Desferrithiocin analogue iron chelators: Iron clearing efficiency, tissue distribution, and renal toxicity

Article abstract of DOI:10.1007/s10534-010-9389-y

The current solution to iron-mediated damage in transfusional iron overload disorders is decorporation of excess unmanaged metal, chelation therapy. The clinical development of the tridentate chelator deferitrin (1, Table 1) was halted due to nephrotoxici

Full text of DOI:10.1007/s10534-010-9389-y

Biometals (2011) 24:239–258
DOI 10.1007/s10534-010-9389-y
Desferrithiocin analogue iron chelators: iron clearing
efficiency, tissue distribution, and renal toxicity
•
Raymond J. Bergeron Jan Wiegand
•
•
Neelam Bharti James S. McManis
•
Shailendra Singh
Received: 19 October 2010 / Accepted: 25 October 2010 / Published online: 20 November 2010
ꢀ Springer Science+Business Media, LLC. 2010
Abstract The current solution to iron-mediated
damage in transfusional iron overload disorders is
decorporation of excess unmanaged metal, chelation
therapy. The clinical development of the tridentate
chelator deferitrin (1, Table 1) was halted due to
nephrotoxicity. It was then shown by replacing the
40-(HO) of 1 with a 3,6,9-trioxadecyloxy group, the
nephrotoxicity could be ameliorated. Further struc-
ture–activity relationship studies have established
that the length and the position of the polyether
backbone controlled: (1) the ligand’s iron clearing
efficiency (ICE), (2) chelator tissue distribution, (3)
biliary ferrokinetics, and (4) tissue iron reduction.
The current investigation compares the ICE and
tissue distribution of a series of (S)-4,5-dihydro-2-
[2-hydroxy-4-(polyether)phenyl]-4-methyl-4-thiazole-
carboxylic acids (Table 1, 3–5) and the (S)-4,5-
dihydro-2-[2-hydroxy-3-(polyether)phenyl]-4-methyl-
4-thiazolecarboxylic acids (Table 1, 8–10). The three
most effective polyether analogues, in terms of
trial on 9, no data were available for ligand 3 or 8.
This is unfortunate, as 3 has many advantages over 9,
e.g., the ICE of 3 in rats is 2.5-fold greater than that
of 9 and analogue 3 achieves very high levels in the
liver, pancreas, and heart, the organs most affected by
iron overload. Finally, the impact of 3 on the urinary
excretion of kidney injury molecule-1 (Kim-1), an
early diagnostic biomarker for monitoring acute
kidney toxicity, has been carried out in rats; no
evidence of nephrotoxicity was found. Overall, the
results suggest that 3 would be a far superior clinical
candidate to 9.
Keywords Iron chelator ꢀ Desazadesferrithiocin
polyether analogues ꢀ Tissue distribution ꢀ
Nephrotoxicity ꢀ Urinary biomarker ꢀ
Kidney injury molecule-1 (Kim-1)
Introduction
performance ratio (PR), defined as mean ICEprimate
/
ICErodent, are 3 (PR 1.1), 8, (PR 1.5), and 9, now in
human trials, (PR 2.2). At the onset of the clinical
Humans have evolved a highly efficient iron man-
agement system in which we absorb and excrete
about 1 mg of the metal daily; there is no mechanism
for the excretion of excess iron. Thus, when iron
accumulates beyond this point, as in transfusional
iron overload (Olivieri and Brittenham 1997; Vichinsky
2001; Kersten et al. 1996), or from increased
absorption of dietary iron, (Conrad et al. 1999;
Lieu et al. 2001), without effective treatment, body
iron progressively increases with deposition in
R. J. Bergeron (&) ꢀ J. Wiegand ꢀ N. Bharti ꢀ
J. S. McManis ꢀ S. Singh
Department of Medicinal Chemistry, University
of Florida, JHMHC, Box 100485, Gainesville,
FL 32610-0485, USA
e-mail: rayb@ufl.edu
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Biometals (2011) 24:239–258
´
the liver, pancreas, heart, and elsewhere. Without
decorporation, the metal buildup eventually leads to
liver disease that may progress to cirrhosis (Angel-
ucci et al. 2000; Bonkovsky and Lambrecht 2000;
Pietrangelo 2002), pancreatic damage, e.g., diabetes
(Cario et al. 2004; Wojcik et al. 2002), and,
ultimately, heart failure (Brittenham et al. 1994;
Brittenham 2000). Iron-induced cardiac damage still
remains the primary cause of death in transfusional
iron overload (Zurlo et al. 1989).
reperfusion damage (Millan et al. 2008), Parkinson’s
(Zecca et al. 2008), and Friedreich’s ataxia (Pietrangelo
2007), or global, as in transfusional iron overload,
e.g., thalassemia (Pippard 1987), sickle cell disease
(Pippard 1987; Olivieri 2001), and myelodysplasia
(Malcovati 2007), with multiple organ involvement,
the solution in both scenarios is the same: chelate and
promote the excretion of the excess unmanaged iron.
The iron-chelating agents now in use or that have
been clinically evaluated (Fig. 1) are desferrioxamine
B mesylate (DFO), 1,2-dimethyl-3-hydroxypyridin-4-
one (deferiprone, L1), 4-[3,5-bis(2-hydroxyphenyl)-
1,2,4-triazol-1-yl]benzoic acid (deferasirox, Exjade),
and the desferrithiocin [(S)-4,5-dihydro-2-(3-hydroxy-
2-pyridinyl)-4-methyl-4-thiazolecarboxylic acid (DFT)]
analogue (S)-4,5-dihydro-2-(2,4-dihydroxyphenyl)-
4-methyl-4-thiazolecarboxylic acid, deferitrin (1,
Fig. 1). Each of these ligands presents with notable
shortcomings. DFO must be given subcutaneously
(s.c.) for protracted periods of time, e.g., 12 h a day,
five or more days a week, resulting in a serious patient
compliance issue (Pippard 1989; Giardina and Grady
2001; Olivieri and Brittenham 1997). Deferiprone,
while orally active, simply does not remove enough
Although iron is a critical micronutrient for all
prokaryotes and eukaryotes, it is not easily accessible.
The low solubility of Fe(III) hydroxide (Ksp=
1 9 10-39) (Raymond and Carrano 1979), the pre-
dominant form of the metal in the biosphere, has led
to the development of sophisticated iron storage and
transport systems in nature. Microorganisms utilize
low molecular weight, virtually ferric-ion-specific
ligands, siderophores (Byers and Arceneaux 1998),
that they secrete into the environment in order to
sequester and incorporate the metal. Higher eukary-
otes tend to employ proteins to transport and
store iron, e.g., transferrin and ferritin, respectively
(Bergeron 1986; Theil and Huynh 1997; Ponka et al.
1998).
In humans, plasma nontransferrin-bound iron
(NTBI), a heterogeneous pool of iron in the circula-
tion, seems to be the principal source of the abnormal
tissue distribution of iron that develops with chronic
iron overload. While plasma NTBI is rapidly taken up
by hepatocytes in the liver, the major iron storage
organ, this mechanism for NTBI management can
become overwhelmed. The toxicity associated with
excess iron, whether a systemic or a focal problem,
derives from its interaction with reactive oxygen
species, e.g., endogenous hydrogen peroxide (H2O2)
(Graf et al. 1984; Halliwell 1994a; Halliwell 1994b;
Koppenol 2000). In the presence of Fe(II), H2O2 is
reduced to the hydroxyl radical (HO•), a very reactive
species, and (HO-), a process known as the Fenton
reaction. The liberated Fe(III) can be reduced back to
Fe(II) via a variety of biological reductants (e.g.,
ascorbate), a problematic cycle. The hydroxyl radical
reacts very quickly with a variety of cellular constit-
uents and can initiate free radicals and radical-
mediated chain processes that damage DNA
and membranes, as well as produce carcinogens
(Halliwell 1994a; Babbs 1992; Hazen et al. 1998).
While iron-mediated damage can be focal, as in
O
O
O
H2N
N
OH
N
N
H
N
CH3
H
N
OH
OH
O
O
Desferrioxamine (DFO)
OH
O
OH
O
OH
N
N
N
N
CH3
CH3
OH
Deferasirox (Exjade)
Deferiprone (L1)
OH
HO
OH
N
CH3
CO2H
N
CH3
CO2H
N
S
S
Deferitrin (1)
Desferrithiocin (DFT)
Fig. 1 Structures ofironchelators thatare now inuse or that have
been clinically evaluated: desferrioxamine B mesylate (DFO),
1,2-dimethyl-3-hydroxypyridin-4-one (deferiprone, L1), 4-[3,
5-bis(2-hydroxyphenyl)-1,2,4-triazol-1-yl]benzoicacid(deferasi-
rox, Exjade), and the desferrithiocin [(S)-4,5-dihydro-2-(3-
hydroxy-2-pyridinyl)-4-methyl-4-thiazolecarboxylic acid (DFT)]
analogue (S)-4,5-dihydro-2-(2,4-dihydroxyphenyl)-4-methyl-4-
thiazolecarboxylic acid, deferitrin (1)
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Biometals (2011) 24:239–258
241
iron to maintain patients in a negative iron balance
(Hoffbrand et al. 1998; Olivieri 1996; Olivieri et al.
1998; Richardson 2001). Exjade did not show nonin-
feriority to DFO (Piga et al. 2006) and is associated
with numerous side effects, including some serious
renal toxicity issues (Nick et al. 2002; Nisbet-Brown
et al. 2003; Exjade Prescribing Information). Finally,
the clinical development of deferitrin (1), although
promising, was recently halted by Genzyme due to
nephrotoxicity (Galanello et al. 2007).
BioAssay Works has recently developed RenaStick, a
direct lateral flow immunochromatographic assay,
which allows for the rapid detection (less than
30 min) and quantitation of Kim-1 (rat) or KIM-1
(human) in urine (Vaidya et al. 2009).
The current investigation provides a comparison
of the iron clearance properties and tissue distribution
of a series of (S)-4,5-dihydro-2-[2-hydroxy-4-(poly-
ether)phenyl]-4-methyl-4-thiazole-carboxylic acids
(Table 1, 3–5) and (S)-4,5-dihydro-2-[2-hydroxy-3-
(polyether)phenyl]-4-methyl-4-thiazolecarboxylic acids
(Table 1, 8–10). The ICE of the ligands was assessed in
rodents and in primates; chelator tissue distribution
was evaluated in rodents. Additional rodent toxicity
studies of the lead candidate, (S)-4,5-dihydro-2-[2-
hydroxy-4-(3,6-dioxaheptyloxy)phenyl]-4-methyl-4-
thiazolecarboxylic acid (3, Table 1), including the
impact of this ligand on urinary Kim-1 excretion, have
also been carried out.
Deferitrin has been reengineered, and extensive
structure–activity relationship (SAR) studies led to the
discovery of a number of polyether analogues with
excellent iron clearing properties with little, if any,
impact on renal function (Bergeron et al. 2006, 2007,
2008, 2010). One of these ligands, (S)-4,5-dihydro-2-
[2-hydroxy-3-(3,6,9-trioxadecyloxy)]-4-methyl-4-
thiazolecarboxylic acid (9, Table 1), is now in clinical
trials. Although the preclinical toxicity profile of 9 was
benign, the clinical results remain to be elucidated.
In the course of the SAR studies, several issues
became clear: the length and the position of the
polyether backbone has a profound effect on (1) the
ligand’s iron clearing efficiency (ICE) (Bergeron
et al. 2007, 2010), (2) chelator tissue distribution
(Bergeron et al. 2007, 2008), (3) biliary ferrokinetics
(Bergeron et al. 2008, 2010), and (4) tissue iron
reduction (Bergeron et al. 2010). It also became
painfully obvious that a method that could provide
information about ligand-induced renal damage early
in the drug discovery process was sorely needed.
Assessment of chelator-induced impaired renal func-
tion has traditionally relied on the detection of a rise
in blood urea nitrogen (BUN) and/or serum creatinine
(SCr). However, because of the functional reserve of
the kidney, these parameters are often unreliable
indicators of acute kidney injury; the ultimate answer
requires histopathology. The Critical Path Institute’s
Preventive Safety Testing Consortium (PSTC) has
identified kidney injury molecule-1 (Kim-1, rat) or
(KIM-1, human) as an early diagnostic biomarker for
monitoring acute kidney tubular toxicity (Goodsaid
et al. 2009; Hoffmann et al. 2010). Kim-1 is a type 1
transmembrane protein located in the epithelial cells
of proximal tubules (Han et al. 2002; Bonventre
2009). After injury, e.g., exposure to a nephrotoxic
agent or ischemia, the ectodomain of Kim-1 is shed
from the proximal tubular kidney epithelial cells into
the urine (Zhou et al. 2008; Vaidya et al. 2006).
Experimental section
Materials and instrumentation
Reagents were purchased from Aldrich Chemical Co.
(Milwaukee, WI), and Fisher Optima-grade solvents
were routinely used. Reactions were run under a
nitrogen atmosphere, and organic extracts were dried
with sodium sulfate. Silica gel 40–63 from SiliCycle,
Inc. (Quebec City, QC, Canada) was used for column
chromatography. Glassware that was presoaked in 3 N
HCl for 15 min, washed with distilled water and
distilled ethanol, and oven-dried was used in the
isolation of 8 and 10. Optical rotations were run at
589 nm (sodium D line) and at 20ꢁC utilizing a Perkin-
Elmer 341 polarimeter, with c being concentration in
grams of compound per 100 ml of a CHCl3 solution.
1
Chemical shifts (d) for H NMR spectra at 400 MHz
are given in parts per million downfield from tetra-
methylsilane for CDCl3. Chemical shifts (d) for 13C
NMR spectra at 100 MHz are given in parts per million
referenced to the residual solvent resonance in CDCl3
(d 77.16). Coupling constants (J) are in hertz. The base
peaks are reported for the ESI-FTICR mass spectra.
Male Sprague–Dawley rats were procured from
Harlan Sprague–Dawley (Indianapolis, IN). Male
Cebus apella monkeys (3.5–4.0 kg) were obtained
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Biometals (2011) 24:239–258
(S)-4,5-Dihydro-2-[2-hydroxy-3-(3,6,9,12-
tetraoxatridecyloxy)phenyl]-4-methyl-4-
thiazolecarboxylic acid (10)
from World Wide Primates (Miami, FL). Ultrapure
salts were obtained from Johnson Matthey Electron-
ics (Royston, UK). Exjade was manufactured by
Novartis (Basel, Switzerland). All hematological and
biochemical studies were performed by Antech
Diagnostics (Tampa, FL). Histopathological analysis
was carried out by Florida Vet Path (Bushnell, FL).
Atomic absorption (AA) measurements were made
on a Perkin-Elmer model 5100 PC (Norwalk, CT).
Techni Ice (Melbourne, Australia) was used to
collect the rat urine chilled. A Rat Kim-1 Rapid
Test R-Rena Strip Kit for the detection of Kim-1 in
rat urine was obtained from BioAssay Works
Degassed phosphate buffer (0.1 M, 40 ml, pH 5.95)
and 16 (2.4 g, 14.0 mmol) were added to 15 (3.56 g,
10.9 mmol) in degassed CH3OH (40 ml). Sodium
bicarbonate (1.47 g, 17.5 mmol) was added in por-
tions, and the reaction mixture (pH 6.2–6.6) was
heated at 75ꢁC for 24 h. After cooling to room
temperature, the bulk of the solvent was removed by
rotary evaporation. The residue was dissolved in 8%
NaHCO3 (50 ml) and was extracted with CHCl3
(3 9 100 ml). The aqueous fraction was cooled
at 0ꢁC, acidified to pH & 1 with 5 N HCl, and
extracted with EtOAc (4 9 40 ml). Combined
EtOAc extracts were washed with saturated NaCl and
concentrated in vacuo. Drying under high vacuum
furnished 4.12 g of 10 (85%) as a yellow-orange oil:
(Ijamsville, MD).
A
Chromatoreader ReaScan
(Otsuka Electronics Co., Japan) was utilized to read
the test strips and to allow for the quantitation of
Kim-1 in rat urine.
1
Chemical synthesis
[a] ?17.5ꢁ (c 0.40). H NMR d: 1.72 (s, 3H), 3.24
(d, 1H, J = 11.6), 3.40 (s, 3H), 3.56–3.59 (m, 2H),
3.62–3.78 (m, 10H), 3.89 (m, 2H), 3.90 (d, 1H,
J = 11.6), 4.16–4.25 (m, 2H), 6.79 (t, 1H, J = 8.0),
7.00–7.06 (m, 2H). 13C NMR d 24.53, 39.94, 58.99,
68.90, 69.75, 70.35, 70.50, 70.52, 70.58, 70.82, 71.94,
83.24, 116.33, 117.57, 118.47, 122.69, 147.61, 150.10,
172.09, 175.18. HRMS m/z calcd for C20H30NO8S,
444.1687 (M ? H); found, 444.1683.
(S)-4,5-Dihydro-2-[2-hydroxy-3-(3,6-
dioxaheptyloxy)phenyl]-4-methyl-4-
thiazolecarboxylic acid (8)
Degassed phosphate buffer (0.1 M, 40 ml, pH 5.95)
and 16 (3.45 g, 20.1 mmol) were added to 13 (3.67 g,
15.5 mmol) in degassed CH3OH (40 ml). Sodium
bicarbonate (2.08 g, 24.8 mmol) was added in por-
tions, and the reaction mixture (pH 6.2–6.6) was
heated at 70ꢁC for 24 h. After cooling to room
temperature, solvent was removed by rotary evapo-
ration. The residue was dissolved in 8% NaHCO3
(50 ml) and was extracted with CHCl3 (3 9 50 ml).
The aqueous portion was cooled in an ice-water bath,
acidified to pH & 1 with 5 N HCl, and extracted
with EtOAc (4 9 50 ml). The EtOAc extracts were
washed with saturated NaCl and were concentrated
in vacuo. Drying under high vacuum furnished
5.11 g of 8 (93%) as an orange oil: [a] ?56.1ꢁ
2-Hydroxy-3-(3,6-dioxaheptyloxy)benzonitrile (13)
Compound 11 (3.2 g, 23.7 mmol) was added to a
suspension of 60% NaH (1.9 g, 47.5 mmol) in
DMSO (30 ml) using oven-dried glassware. After
the reaction mixture was stirred at room temperature
for 1 h, 12 (6.48 g, 23.7 mmol) in DMSO (25 ml)
was added. The reaction mixture was stirred for
24 h at room temperature, poured with stirring into
cold water (50 ml), and extracted with CHCl3
(3 9 50 ml). The aqueous phase was acidified to
pH & 1 with 5 N HCl and was extracted with CHCl3
(4 9 50 ml). The latter CHCl3 extracts were concen-
trated in vacuo. Purification using column chroma-
tography, eluting with 9% CH3OH/CHCl3, gave
1
(c 0.70). H NMR d: 1.72 (s, 3H), 3.26 (d, 1H, J =
11.2), 3.39 (s, 3H), 3.57–3.60 (m, 2H), 3.74–3.76
(m, 2H), 3.88 (d, 1H, J = 11.6), 3.89–3.92 (m, 2H),
4.20–4.23 (m, 2H), 6.80 (t, 1H, J = 8.0), 7.01–7.07
(m, 2H). 13C NMR d 24.52, 39.92, 59.11, 69.07,
69.86, 70.79, 72.02, 83.23, 116.33, 117.87, 118.55,
122.80, 147.70, 150.20, 172.52, 176.71; HRMS m/z
calcd for C16H22NO6S, 356.1162 (M ? H); found,
356.1154.
1
3.98 g of 13 (71%) as a colorless oil. H NMR d:
3.41 (s, 3H), 3.60–3.63 (m, 2H), 3.74–3.77 (m, 2H),
3.85–3.87 (m, 2H), 4.16–4.19 (m, 2H), 6.83 (t, 1H,
J = 8.0), 7.08 (dd, 1H, J = 7.8, 1.6), 7.14 (dd, 1H,
J = 8.2, 1.4). 13C NMR d: 58.99, 69.25, 69.53, 70.43,
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Biometals (2011) 24:239–258
243
71.67, 99.88, 116.48, 118.61, 119.88, 125.55, 146.67,
151.04. HRMS m/z calcd for C12H16NO4, 238.1072
(M ? H); found, 238.1072.
provide about 500 mg of iron per kg of body weight
(Bergeron et al. 1990, 1992); the serum transferrin
iron saturation rose to between 70 and 80%. At least
20 half-lives, 60 days (Wood et al. 1968), elapsed
before any of the animals were used in experiments
evaluating iron-chelating agents.
2-Hydroxy-3-(3,6,9,12-
tetraoxatridecyloxy)benzonitrile (15)
Compound 11 (3.20 g, 23.7 mmol) was added to a
suspension of 60% NaH (1.90 g, 47.5 mmol) in
DMSO (30 ml) and the reaction mixture was stirred
at room temperature for 1 h. After addition of 14
(8.68 g, 24.0 mmol) in DMSO (25 ml), the reaction
mixture was stirred for 24 h at room temperature and
was poured into cold water (60 ml) with constant
stirring and was extracted with CHCl3 (3 9 60 ml).
The aqueous phase was acidified to pH & 1 with 6 N
HCl and was extracted with CHCl3 (5 9 40 ml). The
latter CHCl3 extracts were concentrated in vacuo.
Purification using column chromatography, eluting
with 10% CH3OH/CHCl3, gave 5.40 g of 15 (70%) as
a light brown oil. 1H NMR d: 3.42 (s, 3H), 3.57–3.59
(m, 2H), 3.65–3.71 (m, 10H), 3.74–3.76 (m, 2H),
3.85–3.87 (m, 2H), 4.14–4.16 (m, 2H), 6.80 (t, 1H,
J = 8.0), 7.03 (dd, 1H, J = 8.0, 1.6), 7.13 (dd, 1H,
J = 8.0, 1.6), 9.39 (br s, 1H). 13C NMR d: 59.13,
68.65, 69.30, 70.15, 70.34, 70.53, 70.56, 70.58,
71.64, 100.24, 116.71, 117.41, 119.26, 125.29,
146.95, 151.54. HRMS m/z calcd for C16H24NO6,
326.1598 (M ? H); found, 326.1598.
Primate fecal and urine samples
Fecal and urine sampleswerecollected at 24 h intervals
and processed as described previously (Bergeron et al.
1990, 1993, 1998). Briefly, thecollectionsbegan4 days
priortotheadministrationofthetestdrugandcontinued
for an additional 5 days after the drug was given. Iron
concentrations were determined by flame absorption
spectroscopy as presented in other publications
(Bergeron et al. 1990, 1996).
Drug preparation and administration:
iron clearance and chelator tissue distribution
In the iron clearing experiments, the rats were given 8
and 10 orally (p.o.) at a dose of 300 lmol/kg. An
additional group of rats was given 3 p.o. at a dose of
50 lmol/kg. The primates were given 8 and 10 p.o. at
a dose of 75 lmol/kg. In the chelator tissue distribu-
tion studies, the rats were given 3 and 8 s.c. at a dose
of 300 lmol/kg. Ligand 3 was also given p.o. at the
same dose. The drugs were administered to the rats
and primates as their monosodium salts (prepared by
the addition of 1 equiv of NaOH to a suspension of
the free acid in distilled water). Drug preparation
for the rodent urinary Kim-1 excretion studies
involving 1, 3, and Exjade are described below.
Biological methods
All animal experimental treatment protocols were
reviewed and approved by the University of Florida’s
Institutional Animal Care and Use Committee.
Calculation of iron chelator efficiency
Cannulation of bile duct in non-iron-overloaded rats
In the text below, the term ‘‘iron-clearing efficiency’’
(ICE) is used as a measure of the amount of iron
excretion induced by a chelator. The ICE, expressed
as a percent, is calculated as (ligand-induced iron
excretion/theoretical iron excretion) 9 100. To illus-
trate, the theoretical iron excretion after administra-
tion of one millimole of DFO (Fig. 1), a hexadentate
chelator that forms a 1:1 complex with Fe(III), is one
milli-g-atom of iron. Two millimoles of desferrithio-
cin (DFT, Fig. 1), a tridentate iron chelator that forms
a 2:1 complex with Fe(III), are required for the
theoretical excretion of one milli-g-atom of iron. The
The cannulation has been described previously (Ber-
geron et al. 1990, 1993). Bile samples were collected
from male Sprague–Dawley rats (400–450 g) at 3 h
intervals for up to 48 h. The urine sample(s) was taken
at 24 h intervals. Sample collection and handling are as
previously described (Bergeron et al. 1990, 1993).
Iron loading of C. apella monkeys
The monkeys were iron overloaded with intravenous
iron dextran as specified in earlier publications to
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Biometals (2011) 24:239–258
theoretical iron outputs of the chelators were gener-
ated on the basis of a 2:1 ligand:iron complex. The
efficiencies in the rats and monkeys were calculated
as set forth elsewhere (Bergeron et al. 1998, 1999).
Data are presented as the mean the standard error
of the mean; P-values were generated via a one-tailed
Student’s t-test in which the inequality of variances
was assumed; a P-value of \0.05 was considered
significant.
data are presented as the mean; P-values were
generated via a one-tailed student’s t-test, in which
the inequality of variances was assumed; a P-value of
\0.05 was considered significant.
28-day toxicity assessment of (S)-40-(HO)-DADFT-
norPE (3) in rats
Male Sprague–Dawley rats, 300–350 g, (n = 5) per
group were given 3 p.o. once daily for 28 days at a
dose of 56.9, 113.8 and 170.7 lmol/kg/d. Note that the
doses chosen for 3 are approximately 1, 2, and 3 times
the dose necessary to cause the excretion of 450 lg
Fe/kg in the primates, the suggested iron clearance
required to keep a thalassemia patient in negative iron
balance (Brittenham 1990). Because of the protracted
nature of the experiments (28 days), the animals were
not fasted. To minimize food content in the stomach,
the drug was not given until the afternoon. Additional
animals served as age-matched controls. Urine was
collected from the metabolic cages as described below
to allow for the determination of Kim-1 levels. The rats
were euthanized one day post drug (day 29), and
extensive tissues (Bergeron et al. 1999) were collected
for histopathological analysis.
Collection of chelator tissue distribution samples
from rodents
Male Sprague–Dawley rats (250–350 g) were given a
single s.c. injection of the monosodium salts of 3 and
8 prepared as described above at a dose of 300 lmol/
kg; 3 was also given p.o. at the same dose. At times
0.5, 1, 2, 4 and 8 h after dosing (n = 3) rats per time
point, the animals were euthanized by exposure to
CO2 gas. Blood was obtained via cardiac puncture
into vacutainers containing sodium citrate. The blood
was centrifuged, and the plasma was separated for
analysis. The liver, heart, pancreas, and kidneys were
removed from the animals and frozen.
Tissue analytical methods
Tissue samples of animals treated with (S)-40-(HO)-
DADFT-norPE (3) and (S)-30-(HO)-DADFT-norPE
(8) were prepared for HPLC analysis by homogeniz-
ing them in 0.5 N HClO4 at a ratio of 1:3 (w/v). Then,
as a rinse, CH3OH at a ratio of 1:3 (w/v) was added,
and the mixture was stored at -20ꢁC for 30 min. This
homogenate was centrifuged. The supernatant was
diluted with mobile phase A (95% buffer [25 mM
KH2PO4, pH 3.0]/5% CH3CN), vortexed, and filtered
with a 0.2 lm membrane. Analytical separation was
performed on a Discovery RP Amide C16 HPLC
system with UV detection at 310 nm as described
previously (Bergeron et al. 1998, 2003). Mobile
phase and chromatographic conditions were as
follows: solvent A, 5% CH3CN/95% buffer; solvent
B, 60% CH3CN/40% buffer. The concentrations were
calculated from the peak area fitted to calibration
curves by nonweighted least-squares linear regression
with Rainin Dynamax HPLC Method Manager soft-
ware (Rainin Instrument Co.). The method had a
detection limit of 0.25 lM and was reproducible and
linear over a range of 1–1000 lM. Tissue distribution
Drug preparation and administration: rodent
toxicity/urinary Kim-1 excretion studies
The impact of ligand 3 on urinary Kim-1 excretion was
evaluated in rodents under three chelator dosing
regimens. The drug was given p.o.: (1) once daily
during the course of the 28-day toxicity trial, described
above; (2) once daily at a dose of 384 lmol/kg/
d 9 10 days, and (3) twice daily at adose of 237 lmol/
kg/dose (474 lmol/kg/d) 9 7 days. The studies were
performed on rats with normal iron stores. Untreated
age-matched rats were used as negative controls.
Exjade was used as a positive control for the
384 lmol/kg/d 9 10 days dosing regimen, while def-
eritrin (1) served as a positive control for the 237 lmol/
kg/dose b.i.d. (474 lmol/kg/d) 9 7 days drug expo-
sure. The animals in the 10- and 7-day dosing protocols
were fasted overnight and were given the first dose of
the chelator first thing in the morning. The rats were fed
*3 h post-drug and had access to food for *5 h
before being fasted overnight. Ligands 1 and 3 were
administered to the rats p.o. as their monosodium salts,
123

Biometals (2011) 24:239–258
245
Results
prepared as described above. Exjade was administered
to the rats p.o. as a suspension in water.
Chemical synthesis
Collection of urine for Kim-1 studies
The synthesis of 30-polyethers 8 and 10 (Table 1) was
accomplished by modifying our efficient route to (S)-
30-(HO)-DADFT-PE (9) (Scheme 1) (Bergeron et al.
2008). In this instance, 2,3-dihydroxybenzonitrile
(11) (Bergeron et al. 2008) was converted to its
dianion with sodium hydride (2.0 equiv) in DMSO,
and a molar equivalent of tosylate 12 (Brunner and
Gruber 2004) or tosylate 14 (Brunner and Gruber
2004; Kitto et al. 2008) was added, resulting in 3-O-
alkylated nitrile 13 (71% yield) or 15 (70% yield),
respectively. Cyclocondensation of (S)-a-methyl cys-
teine (16) with precursor 13 at a pH of 6 generated
(S)-4,5-dihydro-2-[2-hydroxy-3-(3,6-dioxaheptyloxy)-
phenyl]-4-methyl-4-thiazolecarboxylic acid (8),
which has a shorter polyether chain than 9, in 93%
yield (66% overall). In like manner, D-amino acid 16
and nitrile 15 gave (S)-4,5-dihydro-2-[2-hydroxy-3-
(3,6,9,12-tetraoxatridecyloxy)phenyl]-4-methyl-4-
thiazolecarboxylic acid (10), which contains a longer
polyether substituent than 9, in 85% yield (60%
overall).
The rats were housed in individual metabolic cages.
Twenty-four hour urine samples were collected from
the metabolic cages-once every 4 days for the
animals in the 28-day toxicity study, and daily for
the rats in the 10- and 7-day drug exposure trials. In
all experiments, a baseline (day 0) urine sample was
collected and assessed for its Kim-1 content; each
animal served as its own control. The urine was
collected chilled as follows: the urine collection tube
was detached from the metabolic cage and inserted
into a 473 ml specimen container containing approx-
imately 240 g of gelled Techni Ice (the powder from
8 pouches/240 ml water). A styrofoam ring was
placed around the collection tube and the container
was frozen overnight at -80ꢁC. The following
morning, a 50-ml centrifuge tube was inserted into
the frozen urine collection tube. The frozen speci-
men containers were placed into hollowed out
styrofoam blocks that were 18 9 18 9 13 cm
(l 9 w 9 h). The styrofoam blocks were positioned
under the metabolic cages to allow for the collection
of the urine.
Biological results
Chelator-induced iron clearance in non-iron-
overloaded, bile duct-cannulated rodents
Performance of rodent urinary Kim-1 studies
The 40- and 30-polyether families of desazadesferri-
thiocin (DADFT) ligands were chosen for evaluation
because they best illustrate how the geometry and
position of a polyether backbone, e.g., (S)-4,5-dihy-
dro-2-[2-hydroxy-4-(3,6,9-trioxadecyloxy)phenyl]-4-
methyl-4-thiazolecarboxylic acid (4, Table 1) versus
(S)-4,5-dihydro-2-[2-hydroxy-3-(3,6,9-trioxadecyloxy)-
phenyl]-4-methyl-4-thiazolecarboxylic acid (9, Table 1),
can influence a chelator’s behavior. We have included
historical data in Table 1 to allow for comparative
purposes (Bergeron et al. 2005, 2006, 2008, 2010).
In the rodents, the polyether analogues are more
effective iron-clearing agents than their phenolic
counterparts, e.g., 3–5 versus 1 and 8–10 versus 6
(Table 1). For example, the ICE of 1 in rodents is
1.1 0.8% (Table 1). Introduction of a 3,6,9-triox-
adecyloxy polyether fragment to yield analogue 4
increased the ICE to 5.5 1.9% (P \ 0.003), a
The chilled urine was collected, vortexed, and
warmed to room temperature; any sediment in the
samples was allowed to settle. Equal amounts of the
sample diluent buffer and test urine (75 ll each) were
added to a clean test tube. The test tube was vortexed,
and 120 ll of the diluted urine was added to the
sample well of an R-Rena Strip (Vaidya et al. 2009).
The result was read 20 min later using a ReaScan
Test Reader. The quantity of Kim-1 excreted in the
urine per day was calculated by multiplying the
concentration of Kim-1 (ng/ml urine) 9 24-h urine
volume, divided by the weight of the animal. The
result is expressed as urinary Kim-1 (ng/kg/24 h).
Data are presented as the mean the standard error
of the mean; P-values were generated via a one-tailed
Student’s t-test in which the inequality of variances
was assumed; a P-value of \0.05 was considered
significant.
123

246
Biometals (2011) 24:239–258
Table 1 Iron-clearing efficiency of desferrithiocin analogues administered orally to rodents and primates
Compound
Rodent ICEa
Primate ICEc
Performance
ratiod
HO
OH
1.1 0.8% [100/0]
16.8 7.2% [88/12]
15.3
N
CH3
CO2H
S
(S)-4′-(HO)-DADFT (1)
CH3O
OH
6.6 2.8% [98/2]
24.4 10.8% [91/9]
3.7
N
CH3
CO2H
S
(S)-4′-(CH3O)-DADFT (2)
26.7 4.7%b [97/3]
26.3 9.9% [93/7] (capsule)
1.0
1.1
O
OH
O
OCH3
N
CH3
28.7 12.4% [83/17] (sodium salt)
CO2H
S
(S)-4′-(HO)-DADFT-norPE (3)
O
OH
5.5 1.9% [90/10]
25.4 7.4% [96/4]
9.8 1.9% [52/48]
23.1 5.9% [83/17]
22.5 7.1% [91/9]
22.5 6.4% [86/14]
23.0 4.1% [95/5]
4.6
0.8
5.0
1.8
1.5
2.2
O
O
N
CH3
OCH3
CO2H
S
(S)-4′-(HO)-DADFT-PE (4)
O
OH
12.0 1.5% [99/1]
4.6 0.9% [98/2]
12.4 3.5%b [99/1]
15.1 2.0% [99/1]
10.6 4.4%b [95/5]
O
O
O
N
CH3
CO2H
OCH3
S
(S)-4′-(HO)-DADFT-homoPE (5)
OH
OH
N
CH3
CO2H
S
(S)-3′-(HO)-DADFT (6)
CH3O
OH
N
CH3
CO2H
S
(S)-3′-(CH3O)-DADFT (7)
O
O
O
CH3
OH
N
CH3
CO2H
S
(S)-3′-(HO)-DADFT-norPE (8)
O
O
O
OH
OCH3
N
CH3
CO2H
S
(S)-3′-(HO)-DADFT-PE (9)
123

Biometals (2011) 24:239–258
247
Table 1 continued
Compound
Rodent ICEa
Primate ICEc
Performance
ratiod
O
12.4 1.7% [98/2]
9.6 4.9% [25/75]
0.8
O
O
OH
O
N
CH3
CO2H
S
OCH3
(S)-3′-(HO)-DADFT-homoPE (10)
a
In the rodents [n = 3 (3), 4 (2, 4–6, 9, 10), 5 (7, 8), 8 (1)], the drugs were given p.o. at a dose of 300 lmol/kg. The drugs were
administered in capsules (3), solubilized in distilled water (4), or were given as their monosodium salts, prepared by the addition of 1
equiv of NaOH to a suspension of the free acid in distilled water (1–2, 5–10). The efficiency of each compound was calculated by
subtracting the 24 or 48-h iron excretion of control animals from the iron excretion of the treated animals. The number was then
divided by the theoretical output; the result is expressed as a percent. The relative percentages of the iron excreted in the bile and
urine are in brackets. The ICE data for 1, 2, and 4 are from Bergeron et al. (2006); 3 and 5 are from Bergeron et al. (2010); 6 and 7 are
from Bergeron et al. (2005); 9 is from Bergeron et al. (2008)
b
ICE is based on a 48 h sample collection period
c
In the primates [n = 3 (9), 4 (2, 3 in capsules, 4–6, 8, 10), 5 (7), or 7 (1, 3 as the monosodium salt)], the chelators were given p.o. at
a dose of 75 lmol/kg (3, 5, 8, 10) or 150 lmol/kg), (1, 2, 4, 6, 7, 9). The drugs were administered in capsules (3), solubilized in either
40% Cremophor RH-40/water (2, 6, 7), distilled water (4), or were given as their monosodium salts, prepared by the addition of 1
equiv of NaOH to a suspension of the free acid in distilled water (1, 3, 5, 8–10). The efficiency was calculated by averaging the iron
output for 4 days before 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 percent. The relative percentages of the iron excreted in the feces
and urine are in brackets. The ICE data for 1 is from Bergeron et al. 1999; 2 is from Bergeron et al. (2003); 3 and 5 are from Bergeron
et al. (2010); 4 is from Bergeron et al. (2006); 6 and 7 are from Bergeron et al. (2005), 9 is from Bergeron et al. (2008)
d
Performance ratio is defined as the mean ICEprimates/ICErodents
five-fold increase (Bergeron et al. 2006). The shorter
ether analogue, the 3,6-dioxaheptyloxy ligand (3),
had an ICE of 26.7 4.7% (Bergeron et al. 2010).
Lengthening of the polyether chain to a 3,6,9,12-
tetraoxatridecyloxy group, 5, resulted in a compound
with an ICE of 12.0 1.5% (Table 1). A similar
trend was also observed with the 30-polyether family
of ligands (Table 1). The ICE of 6 in the rats is
4.6 0.9% (Bergeron et al. 2005), while the ICE of
the corresponding polyether analogue 9 is more than
two-fold greater, 10.6 4.4% (P \ 0.04). The shorter
ether ligand, the 3,6-dioxaheptoxy analogue, 8, had an
ICE (15.1 2.0) that was more than three-fold greater
than that of its phenolic counterpart, 6 (P \ 0.001).
The ICE of the longer 3,6,9,12-tetraoxatridecyloxy
polyether analogue, 10, was essentially the same as
Scheme 1 Synthesis of (S)-4,5-dihydro-2-[2-hydroxy-3-(3,6-
dioxaheptyloxy)phenyl]-4-methyl-4-thiazolecarboxylic Acid
(8) and (S)-4,5-Dihydro-2-[2-hydroxy-3-(3,6,9,12-tetraoxatride-
cyloxy)phenyl]-4-methyl-4-thiazolecarboxylic Acid (10)a.
aReagents and conditions: (a) 60% NaH (2.0 equiv), DMSO,
CH3[O(CH2)2]2OTs (12, 1.0 equiv), 71% (13) or CH3[O(CH2)2]4
OTs (14, 1.0 equiv), 70% (15); (b) CH3OH (aq), pH 6, 70ꢁC,
24 h, 93% (8) or 85% (10)]
123

248
Biometals (2011) 24:239–258
that of 9, 12.4 1.7% versus 10.6 4.4%, respec-
tively (P [ 0.05). Finally, the ICE in rats of the 40-
polyether parent, 4, is approximately half that of 30-
polyether parent, 9 (Table 1). However, the opposite
is true when comparing the ICE of the shorter ether
analogues, 3 versus 8: 3 is nearly twice as effective as 8
(P\ 0.02). In addition, the ICE of 3 is 2.5 times greater
than that of the ligand currently in clinical trials, 9
(P\ 0.005). Interestingly, the ICE of the 40- and 30-
3,6,9,12-tetraoxa analogues 5 and 10 are virtually
identical, 12.0 1.5% and 12.4 1.7%, respectively
(Table 1).
given p.o. to the bile duct-cannulated rats at a dose
of 300 or 50 lmol/kg (Fig. 2). The ICE of 3 was
26.7 4.7% when it was given p.o. at 300 lmol/kg,
and 22.8 5.9% when the dose was reduced to
50 lmol/kg (P [ 0.05). Therefore, in contrast to what
was seen with 4 or 9, the deferration induced by
analogue 3 was not saturable over this dose range. This
suggests much greater flexibility in dosing with 3,
which could be critical in managing patients with acute
iron overload, e.g., iron poisoning.
Chelator-induced iron clearance
in iron-overloaded primates
Chelator-induced iron clearance in non-iron-
overloaded, bile duct-cannulated rodents: dose
response studies
The iron clearance data for ligands 1–10 in the
primates are described in Table 1. Historical data
have been included to allow for comparative purposes
(Bergeron et al. 1999, 2003, 2005, 2006, 2008, 2010).
Ligand 1 had an ICE of 16.8 7.2%, while the
ICE of the 40-parent polyether (4) is 25.4 7.4%
(Bergeron et al. 2006). The shorter 3,6-dioxa ana-
logue, 3, had an ICE of 26.3 9.9% when it was
given to the primates in capsules (Bergeron et al.
2010); the ICE was virtually identical when 3 was
administered by gavage as its sodium salt,
28.7 12.4% (P [ 0.05). The ICE of the longer
3,6,9,12-tetraoxa analogue (5) was significantly less
than that of either 3 or 4, 9.8 1.9% (P \ 0.001).
The ICE of 6 and the 30-polyether ligands, 7–9, were
very uniform, 22–23% (Table 1). However, as with 5,
the ICE the longer 3,6,9,12-tetraoxatridecyloxy
30-polyether analogue (10) was significantly less than
8 or 9, 9.6 4.9% (P \ 0.001).
The ICE of the majority of the ligands in Table 1
are significantly greater in the iron-overloaded pri-
mates than in the non-iron-overloaded rats. In this
regard, the performance ratio (PR), defined as the
mean ICEprimate/ICErodent, provides an excellent
decision-making tool for helping to determine which
drugs to move forward. When iron chelators work
well in both primates and rodents (PR close to 1.0),
the probability of them also performing well in
humans is higher. The three most effective polyether
analogues, in terms of PR (Table 1), are 3 (PR 1.1), 8
(PR 1.5), and 9 (PR 2.2), suggesting that the ligands
would be chosen to move forward in the following
order 3 [ 8 [ 9. At the onset of the clinical trial
focused on 9, no data were available for ligand 3 or 8.
This is unfortunate, as 3 has many advantages over
In previous dose response studies with ligand 1, 4, and
9, given p.o. to bile duct-cannulated rats (Bergeron
et al. 2008), we demonstrated that while the ICE of the
parent drug 1 in rodents was uniformly poor at 300 and
150 lmol/kg (\2%), the polyether analogues 4 and 9
were significantly more efficient at all doses (Fig. 2).
However, their deferration properties were shown to be
saturable (Bergeron et al. 2008). The ICE for 4
increased nearly four-fold, from 5.5 1.9% to
21.7 3.5%, as the dose was decreased from 300 to
50 lmol/kg. The ICE for 9 also increased, nearly two-
fold, on moving from 300 lmol/kg (10.6 4.4%) to
50 lmol/kg (20.7 4.4%). In the current study, 3 was
30
25
20
15
Deferitrin (1)
10
(S)-4'-(HO)-DADFT-norPE (3)
(S)-4'-(HO)-DADFT-PE (4)
5
(S)-3'-(HO)-DADFT-PE (9)
0
300
150
50
Dose (µmol/kg)
Fig. 2 Dose-response curves for compounds 1, 3, 4, and 9
administered p.o. to bile duct-cannulated rats. The ICE data,
expressed as a percentage, (y-axis) for ligands 1, 4, and 9 are
from Bergeron et al. (2008)
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Biometals (2011) 24:239–258
249
ligand 8 or 9. In addition to its superior ICE in rats, 3
also achieves much higher concentrations than 8 or 9
in the organs most adversely affected by excess iron:
the liver, heart, and pancreas (discussed below).
Finally, 3 is a crystalline solid, while 8 and 9 are oils.
(Fig. 4). At 0.5 h, the concentration of ligand 3 in the
heart was 186 54 nmol/g wet weight, while that of 4
was 36 8 nmol/g wet weight (P \ 0.02 versus 3)
and that of 9 was 59 11 nmol/g wet weight
(P \ 0.03 versus 3). By 2 h post-drug, the chelator
concentrations of all of the drugs had decreased to less
than 20 nmol/g wet weight, and were virtually unde-
tectable at the 4 and 8 h time points (Fig. 4).
Chelator tissue distribution in rodents
The tissue distribution description will focus on the
40-DADFT ligands 1, 3, and 4 (Table 1) and on the
30-DADFT analogues 8 and 9 (Table 1). In each
instance, we will consider the liver, heart, plasma,
pancreas, and kidney. In these studies, non-bile duct-
cannulated (intact) rats were given a single 300-
lmol/kg dose of the drug s.c. (Fig. 3) and/or p.o.
(Fig. 4). The rats (n = 3/group) were sacrificed 0.5,
1, 2, 4 or 8 h post-drug. The historical data for
ligands 1, 4, and 9 have been included for compar-
ative purposes (Bergeron et al. 2006, 2007, 2008).
The 40-norpolyether (3) achieved the highest level
in all of the tissues evaluated. This was the case with
3 regardless of the route of administration. In the liver
at 0.5 h, the relative concentrations of the ligands
given to the rats s.c. were 3 [ 4 [ 9 [ 8 [ 1
(Fig. 3). A similar trend was also observed at the
1 h time point, except that the concentrations of 4 and
9 were virtually identical. Although all of the ligands
were still detected in the liver at 8 h, the concentra-
tions had decreased to less than 50 nmol/g wet weight
(Fig. 3). When the chelators were given p.o., the
relative concentration of the drugs in the liver at 0.5
and 1 h were 3 [ 9 [ 4 [ 1 (Fig. 4). Once again, by
8 h, the concentration of the drugs in the liver had
decreased to less than 50 nmol/g wet weight (Fig. 4).
Note that the p.o. tissue distribution of analogue 8 has
not been assessed, simply because of the low chelator
tissue levels observed after s.c. dosing.
The plasma chelator concentration data are con-
sistent with the idea that the ligands are cleared
quickly whether they are given s.c (Fig. 3) or p.o.
(Fig. 4). The highest relative concentration of the
ligands in the plasma are seen at the 0.5 h time point:
3 [ 4 [ 1 & 8 [ 9 when the chelators were given
s.c. (Fig. 3), and (3 ꢁ 4 [ 9 [ 1) when the drugs
were given p.o. (Fig. 4). By 2 h post-drug, the drug
concentrations have decreased to less than 50 lM
when dosed s.c., and less than 20 lM when they were
administered p.o. No drug was detectable at the 8 h
time point (s.c. or p.o.).
In the pancreas, at 0.5 h, ligand 3 achieves a
concentration of 98 6 nmol/g wet weight when the
drug is given s.c. (Fig. 3), and 110 15 nmol/g wet
weight when it is dosed p.o. (Fig. 4). At 1 h, the
concentration of 3 is still elevated, 92 9 nmol/g wet
weight and 67 7 nmol/g wet weight when dosed s.c.
and p.o., respectively. The pancreatic chelator content
of 3 at 0.5 h is significantly greater than that of 9, the
ligand currently undergoing clinical trials: six-fold
greater (P \0.001) when the drugs are given s.c.
(Fig. 3) and nearly three-fold greater (P \0.005) when
they are dosed p.o. (Fig. 4). By 4 h, ligand 1 is no longer
detectable when the ligands are dosed s.c., while the
concentration of the other chelators have decreased to
11–15 nmol/g wet weight. When the chelators are given
p.o., ligand 4 is no longer detectable at 4 h, while the
concentration of the other chelators are less than
6 nmol/g wet weight. Very little if any of the ligands
are detected at the 8 h time point (s.c. or p.o.).
In the heart, at 0.5 h, ligand 3 given s.c. achieved
a concentration of 178 26 nmol/g wet weight
(Fig. 3). This concentration is almost four-fold higher
In the kidney, we previously demonstrated that the
40-polyether (4) was much less nephrotoxic than
deferitrin (1), which, at the time, was consistent with
the relative tissue levels at the 2 h time point: the
concentration of 4 was significantly less than that of 1
(Bergeron et al. 2006). However, renal concentration
data from samples collected earlier than 2 h, e.g., 0.5
and 1 h, revealed that the tissue chelator concentra-
tions of the two drugs were virtually identical to each
other (Bergeron et al. 2007). Thus, the reduced
than that of
4 (47 15 nmol/g wet weight,
P \ 0.002), and more than eight-fold greater than that
of the ligand currently undergoing clinical trials, 9
(22 13 nmol/g wet weight, P \ 0.002). At 1 h, the
order of concentration in the heart is 3 ꢁ 4 [
9 [ 8 & 1. By 2 h post-drug, the chelator concentra-
tions had decreased to less than 20 nmol/g wet weight,
and were undetectable at the 8 h time point. A similar
trend was observed when the drugs were given p.o.
123

250
Biometals (2011) 24:239–258
(S)-4’-(HO)-DADFT (1)
LIVER
800
700
600
500
400
300
200
100
0
(S)-4’-(HO)-DADFT-norPE (3)
(S)-4’-(HO)-DADFT-PE (4)
(S)-3’-(HO)-DADFT-norPE (8)
(
S
)-3’-(HO)-DADFT-PE (9)
0.5
1
2
4
8
Time (h)
PANCREAS
HEART
200
175
150
125
100
200
175
150
125
100
75
75
50
25
0
50
25
0
0.5
1
2
4
8
0.5
1
2
4
8
Time (h)
Time (h)
800
700
600
500
400
300
200
PLASMA
KIDNEY
500
400
300
200
100
0
100
0
0.5
1
2
4
8
0.5
1
2
4
8
Time (h)
Time (h)
Fig. 3 Tissue distribution in the liver, heart, plasma, pancreas,
and kidney of rats treated with DADFT analogues 1, 3, 4, 8,
and 9 given s.c. at a dose of 300 lmol/kg. The concentrations
(y-axis) are reported as nmol compound per g wet weight of
tissue, or as lM (plasma). For all time points, n = 3. The data
for ligands 1, 4 and 9 are from Bergeron et al. (2006, 2007,
2008)
nephrotoxicity of 4 versus 1 could not be explained
simply by lower kidney chelator levels. In the current
study, at 0.5 h, the relative concentration in the
kidney of the drugs given s.c. (Fig. 3) is 3 ꢁ 4
& 1 [ 8 [ 9. At 1 h, the order is 3 [ 9 & 4 [
8 [ 1; at 2 h, the order is 8 & 9 [ 3 [ 1 [ 4.
Finally, at the 8 h time point, the concentration of
all of the chelators are less than 15 nmol/g wet
weight. A similar trend was observed when the drugs
were given p.o. (Fig. 4). At 0.5 h, the relative
concentration of the chelators in the kidney is
3 [ 9 [ 1 [ 4. At 1 h, the order is 3 [ 1 [ 9 [ 4,
while at 2 h, the concentration order is 1 [ 3 [
4 [ 9. At 8 h, the renal concentration of 1 is
31 nmol/g wet weight, while those of the other
chelators are less than 10 nmol/g wet weight (Fig. 4).
123

Biometals (2011) 24:239–258
251
LIVER
800
700
600
500
400
300
200
100
(S)-4’-(HO) -DADFT (1)
(S)-4’-(HO) -DADFT-norPE (3)
(S)-4’-(HO) -DADFT-PE (4)
(S)-3’-(HO) -DADFT-PE (9)
0
0.5
1
2
4
8
Time (h)
PANCREAS
HEART
200
200
175
150
125
100
75
175
150
125
100
75
50
50
25
25
0
0
0.5
1
2
4
8
0.5
1
2
4
8
Time (h)
Time (h)
KIDNEY
800
700
600
500
400
300
200
100
0
PLASMA
500
400
300
200
100
0
0.5
1
2
4
8
0.5
1
2
4
8
Time (h)
Time (h)
Fig. 4 Tissue distribution in liver, heart, plasma, pancreas,
and kidney of rats treated with DADFT analogues 1, 3, 4, and 9
given p.o. at a dose of 300 lmol/kg. The concentrations
(y-axis) are reported as nmol compound per g wet weight of
tissue, or as lM (plasma). For all time points, n = 3. The data
for ligands 1, 4, and 9 are from Bergeron et al. (2008)
Toxicity evaluation of ligand 3 in rodents: impact
on urinary Kim-1 excretion
submitted for histological analysis; no drug-related
abnormalities were found. In addition, the rats’ BUN
and SCr levels were within the normal range.
However, it is well documented that BUN and SCr
are poor predictors of acute kidney injury. Therefore,
in the current study, we elected to assess the affect of
ligand 3 on Kim-1, a novel urinary biomarker that
allows for the detection of very subtle changes in
renal tubule integrity (Han et al. 2002; Bonventre
2009). Kim-1 appears in the urine very early in
response to exposure to a nephrotoxic agent, far
sooner than increases in BUN or SCr can be detected
The high concentration of ligand 3 found in the
kidney during the course of the tissue distribution
studies after s.c. or p.o. administration caused some
concern as to how this could affect the ligand’s
toxicity profile. In a previous experiment, 3 was given
p.o. to rats with normal iron stores once daily at a
dose of 384 lmol/kg/d for 10 d (Bergeron et al.
2010). The drug was found to be well tolerated.
Extensive tissues (Bergeron et al. 1999) were
123

252
Biometals (2011) 24:239–258
(Zhou et al. 2008; Vaidya et al. 2006). Accordingly,
the impact of ligand 3 on urinary Kim-1 excretion
was evaluated in rodents under three chelator dosing
regimens. The drug was given p.o.: (1) once daily
during the course of the 28 d toxicity trial described
above; (2) once daily at a dose of 384 lmol/kg/
d 9 10 d, and (3) twice daily at a dose of 237 lmol/
kg/dose (474 lmol/kg/d) 9 7 d. The studies were
performed on rats with normal iron stores. Untreated
age-matched rats were used as negative controls.
Exjade was used as a positive control for the 384 lmol/
kg/d 9 10 days dosing regimen, while deferitrin (1)
served as a positive control for the 237 lmol/kg/dose
b.i.d. (474 lmol/kg/d) 9 7 d drug exposure.
The urinary Kim-1 content of the untreated, age-
matched negative control rats (n = 5) from the 28-
day toxicity trial of 3 was monitored for 24-h every
4 days, on day 0, 4, 8, 12, 16, 20, 24, and 28. The data
are expressed as urinary Kim-1 (ng/kg/24 h). Very
little Kim-1 was found in the urine of the untreated
animals at any time (Fig. 5a). The rats were eutha-
nized on day 29; their BUN at that time was
19 3 mg/dl, while their SCr was 0.5 0 mg/dl.
Note that these values are well within the normal
range for this species: 9–30 mg/dl for BUN, and
0.4–1.0 mg/dl for SCr (Antech Diagnostics).
from the untreated control animals and the rats
treated with 3 at 170.7 lmol/kg/d 9 28 d were sent
out for histopathology; no drug-related abnormalities
were found.
In a second study, we chose to assess the impact of
Exjade and 3 on urinary Kim-1 excretion when the
chelators were given to the rats p.o. once daily at a
dose of 384 lmol/kg/d 9 10 d. Note that this is our
‘‘standard’’ 10-day toxicity dosing regimen (Bergeron
et al. 1999); the dose is equivalent to 100 mg/kg/d of
the DFT sodium salt, and corresponds to a dose of
143 mg/kg/d of Exjade. Exjade was selected to serve
as a positive control for this study due to its well-
known nephrotoxicity issues (Nick et al. 2002;
Nisbet-Brown et al. 2003). Urinary Kim-1 levels
were assessed at 24-h intervals. None of the rats
(n = 5) survived the planned 10-day exposure to
Exjade. One rat was found dead the morning of day 5.
He had received four doses of the drug. The remaining
rats became moribund and were sacrificed after being
given the drug for 7 days (two rats) or 8 days (two
rats). This is consistent with what had been reported
during the rodent preclinical toxicity assessments of
Exjade (Nick et al. 2002). The rats’ baseline (day 0)
urinary Kim-1 value was 10 8 ng/kg/24 h (Fig. 5c).
After 2 days of Exjade, the Kim-1 had increased to
27 15 ng/kg/24 h (P \ 0.04). After 4 days of
Exjade, the Kim-1 was 76 39 ng/kg/24 h, and by
day 8 the urinary Kim-1 of the two surviving rats was
1010 173 ng/kg/24 h. Blood was taken from the
moribund animals immediately prior to sacrifice; the
serum was assessed for its BUN and creatinine
content. The rats’ BUN was 139 71 mg/dl, while
their SCr was 2.5 0.7 mg/dl. These values are well
outside of the normal range. In fact, the BUN and SCr
of the Exjade treated rats are seven-fold (P \ 0.03)
and five-fold (P \ 0.01) greater, respectively, than
those of the age-matched control rats. In addition, as
no blood was obtained from the animal that was found
dead, these values likely underestimate the actual
impact of Exjade on these parameters.
Comparable results were found when rats (n = 5/
dose) were given ligand 3 p.o. once daily for 28 days
at a dose of 56.9, 113.8, or 170.7 lmol/kg/day. These
doses are approximately 1, 2, and 3 times the dose
necessary to excrete 450 lg Fe/kg in the primates,
the suggested iron clearance required to keep a
thalassemia patient in negative iron balance (Britten-
ham 1990). All of the rats survived the exposure to
the drug. Urine was collected as described above for
the control rats. The urinary Kim-1 data for the
animals in the 170.7 lmol/kg/d group are presented
in Fig. 5b. Their baseline urinary Kim-1 content was
8
2 ng/kg/24 h and did not increase above this
value throughout the course of the study. The rats
were euthanized one day post drug (day 29); their
BUN and SCr at that time were in the normal range.
In fact, their BUN (20 1 mg/dl) and SCr
(0.5 0 mg/dl) were virtually identical to those of
the age-matched control rats. Similar results for
urinary Kim-1 excretion, BUN, and SCr, were also
observed in the rats given 3 at the two lower doses
(data not shown). Extensive tissues (Bergeron et al.
1999) were collected from all of the rats. The tissues
Conversely, a separate group of rats (n = 5) was
given analogue 3 p.o. under an identical dosing
regimen, 384 lmol/kg/d for 10 d. Although this study
had been performed previously (Bergeron et al.
2010), it was repeated to allow for the determination
of urinary Kim-1 levels. All of the rats survived the
chelator dosing period. Urinary Kim-1 levels were
assessed at 24-h intervals. The baseline (day 0)
123

Biometals (2011) 24:239–258
253
B
1400
1400
1200
1000
800
600
400
200
0
A
Age-matched Controls
(S)-4'-(HO)-DADFT-norPE (3)
170.7 µmol/kg/d x 28 d
1200
1000
800
600
400
200
0
0
4
8
12 16 20 24 28
0
4
8
12 16 20 24 28
Day
Day
Exjade
384 µmol/kg/d x 10 d
C
D
1400
1200
1000
800
600
400
200
0
1400
(S)-4'-(HO)-DADFT-norPE (3)
384 µmol/kg/d x 10 d
1200
1000
800
600
400
200
0
Dead
10
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9 10
Day
Day
E
F
1400
1200
1000
800
600
400
200
0
1400
1200
1000
800
600
400
200
0
(S)-4'-(HO)-DADFT-norPE (3)
237 µmol/kg BID x 7 d
Deferitrin (1)
237 µmol/kg BID x 7 d
0
1
2
3
4
5
6
7
0
1
2
3
4
5
6
7
Day
Day
Fig. 5 Urinary Kim-1 excretion, expressed as Kim-1 (ng/kg/
24 h), for group: a untreated age-matched control rats; b rats
treated with 3 p.o. once daily at a dose of 170.7 lmol/kg/
d 9 28 d; c rats given Exjade p.o. once daily at a dose of
384 lmol/kg/d; d rats given 3 p.o. once daily at a dose of
384 lmol/kg/d 9 10 d; e rats given deferitrin (1) p.o. twice
daily at a dose of 237 lmol/kg/dose (474 lmol/kg/d) 9 7 d,
and f rats given 3 p.o. twice daily at a dose of 237 lmol/kg/
dose (474 lmol/kg/d) 9 7 d. Note that none of the rats
survived the planned 10-day exposure to Exjade. For groups
(a–d, f), n = 5; group (e), n = 3
urinary Kim-1 content of the 3-treated rats was
9
(Fig. 5d). The rats were sacrificed on day 11
(one day post exposure). Their BUN and SCr at
that time were 19 1 mg/dl and 0.4 0.1 mg/dl,
9 ng/kg/24 h and remained within error of this
value throughout the chelator dosing regimen
123

254
Biometals (2011) 24:239–258
Discussion
respectively, well within the normal range, and
virtually identical to those of the age-matched control
rats.
Deferitrin (1, Fig. 1) was abandoned in clinical trials
as an iron chelator for the treatment of iron overload
disease because of its nephrotoxicity (Galanello et al.
2007). However, subsequent investigations revealed
that replacing the 40-(HO) of 1 with a 3,6,9-
trioxadecyloxy group, ligand 4 (Table 1), increased
iron clearing efficiency (ICE) and ameliorated the
renal toxicity in rats of 1 (Bergeron et al. 2006). The
ICE in both rodents and primates and the absence of
toxicity seen with the 40-polyether (4) compelled us
to investigate what the impact of fixing the 3,6,9-
polyether chain to the aromatic ring of DADFT at
positions other than the 40-carbon had on ICE and
ligand tissue distribution (Bergeron et al. 2007). One
of these ligands, in which the polyether chain has
been fixed to the 30-carbon, 9 (Table 1), is currently
undergoing clinical trials. Although the preclinical
toxicity profile of 9 was benign, the clinical results
remain to be elucidated.
In a final experiment, we chose to replicate our
renal perfusion chelator dosing protocol (Bergeron
et al. 2006) with deferitrin (1) and analogue 3, in
which rats are given a chelator b.i.d at a dose of
237 lmol/kg/dose (474 lmol/kg/d, equivalent to
120 mg/kg/d of 1) 9 7 d, to determine their impact
on urinary Kim-1 excretion. Deferitrin (1) served as a
positive control. Recall that we had previously
demonstrated that the proximal tubules of rats treated
with 1 p.o. b.i.d. 9 7 d showed regional, moderate to
severe vacuolization, a loss of brush border, and
extrusion toward the lumen (Bergeron et al. 2006). In
the current study, all of the 1-treated rats (n = 3)
survived the 7-day chelator dosing period. Urine
samples were collected at 24-h intervals. The rats’
baseline (day 0) urinary Kim-1 content was
13 8 ng/kg/24 h (Fig. 5e). After 3 days of expo-
sure to 1, the urinary Kim-1 had increased to
69 47 ng/kg/24 h. At the end of the 7-day dosing
period, the urinary Kim-1 had further increased to
189 187 ng/kg/24 h (Fig. 5e). The rats were eutha-
nized on day 8; their BUN at that time was
32 13 mg/dl, while their SCr was 1.3 1.0 mg/dl.
Note that although the rats’ BUN and SCr levels are
only slightly outside of the normal range for this
species, these values are 1.6 and 2.6 times greater
than the BUN and SCr, respectively, of the age-
matched control rats.
These findings encouraged an assessment of how
altering the length of the polyether chain would affect
a ligand’s ICE, lipophilicity, and physiochemical
properties. In a previous study, the 3,6,9-trioxadecyl-
oxy substituent at the 40-position of ligand 4 was both
shortened to a 3,6-dioxaheptyloxy moiety, providing
3, and lengthened to a 3,6,9,12-tetraoxatridecyloxy
group, providing 5 (Bergeron et al. 2010). These
same manipulations have now been carried out with
the corresponding 30-polyether parent (9), resulting in
the shorter 3,6-dioxaheptyloxy polyether analogue
(8), and the longer 3,6,9,12-tetraoxatridecyloxy
ligand (10). Note that while the 40- and 30-polyether
parents, 4, and 9, respectively, and analogues 5, 8,
and 10 are oils, ligand 3 is a crystalline solid
(Bergeron et al. 2010). The iron clearance properties
of 3 and 5 in rodents and primates were reported in a
previous publication (Bergeron et al. 2010). These
measurements have now been made with the corre-
sponding 30-polyether analogues, 8 and 10, in rodents
and primates (Table 1); a dose–response study with 3
in the rats has also been carried out (Fig. 2). In
addition, the chelator tissue distribution of ligand 3
and 8 in rats after s.c. (Fig. 3) and/or p.o. (Fig. 4)
dosing has been assessed. Finally, the impact of the
lead candidate, 3, on rat urinary Kim-1 excretion has
been evaluated under three chelator dosing regimens
(Fig. 5).
A separate group of rats (n = 5) were given
analogue 3 p.o. under an identical dosing regimen,
237 lmol/kg/dose b.i.d. (474 lmol/kg/d) 9 7 d.
All of the rats survived the dosing period. Urinary
Kim-1 levels were assessed at 24-h intervals. The
rats’ baseline (day 0) urinary Kim-1 content was
18 9 ng/kg/24 h and stayed within error of this
value for the duration of the drug exposure (Fig. 5f).
The rats were euthanized on day 8; their BUN at
that time was 16 3 mg/dl, while their SCr was
0.5 0.1 mg/dl. Both values are well within the
normal range for this species, and are virtually
identical to those of the age-matched control rats.
Thus, despite the high levels of 3 observed in the
kidney during the chelator tissue distribution studies,
ligand 3 does not have an adverse affect on urinary
Kim-1 excretion, even when the drug is administered
to the rats for up to 28 d.
123

Biometals (2011) 24:239–258
255
The 40-3,6-dioxaheptyloxy analogue, ligand 3, had
an ICE in rats that was nearly five-fold greater than
that of its parent, 4; it was also very effective
(ICE [ 26%) in the iron-loaded primates (Bergeron
et al. 2010). The longer 3,6,9,12-tetraoxatridecyloxy
analogue of 4, chelator 5, had twice the ICE in
rodents of 4, but its ICE in primates was reduced
relative to that of 4 (Bergeron et al. 2010). In the
current study, the ICE in rats of the shorter 30-
polyether ligand, 8, was 1.4-fold greater than that of
its parent, 9. The ICE of the longer 3,6,9,12-
tetraoxatridecyloxy polyether analogue, 10, in rats
was essentially the same as that of 9. Interestingly,
while the ICE in rats of the 40-polyether parent, 4, is
approximately half that of 30-polyether parent, 9, the
opposite is true when comparing the ICE of the
shorter ether analogues, 3 versus 8: 3 is nearly twice
as effective as 8. In addition, the ICE of 3 in rats is
2.5-fold greater than that of the ligand currently in
clinical trials, 9. Finally, in rodent chelator dose–
response studies, while the deferration induced by 4
and 9 was shown to be saturable as the dose was
decreased from 300 to 50 lmol/kg (Bergeron et al.
2008), this was not the case with ligand 3 (Fig. 2).
This suggests much greater flexibility in dosing with
3, which could be critical in managing patients with
acute iron overload, e.g., iron poisoning. In the
primates, the ICE of 8 was within error of that of
the 30-polyether parent, 9. However, the ICE of
the tetraoxatridecyloxy-substituted analogue, 10, in
primates was significantly less than 8 or 9. The
performance ratio, ICEprimate/ICErodent, suggests that
the ligands would be chosen to move forward in the
following order 3 [ 8 [ 9. However, at the onset of
the clinical trial focused on 9, no data were available
for ligand 3 or 8.
However, high levels of ligand 3 were also found in
the kidney after s.c. or p.o. dosing. This caused some
concern as to how this might affect the ligand’s
toxicity, particularly nephrotoxicity, profile.
Recall that the major hurdle in exploiting the DFT
pharmacophore, e.g., deferitrin (1), as an orally active
iron chelator was its nephrotoxicity (Galanello et al.
2007). Iron chelator-induced proximal tubule damage
is not uncommon. In fact, one of the problems
associated with a currently accepted iron overload
treatment, Exjade, is proximal tubule-derived renal
damage (Nick et al. 2002; Yacobovich et al. 2010;
Exjade Prescribing Information). Unfortunately, bio-
markers, e.g., BUN and SCr levels, often do not
increase until a serious loss of renal function has
unfolded. This results in the performance of long-
term, expensive exposure studies until sufficient
evidence of nephrotoxicity presents to warrant ter-
minating the trial. However, this problem has now
been overcome with the discovery of kidney injury
molecule-1 (Kim-1, rodent) and (KIM-1, human).
Kim-1 is a type 1 transmembrane protein located
in the epithelial cells of proximal tubules (Han et al.
2002). The ectodomain of the Kim-1 proximal tubule
protein is released into the urine very early after
exposure to a nephrotoxic agent or ischemia (Bailly
et al. 2002); it appears far sooner than increases in
BUN or SCr are detected. Although urinary Kim-1
measurements have been performed for several years
using enzyme-linked immunosorbent assays (Bailly
et al. 2002) or a Luminex assay (van Timmeren et al.
2006) involving multi-analyte profiling beads
(xMAP) technologies, these assessments required
3–4 h of assay time and were dependent on a large
analyzer. However, to circumvent these issues, Bio-
Assay Works has recently developed RenaStick, a
direct lateral flow immunochromatographic assay,
which allows for the rapid detection and quantitation
of rat urinary Kim-1 (Vaidya et al. 2009). A similar
test kit is also available to determine the KIM-1
content of human urine. This now makes it possible
in both rodents and patients to uncover potential
chelator-induced renal toxicity early on.
The effect of altering the length and position of the
polyether chain on ligand-tissue concentrations is
significant. The 40-norpolyether (3) achieved the
highest level of all of the ligands in the liver, heart
and pancreas, the organs most affected by iron
overload. This was the case whether the chelators
were given s.c. (Fig. 3) or p.o. (Fig. 4). For example,
in the heart, at 0.5, the concentration of 3 given s.c.
was almost four-fold higher than that of 4, and greater
than eight-fold that of 9. In the pancreas, at 0.5 h, the
concentration of 3 was six-fold greater than 9 when
the drugs were given s.c., and nearly three-fold
greater than 9 when they were given to the rats p.o.
The impact of ligand 3 on urinary Kim-1 excretion
has been evaluated in rodents when the drug was
given p.o.-once daily for 28 d (56.9, 113.8, or
170.7 lmol/kg/day); once daily for 10 d (384 lmol/
kg/d), and twice daily at 237 lmol/kg/dose
(474 lmol/kg/d) 9 7 d. The studies were performed
123

256
Biometals (2011) 24:239–258
Thompson for their technical assistance, and Miranda E. Coger
for her editorial and organizational support. We acknowledge
the spectroscopy services in the Chemistry Department,
University of Florida, for the mass spectrometry analyses.
on rats with normal iron stores. Untreated age-
matched rats were used as negative controls. Exjade
was used as a positive control for the 384 lmol/kg/
d 9 10 d dosing regimen, while deferitrin (1) served
as a positive control for the 237 lmol/kg/dose b.i.d.
(474 lmol/kg/d) 9 7 d drug exposure. Very little
Kim-1 was found in the urine of the age-matched
negative control rats at any time (Fig. 5a). However,
considerable quantities of Kim-1 were found in the
urine of the Exjade-treated rats (Fig. 5c); the rats’
BUN and SCr were also significantly increased. In
fact, none of the Exjade-treated rats survived the
planned 10-day exposure to the drug. The b.i.d. 9 7 d
dosing of deferitrin (1) was also associated with an
increase in urinary Kim-1 excretion (Fig. 5e).
Although the BUN and SCr of the 1-treated rats
were slightly elevated, all of animals survived the
exposure to the drug. In sharp contrast, very little
Kim-1 was found at any time in the urine of any of
the rats exposed to 3 (Fig. 5b, d, f). Finally, the BUN
and SCr of the 3-treated groups of rats were virtually
identical to those of the age-matched control animals.
Our results have provided additional evidence for
the efficacy, safety, and tolerability of the 3,6-
dioxaheptyloxy polyether ligand (3). The ICE of 3
in rats is 2.5-fold that of 9, the drug currently
undergoing clinical trials. In addition, while the
deferration in rats induced by 4 and 9 was shown to
be saturable as the dose was decreased from 300 to
50 lmol/kg (Bergeron et al. 2008), this was not the
case with 3. In rodent chelator tissue distribution
studies, analogue 3 was shown to achieve very high
levels in the liver, pancreas and heart, the organs
most affected by iron overload. Although significant
quantities of 3 were also found in the kidney, no
evidence of nephrotoxicity was found. Overall, these
results indicate the need for prompt completion of the
preclinical evaluation of 3 in preparation for studies
in human volunteers. Analogue 3 may provide an
orally effective alternative to s.c. DFO for the
treatment of both chronic transfusional iron overload
and of acute iron poisoning.
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