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DRUG METABOLISM AND DISPOSITION
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics
Vol. 27, No. 12
Printed in U.S.A.
PHARMACOKINETICS OF ORALLY ADMINISTERED DESFERRITHIOCIN ANALOGS IN
CEBUS APELLA PRIMATES
RAYMOND J. BERGERON, WILLIAM R. WEIMAR,
AND
JAN WIEGAND
Department of Medicinal Chemistry, University of Florida, Gainesville, Florida
(Received June 17, 1999; accepted August 18, 1999)
This paper is available online at http://www.dmd.org
ABSTRACT:
achieves a substantially higher plasma concentration than does 3,
yet the renal clearance of these compounds is similar. The oxazoline analog 4 shows poor iron clearance when administered orally,
although it remains in the plasma for extended periods. Chelator 4
demonstrates a marked capacity to bind to human serum albumin
compared with the thiazoline derivatives. The possible implications for designing ligands for the treatment of transfusional iron
overload are discussed.
Iron metabolism is characterized by a highly efficient recycling
process (Finch et al., 1970; Hallberg, 1981; Finch and Huebers, 1982,
1986) with no specific mechanism for elimination. The introduction of
“excess iron” (O’Connell et al., 1985; Thomas et al., 1985; Seligman
et al., 1987) into this closed metabolic loop leads to chronic overload
and ultimately to peroxidative tissue damage. Iron excretion can be
promoted by therapy with ferric ion-specific chelators such as the
hydroxamate desferrioxamine B (DFO)1, which is a bacterial siderophore and the drug of choice for the treatment of transfusional iron
overload. Because DFO is poorly absorbed from the gastrointestinal
tract and rapidly eliminated from the circulation, prolonged parenteral
infusion is needed (Pippard, 1982, 1989; Lee et al., 1993). Patient
compliance with such a demanding, expensive, and unpleasant regimen is a problem. Thus, considerable interest has developed in the
search for an iron chelator that does not require parenteral administration.
Our laboratory has considerable experience in the study of iron
chelators in the iron-loaded Cebus apella primate model. This model
is highly predictive for the performance of ligands in patients with
transfusional iron overload (Peter et al., 1994). We recently reported
an extensive structure-activity relationship for orally active iron chelators based on the desferrithiocin (DFT) pharmacophore [(S)-4,5dihydro-2-(3-hydroxy-2-pyridinyl)-4-methyl-4-thiazolecarboxylic
acid, 1, Fig. 1] (Bergeron et al., 1999a,b). This and the other studies
(Bergeron et al., 1991, 1993, 1994, 1996b) have made it possible to
construct DFT analogs that are still orally effective, yet have substantially reduced toxicity compared with the parent ligand. Nonetheless,
an even broader therapeutic window would be desirable, and it also
may be advantageous to design chelators that selectively target one of
the body’s two main pools of chelatable iron, the hepatocellular
(parenchymal) and reticuloendothelial (RE) stores. Thus, an understanding of the pharmacokinetic behavior of iron chelators may allow
the development of improved strategies of chelation therapy, including combination regimens targeting specific physiological compartments.
This paper describes the distinctive pharmacokinetic behavior of three
prospective oral iron chelators based on the DFT pharmacophore (Fig. 1):
desmethyldesferrithiocin [(S)-4,5-dihydro-2-(2-hydroxyphenyl)-4-thiazolecarboxylic acid, DMDFT, 2]; 4-(S)-hydroxydesazaDMDFT [(S)-4,5dihydro-2-(2,4-dihydroxyphenyl)-4-thiazolecarboxylic acid, 3]; and the
oxazoline analog of desazaDMDFT [(R)2-(2-hydroxyphenyl)-4-oxazolinecarboxylic acid, 4].
This study was supported by National Institutes of Health Grant DK 49108.
1
Abbreviations used are: DFO, desferrioxamine B; DFT, desferrithiocin; RE,
reticuloendothelial; DMDFT, desmethyldesferrithiocin; 4-(S)-hydroxydesazaDMDFT, (S)-4,5-dihydro-2-(2,4-dihydroxyphenyl)-4-thiazolecarboxylic acid; desazaDMDFT, 2-(2-hydroxyphenyl)-4-thiazolecarboxylic acid; AUC, area under the
time-concentration curve; AUMC, the total area under the first-moment curve;
MRT, mean residence time; CLR, renal clearance.
Send reprint requests to: Raymond J. Bergeron, Ph.D., Box 100485 JHMHC,
Department of Medicinal Chemistry, University of Florida, Gainesville, FL 32610.
E-mail: [email protected]
Experimental Procedures
Materials. Ligands 2, 3, and 4 were synthesized as described previously
(Bergeron et al., 1994, 1999a,b). C. apella monkeys were purchased from
World Wide Primates (Miami, FL). All reagents and standard iron solutions
were obtained from Aldrich Chemical Co. (Milwaukee, WI). All hematological
and serum chemical tests were carried out by Allied Clinical Laboratories
(Gainesville, FL). Cremophor RH-40 was obtained from BASF (Parsippany,
NJ).
Collection of Pharmacokinetic Samples in Monkeys. After an overnight
fast, sedation with either ketamine and scopolamine or Telazol and atropine
(3 and 4), and insertion of a lubricated 5 French, 16-inch urethral catheter, the
compounds were solubilized in 40% (v/v) Cremophor RH-40/water and administered p.o. by gavage at a dose of 300 mmol/kg via an 8 French feeding
tube. Sedation was maintained by either additional i.m. ketamine (2) or
isoflurane gas given by intubation (3 and 4).
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The pharmacokinetic behavior of three iron chelators based on the
desferrithiocin (DFT) pharmacophore, (S)-4,5-dihydro-2-(2-hydroxyphenyl)-4-thiazolecarboxylic acid (desmethyldesferrithiocin,
DMDFT, 2); (S)-4,5-dihydro-2-(2,4-dihydroxyphenyl)-4-thiazolecarboxylic acid [4-(S)-hydroxydesazaDMDFT, 3); and (R)-2-(2-hydroxyphenyl)-4-oxazolinecarboxylic acid, the oxazoline analog of desazaDMDFT, 4, is described. Although 2 and 3 are comparably
effective in inducing iron excretion upon oral administration, they
exhibit markedly different plasma pharmacokinetics. Ligand 2
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PHARMACOKINETICS OF ORAL DFT ANALOGS IN C. APELLA
FIG. 2. Plasma pharmacokinetics of DMDFT (2, M), 4-(S)-hydroxydesazaDMDFT
(3, f), and the oxazoline analog of desazaDMDFT (4, F).
Compounds were administered by oral gavage at 300 mmol/kg to male C. apella
monkeys. For clarity, data are plotted as means of five (M), eight (f), or four (F)
experiments.
At times t 5 0, 0.5, 1, 2, 3, 4, 6, and 8-h postdrug, blood samples were taken
from a leg vein. Urine was collected via catheter followed by rinsing the
bladder with saline before drug administration and at 0.5-h intervals for 4 h
thereafter. In some experiments with 3, 10-, 12-, and 24-h blood and urine
samples were collected. The plasma and urine were stored frozen until HPLC
analysis.
Plasma and Urine Analytical Methods. Plasma and urine pharmacokinetic
samples were prepared for HPLC analysis by mixing with an equal volume of
methanol, chilling at 0°C for 30 min, centrifugation at 3000g, and filtration of
the methanolic supernatant through a 0.2-mm filter before injection.
Analytical separation was performed on a C18 reversed-phase HPLC system
with UV-detection at 310 nm as described previously (Bergeron et al., 1998).
The mobile phases and chromatographic conditions for each compound were
as follows: ligand 2, solvent A, 5% aqueous CH3CN; solvent B, 80% aqueous
CH3CN; isocratic 100% A (5 min) followed by a linear ramp to 100% B (10
min); analog 3, solvent A, 5% CH3CN in 25 mM potassium phosphate buffer
(pH 3.4), solvent B, 60% CH3CN in 25 mM potassium phosphate buffer (pH
3.4); isocratic 100% A (10 min) followed by a linear ramp to 100% B (15 min);
chelator 4, solvent A, 5 mM tetrabutylammonium phosphate in water (pH 7.0);
solvent B, 80% aqueous CH3CN; isocratic 100% A (7 min) followed by a
linear ramp to 100% B (3 min).
Results and Discussion
Compounds 2 (Bergeron et al., 1993) and 3 (Bergeron et al., 1999a)
administered p.o. were at least as active in promoting iron excretion as
an equivalent dose of parenteral DFO, whereas the oxazoline analog
4 was unexpectedly ineffective in inducing iron excretion (Table 1).
Pharmacokinetic analysis provides some insight into the origin of the
differences in iron clearance among the analogs.
TABLE 1
Pharmacokinetic parameters of 2, 3, and 4 in C. apella monkeys dosed orally at 300 mmol/kg
Compound
2
a
Iron clearance efficiency (%)
N
Cmax (mmol/l)
AUC (mmol-h/l)b
AUMC (mmol-h2/l)
MRT (h)
Urinary excretion
(0–4 h) (% total dose)c
CLR (ml/h-kg)
8.0 6 2.5 [42 stool, 58 urine]
5
231 6 90 (122–345)
384 6 88 (250–487)
723 6 318 (494–1255)
1.88 6 0.61 (1.37–2.90)
50.7% 6 4.2%
492 6 55
3
5.3 6 1.7 [90 stool, 10 urine]
8
9.5 6 7.1 (3.1–22.9)
33 6 27 (12–94)
154 6 140 (61–463)
4.63 6 1.23 (2.12–6.13)
5.7 6 2.8%d
312 6 161
4
0.1 6 0.1
4
103 6 22 (76–128)
598 6 62 (506–647)
2788 6 421 (2177–3141)
4.65 6 0.33 (4.30–5.07)b
1.0% 6 0.7%
12 6 7
Iron clearance efficiency is the amount of iron excreted divided by the theoretical amount of 1 g-atom iron/2 mol of chelator (based on a 2:1 complex) 3 100%. The efficiency values for 2
and 3 were taken from Bergeron et al. (1993) and Bergeron et al. (1999a), respectively. An equivalent dose of DFO given s.c. is 5.5 6 0.9% efficient with 55% in stool and 45% in urine (Bergeron
et al., 1992).
b
The values for AUC and AUMC are for 0- to 8-h data for ease of comparison. The MRT value was calculated as AUMC/AUC. Because there was a very substantial level of 4 remaining in
the plasma at 8 h, this value greatly understates that which would be obtained if reliable 0 –` estimates were available for this drug. This is due to the heavy weighting of the AUMC value by
significant plasma concentrations present at later times.
c
Urine samples were obtained from the bladder via catheter followed by saline flush at 30-min intervals during the 4 h after drug administration.
d
Because low plasma levels and urinary output was observed from 0 to 4 h for compound 3 in the first four experiments, four additional experiments were conducted in which urine and plasma
samples were collected over a 24-h interval. The data reported herein for 3 represent total 24-h output.
a
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FIG. 1. Structures of DFT (1), DMDFT (2), 4-(S)-hydroxydesazaDMDFT (3),
and the oxazoline analog of desazaDMDFT (4).
The concentrations of 2, 3, and 4 were calculated from the peak area fitted
to calibration curves by nonweighted least-squares linear regression with
Rainin Dynamax HPLC Method Manager software (Rainin Instrument Co.
Inc., Woburn, MA). The method had a detection limit of 0.5 mg/l and was
reproducible and linear over a range of 1 to 500 mg/l.
Pharmacokinetic Analyses. The model-independent pharmacokinetic parameters, including the area under the time-concentration curve (AUC) from
time zero to the time of the last measured plasma concentration (8 or 24 h), the
total area under the first-moment curve (AUMC), mean residence time (MRT),
and renal clearance (CLR) were estimated from plasma and urine concentration-time data as reported in Bergeron et al. (1995, 1996a, 1998).
Human Serum Albumin Binding Assay. The binding was carried out by
incubation (30 min at 37°C) in 100 mM TRIS-chloride buffer, pH 7.4, such
that the final concentrations of human serum albumin (Sigma Chemical Co.,
St. Louis, MO) and chelator were 1 and 300 mM, respectively. Ultrafiltration
was performed with Centricon 3 filters (Amicon, Beverly, MA) to separate
bound chelator from free ligand.
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BERGERON ET AL.
TABLE 2
Human serum albumin binding of chelators and iron complexes
Free Ligand
Fe(III) Complex
Compound
2
3
4
Fraction unbounda
Relative % unboundb
Fraction unbound
0.0827
0.0784
0.0059
1.05
1.00
0.07
0.343
0.082
0.007
a
Fraction of total chelator (300 mM) remaining unbound after incubation with 1 mM human
serum albumin at 37°C for 30 min and ultrafiltration to remove bound ligand.
b
Relative percentage of unbound figures for free ligand are normalized to the value for
chelator 3.
Acknowledgments. We thank Michael Slusher, Katie RatliffThompson, and Curt Zimmerman for their technical assistance and
Eileen Eiler-Hughes for her editorial comments.
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Although 2 and 3 were comparably effective in inducing iron
excretion, they exhibited markedly different plasma pharmacokinetics
(Fig. 2). The Cmax and AUC0 – 8 h of 3 were ;4 and ,10%, respectively, of the corresponding values for 2 (Table 1). The amount of 3
eliminated in the urine in 24 h was only 10% of the amount of 2
eliminated in the first 4 h; this mirrored the low plasma AUC for 3.
The CLR of the two compounds was similar, 492 6 55 versus 312 6
161 ml/h/kg for 2 and 3, respectively. The low plasma levels observed
for 3 are most plausibly the result of first-pass clearance by the liver.
This explanation is consistent with the efficacy of 3 in inducing iron
excretion and the predominantly fecal mode of that excretion (Table
1). In contrast, compound 2 achieved high plasma levels and was
found to be extensively eliminated in the urine (50.7 6 4.2% in 4 h),
comparing favorably with the 58% urinary mode of excretion observed for 2 in iron clearance studies (Table 1). The different modes
of excretion are not readily attributable to different distribution patterns of loaded iron because liver parenchymal and hepatocyte compartments appear to be loaded to a similar, massive extent in the
primate model (R.J.B., G.M. Brittenham, H. Fujioka, W.R.W., and
J.W., unpublished data).
Given the inability of oxazoline 4 to promote iron excretion (Table
1), the plasma pharmacokinetics of 4 were remarkable (Fig. 2, dashed
line). This compound was well absorbed orally with a Cmax value of
103 mM and a very prolonged presence in the plasma; a terminal
elimination phase was difficult to discern from these 0 to 8 h experiments. Also remarkable was the very poor CLR of 4 compared with
2 and 3 (Table 1). We suspected that plasma protein binding might be
the basis for the prolonged presence of 4 in the plasma. Thus, the
ability of human serum albumin to bind the ligands was evaluated
(Table 2). Compound 4 was .99.5% bound to the albumin; this was
at least a 10-fold greater extent than 2 or 3. Therefore, plasma protein
binding may provide an explanation of the prolonged residence of 4,
its poor CLR, and its lack of clinical efficacy, i.e., such binding may
simply render the drug unavailable to chelatable iron pools in the
body. Alternatively, it may be that 4 is promoting iron excretion, but
the iron chelate is eliminated at such a slow rate as to not be detected
in our iron balance experiments. Certainly, the ultimate fate of 4 in the
body remains to be determined.
Although the three ligands analyzed in these experiments are all
DFT analogs, their iron-clearing properties and pharmacokinetic parameters are markedly different. It is clear that the oral bioavailability
of a ligand does not guarantee iron-clearing efficacy. Because the
pharmacokinetic behavior of the DFT pharmacophore can be manipulated, it follows that the site of iron chelation also can be altered. To
find whether a chelator targets either of the two main pools of
chelatable iron, the selective radiolabeling of hepatocellular and RE
iron stores can be accomplished with a hypertransfused rat model
(Zevin et al., 1992). In studies using this model, iron mobilized from
hepatocellular pools was excreted directly into the bile. About onethird to one-half the iron derived from RE stores was eliminated in the
urine; the remainder of this iron was recycled into the liver and
excreted in the bile (Zevin et al., 1992). If this is the case, then our
findings suggest that 3 particularly targets hepatocellular pools,
whereas 2 promotes excretion of RE iron. Thus, it may be possible to
design nontoxic, orally effective iron chelators that select either of the
two major pools of chelatable iron.