Synthesis and biological evaluation of a series of 99mTc-labeled diphosphonates as novel radiotracers with improved bone imaging

Article abstract of DOI:10.1002/jlcr.2967

Three radiolabeled diphosphonates, ^99mTc-labeled 1-hydroxy-3-(2-propyl-1H-imidazol-1-yl)propane-1,1-diyldiphosphonic acid (PIPrDP), 1-hydroxy-4-(2-propyl-1H-imidazol-1-yl)butane-1,1-diyldiphosphonic acid (PIBDP), and 1-hydroxy-5-(2-propyl-1H-imidazol-1-yl)pentane-1,1- diyldiphosphonic acid (PIPeDP), have been designed and synthesized with good chemical yields and high radiochemical purity. Their in vitro and in vivo biological properties were investigated and compared. All radiotracers evaluated in mice showed substantial retention in bone (8.42 ± 0.53, 9.08 ± 0.65, and 10.3 ± 0.61 ID%/g, respectively) at 1 h post-injection and had rapid clearance in blood (1.9484, 1.3666, and 0.7704 ID%/g/min, respectively). ^99mTc-PIBDP has the highest uptake ratio of bone-to-soft tissue at 1 h post-injection among the three radiotracers. The results indicate that ^99mTc-PIBDP is the most promising bone imaging agent.

Full text of DOI:10.1002/jlcr.2967

Research Article
Received 16 July 2012,
Revised 29 August 2012,
Accepted 31 August 2012
Published online 11 October 2012 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.2967
Synthesis and biological evaluation of a series
of 99mTc-labeled diphosphonates as novel
radiotracers with improved bone imaging
*
Ling Qiu, Wen Cheng, Jianguo Lin, Liping Chen, Jun Yao, and Shineng Luo
Three radiolabeled diphosphonates, 99mTc-labeled 1-hydroxy-3-(2-propyl-1H-imidazol-1-yl)propane-1,1-diyldiphosphonic
acid (PIPrDP), 1-hydroxy-4-(2-propyl-1H-imidazol-1-yl)butane-1,1-diyldiphosphonic acid (PIBDP), and 1-hydroxy-5-(2-propyl-
1H-imidazol-1-yl)pentane-1,1-diyldiphosphonic acid (PIPeDP), have been designed and synthesized with good chemical yields
and high radiochemical purity. Their in vitro and in vivo biological properties were investigated and compared. All radiotracers
evaluated in mice showed substantial retention in bone (8.42Æ 0.53, 9.08 Æ 0.65, and 10.3 Æ 0.61 ID%/g, respectively) at 1 h
post-injection and had rapid clearance in blood (1.9484, 1.3666, and 0.7704 ID%/g/min, respectively). 99mTc-PIBDP has the
highest uptake ratio of bone-to-soft tissue at 1 h post-injection among the three radiotracers. The results indicate that 99mTc-PIBDP
is the most promising bone imaging agent.
Keywords: 99mTc-labeled diphosphonates; bone imaging agent; stability; pharmacokinetics; biodistribution
affinity for bone, lower soft tissue uptake, and more rapid clearance
Introduction
from the blood.16 In the last decade, we have prepared and inves-
tigated a series of 99mTc-labeled DPs with different alkyl substitu-
Diphosphonates (DPs) are synthetic analogs of pyrophosphate,
with the backbone structure P–C–P. They are very stable and
ents at the 2-position of the imidazole ring of ZL as new potential
bone imaging agents.17–23 These studies have showed that optimi-
exhibit high affinity for calcified matrices, such as hydroxyapatite
in bone.1 Therefore, they have been widely used successfully as
zation of the alkyl substituent in the imidazole ring and the carbon
powerful inhibitors of increased bone resorption in several bone
chain between the imidazolyl and geminal DP groups can bring
significant influence on the biological properties of 99mTc-labeled
diseases, such as osteoporosis, Paget’s disease, hypercalcemia of
malignancy, and bone metastases of several cancers.2–5 A number
of DPs have been commercially available, such as neridronate, rise-
dronate, zoledronate (ZL), minodronate, clodronate, pamidronate,
alendronate, and ibandronate. ZL, which is considered a typical
third-generation DP, is the most potent among the DPs. In preclinical
models of bone resorption, ZL is at least 100 times more potent than
either clodronate or pamidronate and at least 1000 times
more potent than etidronate.6 Therefore, the carbon side chains
determine the pharmacological properties of the DPs.
complexes.21–23 In this work, we report on the synthesis and
evaluation of a series of novel DPs with different length of the
carbon chain (Scheme 1), in order to improve the bone uptake
and the clearance rate from blood and soft tissues.
Experimental
Reagents, instruments, and animals
Diphosphonates are good targeting ligands to serve as bone
imaging agents.7 For example, 99mTc-labeled methylenedipho-
sphonate (MDP)8 and hydroxymethylenediphosphonate (HMDP)9
have been widely used for many years in bone scanning, which
is an effective means to diagnose primary bone cancer, metastatic
bone disease, osteoporosis, bone trauma, and so on.10–13 Because
of the ideal nuclear properties (t1/2 = 6.02 h, Eg = 140keV) and low
cost of the radionuclide technetium-99m, technetium-99m is the
element of choice for more than 80% of diagnostic procedures in
nuclear medicine.14
All of the chemical reagents were of analytical reagent grade. Distilled
water was used for solution preparation. Fetal bovine serum (FBS)
(Zhejiang Tianhang Biological Technology Co., Ltd), human serum (HS)
(supplied by the affiliated Jiangyuan Hospital of Jiangsu Institute of
Nuclear Medicine), and mouse serum (MS) were used for in vitro stability
study. Melting points were measured on Yanaco MP-500 melting point
apparatus (Shimadzu, Japan). Elemental analysis was carried out using
Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory
of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi
99mTc-labeled DPs as diagnostic agents have a number of clinical
and chemical limitations,3 such as low selectivity and relatively 214063, China
slow clearance from the blood and soft tissues. These properties
*Correspondence to: Jianguo Lin, Key Laboratory of Nuclear Medicine, Ministry of
Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute
of Nuclear Medicine, Wuxi 214063, China.
usually lead to false negative results and extended intervals
(2–6 h) between injection and bone imaging.15 To address these
problems, we need a radiopharmaceutical agent with higher E-mail: jglin@yahoo.cn
J. Label Compd. Radiopharm 2012, 55 429–435
Copyright © 2012 John Wiley & Sons, Ltd.

L. Qiu et al.
Scheme 1. Syntheses of PIPrDP, PIBDP, and PIPeDP.
an Elementar Vario EL III analyzer. Electrospray ionization mass spectrome- refluxed for 5h. After cooling, charcoal was added to decolor the solution.
try (ESI-MS) was determined using a Waters Platform ZMD4000 LC/MS After filtration, the solvent was removed. Finally, the crude product
(Waters, USA). Proton nuclear magnetic resonance (1H NMR) spectra were was recrystallized from ethanol to give the white crystalline product 2.
obtained on a Bruker DRX-500 spectrometer (Bruker, Germany), and the 1-Hydroxy-3-(2-propyl-1H-imidazol-1-yl)propane-1,1-diyldiphosphonic acid (2a):
chemical shift values were referenced to the internal tetramethylsilane. Yield, 40%. mp 242–245 ꢀC; ESI-MS, m/z (%): 327(100, [M-H]+). 1H NMR
Xinhua grade 1 chromatography paper (Shanghai, China) was used for thin (400 MHz, D2O): d7.39 (d, J= 2.0 Hz, 1H, CH-ring), 7.26 (d, J= 2.0 Hz, 1H,
layer chromatography (TLC) analysis. A Packard-multi-prias g counter CH-ring), 4.39–4.43 (m, 2H, N–CH2), 2.92–2.96 (t, J= 7.6 Hz, 2H, ring-CH2),
(Perkins Elmer, USA) were used for counting the radioactivity. The high 2.34–2.45 (m, 2H, OH–C–CH2), 1.71–1.80 (m, 2H, CH2CH2CH3), 0.91–0.95
performance liquid chromatography (HPLC) system was equipped with a (t, J=7.6Hz, 3H, –CH3). 1-Hydroxy-4-(2-propyl-1 H-imidazol- 1-yl)butane-1,
Waters 1525 binary HPLC pump (Waters, USA), a Waters 2487 dual
1-diyldiphosphonic acid (2b): Yield, 35%. mp 212–214 ꢀC; ESI-MS, m/z (%):
wavelength absorbance detector (Waters, USA), and a Perkin Elmer 341(100, [M-H]+ ). 1H NMR (400MHz, D2O): d7.32 (d, J= 2.0 Hz, 1H, CH-ring),
Radiomatic 610TR radioactivity detector (Perkin Elmer, MA, USA), 7.24 (d, J=2.0Hz, 1H, CH-ring), 4.06–4.10 (t, J=7.2Hz, 2H, N–CH2), 2.87–2.90
which were operated by Breeze and proFSA software. A reversed phase (t, J= 7.2 Hz, 2H, ring-CH2), 1.91–1.98 (m, 2H, OH–C–CH2), 1.76–1.83 (m, 2H,
C
18 (RP-C18) column (4.6Â 250 mm; 10-mm particle size) was used for HPLC CH2CH2CH3), 1.66–1.73 (m, 2H, N–CH2CH2CH2), 1.58–1.62 (m, J=6.8Hz, 2H,
analysis (Elite Analytical Instrument Company, Dalian, China). Na99mTcO4 –CH2), 0.88–0.91 (t, J=7.2Hz, 3H, –CH3). 1-Hydroxy-5-(2-propyl-1H-imidazol-
was eluted from the 99Mo-99mTc generator (radiochemical purity (RCP) : 1-yl)pentane-1,1-diyldiphosphonic acid (2c): Yield, 25%. m.p. 217–221 ꢀC;
99.99%; Jiangyuan Hospital of Jiangsu Institute of Nuclear Medicine).
ESI-MS, m/z (%): 355 (100, [M-H]+). 1H NMR (400 MHz, D2O): d7.34
Normal institute of cancer research (ICR) mice (weighing 18–20 g) used (d, J= 2.0 Hz, 1H, CH-ring), 7.27 (d, J= 2 Hz,1H, CH-ring), 4.08–4.12 (t, 2H,
for the pharmacokinetics and biodistribution studies were supplied by SLAC J=7.2Hz, N–CH2), 3.56–3.62 (m, 2H, OH–C–CH2), 2.89–2.93 (dd, J=7.2Hz,
Laboratory Animal Company (Shanghai, China). The animal experiment was 2H, ring-CH2), 1.81–1.93 (m, 2H, N–CH2–CH2), 1.60–1.76 (m, 2H, –CH2CH2CH3),
approved by the Animal Care and Ethics Committee of Jiangsu Institute of 1.10–1.14 (m, 2H, –CH2), 0.90–0.94 (t, J=7.2Hz, 3H, –CH3).
Nuclear Medicine.
Radiolabeling
Syntheses of diphosphonic acids
Compounds 2a-2c (5 mg, dissolved in 0.1 mL 0.2 M NaOH solution),
Three diphosphonic acids (PIPrDP, PIBDP, and PIPeDP) were synthe-
SnCl2Á2H2O solution (100 mL, freshly prepared, 10 mg SnCl2Á2H2O dis-
solved in 10.0 mL 0.5 M HCl solution), and Na99mTcO4 (74.0 MBq freshly
sized according to the procedure outlined in Scheme 1 as described
previously.23–25
eluted) were added into a penicillin vial in turn. The pH of the mixture
was adjusted to pH = 6.0 by adding 0.2 M phosphate buffer solution
(PBS). The final volume of the solution was adjusted to 2 mL by water.
Syntheses of compound 1
The reaction mixture was heated at 70 ꢀC for 30 min.
2-Propylimidazole (0.1mol) was dissolved in CH2Cl2 (75mL) under stirring
condition. KOH (8.4g, 0.15 mol), K2CO3 (13.8g, 0.0835mol), and N(Bu)4Br
(tetrabutylammonium bromide, TBAB) (0.7g, 0.002mol) were added at
Quality control
room temperature. The homologue of ethyl bromoacetate (0.1mol) was
The radiolabeling yield (RLY) and RCP of 99mTc-DPs were determined by
added, respectively. The reaction mixture was refluxed for 7h. The solution
was filtered and the filtrate was washed with brine solution. The organic
TLC and HPLC analyses, respectively.
phase was dried and evaporated to give the corresponding ester, which
was used without further purification. HCl aqueous solution (1.2M,
100 mL) was added to the crude ester, and the mixture was refluxed for
8 h. The solution was concentrated, and the residue was recrystallized from
isopropanol to give the product 1. 3-(2-Propyl-1H-imidazol-1-yl)propanoic acid
(1a): White solid, Yield, 46%. m.p. 149–151 ꢀC; ESI-MS, m/z (%): 181 (100, [M-
H]+). 4-(2-Propyl-1H-imidazol-1-yl)butanoic acid (1b): White solid, Yield, 27%.
m.p. 126–129 ꢀC; ESI-MS, m/z (%): 195 (100, [M-H]+). 5-(2-Propyl-1H-imidazol-
1-yl)pentanoic acid (1c): Sticky substances, Yield, 24%. ESI-MS, m/z (%): 208
(100, [M-H]+).
Thin layer chromatography analysis
Radiolabeling yields of 99mTc-PIPrDP, 99mTc-PIBDP, and 99mTc-PIPeDP were
determined using TLC analysis. Strips of Xinhua No.1 paper chromatogra-
phy of 13 cm long and 0.5cm wide were marked at a distance of 1.5cm
from the lower end and lined into sections 1cm each up to 10cm. About
3 mL of 99mTc-PIPrDP, 99mTc-PIBDP, or 99mTc-PIPeDP solution was applied
with a syringe at 1.5cm from the bottom of the paper strip, and then the
strip was developed in distilled water and acetone. After complete develop-
ment, the strip was dried and assayed by Mini-Scan radio-TLC.
Syntheses of compound 2
High performance liquid chromatography analysis
Compound 1 (20mmol) was dissolved in chlorobenzene (25mL) and
heated to 120 ꢀC for 30 min, then phosphoric acid (85%, 4.2mL) was added Radiochemical purities of 99mTc-PIPrDP, 99mTc-PIBDP, and 99mTc-PIPeDP
slowly and followed by phosphorus trichloride (7.6mL). The reaction were determined by HPLC analysis. Each sample was filtered through a
mixture was kept at 120 ꢀC for 4 h. The chlorobenzene was decanted, and millipore filter (0.22 mm) carefully and 10 mL of sample was injected into
the yellow residue was redissolved in HCl (20 mL, 9.0M). The mixture was the HPLC column. The elution conditions are as follows: isocratic of
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L. Qiu et al.
70% TBAB (5 mmol/L) aqueous solution and 30% CH3CN, the flow rate of
1 mL/min, and a Cd (Te) detector for radioanalysis.
Statistical analysis
Statistical analysis of the bone uptake and uptake ratios of bone to heart,
liver, spleen, blood, and muscle was performed using the Student’s
t-test for unpaired data (GraphPad Prism 5.0). A 95% confidence level
was chosen to determine the significance between groups, with p < 0.05
being significantly different.
In vitro stability
In vitro stability of 99mTc-DPs was studied in PBS (pH = 7.4), FBS, HS, or MS
for 1–6h. Briefly, 200 mL (3.7MBq) of 99mTc-PIPrDP, 99mTc-PIBDP, and
99mTc-PIPeDP were added into 200mL of PBS, FBS, HS, or MS, respectively.
After incubation at 37ꢀC for 1–6h, an aliquot of the PBS solution was taken
directly, and the radioactivity was analyzed by TLC. For the solution of FBS,
HS, or MS, an aliquot was added into 100mL of 50% trifluoroacetic acid in
water. After centrifugation, the upper solution was taken for TLC analysis.
Results and discussion
Chemistry and radiolabeling
PIPrDP, PIBDP, and PIPeDP were synthesized by four steps
according to the previous method (Scheme 1).23 In the first step,
compound 1 was prepared through the N-alkylation reaction. To
accelerate the reaction rate and achieve a satisfactory yield,
TBAB was added to the reaction system as a phase transfer
catalyst.27 Compound 2 was obtained through the phosphine
acidification reaction of the carboxylic acid. The speed of adding
PCl3 had critical effect on the yield of the reaction. If the addition
is fast, the reaction may be hard to control, and the yield was
also low. Otherwise, the reaction time may be too long. A
moderate speed (about 0.4 mL/min) was selected to control the
reaction temperature and increase the yield.
Octanol–water partition coefficient
Octanol–water partition coefficient (log POW) was determined for 99mTc-
PIPrDP, 99mTc-PIBDP, and 99mTc-PIPeDP at pH= 7.4 by measuring the radio-
activity of the radiolabeled compound in octanol and PBS, respectively, at
equilibrium. 99mTc-PIPrDP, 99mTc-PIBDP, or 99mTc-PIPeDP solution was
diluted with PBS (100 mL + 900 mL), respectively. The solution was mixed
with 1 mL octanol, vortexed for 5 min, and centrifuged at 4000 rpm for
5min to ensure complete separation of layers. An aliquot of organic and
aqueous phases (100mL each) were collected, and the radioactivity was
measured with a g counter. The log POW was calculated using the formula
log POW = log (octanol CPM / PBS CPM).26 The reported value is the average
obtained from three independent experiments.
PIPrDP, PIBDP, and PIPeDP were further labeled with 99mTcO4-
with the reducing agent stannous chloride. The pH value was
very important for the radiolabeling. When the acidity is high
(pH = 1–3), the RLY and RCP are less than 90%, because of
incomplete reduction of 99mTcO-4 under strong acidic conditions.
When the pH is larger than 6, over-reduction of 99mTcO4- into a
colloid of 99mTc predominates, the RLY and RCP decrease signif-
icantly. The best pH range is 4–6, the RLY and RCP are more than
95%. This labeling method meets the clinical requirement
used for the production of other standard 99mTc-labeled DPs,
such as 99mTc-MDP.
Plasma protein binding assay
99mTc-PIPrDP, 99mTc-PIBDP or 99mTc-PIPeDP (100 mL, 37 KBq) was mixed
with a human plasma solution (100 mL) in the centrifuge tube. After the
mixture was incubated at 37 ꢀC for 2 h, the plasma protein was precipi-
tated by adding 1 mL trifluoroacetic acid solution in water (250 g/L).
The supernatant and precipitate were separated by centrifugation at
3000 rpm for 5 min. The radioactivities of both phases were measured
by a g counter separately. The aforementioned procedure was repeated
three times. The percentage of protein binding was determined by the
following equation: Plasma binding % = (Precipitate CPM / [Precipitate
CPM + Supernatant CPM]) *100%.
Quality control
We used TLC to monitor the progress of the radiolabeling reactions
and check the purity of the radiolabeled compounds. All of the
chemical species involved in the radiolabeling reactions were sepa-
rated by TLC method. From TLC analysis, the three radiotracers
showed similar results. With the distilled water as eluate,
99mTcO2ÁnH2O remained at the origin (Rf = 0–0.1), whereas
Na99mTcO4 and 99mTc-DPs both migrated with the solvent
front (Rf = 0.7–0.9). When acetone was used, 99mTcO2ÁnH2O and
99mTc-DPs remained at the origin (Rf = 0–0.1), Na99mTcO4 migrated
with the solvent front (Rf =0.9–1.0). At optimal radiolabeling condi-
tions, TLC showed that Na99mTcO4 was completely reduced and
99mTc-colloidal amount was less than 2% (Figure 1(a)).
We also used HPLC to monitor the progress of the radiolabeling
reactions and to check the purity of the radiolabeled compounds.
Because these 99mTc-DPs are all ionic and highly polar, they
showed no retention on standard reversed phase HPLC column,
such as RP-18.28 A little amount of TBAB was added in the mobile
phase to serve as the ion pair reagent to improve the retention
time of 99mTc-DPs and free technetium. HPLC analysis revealed
that the retention time of free technetium (Na99mTcO4) was
9.8 min, whereas that of 99mTc-DPs was 3.4 Æ 0.2 min. (Figure 1(b)).
For each radiotracer synthesized, only one single peak was
observed. This clearly showed that the complexes were pure, no
residual Na99mTcO4 or other impurities.25 Both TLC and HPLC
showed that RCPs of three 99mTc-DPs were all larger than 98%.
Pharmacokinetic studies
For pharmacokinetics studies, 99mTc-PIPrDP, 99mTc-PIBDP, or 99mTc-PIPeDP
(7.4MBq, 0.2mL) was administered to the mice via intravenous injection
through the tail vein. A series of blood samples (20 mL) were collected in
microcap tubes by nicking the tail with a needle at 2, 5, 10, 15, 30, 60,
120, 180, 240, and 360 min after injection. The radioactivity of each blood
sample was counted and expressed as a percentage of the injected dose
per gram of blood (%ID/g). The radioactivity was expressed as a function
of time, and pharmacokinetics parameters were calculated using the 3P97
program (Chinese Mathematical Pharmacology, 1997, Beijing, China).
Biexponential equation was used to fit the curve: C = AeÀat + BeÀbt, where
C is the plasma level of the tracer at any given time t; A and B are constants;
a and b are rate constants of distribution and elimination phases.
In vivo distribution
99mTc-DPs (7.4 MBq, 0.2 mL) were administered to 30 institute of cancer
research mice (five mice for each group) via the tail vein injection. The
mice were sacrificed by decapitation at 5, 15, 30, 60, 120, and 240 min
post-injection. Interested organs (containing joints and femur) were
collected and weighed, and 200 mL of blood was taken from the carotid
artery. The radioactivity of each sample was measured by a g counter.
The distribution of each radiotracer in different organs was calculated
and expressed as the percentage uptake of the injected dose per gram
of organ (%ID/g) and the uptake rations of bone to other tissues were
obtained from the ID%/g values.
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Copyright © 2012 John Wiley & Sons, Ltd.
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L. Qiu et al.
ring had higher lipophilicity.23,30 Because log POW is an important
parameter for assessing the biological distribution of these com-
plexes,31 we speculate that the new 99mTc-DPs have larger positive
effects on the bone uptake than the previously synthesized DPs.
Almost no difference was observed among the plasma protein
binding of these complexes although the carbon chain increased
from 2 to 4. The percent of plasma protein binding was
25.2 Æ 0.7, 26.2 Æ 0.5, 25.8 Æ 0.9 for 99mTc-PIPrDP, 99mTc-PIBDP,
and 99mTc-PIPeDP, respectively. This may be because of the struc-
tural analogy of these 99mTc-DPs, as observed from log POW study
described earlier. In general, the plasma protein binding efficiency
has significant influence on the bone uptake of the radiotracer32
and analysis of 99mTc-DPs in the whole blood is the key to the
development of novel drugs for clinical use.33
(a)
Pharmacokinetic studies
The time-activity curve of radioactivity in blood of mice for each
99mTc-DP during 6 h post-injection follows a double exponential
curve (Figure 3), with C = 5.39eÀ0.055t + 0.85eÀ0.010t, C = 8.49eÀ0.053t
+
0.85eÀ0.075t, and C = 17.27eÀ0.056t + 0.737eÀ0.0042t. The T1/2a value
is correlated with the molecular size, where the smaller the
compound, the shorter is the distribution half-life (Table 1).34 We
believe that when the molecular weight is smaller, the permeability
into the organs and tissues from the blood is faster. Consequently,
the distribution half-life (T1/2a) is shorter. On the other hand, the
elimination half-life T1/2b basically followed the same trend. The
elimination is primarily through the renal filtration, and larger
molecules would be removed slower than the smaller ones.
(b)
Figure 1. (a) Thin layer chromatography of 99mTc-PIPrDP in water and acetone;
(b) HPLC of 99mTc-PIPrDP and Na99mTcO4.
Hence, these radiolabeled compounds were used immediately with-
out further purification for both in vitro and in vivo studies.
In vitro stability
Biodistribution studies
The stability was evaluated by measuring the RCP of each
radiotracer using TLC analysis. More than 95% of 99mTc-PIPrDP,
99mTc-PIBDP, and 99mTc-PIPeDP remained intact in the PBS, FBS,
HS, and MS after 6 h of incubation (Figure 2). Therefore, 99mTc-
DPs are stable enough for human or rodent studies for up
to 6 h.
log POW and plasma protein binding
Octanol–water partition coefficients (log POW) of 99mTc-PIPrDP,
99mTc-PIBDP, and 99mTc-PIPeDP were determined to be À1.58,
À1.56, and À1.53, respectively. The rather similar log POW values
reflect the structural analogy of three 99mTc-DPs. Usually, higher
lipid molecules have positive effects on the bone resorption in
the bone-binding model.29 In our previous studies, we found
that complexes with longer alkyl substituent in the imidazole
Figure 3. The time activity curve of 99mTc-DPs in the blood of mice (n = 5,
mean Æ SD).
(a)
(b)
Figure 2. In vitro stability of 99mTc-DPs in phosphate buffer solution (PBS), fetal bovine serum (FBS), human serum (HS) and mouse serum (MS) after 1 and 6 h, respectively.
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L. Qiu et al.
Table 1. Pharmacokinetics parameters of 99mTc-DPs
99mTc-DPs
T1/2a (min)
T1/2b (min)
Ke (minÀ1
)
AUC (ID%/g*min)
CL (ID%/g/min)
99mTc-PIPrDP
99mTc-PIBDP
99mTc-PIPeDP
13.467
15.957
21.232
75.294
91.276
164.82
0.0329
0.0344
0.0375
189.89
270.77
480.24
1.9484
1.3666
0.7704
T1/2a, distribution half-life; T1/2b, elimination half-life; Ke, elimination rate constant; AUC, area under the time activity curve; CL,
total clearance rate.
The 99mTc-DPs mainly accumulated in the skeleton, particularly in of 99mTc-PIPeDP is larger than 99mTc-PIPrDP and 99mTc-PIBDP.
the joint (Table 2). All 99mTc-DPs reached the skeleton within 5 min Compared with the clinically widely used bone imaging
post-injection. At 60 min post-injection, in general, the uptake of agent 99mTc-MDP, the bone uptake of these 99mTc-DPs at
99mTc-DPs in the joint reached the peak at 10.3 Æ 1.25 (at 120 min, 30, 60, and 120 min post-injection were all higher than those
13.0 Æ 0.74), 12.3 Æ 1.53, and 13.4 Æ 1.43 ID%/g for 99mTc-PIPrDP, of 99mTc-MDP, which were 3.26, 4.79, and 3.87%ID/g, respec-
99mTc-PIBDP, and 99mTc-PIPeDP, respectively. Throughout the study tively.37 This is consistent with what we have observed
period, no sign of toxicity was observed for these 99mTc-DPs. This is before in analogous systems.31
consistent with the general observation that ZL has low toxicity and
The uptakes of 99mTc-PIBDP in most soft tissues were always
can be used therapeutically at a high dose.35,36 Therefore, the most smaller than 99mTc-PIPrDP and 99mTc-PIPeDP, such as in the heart,
suitable time of the new agents for bone imaging is 60 min post- liver, spleen, kidney, and muscle. However, all these 99mTc-DPs
injection. This represents significant shortening of the time interval have higher uptake in the kidney. At 2 h post-injection, the kidney
between injection and bone scanning.
uptakes of the three radiotracers were still 5.71 Æ 0.17, 3.29 Æ 0.10,
The extension of the carbon chain in this series of 99mTc- and 5.52 Æ 0.28 ID%/g, respectively. One possibility is that the
DPs increased their bone uptake. That is, the bone uptake filtration rate of glomerulus is slow, and the reabsorption is not
Table 2. Biodistribution of 99mTc-DPs in mice as a function of time (Mean Æ SD, n = 5, ID%/g)
Time post-injection
Tissue
5 min
15 min
30 min
60 min
120 min
240 min
99mTc-PIPrDP
Heart
Liver
Spleen
Kidney
Femur
Joint
Muscle
Blood
3.11 Æ 0.19
3.10 Æ 0.24
1.55 Æ 0.10
11.5 Æ 0.17
4.06 Æ 0.19
7.96 Æ 0.38
1.97 Æ 0.02
8.73 Æ 0.24
6.85 Æ 0.07
1.31 Æ 0.08
2.17 Æ 0.08
0.73 Æ 0.01
8.30 Æ 0.34
3.58 Æ 0.16
7.91 Æ 1.54
0.81 Æ 0.01
4.64 Æ 0.07
6.68 Æ 0.25
0.73 Æ 0.02
2.18 Æ 0.21
0.60 Æ 0.05
6.40 Æ 0.25
3.93 Æ 0.41
9.56 Æ 2.05
0.55 Æ 0.01
2.71 Æ 0.12
7.61 Æ 1.12
0.44 Æ 0.01
2.17 Æ 0.04
0.44 Æ 0.03
5.88 Æ 0.06
4.16 Æ 0.19
10.3 Æ 1.25
0.25 Æ 0.02
1.00 Æ 0.02
8.42 Æ 0.53
0.30 Æ 0.02
1.88 Æ 0.10
0.46 Æ 0.03
5.71 Æ 0.17
4.38 Æ 0.15
13.0 Æ 0.74
0.12 Æ 0.02
0.37 Æ 0.04
10.2 Æ 0.03
0.30 Æ 0.01
2.23 Æ 0.11
0.32 Æ 0.02
4.78 Æ 0.22
4.41 Æ 0.19
11.4 Æ 0.85
0.17 Æ 0.00
0.13 Æ 0.01
8.60 Æ 1.00
All bone
99mTc-PIBDP
Heart
2.06 Æ 0.19
1.10 Æ 0.08
1.00 Æ 0.08
3.90 Æ 0.34
3.66 Æ 0.21
6.20 Æ 0.36
1.01 Æ 0.06
6.42 Æ 0.36
4.79 Æ 0.35
0.86 Æ 0.05
0.95 Æ 0.11
0.68 Æ 0.05
3.71 Æ 0.26
3.31 Æ 0.25
7.51 Æ 1.08
0.50 Æ 0.06
2.72 Æ 0.12
6.14 Æ 0.87
0.40 Æ 0.10
0.68 Æ 0.14
0.33 Æ 0.01
4.11 Æ 0.44
4.08 Æ 0.11
7.46 Æ 0.57
0.38 Æ 0.00
1.01 Æ 0.00
5.62 Æ 0.49
0.30 Æ 0.02
0.77 Æ 0.02
0.26 Æ 0.04
3.70 Æ 0.13
4.38 Æ 0.41
12.3 Æ 1.53
0.22 Æ 0.03
0.84 Æ 0.03
9.08 Æ 0.65
0.14 Æ 0.00
0.40 Æ 0.01
0.15 Æ 0.00
3.29 Æ 0.10
4.00 Æ 0.18
7.88 Æ 0.35
0.12 Æ 0.02
0.18 Æ 0.00
6.64 Æ 0.84
0.10 Æ 0.01
0.41 Æ 0.05
0.13 Æ 0.00
3.36 Æ 0.07
3.60 Æ 0.01
9.49 Æ 0.85
0.06 Æ 0.00
0.07 Æ 0.01
6.80 Æ 0.39
Liver
Spleen
Kidney
Femur
Joint
Muscle
Blood
All bone
99mTc-PIPeDP
Heart
1.91 Æ 0.10
2.24 Æ 0.19
1.46 Æ 0.04
10.5 Æ 0.30
4.63 Æ 0.26
7.25 Æ 0.81
1.44 Æ 0.10
6.51 Æ 0.07
6.75 Æ 0.07
1.25 Æ 0.04
2.04 Æ 0.03
0.82 Æ 0.05
7.39 Æ 0.63
4.57 Æ 0.03
7.55 Æ 0.96
0.69 Æ 0.09
3.38 Æ 0.03
7.54 Æ 1.31
0.57 Æ 0.04
1.62 Æ 0.04
0.47 Æ 0.01
6.19 Æ 0.19
4.56 Æ 0.19
10.9 Æ 1.21
0.34 Æ 0.01
1.49 Æ 0.04
7.92 Æ 0.99
0.44 Æ 0.01
1.49 Æ 0.11
0.39 Æ 0.01
5.46 Æ 0.34
5.31 Æ 0.17
13.4 Æ 1.43
0.25 Æ 0.05
0.51 Æ 0.04
10.3 Æ 0.61
0.26 Æ 0.02
1.13 Æ 0.06
0.32 Æ 0.05
5.52 Æ 0.28
4.55 Æ 0.06
11.6 Æ 0.77
0.15 Æ 0.01
0.17 Æ 0.03
10.0 Æ 0.33
0.34 Æ 0.03
1.76 Æ 0.16
0.26 Æ 0.06
5.94 Æ 0.13
6.51 Æ 0.60
12.5 Æ 2.26
0.10 Æ 0.01
0.13 Æ 0.04
10.5 Æ 1.32
Liver
Spleen
Kidney
Femur
Joint
Muscle
Blood
All bone
J. Label Compd. Radiopharm 2012, 55 429–435
Copyright © 2012 John Wiley & Sons, Ltd.
www.jlcr.org

L. Qiu et al.
(a)
(b)
(c)
(d)
Figure 4. Uptake ratios of bone to soft tissues at different time post-injection of 99mTc-DPs. Data were expressed as mean Æ SD. Statistical differences among three
groups were evaluated using unpaired Student’s t-test (*, p < 0.05; **, p < 0.01).
Table 3. Statistical difference among the bone uptake of three 99mTc-DPs assessed by the unpaired Student’s t-test
Time
5 min
15 min
30 min
60 min
120 min
240 min
t
p
9.141
0.0118
16.65
0.0036
9.784
0.0103
16.83
0.0035
7.748
0.0163
8.082
0.015
significant.38 Moreover, the accumulation of 99mTc-DPs in the liver examined, the carbon chain between the imidazolyl and geminal
DP groups determined the radioactivity uptake of bone and soft
tissue. At 1 h post-injection, 99mTc-PIBDP showed a higher selec-
tive bone uptake and lower soft tissue uptake, therefore uptake
ratios of bone to soft tissues. It has the great potential to serve as
bone scanning agent among three designed 99mTc-DPs. We will
continue to explore 99mTc-PIBDP as a novel SPECT imaging agent
in larger animals and animals with bone metastases.
was also higher in the period of investigation, suggesting that
these radiotracers were not only cleared through the kidney but
also eliminated from the liver. This kind of metabolic pathway
may increase the burden on the liver. The uptake of 99mTc-PIPrDP,
99mTc-PIBDP, and 99mTc-PIPeDP in blood cleared rapidly (Table 2).
This is also consistent with the previous pharmacokinetic investiga-
tion of other 99mTc-DPs.23
The uptake ratios of bone to heart, liver, spleen, and muscle for
the complex 99mTc-PIBDP were higher than those of 99mTc-PIPrDP
and 99mTc-PIPeDP at any given time post-injection (Figure 4).
99mTc-PIBDP has both higher selective uptake in the skeletal
system and lower background uptake in soft tissues. Therefore, it
has great potential to shorten the interval between injection and
imaging, lessening the burden on patients in terms of examination
time when compared with the clinically widely used 99mTc-MDP
agent. Statistical analysis of bone uptakes and uptake ratios of
bone to soft tissues (heart, liver, spleen, blood, muscle) of three
radiopharmaceuticals (Table 3 and Figure 4) revealed that they
were significantly different.
Acknowledgements
We are very grateful to the National Natural Science Foundation
of China (20801024 and 21001015), Natural Science Foundation
of Jiangsu Province (BK2009077), and Key Medical Talent Project
of Jiangsu Province (RC2011097) for their financial support.
Conflict of Interest
The authors did not report any conflict of interest.
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