Preparation of radioiodo-metoprolol and its biological evaluation as a possible cardiac imaging agent1

Article abstract of DOI:10.1134/S1066362210020165

¹²⁵I-Metoprolol was prepared by electrophilic substitution reaction in the presence of Chloramine-T (CAT) as an oxidizing agent. The reaction conditions were optimized. The radiochemical yield of ¹²⁵I-metoprolol exceeding 90% was attained in 10 min at ambient temperature (25 ± 1°C). The radiochemical yield and purity of the labeled product were evaluated by electrophoresis, thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC). Biodistribution studies of ¹²⁵I-iodometoprolol were carried out in normal Albino Swiss mice. The results indicate the possibility of using [¹²⁵I]- iodometoprolol for myocardial imaging. Pleiades Publishing, Inc., 2010.

Full text of DOI:10.1134/S1066362210020165

ISSN 1066-3622, Radiochemistry, 2010, Vol. 52, No. 2, pp. 212–216. © Pleiades Publishing, Inc., 2010.
Published in Russian in Radiokhimiya, 2010, Vol. 52, No. 2, pp. 181–185.
Preparation of Radioiodo-Metoprolol and Its Biological
Evaluation as a Possible Cardiac Imaging Agent1
I. T. Ibrahima, A. M. Amina, and K. M. El-Azony*b
a Labeled Compound Department, Hot Laboratories Center, Atomic Energy Authority,
P.O. Box 13759, Cairo, Egypt
b Radioactive Isotopes and Generators Department, Hot Laboratories Center, Atomic Energy Authority,
P.O. Box 13759, Cairo, Egypt; * e-mail: mmenaa_2004@yahoo.com
Received July 6, 2009
Abstract—125I-Metoprolol was prepared by electrophilic substitution reaction in the presence of Chloramine-T
(CAT) as an oxidizing agent. The reaction conditions were optimized. The radiochemical yield of
125I-metoprolol exceeding 90% was attained in 10 min at ambient temperature (25 ± 1°C). The radiochemical
yield and purity of the labeled product were evaluated by electrophoresis, thin-layer chromatography (TLC),
and high-performance liquid chromatography (HPLC). Biodistribution studies of 125I-iodometoprolol were car-
ried out in normal Albino Swiss mice. The results indicate the possibility of using [125I]-iodometoprolol for
myocardial imaging.
Key words : Radioiodination, iodine-125, metoprolol, chromatographic analysis, biodistribution
DOI: 10.1134/S1066362210020165
Radioiodination of organic compounds and bio-
molecules is the subject of numerous studies [1],
thanks to the fact that the iodine atom occupies a vol-
ume similar to that occupied by a methyl or ethyl
group and can substitute an alkyl group in an organic
molecule without unduly perturbing the steric or polar
configuration [2]. The radioiodination of β-agonist or
β-blocker drugs which exert cardioselective pharma-
cological action may be useful in myocardial imaging.
In this study we developed a simple method for
radioiodination of metoprolol and examined the effect
of various parameters on the labeling yield so as to
attain the maximum labeling yield. The purity of the
final product was examined by TLC, electrophoresis,
and HPLC. Biodistribution of 125I-metoprolol in nor-
mal mice was studied to evaluate its suitability
for myocardial imaging and compare with 99mTc-
metoprolol [8].
The suggested structure of 125I-metoprolol, which is
formed by electrophilic substitution in the presence of
an oxidizing agent like CAT, is given below. 125I+ re-
places H+ in the aromatic ring in the ortho position to
alkoxy group.
Radioiodinated myocardial agents like penta-
decanoic acid derivatives [3] was prepared and used to
image myocardial disorders [4].Recently Gungor et al.
[5] prepared iodoatenolol and evaluated its biodistribu-
tion with the aim to assess the applicability of the
tracer to scintgraphy of β-receptors [5].
Metoprolol is a β-adrenergic blocking agent. Its
activity is similar to that of propranolol [6]. It is
classed with carioselective agents and is used to treat
hypertension and some types of arrhythmias [7]. It is
also useful in slowing and regulating certain types of
abnormally rapid heart rates [6, 7]. Metoprolol was la-
beled with techntium-99m, and its suitability for heart
imaging was demonstrated in the previous study [8].
125I-Metoprolol {1-isopropylamino-3-[4-(2-methoxyethyl)-
2-125I-phenoxy]propan-2-ol tartrate}
1 The text was submitted by the authors in English.
212

PREPARATION OF RADIOIODO-METOPROLOL
213
EXPERIMENTAL
Electrophoresis. Electrophoresis technique consists
in applying a radioactive sample onto a paper or poly-
acrylamide gel soaked in a suitable buffer and applying
an appropriate voltage across the paper or gel for a
certain period of time. The components of the sample
move to different positions along the paper or gel me-
dium, depending on their charge and ionic mobility
[10].
Materials and methods. All the chemicals used in
this study were of analytical grade. Metoroplol {1-iso-
propylamino-3-[4-(2-methoxyethyl)phenoxy]-propan-
2-ol tartrate} was presented by the Egyptian Interna-
tional Pharmaceutical Industries Company (EIPICO),
Egypt. Absolute ethanol was used as a solvent without
additional purification. Double-distilled water was
used for all the experiments. Chloramine-T (N-chloro-
p-toluenesulfonamide sodium salt, CAT) was pur-
chased from Aldrich. Thin-layer chromatography
(TLC) was performed on 20 × 25-cm aluminum sheets
SG-60 F254 (Merck). Na125I (185 MBq/100 μl) in dilute
NaOH (pH 7–11) was purchased from Nordion
(Belgium).
Labeling process. 10 μl of a Na125I solution
(3.7 MBq) was applied onto a flask bottom and dried
with a vacuum line, after which 200 μl of an oxidizing
(CAT) solution of definite concentration and 200 μl of
a metoprolol solution in appropriate medium (0.5 M
phosphate buffer, pH 7) were added at room tempera-
ture. After a specified interval of time, the reaction was
quenched by using 0.2 N Na2S2O3 solutions (100 μl) to
ensure that the unchanged iodine is reduced before
chromatographic analysis [9]. The labeling yield of
125I-metoprolol and its purity were determined by pa-
per electrophoresis and thin-layer chromatography.
The influence of various parameters such as CAT con-
centration (0.09–3.7 mM), metoprolol concentration
(0.094–3.75 mM), pH of the reaction mixture (pH 2–
11), and reaction time (1–60 min) was studied using
the trial-and-error method to attain the optimum value.
The experiment was repeated with all factors fixed
except the factor under study, until the optimal condi-
tions were achieved.
Electrophoresis was performed with an EC-3000
p-series programmable device (EC Apparatus Corpora-
tion) and chamber supply units using cellulose acetate
strips. These strips were moistened with 0.05 M phos-
phate buffer at pH 7.2 ± 0.2 and then were introduced
into the chamber. Samples (5 μl) were applied at a dis-
tance of 10 cm from the cathode. Electrophoresis was
performed for 1.5 h. The developed strips were dried
and cut into 1-cm segments which were examined with
a well-type NaI scintillation counter. The radiochemi-
cal yield was calculated as the ratio of the radioactivity
of the labeled product to the total radioactivity [11].
Analysis of the fractions obtained revealed the pres-
ence of three peaks. One of them corresponded to free
iodide which moved toward the anode to a distance of
20 cm. The second peak remained at spotting point,
and the third peak also migrated toward the anode, but
to a lesser extent (10 cm). The species that stayed at
the spotting point was identified as 125I-metoprolol.
HPLC analysis. The radiochemical yield of 125I-
metoprolol was determined by direct injection into a
chromatograph of 5–10 μl of the reaction mixture ob-
tained under the optimum conditions for reaching the
highest radiochemical yield. The column (250 ×
4.6 mm) was packed with Nucleosil phenyl reverse
phase (5 μm). The eluent was methanol : H2O (70 : 30
v/v) at a flow rate of 1 ml min–1 [8]. The labeled com-
pound was collected using a fraction collector, and its
activity was determined with a well-type counter
equipped with a NaI (Tl) crystal and connected to a
single-channel analyzer. The free radioiodide had the
retention time of 4.5 min; metoprolol, 8 min; and the
labeled compound (125I-metoprolol), 10 min as shown
in Fig. 1.
Radiochemical yield and purity. The radiochemi-
cal yield was determined by thin-layer chromatography
(TLC) and by electrophoresis, after which radiochemi-
cal purity was determined by high-performance liquid
chromatography (HPLC).
TLC analysis was performed with silica gel coated
aluminum sheets. The sheets were cut into strips 1 cm
wide and 13 cm long. The spotted point was placed
2 cm above the edge. The solvent used for developing
was chloroform : water : ammonia [90 : 8 : 2 (v/v/v)].
125I-Metoprolol remained near the origin (Rf = 0–0.1),
whereas radioiodide (125I–) moved with the solvent
front (Rf = 0.6).
Animals. Male Swiss Albino mice weighing 20–
25 g were purchased from the Research Center, Cairo,
Egypt. The animals were kept at constant environ-
mental and nutritional conditions throughout the ex-
perimental period and kept at room temperature (22 ±
2°C) with a 12 h on/off light schedule. The food and
water were standard.
RADIOCHEMISTRY Vol. 52 No. 2 2010

214
IBRAHIM et al.
weight, respectively [14]. The percentage of the activ-
ity in the organ relative to the total injected dose was
calculated and divided by the organ weight (%ID/g).
Statistical analysis. The results are expressed as
mean ± SEM for the indicated number of different ex-
periments. The statistical significance of differences
was assessed by unpaired Student’s t-test for P < 0.05.
RESULTS AND DISCUSSION
Our results show that the incorporation of iodonium
ion (125I+) occurs at the reactive ring of the metoprolol
molecule. The reaction can be categorized as electro-
philic substitution and occurs at ambient temperature
(25 ± 1°C). The reagents are Na125I and CAT. The ef-
fect of certain parameters on the reaction was exam-
ined with the aim to achieve the maximum radiochemi-
cal yield of 125I-metoprolol.
Fig. 1. HPLC elution profile of metoprolol.
Effect of CAT concentration. CAT as oxidiz-
ing agent strongly affects the radiochemical yield of
125I-metoprolol, because it is oxidizes iodide ions to
iodonium ions, which are the active species in the reac-
tion. It was observed that the radiochemical yield of
125I-metoprolol increased gradually from 80 to 95%
with an increase in the CAT concentration from 0.09 to
1.8 mM, respectively (10 min, 25°), as shown in
Fig. 2. At a CAT concentration higher than 1.8 mM,
the labeling yield decreased. The low labeling yield
was noted at a low CAT concentration, apparently be-
cause of incomplete oxidation of iodide ions to io-
donium ions [15]. A decrease in the labeling yield at
CAT concentrations above 1.8 mM is due to the occur-
rence of undesirable oxidative side reactions like
chlorination [9, 16], polymerization, and denaturation
of the substrate [17].
Fig. 2. Radiochemical yield of 125I-metoprolol as a func-
tion of CAT concentration [10 μl of Na125I solution
(3.7 MBq) + 200 μl of metoprolol solution (1 mM) +
200 μl of CAT solution (x mM)]. Reaction time 10 min,
room temperature.
Fig. 3. Radiochemical yield of125I-metoprolol as a function
of metoprolol concentration [10 μl of Na125I solution
(3.7 MBq) + 200 μl of metoprolol (x mM) + 200 μl of CAT
(0.5 mM)]. Reaction time 10 min, room temperature.
Influence of metoprolol concentration. Figure 3
illustrates the influence of the metoprolol concentra-
tion on the labeling yield in the presence of CAT as an
oxidizing agent. The labeling yield increased from
85% to 95% with an increase in the metoprolol con-
centration from 0.095 to 1 mM (reaction time 10 min,
25°C). The metoprolol concentration higher than
1 mM has no effect on the labeling yield, apparently,
owing to saturation, i.e., 1 mM of metoprolol was
sufficient to capture all the iodonium ions in the reac-
tion. At the same time, low metoprolol concentration
(0.095 mM) is insufficient to capture all the iodinium
ions in the 125I+–H+ exchange reaction, so that the la-
beling yield decreases.
In vivo biodistribution in normal mice. In vivo
biodistribution studies were performed using three
groups, each comprising six mice. Each animal was
injected in the tail vein with 0.2 ml solution containing
3.7 MBq of 125I-metoprolol. The mice were kept in
metabolic cages for the required time. Each group was
sacrificed by cervical dislocation at the recommended
time (0.25, 1, 3 h) after injection. Organs or tissues of
interest were removed, washed with saline, weighed,
and counted. Correction was made for the background
radiation and physical decay during the experiment
[12, 13]. The weights of blood, bones, and muscles
were assumed to be 7, 10, and 40% of the total body
Influence of pH. The radiochemical yield of 125I-
RADIOCHEMISTRY Vol. 52 No. 2 2010

PREPARATION OF RADIOIODO-METOPROLOL
215
metoprolol depends on pH of the reaction mixture in
presence of CAT as oxidizing agent, as shown in
Fig. 4. The maximum yield was attained at pH 7. This
can be attributed to the fact that CAT works well as
oxidizing agent to generate the iodonium ions around
pH 7.5 [18, 19]. The iodonium ion at these pH values
replaces a proton in the phenyl ring [20]. Acidification
of the reaction medium to pH 2 leads to a decrease in
the yield to 35%, wihch may be attributed to the pre-
dominance of ICl species whose oxidation potential is
lower than that of HOCl species [21]. In alkaline solu-
tions, the yield of 125I-metoprolol is relatively low
(55%) as a result of decreasing concentration of HOI,
the active agent in the electrophilic substitution [17].
Fig. 4. Radiochemical yield of125I-metoprolol as a function
of pH of reaction mixture [10 μl of Na125I solution
(3.7 MBq) + 200 μl of metoprolol (1 mM) + 200 μl of CAT
(0.5 mM) + 100 μl of 0.5 M buffer (pH x)]. Reaction time
10 min, room temperature.
Effect of reaction time. Figure 5 shows the
relationship between the reaction time and the yield of
125I-metoprolol. The radiochemical yield of 125I-
metoprolol significantly increased, from 65 to 94%,
with an increase in the reaction time from 1 to 10 min.
Extending the reaction time to 60 min leads to a slight
decrease in the labeling yield to 90%. The reaction
time can affect the performance of the oxidizing agent,
which affected the labeling yield [22].
In vitro stability of 125I-metoprolol. The stability
of 125I-metoprolol was studied in order to determine
the suitable time for injection to avoid the formation of
the undesired products resulting from the radiolysis of
the labeled compound. These undesired radioactive
products can be accumulated in nontarget organs.
Below are data on the stability of 125I-metoprolol. They
show that 125I-metoprolol is stable for up to 72 h.
Fig. 5. Radiochemical yield of125I-metoprolol as a function
of the reaction time [10 μl of Na125I solution (3.7 MBq) +
200 μl of metoprolol (1 mM) + 200 μl of CAT (0.5 mM) +
100 μl of 0.5 M buffer (pH 7)]. Room temperature.
vivo biodistribution studies carried out in normal Al-
bino Swiss mice at different time intervals after ad-
ministration of 125I-metoprolol are given in the table.
The blood uptake was 15.7% 15 min after the injec-
tion, and it decreased to 4.2% at 3 h, indicating that the
labeled compound cleared from the systematic circula-
tion within 3 h after administration. The heart tissue
uptake was high, as seen from the table, and hence 125I-
metoprolol can be used safely as myocardial imaging
agent. The uptake of the tracer by the liver was 8%
15 min post injection and decreased to 4.6% in 3 h,
because metoprolol is extensively metabolized in the
liver. The renal uptake is higher than that the hepatic
uptake, which indicates that the labeled compound was
excreted mainly through the urinary system. In 3 h af-
ter the injection, the activity held by the collected urine
increased to 36.4% due to the normal excretion of
metoprolol. The thyroid uptake mostly ranged within
0.5–2% in 3 h, indicating that 125I-metoprolol did not
generate free radioiodine and was stable in vivo.
Time, h
Radiochemical purity, %
95.2 ± 2.0
1
4
94.0 ± 2.1
12
24
48
72
93.8 ± 1.5
94.2 ± 1.3
94.5 ± 1.9
93.9 ± 2.2
Isolation of 125I-metoprolol. As seen from Fig. 1,
125I-metoprolol is separated from metoprolol by HPLC
on semipreparative scale incompletely. The peaks
overlap (retention time of metoprolol and 125I-meto-
prolol 8 and 10 min, respectively). The fractions were
collected and evaporated under reduced pressure, dis-
solved in saline solution, and sterilized by Millipore fil-
ter (0.22 μm) under aseptic conditions for injection [23].
Biodistribution in normal mice. The results of in
Adopted procedure. To a lyophilized portion of
RADIOCHEMISTRY Vol. 52 No. 2 2010

216
IBRAHIM et al.
Biodistribution of [125I] iodometoprolol in normal Albino
Swiss mice (%ID/g in indicated time after intravenous ad-
ministration)
3. El-Wetery, A.S., El-Azony, Kh.M., and Raieh, M.,
J. Label. Comp. Radiopharm., 1997, vol. 39, p. 987.
4. Hanson, J., Reynaud, D., Qiao, N., et al., J. Med. Chem.,
2006, vol. 49, p. 3701.
Organ
15 min
1 h
3 h
5. Gungor, T., Chen, Y., Golla, R., et al., J. Med. Chem.,
Blood
Bones
Muscles
Liver
Intestines
Stomach
Lungs
Spleen
Heart
15.7 ± 0.8
1.5 ± 0.1
1.3 ± 0.1
8.0 ± 0.4
6.0 ± 0.3
7.1 ± 0.4
6.7 ± 0.3 4.2 ± 0.2
2.8 ± 0.1 2.6 ± 0.1
4.2 ± 0.2 2.1 ± 0.1
6.5 ± 0.3 4.6 ± 0.2
8.0 ± 0.4 5.1 ± 0.3
7.0 ± 0.3 4.6 ± 0.2
2006, vol. 49, p. 2440.
6. Osipo, C., Meeke, K., Liu, H., et al., Cancer Res., 2005,
vol. 65, p. 8504.
7. Shi, Z., Zhang, J., and Zheng, J., J. Zhejiang Univ. Sci.
B, 2007, p. 8.
8. Amin, A.M., El-Azony, K.M., and Ibrahim, I.T.,
J. Label. Comp. Radiopharm., 2009, vol. 52, pp. 467–472.
9.1 ± 0.5 10.1 ± 0.5 9.8 ± 0.5
2.3 ± 0.1 3.5 ± 0.2 4.5 ± 0.2
12.2 ± 0.6 19.0 ± 1.0 12.5 ± 0.6
0.5 ± 0.03 2.0 ± 0.1 1.5 ± 0.1
10.0 ± 0.5 13.0 ± 0.7 11.7 ± 0.6
20.0 ± 1.0 25.0 ± 1.3 36.4 ± 1.8
9. Knust, E.J., Dutschka, K., and Machulla, H.J., J. Radio-
Thyroid
Kidneys
Urine
anal. Nucl. Chem. Lett., 1990, vol. 144, p. 107.
10. El-Shaboury, G. and El-Tawoosy, M.M., J. Radioanal.
Nucl. Chem., 2002, vol. 252, p. 109.
11. El-Shaboury, G., Farah, K., and El-Tawoosy, M., J. Ra-
Na125I (9.25 MBq) in a V-shaped reaction vial, we
added 200 μl of a metoprolol solution in absolute etha-
nol (1 mM) and then exactly 200 μl of CAT in water
(0.5 mM) and 100 μl of 0.5 M phosphate buffer at
pH 7. The reaction vial was left at ambient temperature
(25 ± 1°C) for 10 min. The aqueous–ethanol solution
was purified by passing it through filter paper prelimi-
narily impregnated with AgCl and then was subjected
to sterilization by filtration through a conjugated bacte-
rial filter (0.22 μm Millipore), after which it was ana-
lyzed as described above.
dioanal. Nucl. Chem., 2001, vol. 249, p. 535.
12. Slabert, J.P., Langerhoven, J.H., and Smit, B.S., J. Ra-
dioanal. Nucl. Chem., 1999, vol. 240, p. 505.
13. Attallah, Kh.M., MSc. Thesis, Pharmaceutics Depart-
ment, Faculty of Pharmacy, Zagazig Univ., 2002.
14. Fraker, P.J. and Speck, J.C., J. Biochem. Biophys. Res.
Commun., 1978, vol. 80, p. 849.
15. Danforth, E. and Himms-Hagen, J., Eur. J. Endocrinol.,
1997, vol. 136, p. 362.
16. Petzold, G. and Coenen, H.H., J. Label. Comp. Radio-
pharm., 1981, vol. 18, p. 139.
17. Saccavini, J.C. and Bruneau, C., IAEA-CN-4519, 1984,
Thus, the procedure we developed allows incorpo-
ration of radioiodine radionuclides into the ring of the
metoprolol molecule at ambient temperature (25 ± 1°C)
in reasonable time (up to 10 min). Metoprolol is taken
in a twofold excess relative to the oxidant (CAT).
125I-Metoprolol is of interest for nuclear medicine as
myocardial imaging agent, due to the high heart uptake
within short time (1 h).
p. 153.
18. Rudinger, J., Discussion in Radioimmunoassay Meth-
ods, Kikham, K.E. and Hunger, W.M., Eds., Edinburgh:
Churchill Livingstone, 1971, p. 104.
19. El-Azony, K.M., El-Mohty, A.A., Killa, H.M., et al.,
J. Label. Comp. Radiopharm., 2008, vol. 52, p. 1.
20. El-Ghany, E.A., Amine, A.M., El-Sayed, A.S., et al.,
J. Radioanal. Nucl. Chem., 2005, vol. 266, p. 117.
21. Baerer, C.F., Knapp, A.J., Kaumann, L.S., et al., J. Mol.
Pharmacol., 1979, vol. 12, p. 328.
REFERENCES
22. El-Shaboury, G., El-Kolaly, M.T., El-Wetery, A., et al.,
in Proc. 6th Conf. on Nuclear Sciences and Appplicai-
tons, Cairo (Egypt), March 15–20, 1996, vol. III, p. 181.
1. Nijsen, M.J.M.A., Croiset, G., Stam, R., et al., Neuro-
psychopharmacology, 2000, vol. 22, p. 388.
2. Pandalai, P.K., Bulcao, C.F., Merrill, W.H., and Akh-
ter, S.A., J. Thorac. Cardiovasc. Surg., 2006, vol. 131,
p. 975.
23. Verbruggen, R.F., Int. J. Appl. Radiat. Isot., 1986,
vol. 37, p. 1249.
RADIOCHEMISTRY Vol. 52 No. 2 2010