Labelling of octreotide using 76Br-prosthetic groups

Article abstract of DOI:10.1002/jlcr.478

A method for labelling the octapeptide octreotide (D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr(ol)) with the positron emitting ⁷⁶Br (T_1/2=16h) is presented, ε-Boc-protected octreotide was conjugated to N-succinimidyl 4-[⁷⁶Br]bromo

Full text of DOI:10.1002/jlcr.478

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Labelled Cpd Radiopharm 2001; 44: 561–573.
DOI: 10.1002/jlcr.478
76
Labelling of octreotide using Br-prosthetic
groups
1,2
Ulrika Yngve , Tanweera S. Khan , Mats Bergstrom and
2,3
2
.
1,2,
(
.
Bengt Langstrom
*
1 Department of Organic Chemistry, Institute of Chemistry, Uppsala University,
Box 531, SE-751 21 Uppsala, Sweden
2 Uppsala University, PET Centre, UAS, SE-75185 Uppsala, Sweden
3 Department of Medical Sciences, University Hospital, Uppsala, Sweden
Summary
A method for labelling the octapeptide octreotide (d^Phe–Cys–Phe–d–Trp–
Lys–Thr–Cys–Thr(ol)) with the positron emitting 76Br (T1=2=16 h) is
presented. e-Boc-protected octreotide was conjugated to Nꢀsuccinimidyl
4-[76Br]bromobenzoate 1 and Nꢀsuccinimidyl5-[ 76Br]bromo-3-pyridinecar-
%
boxylate 3 using microwave heating. The conjugates 4 and 5 were isolated in
%
%
%
50–55% radiochemicalyiedl after the removalof the protecting Boc-group.
Compound 3 was synthesised from the corresponding trimethylstannyl-
%
precursor in 25% radiochemical yield. The synthesis of methyl-4-[76Br]
bromobenzimidate 8 in 40% radiochemicalyiedl from the precursor methyl
%
-4-trimethylstannylbenzimidate is also described. Experiments were performed
to react 8 with Boc-octreotide but no product was obtained. The binding-
%
properties of 76Br-conjugates 4 and 5 to meningiomas were investigated using
%
%
frozen section autoradiography. Compound 5 showed better binding proper-
%
ties than 4. Copyright # 2001 John Wiley & Sons, Ltd.
%
Key Words: 76Br; labelled octreotide; prosthetic groups
Introduction
Radiolabelled analogues of octeotide, a synthetic octapeptide that
has shown high selectivity for the somatostatin receptor, are of
*Correspondence to: B. Langstrom, Uppsala University PET centre, UAS, SE-751 85 Uppsala,
Sweden.
Copyright # 2001 John Wiley & Sons, Ltd.
Received 30 January 2001
Revised 9 February 2001
Accepted 15 February 2001
Published online \ \ \ \

562
U. YNGVE ET AL.
interest for the diagnosis and treatment of somatostatin-positive
tumours. Many of the labelling methods involve conjugation of
metalchaetlors to the
N-terminal of the peptide followed by
labelling with radiometals such as 64Cu,1 67Ga,2 68Ga,3 111In,4 86Y5
and 90Y6,7. Also, Tyr3-octreotide has been labelled with different
isotopes of iodine by oxidative iodination of the tyrosine residue.8
Conjugation of labelled activated esters has been used for the
introduction of 131I or 211At9 and 18F has been introduced using
conjugation to 2-[18F]fluoropropionic acid 4-nitro-phenylester10 or
N-succinimidyl4-[ 18F]fluorobenzoate11. Of these various radio-labelled
derivatives of octreotide, 111In-DTPA-(d)Phe1-octreotide (Octreo-
scan1) and 123I–Tyr3–octreotide are routinely applied in clinical
investigations.12
76Br is an alternative to 18F and 68Ga for labelling radiotracers
with applications in positron emission tomography, PET.13,14 76Br has
a half-life of 16.2 h and decays to 55% by positrons and can be
produced by the 76Se(p, n)76Br reaction. N-Succinimidyl4-[ 76Br]bro-
mobenzoate 1,15 N-succinimidyl5-[ 76Br]bromo-3-pyridine-carboxylate
%
3 and methyl-4-[76Br]bromobenzimidate 8 are candidates for incorpor-
%
%
ating 76Br into octreotide through conjugation labelling. The conjuga-
tion of compounds 1,3 and 8 to octreotide is described in this paper as
% %
%
well as the syntheses of 3 and 8 from the corresponding trimethyl-
%
%
stannyl-precursors. The binding properties of the 76Br-labelled octreo-
tide-conjugates to meningiomas, as determined by frozen section
autoradiography, are also presented.
Results and discussion
N-Succinimidyl4-[ 76Br]bromobenzoate 1 has previously been used for
%
labelling of proteins and oligonucleotides modified with an aminolinker
in the 50-position.15 Using this method for labelling with 76Br, analogous
to one of the methods for [18F]fluorine-labelling, was our first choice
when looking for procedures to label octreotide.
Conjugation of compound 1 and e-Boc-octreotide using mixtures
of buffers and DMF gave low radiochemical yields even at
%
relatively high concentrations of octreotide (10 mg/ml, 8 mmol/ml as
compared to 0.7 mmol/ml used in conjugation with oligonucleotides).15
Due to the good solubility of e-Boc-octreotide in organic solvents, the
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BROMINATION OF OCTREOTIDE
563
conjugation reaction was also performed in solvents such as DMF,
DMSO, acetonitrile, methanol and dichloromethane with or
without the addition of various bases such as triethylamine, 4-
(dimethylamino)pyridine (DMAP) and 1,4-diazabicyclo[2.2.2]octane
(DABCO). Conventionalheating as wellas microwave heating were
applied to the samples, but the radiochemical yield did not exceed
20%. The use of dichloromethane as the solvent and microwave heating
resulted in 70–90% labelling of e-Boc-octreotide after 30 min using
5 mg/mlof e-Boc-octreotide (Scheme 1). The Boc-protective
group was removed by adding TFA to reaction mixture. After
30 min, the solvents were removed by gas purge and the product 4
%
was isolate by semi-preparative HPLC in 55% radiochemical yield. The
advantage of using dichloromethane as a solvent was that the Boc-
protecting group could be removed by the addition of TFA to the
reaction mixture. As a comparison, the method presented for
conjugation of the 131I and 211At-analogues of 1 to octreotide required
%
two HPLC-separations and one solid phase extraction work-up in
addition to extra activating reagents used.9 The specific radioactivity
for compound 1 had earlier been determined 20–200 GBq/mmol15,16 and
%
the specific radioactivity for 4 was determined to 2 GBq/mmolstarting
%
from 70 MBq 76Br.
In order to decrease the lipophilicity of the labelled peptide, the use of
two different prosthetic groups, N-succinimidyl5-[ 76Br]bromo-3-pyr-
idinecarboxylate 3 (Scheme 2) and methyl-4-[76Br]bromobenzimidate 8
%
%
(Scheme 3), that would give protonated octreotide-conjugates at
physiological pH were investigated. Imidates can react with amines to
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U. YNGVE ET AL.
Scheme 1
form amidines (Scheme 4), which are protonated at physiological pH
and the originalcharge on the peptide can be retained. 17 Iminoesters are
reported to be less reactive towards amines than succinimidyl-esters but
they are also less sensitive to hydrolysis.18 For example, methyl-4-
hydroxybenzimidate has been radioiodinated and conjugated to
proteins19 and a fluorinating reagent for proteins, 3-[18F]fluoro-5-
nitrobenzimidate, has been reported20.
N-Succinimidyl5-[ 131I]iodo-3-pyridinecarboxylate21 and N-succini-
midyl5-[ 211At]astato-3-pyridinecarboxylate22 and N-succinimidyl5-
[
211Ar]astato-3-pyridinecarboxylate22 have been used for labelling of
monoclonal antibodies and recently used for the labelling of octreotide.9
N-Succinimidyl-3-pyridinecarboxylate 2 and the reference N-succinimi-
%
dyl5-bromo-3-pyridinecarboxyalte were prepared according to the
literature.21,23 An advantage using N-succinimidyl5- [ 76Br]bromo-3-
pyridinecarboxylate 3 is that the same conjugation conditions could be
applied for reactions using N-succinimidyl[ 76Br]bromobenzoate as
prosthetic group (Scheme 1).
%
Scheme 2
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BROMINATION OF OCTREOTIDE
565
Compound 2 was radiobrominated in 25% radiochemicalyiedl
%
using chloramine-T as an oxidant at room temperature (Scheme 2).
All [76Br]bromide was consumed according to HPLC-analysis and a
very lipophilic labelled side-product was formed, probably due to a
minor impurity in stannyl-compound 2. The use of hexane: ethylacetate
%
as a mobile phase for HPLC-purification of compound 3 required
%
the addition of 2% acetic acid to the mobile phase. The remaining
acetic acid caused problems in the conjugation reaction, giving
hydrolysis of 3, so the mobile phase was changed to dichloromethane
%
containing 1% methanol. Compound 3 was conjugated to e-Boc-
octreotide using the same conditions as for 1 to give a Boc-protected
%
%
octreotide conjugate of 5 (Scheme 1). Deprotection in TFA followed by
%
HPLC-purification resulted in a 50% radiochemical yield of the labelled
octreotide 5 (calculated from 3). The specific radioactivity for the
%
%
labelled octreotide 5 was determined to be 1 GBq/mmolstarting from
%
40 MBq 76Br.
In the syntheses of methyl-4-bromobenzmidate 6, different methods
%
were investigated. The highest yield (74%) was obtained by bubbling
HCl (g) in a methanol solution of 4-bromobenzonitrile (Scheme 3).24
Bubbling HCl (g) into a solution of 4-bromobenzonitrile and methanol
in equimolar amounts in diethyl ether25 or the use of 4-bromo-
benzonitrile and sodium methoxide26 gave lower yields. The stannyl-
compound 7 was prepared in 40% yield using a palladium catalysed
%
metallation reaction.27 Compound 7 was radiobrominated and isolated
%
in 40% radiochemical yield using chloramine-T as an oxidant (Scheme
3). Since the conjugation of 8 to octreotide failed, no further
%
optimisation studies were performed. Removalof the sovl ent from the
purified compound 8 had to be done with care since the radioactivity in
%
the vesselcontaining 8 decreased when purging with helium gas after all
%
Scheme 3
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U. YNGVE ET AL.
Scheme 4
solvent was removed. Adding a small amount of high boiling solvent,
for example DMF, solved this problem.
The conjugation between methyl-4-bromobenzimidate 6 and e-Boc-
octreotide worked well using a large excess of the imidate 6 in methanol/
%
%
dichloromethane at 708C overnight (Scheme 4). The use of these
conditions in the labelling synthesis was not successful. Using
microwave heating and dichloromethane as in the N-succinimidyl
bromobenzoate case, left the methyl-4-[76Br]bromobenzimidate 8
%
unreacted. A variety of conditions using mixtures of borate buffer and
DMF or DMSO at different temperatures were tested. The use of
organic solvents left compound 8 unreacted. With water as solvent, 8
%
%
was consumed, probably due to hydrolysis. Addition of different
organic bases was also explored, but no product was observed. The
reactivity of compound 8 was tested using BSA (10 mg/ml) in borate
%
buffer pH 8.7. The radiochemical yield, determined by size exclusion
chromatography, was 10%.
Meningiomas have high affinity binding sites specific to somatostatin
(SRIH) receptors,28 whereas heart lacks specific binding. High specific
binding of the tracer 76Br-octreotide 5 was observed in meningioma
%
tissue, whereas 76Br-octreotide 4 only demonstrated non-specific
%
binding (Figure 1). The non-specific binding, observed in meningioma
Figure 1. Frozen section autoradiography shows high specific binding in
meningioma with 76Br-octreotide 5 in comparison to 76Br-octreotide 4
%
%
Copyright # 2001 John Wiley & Sons, Ltd.
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BROMINATION OF OCTREOTIDE
567
after blocking and in heart tissue, was of similar magnitude with the two
tracers. The difference in the behaviour of the two tracers might be due
to the difference in lipophilicity.11 The pyridine-moiety in compound 5
%
is protonated at physiological pH, retaining the positive charge
originating from the N-terminalof octreotide and olwering the
lipophilicity compared to 4.
%
Experimental
General
All chemicals used were commercially available and used without
further purification unless otherwise indicated. Boc-octreotide
and octreotide were a gift from Novartis Pharma Ag, Basel, Switzer-
land. Methanol was refluxed over Mg and I2 and distilled onto
molecular sieves under N2-atmosphere. HCl(g) was dried by passing it
through sulphuric acid (conc.) and a drying tower containing CaCl2.
Deionised water (18.2 MO) and 99.5% ethanol were used for labelling
reactions.
1 H NMR and 13C NMR spectra were recorded on a Varian XL-300
(300 MHz) or Varian Gemini (200 MHz) spectrometer with chloroform-
d1 as solvent and as internal standard. Thin-layer chromatography
(TLC) was performed using DC-Alufolien Kiselgel 60 F254 plates
(Merck, Darmstadt, Germany). Ultraviolet (UV) absorbance was
visualised using short- and long-wave ultraviolet light. A Micro Well
10 (Labwell AB, Uppsala, Sweden) was used for the microwave heating.
Microinsert vials (0.2 ml) placed in 1.5 ml glass vials equipped with a
screw cap and double septa were used for the reactions in the microwave
oven. The reported radiochemicalyields are uncorrected with respect to
decay. Autoradiographic imaging was performed using a Phosphor
Imager (Molecular Dynamics, Sunnyvale, CA, USA) and data was
analysed by ImageQuant.
Liquid chromatography
Analytical liquid chromatography separations were performed using a
Beckman (Fullerton, CA, USA) System (a 126 pump and 166 UV
detector) with a bþ-flow detector in series. Data collection was
performed using the Beckman System Gold Noveau Chromatography
Software Package. Semi-preparative HPLC was performed using
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U. YNGVE ET AL.
equipment from Waters (Milford, MA, USA); a modified M 6000 A
pump, a 440 UV detector with a bþ-flow detector in series. The UV-
absorbance was measured at 280 nm and the retention-times of the
radioactive compounds were matched with those of characterised
standards.
Electrospray ionisation mass spectroscopy, ESI-MS, was performed
using a Fisons Platform (Micromass, Manchester, UK). A post-column
split was used and 1% of the flow was delivered to the electrospray
probe.
The following columns and mobile phases were used:
System 1: Ultrasphere ODS C-18 HPLC column (Beckman)
250 Â 4.6 mm ID, 5 mm, flow 1.5 ml/min, A=10 mM TFA, B=MeCN.
(1a) 20% B 0–7 min, linear gradient 20–90% B 7–15 min, linear
gradient 90–20% 18–21 min.
(1b) 10% B 0–2 min, linear gradient 10–50% B 2–22 min, linear
gradient 50–80% B 25–28 min, linear gradient 80–10% 30–34 min.
(1c) 20% B 0–7 min, linear gradient 20–70% B 7–15 min, linear
gradient 70–20% B 18–21 min.
System 2: Ultrasphere Silica HPLC-column (Beckman) 25 Â 10 mm ID,
5 mm, flow 4 ml/min.
(2a) hexane : ethylacetate : acetic acid 70 : 30 : 0.1 isocratic eul tion,
(2b) hexane : ethylacetate : acetic acid 70 : 30 : 2 isocratic eul tion,
(2c) dichloromethane : methanol 100 : 1 isocratic elution.
System 3: Ultrasphere ODS C-18-column (Beckman) 250 Â 10 min ID,
5 mm, flow 4 ml/min, A=50 mM phosphate buffer pH 7, B=MeCN,
10% B 0–2 min, linear gradient 10–50% B 2–22 min, linear gradient
50–80% 35–38 min, 80% B 38–40 min.
Production of 76Br
76Br was produced via the 76Se(p, n)76Br nuclear reaction using a
16 MeV H+ cyclotron (Scanditronix, Uppsala, Sweden) and an 76Se-
enriched Cu2Se-target.13 The 76Br was released from the target-pellet by
a thermochromatographic method and deposited on the walls of a piece
of Teflon-tubing. The [76Br]bromide was recovered from the tubing by
rinsing with water and the resulting 76Br-solution was passed through a
C-18 SPEC (Ansys Inc., Irvine, CA, USA), evaporated to dryness and
redissolved in the solvent of choice prior to use (15).
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BROMINATION OF OCTREOTIDE
569
Synthesis of N-succinimidyl 5-[76Br]bromo-3-pyridinecarboxylate 3
%
0.5–1 mg of stannyl-compound 2, [76Br]bromide in ethanol(200 ml),
%
chloramine-T (10 mg/ml, 20 ml) and acetic acid (2 ml) were mixed and left
at room temperature for 10 min. The solvent was evaporated and the
product 3 was isolated in 25% radiochemical yield using normal phase
%
HPLC, system 2b Rt=15.5 min or system 2c Rt=12.8 min. Analysis by
HPLC system 1 c gave tR=11.8 min.
Synthesis of N-succinimidyl 4-[76Br]bromobenzoyl-octreotide 4
%
N-Succinimidyl4-[ 76Br]bromobenzoate 1 was prepared as described
%
previously.15 The solvent was evaporated by a gas purge in a 0.2 ml
microinsert vial. e-Boc-octreotide (0.25 mg) in dichloromethane (50 ml)
was added to the residue containing 1 and the mixture was heated by
%
microwaves, 10 W for 30 min to give 70–90% of labelled Boc- protected
octreotide-conjugate of 4 (determined by HPLC-analysis). TFA (50 ml)
was added and the reaction was left at room temperature for 30 min.
%
The solvents were removed under a stream of nitrogen, the residue was
redissolved in water: acetonitrile 80 : 20 and 4-[76Br]bromobenzoyl
octreotide 4 was isolated in 55% radiochemical yield (calculated from
%
1) using semi-preparative HPLC system 3, Rt=30.5 min. The products
%
were analysed using HPLC-system 1b. Boc-4 tR=29.3 min, compound 4
%
%
tR=24.1 min.
The identity of the peaks in the chromatogram was confirmed using
LC–MS, HPLC-system 1b, after addition of carrier N-succinimidyl4-
bromobenzoate in the conjugation reaction. LC-MS (ESI+) : (Boc-4)
%
m=z¼ 1303 ½M þ Hþꢁ, compound 4 m=z ¼ 1203 ½M þ Hþꢁ.
%
Synthesis of 5-[76Br]bromo-3-pyridinecarboxy-octreotide 5
%
Using the same conditions as in the synthesis of 4, compound 5 was
%
%
isolated in 50% radiochemical yield (calculated from 3) using HPLC-
%
system 3, tR=24.4 min. Analysis by HPLC system 1b: (Boc-5)
%
tR=28.6 min, compound 5 tR=22.0 min. LC–MS: (Boc-5) m=z¼ 1304
%
%
½M þ Hþꢁ, 652 [M+2H+]; compound 5 m=z ¼ 602:7 ½M þ 2Hþꢁ.
%
Synthesis of methyl-4-bromobenzimidate 6
%
4-Bromobenzonitrile (1.96 g, 10.8 mmol) was dissolved in dry methanol
(100 ml) and the mixture was cooled on ice. Dry hydrogen chloride was
bubbled through the solution for 3 h and the reaction mixture was left
under nitrogen atmosphere at room temperature overnight. The
methanolwas evaporated in vacuo. The solid was recrystallised from
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U. YNGVE ET AL.
methanol: ether to give (2.0 g, 74%) of the hydrochloride of 6. 1 H NMR
%
(200 MHz): d 8.3 (m, 2 H), 7.7(m, 2 H), 4.53 (s,3H), 1,8(s, 2 H).
The hydrochloride of compound 6 was dissolved in 1 M K2CO3 and
%
1
extracted with ether, dried over MgSO4 and evaporated to give 6. H
%
NMR (300 MHz): d 7.6 (m, 2 H), 7.5 (m, 2 H), 3.9 (2, 3 H), 3.9 (s, 3 H).
13C NMR: d 167, 132, 121, 128, 126, 53. IR (CHCl3) 3024 (N¼H stretch).
MS (ESI+): m=z=215.0 [M+H]+ calculated M=214.0.
Synthesis of methyl-4trimethylstannylbenzimidate 7
%
Compound 6 (0.50 g, 2.34 mmol) was dissolved in dry toluene.
%
Hexamethylditin (1.34 g, 4.1 mmol) was added followed by tetrakis
(triphenylphosphine)palladium(0), Pd(PPh3)4, (46 mg, 0.04 mmol). The
mixture was stirred under nitrogen atmosphere at 608C for 2 h. The
temperature was raised to 758C and left for 20 h. The solvent was
removed in vacuo and the product was purified on silica, eluting with
pentane containing 2% triethylamine until the unreacted tin eluted,
followed by pentane: ethyl acetate 10 : 2 containing 2% triethylamine
giving compound 7 in 40% yield. 1 H NMR (300 MHz): d 7.75 (m, 2 H),
%
7.55 (m, 2 H). 3.95 (s, 3 H), 0.35 (s, 9 H). 13C NMR (75 MHz): d
168, 146, 136, 132, 126, 53, ꢀ9.6.
IR: 2980 cmꢀ1 (N¼H stretch). LC–MS (ESI+): m=z 299.3 [M+H]+,
calculated M=299.0 (monoisotopic mass) with isotope distribution
corresponding to the distribution of Sn.
Synthesis of methyl-4-[76Br]bromobenzimidate 8
%
Compound 7 (1.0–1.5 mg) was dissolved in methanol (50 ml). Chlor-
%
amine-T (20 ml, 10 mg/ml in methanol), acetic acid (2 ml) and [76Br] Brꢀ
(150 mlin methano)l were added. The mixture was heated at 50 8C for
20 min, and the methanolwas removed by a gentel stream of He. The
residue was dissolved in hexane: ethyl acetate and the product 8 was
%
isolated in 40% radiochemical yield using semi-preparative HPLC,
system 2a, tR=14.5 min.
The identity and purity of the compound was confirmed using HPLC
system 1a, tR=11.3 min and LC–MS (ESI+): m=z=215.0 [M+H]+.
Synthesis of 4-bromobenzimidate octreotide 9
%
e-Boc-octreotide (2 mg, 1, 8 mmol) and methyl-4-bromobenzimidate 6
%
(3.9 mg, 18 mmol) where dissolved in methanol (100 ml) and CH2Cl2
(100 ml) in a capped vial and heated at 508C overnight. The
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BROMINATION OF OCTREOTIDE
571
reaction-mixture was analysed by LC-MS, HPLC-system 1b,
tR=25.8 min. LC-MS (ESI+): m=z=1302 [M+H]+.
Deprotection of the e-Boc-octreotide-conjugate in TFA (50 ml) gave a
product 9 with m=z=601.3 [M+2 H]2+, calculated for bromobenzimi-
%
date octreotide 9 601.2. HPLC-system 1b, tR=19.6 min.
%
Synthesis of [76Br]bromobenzimidate albumin
BSA (10 mg/ml) in borate buffer 0.1 M, pH 8.7 (100 ml) was added to the
dried compound 8. The mixture was heated at 408C for 1 h and was
analysed by size exclusion chromatography using a Fast Desalting HR
%
10/10 FPLC gel filtration column (Amersham Pharmacia Biotech,
Uppsala, Sweden) and phosphate buffer pH 7. Ten percent of the
radioactivity was incorporated in the high molecular fraction.
Frozen section autoradiography
Frozen human meningiomas and rat heart were cut in 20 mm sections
and mounted on microscope slides. Visualization of SRIH receptors was
performed as described by Reubi et al.28 Sections were preincubated for
10 min at room temperature in 50 mM Tris–HClbuffer, pH 7.4,
containing 2 mM CaCl2 and 5 mM KCland washed twice for 2 min in
50 mM Tris–HClbuffer. To define the totalbinding, sections were
incubated for 2 h at room temperature in 170 mM Tris–HClbuffer, pH
7.4, containing 1% BSA, 40 mg bacitracin and 5 mM MgCl2 in the
presence of brominated ligand 4 and 5 (500 pM). Non-specific binding
%
%
was determined by adding 1 mM octreotide. Incubated sections were
washed thrice for 5 min in cold incubation buffer containing 0.25%
BSA. After a brief dip in distilled water, the sections were dried quickly
under a stream of warm air and apposed to a storage phosphor (SP)
imaging plate.
For quantification, individualcalibration standards were prepared for
each section and exposed to same imaging plate.29 The standard was a
20-mldrop of tracer soul tion on a thin absorbent paper. Using the SP
imaging system, volume of standard and average count/pixel subtract-
ing the background area of each region of interest of a structure was
measured.
Conclusions
N-Succinimidyl4-[ 76Br]bromobenzoate 1 and N-succinimidyl5-
%
[76Br]bromo-3-pyridinecarboxylate 3 were used to label Boc-protected
%
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octreotide by microwave heating. The Boc-group was removed by the
addition of TFA to the reaction mixture and the products were isolated
by HPLC. This method is an improvement over the published use of
astato and iodo-analogues of 1 and 3 in the labelling of octreotide. Also,
%
%
methyl-4-[76Br]bromobenzimidate 8 could be synthesized from the
%
corresponding trimethylstannyl-compound. Compound 8 did not react
%
with Boc-octreotide under the conditions examined. Octeotide-con-
jugate 5 showed better binding-properties to meningiomas than 4 as
%
%
determined by frozen section autoradiography.
References
1. Anderson CJ, Pajeau TS, Edwards BW, Sherman ELC, Rogers BE, Welch
MJ. J NuclMed 1995; 36: 2315–2325.
.
2. Heppeler A, Froidevaux S, Macke HR, et al. Chem Eur J 1999; 5:
1974–1981.
3. Smith-Jones PM, Stolz B, Burns C, et al. J Nucl Med 1994; 35: 317–325.
.
4. Kwekkebom DJ, Kooij PP, Bakker WH, Macke HR, Krenning EP. J Nucl
Med 1999; 40: 762–767.
.
5. Rosch F, Herzog H, Stolz B, et al. J Nucl Med 1999; 26: 358–366.
6. Otte A, Jermann E, Behe M, et al. J Nucl Med 1997; 24: 792–795.
7. Bakker WH, Krenning EP, de Jong M. Eur J Nucl Med 1997; 24: 368–371.
8. Krenning EP, Breeman WA, Bakker WH. J Nucl Med 1990; 31: 1501–1509.
9. Vaidyanathan G, Affleck D, Welsh P, Srinivasan A, Schmidt M, Zalutsky
M. Nucl Med Biol 2000; 27: 329–337.
.
10. Guhlke S, Wester H-J, Bruns C, Stocklin G. Nucl Med Biol 1994; 21:
819–825.
11. Hostetler ED, Edwards WB, Anderson CJ, Welch J. J Labelled Cpd
Radiopharm 1999; 42(Suppl. 1): 720–722.
12. Kwekkeborn DO, Bakker WH, Krenning EP. J Nucl Med 1993; 20: 716–
731.
.
13. Tolmachev V, Lovqvist A, Einarsson L, Schultz J, Lundqvist H. Appl Rad
Isot 1998; 26: 249–258.
.
14. Lovqvist A, Lundqvist H, Lubberink M, Tolmachev V, Carlsson J, Sundin
A, Med Phys 1999; 26: 249–258.
.
15. Yngve U, Hedberg E, Lovqvist A, Tolmachev V, Langstrom B, Acta Chem
(
.
Scand 1999; 53: 508–512.
(
16. Forngren BH, Yngve U, Forngren T, Langstrom B. Nucl Med Biol 2000;
.
27: 851–853.
17. Hunter MJ, Ludwig, ML. J Am Chem Soc 1962; 84: 3491–3503.
18. Wilbur DS. Bioconj Chem 1992; 3: 433–470.
Copyright # 2001 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2001; 44: 561–573

BROMINATION OF OCTREOTIDE
573
19. Wood FT, Wu MM, Gerhart JC. Anal Biochem 1975; 69: 339–349.
20. Kilbourn MR, Dence CS, Welch MJ, Mathias CJ. J Nucl Med 1987; 28:
462–470.
21. Garg S, Garg PK, Zalutsky MR. Bioconj Chem 1991; 2: 50–56.
22. Reist CJ, Foulon CF, Alston K, Bigner DD, Zalutsky MR. Nucl Med Biol
1999; 26: 405–411.
23. Miyagi M, Nakao M, Nakazawa T, Kato I, Tsunasawa S. Rapid Commun
Mass Spectrom 1998; 12: 603–608.
24. Derkach GI, Lepesa AM, Kirsanov AV. Zh Obshch Khim 1962; 32:
171–174.
25. Leschke C, Elz S, Garbarg M, Schunack W. J Med Chem 1995; 38:
1287–1294.
26. Schaefer FC, Peters GA. J Org Chem 1961; 26: 412–417.
27. Azizian H, Eaborn C, Pidcock A, J Organomet Chem 1981; 215: 49–58.
28. Reubi JC, Maurer R, Klijn JG, et al. J Clin Endocrinol Metab 1986; 63:
433–438.
.
29. Sihver S, Sihver W, Bergstrom M, et al. J Pharm Exp Ther 1999; 290:
917–922.
Copyright # 2001 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2001; 44: 561–573