Nuclear Medicine and Biology 31 (2004) 1087–1095
Novel
www.elsevier.com/locate/nucmedbio
99m
Tc radiolabeled quinazolinone derivative [Qn-In]: synthesis,
evaluation and biodistribution studies in mice and rabbit
Saroj Kumaria, Neetu Kalraa, Pushpa Mishrab, Krishna Chutanib, Anil Mishrab,1,
Madhu Chopraa,*
a
b
Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110007, India
Institute of Nuclear Medicine and Allied Sciences, Brigadier Mazumdar Road, Timar Pur, Delhi 110054, India
Received 6 September 2003; received in revised form 9 March 2004; accepted 22 March 2004
Abstract
A quinazolinone derivative as a novel non-peptidic CCK-B receptor antagonist designated as Qn-In, was synthesized, characterized by
spectroscopic techniques and evaluated for radiopharmaceutical potential. The efficiency of labeling with 99mTc was greater than 98% and
the complex was stable for about 7 hours at 37°C in presence of serum. Affinity of Qn-In was determined to be in nanomolar range by
competitive binding studies on cancer cell line MDA-MB-468. Bio-distribution of 99mTc labeled Qn-In in mice was examined by
intravenous administration and time-activity curves were generated. The ligand showed binding to most of the organs, known to express
CCK-B receptor. The lack of uptake in brain may be due to the inability of the complex to cross the blood-brain barrier. Our results show
that 99mTc labeled Qn-In ligand provides a new template for further development of non-peptidic ligands for diagnosis and therapy of
diseases related with CCK-B receptor. © 2004 Elsevier Inc. All rights reserved.
Keywords: Cholecystokinin; Cholecystokinin-B/gastrin receptors; Nonpeptidic ligand
1. Introduction
The gastrointestinal peptides gastrin and/or CCK have
been implicated in various regulatory functions; as neurotransmitters in the brain and as regulators of various functions of the gastrointestinal tract, primarily at the level of the
stomach, pancreas, and gallbladder [1]. In addition, they can
act as physiological growth factors in most parts of the
gastrointestinal tract [2– 4]. Gastrin and CCK possess the
same five amino acids at their COOH terminus, which is
biologically active site; their actions are mediated by two
different receptor types, CCK-A and CCK-B/gastrin [5,6].
CCK-B/gastrin receptors are present in the gut mucosa and
in the brain [1,7,8], whereas CCK-A receptors are present in
the gallbladder, pancreas and brain [1,9].
Several human tumors over express receptors for small
regulatory peptides [10], an observation, which has led to a
* Corresponding author. Tel.: ⫹91-011-2766 6272; fax: ⫹91-0112766 6248.
E-mail address: [email protected] (M. Chopra).
1
Current affiliation: Maxplanck Institute for Biological Cybernetics,
Tuebingen, Germany.
0969-8051/04/$ – see front matter © 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.nucmedbio.2004.03.014
99m
Tc labeled Qn-In; Breast cancer; Biodistribution
number of clinical applications in the diagnosis [11] and
treatment of tumors [12]. Receptor autoradiograhic studies
have shown that cholecystokinin CCK-B/gastrin receptors
are expressed not only in more than 90% of medullary
thyroid carcinomas (MTC) [13] but also in high percentage
of small cell lung cancer, some ovarian cancer, astrocytomas, and potentially a variety of adenocarcinomas, gastrointestinal tumor, and colorectal cell lines [14].
The development of agonists or antagonists as tools to
visualize and more recently to treat malignant tumors has
been an important focus of interest over the past years.
Somatostatin analogues such as 123I-Tyr- or 111In- DTPAD-Phe-Octreotide for human hepatocellular carcinomas
[11,15], gastroenteropancreatic tumors and neuroendocrine
tumors; Substance P [16] for hepatocellular carcinomas,
Vasoactive intestinal peptide [17] for localization of intestinal adenocarcinomas and endocrine tumors, 131I labeleledgastrin and related peptide [14] for human medullary thyroid cancer have emerged as potentially useful candidates
for in vivo scintigraphy and radiotherapy.
However, a number of problems remained to be solved,
such as molecular characteristics that render these peptide
ligands as optimal candidates for in vivo targeting of tumors
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S. Kumari et al. / Nuclear Medicine and Biology 31 (2004) 1087–1095
Fig. 1. Design of novel quinazolinone derivative with heteroatom as linker in place of ethylene.
expressing CCK-B receptor expressing tumors considering
in vivo stability, affinity and selectivity for CCK-B receptor;
or the potentially unfavorable accretion in normal organs
such as liver, bowel, and kidney. The aim and objectives of
present study was to develop a suitable non-peptidic ligand
for targeting CCK-B receptors in vivo and evaluation of the
synthesized ligand for biodistribution in CCK receptor expressing organs to be developed ultimately for diagnosis
and treatment of malignant tumors.
Asperlicin 1 (Fig.1), a week and non-selective CCK
ligand, has been used as a lead for designing distinct series
of novel non-peptidic CCK ligands by many research
groups [18 –25]. Asperlicin has an inhibitory binding constant in the micro molar range for the pancreatic CCKreceptor [25]. The natural ligand possesses elements of
1,4-benzodiazepine ring and also contains a 3-hydroxyindole with similarities to L-Trp, a key amino acid of the
bioactive region of CCK. Computer-assisted modeling was
used to design benzodiazepine derivative L 365,260 which
was selective antagonist of brain cortex CCK-B. Biodistribution of Carbon–11 labeled non-peptidic CCK receptor
antagonists, [11C]L-365,260 and its enantiomer [11C]L365,246, have been evaluated in vivo for use in CCK receptor imaging with PET [26]. These radio-ligands show
high selectivity in vivo to the two distinct CCK receptors.
[11C]L-365,260, however, had no potential as a PET tracer
for imaging brain CCK-B receptors because of its very low
BBB permeability, [11C]L-365,246 on the other hand, may
be useful for imaging peripheral CCK-A receptors with
PET. Researchers at Eli-Lilly Company, using the molecular framework of Asperlicin, detected the presence of a
3-phenyl-4(3H)-quinazoline nucleus common to mecloqualone and methaqualone, both sedatives and hypnotic drugs.
Using quinazoline as a template, a large series of compounds were synthesized of which LY 262691 (compound 2
in Fig. 1) demonstrated a high selectivity for brain cortex
and gastrin CCK-B receptors [22,23]. On the basis of previous studies a series of CCK-B antagonists have been
designed and synthesized [25]. It was shown that linker
between the two aromatic moieties was critical for CCK-B
receptor binding affinity. Compound 2 (Fig. 1) with a methylene group as a linker showed a moderate binding affinity
and selectivity for CCK-B receptors. We predicted that an
optimal linker between the two key moieties, the quinazolinone ring and the aryl ring, would bring them into preferred orientation, thus might provide more potent CCK-B
antagonist. As a part of a search for an optimal linker we
examined the effect of a heteroatom as a linker and an
indole ring as aryl moiety (Fig. 2) linked to the quinazolinone ring. In this paper we present the synthesis, in vitro
and in vivo studies of this novel compound.
2. Material and methods
2.1. Chemicals and reagents used
The compound (3Z)-1H-indole-2,3-dione 3-{[3-(4-methylphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl]hydrazone} (QnIn) was synthesized in our laboratory. Cholecystokinin (CCK)
fragment 26 –33 was synthesized at Dr. T.P. Singh’s Laboratory at All India Institute of Medical Sciences (AIIMS), New
Delhi. The peptide was stored as a lyophilized powder at
⫺20°C. All other chemicals were of analytical grade purchased from Sigma, Aldrich, Fluka and Merck Chemical Co.
All the solvents were used after distillation.
2.2. Instrumentation
Melting points were determined by using Thomas
Hoover apparatus and are uncorrected. The 1H NMR spectra
were obtained by using Brucker spectro spin DPS 300 MHz.
S. Kumari et al. / Nuclear Medicine and Biology 31 (2004) 1087–1095
1089
Fig. 2. Scheme for the synthesis of 3-(4-methylphenyl)-2-[2-(2-oxo-2,3-dihydro-1H-indol-3-yl)hydrazino]quinazolin-4(3H)-one (Qn-In).
at IIT Delhi and tetramethylsilane was used as internal
standard. I.R. spectra were recorded by using Shimadzu
instrument model no. 8300 and MASS spectra on AP 1, PE
SCEIX, LC/MS/MS. Radio imaging and biodisrtibution
studies were done using GE HAWKEYE Dual Head ␥
camera with CT and ␥ scintillation counter (GRS230,
ECIL).
2.3. Synthesis of quinazolinone derivatives (Qn-In):
(Fig.2)
NMR, DMSO-d6, ␦: 7.9250 –7.8995 (d, 1H, J⫽7.65 Hz),
7.7874 –7.7615 (t, 1H, J⫽7.77, Hz), 7.3890 –7.3361 (t, 3H,
J⫽7.92 Hz), 7.1871–7.1615 (d, 3H, J⫽7.68 Hz), 6.7874
(brs, 1H), 4.3913 (brs, 2H), 2.3935 (s, 3H).
2.3.3. Step 3: Synthesis of (3Z)-1H-indole-2,3-dione 3-{[3(4-methylphenyl)-4-oxo-3,4-dihydroquinazolin-2yl]hydrazone} (Qn-In) (5)
An equimolar mixture of 2,3-dioxoindole (0.499 g,
0.0034 mol) and 2-hydrazino-3-(4-methylphenyl)quinazolin-4(3H)-one (1 g, 0.0034 mol) was refluxed in 50 mL of
absolute ethanol for 2 hours. The precipitate thus formed
was filtered, washed with cold ethanol and dried to give title
compound. Yield 0.6 g (68%), m.p. 280°C; IR, ␯max, cm⫺1:
3184.3 (NH), 1708.8 (C⫽O) 1H NMR, DMSO-d6, ␦: 11.53
(s, 1H), 10.43 (s, 1H), 8.0068 –7.9811 (d, 1H, J⫽8.19 Hz),
7.8791–7.8486 (d, 1H, J⫽7.38 Hz), 7.7688 –7.7442 (t, 1H,
J⫽7.38 Hz), 7.4076 –7.3809 (d, 2H, J⫽8.01 Hz), 7.3106 –
7.2557 ( t, 2H, J⫽8.22 Hz), 7.1543–7.1046 (t, 2H, J⫽7.44
Hz), 6.8393– 6.8143 (d, 1H, J⫽7.50 Hz), 6.7406 – 6.7150
(d, 1H, J⫽7.68 Hz), 6.5013– 6.4515 (t, 1H, J⫽7.47 Hz),
2.45 (s, 3H); MASS: m/e M⫹ 395 [calculated 395.414].
2.3.1. Step 1: Synthesis of 3-(4-methylphenyl)-2-thioxo2,3-dihydroquinazolin-4(1H)-one (3)
Anthranilic acid (3.0 g, 0.021 mol) and 4-methylphenylisothiocynate (3.26 g, 0.021 mol) were refluxed in 75 ml
acetic acid for 16 hours. The white lustrous crystals was
separated by filtration and washed with water until the
washings showed neutral pH. The crystalline product was
dried and checked for purity using TLC (ethyl acetate:
petroleum ether) yield was 4.5g (85%), m.p. ⬎300°C ; IR,
␯max, cm⫺1: 3245.02 (NH), 1662.43 (C⫽O), 1200.05
(C⫽S) 1H NMR, DMSO-d6, ␦: 12.965 (Brs, 1H), 7.953–
7.927 (d, 1H, J⫽7.8 Hz), 7.797–7.4699 (t, 1H, J⫽8.4 Hz),
7.4507–7.4238 (d, 1H, J⫽8.07 Hz), 7.3591–7.2547 (m,
4H), 7.1401–7.1201 (m, 1H), 2.3641 (s, 3H).
2.4. 99mTc radio labeling of the compound Qn-In and
determination of in vitro serum stability
2.3.2. Step 2: Synthesis of 2-hydrazino-3-(4methylphenyl)quinazolin-4(3H)-one (4)
A mixture of thioxoquinazolinone (3, 3.0 g, 0.0117 mol)
and anhydrous hydrazine (9 ml, 0.12 mol) in 100 ml of
methanol was refluxed for 18 hours. The solid was separated
by filtration, washed with cold methanol and was dried.
Yield 2.2 g (71%), m.p. 193–194°C; IR, ␯max, cm⫺1:
3497.67, 3311.65, 3209.05 (NH’s), 1673.02 (C⫽O) 1H
100 ␮l of 0.02 nM solution of the compound Qn-In (in
PEG 600:ethanol::4:6) was taken in a vial. 50 ␮l of 1⫻10⫺2
M SnCl2.H2O dissolved in 10 % acetic acid was added to
the compound. pH was adjusted to 6.5 by using sodium
bicarbonate buffer 100 ␮l of 0.02 nM 99mTcO4⫺ was added
to the reaction mixture and incubated for 15 minutes at
room temperature. Labeling of the compound and Rf were
checked by ITLC-SG (Table 1). Labeled complex was in-
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S. Kumari et al. / Nuclear Medicine and Biology 31 (2004) 1087–1095
Table 1
Rf values
Complexes
Saline
(Rf)
Acetone
(Rf)
PAW
(Rf)
Qn–ln
Na99mTcO4
Reduced 99mTc
99m
Tc labeled Qn–ln
0
1
0
0.2
0.7
1
0
0.2
ND
ND
0
1
cubated at 37°C in fresh rabbit serum at a concentration of
1 nM mL⫺1. Serum stability was assessed by ITLC.
2.5. Determination of the composition of the
labeled Qn-In
99m
Tc
Number of the ligand (Qn-In) molecules involved in
complexation with 99mTc was determined by ascending thin
layer chromatography on ITLC-SG (Paul Gelman, Ann Arbor, MI, USA) strips using 100% acetone as developing
solvent and simultaneously in pyridine, acetic acid and
water (PAW) (3:5:1.5). Each TLC was cut in 0.5-cm segments and counts of each segment were taken. By using this
method percentage of free Na99mTcO4⫺, reduced 99mTc and
the complex formed between 99mTc and Qn-In could be
calculated (Tables 1 and 2).
with trypsin (0.05%) in HBSS. The cells were then suspended in 500 ␮L of HBSS and counts were taken in
gamma scintillation counter. Bound and free drug concentration was calculated and data were analyzed using software EQUILIBRATE from graph pad.
2.7. Scintigraphic studies in rabbits and biodistribution in
mice
Albino New-Zealand rabbits and albino mice strain A
were used for the imaging and tissue distribution studies
respectively. Animal handling and experimentation was carried out as per the guidelines of the Institutional Animal
Ethics Committee.
2.7.1. Scintigraphic studies
Scintigraphic studies of the 99mTc labeled Qn-In were
done in presence and absence of the CCK, the natural
agonist. 10 ␮Ci of the labeled compound was injected
intravenously into the animal through ear vein. Results are
shown in Fig. 3. Simultaneously in another animal CCK-8
(1 mg/kg) was injected 10 minutes before injection of the
radio labeled test compound. Serial images of whole body
were taken at different time intervals under the gamma
camera to find out the specificity of labeled products and a
representative image taken after 30 minutes is shown in Fig.
3.
2.6. Affinity determination
Competitive binding studies of the 99mTc labeled ligands
were performed on breast cancer cell line viz. MDA-MB468. The cell lines has been shown to express CCK-B/
gastrin receptor mRNA [27] by RT-PCR technique in our
laboratory. Approximately 1⫻106 cells per plate in Growth
medium DMEM and 10% FCS were incubated for 40 minutes at different concentrations of the labeled ligand in
absence and presence of CCK-8 (10 ␮M, pre-incubation for
10 minutes at 30°C). The cells were washed three times
with buffer HBSS (HEPES/NaOH 7.4 pH) and then treated
Table 2
Determination of composition of the compound Qn–ln with the
99m
Tc
S. No.
Molar ratio
(Qu–ln:
99mTc
)
% Labeling
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
1:1
0.8:1
0.6:1
0.4:1
0.2:1
0:1.
1.2:1
1.4:1
1.6:1
1.8:1
2.0:1
98.69
81.02
62.15
38.00
20.77
100% reduced
98.39
98.23
97.77
96.58
78.65
2.7.2. Biodistribution studies
For biodistribution studies albino mice strain (A) was
used as animal model and complex of 99mTc labeled Qn–In
was used. An equal dose of 10 ␮Ci of labeled test compound was injected in mice through tail vein of each animal.
Similarly in another group of animals CCK-8 (1 mg/kg) was
injected 10 minutes before injection of the radio labeled test
compound and biodistribution was studied under receptor
saturation conditions. At different time intervals mice were
sacrificed, blood was collected and different tissues and
organs were dissected and analyzed. The radioactivity was
measured in a solid gamma counter. The actual amount of
radioactivity administered to each animal was calculated by
subtracting the activity left in the tail from the activity
injected. Radioactivity accumulated in each organ was expressed as percent administered dose per gram of tissue.
Total volume of the blood was calculated as 7% of the body
weight.
2.8. Blood kinetics studies
The blood clearance study was performed in albino NewZealand rabbits weighing approximately 2.5–3.0 kg after
administration of 30 ␮Ci of the 99mTc labeled Qn-In in 0.3
mL via the ear vein. At different time intervals about 0.5 mL
blood samples were withdrawn from the dorsal vein of other
ear and radioactivity was measured in the gamma counter.
S. Kumari et al. / Nuclear Medicine and Biology 31 (2004) 1087–1095
1091
Fig. 3. Saturation analysis of the in vitro binding of 99mTc labeled Qn-In to breast cancer cell line, MDA-MB-468 (I). The specific binding is expressed as
a function of total radio ligand in the incubate. (II) is the representative Scatchard plot of the specific binding data to ratio of bound to free (B/F). Data shown
are the mean of three different experiments.
The data from the experiment were expressed as percentage
of administered dose at each time interval.
3. Results
3.1. Synthesis, labeling, and serum stability of the
quinazolinone derivative
The synthesized compound was characterized by IR,
NMR, and MASS spectroscopic studies. Table 1 shows the
Rf values of the relevant 99mTc complexes. Labeling efficiencies were more than 98%. Results of serum stability
studies showed that the metal ion was intact under physiological conditions. Unbound radioactivity was less than
0.5% in 7 h.
3.2. Composition of the complex between
3.3. In vitro binding affinity determination
Competitive binding affinity constant was determined in
cell suspension using breast cancer cell line (MDA-MB468). Bound and free ligand concentration was calculated
and specifically bound [ligand] was plotted against total
[ligand] (Fig. 4). Each data point is average of three readings. Specific binding increased with increase in concentration and reached saturation.
The specific [bound] and free [ligand] concentration data
were analyzed using the software “EQUILIBRATE” from
Graph Pad and Scatchard analysis was done. The plots and
result are shown in Fig. 4. Kd was calculated to be 17.382
nM.
3.4. Biodistribution studies in mice and scintigraphic
studies in rabbits
99m
Tc and Qn-In
The composition of the complex formed between 99mTc
and quinazolinone derivative [Qn-In] was determined by
using ITLC method (Table 2). Different molar ratios of the
Qn-In and 99mTcO4⫺ were incubated and ITLC was run
simultaneously in two different solvent systems. Difference
between Rf values in acetone medium to that in pyridine
medium could be used to determine the percentage labeling
of the compound. It was observed that on increasing the
concentration of Qn-In in the reaction mixture percentage
labeling increased up to nearly 1:1 molar ratio. Beyond this
ratio percentage of radiolabeling remained almost constant.
Each value is mean of three different test reactions. This
suggested the formation of 1:1 complex between the compound and 99mTc. Solvent molecules might occupy rest of
the valency of the 99mTc.
The 99mTc radiolabeled Qn-In was injected intravenously
into mice and biodistribution was determined as a function
of time (Fig. 5) differentiating those tissues which are
known or expected to express CCK receptors [Fig. 5, upper
panel], organs involved in blood pool and excretion of the
ligand [Fig. 5, lower panel]. Highest tracer uptake was
observed in liver after 1 hour p.i. The uptake decreased with
time but showed appreciable retention and accretion. Significantly higher uptake and long retention time was observed in stomach. Stomach uptake of the ligand increased
initially and was maximized at 1 hour, thereafter-decreased
upto 24 hours with considerable retention as compared to
intestine. This is also reflected by an external scintigraphic
visualization of these tissues in rabbit (Fig. 3). The external
Scientigraphic image depicts high activity regions in stomach and intestine region. The radioactivity decreased in
1092
Fig. 4. Biodistribution of
(lower panel).
S. Kumari et al. / Nuclear Medicine and Biology 31 (2004) 1087–1095
99m
Tc labeled Qn-In in CCK-B receptor expressing tissues (upper panel) and organs involved in excretion and blood pool organs
rabbit pretreated with 1 mg/kg of CCK-8, 10 min before the
injection of the radio- ligand. In the case of intestine (Fig. 5)
the uptake was highest at 1 hour and thereafter decreased up
to 24 hour. Very low activity was observed in brain which
could be due to the inability of the complex to cross the
blood-brain barrier. Spleen showed highest uptake after 4
hour where as uptake in blood decreased in 24 hours. There
was uptake in heart also, which cleared after 24 hour. The
rapid blood clearance was also evident from the blood
kinetics study shown in Fig. 6.
3.5. Inhibition of in vivo
99m
Tc labeled Qn-In
The results of pretreatment studies of 99mTc labeled
Qn-In are shown in Fig. 7. Blocking with 1 mg/kg of
CCK-8, 10 minutes before the injection of the radio-ligand
reduced the accumulation in stomach to about 30% of control where as the activity in the intestine was reduced to
50% at 1 hour p.i. There was increased accumulation of
activity in liver, but reduction in case of rest of the organs
studied.
4. Discussion
A variety of biologically active neuropeptides are known
to exist in the mammalian brain and to have neurotransmitterlike properties. It is also well established that most neuropeptides coexist with classical low molecular weight neurotransmitters, like dopamine, within neurons in the brain.
S. Kumari et al. / Nuclear Medicine and Biology 31 (2004) 1087–1095
Fig 5. Whole body scintigraphic images of rabbit after injection of 10 ␮Ci
99m
Tc labeled Qn-In (dorsoventral) at 30 min (a) untreated rabbit b)
pretreatment using 1 mg/kg CCK-8, injection given 10 minutes before
99m
Tc labeled Qn-In.
Functional studies of these neuropeptides, however, are less
advanced from those of classical transmitters because of
lack of potent agonists and antagonists for these peptide
receptors, especially the non-peptide analogs that have the
ability to cross blood brain barrier. Recently, based on the
development of novel CCK antagonists such as MK-329
and L-365, 260, the central role of CCK in several processes
has been elucidated. Further, the modulation of dopamine
functions, by CCK, through the two distinct CCK receptors
have been reported in mammalian dopamine neurons [26].
Reubi and Waser [13] have been able to show the presence of CCK-B receptors in more that 90% of Medullary
thyroid cancers with the help of receptor autoradiographic
studies. A high percentage of other tumor types such as lung
cancer, stromal ovarian cancer, astrocytomas, gastrointestinal cell lines, colorectal, breast MCF-7, hepatocellular carcinomas also show expression of CCK–B receptor, in which
the peptides appear to have growth promoting effects.
Behr et al. [14] have demonstrated biodistribution of a
1093
variety of CCK-B/gastrin related peptides labeled with
I-131 by iodogen or Bolten hunter reagents and In-111 by
using DTPA bifunctional chelating agents in nude mice
bearing subcutaneous human MTC xenografts. They proposed that CCK-B/gastrin analogues might be useful new
class of receptor binding peptides for the diagnosis as well
as therapy of CCK-B receptor expressing tumors. In the
non-peptide category C-11 labeled L365, 260 and its enantiomer L-365, 346 have been studied for probing CCK
receptors in vivo by PET. Among these two [11C]L-365,
260 a potent CCK-B receptor specific antagonist had very
low brain permeability and low potential for probing brain
CCK-B receptors [26].
We have been able to demonstrate the synthesis of a
non-peptidic 99mTc radio labeled ligand Qn-In and evaluated its in vitro receptor binding affinity to MDA-MB-468
(Kd⫽17.38 nM) cancer cell line. Biodistribution studies of
the ligand in mice showed binding to most of the organs
known to express CCK-B receptor. The lack of uptake in
brain is explained by the inability of the ligand to cross the
blood-brain barrier. The animal distribution data showed
significant targeting of the complex to CCK-B receptor
expressing tissues such as stomach with long retention of
activity. High non-specific activity in liver was later secreted through hepatobiliary excretion to bowel (Fig. 3)
leading to uptake in intestine. The biodistribution studies
showed the uptake of radiolabeled complex in the stomach
and gut was decreased when the animal was pretreated with
CCK-8. In this case the possibility of formation of free Tc
by degradation of the complex could not be ruled out.
Earlier reports using [11C]L-365, 260 and [11C]L-365, 346
showed the specific uptake in the stomach in the case of
[11C]L-365, 260 and in the pancreas and gall bladder in the
case of [11C]L-365, 246. In our case besides uptake of the
radiolabeled complex in stomach, there was uptake in the
heart also, which could not be explained. The high activity
in the spleen could be attributed to possible formation of
aggregates under in vivo conditions. This was also suggested by uptake in the lung observed in one of the experiments (data not shown) in external scintigraphic image
using rabbit as the experimental model.
Fig. 6. Blood kinetics studies in rabbit.
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S. Kumari et al. / Nuclear Medicine and Biology 31 (2004) 1087–1095
Fig. 7. Biodistribution of 99mTc radiolabeled Qn-In in mice after pretreatment using 1 mg/kg CCK-8, injected 10 minutes before the radio ligand (n⫽4). Data
collected 60 minutes after injection.
Thus the quinazolinone ring structure fused to indole
ring via heteroatom as linker might provide lead for new
types of compounds having CCK-B receptor affinity. But
there is non-specific uptake in other organs such as intestine,
liver, heart and spleen, as implicated by biodistribution
results. This limitation can be overcome by changing the
basic ring structure by varying the methyl substitution in the
benzene ring to give more potent CCK-B receptor specific
ligands. Work in this direction is underway in our laboratory. The new non-peptide CCK ligand might be useful for
tumor diagnosis, although a detailed study on the uptake of
99m
Tc labeled Qn-In in various human cancer cells expressing CCK-B/gastrin receptors will be required.
5. Conclusion
In the context of the earlier reports where I-131 and
In-111 label were utilized to carry gastrin related peptides(REF), our non-peptidic ligand labeled with 99mTc appears
to be more convenient for clinical purposes because of easy
labeling formulation and more favorable physical imaging
characteristics of technetium as compared to iodine. The
results of this study indicate that the new tracer binds
CCK-B receptors in vivo in rabbit and mice. But its uptake
in organs such as liver, heart and spleen suggests that its
structure can be further improved to over come the nonspecific binding. The novel ligand synthesized and characterized here, adds to the list of CCK-B receptor ligands and
provides a new template for further improvement as nonpeptidic ligands for targeting CCK-B receptors in vivo. The
future aspect of this study is to improve the structure of the
ligand and to develop appropriate tumor model in nude mice
for targeting to tumor tissues expressing CCK-B receptor.
Acknowledgments
The authors thank Dr. T.P. Singh at All India Institute of
Medical Sciences and Indian Institute of Technology, Delhi,
India, for providing access to their facilities. This study was
supported by grant from Department of Science and Technology, Ministry of Science and Technology, Government
of India to MC. One of the authors SK is thankful to
University Grants Commission, New Delhi for providing
Research Fellowship.
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