Blood, Vol. 91 No. 11 (June 1), 1998:
pp. 4368-4372
Biliary Iron Excretion in Rats Following Treatment With Analogs of
Pyridoxal Isonicotinoyl Hydrazone
By
Karel Bláha,
Miroslav Cikrt,
Jana Nerudová, and
Helena
Forn
sková, and P
emysl Po
ka
From the Centre of Industrial Hygiene and Occupational Diseases,
National Institute of Public Health, Prague, Czech Republic; and the
Lady Davis Institute for Medical Research of the Jewish General
Hospital and Departments of Physiology and Medicine, McGill University,
Montreal, Quebec, Canada.
 |
ABSTRACT |
Iron overload is a major life-threatening complication of
thalassemia major and other iron-loading anemias treated by regular blood transfusions. Although the clinical manifestations of iron overload may be prevented by desferrioxamine, the only iron-chelating drug in routine clinical use, this treatment requires subcutaneous infusion of desferrioxamine for 12 hours each day. New orally effective
iron chelators are urgently needed, and pyridoxal isonicotinoyl hydrazone (PIH), which was first recognized as an effective iron chelator in vitro and subsequently in vivo, shows promise
for the treatment of iron overload. More recently, over 40 analogs of
PIH were synthesized, and some of them proved to be very potent in
mobilizing 59Fe in vitro from 59Fe-labeled
cells. In this study, we show that PIH analogs such as pyridoxal
benzoyl hydrazone, pyridoxal p-methoxybenzoyl hydrazone (PMBH),
pyridoxal m-fluorobenzoyl hydrazone (PFBH), and
pyridoxal-2-thiophenecarboxyl hydrazone, compounds previously shown to
mobilize iron from cells in vitro, are also effective in vivo. All of
these chelators significantly enhanced biliary excretion of iron
(measured by atomic absorption spectrophotometry) following their
intraperitoneal (IP) and/or oral administration to rats. The
most effective was PFBH, which increased iron concentration in the bile
about 150-fold, as compared with basal biliary iron concentration,
within 1 hour following a single IP dose of 0.2 mmol/kg body
weight. In contrast, desferrioxamine increased the biliary
iron concentration only 20-fold to 30-fold under the same conditions.
Moreover, while control rats excreted 
0.8 µg Fe in 2 hours,
treatment with PFBH, PMBH, and desferrioxamine resulted in cumulative
excretions of 87, 59, and 22 µg Fe, respectively, in the same period
of time. Interestingly, PMBH was also quite effective following gastric
administration, resulting in a 6-hour cumulative value of 34 µg Fe.
These compounds are nontoxic and are inexpensive and easy to make.
Their further evaluation as candidate drugs for the treatment of iron
overload is warranted.
| |
INTRODUCTION |
DESFERRIOXAMINE (DFO) is the only iron
chelator in routine clinical use for the treatment of secondary iron
overload, but this drug is very expensive and may cause toxic side
effects. Moreover, desferrioxamine treatment involves a slow
subcutaneous delivery over a 12-hour period each day, resulting in lack
of compliance with the therapy.1-3 Although the new orally
active chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1, deferiprone)
increases urinary Fe excretion,4,5 clinical trials have
shown a number of serious complications with L1 therapy, including
agranulocytosis, arthritis, and gastrointestinal side
effects.6-11 Moreover, recent long-term clinical trials
showed that there is either no overall change in iron
stores12 or that mean body iron increases13 in
the course of L1-treatment. In fact, tissue iron can reach concentrations associated with iron-induced complications in 95% of
patients.13 Hence, there is an urgent need to develop
alternative iron chelators that are economical, orally effective, and
highly efficient.
Pyridoxal isonicotinoyl hydrazone (PIH), produced by the Schiff base
condensation of pyridoxal and isonicotinic acid hydrazide (INH), was
initially identified as an effective iron chelator in
vitro.14,15 Subsequently, PIH was found to be effective in
vivo when given parenterally to mice15 and orally to
rats.16-19 The high activity of PIH both in vitro and in
vivo14-26 has prompted a clinical trial, where PIH showed
no evidence of toxicity and produced significant iron
excretion.27
We have recently synthesized 44 analogues of PIH, which retained the
key iron-binding groups, and tested their effect on hepatocyte iron
uptake from transferrin, as well as on iron mobilization from
intracellular sites of hepatocytes.28 This study identified several PIH analogues, which are relatively simple and inexpensive to
synthesize, that are highly effective in blocking 59Fe
uptake by hepatocytes and/or mobilizing hepatocyte
59Fe. Another study29 confirmed these results
and also documented that the compounds do not cause toxic effects when
added in effective concentrations to isolated rat hepatocytes incubated
in vitro. In the present study, we examined the capacity of these newly identified iron chelators to promote biliary iron excretion following their administration to rats.
| |
MATERIALS AND METHODS |
Animals.
Female Wistar rats (220 ± 20 g) fed on a standard pellet diet were
used. The rats were starved 24 hours before the experiment during which
they had free access to deionized water.
Chelators.
The chelators were synthesized by Schiff base condensation between
appropriate aldehydes and acid hydrazides, using standard procedures.30 The number codes
(Fig 1) for the hydrazones were identical
as in our previous reports.24,28 The compounds were characterized with respect to their carbon-hydrogen analysis, melting
points, and infra-red and nuclear magnetic resonance
spectra.30 The chelators were administered either
intraperitoneally (IP) or by gastric gavage at the dose of 0.2 mmol/kg
body weight, roughly corresponding to concentrations shown to be
effective in in vitro systems.28,29 Solutions of chelators
were prepared within 30 minutes or less before administration, as
follows: the appropriate amount of chelator was dissolved in a minimum
volume (0.3 to 0.5 mL) of 1 mol/L HCl, then adjusted to pH 6 to 7 by
addition of Krebs-Henseleit buffer (pH 7.4). The temperature of the
solution was kept within the range of 35°C to 37°C. Injected
volumes were approximately 1 mL per rat, depending on the body weight.
Experimental procedure.
The bile duct was cannulated using fine tubing (PE-20, Clay-Adams,
internal diameter 0.28 mm), which was then subcutaneously conveyed to the dorsal area, exposed extracorporeally, and connected to
a small test tube fitted to a jacket that was fixed on the back of the
rat. The bile was collected in 12 × 75 mm plastic tubes, which
were replaced as needed to allow assessment of the rate of bile flow
and the iron content in the bile.31 The animals were kept
in metabolic cages and bile samples were collected every hour for 2 hours before and 8 hours after administration of the chelator.
Immediately after the collection, the weight of the bile sample was
determined, and the samples were kept in a refrigerator until analyzed.
The urine was collected for 18 hours before and after the injection of
chelator using the method described earlier.31,32 At the
end of the experiment, the rats were killed by exsanguination from the
heart, and the brains were carefully removed and weighed.
Iron determinations.
All analyses were performed on an AAS-30 Varian spectrophotometer
(Varian Techtron Pty, Limited, Mulgrave, Victoria,
Australia) equipped with Zeeman's background correction
for flameless and deuterium correction for flame technique. The
concentration of iron in the bile was determined directly using
flameless technique. The content of iron in the urine was determined
after extraction of filtration paper with 30 mL of 0.1 mol/L Na-EDTA
(flame technique).
| |
RESULTS AND DISCUSSION |
Our recent study showed that several hydrazones of pyridoxal,
viz those derived from hydrazides of benzoic
(#101), p-methoxybenzoic (#107), m-fluorobenzoic
(#109), and 2-thio-phenecarboxylic (#115) acids, as well as pyridoxal
2-furoyl hydrazone (#114) are highly effective in mobilizing
59Fe from 59Fe-labeled hepatocytes in
vitro.28 Before starting in vivo experiments, toxicities of
these compounds were tested using isolated hepatocytes in
vitro.29 This model was helpful in determining nontoxic and effective concentrations of the chelators.29
In the present study, the bile ducts of normal female rats were
cannulated and the bile was collected for 2 hours before administration of the chelators (either IP or by gastric gavage); bile collection was
continued for an additional 7 to 8 hours after drug administration. Each experimental group comprised eight animals, which before chelator
administration, served as their own controls. Before chelator
treatment, biliary iron concentration was 
1 µg/mL, and dramatic
increases were noted within 1 hour after administration of the
hydrazones (Table 1). The highest biliary
iron concentration was in rats treated with pyridoxal
m-fluorobenzoyl hydrazone (PFBH, #109), which increased the
concentration of iron in the bile more than 100-fold within 1 hour.
Rats treated with this chelator showed a peak of biliary iron excretion
between 1 to 3 hours, but very high iron levels in the bile were still
detectable at 8 hours after the drug administration (Table 1). The
remaining compounds (#101, #107, #114, and #115) were less effective
than PFBH in promoting biliary iron excretion, both in terms of
increasing the biliary iron levels and the duration of their effect
(Table 1). Nevertheless, all of the compounds dramatically increased the cumulative excretion of iron in the bile
(Fig 2). As compared with untreated rats,
which excreted about 1 µg iron in 2 hours, at 6 hours, the cumulative
iron excretion was 69, 74, 122, and 194 µg after a single dose of
pyridoxal 2-thiophenecarboxyl hydrazone, pyridoxal benzoyl hydrazone,
pyridoxal p-methoxybenzoyl hydrazone and pyridoxal
m-fluorobenzoyl hydrazone, respectively (Fig 2). PMBH and, in
particular, PFBH were significantly more effective than the parent
compound, PIH, which gave a 6-hour cumulative value of 59 µg Fe (Fig
2). Pyridoxal 2-furoyl hydrazone was somewhat less effective, leading
to the excretion of 34 µg iron in the bile in 6 hours. The efficacy
of m-fluorobenzoyl hydrazone in stimulating biliary iron
excretion may be related to the high lipophilicity of this compound,
either as a free ligand or in a complex with iron.22 With
the exception of compound #114 (pyridoxal 2-furoyl hydrazone), all acyl
hydrazones tested were significantly more effective than
desferrioxamine (Fig 2). Bile appears to be the primary route of iron
excretion after the administration of these compounds, as none of the
hydrazones tested significantly increased urinary iron excretion
(Table 2).
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| Fig 2.
Cumulative excretion of iron in the bile of rats injected
IP with pyridoxal-derived hydrazones. The bile ducts of normal rats were cannulated and the bile was collected for 2 hours, after which the
hydrazones were injected IP (0.2 mmol/kg body weight) and then bile
collection continued as indicated.
|
|
Three of the most efficient hydrazones (PFBH, PMBH, and PTCH) were
further tested after administration by gastric gavage. The hydrazones
were less effective in increasing biliary iron excretion when
administered by gavage (Table 3) compared
with IP administration (see Table 1). The most effective chelator after
gastric administration was pyridoxal p-methoxybenzoyl hydrazone (PMBH) which, within 1 hour, increased biliary iron concentration more
than 10-fold and maintained a high iron content in the bile for the
additional 5 hours (Table 3). Consequently, PMBH given intragastrically
significantly increased cumulative excretion of iron, resulting in a
6-hour cumulative value of 34 µg Fe (Fig 3). As compared with PFBH, PMBH is more hydrophilic,28 a
property that would seem to make this compound more soluble than PFBH
in gastrointestinal fluids. If so, the availability of PMBH for
absorption would be higher than that of PFBH, and this could explain
the higher efficacy of PMBH, as compared with PFBH, when this compound was administered by gavage. Gastric administration of PMBH, PFBH, and
PTCH did not increase urinary iron excretion
(Table 4).
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| Fig 3.
Cumulative excretion of iron in bile of rats after
gastric administration of pyridoxal-derived hydrazones. The bile ducts of normal rats were cannulated and the bile was collected for 2 hours,
after which the hydrazones were administered by gavage (0.2 mmol/kg
body weight) and then bile collection continued as indicated.
|
|
These results, as well as our previous observations,16
suggest that the limited oral efficacy of PIH and other acyl hydrazones is caused by lower absorption. Similarly, Brittenham's27
evaluation of PIH in patients has yielded lower than expected effect on
iron excretion at doses permissible for clinical use. Because PIH was given as a powder in gelatin capsules, it appears likely that the
chelator was poorly available for absorption. Hence, a more bioavailable formulation of PIH and its analogs should be designed and
evaluated as a means of increasing Fe excretion. One promising strategy
may involve the use of chelators encapsulated into biodegradable polymers.33 However, the bioavailability of PIH and analogs may also be limited by hydrolysis of acyl hydrazones in the stomach, as
it has been reported that these compounds hydrolyze in strongly acidic
solutions.34
A clinically useful iron chelator should have high affinity for iron,
but low affinity for all other biologically important cations. It is
well known that DFO fullfils this criterion. Importantly, PIH and several of its analogs have very low affinity for Ca(II) and
Mg(II) and complexation with these ions only occurs at pH greater than
8.35 Hence, chelation of these biologically important cations should not occur under physiologic conditions. Although the PIH
class of chelators can bind Zn(II) at pH 7.4, their affinity for Zn(II)
is much less than for Fe(III),35 and complexation of Zn(II)
probably does not occur in vivo. In rats, PIH did not increase biliary
excretion of zinc and copper.16
One of the aims of this study was to identify a chelator with the
highest efficacy to promote biliary iron excretion at a relatively low
dose (0.2 mmol/kg). It is possible that at this dose the chelators do
not produce maximum effects, and further experiments are being planned
to examine whether PMBH and PFBH would be more effective at higher
doses administered either IP or orally. DFO is known to promote urinary
iron excretion in iron overloaded rats,36 and it should be
pointed out that in this study we used rats that were not iron
overloaded. The use of noniron overloaded rats can probably explain low
urinary iron excretion after DFO administration.
In conclusion, this study showed that pyridoxal hydrazones with benzoyl
(#101), p-methoxybenzoyl (#107), m-fluorobenzoyl
(#109), and 2-thiophene-carboxyl (#115) substituents show potential as candidate iron chelating drugs. The most promising compounds are pyridoxal m-fluorobenzoyl hydrazone (#109) and pyridoxal
p-methoxybenzoyl hydrazone (#107) that deserve further vigorous
evaluation for iron chelation therapy.
| |
FOOTNOTES |
Submitted January 26, 1996;
accepted January 21, 1998.
Supported by a grant from the Medical Research Council of Canada,
Ottawa, Cananda; and Grant No. 0259-3 from the Czech Ministry of
Health, Prague, Czech Republic.
Presented at the 37th Annual American Society of Hematology Meeting,
Seattle, WA, December 1-5, 1995 and published in abstract form (Blood 86:300a, 1995 [suppl 1]).
Address reprint requests to Premysl Ponka, MD, PhD, Lady Davis
Institute for Medical Research, Jewish General Hospital, 3755 Cote
Ste-Catherine Rd, Montreal, Quebec, Canada H3T 1E2.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The excellent technical assistance of Jana Tumova and Eva Nagy is
gratefully acknowledged. We would like to thank Sandy Fraiberg for her
excellent editorial assistance.
| |
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Abstract
Introduction
Abstract
