Evaluation of a 99mTc-labelled meso-bisphenylporphyrin as a tumour image agent

Article abstract of DOI:10.1002/jlcr.3180

Porphyrins are excellent agents for photodynamic treatment of various types of cancer and also good metal chelators that form highly stable metallo-complexes with different radionuclides. Therefore, radiolabelled porphyrins could also be potentially used

Full text of DOI:10.1002/jlcr.3180

Research Article
Received 26 June 2013,
Revised 20 August 2013,
Accepted 10 December 2013
Published online 7 February 2014 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3180
99m
Evaluation of a Tc-labelled meso-
bisphenylporphyrin as a tumour image agent
Pedro M. Santos,a,b† Mafalda Laranjo,a,c,d † Arménio C. Serra,e
*
Ana Margarida Abrantes,a,c,d Marta Piñeiro,d,e João Casalta-Lopes,a,f
Diná Trindade,a Jorge Maia,b António Rocha-Gonsalves,e,d and
Maria Filomena Botelhoa,c,d
Porphyrins are excellent agents for photodynamic treatment of various types of cancer and also good metal chelators that
form highly stable metallo-complexes with different radionuclides. Therefore, radiolabelled porphyrins could also be
potentially used as tumour imaging agents. In this context, the aim of this work was the radiolabelling of meso-bis[3,4-bis
(carboxymethyleneoxy)phenyl]porphyrin, 2CPP, with Technetium-99 m (99mTc) and the evaluation of its radiochemical and
biological properties in vitro and in vivo.
The labelling procedure was optimized resulting in an efficiency of 92.52 0.48%. The complex 99mTC-2CPP remained stable
for more than 4 h. The biodistribution showed that 99mTc-2CPP is eliminated by gastrointestinal and urinary pathways. The
tumour/muscle ratio increases over time, being 3.33 1.22 and 3.55 1.29 in WiDr-bearing tumours mice and in H1299-
bearing tumours mice, respectively, 6 h post-injection, showing the tumour specificity of the 99mTc-2CPP complex.
The favourable tumour/muscle ratio of 99mTc-2CPP shows that this complex could potentially be used as tumour imaging
agent. Moreover, it could be used to follow the progression or regression of tumours before, during and after the
radiotherapy, chemotherapy and photodynamic therapy.
Keywords: Technetium-99 m (99mTc); photodynamic therapy; photosensitizer; lung cancer; colon adenocarcinoma; imaging
the other non-target tissues.3,21 Furthermore, the lipophilicity/
Introduction
hydrophilicity balance is one key factor in the pharmacokinetic
properties.4,22,23 While the tumour accumulation is mostly
To increase the chances of a successful cancer treatment, it is
essential to have an early diagnosis.1 In the last years, many
advances were achieved in the development of new imaging
agents for tumour detection. Several carrier molecules for these
agents have been developed with improved biochemical
properties based on the receptor and metabolic activity.2 A great
effort has been directed to the development of specific
biomarkers for tumour tissue.3
Photodynamic therapy (PDT) is a promising treatment for a
variety of tumours4 with proven results in several types of
cancer.1,4–7 The photosensitizer (PS) activation with visible light
induces the generation of cytotoxic species, such as reactive
singlet oxygen, leading to irreversible damage of the treated
tissues.8–16 Porphyrins are good PS being also excellent metal
chelators that can form highly stable metallo-complexes with
radionuclides.17,18 Moreover, several authors pointed that some
dependent on the lipophilicity of the compounds,3,5,24
hydrophilicity plays an essential role in the clearance of the
agent.3,21 In this context, meso-bis[3,4-bis(carboxymethyleneoxy)
phenyl]porphyrin, 2CPP, was designed for the present work and
aBiophysics Unit, Faculty of Medicine, University of Coimbra, Azinhaga de Santa
Comba, Celas, 3000-548 Coimbra, Portugal
bUniversity of Beira Interior, 6200-001 Covilhã, Portugal
cIBILI, Faculty of Medicine, University of Coimbra, Azinhaga de Santa Comba,
Celas, 3000-548 Coimbra, Portugal
dCIMAGO, Faculty of Medicine, University of Coimbra, Azinhaga de Santa
Comba, Celas, 3000-548 Coimbra, Portugal
porphyrin PS can be accumulated and retained by tumour eChymiotechnon, Department of Chemistry, University of Coimbra, Rua Larga,
tissue.8–10,12,14,15 Thus, radioactive labelled porphyrins could be
3000 Coimbra, Portugal
used as imaging agents useful to optimize PDT protocols as well
as to monitor therapies.18,19 In theory, it is possible to have in the
same compound the ability for imaging and treating the cancer
lesions by PDT.18–20
fRadiation Oncology Department, Coimbra Hospital and Universitary Centre,
Praceta Mota Pinto, 3000-075 Coimbra, Portugal
*Correspondence to: Mafalda Laranjo, Biophysics Unit, IBILI, CIMAGO, Faculty of
Medicine, University of Coimbra, Azinhaga de Santa Comba, Celas, 3000-548
Coimbra, Portugal.
An ideal tumour imaging porphyrin-based agent should
exhibit a large accumulation/retention by tumour tissue in a
E-mail: mafaldalaranjo@fmed.uc.pt
suitable time as well as it should have a high clearance by † These authors contributed equally to this work.
J. Label Compd. Radiopharm 2014, 57 141–147
Copyright © 2014 John Wiley & Sons, Ltd.

P. M. Santos et al.
99mTc was the chosen radionuclide. Porphyrins bearing only two Quality control of 99mTc-2CPP
phenyl groups are structurally similar to natural porphyrins and
Radiochemical purity of 99mTc-2CPP complex was assessed by ascending
instant thin layer chromatography. After several experiments, we achieved
can be a compromise between a good chelating molecules with
an easy body clearance. Technetium-99 m (99mTc) with an energy
peak at 140 keV is ideal for imaging purposes with gamma-
camera25,26 based imaging systems. Moreover, the type of
radiation emitted and its half-life of 6 hours do not deliver
unnecessary radiation to the patients4,27 and because of its low
cost is easily available.4,19,26
This paper reports the process of synthesis of 2CPP compound
and its radiolabelling with 99mTc, as well as, the evaluation of its
radiochemical and biological properties in vitro and in vivo.
the two microchromatographic systems: Whatman Nº1 paper as
®
stationary phase with acetone as mobile phase and Instant Thin Layer
Chromatography-Silica Gel (ITLC-SG) strips (Agilent Technologies) as
stationary phase with a solvent mixture of acetone, water and ammonium
hydroxide (1; 20; 0.8, v/v) as mobile phase. The retention factor (Rf) of the
different 99mTc radioactive species, 99mTc-2CPP, reduced/hydrolysed (R/H)
99mTc and free 99mTc-pertechnetate are shown on Table 1.
Human serum stability
The stability of 99mTc-2CPP complex was studied in normal human serum.
To a sample of 0.5mL of normal human serum, 1.6MBq of 99mTc-2CPP
complex was added, and incubated at 37°C. Aliquots (5μL) of each sample
were taken at 30, 120 and 360 min and were analysed by ascending instant
thin layer chromatography, using the protocol referred previously.
Experimental
Synthesis and characterization of 2CPP
Dipyrromethane was prepared as described.2,16 The 3,4-dicarbomethoxy-
methyleneoxybenzaldehyde were prepared by reaction of 3,4-
dihydroxybenzaldehyde with methylbromoacetate in reflux of acetone28
and in the presence of anhydrous potassium carbonate. Condensation of
dipyrromethane and the aldehyde was carried out in dichloromethane
and trifluroacetic acid.16 Oxidation with 2,3-dichloro-5,6-dicyanobenzo-
quinone (DDQ) and isolation by column chromatography (CH2Cl2/AcOEt
80/20) originate the dimethyl ester of meso bis [(carboxymethyleneoxy)
Determination of partition coefficient
Partition coefficient (log P) of 99mTc-2CPP complex was determined
subsequently to the determination of P, wherein P is the ratio between
the activity of organic phase and activity of aqueous phase. Therefore,
a sample of 99mTc-2CPP complex with 15.0 2.1 MBq was added to a
mixture of 1 mL of 1-octanol and 1 mL of saline, stirred in vortex during
1 min and left to stand for 5 min. Following centrifugation at 1.2 × 103 g
for 5 min, equal aliquots (40 μL) of each phase were taken and its activity
measured, in counts per min (CPM) using a well-counter (Berthold DPC,
Gamma-C12 multi crystal gamma counter, Berthold, Germany).
1
phenyl]porphyrin in 27% yield. H NMR (300 MHz,CDCl3): δ À3.14 (s, 2H,
NH), 3.70–4.00 (m, 12H, OCH3), 4.92–5.05 (m, 8H, OCH2), 7.29 (m, 2H,
Ph), 7.84 (m, 4H, Ph), 9.09 (d, J = 3.3Hz, 4H, β-H), 9.39 (d, J = 3.3Hz, 4H, β-
H), 10.29 (s, 2H, meso-H), UV–vis (CH2Cl2), λmax in nm (relative intensity);
410 (1), 504 (0.04), 539 (0.01), 576 (0.01), 631 (4.7 × 10À3); ESI
(M+ + Na) = 837.
Cell lines and culture conditions
The hydrolysis of the diester prepared previously was carried out
as followed. The diester (100 mg) is dissolved in 15 mL of THF, and
0.5 mL of a solution of 120 mg of potassium hydroxide in water is
added. The mixture stirred overnight protected from light.
Neutralization with acetic acid and evaporation of the solvent give
a red solid which is washed with MEOH. The material is recrystallized
from aqueous acetone giving 80 mg (yield: 88%). 1H NMR (300 MHz,
D2O/DMSO): δ 4.55 (m, 8H, OCH2), 7.27 (d, 2H, J = 6Hz, Ph), 7.71–
7.62 (m, 4H, Ph), 9.15 (d, J = 3.6Hz, 4H, β-H), 9.58 (d, J= 3.6Hz, 4H,
β-H), 10.53 (s, 2H, meso-H), UV–vis (D2O), λmax in nm; 406, 509, 545,
626; ESI (M+) = 758.
Human cell lines of colorectal adenocarcinoma (WiDr) and non-small cell
lung cancer (H1299) were obtained from American Type Culture Collection
(ATCC) and passaged less than 20 times to perform all studies. The cell
lines were maintained in accordance with ATCC recommendations in
DMEM medium (Sigma) supplemented with 10% FBS (Gibco) at 37°C in
a wet atmosphere of 95% air and 5% CO2. For all studies, cells were
detached using a solution of 0.25% trypsin-EDTA (Gibco).
Photocytotoxicity studies
WiDr and H1299 cells were incubated with several concentrations of
2CPP ranging from 1 to 200 μM. After 24 h incubation, medium was
substituted by PS-free medium, and cultures were irradiated with a flux
of 7.5 mW/cm2 until achieving 10 J, using a light source equipped with
a red filter (λ cut off 560 nm). The evaluation of metabolic activity was
evaluated at 24, 48 and 72 h after irradiation through the MTT assay.29
Briefly, cell cultures were exposed to a solution of 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (0.5mg/mL, Sigma) in phosphate
buffered saline, pH 7.4, in the dark at 37°C for 4 h. Afterwards, a 0.04M
solution of hydrochloric acid in isopropanol was added to solubilise
formazan crystals formed, and the absorbance was measured using an
Radiolabelling of 2CPP with 99mTc
The kit formulation was composed of a solution of 0.4 mg of 2CPP
dissolved in 0.2 mL of water for injection at pH 7.0. Immediately prior
labelling, 15 μg of stannous chloride dihydrate (Sigma 208035) in 15 μL
of 0.1 N HCl was added. Thereafter, 0.5 mL of 99mTcO4Na with an activity
of 162.95 2.83 MBq was added to solution, mixed thoroughly for 10 s
using a vortex and incubated at room temperature (RT) for 20 min in
order to achieve the optimum labelling yield.
To achieve the procedure described previously, several parameters
were tested: pH (5.5–7.5), reducing agent mass (10–60 μg), ligand mass
SLT-Spectra spectrophotometer (Biotek Synergy HT). Cytotoxicity was
®
(0.1–2.0 mg), incubation temperature (RT, 23 3, to 100°C) and expressed as the percentage of inhibition of cultures exposed to PDT
radionuclide activity (37–300 MBq). To evaluate the influence of each
parameter in the labelling efficiency, the others were kept constant.
normalized to untreated cultures. This procedure allowed establishing
dose-response curves.
Table 1. Retention factor (Rf) value of different 99mTc species determined by ascending instant thin layer microchromatography
Stationary phase
Mobile phase
Rf (Free 99mTc)
Rf (R/H 99mTc)
Rf (99mTc-2CPP)
Whatman No
ITLC-SG
1
Acetone
Acetone; water; ammonium hydroxide (1;20;0.8, v/v)
1.0
1.0
0.0
0.0
0.0
1.0
®
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J. Label Compd. Radiopharm 2014, 57 141–147

P. M. Santos et al.
Uptake studies of 99mTc-2CPP
parametric or non-parametric tests. Comparison between cell lines was
performed using one factor ANOVA test (parametric test) or Kruskal–Wallis
test (non-parametric test). Multiple comparisons were performed using
Bonferroni’s correction.
The evaluation of complex stability was performed by comparing the
labelling efficiency at different times using Friedman’s test.
For uptake studies, comparisons between cell lines for each time point
were performed using Student’s t-test (parametric test) or Mann–Whitney
test (non-parametric test). Uptake curves for each cell line were fitted to a
two-parameter exponential function through computer-assisted curve
fitting (OriginPro v.8.0, OriginLab).
The uptake studies of 99mTc-2CPP were performed using suitable
equipment, the LigandTracer Yellow (Ridgeview Instruments AB, Sweden).
®
Cells were plated in Petri dishes and incubated to allow cells adhesion. At
the beginning of each study, 0.925 MBq/mL of 99mTc-2CPP complex was
added. Data analysis was carried out using the homemade software ‘Influx
Studies’ dedicated to the analysis of LigandTracer data files.
Complementarily, uptake studies based on 2CPP fluorescence were
also performed. These studies allow us to evaluate to total concentration
of 2CPP (2CPP compound and 99mTc-2CPP complex) that is internalized
into cells. Cells, 4 × 105, were incubated with 99mTc-2CPP complex
(8.8 mM of 2CPP) for 5, 15, 30, 60, 90 and 120 min. Cells were washed with
cold phosphate buffered saline and lised through MEOH addition. The
resulting suspension was left in the dark, at 4°C for 24 h. After
centrifugation at 200 g, the fluorescence intensity of the supernatant
was determined in a spectrophotometer (Hitachi U-2001, Hitachi, Japan)
using 404 nm excitation wavelength. The intracellular concentration was
determined using a calibration curve obtained from the fluorescence
intensity for different solutions of the PS in MEOH.30
A type I error of 0.05 was considered for all comparisons.
Results and discussion
The 3,4-bis(carboxymethyleneoxy)phenyl group was already
used to complex 99mTc2 or 188Re28, but it is incorporated as parts
of tetrasubstituted porphyrins.2 In this work, we decided to use
the same chelating group but incorporate it into a new
disubstituted porphyrin (2CPP) prepared by synthetic
procedures common for this type of porphyrins.16 The goal is
to have a simple molecule, more similar to the natural
Biodistribution studies
60 mice of 6-8 weeks old male athymic nude mice (Balb/c nu/nu), with porphyrins, aiming for fast clearance while at the same time
18–22 g weight, were purchased from Charles River Laboratories
(Barcelone, Spain). The mice were housed and kept in accordance with
the Institution of Animal Care of the University of Coimbra. The animals
were divided into three groups: group A used to perform biodistribution
and pharmacokinetics studies, group B and C used as animal-model
based in xenografts of colorectal adenocarcinoma (WiDr) and non-small
cell lung cancer (H1299), respectively, in order to evaluate tumour/
muscle (T/M) ratio. Each group of mice was divided into four subgroups
of five animals that were killed at different times after 99mTc-2CPP
complex administration: 30, 120, 240 and 360 min.
maintaining the complexing ability.
The absorbance spectra of the 99mTc-2CPP (data not shown)
are characteristic of an uncomplexed porphyrin (by the central
nitrogen atoms) leading to the conclusion that the binding of
the 99mTc to the 2CPP occurs, as represented in Figure 1, by
the carboxyl moiety, a conclusion already reached by others.2,28
The radiochemical purity of the 99mTc-2CPP complex was
assessed by ascending instant thin layer microchromatography,
a simple, cheap and effective method. The labelling procedure
For biodistribution studies, the animals were anesthetized, according was optimized by varying several conditions. Optimization of
the labelling of 2CPP with 99mTc lead to a procedure where to
to the local animal facility guidelines, using 0.1 mL of a solution of
ketamine (77%) and chlorpromazine (23%) administered subcutaneously.
0.2 mL of 2CPP 2 mg/mL, pH 7, are added to 15 μL of stannous
After that, each animal of group A was injected in the tail vein with
chloride dihydrate (1 μg/μL) in 0.1 N HCl and 162.95 2.83 MBq
3.7 0.74 MBq of 99mTc-2CPP complex after being placed over a low
of 99mTcO4Na in a volume of 0.5 mL at RT. The influence of pH,
energy and high resolution parallel hole collimator of a gamma-camera.
temperature, reducing agent mass in the labelling procedure is
Static images with 256 × 256 matrix were acquired during 2 min, every
30 min after radiopharmaceutical intravenous administration, until
270 min. After image acquisition, mice were killed (by cervical
dislocation) at different times (according to the distribution in subgroups
presented and discussed in the succeeding texts (Figure 2).
The effect of pH on the labelling efficiency of 99mTc-2CPP
complex is shown in Figure 2(A). Solutions of tin (Sn2+) become
insoluble from neutral to alkaline pH values, which may cause
already mentioned) and organ samples were immediately collected to
appropriate plastic tubes that were sealed. All samples were weighed, the formation of colloidal components.32,33 On the other hand,
carboxylic groups bind more strongly to 99mTc in alkaline
counted in a well-counter, and the percentage of the injected activity
per gram (%ID/g) of organ was determined.31 The organs collected were
medium once they are deprotonated. In the case of 2CPP
total spleen, heart, thyroid, and brain, one kidney, one lung, one bone
labelling, the balance of these two parameters proves that the
amount of 99mTc-2CPP complex increases from acidic to pH 7
(tibia) and samples of blood, liver, large intestine and small intestine
and stomach. All organs, except blood, were washed in cold saline and
and begins to decay from this value.
dried with absorbent paper after collection.
As known, when the technetium pertechnetate is obtained
The animals from group B and C were injected with suspensions of
from the 99Mo/99mTc generator in the form of sodium
pertechnetate ion has an oxidation state +7, having low
chemical reactivity. Therefore, it is required to add a reducing
15 × 106 WiDr or H1299 cells, respectively, in the axillary zone. When
tumours reached a volume superior to 200 mm3 the 99mTc-2CPP complex
was administered, and every 30 min, static images were also acquired
during 2 min until 270 min. Afterwards, mice were killed at different time agent in order to reduce the sodium pertechnetate ion.
Stannous chloride dihydrate is the most used,32,34,35 however,
intervals: 30, 120, 240, 360 min and samples of tumour and muscle were
collected, weighed, and their CPM were counted in the well-counter in
it requires some precautions due to the possible oxidation by
order to calculate the T/M ratio.
Statistical analysis
Statistical analysis was performed using IBM SPSS Statistics v.20 software.
Descriptive analysis was reported using mean standard deviation (SD)
for quantitative variables.
For inferential analysis, normal distribution of quantitative variables
was assessed using Shapiro–Wilk test, in order to determine the use of
Figure 1. The inferential structure of 99mTc-2CPP.
J. Label Compd. Radiopharm 2014, 57 141–147
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P. M. Santos et al.
Figure 2. (A) Effect of pH on the labelling efficiency of 99mTc-2CCP complex. (B) Effect of mass of stannous chloride on the labelling efficiency of 99mTc-2CCP. (C) Effect of
incubation temperature on the labelling efficiency of 99mTc-2CCP. The results represent the average standard deviation of three independent experiments.
oxygen35 and hence, it was necessary to fill the vials with argon. evaluated in human serum at 37°C and, as presented in Figure 3(B),
The effect of stannous chloride dihydrate mass, ranging from 10 a high stability of approximately 86.5 2.8% was reached and
to 60 μg at pH 7.0, on the labelling efficiency is presented on remained constant over time with no significant alterations
Figure 2(B). Therefore, after testing different masses of stannous (p = 0.846).
chloride dihydrate, the greater efficiencies of labelling were
The possibility of the 2CPP to have a photodynamic outcome
obtained when 15 μg per 0.4 mg of 2CPP were used. From 10 was evaluated. Figure 4 depicts different dose-response profiles
to 15 μg of stannous chloride dihydrate, there is an increase of of H1299 (Figure 4(A)) and WiDr (Figure 4(B)), human cancer cell
approximately 6% in the amount of 99mTc-2CPP complex, and lines to photodynamic treatment with 2CPP measured at 24, 48
beyond this point, the amount of R/H 99mTc is significantly and 72 h after treatment. Considering the concentrations of
increased. The mass of stannous chloride dihydrate is indivi- 2CPP tested, it was not possible to determine the concentration
dually optimized for each radiopharmaceutical formulation, that inhibits the proliferation 50% of cultures (IC50) for both cell
maintaining the balance between an excess of stannous chloride lines. However, there was a reduction in H1299 cell proliferation
dihydrate in respect to the pertechnetate amount and avoiding until 85 7.44% at 24 h, which becomes sharper at 48 h,
the undesirable reduction of sodium pertechnetate.35
77 12.32%, and of 72 9.13% at 72 h. In WiDr cells, the cell
Temperature also influences the labelling efficiency of 2CPP as proliferation decreases up to 54 9.31% at 24 h, however, there
shown in Figure 2(C). The increase of temperature affects the is a recovery through time, reaching 87 6.68% at 48 h and
labelling, mainly, by favouring the formation of reduced/hydrolysed 89 14.99% at 72 h. You et al. (2003)36 compared several PS
forms of 99mTc. Many radiopharmaceuticals require heating to differing in the number of carboxylic acid of side chains and
favour the complexation kinetics, but excessive temperature can showed that the higher number of carboxylic groups is related
lead to the formation of radiochemical impurities.33
with reduced cellular uptake and, subsequently, with lower
After changes of the referred parameters, the optimized proliferation inhibition.36 Thus, carboxylic acid is indispensable
procedure allowed to reach
92.5 0.48% with pH = 7.0, RT.
a
labelling efficiency of to Tc complexation in 2CPP, however, it can have a negative
effect on the photodynamic process as observed by us in other
The stability of the 99mTc-2CPP complex over time was carboxylic substituted porphyrins.37
checked at RT, and the results are presented in Figure 3(A). The
The uptake of 99mTc-2CPP complex increases over time for
complex remains stable for more than 240 min with a decrease both cells lines (Figure 5(B)). The maximum uptake of WiDr and
at 300 and 360 min (t = 20 vs t = 300: p = 0.022; t = 20 vs t = 360: H1299 cells was 2.44 0.60% and 2.63 0.99%, respectively.
p = 0.022). Stability of the 99mTc-2CPP complex was also Additionally, the uptake studies based on 2CPP are represented
Figure 3. Stability over time of 99mTc-2CPP complex: (A) in vitro and (B) in normal human serum. The results represent the average standard deviation of four (A) and
three (B) independent experiments.
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J. Label Compd. Radiopharm 2014, 57 141–147

P. M. Santos et al.
Figure 4. Dose-response profiles of human lung cancer H1299 cell line (A) and human colorectal adenocarcinoma WiDr cell line (B) to photodynamic therapy with 2CPP
compound and 10 J light delivery. The analysis was performed 24, 48 and 72 h after treatment. The results represent the average standard deviation of at least six
independent experiments.
Figure 5. (A) Uptake profile of 2CPP by WiDr and H1299 cell lines obtained by the fluorimetric studies. (B) Uptake profile of 99mTc-2CPP by WiDr and H1299 cell lines
achieved using the LigandTracer Yellow. The results represent the average standard deviation of five independent experiments.
®
in Figure 5(A). In these studies, the fluorescence of the amount of radioactivity accumulated in the lungs which is in
macrocycle is quantified, which is proportional to the accordance with other experimental results.38 A significant
concentration and was achieved through a calibration curve. In accumulation was also observed in the spleen. Accumulation in
these studies, we verified that the uptake by H1299 cell line this organ appears to vary widely with the structure, charge
was of 4.04 0.77%, and it was superior to WiDr cell line, and hydrophilicity of ligand.38,39 Some studies with porphyrins
2.24 0.32%, at 120 min (p = 0.002). In this kind of measurement, also show large amounts of radioactivity in the spleen when
all the porphyrin presented into the cells is quantified. These compared with other organs.3,4
results show that porphyrin structure determines the uptake of
The high accumulation of activity in liver and kidneys is in
the 99mTc-2CPP complex. The uptake values increases over time agreement with similar results obtained for certain
in both cell lines, being higher by H1299 cell line, with a metalloporphyrin,40,41 such as Photofrin25 and Photosan-3.19
statistical significance (p < 0.05) except for time of 15 min.
Thus, 99mTc-2CPP complex appears to be metabolized in liver
Partition coefficient (log P) is a logarithmic measure of the and partially eliminated by the kidneys and gastrointestinal tract.
distribution of a compound between a two-phase 1-octanol/ The in vivo stability of the 99mTc-2CPP complex is evident from
water system. Compounds that exhibit P-values lower than 10 the negligible thyroid and stomach activity, which are the
are soluble in water, while higher P-values superior to 104 are target-organs for free 99mTcO4- .2,42
considered very hydrophobic.4 With
a
log P-value of
1
In the scintigraphic images performed, as presented in
determined at pH = 7, 99mTc-2CPP is
a
very hydrophilic Figure 6, we can observe the greater accumulation in liver and
compound. This high hydrophilicity is due, mostly, to the bladder (urine), which agrees with the biodistribution studies
existence of the four carboxylic acids groups in 2CPP. and confirms elimination by renal via and gastrointestinal tract.
The results of biodistribution studies of 99mTc-2CPP complex In clinical practice, the fact that this compound has
a
carried out at 30, 120, 240 and 360 min in balb/c mice are gastrointestinal elimination can be a disadvantage considering
summarized in Table 2. The low activity present in almost every imaging of colorectal tumours, because the signal/background
organ at 360 min reveals that 99mTc-2CPP complex has a fast ratio can be unfavourable due to this excretion pathway. On
elimination, which might be related to its hydrophilicity. It was the other hand, considering lung tumours, renal and
found that at 30 and 120 min after injection, there was a large gastrointestinal excretion will not interfere on tumour imaging.
J. Label Compd. Radiopharm 2014, 57 141–147
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P. M. Santos et al.
Table 2. Biodistribution of 99mTc-2CPP complex
30 min
120 min
240 min
360 min
Blood
Liver
Spleen
Lungs
5.75 1.61
18.88 3.83
7.91 1.29
9.53 2.75
1.22 0.31
1.18 0.23
2.80 0.94
0.48 0.04
0.05 0.02
1.47 0.32
1.13 0.33
9.11 0.58
2.76 0.64
17.73 3.38
7.30 2.04
6.33 1.76
0.64 0.13
0.90 0.22
1.40 0.25
0.43 0.04
0.04 0.01
1.21 0.33
0.71 0.11
6.33 0.72
1.54 0.32
17.08 1.58
6.62 1.16
3.79 0.95
0.46 0.16
0.47 0.16
1.40 0.27
0.35 0.08
0.03 0.01
0.79 0.11
0.73 0.22
5.44 1.72
1.02 0.44
14.84 4.92
3.01 1.31
1.25 0.18
0.30 0.04
0.34 0.12
0.69 0.20
0.28 0.07
0.02 0.00
0.44 0.15
0.42 0.04
4.40 1.38
Heart
Large intestine
Small intestine
Thyroid
Brain
Bone
Stomach
Kidneys
The values are the percentage of injected activitiy per gram tissue (%ID/g) in normal balb/c mice. The mice were administered
with (3.7 0.74 MBq) of 99mTc-2CPP complex intravenously, and the activity was measured at 30, 120, 240 and 360 min. Each
value expresses the average standard deviation of five mice.
Figure 7. Tumour/muscle ratio for 99mTc-2CPP complex. The results represent the
average standard deviation of five mice.
3.0.2,28 It was observed (Figure 7) that it increases over time for
both tumour types. For 99mTc-2CPP complex, the T/M ratio at
360 min post-injection in WiDr-bearing mice and H1299-bearing
mice was 3.33 1.22 and 3.55 1.29, respectively. The increase of
T/M ratio over time indicates some tumour specificity of the
99mTc-2CPP complex, therefore, this compound can be a
promising agent to tumour imaging.
Conclusion
The developed radioactive labelling and quality control
protocols for 99mTc-2CPP complex are quite accurate allowing a
high labelling efficiency higher than 90%. This complex has a
quick clearance being eliminated by gastrointestinal and by
urinary pathways. Moreover, the 99mTc-2CPP complex showed a
Figure 6. Static image of 99mTc-2CPP complex acquired with the gamma camera
in H1299-bearing mice at 270 min after administration. This figure is available in
colour online at wileyonlinelibrary.com/journal/jlcr
These elimination pathways might, therefore, be a disadvantage high stability in human blood serum over time and in vivo. Both
concerning the monitorization of abdominal tumours.
in vitro and in vivo studies showed a higher uptake by H1299 cell
The selectivity of 99mTc-2CPP complex for tumour cells was line than WiDr cell line. The 2CPP has a lower activity for PDT
evaluated by the analysis of the T/M ratio in the animals of mainly due to a low intake by cancer cells. However, the 99mTc-
groups B and C. An ideal imaging agent should localize in the 2CPP complex due to a high T/M ratio can potentially be used
target tissue with a target to non-target ratio greater than as tumour imaging agent. 99mTc-2CPP complex has a fast
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Copyright © 2014 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2014, 57 141–147

P. M. Santos et al.
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Acknowledgements
Thanks are due to the Nuclear Medicine Department of the
Coimbra Hospital and University Centre for providing the 99mTc
radioisotope. This work was supported by The Foundation of
Science and Technology (SFRH/BD/44957/2008) to Mafalda Laranjo
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Conflict of Interest
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The authors did not report any conflict of interest.
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