount of sodium
phenylbutyrate administered as urinary phenylacetylglutamine. Plasma glutamine levels, measured before therapy and
again after 2,9, and 21 days on therapy showed no evidence
of glutamine depletion. These results are similar to those
reported in patients with urea-cycle disorders32.”and homozygous sickle cell disease29treated with sodium phenylbutyrate.
Adverse events occurring on therapy. The daily dose of
20 g of sodium phenylbutyrate contributes 2,460 mg (107
mmol) of sodium to the diet, a significant proportion of the
recommended daily intake. While in the hospital, one of the
twelve patients (no. 6) developed ankle edema associated
with a 3.5% increase in body weight, which resolved spontaneously with dietary modification. After discharge from hospital, one patient (no. 1) required intermittent treatment with
a thiazide diuretic and one (no. 8) required an increase in
her previous diuretic dose to control peripheral edema. No
patient developed hypertension. Epigastric discomfort after
80
70
-
60 50
-
F RETICSX
40 -
30
-
20 -
b
10
PRE
DAY 21
POST
RESTART
STOP
Fig 3. The percentage of F reticulocytes in individual patients before sodium phenylbutyrate therapy (PRE), after 21 days of therapy
(DAY 211, at least 2 weeks after the cessation of therapy (POST),
on restarting therapy (RESTART). and after again stopping therapy
(STOP). 1.,
responders; (0).
nonresponders.
From www.bloodjournal.org by guest on November 18, 2014. For personal use only.
THERAPY
PHENYLBUTYRATE
IN
p
THALASSEMIA
47
Table 3. Percent HbF, Absolute HbF, and cu/Non-a Ratios Pre- and Post-Sodium Phenylbutyrate Therapy
AbsHbF (g/dL)
HbF (%)
Patient No.
Pre
Post
Pre
Post
a-/Non-uRatio
Globin
alNon-rr Ratio
mRNA (mean r SD)
Pre
Day 21
Day 270
Pre
2.6 t 0.3
1.0 ? 0.1
2.0 t 0.3
Post
Hb Increase Cg/dU
Nonresponders
13
71
76
21
6
7
11
1
9’
2
2
10’
11’ 2.03.01.1 59 13
3.1
12
l*
2
27
75
86
1.7
1.4
1.0
1.7
4.3
4.3
5.2 4.4
50
2.9
3.2
1.8
14
3.9
0.6
0.5
22.7 0.2 0.1
1
0.2
1.8
5.0 2.90.40.3
f 0.2
2
0.1
t 0.1
0.1
t 1.9
1.8 t 0.2
t 0.2
t 0.3
f 0.8
1.8 ? 0.1
3.1 ? 0.4
1.9 t 0.1
2.6 t 0.2
2.2 t 0.5
0.7 f 0.1
1.7 t 0.1
1.5 f 0.1
-t 0.2
2 0.1
2 0.2
1.0 t 0.1
.3
2.6 f 0+2.8
+1.8
2.0 -c 0.1
+2.8
2
0.1
0.2
2.6
3.1
1.6
1.5
-2.7
-0.2
f0.4
-2.0
+0.5
-1.n
-03
-2.6t
-3.4
-0.1
Responders
4.7 3 3.9
4
5
8
58
57
90
86 85
83 82
84
1.7
8.6
7.5
2.47.55.5
1.5 6.7 4.9
+1.2
1.7 2 0.1
2.1
2.2 2 0.1
2.2
* Patient was transfused before commencing
sodium phenylbutyrate.
t Patient was transfused while on sodiumphenylbutyrate therapy.
the ingestion of the tablets was the most common adverse
effect, being reported by seven of the twelve patients. Two
patients, both splenectomized and not on regular penicillin
prophylaxis, had nonfatal episodes of bacterial septicemia:
patient 4 developed infection with Streptococcus pneumoniae at day 71 and Plesiomoms shigelloides at day 200, and
patient no. 6 developed infection with Staphylococcus epidermidis related to a indwelling central venous catheter at
day 24. Patient 8 suffered a hemorrhage from a gastric ulcer
at day 220 soon after the commencement of aspirin therapy
for long-standing pulmonary hypertension. Patient 1, who
had spun Hb levels between 5.1 and 7.5 g
/
&
associated with
marked erythroblastosis, developed spinal cord compression
requiring irradiation at day 323 and ceased sodium phenylbutyrate therapy. Patient 10 developed a deep venous thrombosis at day 28 with a Hb 5.9 g/dL. Three patients experienced
bad body odor while on therapy, which in one, no. 12, caused
her to be unable to tolerate the medication long-term, even
at half the usual dose. These complaints are probably related
to the in vivo @ oxidation of phenylbutyrate to phenylacetate,
a compound with an offensive odor secreted as a defense
mechanism by the stinkpot turtle.3s
DISCUSSION
This study shows that sodium phenylbutyrate can safely
be administered to patients with homozygous @ thalassemia
and is well tolerated by the majority. The need to take 40
tablets daily, epigastric discomfort, and the body odor created in some patients are problematic. Poor compliance with
this regimen, based on previous experience with this drug,
was expected to be a frequent
but surprisingly
was not, possibly related to the fact that many of these patients had hadprior experience with other cumbersome medical interventions including transfusion schedules and iron
chelation therapy. However, the oral route of administration
has clear advantages over the intravenous route needed for
arginine butyrate, particularly because all the evidence available suggests that in the management of the @ hemoglobinopathies, these therapies, if effective, will be needed long-term.
We found that 36% (4/11) of all patients or 50% (418)of
nontransfused patients responded to sodium phenylbutyrate
when a response was defined asa sustained increase in Hb of
more than 1 gjdL over pretreatment values. Clearly, sodium
phenylbutyrate can increase Hb in some patients with homozygous @ thalassemia, but is not effective in all of them.
Although it seems evident that @-globinmutation alone does
not predict response, the fact thattwo siblings treated in
this study both responded to sodium phenylbutyrate therapy
raises the possibility that some other genetic factor is involved. Other genetic factors linked and unlinked to the @globin locus have been shown to effect HbF levels in normal
individuals and patients with @ hemoglobin~pathies.~~.~~
The failure of Hb to increase in patients showing a decrease in levels of lactate dehydrogenase and indirect bilirubin is disappointing and raises interesting questions as to the
cause of these changes if not related to decreased hemolysis.
Similarly, we have observed increased production in F reticulocytes in all patients treated with this agent to date and
the persistence of levels of F reticulocytes higher than baseline in some patients up to a month or more after the cessation of therapy with an agent known to be rapidly metabolized and excreted. Similar observations have been reported
after the use of arginine b~tyrate.’~.’~
This uniformity of Freticulocyte response, persistence of response in some patients long after the cessation of therapy, and lack of correlation between changes in F reticulocytes and increased total
Hb or increased absolute HbF production may indicate suboptimal increases in HbF insufficient to decrease ineffective
erythropoiesis.
The lack of correlation between changes in a- to non-
From www.bloodjournal.org by guest on November 18, 2014. For personal use only.
COLLINS ET AL
48
a-globin ratios, measured both as mRNA or globin-chain
synthesis, and response to therapy raises more questions as
to the mechanism of action of these fatty acid compounds.
In this study, we were unable to show a correction in a to
non-a ratios in patients experiencing increases in Hb and
observed afallintotal
Hb in one nontransfusedpatient
whose HbF productionclearly increased. The recent demonstration of induction of a - as well as y-globin production in
butyrate-treatedtransgenic mice (G. Stamatoyannopoulos,
personal communication, December 1993) may help explain
some of these observations and suggests that viewing these
compounds as selective inducers
of y-globin production may
have been premature. Similar data exists concerning theuse
of intravenous argininebutyrate, which was initially reported
as a selective stimulator of the human y-globin gene promoter capable of correcting a - to non-a-globin-chain imbalance in patients with thala~semia.'~Although early reports
linking correction of globin-chain imbalance to subsequent
increase in Hb with arginine butyratewere encouraging,
these changes have notbeenreproduced
in other patients
showing a response to therapy.z5 Similarly, the slow nature
of the response to therapy reported in the current study, with
gradual increases in Hb over 200 to 500 days on therapy is
not consistent with a mechanism invoking rapid correction
of globin-chain ratios over a few days or weeks.
We observed an inconsistent response to therapy, a decrease in traditional indicators of hemolysis in all nontransfused patients that were not predictive of an increase in Hb,
and increases in Hb not entirely explained by increased HbF
in those patients who did respond to therapy. This suggests
that classic Hb switching,an increase in y-globin production
with resultant decrease in globin-chain imbalance, could not
explain the increases in Hb seen. Three possible explanations
exist: sodium phenylbutyrate (1) caused nonspecific induction of all globin production,ie, a , p, y , andnotjust y
alone; (2) caused nonspecific expansion of RBC mass by
the release of thalassemic RBCs previously sequestered in
the marrow or by an increase in production of thalassemic
RBCs; or (3) causeda prolongation of RBC survival without
a change in RBC production. There is evidence to support
the first of these (G. Stamatoyannopoulos, personal communication, December 1993) and the latter two lead to testable
hypotheses in further patients.
The positive correlation between baseline serum erythropoietin level and the likelihood of responding to therapy is
very interesting. This observation,together with the fact that
erythropoietin levels in homozygous P thalassemia are generally elevated, but inappropriately so for the degreeof anemia, suggests that clinical trials ofcombination therapy using
erythropoietin with sodium phenylbutyrate may be of value.
However, it mustbe rememberedthat in these patients,erythropoietin levels are related to other factors, such as baseline
HbF%41 and as such, erythropoietin may only be a marker
of some other factor affecting response.
It is also of interest that an increasing number of structurally related compounds are being shown to
increase HbF
production in vitro and in vivo in animals, hematologically
normal individuals, and patients with P hemoglobinopathies.
Most recently, we have discovered that valproic acid, a drug
used for thetreatment of epilepsyforthe past 20 years,
causes increased HbF production in nonanemic individuals4'
just as sodium phenylbutyrate was shown to increase HbF
productioninpatientswith
urea-cycle disorders."Adding
to theintrigueisthe
fact that the compounds reported to
date sharea relatively small part of their structure in common
and appear to be effectiveat vastly different molar dosages.
Although mechanistically intriguing, and clearly of value
to a selected group of patients, butyrate and other fatty acid
compounds remain far from being a panacea for all patients
with p hemoglobinopathies. The pharmacologic manipulation of HbF remains aninexact and as yet unproven therapy
for the P-globin disorders.
ACKNOWLEDGMENT
We thank Dr A.W. Nienhuis
for patient referral and helpful discussion; Dr H.E. Witkowskn for performanceof the globin-chain analysis, C.D.Boehm for a- and,&globinDNA analysis; A.C.Mayes
and S.H. Purvis for F-cell and F-reticulocyte assays; and
Dr T.R.
Bishop and L. Frelin for help with the RNAase protection assays.
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