Synthesis and biodistribution studies of iodine-131 D-amino acid YYK peptide as a potential therapeutic agent for labeling an anti-CD20 antibody

Article abstract of DOI:10.1002/jlcr.1600

A major drawback of conventionally radioiodinated monoclonal antibodies for radioimmunotherapy is in vivo dehalogenation of iodine as a result of deiodinase recognition. To solve this problem we have synthesized a YYK tri-peptide consisting of non-metabol

Full text of DOI:10.1002/jlcr.1600

Research Article
Received 26 July 2008,
Revised 19 January 2009,
Accepted 2 March 2009
Published online 19 May 2009 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1600
Synthesis and biodistribution studies of
iodine-131 D-amino acid YYK peptide as a
potential therapeutic agent for labeling an
anti-CD20 antibody
K. Sadri,a M. Gandomkar,b M. H. Babaei,b R. Najafi,b S. R. Zakavi,c and
Ã
S. E. Sadat Ebrahimia
A major drawback of conventionally radioiodinated monoclonal antibodies for radioimmunotherapy is in vivo
dehalogenation of iodine as a result of deiodinase recognition. To solve this problem we have synthesized a YYK
tri-peptide consisting of non-metabolizable D-amino acids modified with the N-succinimidyl (N-Succ) function. The chemical
purity of the synthesized peptide as assessed by analytical high performance liquid chromatography was 95%. Labeling of
the Fmoc-D-Tyr(tBu)-D-Tyr(tBu)-D-Lys(Boc)-N-Succ was performed using the chloramine-T method and the conventional
extraction, resulting in a radiochemical yield of 50–71% and a radiochemical purity of 495%. Radioiodination of the
peptide was followed by conjugation to anti-CD20 antibody with 65–75% labeling efficiency and 90% radiochemical purity.
The effect of radioiodinated peptide on the biological behavior of the conjugate was evaluated through biodistribution
studies in normal Lewis rats. Thyroid and stomach levels from Rituximab labeled with [131I]-YYK-peptide were two- to four-
fold less than those with directly labeled [131I]-Rituximab, suggesting low recognition of its D-iodotyrosine residue by
endogenous deiodinases. The favorable in vitro/in vivo stability and biodistribution profiles suggest that this radioiodine-
labeled YYK peptide is a good candidate for further exploration of its potential clinical application.
Keywords: radioiodination; radioimmunotherapy; dehalogenation; D-amino acid
Iodine-131 is a relatively inexpensive and readily available
nuclide for labeling of radiopharmaceuticals. It has a long half-
Introduction
Non-Hodgkin lymphoma (NHL) involves a heterogeneous group
of malignancies derived from lymphoid tissues, which have the
ability to spread to other organs, with differences in appearance
behavior and response to therapy.1 Studies indicated that NHL
can be treated effectively by radioimmunotherapy (RIT) for
several reasons, including the inherent radiosensitivity of
lymphocyte, the vascular accessibility of these malignancies
and the large number of target antigens on the surface of
lymphocytes.2
The integral membrane protein CD20 has been identified as an
important therapeutic target in the treatment of NHL.3 CD20 is a
35 kD hydrophobic transmembrane protein expressed on pre-B-
cells and mature B-lymphocyte. It is expressed on more than 90%
of B-cell NHL.4 The absence of antigen expression on stem cells
allows for the recovery of normal B-cell following RIT, which leads
to the destruction of both malignant and normal B-cells.5
The first antibody to be approved in 1997 for treating
NHL was Rituximab, a chimeric anti-CD20 immunoglobulin.6
Rituximab acts via various mechanisms to kill tumor cells,
life of 8.1 days with beta emission of 0.69 MeV for therapy, and a
gamma emission for imaging, which is useful for making
dosimetry estimates.5 The widespread use of iodine-131 for
the labeling of tumor-associated antibodies has shown that this
nuclide suffers substantially from undesirable physical and
biological properties, principally the rapid and persistent in vivo
dehalogenation of the radiolabeled antibody.9 Such in vivo
removal of radioiodine from target cells within the first 24–120 h
postinjection of the labeled antibodies reduces tumor to non-
tumor ratio, which is important for radiodiagnosis. In addition, it
reduces the residence time of radioiodine in target cells, which
significantly affects radiotherapy effectiveness.10
aDepartment of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of
Medical Sciences, Tehran, Iran
bNuclear Science Research School, Nuclear Science and Technology Research
Institute (NSTRI), Atomic Energy Organization of Iran, Tehran, Iran
including complement-dependent cytotoxicity, antibody- cNuclear Medicine Department of Imamreza Hospital, Mashad University of
Medical Sciences, Mashad, Iran
dependent cell cytotoxicity and induction of apoptosis. The
next agent to be approved was 90Y-ibritumomab tiuxetan
*Correspondence to: S. E. Sadat Ebrahimi, Department of Medicinal Chemistry,
Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.
E-mail: sesebrahimi@sina.tums.ac.ir, sesebrahimi@yahoo.com
(Zevalin).7 This was followed approximately 1.5 years later by
131I-tositumomab (Bexxar).8
J. Label Compd. Radiopharm 2009, 52 289–294
Copyright r 2009 John Wiley & Sons, Ltd.

K. Sadri et al.
There has been an ongoing effort to develop new methods of tumor site following Rituximab attachment to the cell surface
radioiodination designed to trap radioiodine inside tumor cells antigen (CD20), matching the fact that mono iodo-D-tyrosine is
following delivery by a labeled antibody. Early work was based more stable to deiodination in vivo than its L enantiomer.16
on the use of non-metabolizable carbohydrates as linking
The YYK peptide was synthesized by standard Fmoc solid-phase
agents. Dilactitol-tyramine11 and tyramine cellobiose12 are two synthesis on trityl chloride resin (substitution, 0.8mmol/g) on a
substrates studied for this purpose. Another technique involves semiautomatic peptide synthesizer.17 The Fmoc-protected first
the use of non-metabolizable peptide adduct.13,14
amino acid D-lys-Boc I was attached via its carboxyl group to the
In this research, we describe the use of YYK-peptide-N-Succ resin. Then the peptide was enlarged by the sequential addition of
containing D-amino acids as an improved radioiodine labeling Fmoc-D-Tyr-tBu (Figure 1). After coupling the last D-tyrosine III, the
method for metabolically stable attachment of radioiodine. This peptide was cleaved from the resin and collected by filtration of
study is based on our knowledge about non-metabolizable the reaction mixture, and solvent removal furnished the protected
peptides, which are not a natural substrate for endogenous peptide IV as a gummy material, which was essentially pure as
enzyme15 including those present in plasma.
determined by analytical high performance liquid chromatogra-
phy (HPLC) (495%) with a yield of 90%.
N-hydroxysuccinimide was coupled to the C-terminal of YYK
Result and discussion
peptide IV using a solution of dicyclohexylcarbodiimide (DCC) in
Fmoc-D-Tyr(tBu)-D-Tyr(tBu)-D-Lys(Boc)-N-Succ was selected as the N,N-dimethlyformamide (DMF). Molarity was the same for all
linking agent between Rituximab and iodine-131 and the effective- materials.18 It gave a brown residue with a yield of 60% and a
ness parameters for labeling and its characters were studied. These retention time of 32 min on analytical HPLC. YYK-peptide-N-Succ
amino acids were connected via non-metabolizable D-amino acid was dissolved in anhydrous DMSO and frozen in aliquots for the
bonds. This peptide promotes the stability of radioiodine at the labeling process.
O
O
Resin
HN
HN
1
Boc
Boc
OH
O
NH
NH
Fmoc
2-3
Fmoc
I
II
O
O
Resin
HN
HN
Boc
O
O
Boc
O
OH
4
O
O
HN
HN
HN
Fmoc
HN
NH
Fmoc
NH
CH2
CH2
CH2
CH2
O
O
tBu
O
5
O
tBu
tBu
tBu
III
IV
O
O
HN
N
O
Boc
O
O
O
HN
HN
Fmoc
NH
CH2
CH2
O
O
tBu
tBu
V
Figure 1. Synthesis of YYK-peptide (IV) on trityl chloride resin and its coupling with NHS: (1) 2-chlorotrityl chloride resin, DIPEA, DCM, 18 h; (2) 20% piperidine/DMF; (3)
Fmoc-D-Tyr(tBu), activated with HOBT and DIC, DIPEA, 18 h, steps 2 and 3; (4) 20% CH3COOH/DMF; and (5) NHS, DCC/DMF, 18 h.
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Copyright r 2009 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2009, 52 289–294

K. Sadri et al.
O
O
O
131I] NaI
O
[
HN
N
O
HN
N
O
Chloramine-T
(pH=7.5)
Boc
O
O
Boc
O
O
O
O
HN
HN
HN
HN
Fmoc
NH
Fmoc
NH
CH2
CH2
CH2
CH2
R1
R2
tBu
O
O
O
O
tBu
tBu
tBu
V
VI
Rituximab
pH= 8.5
O
HN
Rituximab
Boc
O
N
H
O
HN
HN
Fmoc
NH
CH2
CH2
R1
R2
tBu
O
O
tBu
VII
Figure 2. Preparation of [131I]-YYK-peptide-N-Succ and labeling with Rituximab (R1 or R2 = 131I).
Table 1. Labeling yield of [131I]-YYK-peptide-N-Succ after
extraction into benzene/DMF at different time points and
CAT concentrations (n = 3)
Table 2. Stability of [131I]-YYK-peptide-Rituximab at 371C
up to 48 h (n = 3)
Time postlabeling (h)
PBS
Serum media
Time
(s)
Chloramine-T
(2 mg/ml)
Chloramine-T
(3 mg/ml)
Chloramine-T
(4 mg/ml)
4
24
48
87.20%71.51
78.42%70.53
73.62%72.18
85.54%72.21
75.74%71.25
71.25%71.39
15
30
45
60
46.62%70.74
49.39%70.51
55.86%71.70
58.40%70.22
52.25%70.19
66.44%70.83
65.31%70.61
71.84%71.51
61.96%71.21
56.41%70.87
56.28%71.43
63.46%70.59
buffer, 50 mM, pH = 8.5) and incubated for 2 h at room
temperature. Differential protections of amines in D-lysine side
chain and also the peptide’s N-terminus with Fmoc and Boc
Labeling of this peptide V (Figure 2) was performed by the protecting groups19 enabled the specific conjugation of
electrophilic substitution of iodine-131 in the D-tyrosine ring, activated [131I]-YYK-peptide-N-Succ VI to NH2-lysine antibody.
using chloramine-T as the oxidant. Since the NHS group in the The labeled Rituximab VII was purified using a 1 Â 20-cm
YYK peptide is sensitive to hydrolysis, the radioiodination was Sephadex G-50 column. At a Rituximab concentration of 10 mg/
terminated after 60 s by the conventional extraction of labeled ml, coupling efficiencies of 65–75% were obtained with a
peptide into benzene/DMF and evaporation to dryness with a radiochemical purity of 90% and specific radioactivity of
stream of nitrogen.
Labeling conditions such as chloramine-T concentration and
185 MBq/mg (5 mCi/mg).
In vitro stability of the rodioiodinated antibody under
extraction time were optimized to obtain the maximum labeling physiological conditions is an important parameter in the
yield. The isolated radiochemical yield was between 50 and 71% evaluation of in vivo dehalogenation. Such in vivo removal of
with a maximum yield after 60-s incubation time (Table 1). TLC free iodide causes radiation dose exposure of non-target tissues
analysis showed a radiochemical purity of 495%.
limiting the clinical efficacy. Thus, the stability of the labeled
To provide a stable conjugate without affecting the affinity antibody was assessed in the presence of PBS solution and
and specificity of the antibody for CD20 antigen, Rituximab was human serum for up to 48 h at 371C (Table 2). Studies have
added to the labeled peptide under mild conditions (borate demonstrated that the labeled antibody was radiochemically
J. Label Compd. Radiopharm 2009, 52 289–294
Copyright r 2009 John Wiley & Sons, Ltd.
www.jlcr.org

K. Sadri et al.
Table 3. Stability of [131I]-Rituximab at 371C up to 48 h
(n = 3)
Time
postlabeling (h)
PBS
Serum media
4
24
48
92.90%70.13
82.83%70.71
79.29%71.13
79.85%70.83
65.29%71.13
58.10%72.21
stable in both PBS and human serum for 2 days and TLC analysis
showed comparable decrease in both media, which was most
likely because of beta emission radiolysis by iodine-131.20,21 For
Rituximab labeled with direct method, a considerable decrease
of stability in human serum media has been obtained (Table 3).
Immunoreactivity was measured comparatively for two labeling
methods using direct binding on fresh Raji cells according to
the method of Lindmo et al.22 The results showed a statistically
significant higher binding for 131I-labeled YYK-peptide-Rituximab
than for [131I]-Rituximab (86.1%71.4 and 76.3%71.8, respectively;
Po0.05).
Figure 3. Biodistribution studies in normal Lewis rats, expressed as %ID/g organs
[
131I]-YYK-peptide-Rituximab at 4, 24 and 48 h after IV administration
for
(mean7SD).
As previous studies have shown, a potential concern with
labeling antibodies by most conventional methods is that these
tracers are rapidly dehalogenated in vivo, as reflected by high
radioactivity levels in thyroid and stomach.23 Distribution and
radioactivity uptake of 131I-labeled YYK-peptide-Rituximab in
tissues of normal rat are shown in Figure 3 and compared with
the distribution of [131I]-Rituximab labeled via the direct method
(Figure 4). Differences in normal tissue uptake of radioiodine
activity were observed between [131I]-YYK-peptide labeling and
the direct method.
With 131I-labeled YYK-peptide-Rituximab, liver levels were
significantly higher than those for [131I]-Rituximab (5.3370.86
and 2.6670.63 %ID/g at 24 h, respectively; Po0.05), whereas
thyroid and stomach were significantly lower (Po0.05), suggest-
ing the fact that most radioiodine activity eliminated through
liver in the form of [131I]-YYK-peptide-Rituximab and less free
iodine was observed. Thyroid and stomach levels from
Figure 4. Biodistribution studies in normal Lewis rats, expressed as %ID/g organ
for [131I]-Rituximab at 4, 24 and 48 h after IV administration (mean7SD).
bombardment mass spectroscopy was performed using Finni-
gan Mat-TSQ 70, USA. A well counter EG & G, ORTEC, Model
4001M was used. Analytical reverse-phase HPLC was carried out
on a JASCO 880-PU intelligent pump HPLC system (Tokyo,
Japan). CC 250/4.6 Nucleosil 120-3 C18 columns from Macherey-
Nagel (Germany) were used for analytical HPLC. The gradient
systems consisted of 0.1% trifluoroacetic acid/water (solvent A)
and acetonitrile (solvent B). For analytical HPLC, this gradient
was used: 0 min 95%A (5%B), 5 min 95%A (5%B), 30 min
0%A (100%B), 35 min 0%A (100%B), 40 min 95%A (5%B), flow:
1 ml/min, l = 280 nm.
Rituximab labeled with
3.6770.31 %ID/g at 24 h, respectively) were two- to four-fold
less than the levels for directly labeled
131I]-Rituximab
[
131I]-YYK-peptide (1.170.15 and
[
(2.870.39 and 6.770.85 %ID/g at 24 h, respectively), suggesting
low recognition of its D-iodotyrosine residue by endogenous
deiodinases.15 Thyroid levels for [131I]-YYK-peptide-Rituximab
were 57, 39 and 26% of the levels for the directly labeled [131I]-
Rituximab at 4, 24 and 48 h, respectively.
Iodine-131 was prepared by the radioisotope division (Atomic
Energy Organization of Iran (AEOI)).
Experimental
All chemicals were obtained from known commercial sources
and used without further purification. Tritylchloride resin and
Synthesis of YYK tri-peptide
9-fluorenylmethoxy carbonyl (Fmoc)-protected amino acids Fmoc-D-Lys-Boc (0.423 g; 0.903 mmol) I (Figure 1) was dissolved
were purchased from Nova Biochem (Germany). The reactive in anhydrous DCM (3 ml) and mixed with DIPEA (0.617 ml). The
side chains of the amino acids were masked with one of the solution was stirred with 0.215 g of 2-chlorotrityl chloride resin in
following groups: lysine, t-butoxycarbonyl (Lys-Boc), tyrosine, a 10 ml filter cartridge and the content shaken vigorously for
t-butyl (Tyr-tBu). 1-Hydroxybenzotriazole (HOBT), diisopropylcar- 18 h. The combined resin II was filtered and the resin was
bodiimide (DIC), N-methylpyrrolidone (NMP), piperidine, DMF, washed with solvents: 3 Â 2 ml of DCM, 3 Â 2 ml of DCM/MeOH
dichloromethane (DCM) and DCC were purchased from Fluka. (17/3 v/v) and 3 Â 2 ml DMF to remove unbound amino acids.
Rituximab, a mouse–human chimeric anti-CD20 antibody, was The reaction was monitored by the ninhydrin test and the Fmoc
commercially obtained from Roche (Mabthera, Switzerland, groups were removed by adding 2 ml of 20% piperidine in DMF
100 mg/10 ml).
for 5 min, draining the solution off and continuing cleavage with
1H NMR spectra were obtained with BRUKER (500 MHz) 20% piperidine in DMF for 15 min. This was followed by a wash
instrument using CDCl3 as the solvent. High-resolution fast with 6 Â 2 ml DMF. The peptide was prepared using activated
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Copyright r 2009 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2009, 52 289–294

K. Sadri et al.
Fmoc-D-Tyr-tBu. The activation was carried out using 0.414 g by TLC using silica gel (Silica gel 60; Merck) and acetate–metha-
(0.9 mmol) of Fmoc-D-Tyr-tBu, 0.138 g of HOBT in 2 ml of NMP, by nol (9/1 v/v) as the mobile phase ([131I]-YYK-peptide-N-Succ:
adding to the clear solution of 0.14 ml of DIC and keeping at Rf = 0.82, YYK-peptide-N-Succ: Rf = 0.16).
room temperature for 30 min. After this period, 0.9mmol
[
131I]-YYK-peptide-N-Succ and
(0.154 ml) of DIPEA was added, and the mixture was shaken for
Labeling Rituximab with
Na131
18 h. The wash sequence following Fmoc cleavage and
subsequent wash sequences were done as described above. A
second coupling using activated Fmoc-D-Tyr-tBu 0.9 mmol was
carried out in the same manner. After coupling the last amino
acid III, the peptide chain with all protecting groups IV was
cleaved from the resin by 2-h treatment in a solution of 20%
acetic acid in DMF. For further exploration, removal of lateral
protecting groups was performed by treatment with a solution
of trifluoroacetic acid (90%), water (5%) and thioanisole (5%)
at room temperature for 10 min. The solution was then
concentrated to a small volume and the peptide was precipi-
tated by the addition of ether. The side product with formula
of C24H32N4O6 and calculated Mw of 472.53 exhibited a single
peak with a retention time of 14 min on analytical reverse-phase
HPLC with a yield of 90%. The electrospray mass spectrum
showed an [M1H]1 peak at m/e 473.2 and an [MÀH]À at
m/e 471.1. 1H NMR: (D2O) d: 0.711 (tt, 2H, J = 11 Hz); 1.358 (tt, 2H,
J = 14.5 Hz); 1.436 (tt, 2H, J = 8.5Hz); 2.755 (t, 2H, J = 7.5 Hz); 2.979
(m, 4H, J = 6.5Hz); 4.023 (t, 1H, J = 4.5Hz); 4.065 (t, 1H, J = 6.5 Hz);
4.475 (t, 1H, J = 5 Hz); 6.697 (2d, 4H, J = 7.75 Hz); 6.972 (2d, 4H,
J = 13.25 Hz).
I
The solvent of [131I]-YYK-peptide-N-Succ VI extract was com-
pletely removed with a gentle stream of nitrogen. One microliter
of Rituximab (10 mg, 6.6 Â 10À5 mmol) was mixed with 100 ml of
borate buffer (50 mM, pH = 8.5) and incubated with [131I]-YYK-
peptide-N-Succ (5 mg, 0.55 Â 10À5 mmol) at room temperature
for 2 h. The labeled antibody was isolated by gel filtration
column (Sephadex G-50 Pharmacia). The column was eluted
with PBS buffer (0.2 M, pH = 7.5) in 20 aliquots of 1 ml.
Radioactivity in each fraction was determined by well counter,
and the fraction containing the labeled antibody VII was
separated.
In the direct labeling method, 1 ml of Rituximab (10 mg,
6.6 Â 10À5 mmol) was added to 50 ml of sodium phosphate buffer
(0.5 M, pH = 7.5), followed by 200 mCi Na131I (10 ml, 7.4 MBq in
0.1 N NaOH) and 25 ml of chloramine-T (4 mg/ml PBS 50 mM,
pH = 7.5). The mixture was stirred for 1 min and 50 ml of sodium
metabisulfite (4 mg/ml PBS 50 mM, pH = 7.5) was added
followed by 100 ml of KI (10 mg/ml). The reaction mixture was
immediately applied to a gel filtration column (Sephadex G-50
Pharmacia) and the fraction containing [131I]-Rituximab was
isolated as described above.
Synthesis of YYK tri-peptide-N-Succ (V)
Biodistribution studies of Rituximab labeled with [131I]-YYK-
The protected peptide cleaved from the resin IV was derivatized
with N-succinimidyl function. The solution of NHS (0.045 g,
0.39 mmol) and DCC (0.08 g, 0.39 mmol) in DMF (10 ml) was
added to the precipitated peptide (0.355 mg, 0.39 mmol). The
reaction mixture was stirred for 18 h at room temperature. The
reaction mixture was filtered and the filtrate was concentrated
to dryness to give a brown residue. The product with formula
of C56H69N5O12 and calculated Mw of 1004.17 exhibited a
peak with a retention time of 32 min on analytical HPLC with a
yield of 60%; m.p. 155–1581C; electrospray mass spectrum
shows an [M1H]1 peak at m/e 1005.2 and an [MÀH]À peak
at m/e 1003.4. 1H NMR: (CHCl3) d: 1.31 (t, 2H, J = 4 Hz); 1.39
(s, 9H); 1.431 (s, 18H); 1.596 (t, 2H, J = 3.5 Hz); 1.725 (t, 2H,
J = 3.5 Hz); 2.705 (s, 4H); 2.931 (t, 2 H, J = 6 Hz); 3.051 (m, 4H,
J = 10.5 Hz); 4.149 (m, 1H); 4.163 (t, 1H); 4.265 (m, 2H); 4.314 (t, 1H);
4.328 (t, 1H), 6.912 (dd, 4H, J = 17.5Hz); 7.032 (dd, 4H, J = 10 Hz);
7.295–7.544 (m, 6H).
peptide-N-Succ and Na131
I
Animal experiment was performed in compliance with the
regulation of our institution and with generally accepted
guidelines governing such work. Eighteen normal male Lewis
rats (150–200 g) were administered with 0.37 MBq (2 mg,
1.3 Â 10À5 mmol) of [131I]-YYK-peptide-Rituximab and 0.37 MBq
(2 mg, 1.3 Â 10À5 mmol) of [131I]-Rituximab in 0.2 ml via lateral tail
vein. Animals were divided into three groups of three for each
time studied, 4, 24 and 48 h, respectively. The rats were
sacrificed, the blood was collected and organs of interest were
dissected, weighed and counted for 131I activity in well gamma
counter. The percent injected dose per gram was calculated
(%ID/g). All mean values are given as mean7SD. Statistical
analysis was performed using the t-test. The level of significance
was set at Po0.05.
Preparation of [131I]-YYK-peptide-N-Succ (VI)
Conclusion
The favorable biodistribution data obtained with [131I]-YYK-
peptide-Rituximab suggest further investigation of this labeled
antibody for RIT of NHL. We do not know at this time whether
improved intracellular retention of radioiodine activity would
also be obtained with this D-amino acid peptide. In near future
we are planning to study internalization of this peptide with
other antibodies.
YYK-peptide-N-Succ (20 mg, 0.024 mmol) was dissolved in DMSO
(2 ml) and frozen in aliquots. Two microliters of YYK-peptide-N-
Succ (20 mg, 2.2 Â 10À5 mmol) V (Figure 2) was added to a
solution of 200 mCi Na131I (10 ml, 7.4 MBq in 0.1 N NaOH) in buffer
PBS (50 ml, 0.5 M, pH = 7.5), followed by 25 ml chloramine-T
(4 mg/ml PBS 50 mM, pH = 7.5). The component was mixed by a
pipette for 1 min and the reaction was terminated by extracting
the iodine-131-labeled YYK-peptide-N-Succ by adding 100 ml
extraction solvent: benzene/DMF (100/5 v/v). After mixing with a
pipette, the organic phase was removed to a glass vial and dried
with a gentle stream of nitrogen. The extraction of the aqueous
phase was repeated twice following the steps described above.
The extract fraction of [131I]-YYK-peptide-N-Succ was analyzed
Acknowledgement
The authors wish to thank Mr Mirfallah, Mr Mazidi and
Mr Gourani of the Radioisotope Division (AEOI) for their
assistance.
J. Label Compd. Radiopharm 2009, 52 289–294
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www.jlcr.org

K. Sadri et al.
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