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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 370-375
HBED: The Continuing Development of a Potential Alternative to
Deferoxamine for Iron-Chelating Therapy
By
Raymond J. Bergeron,
Jan Wiegand, and
Gary M. Brittenham
From the Department of Medicinal Chemistry, University of Florida,
Gainesville, FL; and the Department of Medicine, MetroHealth Medical
Center, Case Western Reserve University, Cleveland, OH.
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ABSTRACT |
To further examine the potential clinical usefulness of the
hexadentate phenolic aminocarboxylate iron chelator
N,N -bis(2-hydroxybenzyl)ethylenediamine-N,N-diacetic acid (HBED) for the chronic treatment of transfusional iron
overload, we performed a subchronic toxicity study of the HBED
monosodium salt in rodents and have evaluated the iron excretion in
primates induced by HBED. The HBED-induced iron excretion was
determined for the monohydrochloride dihydrate that was first dissolved
in a 0.1-mmol/L sodium phosphate buffer at pH 7.6 and administered to
the primates either orally (PO) at a dose of 324 µmol/kg (149.3 mg/kg, n = 5), subcutaneously (sc) at a dose of 81 µmol/kg (37.3 mg/kg, n = 5), sc at 324 µmol/kg (n = 5), and sc
at 162 µmol/kg (74.7 mg/kg) for 2 consecutive days for a total dose
of 324 µmol/kg (n = 3). In addition, the monosodium salt of HBED in
saline was administered to the monkeys sc at a single dose of 150 µmol/kg (64.9 mg/kg, n = 5) or at a dose of 75 µmol/kg every
other day for three doses, for a total dose of 225 µmol/kg (n = 4).
For comparative purposes, we have also administered deferoxamine (DFO) PO and sc in aqueous solution at a dose of 300 µmol/kg (200 mg/kg). In the iron-loaded Cebus apella monkey, whereas the PO
administration of DFO or HBED even at a dose of 300 to 324 µmol/kg
was ineffective, the sc injection of HBED in buffer or its monosodium
salt, 75 to 324 µmol/kg, produced a net iron excretion that was
nearly three times that observed after similar doses of sc DFO. In
patients with transfusional iron overload, sc injections of HBED may
provide a much needed alternative to the use of prolonged parenteral
infusions of DFO. Note: After the publication of our previous
paper (Blood, 91:1446, 1998) and the completion of the studies
described here, it was discovered that the HBED obtained from Strem
Chemical Co (Newburyport, MA) that was labeled and sold as a
dihydrochloride dihydrate was in fact the monohydrochloride
dihydrate. Therefore, the actual administered doses were 81, 162, or
324 µmol/kg; not 75, 150, or 300 µmol/kg as was previously
reported. The new data have been recalculated accordingly, and the data
from our earlier study, corrected where applicable, are shown in
parentheses.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
BECAUSE THERE IS NO mechanism for the
excretion of iron, patients with transfusion-dependent chronic
hemolytic anemias will ultimately develop toxic iron overload, and
without adequate chelation therapy, will frequently die in their second
decade.1-3 For the past 30 years, deferoxamine B (DFO),
produced in large-scale fermentation by a strain of Streptomyces
pilosus,4 has been regarded as the drug of choice for
the treatment of transfusional iron overload. DFO mesylate
(Fig 1) has been shown to be a generally safe and efficacious means of controlling body iron that can prolong survival and prevent or ameliorate organ dysfunction.3,5-9 Although the drug's efficacy and long-term tolerability have been well
documented, there are still problems associated with treatment with
DFO. Because DFO is poorly absorbed from the gastrointestinal tract and
rapidly eliminated from the circulation, prolonged parenteral infusion
is needed; effective therapy usually requires subcutaneous (sc) or
intravenous administration by a portable infusion pump for 9 to 12 hours daily.10-12
Although the experience with DFO has shown that adequate control of
body iron can avert complications of transfusional iron overload,
drug-related toxicities13-19 and patient compliance have prompted an ongoing search for safe, inexpensive alternatives to DFO.
To date, most efforts to develop substitutes for DFO for iron-chelating
therapy have concentrated on bi- or tridentate iron-chelating agents
that remain active after oral administration.20,21 However,
because of concerns that these partial ligands might exacerbate iron
toxicity and the realization that oral formulations are likely to
require taking a large number of tablets on multiple occasions each
day, we have examined the possibility of the administration of a
hexadentate iron chelator,
N,N-bis(2-hydroxybenzyl)ethylenediamine-N,N-diacetic acid22 (HBED) by sc injection. HBED is a synthetic
hexadentate ligand that, like DFO, forms a 1:1 complex with iron with
high affinity and selectivity. Because HBED is a synthetic product, problems of local reactions to potential fermentation by-products not
removed during purification would be avoided. For patients allergic to
DFO,23-25 HBED, a member of a different family of
chelators, would be unlikely to provoke a similar response.
In a recently published study,26 we showed that HBED
administered sc to iron-loaded monkeys consistently induced iron
clearance that was two to three times greater than that induced by a
similar dose of DFO. No adverse effects of chelator administration were noted in the monkeys; all hematologic and biochemical tests remained within normal ranges. On the basis of those findings, we have now
expanded the preclinical investigation of the compound in both rodent
and primate models. In the studies described here, we have performed a
2-week toxicity study in rodents and have assessed the iron excretion
induced by additional dosages and formulations of both orally (PO) and
sc administered HBED to monkeys. Based on the data available thus far,
sc HBED has emerged as a strong candidate for a much needed alternative
to DFO and could potentially provide patients with a clinically
effective form of iron-chelating therapy.
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MATERIALS AND METHODS |
Materials.
DFO in the form of the methanesulfonate salt, Desferal, was obtained
from Ciba-Geigy Ltd (Basel, Switzerland). HBED monohydrochloride dihydrate (Fig 1) was obtained from Strem Chemical Co (Newburyport, MA). Conversion to its monosodium salt (Fig 1) was performed by SRI
International (Menlo Park, CA). Sprague-Dawley rats (Crl:CD[SD]BR-CD) were purchased from Charles River (Wilmington, MA). Cebus apella monkeys were obtained from World Wide Primates (Miami, FL). All reagents and standard iron solutions were obtained from Aldrich Chemical Co (Milwaukee, WI). Atomic absorption (AA) measurements were
made on a Perkin-Elmer model 5100 PC (Norwalk, CT). Ultrapure salts
were obtained from Johnson Matthey Electronics (Royston, UK). All
hematologic and biochemical studies27 were performed by
Antech Diagnostics (Tampa, FL). Histological evaluation of necropsy
tissues was performed by Florida Animal Resources (Ocala, FL).
Rodent toxicity studies.
Male Sprague-Dawley rats averaging 450 g were housed in polycarbonate
cages with Beta-chips (Northeastern Products Corp, Warrensburg, NY)
provided as bedding. Before the first drug administration, the rats were weighed and evaluated for their general condition. To
allow for better visualization of the injection sites, the dorsal
region of each rat was shaved free of hair. The injection sites were
rotated and were closely monitored for any signs of inflammation or
irritation. The HBED monosodium salt was put into solution with sterile
normal saline and filtered via a 0.2-µ syringe filter. The drug was
administered at a concentration of 60 mg/mL, and the solution had an
unadjusted pH of 7.3. The rats (n = 5/group) were administered the HBED
at a dose of 75, 150, or 300 µmol/kg (32.4, 64.9, or 129.7 mg/kg,
respectively) as a single sc injection every other day for 14 days (7 doses). Control rats were administered saline by sc injection. Before
each drug administration, the rats were weighed and evaluated for their
general condition. Food and water intake were determined, and a
necropsy was performed 1 to 2 days after the last dose of the drug had
been administered. Extensive tissues, obtained from all animals,
included the following: adrenal gland, aorta, bone marrow, brain, eye,
esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum,
testicle, heart, kidney, liver, lung, mesenteric lymph node, pancreas,
prostate, mandibular salivary gland, skeletal muscle, spleen, thymus,
thyroid, trachea, bladder, and skin from injection and noninjection
sites. The tissues from the control animals and the high-dose (300 µmol/kg) group were examined by an outside pathologist.
Iron loading of C apella monkeys.
The monkeys were iron overloaded as previously described to provide
about 500 mg iron/kg body weight.28 After administration of
iron dextran, the serum transferrin iron saturation rose to between 70% and 80%. The serum half-life of iron dextran in humans is
2.5 to 3.0 days.29 We waited at least 20 half-lives, 60 days, before using any of the animals in experiments evaluating
iron-chelating agents.
Iron-balance studies in C apella monkeys.
Seven days before the administration of the drug, the animals were
placed in metabolic cages30 and started on a low-iron liquid diet.27 The monkeys were maintained on the low-iron
liquid diet for the duration of the experiment. They were administered food according to their body weight, and intake was very carefully monitored.
The total amount of iron intake was compared with the total amount of
iron excreted. Net iron balance = dietary iron intake  (urinary + fecal iron excretion); animals in a negative iron balance are
excreting more iron than they are absorbing.
Primate fecal and urine samples.
Fecal and urine samples were collected at 24-hour intervals. The
collections began 4 days before the administration of the test drug and
continued for an additional 5 to 7 days after the first dose of the
drug was administered. Fecal samples were assayed for the presence of
occult blood, weighed, and mixed with distilled deionized water and
autoclaved for 30 minutes. The mixture was then freeze-dried, and a
known portion of the powder was mixed with low-iron nitric acid and
refluxed for 48 hours. Once any particulate matter in the digested
samples was removed by centrifugation, iron concentrations were
determined by flame AA. Monkey urine samples were acidified and
reconstituted to initial volume after sterilization, if necessary.
Drug preparation and administration.
Rodents were administered the HBED monosodium salt in saline at a
concentration of 60 mg/mL. The rats (n = 5/group) were administered the
HBED at a dose of 75, 150, or 300 µmol/kg (32.4, 64.9, or 129.7 mg/kg, respectively) as a single sc injection every other day for 14 days (seven doses). In the primates, DFO was administered PO or sc in
sterile water for injection at a dose of 300 µmol/kg (129.7 mg/kg).
The HBED monohydrochloride dihydrate was first dissolved in a phosphate
buffer and administered (1) PO at a dose of 324 µmol/kg (149.3 mg/kg,
n = 5), (2) sc at a dose of 81 µmol/kg (37.3 mg/kg, n = 5), (3) sc at
162 µmol/kg (74.7 mg/kg) for 2 consecutive days for a total dose of
324 µmol/kg (n = 3), and (4) 324 µmol/kg as a single sc injection
(149.3 mg/kg, n = 5). In addition, the monosodium salt of HBED in
saline was administered to the monkeys sc at a single dose of 150 µmol/kg (64.9 mg/kg, n = 5) or at a dose of 75 µmol/kg (32.4 mg/kg)
every other day for three doses, for a total dose of 225 µmol/kg (n = 4). Before each drug administration the monkeys were anesthetized with
Telazol (Elkins-Sinn, Inc, Cherry Hill, NJ), 0.03 mg/kg
intramuscularly (IM), and given a single injection of
atropine, 0.1 mg/kg IM.
Calculation of iron chelator efficiency.
The efficiency of each chelator was calculated on the basis of a 1:1
ligand-iron complex. For animals administered a single dose, the
numbers were generated by averaging the iron output for 4 days before
the administration of the drug, subtracting these numbers from the
2-day iron clearance after the administration of the drug, and then
dividing by the theoretical output; the result is expressed as a
percentage. If two or more doses were administered, the efficiency was
calculated by averaging the iron output for 4 days before the
administration of the drug, subtracting these numbers from the daily
iron clearance after the administration of the drug, and then dividing
by the theoretical output; the result is expressed as a percentage.
Statistical analysis.
Data are presented as the mean ± standard error of the mean. For
comparisons of the means of two groups, the two-sample t-test (without the assumption of equality of variances) was used for analyzing the primate data. All tests were two-tailed, and a
significance level of P < .05 was used.
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RESULTS |
Rodent drug toxicity.
A subchronic toxicity study of the monosodium salt of HBED was
performed in normal rodents. The animals were administered the drug as
a single sc injection at a dose of 75, 150, or 300 µmol/kg every
other day for 14 days (seven doses). Control animals were administered
sc injections of saline. At the doses investigated, no toxicity was
noted in any of the animals. All the animals ate and drank well and
gained weight at a rate that was indistinguishable from the control
animals. In addition, no erythema was noted at any of the injection
sites, either grossly or histologically. At necropsy, all tissues (see
above list) appeared grossly normal; histological evaluation of tissues
from HBED 300 µmol/kg-treated versus control animals showed no
drug-related abnormalities.
Chelator-induced iron excretion in C apella monkeys.
The studies were conducted with C apella monkeys who had been
administered intravenous iron dextran to provide about 500 mg iron/kg
body weight.28 We recently reported that DFO administered sc in aqueous solution at a dose of 150 µmol/kg was found to have an
efficiency of 5.1% ± 1.3% and induced the excretion of 435 ± 115 µg iron/kg body weight.26 About 65% of the
chelator-induced iron excretion was in the stool and about 35% in the
urine. For comparative purposes, we have now also administered DFO in
aqueous solution both PO and sc at a dose of 300 µmol/kg
(Table 1). When DFO is administered PO at
300 µmol/kg, very little iron is excreted in either the urine or the
feces (Fig 2). The efficiency of the drug
by this route is only 0.1% ± 0.4%. However, when the drug is
administered sc at 300 µmol/kg, it induced the excretion of 716 ± 244 µg/kg of iron (Fig 2) and had an efficiency of 4.2% ± 1.4%.
The majority of iron, 60%, was excreted in the feces, and about 40%
was excreted in the urine.
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| Fig 2.
Mean net iron excretion in C apella monkeys with
iron overload (see text) after administration of DFO or HBED. The
primates were administered (A) DFO 300 µmol/kg PO, (B) HBED 324 µmol/kg PO, (C) DFO 150 µmol/kg sc, (D) HBED 150 µmol/kg sc, (E)
DFO 300 µmol/kg sc, (F) HBED 324 µmol/kg sc, (G) HBED 81 µmol/kg
sc, (H) HBED 75 µmol/kg sc for three doses (225 µmol/kg total), and
(I) HBED 162 µmol/kg sc for two doses (324 µmol/kg total). Animals
in groups B, F, G, and I received the HBED monohydrochloride dihydrate
in 0.1 mmol/L sodium phosphate buffer at pH 7.6, whereas those in
groups D and H were administered the HBED monosodium salt in saline at
pH 7.3. Excretion is shown as µg iron/kg body weight on the scale of
the left vertical axis. For comparative purposes, the result of our
previously published study26 of the sc administration of
DFO 150 µmol/kg to C apella monkeys with a similar magnitude
of iron overload is shown and is indicated by an asterisk.
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Because of its poor solubility, the HBED monohydrochloride dihydrate
was first dissolved in a 0.1-mmol/L sodium phosphate buffer adjusted to
pH 7.6 (no Cremophor vehicle was used) and was
administered to the primates as described in Materials and Methods.
Although HBED administered PO at a dose of 150 µmol/kg was
ineffective, only 0.5% ± 0.5% efficient,31 we had
hoped that increasing the dose to 324 µmol/kg would result in the
excretion of a clinically beneficial amount of iron. Unfortunately,
very little iron was excreted (Fig 2); the efficiency of the drug at this dose was only 0.3% ± 0.3%. However, the sc injection of HBED in a buffer at a dose of 81 µmol/kg did result in the excretion of a
significant amount of iron. In this case, the drug induced the
excretion of 608 ± 175 µg/kg of iron (Fig 2) and had an
efficiency of 13.0% ± 4.6% (Table 1). This is very similar to
what was observed when HBED-Cremophor was administered sc to the
primates at this same dose26; HBED-Cremophor induced the
clearance of 793 ± 410 µg/kg of iron and had an efficiency of
18.4% ± 9.1% (17.5% ± 9.1%) (P > .3).
In both experiments, the majority of the iron was excreted in the
feces 78% when the drug was administered in buffer and 92% when it
was administered in Cremophor.
To more closely mimic potential clinical applications, we have also
performed a study in which the HBED monohydrochloride dihydrate in
buffer was administered sc at a dose of 162 µmol/kg on 2 consecutive
days for a total dose of 324 µmol/kg (Fig
3). In this study, the first dose of the drug induced the excretion of
820 ± 215 µg/kg of iron with a 24-hour efficiency of 9.1% ± 2.4%. The injection of the same dose of the drug on the following day
induced the excretion of an additional 978 ± 213 µg/kg of iron
with a 24-hour efficiency of 10.8% ± 2.4%. The total iron excreted over the 2-day period was 1,798 ± 210 µg/kg of iron (Fig 2) with an overall efficiency of 9.9% ± 2.1% (Table 1). These values are very similar to what is observed when the drug is
administered as a single dose of 324 µmol/kg; in this case, the iron
excretion is 2,400 ± 808 µg/kg of iron (Fig 2) with an efficiency
of 13.3% ± 4.5% (P > .2). In each experiment, the
majority of the iron was excreted in the feces, 65% to 75%, with the
remainder being excreted in the urine.
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| Fig 3.
Urinary and fecal iron excretion (µg/kg) induced by the
sc administration of HBED monohydrochloride dihydrate in 0.1 mmol/L
sodium phosphate buffer at pH 7.6 at a dose of 162 µmol/kg on 2 consecutive days for a total dose of 324 µmol/kg. The baseline iron
levels in the urine and stool have not been subtracted.
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Because the poor solubility of the HBED monohydrochloride dihydrate and
the necessary manipulations to prepare it for injection could be
problematic in a clinical setting, we have also examined the iron
clearing efficiency of the HBED monosodium salt. The salt is much more
soluble than the monohydrochloride dihydrate, with a solubility in
water that is in excess of 30% (wt/vol). In addition, when the drug is
dissolved in saline the unadjusted pH of the resulting solution is 7.3. When the drug was administered to the primates at a single dose of 150 µmol/kg, it induced the excretion of 1,139 ± 383 µg/kg of iron
(Fig 2) and had an efficiency of 13.6% ± 4.5% (Table 1). This is
well within error of the efficiency observed after the sc injection of
162 µmol/kg of the HBED monohydrochloride dihydrate in buffer or in
Cremophor, 10.7% ± 2.3% (9.9% ± 2.1%)(P > .2) and
16.1% ± 5.6% (14.9% ± 5.2%) (P > .7),
respectively.26
Finally, we have also administered the HBED monosodium salt at a dose
of 75 µmol/kg every other day for three doses
(Fig 4), for a total dose of 225 µmol/kg (n = 4). In this study, the first dose of the drug induced
the excretion of 597 ± 91 µg/kg of iron with a 24-hour efficiency
of 14.2% ± 2.2%. The second injection of the drug induced the
excretion of 616 ± 90 µg/kg of iron with a 24-hour efficiency of
14.7% ± 2.2%. The third injection of the same dose of the drug
induced the excretion of 625 ± 156 µg/kg of iron with a 24-hour
efficiency of 14.9% ± 3.7%. The total iron excreted as a result
of the three injections was 1,837 ± 301 µg/kg of iron (Fig 2)
with an overall efficiency of 14.6% ± 2.4% (Table 1). These data
are virtually identical to the iron excretion induced after a single sc
injection of HBED in a buffer administered at a dose of 81 µmol/kg,
608 ± 175 µg/kg, and an efficiency of 13.0% ± 4.6%
(P > .6, P > .5, and P > .5 for doses 1 through 3, respectively).
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| Fig 4.
Urinary and fecal iron excretion (µg/kg) induced by the
sc administration of HBED monosodium salt, 75 µmol/kg for three doses
(225 µmol/kg total). Drug was administered on days 0, 2, and 4. Note
the prompt return to baseline levels within 24 hours of each dose;
baseline iron levels in the urine and stool have not been subtracted.
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Interestingly, with the monosodium salt, a greater proportion of the
iron was excreted in the urine than was observed with the
monohydrochloride dihydrate administered in a buffer. At a single sc
dose of 150 µmol/kg HBED monosodium salt, 32% of the iron was
excreted in the urine with the remaining 68% being excreted in the
feces. When the drug was administered sc at a dose of 75 µmol/kg
every other day for three doses, the amount of iron excreted via the
urine was even higher, 54%, 43%, and 68% for doses 1 through 3, respectively. The apparent difference in urinary versus biliary excretion may be caused by individual animal variability, or it may be
that the more water-soluble monosodium salt is more readily cleared by
the kidneys than the much less soluble monohydrochloride dihydrate.
Primate iron balance studies.
The total amount of iron intake was compared with the total amount of
iron excreted. Net iron balance = dietary iron intake (urinary + fecal iron excretion); animals in a negative iron balance are
excreting more iron than they are absorbing. We have previously shown
that monkeys treated with sc DFO 150 µmol/kg, or HBED 75 (81) or 150 (162) µmol/kg sc with or without the Cremophor vehicle were able to
hold the monkeys in negative iron balance.26
In the current studies, whereas the doubling of the sc dose of DFO to
300 µmol/kg resulted in the excretion of 711 ± 230 µg/kg more
iron than the primates absorbed (Table 1), the PO administration of 300 µmol/kg of DFO or 324 µmol/kg HBED to the monkeys did not result in
a negative iron balance (data not shown). However, animals treated with
a single sc dose of 81 µmol/kg of HBED in buffer excreted 259 ± 209 µg/kg more iron than they absorbed. This is in good agreement
with what we previously observed with the administration of the same sc
dose of HBED-Cremophor, 606 ± 406 µg/kg more iron than they
absorbed (P > .1).26 The sc administration of
HBED, 162 µmol/kg/day for 2 consecutive days, also resulted in a
negative iron balance. The animals excreted 1,607 ± 372 µg/kg of
iron more than they absorbed, which is within error of what is observed after a single sc dose of HBED of 324 µmol/kg, 2,346 ± 830 µg/kg more iron than they absorbed (P > .2).
The sc administration of the monosodium salt of HBED was also able to
hold the monkeys in a negative iron balance (Table 1). Monkeys
treated with the drug at a single dose of 150 µmol/kg excreted 899 ± 365 µg/kg of iron more than they absorbed. This is in keeping
with what we have noted previously when the HBED was administered sc to
the primates in either a buffer or in Cremophor at a similar
dose26; 1,141 ± 456 µg/kg more than they absorbed when the compound was dosed with the Cremophor vehicle (P > .4), and 689 ± 158 µg/kg when the drug was administered without
the Cremophor vehicle (P > .3). Finally, the HBED monosodium
salt administered sc every other day for three doses (75 µmol/kg/dose) also resulted in a negative iron balance (Table 1). The
iron excreted during a 7-day period after the administration of the first dose amounted to 1,578 ± 345 µg/kg of iron more than they absorbed.
As was observed with the urinary and fecal iron clearance data, animals
treated with sc HBED consistently have a negative iron balance that is
two to three times greater than that observed with DFO. The results of
these studies clearly indicate that although PO administered DFO and
HBED are unable to hold the monkeys in a negative iron balance (data
not shown), both sc DFO and HBED administered sc as its
monohydrochloride dihydrate in a buffer or as its monosodium salt can
hold the monkeys in a negative iron balance (Table 1).
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DISCUSSION |
HBED (Mr 388) is a phenolic aminocarboxylate chelator that,
like DFO, forms a 1:1 hexadentate complex with ferric iron. It was
first synthesized by L'Eplattenier et al22 some 3 decades ago, and, in rats, has an LD50 (PO or intraperitoneally
[IP]) that is in excess of 800 mg/kg.32 The
ligand was originally chosen for further development as an
iron-chelating agent after studies in rodents suggested that (1) it
cleared radiolabeled iron from overloaded animals when administered
parenterally,33,34 and (2) it was well absorbed from the
gastrointestinal tract and remained active as an iron chelator after PO
administration.32 Unfortunately, subsequent evaluations in
both iron-loaded primates31 and human
volunteers35,36 showed that although the PO administration of the drug did result in the excretion of some iron, the amount was
insufficient for clinical use in transfusional iron overload.
The potential therapeutic usefulness of HBED administered parenterally
was evaluated in the monkeys26 after recalling that IP
injection of the drug in the hypertranfused rat produced an iron
excretion that was significantly greater than that after injection of
DFO.33 Recently, we showed that the iron excretion induced
by sc injection of HBED in monkeys was at least twice that induced by
sc injection of DFO.26 We reported the iron clearance
values for the drug administered sc in 40% Cremophor at doses of 75 (81) and 150 (162) µmol/kg, as well as the efficiency of the drug
administered PO or sc in a phosphate buffer dosed at 150 or 162 µmol/kg, respectively.26,31 At a dose of 75 (81) µmol/kg, HBED-Cremophor sc induced the clearance of 793 ± 410 µg/kg of iron and had an efficiency of 18.4% ± 9.1% (17.5% ± 9.1%).26 Increasing the sc dose to 150 (162)
µmol/kg resulted in the clearance of 1,349 ± 475 µg/kg of iron
and an efficiency of 16.1% ± 5.6% (14.9% ± 5.2%).26 However, HBED administered PO in a phosphate buffer (no Cremophor) at a dose of 150 µmol/kg had an efficiency of
only 0.5% ± 0.5%,31 whereas a similar dose of the
drug administered sc in the same vehicle induced the excretion of 899 ± 193 µg iron/kg body weight and was found to have an efficiency
of 10.7% ± 2.3% (9.9% ± 2.1%).26 In all three
sc experiments, approximately 90% of the iron was excreted in the
feces with only 10% being excreted in the urine. At the dose of 150 (162) µmol/kg, no significant difference (corrected, P > .1) was found between the mean net iron excretion induced by HBED
prepared in phosphate buffer or in Cremophor.
Given these encouraging results, additional experiments were clearly in
order. Accordingly, in the current studies, we have greatly expanded
the preclinical testing of HBED to include subchronic rodent toxicity
studies, as well as more clinically applicable dosing regimens in the
primates. HBED monosodium salt administered sc to rodents at doses up
to 300 µmol/kg every other day for 2 weeks was found to be virtually
nontoxic, ie, no drug-related toxicities were identifiable either
grossly or histologically. In addition, the net iron excretion after sc
HBED (monohydrochloride dihydrate in phosphate buffer or monosodium
salt) in the primate model with iron overload produced iron excretion
that was at least twice that observed after sc DFO and the compound
retained its effectiveness when administered under a multiple-dosing
protocol.
In patients with a high transfusion regimen, erythropoiesis is
suppressed and iron absorption may be near normal, but each unit of
transfused red blood cells contains 200 to 250 mg of iron. Most
patients with thalassemia major require 200 to 300 mL/kg/year of blood,
an amount equivalent to 250 to 400 µg iron/kg/day.37 Thus, to maintain iron balance, a chelator must be able to remove a
minimum of between 250 and 400 µg of iron/kg/day. The iron excretion observed after a single sc 150 µmol/kg injection of DFO in the C
apella monkey with iron overload, 435 ± 115 µg iron/kg body weight, is consistent with the established ability of the daily use of
DFO to control body iron. Under the same experimental conditions, sc
HBED induces the excretion of more than twice as much iron as sc DFO,
ie, 1,139 ± 383 µg/kg versus 435 ± 115 µg/kg of iron when
both drugs are administered at 150 µmol/kg, and 2,400 ± 808 µg/kg versus 716 ± 244 µg/kg of iron when they are administered at a dose of 324 µmol/kg and 300 µmol/kg, respectively. Although the iron-loaded primate model26-28,30-31,38 has proven
itself to be an excellent predictor of a potential chelator's
effectiveness in a clinical setting, caution is clearly needed in
extrapolating from the primate model to patients with iron overload.
For example, the C apella monkey with iron overload has normal
erythropoiesis, whereas in patients with thalassemia major,
erythropoietic activity is increased, even with regular transfusion.
This difference could influence the relative proportions of iron
excretion via the urinary and biliary routes, because at least with
DFO,11 erythroid hyperplasia is associated with enhanced
urinary iron excretion. Nevertheless, the overall pattern of iron
excretion (urinary v fecal) in the primate model seems to be a
reliable indicator of iron excretion in patients. Accordingly, the
increased iron excretion observed after single or multiple sc
injections of HBED in a phosphate buffer or as its monosodium salt
suggests that a regimen in which sc HBED was used every other day (75 to 150 µmol/kg) might be as effective in maintaining iron balance as
daily sc infusions of DFO. The prospect of using higher doses of HBED
administered less frequently to maintain iron balance, eg, 300 to 324 µmol/kg only once or twice weekly, is also a possibility. Overall,
these findings suggest that prompt completion of preclinical evaluation of parenteral HBED is in order; iron balance studies in human volunteers would be a subsequent step. The sc injection of HBED may
provide patients with transfusional iron overload with a more effective, less demanding alternative iron chelation therapy to the use
of prolonged parenteral infusions of DFO.
| |
FOOTNOTES |
Submitted June 29, 1998;
accepted September 4, 1998.
Supported in part by research grants from the National Institutes of
Health (DK49108, AI35827, and HL61219) and by SunPharm Corporation,
Jacksonville, FL.
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.
Address reprint requests to Raymond J. Bergeron, PhD, Box
100485 JHMHC, Department of Medicinal Chemistry, University of Florida,
Gainesville, FL 32610.
| |
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Abstract
Introduction
