Spacer Effects on in vivo Properties of DOTA-Conjugated Dimeric [Tyr3]Octreotate Peptides Synthesized by a "CuI-Click" and "Sulfo-Click" Ligation Method

Article abstract of DOI:10.1002/cbic.201000639

We report on the SSTR2-binding properties of a series of four dimeric [Tyr3]octreotate analogues with different spacer lengths (nine, 19, 41, and 57 atoms) between the peptides. Two analogues (9 and 57 atoms) were selected as precursors for the design, sy

Full text of DOI:10.1002/cbic.201000639

DOI: 10.1002/cbic.201000639
Spacer Effects on in vivo Properties of DOTA-Conjugated
Dimeric [Tyr3]Octreotate Peptides Synthesized by a “CuI-
Click” and “Sulfo-Click” Ligation Method
Cheng-Bin Yim,[a, b] Berend van der Wildt,[a] Ingrid Dijkgraaf,[b] Lieke Joosten,[b]
Annemarie Eek,[b] Cees Versluis,[c] Dirk T. S. Rijkers,[a] Otto C. Boerman,[b] and
Rob M. J. Liskamp*[a]
We report on the SSTR2-binding properties of a series of four
dimeric [Tyr3]octreotate analogues with different spacer
lengths (nine, 19, 41, and 57 atoms) between the peptides.
Two analogues (9 and 57 atoms) were selected as precursors
for the design, synthesis, and biological evaluation of DOTA-
conjugated dimeric [Tyr3]octreotate analogues for tumor tar-
geting. These compounds were synthesized by using a two-
stage click ligation procedure: a CuI-catalyzed 1,3-dipolar cyclo-
addition (“copper-click” reaction) and a thio acid/sulfonyl azide
amidation (“sulfo-click” reaction). The IC50 values of these
DOTA-conjugated [Tyr3]octreotate analogues were compara-
ble, and internalization studies showed that the nine-atom
111In-DOTA-labeled [Tyr3]octreotate dimer had rapid and high
receptor binding. Biodistribution studies with BALB/c nude
mice bearing subcutaneous AR42J tumors showed that the
111In-labeled [Tyr3]octreotate dimer (nine atoms) had a high
tumor uptake at 1 h p.i. (38.8Æ8.3% IDgÀ1), and excellent
tumor retention at 4 h p.i. (40.9Æ2.5% IDgÀ1). However, the in-
troduction of the extended hydrophilic 57 atoms spacer led to
rapid clearance from the circulation; this limited tumor ac-
cumulation of the radiotracer (21.4Æ4.9% IDgÀ1 at 1 h p.i.).
These findings provide important insight on dimerization and
spacer effects on the in vivo properties of DOTA-conjugated
[Tyr3]octreotate dimers.
Introduction
Somatostatin is a regulatory hormone with diverse physiologi-
cal inhibitory actions on various gastrointestinal functions, in-
cluding the secretion of gastrin, insulin, glucagon, and growth
hormone.[1] These biological effects are mediated by a family
of five specific G protein-coupled cell-surface receptors, the so-
matostatin receptors (SSTRs).[2] As some of these receptors are
overexpressed, in particular subtype 2 (SSTR2) on most neuro-
endocrine tumor cells, native somatostatin has been viewed as
an ideal candidate for the treatment of these tumors. However,
its rapid proteolytic degradation (plasma half-life <3 min) has
limited its clinical usefulness. Structure–activity studies have
been carried out with a variety of synthetic analogues that
have enhanced stability towards enzymatic degradation, and
different affinities for each receptor subtype.[3] Clinical studies
have shown that [Tyr3]octreotate, an octapeptide analogue of
somatostatin, is effective in the treatment of endocrine pancre-
atic tumors.[4]
quate spacing between two RGD motifs.[7] Presently, there are
no reports that identify whether dimeric [Tyr3]octreotate binds
in a similar fashion. This is particularly relevant because recent
studies have established that some somatostatin receptors as-
sociate as homo- and heterodimers.[8] However, the design of
bivalent ligands (where the spacing of two tailored targeting
vectors is suited for simultaneous binding to adjacent receptor
sites) is for the most part a highly empirical endeavor. The
main objective of this study was to determine the SSTR2-bind-
ing properties of dimeric [Tyr3]octreotate analogues, and to
[a] C.-B. Yim, B. van der Wildt, Dr. D. T. S. Rijkers, Prof. Dr. R. M. J. Liskamp
Division of Medicinal Chemistry and Chemical Biology
Utrecht Institute for Pharmaceutical Sciences
Department of Pharmaceutical Sciences, Utrecht University
P.O. Box 80082, 3508 TB Utrecht (The Netherlands)
Fax: (+31)30-2536655
E-mail: r.m.j.liskamp@uu.nl
[b] C.-B. Yim, Dr. I. Dijkgraaf, L. Joosten, A. Eek, Prof. Dr. O. C. Boerman
Department of Nuclear Medicine, Radboud University Nijmegen Medical
Centre
There is growing interest in the development of multimeric
peptide and carbohydrate conjugates, because they have en-
hanced receptor affinity when compared to their correspond-
ing monomeric analogues.[5] For example, it has been demon-
strated that radiolabeled (68Ga, 99mTc, and 111In) dimeric, cyclic
arginine–glycine–aspartic acid (RGD) peptides have higher
tumor uptake and longer tumor retention than their corre-
sponding monomeric counterparts.[6,7] In vitro assays and in
vivo studies have shown that several cyclic RGD-peptide
dimers bind bivalently to their avb3 receptors because of ade-
P.O. Box 9101, 6500 HB Nijmegen (The Netherlands)
[c] C. Versluis
Biomolecular Mass Spectrometry and Proteomics Group
Bijvoet Center for Biomolecular Research and
Utrecht Institute for Pharmaceutical Sciences
Department of Pharmaceutical Sciences, Utrecht University
P.O. Box 80082, 3508 TB Utrecht (The Netherlands)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201000639.
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[Tyr3]Octreotate Dimers for Tumor Targeting
assess the effects of different spacers between the two target-
ing ligands on the in vitro and in vivo characteristics.
Results and Discussion
Rationale for the design
A series of dimeric [Tyr3]octreotate peptides, with different
spacers, has been synthesized and evaluated for their receptor
binding. These conjugates were prepared by CuI-catalyzed 1,3-
dipolar cycloaddition[9] between peptidyl azides and dimeric
alkynes. The investigated spacer
lengths were nine, 19, 41, and 57
atomic units, as measured be-
tween the two alkyne moieties. As
an example, Scheme 1 depicts the
dimeric alkyne building block with
the shortest (nine-atom) spacer.
Scheme 2. Synthesis of [Ahx0,Tyr3]octreotate 1 (Ahx-d-Phe-cyclo(Cys-Tyr-d-
Trp-Lys-Thr-Cys)-Thr-OH). a) SPPS (HBTU/HOBt/DiPEA); b) TFA/TiPS/H2O;
c) 10% DMSO, H2O/CH3CN, pH 8.
The IC50 values of the dimeric
[Tyr3]octreotate derivatives 7–11,
obtained from competitive binding
assays with AR42J tumor cells,
were in the low-nanomolar region.
Scheme 1. Molecular structure
of the dimeric alkyne building
block. Numbers indicate the
atoms involved in the spacer.
formed by using the esterification approach described by
Sieber.[13] The synthesis of the linear peptide followed estab-
lished Fmoc/tBu chemistry protocols.[14] To enable “copper-
click” chemistry, an azide functionality was introduced at the
N terminus by HBTU-coupling with 6-azidohexanoic acid. After
cleavage from the resin, cyclization of the linear peptide was
achieved by DMSO-mediated oxidation of the cysteine
thiols.[15] The cyclic [Ahx0,Tyr3]octreotate 1 was obtained in an
overall yield of 24%, after preparative HPLC, and identified by
mass spectrometry.
The dimer with the longest spacer (57 atoms) 11, which ex-
erted the highest receptor affinity, and the dimer with the
shortest spacer (nine atoms) 7 were selected as candidates for
DOTA
(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic
acid)-conjugation, radiolabeling, and subsequent in vivo evalu-
ation. These DOTA-conjugated dimeric [Tyr3]octreotate deriva-
tives were synthesized by a combination of CuI-catalyzed 1,3-
dipolar cycloaddition and thio acid/sulfonyl azide amida-
tion,[10,11] which have been denoted as “copper-click” and
“sulfo-click”, respectively. This two-stage click-ligation method
has been shown to be very useful for the metal-free and con-
venient introduction of a metal chelator, like DOTA, in the
design of multimeric peptide conjugates for in vivo research
applications.[12] A strategy based on sequential click reactions
could serve as a powerful process for incorporating virtually
any peptide sequence, without interference from other func-
tional groups of the peptide. This ligation procedure avoids
the use of protecting groups, and enables versatile conjuga-
tion with unprotected peptides. Herein, we describe the appli-
cation of this two-stage click approach in the design, synthesis,
and in vivo characterization of DOTA-conjugated dimeric [Ty-
r3]octreotate analogues. Biodistribution studies with 111In-la-
beled compounds were evaluated in a BALB/c nude mouse
model with subcutaneous AR42J pancreatic tumors.
We have reported the convergent synthesis of amino acid
based dendrimers with peripheral propargyl groups to enable
the “copper-click” reaction with peptide-derived azides.[16] This
flexible synthetic strategy, based on the dihydroxybenzoic acid
moiety, can be easily modified with a variety of branching
units, rigidities, cavity sizes, and tunable surface functionali-
ties.[17] Benzoic acid-based monomeric alkyne 2 and dimeric al-
kynes 3–6 (Scheme 3) were prepared following a procedure
which was previously described.[18] In addition, to enable the
“sulfo-click” reaction with the DOTA metal chelator, a thio acid
functionality was introduced. This thio acid moiety can be
readily prepared from a 2,4,6-trimethoxybenzyl (TMOB) thio
ester precursor, as previously reported.[12] Hence, a selection of
mono- and dimeric alkynes 2b, 3b and 6b with TMOB-protect-
ed thio acids was synthesized from their corresponding car-
boxylic acids 2a, 3a and 6a, respectively, as depicted in
Scheme 3.
Synthesis
Conjugation of [Ahx0,Tyr3]octreotate to mono- and dimeric al-
kynes by the “copper-click” reaction procedure: The synthesis
route towards the monomeric [Tyr3]octreotate analogue 7 and
dimeric [Tyr3]octreotate conjugates 8–11 is outlined in
Scheme 4. Following general procedures for microwave-assist-
ed “copper-click” chemistry,[12,16,18b,c] azide 1 was reacted with
acetylenes 2a–6a in the presence of CuSO4/Na ascorbate in
THF/H2O (1:1, v/v) under microwave irradiation at 1008C. HPLC
Syntheses of [Ahx0,Tyr3]octreotate and dimeric alkynes: The syn-
thesis of the [Tyr3]octreotate analogue with an N-terminal 6-
azidohexanoyl (Ahx) linker on solid phase, and its oxidation
towards [Ahx0,Tyr3]octreotate 1 is depicted in Scheme 2. This
peptide required a C-terminal acid functionality; this was ach-
ieved by using the hydroxymethyl-based Wang (HMP) resin.
The loading of the first amino acid, Fmoc-Thr(tBu)-OH, was per-
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R. M. J. Liskamp et al.
pling was allowed to run for 1 h at room temperature, and
subsequent treatment with TFA/TiPS/H2O removed the tBu
groups of DOTA. After purification by HPLC (twice), the N-acyl-
sulfonamides 16, 17, and 18 were obtained in overall yields of
43, 22, and 12%, respectively. These DOTA-conjugated [Tyr3]-
octreotate conjugates were characterized by a nano-ESI LC-MS
system. As an example, Figure 1 shows the high-resolution
Figure 1. High-resolution ESI-MS mass spectrum of DOTA-conjugated dimer-
ic [Tyr3]octreotate N-acylsulfonamide 18: m/z observed, 3903.968; calculat-
ed, 3903.700 (monoisotopic).
Scheme 3. Dihydroxyenzoic acid-based mono- and dimeric alkynes 2a–6a,
and synthesis of TMOB-protected mono- and dimeric thio esters 2b, 3b,
and 6b from their carboxcylic acids 2a, 3a, and 6a. a) TMOB-thiol, EDCI,
DMAP, DMF, 16 h, RT; yields: 2b (27%), 3b (59%), 6b (58%).
mass spectrum of DOTA-conjugated dimeric [Tyr3]octreotate
analogue 18. We also tried a “sulfo-click” ligation in which the
DOTA moiety was unprotected; however, the isolated yield of
this reaction was not improved. Moreover, to avoid any prema-
ture complexation of metal atoms by the unprotected DOTA
moiety, we preferred a tBu-protected DOTA moiety during the
whole synthesis of the molecular constructs.
analysis showed complete conversion within 5 min. Optimiza-
tion studies showed that 0.5 equivalent CuSO4 per alkyne
moiety, accompanied by 2.5 equivalent Na ascorbate, gave the
best results for the examined click reactions. After semi-prepa-
rative HPLC, 7–11 were obtained in acceptable to very good
yields (33–90%). The identities of the cycloadducts 7–11 were
confirmed by mass spectrometry. These compounds were not
contaminated with any copper species.
To summarize, the four dimeric [Tyr3]octreotate analogues
8–11 (with spacers ranging from 9 to 57 atoms) were success-
fully synthesized using the “copper-click” method between
peptidyl azides and alkyne-hydroxy benzoic acid derivatives.
The receptor affinities of these dimers were determined by
using an in vitro binding assay, and compared to their mono-
meric counterpart 7. For subsequent biological characteriza-
tion, a selection of three DOTA-conjugated [Tyr3]octreotate an-
alogues (16–18) were prepared by a combination of “copper-
click” and “sulfo-click” chemistry. In addition to their in vitro
characterization, these compounds were also evaluated for
their tumor targeting and pharmacokinetic profiles in an in
vivo tumor model.
Synthesis of mono- and dimeric DOTA-conjugated [Tyr3]octreo-
tate conjugates by the two-stage click ligation method: The
preparation of DOTA-conjugated peptide conjugates 16–18 by
using a combination of “copper-click” chemistry and the
“sulfo-click” procedure is shown in Schemes 5 and 6. Analo-
gous to the synthesis of peptide triazoles 7–11, the “copper-
click” reaction between azide 1 and alkynes 2b, 3b or 6b was
performed in the presence of 0.5 equivalent CuSO4 per alkyne
functionality. Under optimized conditions these reactions were
allowed to proceed for 16 h at room temperature. After semi-
preparative HPLC, triazoles 12–14 were isolated in 31–66%
yields (Scheme 5). Under these conditions, triazoles 12–14
were found to be stable against hydrolysis. These protected
thio esters were treated with trifluoroacetic acid/triisopropyl-
silane (TFA/TiPS) to give the free thio acids that were used
directly in the subsequent thio acid/sulfonyl azide amidation
(“sulfo-click”) with DOTA derivative 15 (Scheme 6).[12] This cou-
Receptor binding studies
Binding affinities for the somatostatin receptor of the dimeric
[Tyr3]octreotate conjugates 8–11, were determined by using a
competitive binding assay on AR42J tumor cells, by using
[
111In-DOTA0,Tyr3]octreotate as a radiotracer. The IC50 values
were calculated from fitted sigmoidal displacement curves as
depicted in Figure 2A, and summarized in Table 1. For compar-
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[Tyr3]Octreotate Dimers for Tumor Targeting
spacer (nine atoms) were select-
ed as precursors for further in
vitro and in vivo evaluation, to
assess spacer effects on pharma-
cokinetic properties. Therefore,
DOTA-conjugated derivatives 17
and 18, synthesized by the two-
stage click method (vide supra),
were tested for their binding
properties in a competitive bind-
ing assay (Figure 2B). As a refer-
ence, DOTA-conjugated mono-
meric [Tyr3]octreotate analogue
16 was also includeed. The IC50
values of these DOTA-containing
compounds, labeled with natIn,
are reported in Table 1. With IC50
values around 1 nm, the natIn-
DOTA-labeled
[Tyr3]octreotate
monomer 16, and dimers 17 and
18 exerted comparable receptor
binding. The incorporation of
natIn-DOTA, as in 16–18, led to
an overall increase in receptor
affinity compared to [Tyr3]oc-
treotate dimers 8–11 without
the DOTA metal chelator; this
was advantageous for the cur-
rent study.
Lipophilicity studies
The logD values of 111In-labeled
DOTA-conjugated mono- and di-
meric [Tyr3]octreotate sulfona-
mides 16, 17, and 18 were
À2.79Æ0.05, À2.04Æ0.05 and
À1.89Æ0.13, respectively, and
were obtained from n-octanol/
saline partition coefficient meas-
urements. Compared to [111In-
DOTA0,Tyr3]octreotate (logD=
À3.63Æ0.06), 111In-labeled com-
pound 16 was slightly more lipo-
philic (factor: 7ꢁ), which may be
attributed to the synthetic core
Scheme 4. Synthesis of mono- and dimeric [Tyr3]octreotate triazoles 7–11. a) Azide 1 and CuSO4/Na ascorbate,
THF/H2O, microwave (1008C), 5 min.
ison, the [Tyr3]octreotate conjugate 7 was also included as one
of the competing ligands in the same in vitro assay. All the
synthesized compounds showed high receptor binding for the
AR42J cell line, as is apparent from the low IC50 values: be-
tween 1 and 5 nm. Remarkably, the incorporation of different
spacers, ranging from 9 to 57 atoms in length, between the
two [Tyr3]octreotate motifs resulted in only minor changes to
in vitro receptor affinity.
moiety (benzoyl core, sulfonamide, triazole, and spacer).
Serum stability studies
To determine the in vitro stability, [Tyr3]octreotate analogues
were incubated in human serum, and analyzed at several time
intervals. After incubation in human serum at 378C for 24 h,
111In-labeled compounds 16, 17, and 18 remained intact, as in-
ferred from RP-HPLC. The retention times were 16.2, 17.1, and
16.5 min for 16, 17, and 18, respectively. Fortunately, these
111In-labeled compounds showed high stability: >99% intact
However, dimeric compound 11 had the highest affinity.
Therefore, dimeric [Tyr3]octreotate conjugate 11 with the lon-
gest spacer (57 atoms), and dimer 8 containing the shortest
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753

R. M. J. Liskamp et al.
peptide, even after 48 h incuba-
tion in human serum. These data
indicated that the synthetic
compounds, with both sulfona-
mide and triazole linkers, are
highly stable in human serum.
Internalization kinetics
Figure 3A and B shows the re-
ceptor binding and internaliza-
tion rates, respectively, of 111In-la-
beled 16–18 after incubation at
378C with AR42J rat pancreatic
tumor cells. These radiolabeled
[Tyr3]octreotate
analogues
showed specific and time-de-
pendent cellular uptake. 111In-la-
beled 16 and 18 exerted a pro-
gressive increase in receptor
binding, while 111In-labeled 17
had a rapid and high mem-
brane-bound uptake. At 1 h, the
membrane-bound activity of
111In-labeled 17 (14.0Æ0.4%)
was twice as high as 16 (6.0Æ
0.1%) or 18 (7.4Æ0.7%), and re-
mained high after 2 and 4 h of
incubation. As shown in Fig-
ure 3B, 111In-labeled 16 had the
highest
internalization
rate
(16.7Æ0.4% at 30 min and
45.7Æ3.2% at 4 h), followed by
17 (14.7Æ0.4% at 30 min and
40.1Æ1.3% at 4 h) and 18 (9.4Æ
0.2% at 30 min and 32.4Æ3.1%
at 4 h).
Scheme 5. Synthesis of mono- and dimeric [Tyr3]octreotate triazoles 12–14 by the “copper-click” method. a) azide
1, CuSO4/Na ascorbate, THF/H2O, 24 h, RT.
Biodistribution study
The in vivo tumor targeting of
111In-labeled 16–18 was investi-
Table 1. IC50 values for monomeric and dimeric [Tyr3]octreotate ana-
logues 7–11 and for natIn-labeled monomeric and dimeric [Tyr3]octreotate
gated in BALB/c nude mice with subcutaneous SSTR2-express-
ing AR42J tumors. The organ distribution of 111In-labeled 16,
17, and 18 after 1, 4, and 24 h post-injection (p.i.) are summar-
ized in Figure 4 and Table 2. 111In-labeled 16 (Figure 4A) had a
rapid and high tumor uptake (35.99Æ6.89% IDgÀ1 at 1 h p.i.);
this could be blocked by co-injection of an excess of octreo-
tide, which indicates that tumor accumulation was a receptor-
mediated process. 111In-labeled [DOTA0, Tyr3]octreotate, the
reference compound (Figure S11, and Table S1 in the Support-
ing Information), had a tumor uptake of 32.28Æ6.95% IDgÀ1
at 1 h p.i., and 11.63Æ1.24% IDgÀ1 at 24 h p.i. Compared to
DOTA-conjugated analogues 16–18.[a]
Ligand
Monomer/dimer
IC50 [nm]
95% CI[b] [nm]
Non-DOTA
7
8
9
monomer
dimer
dimer
dimer
dimer
1.8
3.4
4.6
3.3
2.1
1.6–2.0
3.0–3.9
3.6–5.8
2.9–3.8
1.7–2.7
10
11
natIn]DOTA
[
16
17
18
monomer
dimer
dimer
0.7
1.0
1.0
0.6–0.9
0.8–1.2
0.9–1.2
[
111In-DOTA0,Tyr3]octreotate, 111In-labeled 16 showed a higher
[a] Values were determined by a competitive binding assay with AR42J
tumor cells, using [111In-DOTA0,Tyr3]octreotate as a radiotracer. (n=2 for
each data point). [b] Confidence interval.
uptake in the pancreas, stomach and kidney at 1 h p.i. (15.77Æ
3.44, 19.27Æ0.93, and 39.65Æ6.20% IDgÀ1 respectively). Nota-
bly, due to fast wash-out from the tissue, the radioactivity in
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[Tyr3]Octreotate Dimers for Tumor Targeting
ly, the activity in the tumor re-
mained high at 4 h p.i. (40.87Æ
2.51% IDgÀ1); this indicates pro-
longed retention of this com-
pound in the tumor. This en-
hanced retention can be ex-
plained by the increased recep-
tor binding rate of 111In-labeled
dimer 17, observed during in
vitro experiment (Figure 3A). At
24 h p.i., however, the accumu-
lated radioactivity in tumor of
111In-
labeled 17 (13.45Æ2.23% IDgÀ1)
was similar to that of 111In-
labeled [Tyr3]octreotate mono-
mer 16 (9.89Æ1.20% IDgÀ1).
A number of studies have re-
ported the effects of specific hy-
drophilic, acidic, or basic linkers
on the in vivo characteristics,
and it was found that these link-
ers are important in reducing ra-
diotracer accumulation in nontu-
mor tissue, and consequently in-
creasing the tumor/background
ratios.[6e,19] The introduction of
the extended hydrophilic 57-
atom spacer, as in the case of
111In-labeled dimer 18, led to
rapid clearance from the blood
pool, and decreased uptake in
the lung, spleen, pancreas, stom-
ach, and liver, when compared
to [Tyr3]octreotate dimer 17
(Figure 4C). Unfortunately, the
rapid wash-out of 111In-labeled
18 from the circulation also de-
creased the radioactivity in the
tumor to half the value of that
of 111In-labeled 17. Apart from
the low tumor uptake (21.36Æ
4.87% IDgÀ1 at 1 h p.i.), radiola-
beled 18 had a very high kidney
accumulation (97.22Æ3.70 at 1 h
Scheme 6. Synthesis of mono- and dimeric DOTA-conjugated [Tyr3]octreotate sulfonamides 16–18 by the “sulfo-
click” method. a) TFA/TiPS (95:5, v/v), 3 h, RT; b) 15, 2,6-lutidine, DMF (4 ꢂ molecular sieves), 1 h, RT; c) TFA/TiPS/
H2O (95:2.5:2.5, v/v/v), 3 h, RT.
pancreas, stomach, and kidney declined to 2.94Æ0.60, 8.43Æ
1.13, and 13.09Æ1.05% IDgÀ1, respectively, at 4 h p.i. Its blood
clearance was fast (1.23Æ0.27% IDgÀ1 at 1 h p.i., 0.04Æ0.01%
IDgÀ1 at 24 h p.i.) with the tumor/blood ratios increasing from
29.2Æ25.2 at 1 h p.i. to 268Æ204 at 24 h p.i. The incorporated
synthetic core with both the sulfonamide and triazole linker
did not influence tumor targeting, as was demonstrated by the
similar values for tumor uptake of [111In-DOTA0,Tyr3]octreotate
and 111In-labeled 16.
and 80.21Æ4.70 at 24 h p.i.).
The comparable tumor uptake at 1 h p.i. of [111In-DOTA0,-
Tyr3]octreotate, 111In-labeled 16, and 111In-labeled 17 were in
accordance with the IC50 values determined in vitro. The unfav-
orable tumor accumulation of 111In-labeled 18 could be due to
a combination of rapid wash-out from the circulation, high
kidney accumulation, and a relatively lower internalization rate
(Figure 3B).
111In-labeled dimeric [Tyr3]octreotate 17, which includes a
nine-atom spacer, showed a high and specific tumor uptake of
38.80Æ8.33% IDgÀ1 at 1 h p.i. (Figure 4B, Table 2). Interesting-
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R. M. J. Liskamp et al.
Figure 3. Time-dependent A) receptor binding and B) internalization of 111In-
labeled 16–18 by AR42J rat pancreatic tumor cells incubated for 30 min, 1, 2
and 4 h at 378C.
Figure 2. Displacement of [111In-DOTA0,Tyr3]octreotate from binding sites on
AR42J tumor cells by increasing concentration of non-radioactive competi-
tor. A) Displacement by dimeric [Tyr3]octreotate conjugates 8, 9, 10, and 11.
Monomeric [Tyr3]octreotate conjugate 7 was used as a reference. B) Dis-
placement by mono- and dimeric natIn-DOTA-conjugated [Tyr3]octreotate 16,
lar “copper-click” cycloaddition followed by thio acid/sulfonyla-
zide “sulfo-click” amidation. Biodistribution studies with 111In-la-
beled compounds were evaluated in a BALB/c nude mouse
model with subcutaneous inoculated AR42J pancreatic tumors.
The 111Inlabeled dimeric [Tyr3]octreotate analogue with a nine-
atom spacer had a high tumor uptake (38.8Æ8.3% IDgÀ1) at
1 h p.i., which was maintained at 4 h p.i. (40.9Æ2.5% IDgÀ1).
However, the introduction of the long hydrophilic 57-atom
spacer into our dimeric [Tyr3]octreotate design resulted in
higher kidney uptake and faster clearance from the circulation,
with concomitantly lower tumor accumulation (21.36Æ4.87%
IDgÀ1 at 1 h p.i.). These findings provide important information
about spacer length effects of dimeric [Tyr3]octreotate pep-
tides on tumor targeting and pharmacokinetics. This can be
translated into future research towards applications of
[Tyr3]octreotate-based conjugates.
17, and 18. [natIn-DOTA0,Tyr3]octreotate was used as a reference (IC50
:
0.7 nm). The error bars indicate the range of the 95% confidence interval
(n=2 for each data point).
Conclusions
We have developed a versatile synthesis method, and have
systemically investigated the binding properties of a series of
dimeric [Tyr3]octreotate analogues, and evaluated the effects
of different spacers between two cyclic [Tyr3]octreotate se-
quences on in vitro characteristics. From a competitive binding
assay, the strongest dimeric binder—the [Tyr3]octreotate dimer
with the longest spacer (57 atoms)—was selected for further
DOTA-functionalization, radiolabeling, and in vivo evaluation.
In addition, the DOTA-conjugated [Tyr3]octreotate dimer with
the shortest spacer (nine atoms), and the corresponding mon-
omeric DOTA-conjugated [Tyr3]octreotate analogue were in-
cluded in the study to assess the effects of spacer length and
dimerization, respectively, on tumor targeting and pharmacoki-
netic properties. These DOTA-conjugated mono- and dimeric
[Tyr3]octreotate-based analogues were successfully prepared
by using the two-stage click chemistry: CuI-catalyzed 1,3-dipo-
Experimental Section
Reagents, materials and analysis methods: Unless stated other-
wise, chemicals were obtained from commercial sources and used
without purification. Peptide-grade solvents were purchased from
Biosolve (Valkenswaard, the Netherlands) and used directly, except
for N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), and
dichloromethane (DCM), which were dried on 4 ꢂ molecular sieves
756
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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2011, 12, 750 – 760

[Tyr3]Octreotate Dimers for Tumor Targeting
Table 2. Biodistribution of 111In-labeled monomeric (16) and dimeric (17
and 18) [Tyr3]octreotate DOTA-conjugated analogues in AR42J tumor-
bearing BALB/c nude mice at 1, 4, and 24 h p.i.[a]
Time after injection
1 h
4 h
24 h
1 h+cold
111In-labeled monomer 16
blood
1.23Æ0.27
35.99Æ6.89
15.77Æ3.44
19.27Æ0.93
39.65Æ6.20
0.83Æ0.18
0.29Æ0.10
0.04Æ0.01
2.91Æ0.55
4.69Æ0.43
1.62Æ0.21
1.82Æ0.29
50.06Æ8.71
1.51Æ0.53
tumor
pancreas
stomach
kidney
liver
26.62Æ5.62
2.94Æ0.60
8.43Æ1.13
13.09Æ1.05
0.26Æ0.03
9.89Æ1.20
0.29Æ0.02
2.97Æ0.49
2.13Æ0.94
0.11Æ0.01
111In-labeled dimer 17
blood
3.24Æ0.69
1.46Æ0.31
40.87Æ2.51
9.24Æ1.55
20.52Æ3.20
52.34Æ4.35
1.94Æ0.30
0.16Æ0.04
13.45Æ2.23
0.54Æ0.05
5.82Æ0.43
9.18Æ0.55
0.72Æ0.04
7.60Æ1.00
8.31Æ1.02
4.19Æ0.41
4.36Æ0.18
79.7Æ11.1
7.23Æ0.64
tumor
pancreas
stomach
kidney
liver
38.80Æ8.33
19.92Æ2.19
25.28Æ2.92
57.71Æ7.90
4.03Æ0.63
111In-labeled dimer 18
blood
0.58Æ0.11
0.16Æ0.17
19.46Æ6.14
7.58Æ1.33
10.42Æ2.01
118.5Æ5.7
0.67Æ0.16
0.03Æ0.001
9.09Æ1.12
2.47Æ0.36
4.15Æ0.26
80.21Æ4.70
0.38Æ0.04
2.08Æ0.27
4.08Æ0.77
9.20Æ2.04
4.77Æ0.69
129.6Æ14.8
2.81Æ0.39
tumor
pancreas
stomach
kidney
liver
21.36Æ4.87
13.03Æ1.75
11.60Æ1.30
97.22Æ3.70
1.06Æ0.24
[a] Each value represents the mean % injected dose per g of tissue ÆSD
of five animals. Mean tumor weight was 0.20 g.
prior to (solid-phase) synthesis. Tentagel S PHB resin was pur-
chased from Rapp Polymere (Tꢃbingen, Germany), and Na-fluore-
nylmethyloxycarbonyl (Fmoc) amino acids were purchased from
Advanced ChemTech, Alexis, or Novabiochem. [DOTA0,Tyr3]octreo-
tate was purchased from Bachem (Bubendorf, Switzerland). Com-
mercially available (Novartis) octreotide (“Sandostatin”) was used as
a receptor-selective ligand. Solid-phase synthesis was carried out in
plastic syringes with polyethylene frits (20 mm) obtained from Ap-
plied Separations Inc. Resin loading was determined by measuring
the UV absorbance of the piperidine–dibenzofulvene adduct
(lmax =300 nm).[20] Analytical thin-layer chromatography (TLC) and
Rf values were determined on Merck precoated silica-gel 60 F-254
(0.25 mm) plates. Spots were visualized with UV light, ninhydrin, or
Cl2/N,N,N’,N’-tetramethyl-4,4’-diaminodiphenylmethane
(TDM).[21]
Column chromatography was performed on Silicycle silica-gel
(60 ꢂ, particle size 41–63 mm) by using distilled solvents. Micro-
wave irradiation was performed in a Biotage Initiator (300 W).
1H NMR spectra were recorded on a Varian G-300 spectrometer
(300 MHz), and chemical shifts are given in ppm (d) relative to
TMS (0.00 ppm). 13C NMR spectra were recorded on a Varian G-300
spectrometer (75.5 MHz), and chemical shifts are given in ppm (d)
relative to CDCl3 (77.0 ppm). The 13C NMR spectra were recorded
by using the attached proton test (APT) sequence. Analytical
reversed-phase HPLC was accomplished on a Shimadzu HPLC
system, and (semi)-preparative reversed-phase HPLC was accom-
plished on a Kratos HPLC workstation. The mobile phase was 0.1%
trifluoroacetic acid (TFA) in CH3CN/H2O (5:95, v/v; solvent A), and
0.1% TFA in CH3CN/H2O (95:5, v/v; solvent B). Analytical HPLC runs
were performed on an Alltech Alltima C8 column (250ꢁ4.6 mm,
pore size 90 ꢂ, particle size 5 mm) by using a linear gradient of
solvent B (0–100% in 20 min) in solvent A at a flow rate of
1.0 mLminÀ1. All preparative HPLC runs were performed on an All-
tech Adsorbosphere XL C8 column (250ꢁ22 mm, pore size 90 ꢂ,
particle size 10 mm, 12.0 mLminÀ1), and semi-preparative HPLC
Figure 4. Biodistribution of A) 111In-labeled [Tyr3]octreotate analogue 16,
B) [Tyr3]octreotate dimer 17 (nine-atom spacer), and C) [Tyr3]octreotate
dimer 18 (57-atom spacer) in BALB/c nude mice bearing subcutaneous
AR42J-expressing tumors in the right flank. Values are given as percentages
of the injected dose per gram of tissue (n=5 mice/group). Blocking was per-
formed by coinjection of octreotide. Mice were dissected at 1, 4, and 24 h
p.i. ID: injected dose.
ChemBioChem 2011, 12, 750 – 760
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
757

R. M. J. Liskamp et al.
runs were performed on an Alltech Alltima C8 column (250ꢁ
10 mm, pore size 90 ꢂ, particle size 10 mm, 4.8 mLminÀ1). The gra-
dient consisted of 100% solvent A for 5 min, to 100% solvent B in
120 min. The purity of all newly synthesized compounds was
judged by analytical HPLC and was found to be at least 98%.
MALDI-ToF analysis was performed on a Kratos Axima CFR appara-
tus with human ACTH(18–39) (2465.2 [M+H]+), or bovine insulin
oxidized B chain (3494.7 [M+H]+) as the external reference, and a-
cyano-4-hydroxycinnamic acid as matrix. High-resolution electro-
spray ionization (ESI) mass spectra were measured on a Micromass
LCT mass spectrometer calibrated with CsI by using nano-ESI at
1200 V capillary voltage and 50 V at the sample cone. All reported
mass values are monoisotopic.
2C(O)CH2CH2), 3.25–3.48 (m, 8H; 2OCH2CH2NH, 2CH2CH2C(O)CH2),
3.50–3.70 (m, 28H; 12OCH2, 2OCH2C(O)NHCH2), 3.83–3.84 (m, 9H;
3OCH3), 4.05 (s, 4H; 2C(O)CH2O), 4.10 (s, 4H; 2C(O)CH2O), 4.14 (t,
J=5.4 Hz, 4H; 2OCH2CH2NH), 4.34 (s, 2H; SCH2), 6.23 (s, 2H;
2CH3OCCH), 6.79 (t, J=2.2 Hz, 1H; CH2OCCH), 7.09 (d, J=2.5 Hz,
2H; 2C(O)CCH); 13C NMR (CDCl3): d=15.8 (CH2CCH), 23.6 (SCH2),
30.4 (CH2CH2CH2), 36.1 (C(O)CH2CH2), 37.7 and 37.8
(C(O)NHCH2CH2CH2), 39.6 (OCH2CH2NH), 55.9 and 56.4 (OCH3), 67.9
(OCH2CH2NH), 69.8 and 70.0 (C(O)CH2O), 70.4 (CH2CCH), 71.2 and
71.5 (OCH2), 83.7 (CH2CCH), 91.6 (CH3OCCH), 105.2 (SCH2C), 106.7
(CH2OCCH), 107.3 (SC(O)CCH), 140.7 (SC(O)C), 160.6 (CH3OC), 161.4
(CH2OC), 162.8 (CH3OC), 171.5, 172.0 and 174.0 (C(O)NH), 193.9
(SC(O)).
Chemistry: The syntheses of dihydroxybenzoic acid-based alkynes
2a–6a have been described previously.[18] The syntheses of 2b,
3b, and 15 have been reported earlier by Yim et al.[12] Details of
the synthesis and characterization of 7–11, 13, and 14 are de-
scribed in the Supporting Information.
Monomeric [Tyr3]octreotate peptide thio ester (12): Aqueous CuSO4
(47 mL, 4.8 mmol) and Na ascorbate (47 mL, 23 mmol) were added to
a solution of azide 1 (10.8 mg, 9.1 mmol) and alkyne 2b (3.5 mg,
9.5 mmol) in THF/H2O (3:1, v/v, 0.3 mL). The reaction mixture was
stirred for 16 h at room temperature. Then, the solvents were
removed under reduced pressure and the residue was taken up in
CH3CN/H2O and purified by semipreparative HPLC (C4). Com-
pound 12 was obtained in 66% yield (6.0 mmol, 9.4 mg). Rt(C4):
32.5 min. ESI-MS: m/z 1560.8 [M+H]+, 1582.8 [M+Na]+, 780.9
[M+2H]2+; calcd for C75H93N13O18S3: 1559.59.
Ahx-d-Phe-cyclo(Cys-Tyr-d-Trp-Lys-Thr-Cys)-Thr-OH (1): All reagents
and glassware were dried in vacuo for 18 h before use. Wang resin
(3.7 g (1.0 mmol), 0.27 mmolgÀ1) was swollen (DCM) and washed
(DMF) prior to loading of the first amino acid. Fmoc-Thr(tBu)-OH
(2.0 g, 5 mmol) was dissolved in DMF (5 mL), dried on 4 ꢂ molecu-
lar sieves, and added to the resin, followed by pyridine (0.7 mL,
8 mmol). The obtained slurry was gently swirled for 30 min until
the amino acid derivative dissolved completely. Then, 2,6-dichloro-
benzoyl chloride (DCBC: 0.7 mL, 5 mmol) was added, and the mix-
ture was gently swirled for 18 h at room temperature. The resin
was subsequently washed (DMF, DCM) and dried. The resin loading
was 68% (0.18 mmolgÀ1), as calculated from an Fmoc determina-
tion. The linear peptide sequence was synthesized according to
Fmoc/tBu solid phase peptide synthesis protocols. After coupling
of N-terminal 6-azido-hexanoic acid, the resin was washed (NMP,
DCM), dried, and suspended in TFA/H2O/TiPS (20 mL, 95:2.5:2.5,
v/v/v), and stirred for 3 h to cleave the peptide from the resin, and
to remove the side chain protecting groups. The crude peptide
was isolated by precipitation (3ꢁ) with cold (À208C) MTBE/hexane
(1:1, v/v). After centrifugation, the pellet was dissolved in acetoni-
trile/water (1:1, v/v), lyophilized, and purified by HPLC. This linear
peptide (45 mg, 38 mmol) was subsequently dissolved in acetoni-
trile/water (1:1, v/v), and DMSO (10%) was added to this solution.
The pH was adjusted to 7.5 with 5% aqueous ammonia. After stir-
ring overnight at room temperature, the solution was partially con-
centrated in vacuo, and the remaining DMSO was evaporated with
a rotational vacuum concentrator (Christ, Alpha-RVC). Following
preparative HPLC, the cyclic peptide was obtained in an overall
yield of 24% (36 mg). Rt: 20.07 min (C8); ESI-MS: m/z 1188.35
[M+H]+, 1210.40 [M+Na]+; calcd for C55H73N13O13S2: 1187.49.
DOTA-conjugated monomeric [Tyr3]octreotate sulfonamide (16): Pep-
tide thio ester 12 (3.7 mg, 2.3 mmol) was treated with TFA/TiPS
(95:5, v/v; 400 mL) for 3 h at room temperature. After concentration
in vacuo, the residue was dissolved in dry DMF (120 mL), then sulfo-
nyl azide 15 (1.7 mg, 2.4 mmol) followed by 2,6-lutidine (2.0 mL,
17 mmol) were added. The reaction mixture was stirred for 1 h at
room temperature and subsequently concentrated in vacuo. This
residue was treated with TFA/TiPS/H2O (95:2.5:2.5, v/v/v) for 3 h at
room temperature. Compound 16 was obtained in 65% yield
(2.8 mg, 1.51 mmol) after purification by semipreparative HPLC.
Rt(C8): 18.28 min; ESI-MS: m/z 1857.0 [M+H]+, 929.0 [M+2H]2+
619.6 [M+3H]3+; calcd for C83H113N19O24S3: 1855.74.
,
DOTA-conjugated dimeric [Tyr3]octreotate C9-spacer sulfonamide (17):
The synthesis of 17 was performed as described for 16, but start-
ing from thio ester 13 (1.7 mg, 0.6 mmol) and sulfonyl azide 15
(0.9 mg, 1.3 mmol). Compound 17 was obtained in 71% yield
(1.3 mg, 0.42 mmol) after purification. Rt(C8): 18.82 min; ESI-MS: m/z
1549.7 [M+2H]2+, 1033.5 [M+3H]3+, 775.4 [M+4H]4+; calcd for
C141H188N32O38S5: 3097.24.
DOTA-conjugated dimeric [Tyr3]octreotate C57-spacer sulfonamide
(18): The same procedure as described for 16 was used, but start-
ing from thio ester 14 (8.4 mg, 2.3 mmol) and sulfonyl azide 15
(1.7 mg, 2.4 mmol). Compound 18 was isolated after column chro-
matography in 22% yield (2.0 mg, 0.51 mmol). Rt(C8): 18.50 min;
ESI-MS: m/z 1953.0 [M+2H]2+, 1302.3 [M+3H]3+, 977.0 [M+4H]4+
781.8 [M+5H]5+; calcd for C177H254N38O52S5: 3903.70.
,
S-2,4,6-Trimethoxybenzyl
3,5-bis((4,8,24-trioxo-6,13,16,19-tetraoxa-
3,9,23-triazaoctacos-27-yn-1-yl)oxy)benzothioate (6b): Ethyl-3-(3-di-
methylaminopropyl)carbodiimide (EDCI, 47 mg, 0.245 mmol), 2,4,6-
trimethoxybenzylthiol (35 mg, 0.163 mmol) and a catalytic amount
of 4-dimethylaminopyridine (DMAP, 2 mg, 0.002 mmol) were added
to a solution of 6a (170 mg, 0.163 mmol) in dry DMF (10 mL). The
yellowish solution was stirred overnight under nitrogen atmos-
phere at room temperature. After evaporating the solvent, the
product was isolated by silica column chromatography by using a
gradient of DCM/MeOH (95:5 to 90:10, v/v). The 2,4,6-trimethoxy-
benzyl-protected dialkyne was obtained as a colorless oil in 58%
yield (117 mg, 94.9 mmol). Rf =0.40 (methanol/dichloromethane,
Cell line and culture conditions: The AR42J cell-line was kindly
provided by the Erasmus Medical Center (Rotterdam, The Nether-
lands). These cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM, Gibco) supplemented with 10% fetal calf serum,
1% penicillin/streptomycin and 1% glutamine. The cultures were
maintained in a humidified atmosphere (5% CO2/95% air, 378C)
and routinely passed by using a trypsin-ethylenediaminetetraacetic
acid solution (Invitrogen).
Receptor binding studies: The half-maximal inhibitory concentra-
tion (IC50) of the peptides for binding to SSTR2 was determined on
AR42J tumor cells in a competitive binding assay with [111In-DO-
1
1:9); H NMR (CD3OD): d=1.74–1.83 (m, 8H; 4CH2CH2CH2), 2.30 (t,
J=2.6 Hz, 2H; 2CCH), 2.39 (m, 4H; 2CHCCH2), 2.47 (m, 4H;
758
www.chembiochem.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2011, 12, 750 – 760

[Tyr3]Octreotate Dimers for Tumor Targeting
TA0,Tyr3]octreotate as a tracer. AR42J cells were seeded into six-
well plates at 8ꢁ105 cells/well and cultured until confluent. Cells
were washed twice with binding buffer (DMEM with 0.1% w/v
bovine serum albumin). Subsequently, binding buffer (1.5 mL), non-
radioactive competitor (from 0.1 to 300 nm), and a trace amount
of radiotracer (100 Bq) were added to each well. After incubation
(378C for 3 h), the medium was removed, and cells were washed
twice with binding buffer, extracted from the wells, and cell-associ-
ated radioactivity was determined in a g-counter. For the “cold” la-
beling of 16–18 with natIn3+, each of the peptides was dissolved in
an aqueous solution (25 mL, 10 mm NH4OAc). Subsequently, a three
molar excess of InCl3 (Aldrich) was added. The InIII complexation
was performed at 958C for 15 min. GraphPad Prism software (ver-
sion 4.00 for Windows, GraphPad Software) was used to calculate
IC50 values and to determine statistical significance at the 95% con-
fidence interval, with P<0.05 being considered significantly differ-
ent.
Biodistribution: All animal experiments were conducted in compli-
ance with the animal welfare committee requirements of our insti-
tution, and performed according to national regulations. Six- to
seven-week-old male BALB/c nude mice were inoculated subcuta-
neously with AR42J cells (107) in the right flank. Mice received irra-
diated chow and acidified drinking water ad libitum. After eleven
days, when tumors had an average mass of 0.2 g, mice were ran-
domly divided into groups of five. Each group received intravenous
injections of 0.37 MBq (0.1 mg) 111In-labeled peptides 16, 17, or 18
into the tail vein. At 1, 4, or 24 h p.i. the animals were euthanized
by CO2/O2 asphyxiation. Blood, tumor and tissues of interest were
dissected, weighed and counted in a g-counter along with three
aliquots (100 mL) of the diluted standard (representing 1% of the
injected activity). From this, the percentage injected dose per
gram (%IDgÀ1) for each tissue was calculated. Additional groups of
five mice were coinjected intravenously with 50 mg octreotide and
euthanized after 1 h to determine nonspecific binding of the radio-
tracers.
Radiolabeling: DOTA-conjugated peptides were labeled with
111InCl3 (Covidien) in 2-(N-morpholino)ethanesulfonic acid (MES) so-
lution (0.1m, pH 5.5) for 15 min at 958C. Labeling reactions were
performed in acid-washed Protein LoBind safe-lock tubes (Eppen-
dorf GmbH). Radiochemical purity was determined by instant thin-
layer chromatography (ITLC) on silica gel (Tec-Control Chromatog-
raphy Strips, Biodex Medical Systems, Inc). The ITLC analysis was
performed by using two different mobile phases: 0.1m ethylene-
diaminetetraacetic acid (EDTA)/0.1m NH4OAc (1:1, v/v) (Rf bound
111In=0, Rf unbound 111In=1) and THF/0.25m NH4OAc (1:1, v/v; Rf
colloid=0, Rf unbound 111In or 111In-labeled compound >0.5).
Acknowledgements
The authors thank Prof. Marion de Jong for providing the rat
pancreatic AR42J tumor cell-line. Bianca Lemmers, Kitty Lem-
mens, Gerben Franssen, Sandra Heskamp, and Cathelijne Frielink
are gratefully acknowledged for biotechnical assistance during in
vivo experiments, and Hans Jacobs is thanked for technical sup-
port and scientific discussions during the synthesis of dimeric
[Tyr3]octreotate peptides. Hans Hilbers is thanked for MALDI-ToF
measurements.
Octanol–saline partition coefficient: 111In-labeled 16, 17, or 18 (ca.
370 kBq, ~10 mL) was added to a mixture of saline (500 mL, pH 7.4)
and n-octanol (500 mL). The resulting biphasic system was shaken
vigorously for 2 min, and the two layers were subsequently sepa-
rated by centrifugation (5 min, 250 g). The radioactivity levels in
100 mL aliquots (n=3) of both the organic and the aqueous layers
were measured in a g-counter (Perkin–Elmer). The partition coeffi-
cient (logD) was determined from at least two independent exper-
iments, and calculated from the formula logD=log10 (counts in
octanol layer/counts in aqueous layer).
Keywords: click
radiopharmaceuticals
receptors
chemistry
·
dimerization
·
·
receptor binding
·
somatostatin
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In vitro stability in serum: 111In-labeled 16, 17, or 18 (~370 kBq,
~10 mL) was incubated with human serum (300 mL) at 378C for up
to three days. Following incubation at selected time points, the
serum proteins were precipitated with acetonitrile (1:1, v/v), and
the sample was centrifuged (5 min, 5000g). The supernatant layer
was filtered and analyzed on an Agilent HPLC system with an in-
line NaI radiodetector (Raytest GmbH) by using an Alltech Alltima
C18 column (250ꢁ4.6 mm, pore size: 100 ꢂ, particle size: 5 mm,
1.0 mLminÀ1) with a linear gradient of buffer B (5–100% over
20 min) in buffer A. (Buffer A: 0.1% TFA in water; buffer B: 0.1%
TFA in acetonitrile.)
Internalization: Internalization of 111In-labeled 16–18 by AR42J
cells was studied as described earlier.[22] Cells were grown to con-
fluency in six-well plates, and were incubated with trace amounts
(ca. 0.1 nm) of radiolabeled peptide for 30 min, 1, 2, and 4 h at
378C. Cell surface-bound radioligand was removed by washing the
cells with cold acidic buffer (0.1m acetic acid, 154 mm NaCl,
pH 2.6). Internalized radioligand was determined as the cell-associ-
ated radioactivity which was not removed by this procedure. The
experiment was performed in triplicate, and nonspecific binding
was determined from a parallel series containing 0.4 mm octreotide
(“Sandostatin”).
ChemBioChem 2011, 12, 750 – 760
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
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Published online on February 15, 2011
760
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ChemBioChem 2011, 12, 750 – 760