99mTc-EC-guanine: Synthesis, biodistribution, and tumor imaging in animals

Article abstract of DOI:10.1007/s11095-005-6157-8

Purpose. DNA markers are useful in assessing cell proliferation. The purpose of this study was to synthesize ^99mTc-ethylenedicysteine- guanine (EC-Guan) for evaluation of cell proliferation. Methods. Tumor cells were incubated with ^99m

Full text of DOI:10.1007/s11095-005-6157-8

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Pharmaceutical Research, Vol. 22, No. 9, September 2005 ( 2005)
DOI: 10.1007/s11095-005-6157-8
Research Paper
99mTc-EC-Guanine: Synthesis, Biodistribution, and Tumor Imaging in Animals
David J. Yang,1,2,3 Kaoru Ozaki,1 Chang-Sok Oh,1 Ali Azhdarinia,1 Thomas Yang,1 Megumi Ito,1
Allison Greenwell,1 Jerry Bryant,1 Saady Kohanim,1 Vincenzo K. Wong,1 and E. Edmund Kim1
Received February 23, 2005; accepted May 26, 2005
Purpose. DNA markers are useful in assessing cell proliferation. The purpose of this study was to
synthesize 99mTc-ethylenedicysteine-guanine (EC-Guan) for evaluation of cell proliferation.
Methods. Tumor cells were incubated with 99mTc-EC-Guan for cell cycle analysis. Prostate tumor cells
that were overexpressing the HSV thymidine kinase gene, or various tumor cells were incubated with
99mTc-EC-Guan at 0.5Y2 h. Thymidine incorporation assays were performed in lung cancer cells
incubated with EC-Guan at 0.1Y1 mg/well. Tissue distribution, autoradiography, and planar scintigraphy
of 99mTc-EC-Guan and 99mTc-EC (control) were determined in tumor-bearing rodents at 0.5Y4 h.
Results. Cell culture assays indicated that EC-Guan was incorporated in DNA, and there was no
significant uptake difference between HSVTK overexpressed and normal groups. Biodistribution and
scintigraphic imaging studies of 99mTc-EC-Guan showed increased tumor/tissue count density ratios as a
function of time.
Conclusions. Our results indicate that 99mTc-EC-Guan may be useful as a tumor proliferation imaging
agent.
KEY WORDS: biodistribution; EC-Guan; proliferation; SPECT.
INTRODUCTION
porter genes. For example, pyrimidine nucleoside [e.g., 20-
fluoro-20-deoxy-5-iodo-1-b-D-arabinofuranosyluracil (FIAU);
Assessment of tumor cell proliferation by positron emission 20-fluoro-20-deoxy-5-iodo-1-b-D-ribofuranosyl-uracil (FIRU);
tomography (PET) and single photon emission computed 20-fluoro-20-5-methyl-1-b-D-arabinofuranosyluracil (FMAU);
tomography (SPECT) could be helpful in the evaluation of 20-fluoro-20-deoxy-5-iodovinyl-1-b-D-ribofuranosyluracil
tumor growth potential, the degree of malignancy, and could (IVFRU)] and acycloguanosine 9-[(2-hydroxy-1-(hydroxy-
provide an early assessment of treatment response (1). Several methyl)ethoxy)methyl]guanine (GCV) and 9-[4-hydroxy-3- (hy-
attempts have been made to assess tumor proliferative activity. droxy-methyl)butyl]guanine (PCV) (6) and other 18F-labeled
It has been reported that 20-fluorodeoxyglucose ([18F]FDG) acycloguanosine analogs (FGCV, FPCV, FHPG, FHBG) (7Y10)
uptake is an indicator of tumor proliferative activity (2). Higashi have been developed as reporter substrates for imaging wild-type
et al. (3) have shown that [18F]FDG uptake is strongly related to and mutant HSV1-tk expression. It has been reported
the number of viable cells. Another approach was to use a that thymidine kinase activity of pyrimidine analogs was linked
radiolabeled amino acid as a tumor cell proliferative marker to G1 and S-phase of cell cycle progression (11). HSV1-tk
(4,5). However, the structures of these agents are not purine- or phosphorylates 18F-labeled acycloguanosine analogs ([18F]GCV,
pyrimidine-based, which are essential building blocks of DNA. [18F]PCV, [18F]FHBG, FIAU). The expression of these reporter
Although several radiolabeled pyrimidine and purine have been genes can be linked to the expression of a therapeutic trans-
developed, they were used as probes for imaging herpes virus gene and then, using PET, can be used to track the expression
type 1 thymidine kinase (HSV1-tk) expression and other re- of the therapeutic transgene in living subjects. The difficulty
with these probes is that HSV1-tk enzyme expression depends on
HSV1-tk gene transduction with adenoviral vectors. The level
1 Division of Diagnostic Imaging, The University of Texas MD and activity of HSV1-tk enzyme expression are likely to
Anderson Cancer Center, 1515 Holcombe Boulevard, Houston,
Texas 77030, USA.
be altered in different transduced cells and tissue; thus, the ap-
plication of HSV1-tk probe is limited.
2 Department of Experimental Diagnostic Imaging, The University of
Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard,
Houston, Texas 77030, USA.
To overcome the sensitivity of HSV1-tk enzyme expres-
sion, it would be advantageous to develop a novel tracer that
interacts with endogenous thymidine kinase, subsequently
targeting S-phase of the cell cycle. Such an agent would be
useful in assessing the efficacy of tumor therapy by measuring
proliferative activity. Synthesis and biological activity of
labeled thymidine or uridine, which were incorporated into
DNA/RNA, have been reported (12Y14). However, either the
3 To whom correspondence should be addressed. (e-mail: dyang@
di.mdacc.tmc.edu)
ABBREVIATIONS: EC, ethylenedicysteine; FDG, Fluorodeoxyglucose;
FLT, Fluoro-L thymidine; Guan, guanine; PET, positron emission
tomography; SPECT, single photon emission computed tomography; Tk,
thymidine kinase.
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0724-8741/05/0900-1471/0
2005 Springer Science + Business Media, Inc.

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Yang et al.
complex chemistry, shorter half-life of radioisotope, or cooled to room temperature and evaporated under reduced
stability of the agent was involved that limited their practical pressure to reduce to 1/4 of the solvent volume. Ethyl
uses. We have previously reported that 18F-labeled aden< acetate (30 mL) and water (25 mL) were added to the
osine, a purine analog, could measure proliferative activity by residue, and the mixture was agitated. Next, the water layer
PET (15). Because of cyclotron availability and the cost of was extracted with ethyl acetate (2 ꢀ 30 mL). Then the
producing K18F for PET, we have also explored the solvent mixture was washed with distilled water (2 ꢀ 25
possibility of using generator-produced isotopes. 99mTc is mL). The gathered solvent layer was dried over magnesium
preferred for labeling radiopharmaceuticals because of its sulfate (anhydrous). After filtration, the solvent was
favorable low energy, inexpensive isotope cost, and efficient evaporated to dryness, the desired azido compound
chemistry.
weighed 453 mg. 1H NMR (CDCl3): d 8.04 (s, 1H),
Several 99mTc-labeling techniques have been reported 7.22Y7.45 (m, 20H), 6.86 (d, 4H, J = 9.0 Hz), 6.78 (d, 4H, J =
such as N4 (e.g., DOTA), N3S (e.g., MAG-3), N2S2 (e.g., 9.0 Hz), 3.79 (s, 3H), 3.76 (s, 3H), 3.55Y3.65 (m, 2H), 3.25Y3.35
ECD), NS3, S4 (e.g., sulfur colloid), diethylenetriamine (m, 2H), 1.63Y1.73 (m, 2H), 1.32Y1.42 (m, 1H), 1.16 (d, 2H, J =
pentaacetic acid (DTPA), O2S2 (e.g., DMSA), and 3.6 Hz). Exact mass calculated for C50H46N8O4 (M + H)+
hydrazinenicotinamide (HYNIC) (17Y19). The nitrogen-and- 823.36 found 823.4.
sulfur combination was shown to be a stable chelator for
The crude azido compound (453 mg) was dissolved in
99mTc-bis-aminoethanethiol tetradentate ligands, also called dried tetrahydrofuran (30 mL) and triphenylphosphine
diaminodithiol compounds, which are known to form very (655 mg, 2.5 mmol) was added. The reaction mixture was
stable Tc(V)O complexes on the basis of efficient binding of stirred at room temperature overnight. Hydrochloric acid
the oxotechnetium group to two thiol and two amine nitrogen (HCl 5 N, 1 mL) was added to the solution and the mixture
atoms. 99mTc-L,L-ethylenedicysteine (99mTc-EC) is the most was heated under reflux for 5 h. The reaction mixture was
successful example of N2S2 chelates. Ethylenedicysteine can filtered and the solution was evaporated to dryness. The
be labeled with 99mTc easily and efficiently with high residue was mixed with water (25 mL) and washed with ethyl
radiochemical purity and stability. In addition, EC can also acetate (2 ꢀ 25 mL). The pH was adjusted to 7Y8 by adding
be labeled with 68Ga for PET imaging, and 68Ga can be sodium hydroxide (1 N), and the product solution was
obtained from a 68Ge/68Ga generator (20).
isolated a through the column chromatography using Sepha-
To continuously explore other purine-based analogs dex-G15 as a packing material. After freeze-drying (Lab-
using chelation radiochemistry, we synthesized a guanine conco, Kansas City, MO, USA), compound I was obtained
analog using EC as a chelator. In this report, the synthesis and (100 mg, 76.2% yield). Ninhydrin (2% in methanol) spray test
1
assessment of tumor growth using 99mTc-EC-guanine (EC- showed the presence of amino group. H NMR (CDCl3): d
Guan) were evaluated.
7.60 (s, 1H), 3.98 (t, 2H, J = 7.2 Hz), 3.46Y3.61 (m, 2H), 2.90
(d, 2H, J = 6.0 Hz), 1.72 (m, 2H), 1.43 (m, 1H). FAB MS: 253
(M + H)+.
MATERIALS AND METHODS
Chemicals and Analysis
Synthesis of 99mTc-ethylenedicysteine-9-[4-amino-3-
(hydroxymethyl)butyl] guanine conjugate (99mTc-EC-Guan)
Mass spectral analyses were conducted at the University
of Texas Health Science Center (Houston, TX, USA). The
Ethylenedicysteine (EC) was conjugated to Guan
mass data were obtained by fast atom bombardment on a according to the established method using water-soluble
Kratos MS 50 instrument (Kratos Instrument, Ltd., Man- carbodiimide as a coupling agent. The general process was
chester, UK). 1H NMR and 13C NMR studies were performed described in our previous reports (21Y28). Briefly, sodium
on Bruker 300 MHz spectrometer at the NMR core facility hydroxide (1 N, 0.2 mL) and sodium bicarbonate (1 N, 0.2
at MD Anderson Cancer Center (Houston, TX, USA). N- mL) were added to a stirred solution of EC (50 mg, 0.19
Hydroxysulfosuccinimide (Sulfo-NHS) and 1-ethyl-3-(3- mmol, m.p. 253-C, reported 251Y253-C) in water (5 mL). To
dimethylaminopropyl) carbodiimide-HCl (EDAC) were this colorless solution, sulfo-NHS (95.5 mg, 0.44 mmol),
purchased from Pierce Chemical (Rockford, IL, USA). 9-[4- EDAC (84.5 mg, 0.44 mmol), and compound I (50 mg, 0.20
Hydroxy-3-(hydroxymethyl)butyl]guanine (penciclovir) was mmol) were added. The mixture was stirred at room tem-
purchased from LKT Laboratories (St. Paul, MN, USA). All perature overnight. The mixture was dialyzed for 24 h using
other chemicals were purchased from Aldrich (Milwaukee, Spectra/POR dialysis membrane with molecule cut-off at 500
WI, USA). Silica gel coated thin-layer chromatography (TLC) (Spectrum Medical Industries, Houston, TX, USA). After
plates were purchased from Whatman (Clifton, NJ, USA).
dialysis, the product was freeze-dried to afford EC-Guan
1
(50 mg, 0.08 mmol, yield 40%). H NMR (CDCl3): d 8.04 (s,
Synthesis of 9-[4-amino-3-(hydroxymethyl)butyl]guanine
(Guan, compound I)
1H), 7.22Y7.45 (m, 20H), 6.86 (d, 4H, J = 9.0 Hz), 6.78 (d, 4H,
J = 9.0 Hz), 3.79 (s, 3H), 3.76 (s, 3H), 3.55Y3.65 (m, 2H),
3.25Y3.35 (m, 2H), 1.63Y1.73 (m, 2H), 1.32Y1.42 (m, 1H), 1.16
N2-(p-anisyldiphenylmethyl)-9-[(4-tosyl)-3-p- (d, 2H, J = 3.6 Hz). FAB MS: 569 (M + H)+. Exact mass
anisyldiphenylmethoxymethylbutyl]guanine (500 mg, 0.52 calculated for C18H27N8Na3O5S2 (M + H)+ 568.56 found
mmol, exact mass C57H54N5O5S (M + H)+ 952.37) (10) was 568.93. The synthetic scheme is shown in Fig. 1.
dissolved in N,N-dimethylformamide (15 mL) and sodium
azide (160 mg, 2.5 mmol) was added. The reaction mixture 0.2 mL water. Tin (II) chloride solution (0.1 mL, 1 mg/mL)
Eethylenedicysteine-Guan (5 mg) was dissolved in
was heated at 100-C for 2 h with stirring. The mixture was was added. Sodium pertechnetate (Na99mTcO4, 37Y370

99mTc-EC-Guanine
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Fig. 1. 99mTc-EC-Guan was synthesized by reacting aminoguanine analog (I) and ethylenedicysteine (EC) in the presence
of coupling agents, followed by tin (II) chloride and pertechnetate.
MBq; Mallinckrodt, Houston, TX, USA) was added. Finally, cose (0.1Y1 mg/well) and saline (control) were added to this
water was added to this solution to adjust the volume to 96-well culture plate and incubated in 5% CO2/air at 37-C.
1 mL. Radiochemical purity was assessed by radio-TLC (ITLC After 24 h, each well was pulsed with 0.5 mCi/10 mL
SG; Gelman Sciences, Ann Arbor, MI, USA) using 1 M [3H]thymidine and incubated for 24 h. Cells were then
ammonium acetate/methanol (4:1) as an eluant.
harvested, trypsinized with 100 mL of trypsin, and incubated
for 10 min in the incubator. Cells were counted using a
Beckman LS380 liquid scintillation counter. Cellular uptake
of [3H]thymidine in the control group was unified to be 100
(baseline).
Cell Cycle Uptake Assays
Human lung cancer cells (20 million, adherent) were
cultured in RPMI in T75 flasks. The cells were washed with
phosphate-buffered saline (PBS) twice and serum-free media
was added. A 5-mL portion of Hoechst 33342 (20-(4-ethoxy-
phenyl)-5-(4-methyl-1-peperazinyl)-2,50-bi-1H-benzimid-
azole, stock solution: 1 mg/mL) was added per 1 mL of
RPMI. The cells were incubated at 37-C for 90 min. The
media was aspirated and washed with PBS. The cells were
trypsinized and collected in a 15-mL test tube. The cells
were sorted using a fluorescence activated cell sorter
(FACS). The cells were plated to a 12-well tissue culture
plate containing 50,000 cells/well and it was incubated for
24 h. To each group consisting of three wells, 4 mCi (0.148
MBq) of 99mTc-EC-Guan or 99mTc-EC was added. After
incubation at 0.5Y4 h, cells were washed with ice-cold PBS
twice and trypsinized with 0.5 mL of trypsin solution. Next,
cells were collected, and radioactivity, which was
incorporated into cells, was measured by gamma counter
(Packard Instrument, Downers Grove, IL). Data are
expressed in mean T SD percent uptake ratio.
In Vitro Cellular Uptake of 99mTc-EC-Guan
To determine whether 99mTc-EC-Guan was a broad
marker for assessment of tumor growth, three different
human cancer cell lines (lung NSCLC A549, ovarian OCA3,
and prostate PC-3) were selected for cellular uptake
assays. To determine the amount of cellular uptake at
early time intervals, shorter time intervals (30 min) were
included in all three-cell lines as a standard. Longer and
immediate time intervals were only included in some of
the cell lines for comparison to 30-min intervals. The cell
lines were obtained from American Type Culture
Collection (Rockville, MD). The cells were plated to a
12-well tissue culture plate containing 50,000 cells/well. To
each well, 4 mCi (0.148 MBq) of 99mTc-EC-Guan or 99mTc-
EC (0.1 mg/well) was added. Cells were incubated with
radiotracers at 37-C at different time intervals. To demon-
strate whether the cellular uptake of 99mTc-EC-Guan was
independent from herpes simplex virus type 1 thymidine
kinase (HSV-TK) gene-mediated process, prostate tumor
cells were transfected with the adeno-associated virus
[3H]Thymidine Incorporation Assay
To demonstrate whether EC-Guan interacts with endo- vector at an input multiplicity of 50 particles/cell contain-
genous thymidine kinase and subsequently measuring the ing HSV-tk gene and beta-galactosidase gene (beta-Gal,
proliferative activity of the cell cycle, a thymidine incorpo- control). After incubation, cells were washed with ice-cold
ration assay was performed. Fluorodeoxyglucose was se- PBS twice and trypsinized with 0.5 nbsp;mL of trypsin
lected as control because it is a gold standard for PET. solution. Then cells were collected and the radioactivity
Human lung tumor cells (A549) were plated at 50,000 cells/ was measured by gamma counter. Data was expressed in
well in 200 mL RPMI, 10%FCS. EC-Guan, FDG, and D-glu- mean T SD percent uptake ratio of three measurements.

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Yang et al.
Fig. 4. In vitro cellular uptake using lung cancer cells showed that
there was a markedly increased uptake of 99mTc-EC-Guan as a
function of time compared to 99mTc-EC control groups. Data are
reported as mean T SEM (n = 3). The data points were calculated as
percentage of uptake.
Fig. 2. Cell cycle phases were sorted by fluorescence activated cell
sorter. Cell cycle uptake assays using lung tumor cells indicated that
high uptake of 99mTc-EC-Guan was observed in cell cycle S-phase,
whereas 18F-FDG had poor uptake.
Biodistribution and Dosimetry of 99mTc-EC-Guan in Tumor-
bearing Rats
Planar Scintigraphic Imaging
The animals were housed in The University of Texas
M. D. Anderson Cancer Center facility. All protocols in-
volving animals [rats and rabbits (see below)] were approved
by the M. D. Anderson Animal Use and Care Committee.
Fischer-344 rats (150 T 25 g) (Harlan SpragueYDawley,
Indianapolis, IN USA) (n = 18) were inoculated subcutane-
ously with rat breast adenocarcinoma cells (106 cells/rodent)
into the lumber region in legs using 25-gauge needles. The
studies were performed 12Y15 days after inoculation. Tumor
sizes of approximately 1 cm were measured. Separate
biodistribution studies using 99mTc-EC-Guan (Study 1, nine
rats) and 99mTc-EC (Study 2, nine rats) were conducted. For
each compound, the rodents were divided into three groups
for three time intervals (0.5, 2, and 4 h, n = 3/time point). The
injection activity was 25 T 0.5 mCi (0.925 T 0.019 MBq) / rat.
The injected mass of 99mTc-EC-Guan was 0.1 mg/rodent.
After the administration of radiotracers, the rats were sac-
rificed, and the selected tissues were excised, weighed, and
counted for radioactivity. The biodistribution of tracer in each
sample was calculated as a percentage of the injected dose per
gram of tissue wet weight (%ID/g). Tumor/nontarget tissue
count density ratios were calculated from the corresponding
%ID/g values.
Female New Zealand rabbits (2Y3 kg) were inoculated
intramuscularly with VX2 squamous carcinoma cells into the
hind thigh region in legs using 25-gauge needles. Imaging
studies were performed 12Y15 days after inoculation. Tumor
sizes of approximately 1Y1.5 cm were measured. Each New
Zealand rabbit was placed in a supine position and anesthe-
tized with ketamine (50 mg/kg, i.m.), xylazine (3 mg/kg, i.m.),
and acepromazine (1 mg/kg, s.c.). The supplemental dose
used was ketamine (10 mg/kg, i.m.) and xylazine (0.8 mg/kg,
i.m.). Planar scintigraphy was acquired for 500 k counts at
immediate, 0.5Y4 h after intravenous injection of 99mTc-EC-
Guan (n = 3) or 99mTc-EC (1 mCi/rabbit; 2 mg mass/rabbit,
n = 3). To compare the radiotracer accumulation, regions of
interest (ROIs in counts per pixel) were determined. The
ROIs count between tumor and muscle was used to calculate
tumor/nontumor ratios.
Scintigraphic images were obtained using a M-camera
from Siemens Medical Systems (Hoffman Estates, IL, USA).
The camera was equipped with a low-energy parallel-hole
collimator. The field of view was 53.3 ꢀ 38.7 cm. The intrinsic
spatial resolution was 3.2 mm and the pixel size was 19.18 mm
(32 ꢀ 32, zoom = 1) to 0.187 mm (1,024 ꢀ 1,024, zoom = 3.2).
With a low-energy, high-resolution collimator (as required
with 99mTc), the system has a resultant sensitivity of 172
counts/min (cpm)/mCi and spatial resolution of 4 mm.
Fig. 3. Thymidine incorporation assays were conducted in lung
cancer cells. The data showed that EC-Guan was involved in cellular Fig. 5. In vitro cellular uptake using ovarian cancer cells showed that
proliferation. Fluorodeoxyglucose showed a dose-dependent behav- an increased uptake of 99mTc-EC-Guan as a function of incubation
ior and had less involvement in cell nuclei activity.
time was observed. Data are reported as mean T SEM (n = 3).

99mTc-EC-Guanine
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Table II. Biodistribution of 99mTc-EC-Guan in Breast Tumor
Bearing Rats
Percent of injected 99mTc-EC-Guan dose
per gram of tissue weighta
30 min
2 h
4 h
Blood
Lung
0.953 T 0.113b
0.683 T 0.071b
5.265 T 0.681b
7.041 T 0.682
0.309 T 0.084b
0.105 T 0.014b
0.436 T 0.019b
0.448 T 0.087
0.301 T 0.040
0.457 T 0.043
4.150 T 0.473
0.421 T 0.022b
0.382 T 0.071b
6.718 T 0.416b
7.637 T 0.961
0.155 T 0.029b
0.042 T 0.006b
0.267 T 0.018b
0.219 T 0.020b
0.083 T 0.035
0.635 T 0.036b
6.409 T 0.614b
0.323 T 0.028b
0.239 T 0.024b
5.692 T 0.153b
8.361 T 1.070
0.102 T 0.009
0.026 T 0.001b
0.240 T 0.032b
0.114 T 0.026
0.052 T 0.020
0.736 T 0.048
9.350 T 0.307b
Liver
Kidney
Intestine
Muscle
Tumor
Thyroid
Stomach
Tumor/Blood
Tumor/Muscle
Fig. 6. In vitro cellular uptake using prostate cancer cells showed that
there were no differences among HSV-tk gene, b-gal gene, and
uninfected groups at 2 h incubation. The data indicate that 99mTc-EC-
Guan, a cell cycle specific and an HSV-tk gene independent marker,
could assess cellular proliferation.
a Values shown represent the mean T SD from three animals at each
time point.
b Significantly different (p < 0.05, t-test) compared to 99m Tc-EC at the
corresponding time interval.
Autoradiography
Whole-body autoradiography was performed using fe-
male ovarian tumor-bearing nude mice. When the tumor
reached 8 mm, tumor-bearing mice were treated with saline
(n = 3) and paclitaxel at (60 mg/kg, single dose, n = 3, i.v.). To
demonstrate whether 99mTc-EC-Guan could assess tumor
growth after chemotherapy, a similar tumor volume (8 mm)
at baseline was selected. After pretreatment and 4 days
posttreatment with paclitaxel, mice were sacrificed at 1 h
following 99mTc-EC-Guan injection (100 mCi/mouse, i.v.);
immediately the body was frozen and fixed in carboxymethyl
cellulose (4%), and further frozen on dry ice to solidify the
block. The block was mounted onto a cryostat and cut into
100-mm coronal sections. Autoradiograms were viewed by a
quantitative image analyzer.
errors of means. To compare differences in %ID/g and tumor/
tissue ratios between control and ECYconjugate groups, a
Student’s t test was used. Statistical significance was assigned
if p < 0.05. All statistical computations were processed using
Statview software 4.1 (Abacus, Berkeley, CA, USA).
RESULTS
Chemistry
Ethylenedicysteine was prepared in a two-step manner
and yielded a pure L,L-EC form. It was conjugated to an
amino analog of guanine under mild basic condition (pH 8.5).
Although alternative structures such as EC conjugate to YOH
group of penciclovir or EC conjugate to NH2 at position 2 of
guanine nucleus may occur, the reaction was conducted in
aqueous condition. Thus, it is unlikely to form an ester
conjugation. The amino (NH2) at position 2 of guanine
nucleus has a stable resonance and the alkyl NH2 is more
reactive than NH2 at position 2; thus, we identified that EC
was conjugated to the alkyl NH2 group. Ethylenedicysteine-
Guan was isolated using simple dialysis with molecule cut-off
at 500. After dialysis, there was no dimeric EC species found
Statistical Analysis
Percent of injected dose per gram of tissueweight (%ID/g)
and tumor/tissue ratios were presented as means and standard
Table I. Biodistribution of 99mTc-EC in Breast Tumor-Bearing Rats
Percent of injected 99mTc-EC dose
per gram of tissue weighta
30 min
2 h
4 h
Blood
Lung
0.273 T 0.039
0.187 T 0.029
0.367 T 0.006
8.991 T 0.268
0.787 T 0.106
0.043 T 0.002
0.149 T 0.020
0.229 T 0.118
0.127 T 0.106
0.544 T 0.004
3.414 T 0.325
0.211 T 0.001
0.144 T 0.002
0.286 T 0.073
9.116 T 0.053
0.401 T 0.093
0.028 T 0.009
0.115 T 0.002
0.106 T 0.003
0.037 T 0.027
0.546 T 0.010
4.425 T 0.397
0.149 T 0.008
0.120 T 0.012
0.234 T 0.016
7.834 T 1.018
0.103 T 0.009
0.019 T 0.001
0.096 T 0.005
0.083 T 0.005
0.043 T 0.014
0.649 T 0.005
5.093 T 0.223
Liver
Kidney
Intestine
Muscle
Tumor
Thyroid
Stomach
Tumor/Blood
Tumor/Muscle
a
Each rat was given 25 mCi of 99m Tc-EC. Values shown represent Fig. 7. Blood was collected from rats at 0.5, 2, and 4 h postinjection of
the mean T standard deviation from three animals at each time 99mTc-EC-Guan and measured for radioactivity. Biological half-life
point.
was determined to be approximately 74 min.

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Yang et al.
Fig. 8. Planar scintigraphy of 99mTc-EC-Guan in VX2 tumor-bearing rabbits (1 mCi/
rabbit, i.v. injection) demonstrated that tumor could be well-visualized (arrow) and
an increased tumor/nontumor (opposite leg, muscle) ratios (3.1Y4.0) were obtained at
1Y3.5 h.
in the finished product. From radio-TLC analysis (Bioscan, In Vitro Cellular Uptake of 99mTc-EC-Guan
Washington, DC, USA), the Rf value and radiochemical
purity were 0.8 and >97%, respectively.
There was an increased uptake of 99mTc-EC-Guan as a
function of incubation time in the cancer cell lines tested
(Figs. 4Y6). Uptake of 99mTc-EC as the control group was less
that 0.1% at any time point. There is a twofold difference at
Cell Cycle Uptake Assays
Cell cycle uptake assays using lung tumor cells indicated 30 min, whereas there were no differences among HSV-tk
that there was a high uptake of 99mTc-EC-Guan, whereas 18F- gene,b-galgene,anduninfectedgroupsat2hincubation(Fig.6).
FDG and 99mTc-EC (control) had poor uptake (Fig. 2). The The data indicated that 99mTc-EC-Guan, a cell cycle specific
FACS analysis of 99mTc-EC-Guan revealed that the uptake in and an HSV-tk gene independent marker, could assess
S-phase was higher than in G2/M and G0/G1 phases. The cellular proliferation.
findings suggest that 99mTc-EC-Guan is involved in cell nuclei
DNA synthesis.
Biodistribution and Dosimetry of 99mTc-EC-Guan in Tumor-
Bearing Rats
[3H]Thymidine Incorporation Assay
Biodistribution of 99mTc-EC and 99mTc-EC-Guan in
Thymidine incorporation assays indicated that EC-Guan tumor bearing rats and mice is shown in Tables I and II.
and glucose did not block cellular uptake of [3H]thymidine, Blood samples was collected from rats at 0.5, 2, and 4
whereas FDG showed dose-dependent effects (Fig. 3). The h postinjection of 99mTc-EC-Guan and measured for radioac-
findings suggest that EC-Guan and glucose interact with tivity. Biological half-life was determined to be approximately
endogenous thymidine kinase, subsequently measuring the 74 min (Fig. 7). Although tumor/blood and tumor/muscle
proliferative activity of the cell cycle; yet FDG had less count density ratios were increased as a function of time in
involvement in cell nuclei activity.
99mTc-EC and 99mTc-EC-Guan groups, accumulation of
Fig. 9. Planar scintigraphy of 99mTc-EC in VX2 tumor-bearing rabbits (1 mCi/rabbit,
i.v. injection) showed that tumor/nontumor (opposite leg, muscle) ratios were 2.5Y2.7
at 1Y3.5 h.

99mTc-EC-Guanine
1477
Fig. 10. Ovarian tumor-bearing nude mice were injected with 100 mCi of 99mTc-EC-
Guan pre- (left, panels 1 and 2) and post- (right, panels 3 and 4) paclitaxel treatment
(60 mg/kg, i.v.), and sacrificed 60 min postinjection. Although different mice were
used for evaluation of paclitaxel treatment response using autoradiographic
technique, the tumor volume selected at baseline are very similar. Autoradiograms
are shown in panels 1 and 3. The corresponding anatomical pictures are shown in
panels 2 and 4. Sections were cut at 100 mm and exposed for 16 h. Arrow designates
the tumor site. There was no enlargement of the tumor in the mouse treated with
paclitaxel. The findings indicated the feasibility of using 99mTc-EC-Guan to assess
tumor growth.
99mTc-EC-Guan in tumors was significantly different from structural alteration; however, the procedure may be lengthy,
99mTc-EC at any time point (p < 0.05, t test).
tedious, costly, and produce low yield. Isotopes commonly
used in covalent chemistry include 18F, 123I, 131I, 75Br, 77Br,
and 11C. In ionic chemistry, a chelator is required to trap
metal isotopes. This type of chemistry is simple and produces
Planar Scintigraphic Imaging
In gamma-scintigraphic imaging studies in tumor-bearing high yield. The isotopes may be obtained from generators.
rabbits, tumor/muscle (T/M) ratios in 99mTc-EC-Guan and Although ionic chemistry is attractive, the chemical properties
99mTc-EC groups at 1Y3.5 h post injection were 3.1Y4.0 and may be altered due to addition of a chelator. We have
2.5Y2.7, respectively (Figs. 8 and 9). Although tumor de- previously reported that a series of ECYagent conjugates
tection was observable in the leg, detection in the body cavity could target the tumor targets (21Y28). Ethylenedicysteine is a
may be hampered by the background intensity of the internal small molecule and has been shown to be a stable chelator with
organs. The 99mTc-EC alone allowed detection although at 111In, 67/68Ga, and other metals (29). Here, we used 9-[4-
a lower ratio. Increased T/M ratios were observed in the hydroxy-3-(hydroxymethyl)butyl]guanine (penciclovir) as a
99mTc-EC-Guan group compared to the 99mTc-EC group at target agent. Penciclovir and other guanine agents were used
the corresponding time interval.
to assess reporter gene expression (7Y10). Interestingly, our data
revealed that conjugation of EC to penciclovir (EC-Guan)
altered penciclovir affinity for assessing the efficiency of gene
transfer. Our data showed that there was no difference in
Autoradiography
Autoradiographic studies in ovarian tumor-bearing cellular uptake between transfected and uninfected tumor cells.
nude mice with 99mTc-EC-Guan showed that tumor volume Others have also shown that even a small modification in the
changes could be assessed at pre- and posttreatment with sugar moiety of the pyrimidine analog can dramatically change
paclitaxel. Although different mice were used for evalua- the biological activity (30). Our hypothesis states that EC-Guan
tion of paclitaxel treatment response using autoradiographic interacts with endogenous thymidine kinase, subsequently tar-
technique, the tumor volume selected at baseline was very geting S-phase of the cell cycle. The cellular uptake of 99mTc-
similar. There was no enlargement of the tumor in the mouse EC-Guan was shown to be S-phase-specific in sorted cells.
treated with paclitaxel (Fig. 10). The findings indicated the Phosphorylation of 99mTc-EC-Guan might have occurred in
feasibility of using 99mTc-EC-Guan to assess tumor growth.
cytosol through various kinases. Subsequently, this agent was
translocated to the cell nuclei as evidenced in thymidine incor-
poration assays. 99mTc-EC-Guan overcomes the sensitivity
deficiencies of HSV1-tk enzyme expression. 99mTc-EC-Guan
may be useful in assessing the endpoints of cancer therapy (e.g.,
DISCUSSION
To develop novel or clinically used tracers, two types of cell proliferation).
chemistries are frequently used in the preparation of radio-
Although FDG-PET imaging demonstrates the increased
tracers: covalent and ionic. In covalent chemistry, either glucose consumption of malignant cells, problems with
displacement or addition reactions are used to place an iso- specificity for cell proliferation have led to the development
tope in the molecule. The labeled product provides minimal of new PET tracers. 30-Deoxy-30-18F-fluorothymidine (18F-

1478
Yang et al.
[(18)F]fluoro-2-methylbutanoic acid (FAMB): relationship of
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M. M. Alauddin, R. Finn, W. Bornmann, H. Thaler, P. S. Conti,
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FLT) is a new tracer that images cellular proliferation by
entering the salvage pathway of DNA synthesis. Although
FLT was shown to be a promising agent to assess cell pro-
liferation, its DNA incorporation rate is low and its chemistry
is complex (31). Deep-seated tumors in blood-rich organs
may require significantly higher ratios for the assessment of
proliferation. To enhance biological activity and increase
chemical or metabolic stability, fluorine substitution at the
C20 position of the sugar moiety (arabino configuration) has
been widely investigated in drug research (32,33). However,
its chemistry is complex, involving several steps in the syn-
thesis. To assess tumor growth, we selected guanine because it
is involved in both mRNA and DNA pathways, unlike
thymidine, which mainly involves DNA pathways. The bio-
logical half-life of 99mTc-EC-Guan was determined to be
approximately 74 min, which is three to four times longer than
known purine- and pyrimidine-based agents (34).
Our preclinical data indicated that EC-Guan was
involved in the cell cycle S-phase. Its simple chemistry also
overcomes the complex chemistry of 18F-FLT. Although
many other radiopharmaceuticals could be used for
assessment of tumor proliferation and/or metabolic activity
(3), the choice should be determined not only by the
biological behavior of radiopharmaceuticals, but also by its
ease of preparation.
9. M. Iyer, J. R. Barrio, M. Namavari, E. Bauer, N. Satyamurthy,
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CONCLUSIONS
In summary, we have developed 99mTc-EC-Guan, and
our findings suggest that 99mTc-EC-Guan incorporates into
tumor cell DNA/RNA, which can be used as a predictor of
tumor proliferative activity.
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ACKNOWLEDGMENTS
The authors wish to thank Eloise Daigle for her
secretarial support. This work was supported in part by
Cell>Point L.L.C (MDA LS01-212) and the John S. Dunn
Foundation. The animal research and NMR facility was
supported by M. D. Anderson Cancer Center (CORE) Grant
NIH CA-16672.
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