Validation of a novel CHX-A″ derivative suitable for peptide conjugation: Small animal PET/CT imaging using yttrium-86-CHX-A″- octreotide

Article abstract of DOI:10.1021/jm060317v

A versatile bifunctional chelating reagent based on a preorganized cyclohexyl derivative of DTPA (CHX-A′) has been developed for the convenient N-terminal labeling of peptides with metal ion radionuclides of Bi(III), In(III), Lu(III), or Y(III). This was

Full text of DOI:10.1021/jm060317v

J. Med. Chem. 2006, 49, 4297-4304
4297
Validation of a Novel CHX-A′′ Derivative Suitable for Peptide Conjugation: Small Animal
PET/CT Imaging Using Yttrium-86-CHX-A′′-Octreotide
Thomas Clifford,†,§,| C. Andrew Boswell,†,| Gra´inne B. Biddlecombe,‡ Jason S. Lewis,‡ and Martin W. Brechbiel*,†
Radioimmune & Inorganic Chemistry Section, Radiation Oncology Branch, National Cancer Institute, Building 10 Center DriVe,
Bethesda, Maryland 20892-1088, and Mallinckrodt Institute of Radiology, Washington UniVersity School of Medicine,
St. Louis, Missouri 63110
ReceiVed March 20, 2006
A versatile bifunctional chelating reagent based on a preorganized cyclohexyl derivative of DTPA (CHX-
A′′) has been developed for the convenient N-terminal labeling of peptides with metal ion radionuclides of
Bi(III), In(III), Lu(III), or Y(III). This was achieved via the synthesis of a mono-N-hydroxysuccinimidyl
penta-tert-butyl ester derivative of CHX-A′′ (trans-cyclohexyldiethylenetriaminepenta-acetic acid) featuring
a glutaric acid spacer. Commercially obtained octreotide was modified at its N-terminus by this reagent in
the solution phase, and its subsequent radiolabeling with 111In (T1/2 ) 2.8 d) and 86Y (T1/2 ) 14.7 h)
demonstrated. Small animal PET/CT imaging results of 86Y-CHX-A′′-octreotide in a somatostatin receptor-
positive tumor-bearing rat model are presented for the validation of the novel agent.
Introduction
The development of improved bifunctional chelating agents
for molecular imaging and targeted radiotherapy of cancer
continues to progress the field of nuclear medicine. Research
by Brechbiel and co-workers led to the establishment of a
preorganized DTPAa analogue, CHX-A′′ (trans-cyclohexyldi-
ethylenetriaminepenta-acetic acid) (Figure 1), as the premier
chelator for targeted R-particle therapy using the bismuth
radionuclide, 213Bi, which has shown impressive results in cancer
therapy studies.1 Furthermore, CHX-A′′ forms stable and
Figure 1. Structures of the parent compound (DTPA) (left) and the
previously reported isothiocyanate derivative of CHX-A′′ (CHX-A′′-
DTPA) (right).
biocompatible metal complexes with the γ and Auger emitter,
111In (T1/2 ) 2.8 d),2,3 a high-energy â--emitter, 90Y (T1/2
)
64.06 h),2-4 the â+-emitter, 86Y (T1/2 ) 14.7 h),3,5 and a lower-
energy â--emitter, 177Lu (T1/2 ) 6.65 d),5 making it a highly
versatile chelator for radiopharmaceutical applications. Due in
part to its overall acyclic structure, CHX-A′′ is capable of
achieving rapid complex formation with a variety of radiometals
at room temperature, offering a distinct advantage over the
macrocyclic chelator, DOTA (1,4,7,10-tetraazacyclododecane-
1,4,7,10-tetraacetic acid).6 Furthermore, Bi(III) complex forma-
tion kinetics with DOTA are slow and somewhat impractical
for use with 213Bi (T1/2 ) 45.6 min).7,8 The characterization of
the Bi(III) complex of a preorganized cyclohexyl analogue of
DTPA (H5CyDTPA) was described in 1996.9
An evaluation of antibodies labeled with 88Y via conjugation
to the four stereoisomers of 2-(p-nitrobenzyl)-trans-CyDTPA
confirmed that the CHX-A isomers are superior to the CHX-B
isomers with the CHX-A′′ diastereomer clearing from the
circulation slightly faster than CHX-A′, while also showing the
lowest uptake by bone of any of the four diastereomers.10,11
Thereafter, the versatility of the CHX-A′′ chelate has been
widely demonstrated through various applications including the
stable complexation of the high-linear energy transfer (high-
LET) R-emitter, 213Bi,12,13 and a variety of other radiometals
(vide supra). These applications, however, feature CHX-A′′
coupled exclusively to antibodies, illustrating the need for a
CHX-A′′-derivative that is amenable to coupling with peptides.
The targeting of somatostatin receptors (SSR) has been a goal
in cancer treatment and diagnosis since the 1980s.14-17 Soma-
tostatin is a 14-amino acid peptide with an inhibitory role in
the normal regulation of the central nervous system, the
hypothalamus, the pituitary gland, the gastrointestinal tract, and
the exocrine and endocrine pancreas.18,19 In addition, SSRs are
overexpressed by a number of neuroendocrine and breast tumor
cells20 and have more recently been linked to cellular prolifera-
tion and angiogenesis.21 Octreotide is an eight-amino acid
analogue of somatostatin that is highly resistant to enzymatic
degradation and useful in the management of cancer.14-17 We
chose to target and image SSR expression using the â+-emitter,
86Y (T1/2 ) 14.7 h), whose intermediate half-life is well suited
to octreotide-based imaging as the radioactive half-life of the
isotope is reasonably matched to the biological half-life of the
radiolabeled peptide conjugate.
* To whom correspondence should be addressed. Tel: 301-496-0591.
Fax: (301) 402-1923. E-mail: martinwb@mail.nih.gov.
† National Cancer Institute.
‡ Washington University School of Medicine.
§ Current address: Morphix Technologies, 2557 Production Road,
Virginia Beach, VA 23454.
| These authors contributed equally to the execution of these studies.
a Abbreviations: DTPA, diethylenetriaminepentaacetic acid; CHX-A′′,
trans-cyclohexyldiethylenetriaminepenta-acetic acid; DOTA, 1,4,7,10-tetra-
azacyclododecane-1,4,7,10-tetraacetic acid; SSR, somatostatin receptor; PET,
positron emission tomography; CT, computed tomography; 1B4M, 2-(4-
aminobenzyl)-6-methyl-diethylenetriaminepentaacetic acid; SPPS, solid-
phase peptide synthesis; OC, octreotide; Cbz, carbamazepine; Fmoc,
9-fluorenylmethyloxycarbonyl; Boc, N-tert-butyloxycarbonyl; EDTA, eth-
ylenediaminetetraacetic acid; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3,-
tetramethyluronium hexafluorophosphate; DMF, dimethylformamide; KOH,
potassium hydroxide; TFA, trifluoroacetic acid; TLC, thin-layer chroma-
tography; HPLC, high-pressure liquid chromatography; TMS, tetrameth-
ylsilane; TSP, sodium 3-trimethylsilyl-2,2′,3,3′-tetradeuteriopropionate; ESI,
electrospray ionization; TOF, time-of-flight; MS, mass spectrometry; EDT,
1,2-ethanediol.
10.1021/jm060317v CCC: $33.50 © 2006 American Chemical Society
Published on Web 06/20/2006

4298 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
Clifford et al.
Results
Synthesis. Characterization and structural confirmation of all
key intermediates and the succinimidyl ester-containing CHX-
A′′ bifunctional chelating agent (Figure 3) were performed by
1H NMR, 13C NMR, and electrospray mass spectrometry
(Supporting Information). Two-dimensional COSY (correlation
spectroscopy) NMR spectra were collected for the nitro-
precursor (1) and for the acid (3) (Supporting Information).
Electrospray mass spectra were obtained for the protected and
deprotected CHX-A′′-octreotide conjugates (Supporting Infor-
Figure 2. Schematic representation of the conjugation reaction between
the novel glutaric acid succinimidyl ester derivative of CHX-A′′ (4)
and a generic N-terminal peptide.
1
mation). The H and 13C NMR spectra of both the protected
and deprotected CHX-A′′-octreotide conjugate indicated the
presence of the expected product; however, a complete peak
assignment of these complicated spectra was not performed
(Supporting Information). NMR spectra of simpler octreotide-
chelator conjugates with complexed metal ions have been
previously reported, including an NMR of Y(III)-DOTA-D-Phe1-
Tyr3-octreotide.22 The protected CHX-A′′-octreotide conjugate
was analyzed by C-18 reversed-phase HPLC and eluted in a
single peak at 30 min with aqueous trifluoroacetic acid (TFA)
with a CH3CN gradient (Figure 4).
Radiolabeling and Reversed-Phase HPLC of 111In-CHX-
A′′-octreotide. Metal ion complexation of 111In was demon-
strated by reversed-phase HPLC of the radiolabeled peptide
under neutral pH conditions. A doublet of peaks at 23.5 and 24
min was observed for 111In-CHX-A′′-octreotide on a reversed-
phase HPLC eluting with aqueous ammonium acetate and an
increasing acetonitrile gradient (Figure 5). The time-resolution
of two distinct radioactive species may be attributed to the
emergence of two structural isomers of CHX-A′′-octreotide upon
In(III) complexation. Reversed-phase HPLC resolution of two
isomeric forms of a similar In(III)-DTPA-peptide conjugate has
been recently documented.23
Radiosynthesis and Characterization of 86Y-CHX-A′′-
octreotide. Radiolabeling was achieved via the incubation of
CHX-A′′-octreotide with 86YCl3 in 0.5 M NH4OAc at 25 °C.
Quality control of the product was performed using radio thin-
layer chromatography on MKC18F silica gel plates (2.5 × 7.5
cm) using NH4OAc/methanol (30:70) as the eluent. Radio-
chemical yields greater than 97% (0.1 mCi/µg; 6.49 × 106 MBq/
mmole) for 86Y-CHX-A′′-octreotide were obtained by this
method.
Figure 3. Schematic representation of the synthesis of 4.
Herein, we report on the development of an improved CHX-
A′′ analogue widely applicable to the modification of peptides,
antibodies, and other molecules via standard succinimidyl ester
coupling chemistry (Figure 2). This has been accomplished by
the design and synthesis of a mono-N-hydroxysuccinimidyl
penta-tert-butyl ester derivative of CHX-A′′ with a glutaric acid
spacer moiety to lessen the steric effects and interactions of the
chelator-metal complex on peptide-receptor binding affinity.
The succinimidyl ester coupling chemistry is clean, efficient,
and compatible with both solution and solid-phase peptide
coupling techniques. This extension of the versatility of CHX-
A′′ bridges a gap between large macromolecules (e.g., antibod-
ies) and smaller biomolecules (e.g., peptides) as exemplified
by the targeting and imaging using the highly successful SSR-
targeted peptide, octreotide.
Small Animal PET/CT Co-Registration Imaging. The 86Y-
dependent PET images were co-registered with computed
tomography (CT) images to aid in anatomical interpretation of
the imaging results. The uptake of 86Y-CHX-A′′-octreotide in
SSR-bearing AR42J tumors is clearly visible in both axial
Figure 4. Structural representation (left) and UV elution profile (right) of protected CHX-A′′-octreotide resulting from the reaction of 4 with
octreotide.

Yttrium-86-CHX-A′′-octreotide
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4299
Figure 5. Radioactive (top) and UV (bottom) elution profiles of 111In-CHX-A′′-octreotide. A structural representation of deprotected CHX-A′′-
octreotide is also shown (right).
(Figure 6) and coronal (Figure 7) views at 4 h postinjection.
Significant accumulation of 86Y-CHX-A′′-octreotide was also
observed in the kidneys (Figures 6 and 7). The images of
animals receiving a blocking injection of octreotide still
demonstrated an uptake in clearance organs (kidneys) but not
in tumors (Figures 6 and 7).
solution phase following cleavage from hyper-acid sensitive
resins with peptides still having protection intact on reactive
residues (e.g., BOC-protected lysines) but whose N-terminus
is available for reaction. In this specific article, we chose to
highlight this latter scenario using the eight-amino acid soma-
tostatin analogue, octreotide, as a prototype.
Biodistribution Studies of 86Y-CHX-A′′-octreotide. The
uptake of 86Y-CHX-A′′-octreotide in SSR-positive organs at 4
h postinjection expressed as percent injected dose per gram
(%ID/g) are as follows: the pituitary gland (1.70 ( 0.08),
adrenal glands (0.95 ( 0.30), and pancreas (0.65 ( 0.24) (Figure
8; Supporting Information). Accumulation was also observed
in the AR42J tumor (0.94 ( 0.22) and the kidneys (20.38 (
5.62) (Figure 8; Supporting Information). At 24 h postinjection,
the majority of the agent had mostly cleared the kidneys
(3.90 ( 0.04), pituitary (0.22 ( 0.01), pancreas (0.12 ( 0.03),
adrenals (0.06 ( 0.01), and the tumor (0.07 ( 0.01) (Figure 8;
Supporting Information).
Solid-phase labeling strategies for the construction of peptides
with inherent metal ion chelators can facilitate the high-
throughput screening of putative molecular imaging agents
generated from peptide libraries. An amino acid analogue
designed to incorporate p-aminobenzyl-EDTA into proteins has
been previously described.26 Similar strategies for the solid-
phase introduction of Re and/or 99mTc chelators have also been
explored.27,28 A more recent report describes the convenient
solid-phase synthesis of a DTPA-conjugated peptide and labeling
with the γ-emitter, 111In.23 Herein, we have described a novel
CHX-A′′ agent that serves to extend this concept to real-time
imaging with improved resolution by PET using 86Y. In addition,
this CHX-A′′ DPTA analogue potentially creates a facile
synthetic route to a variety of R- and â-emitting targeted agents
for radiotherapeutic applications.
The structures of both peptide and chelate play critical roles
in determining the success of radiolabeled peptides in nuclear
medicine. The chemical properties of the chelate-metal complex
(e.g., lipophilicity, charge, size, steric bulk, etc.) do indeed have
an important impact, especially for small peptides. In addition,
the requirement for rapid and kinetically stable chelation is
necessary to prevent the escape of radiometal ions and subse-
quent accumulation into nontarget organs.29 The promising small
animal PET imaging results presented in this article suggest
(1) that this peptide-CHX-A′′ conjugate formed a stable 86Y
complex that allowed for tumor specific targeting and next-
day clearance and (2) that the structure of this CHX-A′′
derivative did not adversely affect the SSR targeting properties
of octreotide.
Discussion
The radiolabeling of antibodies following conjugation with
isothiocyanate derivatives of 1B4M (2-(4-aminobenzyl)-6-
methyl-diethylenetriaminepentaacetic acid) and CHX-A′′ (Figure
1) has been thoroughly investigated in our laboratory.1,2,10-13,15,24
This strategy, however, was not readily applicable to solid-phase
peptide synthesis (SPPS) because there were concerns that the
thiourea conjugate would undergo Edman degradation under
TFA cleavage and deprotection conditions during conventional
Fmoc SPPS. The penta-tert-butyl ester derivative of CHX-A′′
with a glutaric acid spacer moiety was chosen as an alternate
chelator precursor. After converting the terminal carboxylate
to an activated ester (Figure 3), the reagent could be used in a
conventional peptide synthesizer to modify a peptide at the
N-terminus (Figure 2) prior to cleavage of the peptide from the
polymer support. Alternatively, the reagent may be reacted in

4300 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
Clifford et al.
Figure 7. Coronal slices of small animal PET/CT coregistration images
of an AR42J-bearing rat at 4 h following the intravenous injection of
86Y-CHX-A′′-octreotide with (left, center) and without (right) an
octreotide block. The images shown are 1 mm slices with the animal
in the supine position, and images A and C are slices through the center
of the tumor volume. Image B is a slice chosen through the center of
the kidneys. The PET images show that the administration of a blockade
dose substantially reduces the tumor uptake of the agent by such an
extent that the tumor cannot be delineated (A). It is also evident that
background accumulation is very low (i.e., low nontarget tissue uptake)
except for activity in the kidneys (B), consistent with the biodistribution
data. The acute biodistribution data did show accumulation of the agent
into other SSR-rich tissues, such as the adrenals, pituitary, and pancreas;
however, these were not visualized by the imaging because of the
pituitary not being in the field of view, the diffuse nature of the
pancreas, and the inability to resolve the adrenals because of their
proximity to the kidneys. (T, tumor; K, kidney)
Figure 6. Axial slices of small animal PET/CT coregistration images
of an AR42J-bearing rat at 1, 4, and 24 h following the intravenous
injection of 86Y-CHX-A′′-octreotide with (left) and without (right) an
octreotide block. The images shown are 1 mm slices with the animal
in the supine position, and the slices shown are through the center of
the tumor volume. Because tumors do not grow in exactly the same
location from animal to animal, the slices may show different tissues
in addition to the tumors. The PET images show that the administration
of a blockade dose substantially reduces the tumor uptake of the agent
by such an extent that the tumor cannot be delineated. It is also evident
that the background accumulation is very low (i.e., low nontarget tissue
uptake) except for activity in the kidneys, consistent with the biodis-
tribution data. Very little activity was delineated in any other tissues
in the imaging studies. The acute biodistribution data did indicate
accumulation of the agent into other SSR-rich tissues, such as the
adrenals, pituitary, and pancreas; however, these were not visualized
by the imaging because of the pituitary not being in the field of view,
the diffuse nature of the pancreas, and the inability to resolve the
adrenals because of their proximity to the kidneys. (T, tumor; K, kidney;
F, fiducial marker).
One of the most widely used γ-scintigraphy radioisotopes in
radiopharmaceutical studies is 111In, which decays by electron
capture with the emission of γ photons of 173 and 247 keV
(89% and 95% abundance, respectively). Targeted tumor
radiotherapy may be achieved because of the subcellular-range
Auger electron emissions that are a consequence of 111In electron
capture decay.30-32 Octreotide has been labeled with both 123I
Figure 8. Biodistribution profile of AR42J-bearing rats at 4 and 24 h
following the intravenous injection of 86Y-CHX-A′′-octreotide.
octreotides for therapy planning by comparing biodistribution
and dosimetry data.40
The advantages of â+-emitting somatostatin analogues include
increased image resolution with positron emission tomography
(PET) compared to γ-scintigraphy, improved quantification
capabilities with PET, and the ability to use â+-emitters as
33
and 111In34 and used to image SSR-positive tumors in
humans.35 Iodine-123-Tyr3-octreotide has a high hepatobiliary
excretion that hinders the visualization of tumors in the
abdomen, whereas 111In-DTPA-D-Phe1-octreotide (111In-DTPA-
OC) clears primarily through the kidneys35 and is currently used
routinely as an imaging agent for neuroendocrine cancer. The
infusion of lysine and arginine may be used to lower the
radiation dose to the kidneys.36
There have also been reports of targeted radiotherapy studies
in animal models using 90Y-labeled octreotide conjugates37,38
and 188Re labeled to another SSR ligand, RC-160. 90Y-DOTA-
Tyr3-octreotide (90Y-DOTA-Y3-OC) was found to induce the
complete remission of a rat pancreatic tumor in 70% of rats
administered 10 mCi/kg38 and has been in clinical trials in the
U. S. and Europe with favorable results.39 Recent studies have
demonstrated the advantages of 86Y-labeled over 111In-labeled
companion isotopes to therapeutic radionuclides such as the 86
Y
(PET)/90Y (therapy) pair.41 Yttrium-90 has shown therapeutic
efficacy as a radionuclide for both radioimmunotherapy and
peptide radiotherapy. The high energy and long range of the
â- particle are probably most ideal for large tumor burdens and
may not be ideal for smaller tumors.30,42 Furthermore, because
90Y emits only â- particles (Emax ) 2.27 MeV), tumor imaging
and dosimetry calculations have usually been performed using
the γ-emitter, 111In. Subtle differences between the biodistri-
butions of 111In- and 90Y-labeled monoclonal antibodies have
been noted that may lead to discrepancies in dosimetry
calculations.43,44 In the case of targeted radiotherapeutic agents
with short biological half-lives, the 90Y uptake and dosimetry

Yttrium-86-CHX-A′′-octreotide
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4301
could be better quantitated by PET by the prior or coadminis-
tration of the analogous 86Y-labeled compound.45 Yttrium-86
(â+ 34% (1.248 MeV, 14%); EC 66%) has been produced by
the irradiation of an enriched 86SrCO3 target, followed by
purification by a combined coprecipitation and ion-exchange
purification process.46 A Sr(II)-selective resin has also been
employed for 86Y purification,47,48 and an electrochemical
purification was recently reported that results in even higher
chemical purity.49 Recently, no-carrier-added, high-specific
activity 86Y has been produced in high yield from SrO, followed
temperature.7,8 Future studies involving CHX-A′′-peptide con-
jugates labeled with R-emitting isotopes such as 213Bi may lead
to the development of improved targeted radiotherapeutic agents.
Experimental Methods
Materials. Mono-Boc-1,2-trans-(S,S)-diaminocyclohexane was
prepared via bis(Cbz)-mono-Boc-1,2-trans-(S,S)-diaminocyclohex-
ane as previously described73 and was used in the synthesis of the
triamine N-[(R)-2-amino-3-(p-nitrophenyl)propyl]-trans-(S,S)-cy-
clohexane-1,2-diamine as previously reported.19 Tert-butyl bro-
moacetate was purchased from Fluka (Buchs, Switzerland). The
HBTU coupling reagent was purchased from EMD Biosystems (San
Diego, CA). DMF (Aldrich, 99%) was distilled at reduced pressure
prior to use in peptide couplings. Piperidine and diisopropylethyl-
amine were distilled from KOH. Lys(Boc) octreotide, and (Cys2-
Cys7)H-D-Phe-Cys-Phe-D-Trp-Lys(Boc)-Thr-Cys-Thr-ol was pur-
chased from Advanced ChemTech (Louisville, KY). Reagent K
cleavage solution (TFA-phenol-thioanisole-EDT-H2O, 82.5:5:
5:2.5:5) was prepared immediately prior to use in the deprotection
and cleavage of peptides from resin support. All other reagents were
purchased from Aldrich (St. Louis, MO) and used as received.
Chemistry. Column chromatography was performed on Merck
60 Silica Gel deactivated by washing with 10% aqueous EtOH
followed by air-drying to a free flowing powder over 7 days. TLC
was performed on Merck Silica Gel 60 F254 glass plates visualized
by UV fluorescence or development by I2. HPLC was performed
using a Beckman System Gold (Fullerton, CA) equipped with a
Model 126 Solvent Delivery Module and a Model 168 UV detector
(λ 254 and 280 nm) controlled by 32 Karat software.
by electrochemical separation on a biomedical cyclotron.50
A
86Y-labeled cyclic peptide analogue of R-MSH, 86Y-DOTA-
ReCCMSH(Arg11), demonstrated a much higher tumor-to-
background ratio than the corresponding 64Cu-labeled peptide,51
a result attributed to the relative kinetic stability of Y(III)-
DOTA7 versus Cu(II)-DOTA.52
In the small animal imaging and biodistribution studies, 86Y-
CHX-A′′-OC demonstrated high tumor concentration along with
uptake in the kidney associated with the renal clearance of the
agent (Figures 6-8; Supporting Information). Very little activity
was delineated in any other tissues in the imaging studies. The
biodistribution data indicated the accumulation of the agent into
other SSR-rich tissues, such as the adrenal, pituitary, and
pancreas (Figure 8; Supporting Information), although these
were not visualized by the imaging because of the pituitary not
being in the field of view, the diffuse nature of the pancreas,
and the inability to resolve the adrenals because of their
proximity to the kidneys. Tumor uptake was significantly
reduced by the co-injection of a block due to the saturation of
the SSR binding sites (Figure 6). This reduction in tumor uptake
confirms that tumor concentration is receptor-mediated. The
mass of agent administered (biodistribution ) 100 ng; small
animal PET ) 3000 ng) was not optimal because of the final
labeling specific activity of only 0.1 mCi/µg of 86Y-CHX-A′′-
OC. In the experiments described herein, the separation of the
86Y-CHX-A′′-OC from unlabeled CHX-A′′-OC was not at-
tempted because of the high radiochemical purity (97%)
achieved in the labeling. However, even at this level of specific
activity, significant blocking of the receptor is not anticipated
until higher masses of the agent are administered53 as demon-
strated by good tumor accumulation and visualization. The
uptake of somatostatin peptides into tumor and other receptor-
rich tissues often follows a bell-shaped relationship,54 and at
the levels given in this study, the uptake is thought to be on the
fall-slide of this curve. The optimization of tumor accumulation
and, as a consequence, improved delineation of the tumor in
PET imaging could be improved by increasing the specific
activity of the 86Y-labeled peptide by the separation of the
radiolabeled peptide from the nonlabeled one.
1H and 13C NMR data were obtained using a Varian Gemini
300 MHz instrument, and the chemical shifts are reported in ppm
on the δ scale relative to those of TMS, TSP, or the solvent. Proton
chemical shifts are annotated as follows: ppm (multiplicity, integral,
coupling constant (Hz)). Mass spectra were obtained on a Waters
LCT Premier Time-of-Flight Mass Spectrometer using electrospray
ionization (ESI/TOF/MS) operated in positive ion mode. The
electrospray capillary voltage was 3 kV, and the sample cone
voltage was 60 V. The desolvation temperature was 225 °C, and
the desolvation gas flow rate was nitrogen at 300 L/h. Accurate
masses were obtained using the lock spray mode with Leu-
Enkephalin as the external reference compound.
The peptides were purified on a Waters Deltaprep 3000 prepara-
tive HPLC system using a Waters DeltaPak 15 µm C18 100A 30
mm × 300 mm column 30 × 150 mm C18 reversed-phase connected
to a Waters 750 UV/vis detector monitoring at 254 nm. The
purification of tert-butyl-ester-protected CHX-A′′-octreotide was
achieved using a binary solvent gradient of 50-100% B/25 min
(A ) 1% aqueous TFA, B ) 1% TFA in CH3CN) at 40 mL/min.
The purification of deprotected CHX-A′′-octreotide was achieved
using a binary solvent gradient of 10-100% B/25 min (A ) 1%
aqueous TFA, B ) 1% TFA in CH3CN) at 40 mL/min. Data were
output to a strip chart recorder, and the relevant aliquots of the
major products were collected by hand.
Analytical HPLC of tert-butyl-ester-protected CHX-A′′-octreotide
was performed using a Beckmann C18 reversed-phase column and
a binary gradient system of 50-100% B/25 min (solvent A ) 1%
aqueous TFA, solvent B ) 1% TFA in CH3CN). The deprotected
peptides were analyzed using a Beckmann C18 reversed-phase
column and either of two binary solvent gradient systems of
0-100% B/40 min at 1 mL/min: (1) an acidic system where A )
1% aqueous TFA, B ) 1% TFA in CH3CN or (2) a neutral system
where A ) aqueous 15 mM NH4OAc at pH 7.0, B ) CH3CN. The
latter solvent gradient system was also utilized for the chromato-
graphic analysis of peptides labeled with radiometals (vide infra).
General Procedure for Solution Phase Tagging. In brief,
peptides with side chain protection intact were purchased, and the
N-terminus appended with chelate 4 by simply mixing 1.5
equivalents of chelate 4 with the peptide in DMF. Typically ∼100
mg of peptide would be suspended in DMF (∼1 mL), and chelate
4 was added in one portion. The suspension would clear after 10-
Conclusion
In summary, a novel somatostatin analogue developed in our
laboratory, CHX-A′′-octreotide, permits the use of the â+-emitter
86Y as an elementally matched companion isotope with the
therapeutic radionuclide 90Y (therapy).41 Like the previously
reported DOTA-octreotide derivatives, CHX-A′′-octreotide al-
lows for the complexation of In(III), Lu(III), or Y(III). In
addition, CHX-A′′-octreotide provides direct access to a selec-
tion of R-emitting radionuclides including 213Bi, which has
shown impressive results1,12 in targeted R-particle therapy. The
preclinical evaluation of a 213Bi-labeled DOTA-octreotide
derivative has already been reported;55 however, CHX-A′′ is
superior to DOTA for the complexation of 213Bi (T1/2 ) 45.6
min) because of more rapid complexation kinetics at ambient

4302 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
Clifford et al.
30 min. The reaction was allowed to proceed for a total of 6-8 h,
and the crude product was isolated by precipitation from diethyl
ether. The Boc and tert-butyl ester groups were cleaved using TFA-
phenol-thioanisole-EDT-H2O following the same procedure used
for resin cleaved products. The crude products were purified by
reversed-phase C18 chromatography using aqueous TFA (1%) with
an increasing CH3CN gradient in the mobile phase. This general
strategy was applied to a 100-mg sample of commercially obtained
octreotide with Boc-protection on the lysine residue intact.
Radiosynthesis and Characterization of 111In-CHX-A′′-octreo-
tide. A 200-µCi portion of 111In (Perkin-Elmer, Wellesley, MA) in
0.05 N HCl was added to a 100-µg portion of CHX-A′′-octreotide
dissolved in 0.15 M NH4OAc at pH 7. The purpose of this low
specific-activity labeling was to correlate the UV and radiometric
peaks of CHX-A′′-octreotide and 111In-CHX-A′′-octreotide, respec-
tively. The reaction mixture was incubated at 25 °C for 30 min.
An aliquot of the resulting solution was analyzed by RP-HPLC
using a Vydac Protein & Peptide C18 column equilibrated with 0.015
M NH4OAc (pH 7). A gradient of CH3CN that increased from 0%
at 0 min to 100% at 40 min was employed, followed by an
additional 10-min plateau at 100% CH3CN. A UV detector and
radiometric detector were coupled to measure absorbance at 254
nm and radioactivity, respectively.
(s, 2H), 3.46 (dd, Ja ) 14.4 Hz, Jb ) 2.7 Hz, 2H), 3.53 (s, 4H),
7.55 (d, J ) 9 Hz, 2H), 8.07 (d, J ) 8.7 Hz, 2H, Ph); 13C NMR
(75 Hz, CDCl3) δ 26.114, 26.22, 26.83, 36.79, 42.16, 51.60, 52.74,
53.43, 54.12, 60.12, 62.48, 63.99, 80.39, 80.58, 80.65, 123.10,
130.77, 146.14, 151.17, 171.40, 171.76, 172.05. Anal. Calcd. for
C45H74N4O12: C, 62.62; H, 8.64; N, 6.49. Found: C, 62.84; H,
8.85; N, 6.43. ES-MS: [M + H+] requires, 863.53812; found,
863.5381.
N-[(R)-2-Amino-3-(p-aminophenyl)propyl]-trans-(S,S)-cyclo-
hexane-1,2-diamine-N,N,N′,N′′,N′′ -penta-tert-butyl Acetate (2).
Pd on carbon (10%) was placed in an all glass hydrogenation vessel
with EtOH (20 mL) charged with H2. After the saturation of the
catalyst with H2, a solution of (1) (2.4 g, 2.82 mmol) in EtOH (25
mL) was injected and the pressure maintained at room pressure
over 8 h by the periodic refilling with H2. The mixture was left
vigorously stirring overnight and then filtered through Celite
washing with EtOH (5 × 5 mL). The filtrate was evaporated at
reduced pressure to give a pale, yellow oil. The product was purified
on neutral alumina eluting with hexane/EtOAc/NH3 in EtOH in a
18:2:1 ration yielding a colorless oil after evaporation (2.29 g, 97%).
1H NMR (300 MHz, CDCl3) δ 1.09 (d, J ) 6.9 Hz, 4H), 1.26 (d,
J ) 2.3 Hz, 2H), 1.43 (br s, 45H, tBu), 1.66 (br s, 2H), 2.03 (br t,
J ) 12 Hz, 2H), 2.5-2.7 (m, 4H), 2.75-2.9 (m, 2H), 3.05 (p, J )
6 Hz, 1H), 3.20-3.65 (m, 11H); 13C NMR (75 Hz, CDCl3) δ 14.26,
22.78, 25.95, 26.14, 27.35, 28.27, 28.30, 29.89, 31.72, 36.08, 52.29,
53.38, 53.71, 54.09, 62.97, 63.02, 64.34, 80.15, 80.31, 115.26,
130.34, 131.52, 144.104, 171.67, 171.98, 172.40. Anal. Calcd. for
C45H76N4O10: C, 64.88; H, 9.19; N, 6.73. Found: C, 64.96; H,
9.19; N, 6.87. ES-MS: [M + H+] requires, 833.56394; found,
833.5640.
86Y Production. 86Y was produced on a CS-15 biomedical
cyclotron at Washington University School of Medicine using
published methods.64 Radioactivity was measured with a Capintec
radioisotope calibrator and a Beckman 8000 gamma counter.
Radiosynthesis and Characterization of 86Y-CHX-A′′-octreo-
tide. Radiolabeling was achieved via the incubation of CHX-A′′-
octreotide (20 µg) with 86YCl3 (2.0 mCi) in a 100-µL aliquot of
0.5 M NH4OAc (pH 5.5) at 25 °C for 1 h. Quality control of the
product was performed using radio thin-layer chromatography on
MKC18F silica gel plates (2.5 × 7.5 cm) using NH4OAc/methanol
(30:70) as the eluent.
N-[(R)-2-Amino-3-(p-aminophenyl-N-{5-oxopentanoic acid})-
propyl]-trans-(S,S)-cyclohexane-1,2-diamine-N,N,N′,N′′,N′′-penta-
tert-butyl Acetate (3). Compound 2 (11.2 g, 12.98 mmol) and
excess glutaric anhydride (2.96 g, 25.96 mmol) were mixed in
benzene (60 mL) with stirring overnight. A further portion of
benzene was added (60 mL), and the organic layer was washed
with 0.1 M Na2HPO3 (1 × 30 mL) followed by washing with 0.1
M NaH2PO3 (2 × 30 mL). The organic fraction was dried over
Na2SO4, filtered, and then evaporated at reduced pressure to yield
a pale brown oil. The product was further purified by chromatog-
raphy on a silica gel column (prepared as described above) eluting
with EtOH/hexane 1:5 to 1:1 gradient yielding after evaporation at
Biodistribution Studies of 86Y-CHX-A′′-octreotide. All animal
experiments were conducted in compliance with the Guidelines for
the Care and Use of Research Animals established by Washington
University’s Animal Studies Committee. The biodistribution studies
were conducted in male (144-163 g) Lewis rats (Charles River
Laboratories, Wilmington, MA) that had been subcutaneously
implanted with 2-3 mm AR42J tumor pieces into the right flank.
The tumors were allowed to grow for 10 days postimplantation, at
which time, the animals received 10 µCi (100 ng) of 86Y-CHX-
A′′-octreotide in 100 µL of saline via lateral tail-vein injection.
The animals were euthanized at desired time points (4 and 24 h).
Imaging Studies. Small animal PET images in AR42J-bearing
rats were obtained on a microPET-Focus 220 (Concorde Micro-
Systems Inc., Knoxville, TN)74 and were coregistered with CT
images from a MicroCAT II System (ImTek Inc., Knoxville, TN).
The animals received 300 µCi (3000 ng) of 86Y-CHX-A′′-octreotide
in 100 µL of saline via lateral tail-vein injection. A second group
of animals were pretreated with a blocking dose of 125 µg of [Tyr1]-
somatostatin (Sigma, St. Louis, MO) in 150 µL of saline im-
mediately prior to injection with 86Y-CHX-A′′-octreotide.
1
reduced pressure a glassy colorless solid (8.61 g, 71%). H NMR
(300 MHz, DMSO-d6, TMS) δ 1.04 (σ, 4H), 1.37 (s, 45H), 1.58
(br s, 2H), 1.78 (p, J ) 7.3 Hz, 2H), 1.90 (br s, 2H), 2.21 (t, J )
7.4 Hz, 2H), 2.30 (t, J ) 7.4 Hz, 2H), 2.40 (dd, Ja ) 12.9 Hz,
Jb ) 6.6 Hz, 1H), 2.58 (m, 3H), 2.73 (br d, J ) 9.3 Hz, 1H), 2.8-
3.0 (m, 2H), 3.12 (br d, J ) 17.1 Hz, 1H), 3.28 (d, J ) 9.6 Hz,
1H), 3.32 (s, 2H), 3.34 (s, 2H), 3.42 (s, 4H), 4.0 (br s, H2O), 7.14
(d, J ) 8.4, 2H) 7.45 (d, J ) 8.4 Hz, 2H), 9.81 (s, 1H); 13C NMR
(75 Hz, DMSO-d6, TMS) δ 20.90, 25.28, 25.47, 26.48, 27.69, 27.73,
27.87, 28.83, 31.28, 33.77, 35.31, 35.66, 52.77, 53.13, 53.34, 54.71,
62.27, 62.42, 63.01, 79.57, 79.69, 79.83, 118.49, 129.20, 135.53,
136.98, 107.55, 170.04, 171.67, 174.67. Anal. Calcd. for C50H82-
N4O13‚(C2H5OH): C, 62.88; H, 8.93; N, 5.64. Found C, 62.91; H,
8.78; N, 5.59. ES-MS: [M + H+] requires, 947.59563; found,
947.5957.
N-[(R)-2-Amino-3-(p-5-[(2,5-dioxopyrrolidin-1-yl)oxy]-5-oxo-
N-phenylpentanamide) propyl]-trans-(S,S)-cyclohexane-1,2-di-
amine- N,N,N′,N′′,N′′-penta-tert-butyl Acetate. (4). 1-[3-(dimeth-
ylamino)propyl]-3-ethylcarbodiimide hydrochloride (0.47 g, 2.45
mmol), N-hydroxysuccinimide (0.25 g, 2.16 mmol), and 3 (1.86 g,
1.96 mmol) were stirred in a mixture of EtOAc (90 mL) and DMF
(30 mL) overnight. The reaction mixture was then diluted with ethyl
acetate (30 mL) and cooled in an ice bath. The cooled mixture
was washed with ice-cold 5% w/v aqueous NaHCO3 (2 × 20 mL)
and then with ice-cold water (2 × 20 mL). The organic fraction
was dried over Na2SO4, filtered, and evaporated at reduced pressure
to yield a glassy pale yellow solid (1.61 g, 84%). No further
purification was performed on this compound. 1H NMR (300 MHz,
DMSO-d6, TMS) δ1.05 (br s, 4H), 1.38 (s 36H), 1.38 (s, 9H), 1.60
N-[(R)-2-Amino-3-(p-nitrophenyl)propyl]-trans-(S,S)-cyclo-
hexane-1,2-diamine-N,N,N′,N′′,N′′-penta-tert-butyl Acetate (1).
N-[(R)-2-Amino-3-(p-nitrophenyl)propyl]-trans-(S,S)-cyclohexane-
1,2-diamine‚3HCl (2.00 g, 5 mmol) stirred in acetonitrile (50 mL)
with K2CO3 (6.21 g, 45 mmol) was treated with tert-butyl
bromoacetate (5.17 mL, 6.83 g, 35 mmol) and stirred vigorously
for 3 days. The solvent was evaporated at reduced pressure, and
diethyl ether (100 mL) was added and the mixture filtered. The
inorganic salts were washed with additional portions of diethyl ether
(3 × 10 mL) and the filtrate evaporated at reduced pressure to
produce a viscous orange oil. Purification was achieved on a silica
gel column (previously treated with 10% aqueous ethanol and then
rinsed with 100% ethanol followed by ethyl acetate) eluting with
30% ammonia in EtOH/EtOAc/Hexane 1:1:16 to 1:1:8 gradient
1
(2.67 g, 62%). H NMR (300 MHz, CDCl3) δ 1.15-1.0 (br m,
4H), 1.40 (s 18H), 1.43 (s, 18H), 1.44 (s, 9H), 1.7 (br s, 4H), 2.5-
2.9 (m, 4H), 2.95-3.05 (m, 1H), 3.24 (s, 2H), 3.40 (s, 2H), 3.41

Yttrium-86-CHX-A′′-octreotide
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4303
(9) Brechbiel, M. W.; Gansow, O. A.; Pippen, C. G.; Rogers, R. D.;
Planalp, R. P. Preparation of the novel chelating agent N-(2-
aminoethyl)-trans-1,2-diaminocyclohexane-N,N′,N′′-pentaacetic acid
(H5CyDTPA), a preorganized analogue of diethylenetriaminepen-
taacetic acid (H5DTPA), and the structures of BiIII(CyDTPA)2- and
BiIII(H2DTPA) complexes. Inorg. Chem. 1996, 35, 6343-6348.
(10) Wu, C.; Kobayashi, H.; Sun, B.; Yoo, T. M.; Paik, C. H.; Gansow,
O. A.; Carrasquillo, J. A.; Pastan, I.; Brechbiel, M. W. Stereochemical
influence on the stability of radio-metal complexes in vivo. Synthesis
and evaluation of the four stereoisomers of 2-(p-nitrobenzyl)-trans-
CyDTPA. Bioorg. Med. Chem. Lett. 1997, 5, 1925-1934.
(11) Kobayashi, H.; Wu, C. C.; Yoo, T. M.; Sun, B. F.; Drumm, D.;
Pastan, I.; Paik, C. H.; Gansow, O. A.; Carrasquillo, J. A.; Brechbiel,
M. W. Evaluation of the in vivo biodistribution of yttrium-labeled
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(br s, 2H), 1.92 (p, J ) 9 Hz, 2H), 2.41 (t, J ) 7.2 Hz, 2H), 2.52-
2.66 (m, 2H), 2.75 (t, J ) 7.2 Hz, 2H), 2.82 (s, 4H), 2.85-3.04
(m, 2H), 3.1-3.5 (m, 12H), 7.176 (d, J ) 8.4 Hz, 2H), 7.45 (d,
J ) 8.4 Hz, 2H), 9.80 (s, 1H); 13C NMR (75 Hz, DMSO-d6, TMS)
δ 20.14, 25.27, 25.43, 26.60, 27.68, 27.72, 29.60, 34.59, 35.35,
52.71, 53.02, 53.36, 62.12, 62.32, 62.82, 79.44, 79.56, 79.70,
118.46, 129.212, 135.77, 136.76, 168.74, 169.80, 170.19, 170.96,
171.50. Anal. Calcd. for C54H85N5O15‚(C2H5OH): C, 61.69; H, 8.41;
N, 6.42. Found C, 61.51 H, 8.40 N, 6.63. ES-MS: [M + H+]
requires, 1044.61201; found, 1044.6120.
N-[CHX-A′′ penta-tert-butyl ester]-(Cys2-Cys7)-D-Phe-Cys-
Phe-D-Trp-Lys(Boc)-Thr-Cys-Thr-ol (Protected CHX-A′′-oct-
reotide). Prepared by the general solution-phase method; a retention
time of 30 min on RP-HPLC under acidic solvent gradient system
(aqeuous 1% TFA with increasing CH3CN gradient) (Figure 4);
ES-MS of C104H154N14O24S2: [M + 2H+] requires, 1024.54; found,
1025.10.
(12) Milenic, D. E.; Garmestani, K.; Brady, E. D.; Albert, P. S.; Ma, D.
S.; Abdulla, A.; Brechbiel, M. W. Targeting of HER2 antigen for
the treatment of disseminated peritoneal disease. Clin. Cancer Res.
2004, 10, 7834-7841.
N-[CHX-A′′]-(Cys2-Cys7)-D-Phe-Cys-Phe-D-Trp-Lys-Thr-
Cys-Thr-ol (CHX-A′′-octreotide). After TFA deprotection; reten-
tion time of 24 min on RP-HPLC under neutral solvent gradient
system (aqueous NH4OAc with increasing CH3CN gradient) (Figure
5); ES-MS of C79H106N14O22S2: [M + Na+ + K+] requires, 864.33;
found, 864.54.
(13) Milenic, D. E.; Garmestani, K.; Brady, E. D.; Albert, P. S.; Ma, D.;
Abdulla, A.; Brechbiel, M. W. R-Particle radioimmunotherapy of
disseminated peritoneal disease using a 212Pb-labeled radioimmuno-
conjugate targeting HER2. Cancer Biother. Radiopharm. 2005, 20,
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Acknowledgment. This research was supported in part by
the Intramural Research Program of the NIH, National Cancer
Institute, and the Center for Cancer Research. The production
of 86Y at Washington University was supported by a grant from
the National Cancer Institute (R24 CA86307; M.J. Welch, P.I.).
Supporting Information Available: 1H, 13C, and 2D-COSY
NMR spectra, ESI mass spectra, elemental analysis results of
relevant intermediates and compounds, and biodistribution data (4
and 24 h) for 86Y-CHX-A′′-octreotide in AR42J tumor-bearing rats
in tabular format. This information is available free of charge via
the Internet at http://pubs.acs.org.
(19) Reichlin, S. Somatostatin (part 2). N. Engl. J. Med. 1983, 309, 1556-
1563.
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