J Radioanal Nucl Chem

DOI 10.1007/s10967-014-3743-4

Synthesis, characterization and biological evaluation

of 99mTc-labeled Mitomycin C
Tanveer Hussain Bokhari Muhammad Usman Akbar
Samina Roohi Saira Hina Muhammad Sohaib
Faheem Askari Rizvi

Received: 21 May 2014

Akademiai Kiado, Budapest, Hungary 2014

Abstract Mitomycin-C is used for the treatment of different types of tumours. In present work, a reliable method
was developed for radiolabeling of Mitomycin-C with
99m
Tc for diagnostic purpose. 99mTc-Mitomycin-C was
obtained with radiochemical yield of 100 % by adding
200 lg Mitomycin-C, 1 mL (15 mCi) of pertechnetate in
25 lg SnCl22H2O at pH 7. Labeling efficiency was
determined by paper chromatography and ITLC. The
charge on 99mTc-Mitomycin-C was determined by electrophoresis technique. HPLC analyses were performed for
the determination of purity of Mitomycin-C and radiochemical purity of labeled complex. Evaluation of 99mTcMitomycin-C, in vitro stability, biodistribution and scintigraphic images in normal mice were performed.
Keywords 99mTcO-4  Mitomycin C  Quality control 
HPLC  Electrophoresis  Biodistribution and scintigraphy

T. H. Bokhari (&)  M. U. Akbar  F. A. Rizvi

Department of Chemistry, Government College University,
Faisalabad, Faisalabad 38000, Pakistan
e-mail: tanveerhussain@gcuf.edu.pk
S. Roohi
Isotope Production Division, Pakistan Institute of Nuclear
Science and Technology, P.O. Nilore, Islamabad, Pakistan
S. Hina
Department of Bioinformatics and Biotechnology, Government
College University, Faisalabad, Faisalabad, Pakistan
M. Sohaib
Pakistan Institute of Engineering and Applied Sciences,
P.O. Nilore, Islamabad, Pakistan

Introduction
99m

Tc is the most important and frequently used radionuclide in the diagnostic field of nuclear medicine [13].
99m
Tc combine with a wide variety of ligands and produce
a variety of complexes which is the main benefit of 99mTc
for the development of new radiopharmaceuticals [47].
Today radiopharmaceutical therapy or imaging is the
fastest growing field of nuclear medicine [6, 8]. Radiopharmaceuticals have been widely used in many clinical
and non-clinical applications, such as in vivo and noninvasive diagnosis or treatment of human diseases [911].
So, a new radiopharmaceutical is designed for the diagnose
purpose. Mitomycin C is an important antitumor drug
originally isolated from the gram negative bacteria Streptomyces caespitosus [12]. It is routinely used as a chemotherapeutic agent in the treatment of several types of cancer
[1316]. In this research work, labelling of Mitomycin C
was performed and examined the factors that affecting the
radiochemical yield of 99mTc-Mitomycin C such as MMC
concentration, pH, reaction rate, amount of SnCl22H2O
and evaluates the stability, quality control and characterization of labelled compound with chromatographic
methods. We also studied biodistribution and scintigraphy
of 99mTc-Mitomycin in normal mice.

Experimental
Materials and methods
Mitomycin C injection was purchased from medicine
market Faisalabad Pakistan. 99mTc was obtained from a
locally produced fission based PAKGEN 99Mo/99mTc
generator. All chemicals used were AR grade.

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J Radioanal Nucl Chem

Synthesis of

99m

Tc-labeled Mitomycin C

Effect of SnCl22H2O

For the synthesis of 99mTc-labeled Mitomycin C, the

amount of Mitomycin C was 200 lg, while 2025 lg of
SnCl22H2O was used as reducing agent and pH 7 was
adjusted by using 0.5 M NaOH and 0.5 M HCl. Reaction
mixture volume used in all experiments was 1.5 0.2 mL.
After addition of all reagents *15 mCi 99mTcO-4 in saline
was injected into the vial and incubated for 15 min at room
temperature. All experiments were carried out at room
temperature of 25 2 C and in darkness due to the
photosensitive nature of ligand.

To set the optimum amount of reducing agent (SnCl2

2H2O) and to see its effect on labeling, it was used in the
range of 1040 lg (10, 15, 25, 30 and 40 lg) while the
other factors were kept constant. Sn oxidation is necessary
part to convert the 99mTc into low oxidation state which is
necessary to combine the stable complex with MMC. The
amount of SnCl22H2O that produced the maximum
labelling yield was considered as optimum concentration.

Quality control

To set the optimum amount of ligand (Mitomycin C) and to

see the effect of different amount of Mitomycin C on
labeling, it was used in the range of 50300 lg (50, 100,
150, 175, 200, 250 and 300 lg).

Radiochemical yield of 99mTc-labeled Mitomycin C was

checked by ascending paper chromatography and ITLC.
Free 99mTcO-4 in the preparation was determined by
using Whatman No. 3 paper as the stationary phase and
acetone as mobile phase. Reduced and hydrolysed
activity was determined by using instant thin layer paper
chromatography (ITLC-SG strips) as the stationary phase
and 0.05 M NaOH as a mobile phase. The results of
paper chromatography shows that reduced/hydrolysed
99m
Tc and labelled 99mTc-MMC remained at the origin
where as free 99mTcO-4 moved toward the solvent front.
The Rf in each case was calculated by using the following formula,
Distance travelled by the sample
:
Rf
Distance travelled by the solvent
Similarly reduced and hydrolysed activity was determined by using instant thin layer paper chromatography
(ITLC-SG strips) as the stationary phase and 0.05 M NaOH
as a mobile phase and Rf value calculated as above formula. Radiocolloids were separated by passing the preparation through 0.22 lm bacteria filter (Millipore Filter
Corp). Activity remaining on the filter and in the solution
was counted by a gamma-counter (Ludlum). The stability
of 99mTc-labeled Mitomycin C was checked for 6 h at
room temperature. The distribution of labeled, free and
hydrolysed on chromatographic strips was measured by a
2p Scanner (Berthold, Germany). Alternatively, the strips
were cut into 1 cm segments and counted by a gammacounter.

Effect of MMC amount

Radiolabelling
The labeling of Mitomycin C with 99mTc was done at room
temperature and carried out at different intervals of time.
To determine the optimum time, we performed the reaction
for 1, 5, 10, 15, 20, 25 and 30 min while the other factors
were kept constant.
Stability of

99m

Tc-Mitomycin C

To evaluate the stability of the 99mTc-Mitomycin C after

labelling, we studied at different time intervals (0.5, 1, 2, 3,
4, 5 and 6 h) at room temperature. Change in stability of
the radiolabeled complex was analysed at each time
interval by ITLC-SG and PC to determine any dissociation
of the complex. The percentage (%) of free pertechnetate at
a particular time present percentage dissociation of the
complex at that time.
In vitro stability
In vitro stability of the 99mTc-Mitomycin C was also
studied. The 1.8 mL of normal human serum was mixed
with 0.2 mL of 99mTc-Mitomycin C and incubated at
37 C. The 0.2 mL aliquots were withdrawn during the
incubation at different time intervals up to 24 h and subjected to chromatography for determination of 99mTcMMC, reduced/hydrolysed 99mTc and free 99mTcO-4.

Effect of pH
Electrophoresis of
We carried out the labelling reaction at different pH 2, 4, 6,
7, 8, 10, and 11 using appropriate pH solutions. The
labelling yield for each pH value was measured, and the
optimum pH was found.

123

99m

Tc-MMC

The charge on 99mTc-labeled Mitomycin C was studied by

using Deluxe electrophoresis chamber (Gelman) system.
The phosphate buffer of pH 6.0 was used in this experiment.

J Radioanal Nucl Chem

Labelling efficiency %

120
100
80
60
40
20
0
0

10

15

20

25

101

10

12

Fig. 2 Effect of pH on labelling of

100
99
98
97
96
95
94
93
92

pH

99m

Time in Hours

Tc with Mitomycin C
Fig. 6 Stability of

99m

Tc-MMC complex at room temperature

120
100
80
60
40
20
0

10

15

20

25

30

35

40

45

Labeling Efficiency %

Labelling efficiency %

35

Fig. 5 Effect of the incubation time on labelling

110
100
90
80
70
60
50
40
30
20
10
0

30

Incubation Time (Mintues)

Labelling efficiency %

Labelling efficiency %

Fig. 1 Structural formula of Mitomycin C

100

99mTc Mitomycin

90

Free Pertechnetate

80

Colloid\ Hydrolysed

70
60
50
40
30
20
10

Amount of SnCl 2.2H20

Fig. 3 Effect of reducing agent on labelling of

cin C

99m

1 Hour

Tc with Mitomy-

Fig. 7 Stability of

80

Activity % age

Labelling efficiency %

100

60
40
20
0
0

50

100

150

200

250

300

350

Amount of Mitomycin C (g)

Fig. 4 Effect of the amount of ligand on labelling of

Mitomycin C

99m

Tc with

4 Hour

99m

Tc-Mitomycin in normal human serum

110
100
90
80
70
60
50
40
30
20
10
0

99mTc-Mitomycin

-4

-3

-2

-1

cm

Fig. 8 Electrophoresis of the

Whatman No. 1 paper of 30 cm long strip was used. A drop

of 99mTc-Mitomycin mixture was placed at 12 cm far from
the cathode edge of the paper sheet and electrophoresis

24 Hour

Incubation Time( Hours)

99m

Tc-MMC

apparatus was run for 1 h at a voltage of 300 V. After

completion of electrophoresis, the strip was scanned by
using 2p scanner to know the charge on labelled MMC.

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J Radioanal Nucl Chem

HPLC of Mitomycin C
The ligand (Mitomycin C) was analysed on a high performance liquid chromatography (HPLC) instrument to
check the purity of Mitomycin C. HPLC of MMC was
studied by using D-200 Elite HPLC system. The column of
C-18 was used as stationary phase, 20 lL sample volume
was injected and a mixture of water and acetonitrile ratio
85:15 (v/v%) was used as mobile phase in the isocratic
mode. The flow rate was adjusted up to 1 mL/min at a
wavelength of 365 nm. The total run time was 30 min and
temperature of the column was maintained.

Albino mice were used for biodistribution and scintigraphy

purpose having weight ranges 6070 g (48 weeks) and
was obtained from the National Institute of Health (NIH),
Islamabad, Pakistan. They were anesthetized with diazepam injection. 99mTc-labeled Mitomycin C was prepared
and then injected into a tail vein. After injection of 99mTclabeled Mitomycin C (2 mCi/mouse), accumulation of
radioactivity in various organs was determined. The various organs were dissected, weighed and counted for
radioactivity at the various time intervals (1, 4, and 24 h).
Data was expressed as the percentage of injected dose per
gram of tissue (% ID/g).

Biodistribution and scintigraphy in mice

Result and discussion
The animal studies were approved by the animal ethics
committee, Pakistan Institute of Nuclear Science and
Technology (PINSTECH) according to the guidelines set
out by Pakistan atomic energy commission. Male Swiss
Fig. 9 HPLC analysis of
Mitomycin C shows 94.6 %
purity

Fig. 10 HPLC analysis

illustrates the percentage
labeling of mitomycin C with
99m
Tc

123

Mitomycin C is an anti-neoplastic agent obtained from

Streptomyces caespitosus. The chemical structure of
Mitomycin C is presented in Fig. 1. The effects of pH are

J Radioanal Nucl Chem

Activity Accomulation in organ (%)

35

1 Hour

30

4 Hour

25

24 Hour

20
15
10
5
0

Body Organs

Fig. 11 After the injection of

99m

Tc-Mitomycin C at 1, 4, and, 24 h

shown in Fig. 2. At low pH 2, 4 and 6 the minimum

labeling efficiency is 27 3.5, 47 3 and 96 1.5 %
respectively, while at pH 7 the labeling efficiency of
99m
Tc-MMC is 100 %. In basic media at pH 810 the
labeling efficiency is decreased (8610 3.1 %). It means
changing the pH from 7 the labelling yield of 99mTc-MMC
was always decreased. During the studied the effect of pH
on labelling %age of MMC with 99mTc, the other factors
were kept constant. Hence further experiments were performed at pH 7 to obtain the highest radiochemical yield of
99m
Tc-Mitomycin C. To avoid the colloid formation, the
optimum amount of reducing agent was used. The amount
of the reducing agent, SnCl22H2O, which gave the highest
labeling efficiency, was 25 lg (Fig. 3). Below the value of
SnCl22H2O from 25 lg is not sufficient for the complete
reduction of 99mTcO-4. Therefore this amount of SnCl2
2H2O is necessary to form the stable complex of 99mTc
with Mitomycin C. The influence of ligand amount
(Mitomycin C) on the labelling yield 99mTc-Mitomycin C
was shown in Fig. 4. Many experiments were performed
with varying amount of the ligand (MMC), and 200 lg
gave the maximum labeling efficiency of 100 % 1

(Fig. 4). Increase and decrease the amount of Mitomycin

from this, there will be a significant decrease in the
radiochemical yield of 99mTc-labeled Mitomycin C. The
complexation of 99mTc with MMC was not rapid and
maximum labeling efficiency was achieved after 15 min
(Fig. 5). The radiochemical yield was significantly
increased with increasing the reaction time from 1 to
15 min which is shown in Fig. 5. The resulting complex of
99m
Tc-MMC was quite stable and labeling of [93 % was
maintained for up to 6 h (Fig. 6) which was confirmed by
different chromatography technique, no significant dissociation of the complex was observed up to 6 h (Fig. 7). The
99m
Tc-labeled Mitomycin C complex displayed no charge
on paper electrophoresis as shown in Fig. 8. It means, the
prepared complex (99mTc-Mitomycin C) is neutral complex
because the sample drop of 99m Tc-MMC does not moved
towards anode or cathode from the point of origin at the
applied condition. The purity of Mitomycin C and 99mTcMitomycin C were determined by using HPLC as shown in
Figs. 9 and 10. The first peak was observed at retention
time of 12 min which corresponds any impurities (may be
any preservative) present in Mitomycin C and the main
peak was observed at retention time of 15 min which was
the actual peak of Mitomycin C and the this peal told us
that Mitomycin C was 94.6 % pure. No evidence was
found for the presence of significant amount of impurities
in Mitomycin C. 99mTc-Mitomycin C is a neutral radiopharmaceutical that may be used for the diagnosis purpose.
Radiochemical purity of the product was C99 % with a
good stability at room temperature (Fig. 6) and in normal
human serum (Fig. 7). The biodistribution results showed
that the maximum activity was seen in the liver, spleen,
lungs and bladder (Fig. 11). No radioactive accumulation
was found in any specific organ except in these organs as
shown in scintigraphic images (Fig. 12). These results
strongly suggest that 99mTc-MMC can be used for the
medical diagnostic purpose.

Fig. 12 Whole body gamma

camera image of mice injected
with 99mTc-Mitomycin C at 1-h
post administration (a), 4-h post
administration (b), and 24-h
post administration (c)

123

J Radioanal Nucl Chem

Conclusion
Labelling of Mitomycin C with 99mTc by a direct method
was simple, inexpensive and efficient with more than 99 %
radiochemical yield. The electrophoresis analysis confirmed that 99mTc-Mitomycin C complex has no charge.
Based on the data obtained from this study, 99mTc-Mitomycin C has good stability both in isotonic saline and
human serum. The biodistribution and scintigraphic results
showed that 30, 22, 5.89 % ID/g activity at 1, 4 and 24 h
respectively was found in liver which it is metabolized
route. Similarly 8.5, 2.83, 0.44 % in spleen, 4.6, 3.7, 1.09
in lungs and 5.9, 7.4 and 0.07 % were observed in bladder
which suggested that it may be used as an ideal candidate
for diagnostic applications.

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