An osteoclast-targeting agent for imaging and therapy of bone metastasis

Article abstract of DOI:10.1016/j.bmcl.2008.07.092

A hybrid compound (DO3A-BP) featuring a radiometal bifunctional chelator (1,4,7,10-tetraazacyclotetradecane-N,N′,N″,N?-tetraac etic acid, DOTA) and an osteoclast-targeting moiety (bisphosphonate) was designed and synthesized. The ¹¹¹In-labeled complex of DO3A-BP showed significantly elevated uptake in osteoclasts compared to the undifferentiated adherent bone marrow derived cells. Biodistribution studies revealed a favorable tissue distribution profile in normal mice with high bone uptake and long retention, and low or negligible accumulation in non-target organs.

Full text of DOI:10.1016/j.bmcl.2008.07.092

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4789–4793
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
An osteoclast-targeting agent for imaging and therapy of bone metastasis
Wei Liu a, Asghar Hajibeigi a, Mai Lin a, Cynthia L. Rostollan a, Zoltan Kovacs b, Orhan K. Öz a, Xiankai Sun a,
*
a Department of Radiology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, TX 75390, USA
b Advanced Imaging Research Center, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, TX 75390, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A hybrid compound (DO3A-BP) featuring a radiometal bifunctional chelator (1,4,7,10-tetraazacyclotetra-
decane-N,N0,N00,N000-tetraacetic acid, DOTA) and an osteoclast-targeting moiety (bisphosphonate) was
designed and synthesized. The 111In-labeled complex of DO3A-BP showed significantly elevated uptake
in osteoclasts compared to the undifferentiated adherent bone marrow derived cells. Biodistribution
studies revealed a favorable tissue distribution profile in normal mice with high bone uptake and long
retention, and low or negligible accumulation in non-target organs.
Received 24 June 2008
Revised 21 July 2008
Accepted 24 July 2008
Available online 27 July 2008
Keywords:
Ó 2008 Elsevier Ltd. All rights reserved.
Bone metastasis
Imaging agent
Osteoclast
Indium-111
Bifunctional chelator
Bisphosphonate
A variety of cancers preferentially metastasize to the skeleton at
their advanced stages. Whole-body scan using 99mTc-MDP (MDP:
methylene diphosphonate) is currently the standard clinical prac-
tice for the detection of bone metastases.1,2 However, due to its
low specificity a final diagnosis is often aided by other imaging
modalities, such as X-ray radiography, magnetic resonance imag-
ing (MRI), computed tomography (CT), positron emission tomogra-
phy, and/or bone marrow biopsy.3,4 Of the clinical methods to treat
bone metastases, therapies utilizing bisphosphonates (BPs) and
radiopharmaceuticals play critical roles. To date, several BPs have
been approved by the FDA5 and they are commonly used to treat
skeletal complications caused by metastases and other bone dis-
eases, including tumor-associated osteolysis,6,7 hypercalcemia,8
Paget’s disease,9,10 and osteoporosis.11 As shown in Figure 1, bis-
phosphonates are a group of compounds with a chemical structure
similar to that of the natural inorganic pyrophosphate (PPi), an
endogenous regulator of bone mineralization, but differing in the
central atom where BPs have a methylene carbon rather than an
oxygen atom in PPi. This structural feature renders BPs resistant
to hydrolysis under acidic conditions or by pyrophosphatases.
Varieties of BPs could be obtained by tuning the R2 side chain while
leaving the R1 group intact as either –OH or –H.12 It has recently
become clear that BPs first bind avidly to the bone mineral surface
and are subsequently internalized selectively by osteoclasts, where
they inhibit the osteoclastic activity and induce apoptosis. In
addition, BPs have been found to inhibit tumor cell adhesion to
mineralized bone as well as tumor cell invasion and
proliferation.13,14
The binding of the hydroxyl groups of BPs to Ca2+ in hydroxya-
pitate of bone is responsible for the accumulation of BPs in bone.
However, it reduces the coordination sites of 99mTc-MDP in vivo
and subsequently 99mTc-MDP decomposes into 99mTcO4 and BP
components. Therefore the bone uptake of 99mTc-MDP is mainly
dependent on the osteoblastic activity, and the purely osteolytic le-
sion is poorly detectable.15 DOTA (1,4,7,10-tetraazacyclododecane-
N,N0,N00,N000-tetraacetic acid) is a commonly used bifunctional chela-
tor for radioimmunodiagnosis and radioimmunotherapy because it
is able to form thermodynamically stable and kinetically inert
complexes with many divalent or trivalent metal ions.16–18 To date,
few conjugates of DOTA and BPs have been reported but recent re-
search has focused on the improvement of the binding affinity of
BP to bone for the applications as delivery vehicles of MRI contrast
agents and palliation agents for certain bone diseases.19,20 Given
the high expression of osteoclasts in both osteoblastic and osteo-
lytic lesions, here we report the design and synthesis of an osteo-
* Corresponding author. Tel.: +1 214 645 5978; fax: +1 214 645 5885.
E-mail address: Xiankai.Sun@UTSouthwestern.edu (X. Sun).
Figure 1. Structures of Pyrophosphate and bisphosphonate.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.092

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W. Liu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4789–4793
Scheme 1. Synthesis of derivatives of (a) a bisphosphonate and (b) DOTA.
clast-targeting compound by conjugating DOTA to an osteoclast-
targeting BP moiety through an ethylenediamine linker, and its
in vitro and in vivo evaluation.
A convergent approach was chosen to synthesize the hybrid
ligand (DO3A-BP), in which the derivatives of a bisphosphonate
(3) and DOTA metal chelator (7) were synthesized separately as
shown in Schemes 1 and 2.
ture procedure with an overall yield of 27% (Scheme 1a).21 The
synthesis of 7 was previously reported.22 However, an alternative
route was used in this work (Scheme 2), in which a commercially
available intermediate, 5 (1,4,7,10-tetraazacyclododecane-1,4,7-
tris(t-butyl)acetate: DO3A-t-Bu-ester) was used as starting mate-
rial instead of cyclen and a higher overall yield (77%) was achieved.
Alkylation of 5 with methyl chloroacetate afforded 6, and then 7
The carboxylate acid substituted bisphosphonate, 3-bis-(diben-
zyloxyphosphoryl)propanoic acid (3), was synthesized by a litera-
was obtained by reacting
Subsequent conjugation of 3 with 7 via the standard DCC/HOBt
6 with excess ethylene diamine.
Scheme 2. Synthesis of DO3A-BP (9).

W. Liu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4789–4793
4791
procedure (DCC: dicyclo-hexylcarbodiimide; HOBt: 1-hydrox-
ybenzotriazol) (Scheme 2) afforded an orthogonally protected
DO3A-BP (8) in a yield of 50%. Compound 8 was obtained with high
chemical purity via the column chromatography eluted with
CHCl3/MeOH (10:1). The 1H NMR spectrum of 8 showed two
well-separated peaks between 8.6 and 9.0 ppm, which can be as-
cribed to the amide protons on the linkage between the DOTA
and BP moieties. A broad peak between 1.8 and 3.5 ppm was ob-
served for the ethylene protons of the macrocycle and it over-
lapped with the methylene proton signals on the pendent arms.
The product, 9 (DO3A-BP), was obtained in nearly quantitative
yield after a two-step deprotection, where the t-butyl and benzyl
protecting groups were removed by trifluoroacetic acid and Pd/C-
catalyzed hydrogenation, respectively. Bisphosphonates are typi-
cally protected in the form of tetraalkyl bisphosphonate esters
and deprotection was conducted by either acid hydrolysis or sily-
lation–dealkylation, however low yields were commonly seen
due to the decomposition under the acidic condition of the hydro-
lysis.19,23 Our choice of using benzyl rather than alkyl as the BP
protecting group enabled us to selectively deprotect 8 in quantita-
tive yield by two separate procedures, which is advantageous to
the formation of macrocyclic metal complexes; and provide a UV
chromophore for the monitoring of the synthetic procedures.24
The radiolabeling of DO3A-BP with 111In was carried out in
0.4 M NH4OAc buffer, pH 7.5, and monitored by radio-TLC every
4 h via reversed-phase C18 TLC eluted with 10% NH4OAc/MeOH
(v/v: 3:1). Under this TLC condition, free 111In stays at the origin,
while the labeled complex migrates with the mobile phase to a cer-
tain distance. Although it is well documented that DOTA and its
derivatives can be labeled in nearly quantitative radiochemical
yields within 2 h under mild conditions,25,26 such a yield (>95%)
could only be obtained after 44 h under similar conditions in the
formation of 111In-DO3A-BP.27 The slow kinetics of DO3A-BP is
likely due to the 111In binding competition between the bisphosph-
onate and the DOTA moieties.20 An efficient radiolabeling method
should be developed for the further application of this agent.
We found that the ability of bisphosphonate to chelate metal
ions was dramatically reduced at low pH values due to the proton-
ation of the phosphonate groups, which is consistent with the lit-
erature reports.28,29 Based on this pH-dependent binding manner
of BP to metal ions, the 111In-labeled complex was challenged by
reducing the pH from 7.5 to 4.0. The low pH was maintained for
3 days, no free 111In was observed. This clearly indicates that the
radiometal ion, 111In(III), was bound to the DOTA moiety.
centration in rat serum was in large excess. The 111In-DO3A-BP
complex remained nearly 100% intact within 2 days of incubation
with rat serum at 37 °C (n = 4). The high in vitro stability of 111In-
labeled complex warrants further in vitro and in vivo evaluation.27
The osteoclast-targeting property of 111In-DO3A-BP was evalu-
ated on mouse bone marrow derived cells grown for 7 days in
the presence or absence of receptor activator for nuclear factor
j
B (RANK) and macrophage colony-stimulating factor (M-CSF) li-
gands according to an established method.30,31 Osteoclasts are
large multinucleate cells formed by the fusion of cells of the mono-
cyte-macrophage lineage in the presence of RANK ligand and M-
CSF, which are characterized by high expression of tartrate resis-
tant acid phosphatase (TRAP) and cathepsin K. After incubation
for 5–8 days, osteoclasts were formed as seen by cell morphology
microscope and TRAP staining (Fig. 2, right). In contrast, bone mar-
row adherent cells showed negative response to TRAP staining (Fig.
2, left).
For comparison, the uptake in original bone marrow adherent
cells (BMCs) and bone marrow macrophages (BMMs) were used
as controls. Bone marrow macrophages are considered as the inter-
mediates between BMC and OCs and are formed by treatment with
M-CSF only. After a 1-h incubation of 111In-DO3A-BP with the cells,
the cells were lysed and the lysates were counted by a c-counter.
The cell uptake of 111In-DO3A-BP was normalized to protein con-
tent in each well, which was determined by a commercially avail-
able kit. Shown in Figure 3 the osteoclast uptake of 111In-DO3A-BP
was about twofold higher than either BMC or BMM, demonstrating
its preferential uptake by osteoclasts.32
The biodistribution of 111In-DO3A-BP in normal Balb/c mice is
presented in Table 1.33 As anticipated, the 111In-DO3A-BP complex
showed high accumulation within 1 h post-injection (pi) in the
bone and long residence, which is reflected by only a 20% decrease
over 48 h.
In agreement with the reported BP-derivatized compounds,34
the radiolabeled DO3A-BP showed a relatively high renal uptake,
which, however, was rather efficiently cleared. The rapid blood
clearance was a result of the high affinity of BPs for bone mineral
in vivo.35,36 The low liver uptake and efficient clearance reflects
the high in vivo stability of 111In-DO3A-BP. Impressively, this com-
pound showed no significant uptake in other non-target organs
(e.g. lungs, spleen, heart, or brain) and remained intact in the ex-
creted urine within 48 h pi as determined by radio-TLC. The high
bone/blood and bone/muscle contrasts further demonstrate the
potential of this hybrid compound as a bone-seeking agent.
Taken together, we have synthesized an osteoclast-targeting
compound by conjugating DOTA and a BP moiety via an ethylene-
diamine linker in high overall yields. Our preliminary in vitro and
in vivo evaluation results indicate that this compound may find
applications in osteoclast-targeted radiotherapy and nuclear imag-
The octanol–water partition coefficient (logP) of 111In-DO3A-BP
was determined to be ꢀ4.05 0.44 (n = 10), indicative of the highly
hydrophilic nature of the compound. The in vitro serum stability of
111In-DO3A-BP was evaluated out to 48 h by radio-TLC. The con-
centration of 111In-DO3A-BP was 0.05 mM, while the protein con-
Figure 2. TRAP staining of bone marrow adherent cells (left) and osteoclasts (right).

4792
W. Liu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4789–4793
18. Li, M.; Meares, C. F.; Salako, Q.; Kukis, D. L.; Zhong, G. R.; Miers, L.; DeNardo, S. J.
Cancer Res. 1995, 55, 5726s.
19. Kubicek, V.; Rudovsky, J.; Kotek, J.; Hermann, P.; Vander Elst, L.; Muller, R. N.;
Kolar, Z. I.; Wolterbeek, H. T.; Peters, J. A.; Lukes, I. J. Am. Chem. Soc. 2005, 127,
16477.
20. Vitha, T.; Kubicek, V.; Hermann, P.; Elst, L. V.; Muller, R. N.; Kolar, Z. I.; Wolterbeek,
H. T.; Breeman, W. A.; Lukes, I.; Peters, J. A. J. Med. Chem. 2008, 51, 677.
21. Page, P. C.; McKenzie, M. J.; Gallagher, J. A. J. Org. Chem. 2001, 66, 3704.
22. Andre, J. P.; Geraldes, C. F.; Martins, J. A.; Merbach, A. E.; Prata, M. I.; Santos, A.
C.; de Lima, J. J.; Toth, E. Chemistry 2004, 10, 5804.
23. Vitha, T.; Kubicek, V.; Hermann, P.; Kolar, Z. I.; Wolterbeek, H. T.; Peters, J. A.;
Lukes, I. Langmuir 2008, 24, 1952.
24. Chemical synthesis and characterization data.
(a) General methods. All chemicals, solvents, and reagents were purchased from
Sigma–Aldrich (St. Louis, MO) and used directly without further purification
unless otherwise noted. 1,4,7,10-Tetraazacyclododecane-1,4,7-tris-(t-butyl
acetate) (DO3A-t-Bu-ester) was obtained from Macrocylic Inc. (Dallas, TX).
Silica gel 60 (70–230 mesh) used for column chromatography was obtained
from Sigma–Aldrich. Analytical thin-layer chromatography (TLC) was
performed using Merck 60 F254 silica gel (precoated sheets, 0.2 mm thick)
(Lawrence, KS) and Whatman MCK 18F reversed-phase plates (Maidstone,
Kent, UK). The spectra of 1H NMR, 13C NMR, and 31P NMR were recorded on a
Varian 400 or 500 MHz spectrometer; chemical shifts are expressed in ppm
relative to TMS (0 ppm), or chloroform (7.26 ppm), and phosphoric acid was
used as the external standard. Matrix-assisted laser desorption/ionization
(MALDI) mass spectra were acquired on an Applied Biosystems Voyager-6115
mass spectrometer. All reactions were carried out under a nitrogen atmosphere
in degassed dried solvents with magnetic stirring. Bulk solvent removal was
done by rotary evaporation under reduced pressure, and trace solvent was
removed by vacuum pump.
Figure 3. Comparative uptake of 111In-DO3A-BP by osteoclasts (OCs), bone marrow
adherent cell (BMC), and bone marrow macrophages (BMMs) at 1 h incubation.
Data were obtained from five independent experiments (n > 6), and are presented
as uptake ratios versus BMC. Gamma counts of the cell lysates were normalized to
the protein content of each well for the uptake ratio calculation.
Table 1
Biodistribution of 111In-labeled DO3A-BP in normal Balb/c mice (n = 4)
1 h pi
4 h pi
0.01 0.00
0.16 0.04
0.33 0.05
0.15 0.04
3.93 1.01
0.04 0.01
2.66 0.81
24 h pi
0.00 0.00
0.08 0.02
0.16 0.01
0.08 0.01
1.16 0.16
0.03 0.01
2.02 0.59
48 h pi
0.00 0.00
0.08 0.02
0.14 0.01
0.09 0.01
0.70 0.09
0.05 0.01
2.60 0.56
Blood
Lung
Liver
Spleen
Kidney
Muscle
Bone
0.34 0.08
0.52 0.06
0.41 0.04
0.23 0.02
4.61 0.42
0.49 0.05
3.39 0.49
(b) Compound 3. To a suspension of methylene bis(phosphonic dichloride)
(5.00 g, 20.0 mmol) in dry toluene (10 mL),
a mixture of benzyl alcohol
(8.70 mL, 84.0 mmol) and pyridine (6.79 mL, 84.0 mmol) was added dropwise
by an addition funnel while the temperature was maintained at 0 °C. The
reaction was allowed to reach room temperature and the mixture was stirred
for 16 h. The solids formed during the reaction were removed by filtration and
washed twice with toluene. The filtrate was washed twice with 2 M NaOH and
then once with water. After the removal of bulk solvent, the residue was
submitted to column chromatography on silica gel eluting with 100% EtOAc.
Evaporation of appropriate fractions afforded 1 as colorless oil. Compound 1
(5.80 g, 10.8 mmol) in THF (50 mL) was slowly added to a suspension of NaH
(0.27 g, 11.3 mmol) in THF (100 mL) at 0 °C. After the addition was completed,
the reaction mixture was allowed to reach 20 °C and stirred for 30 min. Ethyl
B/B
B/M
10.12
6.94
393.12
72.91
644.90
69.90
816.49
49.06
Data are presented as %ID/g standard deviation. B/B, bone/blood; B/M, bone/
muscle.
bromoacetate (1.91 g, 11.4 mmol) was then added.
A white precipitate
appeared during the addition. After the mixture was stirred for 48 h, the
white solids were removed by filtration and the concentrated residue was
submitted to column chromatography on silica gel eluting with EtOAc/Hexane
(v/v: 7:3). Evaporation of appropriate fractions gave 2 as colorless oil. A
solution of 2 (4.1 g, 6.60 mmol) in methanol (20 mL) was added to 40 mL of a
KOH solution (0.46 g, 8.21 mmol) in 50% methanol (aq) at 0 °C. The mixture
was stirred at room temperature until 2 disappeared as monitored by TLC.
After removal of methanol, the residual was extracted with ethyl ether. The
aqueous layer was acidified to pH 2 by adding aqueous KHSO4 (1 M) and then
extracted with of CHCl3. The extract was dried over MgSO4 and concentrated in
vacuo to give 3 as colorless oil (over all yield 27%): 1H NMR (CDCl3, 500 MHz): d
2.90 (2H, td, J = 6.5, 16.5 Hz), 3.28 (1H, tt, J = 5.5, 24.0 Hz), 4.95–5.05 (8H, m),
7.20–7.35 (20H, m). MS (MALDI-TOF) 617.6 (M+Na+).
(c) Compound 7. Compound 4 (11.52 g, 19.36 mmol) was dissolved in 100 mL of
acetonitrile containing 2 equiv of K2CO3. The resulting mixture was stirred at
50 °C for 16 h. After removal of the solids, the filtrate was dried under vacuum.
The residue was redissolved in CHCl3 and washed with water thoroughly. The
evaporation of CHCl3 afforded 5 in quantitative yield. To 50 mL of 5 (5.26 g,
10.2 mmol) in acetonitrile, K2CO3 (3.37 g, 24 mmol) was added followed by
methyl chloroacetate (1.14 g, 10.5 mmol) in 5 mL of acetonitrile. The resulting
mixture was stirred at 55 °C for 2 days. After removal of the solids and
evaporation of the solvent, the residue was redissolved in CHCl3 and washed
thoroughly by water. The organic layer was then dried over sodium sulfate.
Removal of the solvent under vacuum gave 6 as brown oil, which was used for
the next step without purification. A fivefold excess of ethylenediamine was
cooled in an ice bath and then mixed with a concentrated solution of 6 in THF.
The reaction mixture was stirred at room temperature for 72 h. After
evaporation of THF, the excess amine was distilled off at 50 °C under high
vacuum. Compound 7 was obtained as a white foam with an overall yield of
77%, which showed high chemical purity as evidenced by: 1H NMR (CDCl3,
400 MHz) d 1.45 (27 H, s), 2.52–3.33 (30H, m), 8.76 (1H, br); 13C NMR (CDCl3,
100 MHz) d 28.45, 42.33, 43.03, 52.22, 52.71, 53.79, 55.15, 56.50, 57.15, 58.42,
81.10, 81.22, 170.84, 170.87, 172.68; MS (MALDI-TOF) 615.8 (M+H+).
ing of bone diseases, which over-expresses osteoclasts relative to
osteoblasts or osteoblastic activity, such as multiple myeloma.
Acknowledgments
This work was partially supported by an NIH R21 grant
(CA119219; XS) and an unrestricted fund from the endowment
of the Effie and Wofford Cain Distinguished Chair in Diagnostic
Imaging provided by Dr. Robert W. Parkey. The authors like to
thank Ms. Chandima Siyambalapitiyage for her assistance in the
cell studies.
References and notes
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2. Moore, A. E.; Blake, G. M.; Fogelman, I. J. Nucl. Med. 2008, 49, 375.
3. Peterson, J. J.; Kransdorf, M. J.; O’Connor, M. I. Clin. Orthop. Relat. Res. 2003,
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5. Silverman, S. L.; Maricic, M. Semin. Arthritis Rheum. 2007, 37, 1.
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Dreicer, R.; Kuross, S. A.; Lipton, A.; Seaman, J. J. Cancer 2001, 91, 1191.
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8. Body, J. J. Cancer 2000, 88, 3054.
9. Kohno, N. Int. J. Clin. Oncol. 2008, 13, 18.
10. Penel, N.; Dewas, S.; Doutrelant, P.; Clisant, S.; Yazdanpanah, Y.; Adenis, A.
Support Care Cancer 2008, 16, 387.
11. Pinkerton, J. V.; Dalkin, A. C. Am. J. Obstet. Gynecol. 2007, 197, 559.
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13. Clezardin, P.; Fournier, P.; Boissier, S.; Peyruchaud, O. Curr. Med. Chem. 2003,
10, 173.
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Compound 8.In a round-bottomed flask, compound 7 (200 mg, 0.33 mmol),
compound 3 (212 mg, 0.36 mmol), DCC (74 mg, 0.36 mmol), and HOBt (48 mg,
0.36 mmol) were stirred in 20 mL of dry CH2Cl2 while cooling in an ice bath.
The mixture was then allowed to stand at room temperature with constant
stirring for 48 h. The precipitate, dicyclohexylurea, was filtered off, and the
filtrate was washed sequentially with aqueous KHCO3 solution 3 times and
brine 1 time, and concentrated under reduced pressure to give a white foam.
The residue was purified by column chromatography (silica gel 60–230 mesh)
using 100% EtOAc to 10:1 CHCl3/MeOH for elution. Compound 8 was obtained
15. Schwartz, Z.; Shani, J.; Soskolne, W. A.; Touma, H.; Amir, D.; Sela, J. J. Nucl. Med.
1993, 34, 104.
16. De Leon-Rodriguez, L. M.; Kovacs, Z. Bioconjug. Chem. 2008, 19, 391.
17. Forster, G. J.; Santos, E. B.; Smith-Jones, P. M.; Zanzonico, P.; Larson, S. M. J. Nucl.
Med. 2006, 47, 140.

W. Liu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4789–4793
4793
as a sticky oil (192 mg, 50%): 1H NMR (CDCl3, 500 MHz) d 1.36 (27H, multiple),
30. Holtrop, M. E.; King, G. J. Clin. Orthop. Relat. Res. 1977, 123.
31. Takeshita, S.; Kaji, K.; Kudo, A. J. Bone Miner. Res. 2000, 15, 1477.
32. Osteoclast-targeting evaluation procedures. M-CSF and soluble RANK were
1.80–3.45 (28H, br), 3.70 (1H, tt, J = 6.0, 24.0 Hz), 4.97 (8H, m), 7.18 (20H, m),
8.71 (1H, br), 8.84 (1H, br); 13C NMR (CDCl3, 100 MHz) d 28.08, 28.17, 31.61 (t,
J = 4 Hz), 33.08 (t, J = 135 Hz), 39.54, 46.45, 48.71 (br), 52.23 (br), 55.69, 55.72,
55.83, 56.26, 68.17, 68.23, 82.00, 128.14, 128.52, 128.63, 136.58, 169.81 (t,
J = 8 Hz), 171.67, 172.55; 31P NMR (CDCl3, 162 MHz): d 25.89 (s); Anal. Calcd
for C61H88N6O14P2ꢁ6.5H2O: C 55.99, H 7.78, N 6.42. Found: C 55.80, H 7.62, N
6.68; MS (MALDI-TOF): 1191.6 (M+).
purchased fromR&D Systems (Minneapolis, MN); fetal bovine serum (FBS) and
heat-inactivated (D) FBS were from Atlanta Biologicals (Lawrenceville, GA); the
protein assay kit for protein content measurement was from Bio-Rad
(Hercules, CA); TRAP staining kit of osteoclastic cells was from Sigma–
Aldrich (Diagnostics Acid Phosphatase kit, Procedure No. 387).
(e) Compound 9 (DO3A-BP). Compound 8 (40 mg, 0.03 mmol) was dissolved in
3 mL of a mixed solvent of trifluoroacetic acid (TFA) and CHCl3 (v/v: 1:2). The
reaction mixture was stirred at room temperature overnight. After removal of
bulk solvent under reduced pressure, the residue was dissolved in deionized
waterand thenmixedwith40 mgof Pd/C. Thereaction wasallowed toproceed in
a hydrogenation shaker for 24 h. After removal of the solids, evaporation of water
under vacuum gave the target compound (9) as a colorless thick oil in nearly
quantitative yield: 1H NMR (D2O, 400 MHz) d 4.02–2.50 (br); 13C NMR (D2O,
100 MHz): d 31.14, 32.35, 33.93, 35.17, 36.41, 38.96, 39.0, 46.0–52.10 (br),
52.10–54.24 (br), 55.0, 174.10; 31P NMR (D2O, 162 MHz): d 20.54. MS (MALDI-
TOF) 701.8 (M+K+).
(a) Generation of osteoclasts and macrophages from bone marrow cells. Male
C57B16 mice were anesthetized and sacrificed by cervical dislocation. The
femurs were removed. Both ends of femurs were cut off and bone marrow was
centrifuge out from the bone matrix in Hank’s Balance Salt Solution (HBSS).
Cell clumps were resuspended by pipetting and then filtered through a 70 lm
cell strainer. Cells were diluted into 20 mL HBSS and then washed once with
HBSS. The cells were resuspended in phenol-free alpha minimal essential
medium (a-MEM) containing 10% DFBS. After cell counting (trypan blue
assay), the cells were seeded into 24-well plates at 5 ꢂ 105 cells per well
(n = 8). Bone marrow adherent cells were maintained in 1 mL/well of
a-MEM
containing 10%
atmosphere. Macrophages were generated in
20 ng/mL M-CSF, and 1ꢂ antibiotic, while osteoclasts were generated in
MEM containing 10%
antibiotic (1 mL/well) at 37 °C in a humidified 5% CO2 atmosphere. The media
were changed every 2 days. Multi-nucleated giant cells were formed in 5–8
days, which was verified by TRAP staining.
D
FBS and 1 ꢂ antibiotic at 37 °C in a humidified 5% CO2
25. Liu, S.; Pietryka, J.; Ellars, C. E.; Edwards, D. S. Bioconjug. Chem. 2002, 13, 902.
26. Sun, X.; Fang, H.; Li, X.; Rossin, R.; Welch, M. J.; Taylor, J. S. Bioconjug. Chem.
2005, 16, 294.
a
-MEM containing 10% FBS,
D
a-
D
FBS, 20 ng/mL M-CSF, 50 ng/mL sRANKL, and 1ꢂ
27. Radiochemistry procedures.
(a) General methods. All buffers used in radiochemical procedures and in vitro
or in vivo experiments were prepared by dissolving salts in Milli-Q water (18
M
X
cm) and then treated with Bio-Rad Chelex 100 resin for removal of trace
(b) In vitro cell uptake. In a 24-well plate with cell culture as described above,
metal ions. In-111 in 0.05 N HCl was purchased from Trace Life sciences
(Denton, Texas). Radio-TLC was performed using a Rita Star Radioisotope TLC
Analyzer (Straubenhardt, Germany) on Merck 60 F254 silica gel plates
developed by 10% NH4OAc: MeOH (v/v 3:1) as the mobile phase. Radioactive
medium was removed and 0.5 mL fresh
DO3A-BP (1 Ci/mL) was added to each well. After 1 h incubation, the medium
was removed and the cells were washed with serum free medium (1 mL)
twice. The cells were lysed by the addition of 150 L of Tris-buffered Saline
a
-MEM plus 10% FBS containing 111In-
l
l
samples were counted by a Perkin-Elmer
from Sigma–Aldrich (St. Louis, MO).
c
-counter. Rat serum was purchased
(TBS) containing detergent. The lysates were then transferred to
microcentrifuge tubes. Cell uptake of 111In-DO3A-BP was measured by
(b) Radiolabeling of DO3A-BP with 111In. To 500
lL of the DO3A-BP ligand
counting the cell lysates in a c-counter. The protein concentration in each
lysate was determined using the Bio-Rad Protein Assay. The assay was
repeated 5 times, and all data were pooled for analysis.
solution (0.05 mM) in 0.1 or 0.4 M NH4OAc buffer (pH 7.5), 111InCl3 (ca.
0.5 mCi) in 0.05 N HCl was added. The reaction mixture was incubated at 90 °C
in an Eppendorf thermomixer. The formation of 111In-DO3A-BP was monitored
by radio-TLC on C18 silica plates eluting with 10% NH4OAc/MeOH (v/v 3:1).
(c) Serum stability. The in vitro serum stability experiment was conducted by
33. Biodistribution. All animal studies were performed in compliance with
guidelines set by the UT Southwestern Institutional Animal Care and Use
Committee. Normal male Balb/c mice (18–22 g) were purchased from Harlan
(Indianapolis, IN). The solution of 111In-DO3A-BP was diluted with PBS
(10 mM) to prepare the injection doses. Each mouse was injected with ca.
adding 10 l lCi) to 100 lL of rat serum. The solution
L of 111In-DO3A-BP (ca. 50
was incubated at 37 °C, and sampled for radio-TLC analysis at 1, 4, 24 and 48 h
post-addition to rat serum.
5
l
Ci of radioactivity in 100
prior to sacrifice at each time point (1, 4, 24, and 48 h; n = 4 per time point).
Organs of interest were removed, weighed, and counted by -counter.
lL via the tail vein. The mice were anesthetized
(d) Determination of the partition coefficient (logP) of 111In-DO3A-BP was
determined by adding 5
Milli-Q water and 500 L of octanol (obtained from a saturated octanol/water
solution) (n = 10). The sample vials were shaken for 1 h at room temperature.
Fromeach vial, aliquots of 100 Leach were removed from the octanol phase and
lL of the complex to a solution containing 500
lL of
a c
l
Standards were prepared and counted along with the samples to calculate the
percent injected dose per gram (%ID/g) and percent injected dose per organ
(%ID/organ). The animals of the last time point groups were housed in
metabolic cages (4 mice per cage) to collect urine and feces at 1, 4, 24, and 48 h
pi to evaluate the excretion route and stability of the compound.
34. Ogawa, K.; Mukai, T.; Inoue, Y.; Ono, M.; Saji, H. J. Nucl. Med. 2006, 47, 2042.
35. Santini, D.; Galluzzo, S.; Vincenzi, B.; Schiavon, G.; Fratto, E.; Pantano, F.;
Tonini, G. Ann. Oncol. 2007, 18, vi164.
l
the water phase, respectively, and counted separately. The partition coefficient
was calculated as the ratio of counts in the octanol fraction to the counts in the
water fraction. An average of logP value was obtained from the 10 samples.
28. Rogers, M. J.; Gordon, S.; Benford, H. L.; Coxon, F. P.; Luckman, S. P.;
Monkkonen, J.; Frith, J. C. Cancer 2000, 88, 2961.
29. Sato, M.; Grasser, W.; Endo, N.; Akins, R.; Simmons, H.; Thompson, D. D.; Golub,
E.; Rodan, G. A. J. Clin. Invest. 1991, 88, 2095.
36. Whyte, M. P.; McAlister, W. H.; Novack, D. V.; Clements, K. L.; Schoenecker, P.
L.; Wenkert, D. J. Bone Miner. Res. 2008.