Radiosynthesis and preliminary biodistribution in mice of 6-deoxy-6-[ 131I]iodo-L-ascorbic acid

Article abstract of DOI:10.1002/jlcr.1610

An ascorbate analog labeled with iodine-131, 6-deoxy- 6-[ ¹³¹I]iodo-L-ascorbic acid was prepared for evaluation as an in vivo tracer of L-ascorbic acid. The no-carrier-added radiosynthesis was conducted by nucleophilic bromine-iodine exchange between the brominated precursor and sodium [¹³¹I]iodide in 2-pentanone at 130-140°C. HPLC purification using a reversephase column gave 6-deoxy-6-[¹³¹I]iodo-L-ascorbic acid in radiochemical yield of 36-60% with high radiochemical purity and satisfactory-specific radioactivity in a total preparation time of 90 min. Biodistribution studies in fibrosarcoma-bearing mice showed a high uptake in the adrenal glands, accompanied by low activity of tumor accumulation, accumulation properties similar to previous results obtained with ¹⁴C-labeled ascorbic acid and 6-deoxy-6-[¹⁸F]fluoro-L-ascorbic acid, in spite of high level of deiodination. Copyright

Full text of DOI:10.1002/jlcr.1610

Research Article
Received 15 January 2009,
Revised 21 April 2009,
Accepted 21 April 2009
Published online 16 June 2009 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1610
Radiosynthesis and preliminary
biodistribution in mice of 6-deoxy-
6-[131I]iodo-L-ascorbic acid
Jintaek Kim, Fumihiko Yamamoto, Satoru Karasawa, Takahiro Mukai, and
Minoru Maeda
Ã
An ascorbate analog labeled with iodine-131, 6-deoxy- 6-[131I]iodo-L-ascorbic acid was prepared for evaluation as an in vivo
tracer of L-ascorbic acid. The no-carrier-added radiosynthesis was conducted by nucleophilic bromine–iodine exchange
between the brominated precursor and sodium [131I]iodide in 2-pentanone at 130–1401C. HPLC purification using a reverse-
phase column gave 6-deoxy-6-[131I]iodo-L-ascorbic acid in radiochemical yield of 36–60% with high radiochemical purity
and satisfactory-specific radioactivity in a total preparation time of 90 min. Biodistribution studies in fibrosarcoma-bearing
mice showed a high uptake in the adrenal glands, accompanied by low activity of tumor accumulation, accumulation
properties similar to previous results obtained with 14C-labeled ascorbic acid and 6-deoxy-6-[18F]fluoro-L-ascorbic acid, in
spite of high level of deiodination.
Keywords: 6-deoxy-6-[131I]iodo-L-ascorbic acid; nucleophilic bromine–iodine exchange; mice; adrenal glands; fibrosarcoma
transport characteristics and favorable in vivo properties may
provide a new tool for assessment of tissue redox status induced
Introduction
Disruption of redox homeostasis in the living body may lead to
oxidative stress capable of inflicting biological damage. Oxidant
damage of biological molecules is thought to be of great
significance in the development and progress of numerous
disease states such as cancer, inflammatory and degenerative
diseases.1 Noninvasive assessment of oxidative stress could
therefore be potentially useful in the diagnosis of disease states
and in the assessment of treatment response. A variety of
intracellular molecules contribute to tissue redox status and,
among them, L-ascorbic acid (AsA) plays important physiological
roles in cells as a reducing agent, antioxidant, free radical
scavenger, and enzyme cofactor.2 In recent years, it has been
demonstrated that AsA is accumulated in mammalian cells by
two types of Na1-dependent transporters, SVCT1 and SVCT2,
which show distinct tissue distribution and functional character-
istics.3 The SVCT1 isoform is expressed primarily in the intestine,
liver, and kidney, whereas the SVCT2 isoform is found in the
adrenal gland, pancreas and choroid plexus. Thus, it is most
likely that the activities of these specific membrane transporters
crucially influence transport, intracellular accumulation, and
recycling of AsA in the living body.4
by oxidative stress, utilizing the nuclear imaging technique.
Among structurally similar AsA analogs, 6-deoxy-6-halo-L-
ascorbic acid are very attractive analogs of AsA for the study of
the transport and function of the vitamin, without loss of
prominent antioxidant activity.9–15 In earlier studies, we
reported the synthesis and in vivo biodistribution profile of 6-
deoxy-6-[18F]fluoro-L-ascorbic acid(6-18FAsA), in which the up-
take and distribution pattern of this agent were found to have a
remarkable resemblance to that for 14C-labeled AsA, with
preferential uptake in the adrenal glands and slow brain uptake
kinetics.16,17 Additional studies in rats have demonstrated that
6-18FAsA increasingly accumulates in damaged regions of the
postischemic reperfusion brain,18 and that tissue glutathione is a
factor affecting the in vivo accumulation of this agent.19 On the
other hand, the oxidized form of 6-18FAsA, 6-[18F]fluoro-
dehydro-L-ascorbic acid, revealed rapid and extensive defluor-
ination in mice.20 Moreover, it has also been reported by us that
the 125I-labeled 2-O- and 3-O-m-iodobenzyl and 6-O-m-iodo-
phenyl derivatives of AsA displayed quite different biodistribu-
tion properties from those of 14C-labeled AsA and 6-18FAsA.21
6-Deoxy-6-iodo-L-ascorbic acid (6-IAsA) has already been
described in the literature9 and its 125I-labeled form was utilized
Studies of tissue distribution of the radioactivity of 14C-labeled
AsA in animals were initially carried out mainly using whole-
body autoradiography, and high levels of radioactivity were
observed in the adrenal glands, pituitary glands and parotid
glands, depending on the time after injection.5–7 Colombetti Higashi-ku, Fukuoka 812-8582, Japan
et al. proposed that AsA might be useful as a potential imaging
Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi,
*Correspondence to: Minoru Maeda, Graduate School of Pharmaceutical
Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582,
Japan.
agent of the pituitary gland if labeled with
a suitable
radionuclide.8 It seems reasonable to anticipate that the
development of radiolabeled analogs of AsA with selective E-mail: maeda@phar.kyushu-u.ac.jp
J. Label Compd. Radiopharm 2009, 52 366–371
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J. Kim et al.
for studying the transport properties in human fibroblasts.10 It with the great difficulty and was always an obvious complica-
has been proved that 6-IAsA is an effective inhibitor of AsA tion. Likewise, an alternate attempt to prepare 6-IAsA by
transport, with transport affinity similar to that of AsA, and is involving the nucleophilic opening of the cyclic sulfate
taken up into cells by a Na1-dependent AsA transporter.11 Thus, compound with iodide ion, on analogy with our previous
6-IAsA appeared to be a potential candidate to be developed synthesis of 6-deoxy-6-fluoro-L-ascorbic acid (6-FAsA),16 was also
into an in vivo tracer for the study of the uptake and fruitless (23%), although it was obtained with high purity. It is
accumulation of AsA due to its distinctive transport character- important to note that 6-IAsA has low stability in solution, like
istics. This paper describes the synthesis and radiochemical ascorbate itself, and care must be taken to avoid unfavorable
synthesis of 6-IAsA, and the preliminary biodistribution in oxidative decomposition and byproduct formation, thus limiting
tumor-bearing mice was also evaluated for its 131I-labeled the repeated or time-consuming use of HPLC or column
analog.
chromatography runs. In seeking an additional approach to
6-IAsA, we were prompted to examine the response toward
halogen exchange. According to the procedure described by
Bock et al.,22 6-bromo-6-deoxy-L-ascorbic acid (6-BrAsA) was
prepared in 56% yield by the reaction of L-ascorbic acid with HBr
in AcOH. Subsequent refluxing of 6-BrAsA with NaI in
2-pentanone for 6 h (oil bath: 1301C) resulted in the isolation
of 6-IAsA in 73% yield, with slight contamination (less than 5%)
of 6-BrAsA. The required heating for a long time reflects
the stability of the carbon–bromine bond in regard to the
displacement reaction. The 1H-NMR, mass spectra, and the
melting point of the compounds obtained by the three
procedures described above were in agreement with those
reported in the literature. Additional proof of the structure was
proved by X-ray crystallography, as shown in Figure 1. Crystals
Results and discussion
Chemistry
In order to use it as an authentic standard, as well as the aim of
finding a procedure applicable for labeling with iodine-131, the
synthesis of 6-deoxy-6-iodo-L-ascorbic acid (6-IAsA) was initially
investigated by using three different approaches (Scheme 1),
one of which was employed for the preparation of 6-deoxy-6-
[
131I]iodo-L-ascorbic acid (6-131IAsA) in this study. The synthesis
of 6-IAsA was reported by Kiss et al.9 and this procedure
involved nucleophilic substitution of methyl 2,3-isopropylidene-
6-O-(toluene-p-sulfonyl)-L-gulonate with iodide ion in 2-penta-
none, followed by acid hydrolysis. However, in our experience,
this two-step procedure gave only 26% of the desired
compound. We found that the acid-treatment step proceeded
suitable for X-ray analysis were grown from CH3NO2 solution as
23
a precipitant at 41C for one day
.
Radiochemistry
A previous report by Rumsey et al.11 on the preparation of
125I-labeled form of 6-IAsA involved a halogen exchange
between methyl 2,3-isopropylidene-6-deoxy-6-iodo-L-gulonate
and Na125I in boiling acetone for three days followed by HCl-
catalyzed hydrolysis at 601C for 2 h. A major disadvantage of this
radioiodination procedure is the requirement of a prolonged
reaction time, although further improvement may be possible,
and thus we preferred to develop a single-step labeling
procedure, based on the exchange of radioiodine for bromide,
because of its simplicity and potential for greater practical yield.
Radioiodination was carried out by heating a solution of
6-BrAsA in 2-pentanone with no-carrier-added Na131I, which was
dried beforehand (Scheme 2). Because in preliminary experi-
ments the direct use of an aqueous radioiodide dispensing
solution (0.1 M NaOH) provided poor incorporation of 131I as had
Figure 1. ORTEP drawing of the molecule (Crystal structure of 6-deoxy-6-iodo-
L-ascorbic acid).
Scheme 1. Synthetic route for 6-deoxy-6-iodo-L-ascorbic acid (6-IAsA).
J. Label Compd. Radiopharm 2009, 52 366–371
Copyright r 2009 John Wiley & Sons, Ltd.
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J. Kim et al.
Figure 2. (A) Preparative HPLC-chromatogram of the 6-deoxy-6-[131I]iodo-L-ascorbic acid mixture. HPLC conditions: Nacalai tesque COSMOSIL 5C18 AR-II, 10 ꢀ 250 mm);
Eluent:0.04 M Na2HPO4 with 0.1% triethylamine (pH = 5.65); Flow rate: 2.8 mL/min. HPLC analysis shows (a) the product, 6-deoxy-6-[131I]iodo-L-ascorbic acid (6-131IAsA),
(b) free [131I]iodide, (c) and (d) unknown byproducts, and (e) the precursor, 6-deoxy-6-bromo-L-ascorbic acid. (B) Analytical HPLC chromatogram of the isolated
6-131IAsA fraction.
of 95% in a total preparation time of 90 min. The radioactive
impurities were still found in the final sample after HPLC
purification, as shown in Figure 2(B). The specific activities of the
obtained 6-131IAsA varied from 18 to 74 GBq/mmol as deter-
mined by direct measurement of the HPLC eluate using a UV
detector. The preparations were chemically pure except for
occasionally low-level contamination from 6-BrAsA (3–6 nmol in
the final solution) due to tailing during HPLC isolation,
Scheme 2. Radiochemical synthesis of 6-deoxy-6-[131I]iodo-L-ascorbic acid
depending on the amount of the precursor used. We found
that complete removal of the precursor from the final product
(6-131IAsA) by exchange of radioiodine for bromide.
could be attained when less than 30 mg of the precursor was
used for the exchange reaction. When the HPLC-collected
been noted earlier for some other radiolabelings,24 the water
was removed prior to the reaction. The reaction was allowed to
fraction containing 6-131IAsA was allowed to stand at 01C,
proceed by heating in a closed vessel. The incorporation of
radiochemical purity was maintained for up to 30 min, after
which it dropped to about 85% at 4 h, due to increased
and amount of precursor used, and the optimum results were
formation of radioactive impurities eluted prior to 6-131IAsA as
radioiodine was dependent on the reaction time, temperature
obtained when a concentration of 30 mg–1 mg of 6-BrAsA/
300 mL was allowed to react at temperatures approaching
assessed by HPLC.
130–1401C for 20–40 min. Neither prolonged heating nor
temperatures higher than 1401C significantly led to improved
Biodistribution
Table 1 summarizes the results of the biodistribution of
6-131IAsA to a range of tissues in C3H/He mice bearing
fibrosarcoma, which were used as a tumor model due to our
additional interest in ascorbate transport and uptake in tumors.
There was a somewhat slower clearance of the radioactivity
from the blood pool in comparison with 6-18FAsA, but,
as expected, the highest concentration of radioactivity was
radiolabeling yields. The product was purified by reverse-phase
HPLC, the conditions of which were selected to ensure good
separation of the precursor, 6-BrAsA, from the radioiodinated
product. A solvent system of 0.04 M Na2HPO4 with 0.1%
triethylamine (pH = 5.65),25 which was bubbled with nitrogen
gas beforehand, was used as the HPLC eluent in order to
eliminate solvent evaporation in isolation and also to serve as
a formulation medium for intravenous injection. A typical
HPLC chromatogram of the hot reaction mixture (Figure 2(A))
had two major radioactive peaks, one due to free radioiodide
(peak b) and the other consistent with the required product
(peak a) plus two additional very small peaks (c and d).
Unreacted 6-BrAsA (peak e) appeared at a retention time of
10 min. Isolated radiochemical yields of 6-131IAsA after HPLC
purification ranged from 36 to 60% with a radiochemical purity
found in the adrenal glands, with
a maximum uptake
(109.37728.18%ID/g) at 30 min after injection, which gradually
decreased with time. Thus, the results indicate that the
preferential in vivo uptake in the adrenal glands does not
change significantly when iodine atom is substituted for the
primary 6-hydroxyl group of the ascorbate molecule.
On the other hand, the thyroid uptake for 6-131IAsA increased
from 15.01 %ID/g at 2 min postinjection to 30.39%ID/g
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J. Kim et al.
Table 1. Distribution of radioactivity in tissues of fibrosarcoma-bearing mice following intravenous administration of 6-deoxy-
6-[131I]iodo-L-ascorbic acid (6-131IAsA)
Organ
Uptake (%dose/g)a
6-131IAsA
6-18FAsAb
2 min
10 min
30 min
4.3870.59
60 min
2 min
60 min
Blood
Lung
Liver
Small intestine
Adrenals
Kidneys
Heart
Muscle
Bone
Brain
Thyroid
Tumor
16.3775.30
44.26724.74
10.9875.65
24.9276.77
47.39718.19
28.4378.88
13.7876.30
3.4670.07
5.6070.87
1.0470.29
15.0174.17
3.5570.31
6.6873.33
22.1275.29
11.3571.46
17.0172.39
48.34716.94
17.5072.83
6.3271.13
3.4570.70
5.4271.81
0.6670.04
12.3375.38
6.0972.61
2.9670.18
15.0974.27
3.2670.37
9.2072.55
56.82720.70
5.7171.00
3.7570.56
1.9970.50
4.0371.34
0.6470.10
30.3979.78
2.9771.05
4.0170.44
30.1072.69
21.2171.82
29.8872.53
23.4378.20
41.4276.64
4.7870.17
1.8670.41
2.0470.18
0.7370.15
—
1.9071.11
7.2772.49
6.2372.20
9.7174.34
12.9077.83
8.3172.55
1.7370.39
0.7670.17
1.6271.06
0.4970.08
—
22.3772.39
5.6670.78
17.4572.66
109.37728.18
10.8770.25
5.1970.34
2.4270.16
3.7370.25
0.6870.03
19.8972.47
5.3670.74
4.3170.05
3.2170.70
aAll data were shown as means7SD (n = 3).
bData from References 16, 20 for comparison.
at 60 min, suggesting a high level of in vivo deiodination monitored by TLC on Silica gel 60F 254 plates (Merck, Germany),
for 6-131IAsA. Compared with the previous results of 6-18FAsA, and the spots were visualized with UV light or by spraying with
the tissues that exhibited lower uptake of radioactivity were the 5% alcoholic molybdophosphoric acid. In the synthetic proce-
liver and kidneys while the lungs had much higher uptake. The dures, the organic extracts were routinely dried over anhydrous
accumulation of the radioactivity of 6-131IAsA in the brain was Na2SO4 and evaporated with a rotary evaporator under reduced
low throughout the course of the study, demonstrating the pressure. Elemental analyses were performed by the staff of the
difficulty to penetrate the blood–brain barrier, as for 6-18FAsA. micro-analysis department of Kyushu University. Analysis of the
Moreover, tumor accumulation in the fibrosarcoma was not radioactivity of the TLC plates was performed with an Aloka
significant, similar to that of 6-18FAsA. Considering the reported radio chromatogram scanner. HPLC was done by using a
similar transport affinities (Michaelis constant) of 6-BrAsA Shimadzu Liquid Chromatograph system (SCL/SIL/SPD-6A)
(Km = 15.8 mM for SVCT2)15 and 6-IAsA (Km = 5.2 mM)11 having (Cosmosil 5C18-AR-II Packed column, 250 ꢀ 10 mm, Nacalai
better affinities than AsA itself, the presence of a small amount Tesque, Japan) and by monitoring the radioactivity (NaI (Tl)
(1 nmol/mouse) of the precursor (6-BrAsA) in the injectable detector) as well as the UV absorption (at 254 nm). The
solution may have caused no little influence on the overall radioactivity was quantified with an auto-well gamma counter
biodistribution of 6-131IAsA, although there is no evidence of (ARC-370, Aloka, Japan). Identity of the labeled compound was
this. Nevertheless, the tumor-bearing mouse biodistribution of confirmed from co-injection with authentic samples by HPLC
6-131IAsA reflected that of 6-18FAsA or 14C-labeled ascorbic acid under the same conditions and also by TLC. Specific radioactivity
in that it is rapidly taken up in the adrenal glands with a high and radiochemical purity were determined by the same HPLC
level of the vitamin C transporter, SVCT2.
system. No-carrier-added sodium [
131I]iodide (0.1 M NaOH
solution; ꢁ100 GBq/mmol) was purchased from Amersham
Pharmacia Biotech (Buckinghamshire, UK). Animal experiments
were carried out in accordance with our institutional guidelines
and were approved by the Animal Care and Use Committee,
Kyushu University.
Experimental
General
Chemical reagents and solvents were of commercial quality and
were used without further purification unless otherwise noted.
Nitromethane from CaCl2 and CH3CN from CaH2 was distilled
before use. 2-Pentanone (methyl n-propyl ketone) was refluxed
with a small amount of KMnO4, then filtered and dried with
Chemistry
6-Deoxy-6-iodo-L-ascorbic acid (6-IAsA)
Na2SO4, followed by distillation before use. 1H NMR spectra were (a) 6-IAsA was prepared in 26% yield as a white solid by
obtained on a Varian Inova 400 (400 MHz) and are referenced to nucleophilic substitution of methyl 2,3-isopropylidene-6-O-
TMS (d = 0 ppm). Infrared (IR) spectra were recorded with a (toluene-p-sulfonyl)-L-gulonate with NaI in 2-pentanone, fol-
Shimadzu FTIR-8400 spectrometer and the mass spectra were lowed by acid hydrolysis with Amberlite IR-120 (plus) ion
obtained with a JEOL JMS DX-610 (FAB Mass). All melting points exchange resin (Aldrich) according to the literature procedur-
were determined on
(Yanagimoto Ind. Co. Japan) and are uncorrected. Column 1666. FAB-MS (m/z): 287(M111). 1H NMR(400 MHz, D2O):dppm
chromatography was performed on Silica gel 60 N (63–210mesh, 5.06(d, 1H, J4,5 = 2.0 Hz); 4.11(ddd, 1H, J5,6 = 5.7 Hz); 3.43(dd, 1 H,
a
Yanaco melting point apparatus e.9Mp = 201–2021C (lit.9 203–2051C). IR(KBr) cmꢂ1: 3366, 1744,
0
Kanto Chemical Co. Inc., Japan), the progress of the reaction was J6,6 = 10.5 Hz); 3.36(dd, 1 H, J6 ,5 = 8.0 Hz).
0
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J. Kim et al.
(b) A mixture of methyl 2,3-isopropylidene-2-keto-L-gulonate the exchange reaction. The radiochemical purity was maintained
4,6-cyclic sulfate16 (1 g) and dried NaI (2.4g) in 2-pentanone for up to 30 min at 01C, after which it dropped to about 85% at
(20 mL) was refluxed for 6 h under argon. After cooling to room 4 h, due to increased formation of radioactive impurities eluted
temperature, the solvent was evaporated under reduced pressure prior to 6-131IAsA as assessed by HPLC.
to give a crude mass, which was chromatographed on silica gel
(CH2Cl2:MeOH = 1:1) to afford a crude product. A mixture of the Biodistribution
above crude iodinated compound and 40% aqueous H2SO4
NFSa-fibrosarcoma was inoculated s.c. into the right hind leg
muscle of female C3H/He mice (5 weeks old, 18–20 g). These
to room temperature and adding water (20 mL), the resulting
(10 mL) in CH3CN (10 mL) was heated at 801C for 1 h. After cooling
mice, which developed tumors with a diameter of about 1 cm at
9–14 days after inoculation, were used for in vivo biodistribution
mixture was extracted with AcOEt (4ꢀ 5 mL) and dried. Evapora-
tion under reduced pressure of the combined organic layers gave
studies. The animals were given free access to food and water
6-IAsA (215mg, 23%) as a pale yellow solid, which was
during the investigation.
The fibrosarcoma-bearing mice were intravenously injected
recrystallized from CH3NO2 to give colorless needles.
Mp = 204–2051C (lit.9 203–2051C). IR(KBr) cmꢂ1:3366,1744,1666.
through the tail vein with a solution of 6-131IAsA (10 kBq/g
of body weight) in an HPLC eluent (0.3 mL), immediately after
FAB-MS(m/z): 287(M111). 1H NMR(400 MHz, D2O):dppm
5.08(d, 1H, J4,5 = 2.1 Hz); 4.12(ddd, 1 H, J5,6 = 5.6 Hz);3.44(dd, 1 H,
HPLC purification. The maximum amount of the precursor
0
0
J6,6 = 10.5 Hz); 3.34(dd, 1 H, J6 ,5 = 8.1 Hz). Anal. Calcd for C6H7IO5:
C, 25.20; H, 2.47. Found: C, 25.20; H, 2.49.
(6-BrAsA) in the 6-131IAsA samples was estimated to be about
1 nmol. The mice were sacrificed by exsanguination under
anesthesia at a predetermined time after injection of 6-131IAsA.
A sample (0.2–0.5 mL) of blood was collected at the time of
euthanasia. Samples of the tissues and tumors were excised
immediately and weighed. The tissue radioactivity was counted
by a gamma counter (Aloka, ARC-370). The tissue radioactivity
levels were calculated as a percentage of the injected dose per
gram of tissue (%ID/g of tissue).
(c) A mixture of 6-bromo-6-deoxy-L-ascorbic acid (6-BrAsA,
478 mg), which was obtained by the reaction of L-ascorbic acid
with HBr in AcOH according to the literature procedure22 and
dried NaI (370 mg) in 2-pentanone (15 mL), was refluxed for 6 h.
After cooling to room temperature, the solvent was evaporated
under reduced pressure and water (10 mL) was added to the
residue. The mixture was extracted with AcOEt (3 ꢀ 10 mL). The
organic layer was dried and evaporated to give a crude mass.
Addition of CH2Cl2 (20 mL) to the mass allowed crystallization.
Filtration gave a pale yellow crystal (417 mg, 73%). Recrystalliza-
tion from CH3NO2 gave a colorless crystal. Mp = 206–2081C
(lit.9 203–2051C). IR(KBr) cmꢂ1: 3366, 1744, 1666. FAB-MS(m/z):
287(M111). 1H NMR(400 MHz, D2O):dppm 5.06(d, 1H,
Conclusion
We prepared 6-deoxy-6-[131I]iodo-L-ascorbic acid (6-131IAsA) by
exchanging the corresponding brominated precursor with
Na131I in 2-pentanone at 130–1401C. Isolated radiochemical
yields of 36–60% with a radiochemical purity of 95% was
obtained after HPLC purification in 90 min, and the specific
activity (18–74 GBq/mmol) was adequate for in vivo studies in
small animals. Tissue distribution from tumor-bearing mice with
6-131IAsA is similar to the reported distribution of ascorbic acid
and 6-deoxy-6-[18F]fluoro-L-ascorbic acid (6-18FAsA), particularly
in light of the significant accumulation of radioactivity in adrenal
glands, in spite of high level of in vivo deiodination. It appeared
that 6-131IAsA has limited in vitro stability even in the buffer
solution used for injection. However, 6-131IAsA showed an
increase in the adrenal radioactivity levels up to 30 min post-
injection in the biodistribution study. The presence of carrier
molecule, as well as 6-BrAsA in the injection, or endogenous
molecules in the body might have contributed partially as
antioxidants to the in vivo stability for 6-131IAsA. It may be also
necessary to develop a suitable method for stabilization of the
6-131IAsA post preparation. Further studies in animal models are
in progress to determine whether uptake and accumulation of
radioactivity of this agent in the adrenal glands is associated
with adrenal function.
J4,5 = 2.0 Hz); 4.11(ddd, 1H, J5,6 = 5.6 Hz); 3.42(dd, 1H,
0
0
J6,6 = 10.5 Hz); 3.36(dd, 1H, J6 ,5 = 8.0 Hz). 1H NMR and HPLC
analysis showed that the recrystallized sample contained a small
amount of 6-BrAsA (less than 5%).
Radiochemistry
6-Deoxy-6-[131I]iodo-L-ascorbic acid (6-131IAsA)
No-carrier-added Na131I(3.7–37 MBq) was added to
containing solution of 6-bromo-6-deoxy-L-ascorbic acid
a vial
a
(6-BrAsA, 30 mg-1 mg) in CH3CN (300 mL). The solvent was
evaporated to dryness under a stream of nitrogen at 601C.
Residual water was further removed by azeotropic distillation
with additional CH3CN (2 ꢀ 300 mL). To this residue was added
anhydrous 2-pentanone (300 mL), and the vial was sealed and
then heated at 130–1401C for 20–40 min. After evaporation of
the solvent, the residue was reconstituted in the HPLC mobile
phase and injected into the HPLC column (Nacalai Tesque
COSMOSIL 5C18 AR-II, 10 ꢀ 250 mm, 0.04 M Na2HPO4 with 0.1%
triethylamine (pH = 5.65),25 flow rate 2.8 mL/min). The product
fraction was collected after a retention time of 17 min. The
isolated radiochemical yield of 6-131IAsA ranged from 36 to 60%
with a radiochemical purity of 95% in a total preparation time of
90 min. The specific activities of the obtained 6-131IAsA varied
from 18 to 74 GBq/mmol as determined by direct measurement
of the HPLC eluate using a UV detector. The HPLC-collected
fraction containing the radiotracer occasionally had low-level
contamination from 6-BrAsA (3–6 nmol in the final solution).
Complete removal of the precursor from the final product could
be attained when less than 30 mg of the precursor was used for
Acknowledgement
We appreciate Dr Koichi Ando and Dr Sachiko Koike at the
National Institute of Radiological Sciences (Chiba, Japan) who
provided us with NFSa-fibrosarcoma cells. Supports for this
study by the Takeda Science Foundation and a Grant-in-Aid
for Scientific Research from the Ministry of Education,
Culture, Sports, Science and Technology of Japan are gratefully
acknowledged.
www.jlcr.org
Copyright r 2009 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2009, 52 366–371

J. Kim et al.
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J. Label Compd. Radiopharm 2009, 52 366–371
Copyright r 2009 John Wiley & Sons, Ltd.
www.jlcr.org