Preparation and evaluation of 99mTc-labeled porphyrin complexes prepared using PNP and HYNIC cores: Studying the effects of core selection on pharmacokinetics and tumor uptake in a mouse model

Article abstract of DOI:10.1039/c8md00559a

Porphyrins are tetrapyrrolic macrocyclic ligands known for their affinity towards neoplastic tissues and once radiolabeled with a suitable diagnostic radioisotope could potentially be used for the imaging of tumorous lesions. In the present study, an unsy

Full text of DOI:10.1039/c8md00559a

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Preparation and evaluation of 99mTc-labeled porphyrin complexes prepared using PNP and
HYNIC cores: studying the effects of core selection on pharmacokinetics and tumor uptake
in mouse model
a
a,b
a
a
c
Mohini Guleria, Tapas Das,* Kusum Vats, Jeyachitra Amirdhanayagam, Anupam Mathur,
d
a,b
Haladhar D. Sarma, Ashutosh Dash
aRadiopharmaceuticals Division, Bhabha Atomic Research Centre,
Trombay, Mumbai - 400085, India
bHomi Bhabha National Institute, Anushaktinagar, Mumbai - 400094, India
cRadiopharmaceuticals Program, Board of Radiation and Isotope Technology,
Vashi, Navi Mumbai - 400703, India
dRadiation Biology and Health Sciences Division,
Bhabha Atomic Research Centre, Trombay, Mumbai - 400085, India
#Author for Correspondence
Tapas Das
Radiopharmaceuticals Division
Bhabha Atomic Research Centre
Trombay, Mumbai - 400085, INDIA
91-22-2559 0613
Telephone:
Fax:
91-22-2550 5151
e-mail:
tdas@barc.gov.in
1

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Abstract
Porphyrins are tetrapyrrolic macrocyclic ligands known for their affinity towards
neoplastic tissues and once radiolabeled with a suitable diagnostic radioisotope could potentially
be used for the imaging of the tumorous lesions. In the present study, an unsymmetrically
substituted
porphyrin
derivative
namely
5-(p-amino-propyloxyphenyl)-10,15,20-
tris(carboxymethyleneoxyphenyl)-porphyrin was synthesized and modified further to enable
radiolabeling with 99mTc using two different 99mTc-cores viz. 99mTc-HYNIC (hydrazino nicotinic
acid) and 99mTc(N)PNP2 (PNP2 = Bis-[(2-dimethylphosphino)ethyl]-methoxy-ethylamine) in
order to study the effect of employing different 99mTc-cores on the tumor affinity and
pharmacokinetic behavior of the resultant 99mTc-labeled porphyrin complexes. 99mTc-porphyrin
complexes were characterized by reversed phase HPLC studies and could be prepared with
>
95% radiochemical purity under the optimized radiolabeling conditions. Both the 99mTc-
complexes were found to be adequately stable in human blood serum till 3 h post-preparation.
Bio-distribution studies, carried out in Swiss mice bearing fibrosarcoma tumors, revealed
relatively higher tumor uptake for 99mTc-HYNIC-porphyrin complex (3.95±1.42 and 3.28±0.27
%
IA/g) compared to that exhibited by 99mTc(N)PNP-DTC-porphyrin complex (1.52±0.53 and
1
.56±0.10 % IA/g) at 1.5 and 3 h post-administration, although the former complex exhibited
comparatively lower lipophilicity in octanol-water system. Higher uptake and longer retention in
the blood was observed for 99mTc-HYNIC-porphyrin complex (6.63±0.75 and 4.36±0.25 % IA/g)
compared to that exhibited by 99mTc(N)PNP-DTC-porphyrin complex (2.41±0.54 and 2.30±0.16
%
IA/g) at both 1.5 and 3 h post-administration. However relatively lesser liver uptake was
observed for the former complex (19.26±3.48 and 18.45±1.05 % IA/g) than that exhibited by the
latter one (39.37±3.88 and 34.15±8.25 % IA/g) at both 1.5 and 3 h post-administration. The
2

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study indicates that the in-vivo behavior exhibited by the 99mTc-labeled porphyrins not only
depend on their lipophilicity/hydrophilicity but also governed by the Tc-cores employed for the
radiolabeling.
Keywords: Porphyrin, PNP2, HYNIC, 99mTc, 99mTc-cores, Pharmacokinetics
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Introduction
Over the last several years, porphyrin and its derivatives have attracted considerable
attention and exhibited great promise for developing agents suitable for tumor diagnosis and
targeted tumor therapy.1-6 The growing interest in the use of porphyrin and its derivatives has
been primarily due to their low toxicity, biocompatibility, water solubility, ability to form both
thermodynamically and kinetically stable metal complexes, and intrinsic specificity for tumours,
7
both with and without the presence of a coordinate metal ion in the central core. The utility of
porphyrin derivatives has already been approved by the US-FDA (Food and Drug
Administration of United States of America) for carrying out photodynamic therapy (PDT), a
therapeutic modality used for the treatment of certain types of tumors, accessible either
superficially or endoscopically.8-13 While PDT is an established procedure and has made
significant inroads and undergone phenomenal expansion and growth, it has its own shares of
limitations which include inability to treat or image deep-seated tumors, masking of the tumors
due to the internal haemorrhage, low sensitivity, low repeatability due to fluorescence
quenching, inability to record photographically the fluorescence observed endoscopically.8-14
A
promising approach to circumvent some of these disadvantages is to incorporate a suitable
radionuclide in the porphyrin moiety and using the radiolabeled porphyrin for either tumor
diagnosis or targeted tumor therapy.15,16
Although a plethora of studies have been conducted for developing suitable radiolabeled
porphyrin derivatives, none of these agents has reached the pinnacle of regular clinical
exploitation. In this context, studying the effect of structural modifications of radiolabeled
porphyrins on the tumor affinity and pharmacokinetic behavior still constitutes an active field of
research in radiopharmaceutical sciences.
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The remarkable prospects associated with the use of radiolabeled porphyrin derivatives
for tumor imaging led to a considerable amount of fascinating research and development of
innovative strategies for radiolabeling a variety of porphyrin derivatives with a range of
diagnostic radionuclides, including 18F, 68Ga, 99mTc, and 67Ga.17-21 Among the diagnostic
radionuclides used for radiolabeling, the possibility of using 99mTc seemed enticing due to its
favorable nuclear decay characteristics, well established polyvalent redox chemistry, ready cost-
effective accessibility through 99Mo/99mTc radionuclide generators and widespread clinical
utility.22-24 Additionally, availability of several novel 99mTc synthons as precursors including
[99m
Tc(CO) (H O) ] , [99mTc(N)]2+, [99mTc(N)PNP]2+ (Bis-[(2-dimethylphosphino)ethyl]-
+
3
2
3
methoxyethylamine) and 99mTc-HYNIC (hydrazinonicotinamide) offer the scope to fine tune the
chemical properties of the radiolabeled species.25,26 Each of these complexes, although having
the same set of radioisotope and carrier moiety (but having different 99mTc-cores) has different
chemical properties owing to the difference in their size, hydrophilicity/lipophilicity, charge etc.
2
2
and therefore, expected to have different tumor affinity and pharmacokinetic behavior. Thus
99mTc-labeling employing different cores not only provide the unique flexibility of maneuvering
the chemical properties of the resultant 99mTc-complexes, but also enable to tune their biological
behavior. Towards this, in the present study, an attempt has been made to radiolabel an in-house
synthesized porphyrin derivative namely, 5-(p-aminopropyleneoxyphenyl)-10,15,20-tris-(p-
carboethoxymethyleneoxyphenyl)-porphyrin with 99mTc using two different cores and study the
effects of using two different kinds of 99mTc-cores on the tumor uptake and pharmacokinetic
behavior in animal model. Herein, we describe synthesis of the modified porphrin derivatives,
99mTc-labeling of the porphyrin derivatives using 99m
Tc-HYNIC as well as 99mTc(N)PNP core
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and biological behavior of the 99mTc-labeled porphyrins, formulated using the two different
cores, in the small tumor bearing animal model.
6

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Experimental
Materials and methods
Pyrrole, 4-hydroxybenzaldehyde, ethylbromoacetate, 3-(Boc-amino)propylbromide and
trifluoroacetic acid used for the synthesis of the porphyrin derivative were purchased from
Aldrich Chemical Company (USA). Propionic acid, nitrobenzene and anhydrous potassium
carbonate were obtained from S.D. Fine Chemicals (India). Column chromatography was
performed with silica gel (60-120 mesh size) obtained from Merck (India). Analytical thin-layer
chromatography (TLC) was performed with silica gel plates (Silica Gel 60 F ), procured from
2
54
Merck (India). Whatman 3 MM cellulose chromatography paper, procured from Aldrich
Chemical Company (USA), was used for the paper chromatography (PC) studies. All other
chemicals used were purchased from reputed local manufacturers and were of analytical grade.
Fourier Transform Infra-Red (FT-IR) spectra were recorded in a JASCO FT/IR-420
1
spectrophotometer (Japan). Proton nuclear magnetic resonance ( H-NMR) spectra were acquired
using a 300 MHz Varian VXR 300s spectrometer (USA). Mass spectra were recorded using
Varian 410 Prostar Binary LC mass spectrometer (USA) employing electron spray ionization
(ESI) technique. All radioactive countings, except the coutings related to the biodistribution
studies, were done by using a well-type NaI(Tl) scintillation counter, procured from Electronic
Corporation of India Limited (India), setting the baseline at 100 keV and using a window of 100
keV, so as to utilize the 140 keV gamma photon emission of 99mTc.
The high performance liquid chromatography (HPLC) system (PU 1580) used for the
present study was obtained from JASCO (Japan). A C-18 reverse phase HiQSil (250×4 mmol)
column was used and the elution profile was monitored by detecting the radioactivity signal
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using a NaI(Tl) detector coupled with the HPLC system. All the solvents used for HPLC
analyses were of HPLC grade and degassed as well as filtered prior to use.
All experiments related to human subjects were performed in accordance with the
Guidelines of Institutional Medical Ethics Committee (IMEC) of Bhabha Atomic Research
Centre (BARC) and these experiments were approved by the IMEC, BARC. Informed consents
were obtained from all the human participants involved in this study.
Swiss mice (6-8 weeks age) bearing fibrosarcoma tumors were used as animal models for
the biological studies. All the animals used for the present study were bred and reared in the
laboratory animal house facility of our Institute following the standard management practice.
Radioactive countings associated with the animal studies were performed using a flat-type
NaI(Tl) scintillation detector, procured from Electronic Corporation of India Limited (India)
employing the same counting set-up mentioned earlier. Animal studies reported in the present
article were approved by the Institutional Animal Ethics Committee (IAEC) of BARC and all the
animal experiments were carried out in strict compliance with the institutional (IAEC, BARC)
guidelines following the relevant national laws (Prevention of Cruelty to Animals Act, 1960)
related to the conduct of animal experimentation.
Synthesis of modified porphyrin derivatives
Synthesis of 4-carboethoxymethyleneoxybenzaldehyde (i)
4
-carboethoxymethyleneoxybenzaldehyde was synthesized by following the procedure
2
7
reported in the literature. The crude product was purified by silica gel column chromatography
using CHCl as eluting solvent (R =0.5) whereby pure 4-carboethoxymethyleneoxybenzaldehyde
3
f
(i) was obtained as a colorless viscous liquid (5.7 g, yield 70%).
8

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-1
1
FT-IR (neat, ν cm ) = 1754 (>C=O). H-NMR (CDCl δ ppm): 1.24 (t, 3H, J = 7.8 Hz, -
3
,
COOCH CH ), 4.20 (q, 2H, J = 7.8 Hz, -COOCH CH ), 4.75 (s, 2H, -OCH ), 6.97 (d, 2H, J =
2
3
2
3
2
1
3
7
.2 Hz, ArH), 7.85 (d, 2H, J = 7.1 Hz, ArH), 9.90 (s, 1H, >CHO). C-NMR ([D ]DMSO, δ
6
ppm): 14.02 (CH -CH ), 60.84 (O-CH -CH ), 64.75 (O-CH -C=O), 115.06 (m-phenyl), 130.16
2
3
2
3
2
(p-phenyl), 131.72 (o-phenyl), 162.48 (p-O-phenyl), 168.21 (O=C-O), 191.36 (O=CH-). ESI-MS
(
m/z): (calc.) C H O : 208.21; (obs.) [M+H] 209.15
1
1
12
4
5
,10,15-tris-(p-carboethoxymethyleneoxyphenyl)-20-(p-hydroxyphenyl)porphyrin (ii)
Pyrrole (1.50 g, 22.4 mmol) was added to the refluxing mixture of 4-
carboethoxymethyleneoxybenzaldehyde (i) (3.5 g, 16.8 mmol), 4-hydroxybenzadehyde (0.68 g,
.6 mmol), propionic acid (25 mL) and nitrobenzene (8 mL) in a drop-wise manner and the
5
2
8
refluxing was continued for one hour after the addition of pyrrole was completed. The resultant
reaction mixture was allowed to cool overnight at 4 °C. Subsequently nitrobenzene and
propionic acid were removed from the reaction mixture by distillation under reduced pressure
which gave the crude product. The crude product was purified by silica gel column
chromatography using 0.2% methanol in chloroform as eluting solvent whereby 122 mg of pure
5
,10,15-tris-(p-carboethoxymethyleneoxyphenyl)-20-(p-hydroxyphenyl)porphyrin
(ii)
was
obtained (yield 4%).
−
1
UV-Vis (λmax, nm): 418, 515, 552, 594, 650. FT-IR (KBr, ν cm ): 3210-3080 (-OH), 1754
1
(
>C=O). H-NMR (CDCl , δ ppm): -2.75 (s, 2H, >NH), 1.40 (t, 9H, J = 6.0 Hz, -COOCH CH ),
3
2
3
4
.45 (q, 6H, J = 6.0 Hz, -COOCH CH ), 4.90 (s, 6H, -OCH -), 7.17-7.31 (m, 8H, ArH), 8.03-
2 3 2
8
.14 (m, 8H, ArH), 8.85 (8H, s, pyrrole). ESI-MS (m/z): (calc.) C H O N 936.34; (obs.)
5
6
48 10
4
[M+H] 937.25
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5
-(p-Boc-aminopropyleneoxyphenyl)-10,15,20-tris-(p-carboethoxymethyleneoxyphenyl)-
porphyrin (iii)
N-Boc-3-bromopropylamine (48 mg, 0.20 mmol) was added to a solution of 5,10,15-tris-
p-carboethoxymethyleneoxyphenyl)-20-(p-hydroxyphenyl)porphyrin (ii) (120 mg, 0.13 mmol),
and anhydrous K CO (28 mg, 0.20 mmol) in dry acetone and was kept under refluxing
(
2
3
conditions for 8 h. The progress of the reaction was monitored by TLC using 0.1% of methanol
in chloroform as eluting solvent. After completion of the reaction, solvent was evaporated and
the residue was dissolved in chloroform. The chloroform layer was washed with brine and
subsequently evaporated in a rotary evaporator. The crude product (iii) thus obtained was
purified by silica gel column chromatography using 0.05% methanol in chloroform as eluting
solvent which resulted formation of 121 mg (yield 86%) of pure (iii).
−
1
1
FT-IR (KBr, ν cm ): 1754 (>C=O). H-NMR (CDCl , δ ppm): 1.39-1.44 (m, 9H, -
3
COOCH CH ), 1.49 (s, 9H, -C(CH ) ), 2.12-2.13 (m, 2H, -OCH CH CH -), 2.85 (m, 2H, -
2
3
3 3
2
2
2
OCH CH CH -NH-), 4.38-4.45 (m, 2H, -OCH CH CH -), 4.38-4.45 (m, 6H, -COOCH CH ),
2
2
2
2
2
2
2
3
4
.90 (s, 6H, -OCH -), 7.27-7.53 (m, 8H, ArH), 8.11-8.53 (m, 8H, ArH), 8.84-8.94 (m, 8H,
2
pyrrole). ESI-MS (m/z): (calc.) C H O N 1094.2; (obs.) [M] 1094.2
6
4
63 12
5
5
-(p-aminopropyleneoxyphenyl)-10,15,20-tris-(p-carboethoxymethyleneoxyphenyl)-porphyrin
(iv)
5
-(p-aminopropyleneoxyphenyl)-10,15,20-tris-(p-carboethoxymethyleneoxyphenyl)-
porphyrin (iv) was prepared by Boc-deprotection of compound (iii), which was carried out by
stirring a mixture of (iii) (120 mg, 0.11 mmol) with trifluoroacetic acid (2 mL) in dry
1
0

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dichloromethane (2 mL) for a period of 3 h at room temperature. The reaction was subsequently
quenched by the addition of saturated aqueous NaHCO (10 mL) solution. The organic layer was
3
separated, dried and concentrated under vacuum whereby 110 mg of the 5-(p-
aminopropyleneoxyphenyl)-10,15,20-tris-(p-carboethoxymethyleneoxyphenyl)-porphyrin
yield 90 %) was obtained.
(iv)
(
-1
1
FT-IR (KBr, ν cm ): 3310 (-NH ), 1754 (>C=O), 1650(-NH bending). H-NMR (CDCl , δ
2
3
ppm): -2.89 (s, 2H, >NH), 1.28 (t, 9H, J = 7.4 Hz, -COOCH CH ), 2.10 (m, 2H, -OCH CH CH -
2
3
2
2
2
)
, 2.85 (t, 2H, J = 7.8 Hz, -OCH CH CH -), 3.95 (t, 2H, J = 7.4 Hz, -OCH CH CH -), 4.28 (q,
2 2 2 2 2 2
6
8
H, J = 7.4 Hz, -COOCH CH ), 4.80 (s, 6H, -OCH -), 7.18-7.30 (m, 8H, ArH), 8.02-8.10 (m,
2 3 2
H, ArH), 8.90 (m, 8H, pyrrole). ESI-MS (m/z): (calc.) C H O N 994.10; (obs.) (M) 993.8.
5
9
55 10
5
5
,10,15-tris-(p-carboxymethyleneoxyphenyl)-20-(p-dithiocarbamoylpropyleneoxyphenyl)-
porphyrin (v)
5
,10,15-tris-(p-carboxymethyleneoxyphenyl)-20-(p-dithiocarbamoylpropyleneoxyphenyl)
-porphyrin (v) was prepared following a two-step reaction procedure. In the first step, NaOH (2
N, 5 mL) solution was added to the porphyrin derivative (iv) (50 mg, 0.05 mmol) dissolved in
tetrahydrofuran (5 mL) and the reaction mixture was stirred for 48 h at room temperature. After
completion of the reaction, solvent was evaporated and the crude product, thus obtained, was
directly used for the subsequent step. The hydrolyzed product was allowed to react with slight
excess of CS under ice cold conditions and subsequently stirred for 24 h at room temperature.
2
The solvent was evaporated under vacuum after the completion of stirring and the crude product
thus obtained was purified by semi-preparative HPLC employing 0.1% TFA in water (A) and 0.1
%
TFA in acetonitrile (B) as the mobile phase (0-28 min 90% A to 10% A, 28-30 min 10% A,
1
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3
0-32 min 10% A to 90% A). Flow rate of the mobile phase was maintained at 2 mL/min. Post-
purification ~ 25 mg (yield 50%) of compound (v) was obtained.
-1
1
FT-IR (neat, ν cm ): 3436 (-COOH), 1754 (>C=O), 1447 (-C=S). H-NMR (CD OD, δ ppm):
3
2
.12 (m, 2H, -OCH CH CH -), 3.00 (t, 2H, J = 6 Hz, -OCH CH CH -), 3.80-3.91 (m, 2H, -
2 2 2 2 2 2
OCH CH CH -NH-), 4.71 (s, 6H, -OCH -), 7.33-7.39 (m, 8H, ArH), 8.04-8.10 (m, 8H, ArH),
2
2
2
2
8
.87 (m, 8H, pyrrole). ESI-MS (m/z): (calc.) C H O N S 985.0; (obs.) [M-S-2H+Na] 973.4
54 42 10 5 2
Synthesis of HYNIC-Boc (6-Boc-hydrazinopyridine-3-carboxylic acid)
Synthesis of HYNIC-Boc i.e. 6-Boc-hydrazinopyridine-3-carboxylic acid was achieved
by a two-step procedure. In the first step, hydrazine hydrate (8 mL, 80%) was added to 6-
chloronicotinic acid (1 g, 6.35 mmol) and the reaction mixture was stirred at 100 C for 4 h.
After the completion of the reaction, the reaction mixture was allowed to attain the room
temperature and the solution was concentrated under reduced pressure which resulted formation
of a white solid. The solid was subsequently dissolved in water and pH of the solution was
adjusted to ~5.5. The precipitate formed were filtered, washed with ethanol and dried in vacuum
to give 845 mg of 6-hydrazinopyridine-3-carboxylic acid. In the next step, Boc protection of
hydrazine group was carried out by stirring a solution of 6-hydrazinopyridine-3-carboxylic acid
(
200 mg, 1.31 mmol) with triethylamine (0.365 mL, 2.62 mmol) and di-tert-butyl-dicarbonate
285 mg, 1.31 mmol) in DMF (N,N’-dimethyl formamide) for 16 h at room temperature. The
(
reaction mixture was concentrated under reduced pressure to give a brown solid. The crude
product was purified by silica gel column chromatography using ethyl acetate (R = 0.2) as the
f
eluting solvent which resulted formation of pure 6-Boc-hydrazinopyridine-3-carboxylic acid
(230 mg, 69.5% yield).
1
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1
H-NMR (CD OD, δ ppm): 1.42 (s, 9H, -CH ), 6.53 (d, 1H, J = 8.7 Hz, m-ArH), 7.96 (d, 1H, J =
3
3
+
8
2
.7 Hz, p-ArH) 8.57 (s, 1H, o-ArH). ESI-MS (m/z): (calc.) C H N O 253.8; (obs.) [M+Na]
11 15 3 4
75.9
Conjugation
oxyphenyl)-porphyrin (iv) with 6-Boc-hydrazinopyridine-3-carboxylic acid (vi)
Conjugation of 5-(p-aminopropyleneoxyphenyl)-10,15,20-tris-(p-
of
5-(p-aminopropyleneoxyphenyl)-10,15,20-tris-(p-carboethoxymethylene-
carboethoxymethyleneoxyphenyl)-porphyrin (iv) with 6-Boc-hydrazinopyridine-3-carboxylic
acid was carried out by room temperature stirring of a mixture of compound (iv) (40 mg, 0.04
mmol) and 6-Boc-hydrazinopyridine-3-carboxylic acid (11 mg, 0.04) in presence of HATU (1-
[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid
hexafluorophosphate, 0.08 mmol, 30 mg), and diisopropylethylamine (14 μL, 0.08 mmol) in dry
dimethylformamide (3 mL) for 12 h. The crude reaction mixture was purified by preparative
TLC using 7% MeOH in CHCl (R = 0.3) as mobile phase which resulted formation of 34.0 mg
3
f
(yield 70%) of purified (vi).
1
H-NMR (CDCl , δ ppm): 1.27 (s, 9H, -C(CH ) ), 1.46 (t, 9H, J = 6 Hz, -COOCH CH ), 3.80 (t,
3
3 3
2
3
2
H, J = 6Hz, -OCH CH CH -NH-), 4.44-4.54 (m, 2H, -OCH CH CH -NH), 5.00 (s, 6H, -OCH -
2 2 2 2 2 2 2
), 6.76 (s, 1H, ArH), 7.81 (s, 1H, ArH), 7.54-7.58 (m, 8H, ArH), 8.08 (m, 8H, ArH), 8.51-8.56
(
m, 8H, pyrrole). ESI-MS (m/z): (calc.) C H O N 1229.3; (obs.) [M] 1229.6
7
0
68 13
8
Preparation of hydrolyzed product of compound vi (vii)
Compound (vi) (30 mg, 0.02 mmol) was hydrolyzed in acidic medium using TFA
(trifluoro acetic acid) and dichloromethane (1:1 v/v, 3 mL) by room temperature stirring for 3 h
1
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to remove the Boc protection of the hydrazine moiety. The reaction mixture was subsequently
evaporated using rotary evaporator and was directly used for base hydrolysis which was carried
out by room temperature stirring of Boc de-protected product with a solvent mixture of NaOH (2
N) and tetrahydrofuran (1:1 v/v) for 48 h. After the completion of hydrolysis, absolute separation
of the layers was observed with hydrolyzed product being present in the aqueous layer. The
organic layer was decanted and the aqueous reaction mixture was dried under vacuum, which
provided compound 22 mg (yield 90%) of (vii).
1
H-NMR (D O, δ ppm): 3.47-3.66 (m, 2H, -OCH CH CH -NH-), 6.88 (d, J = 10Hz, 2H, ArH),
2
2
2
2
7
.42 (m, 8H, ArH), 7.82 (d, J = 10Hz, 2H, ArH), 7.42 (m, 8H, ArH), 8.86 (m, 8H, ArH), 8.51
(
m, 8H, pyrrole). ESI-MS (m/z): (calc.) C H O N 1045.0; (obs.) [M] 1044.6
5
9
48 11
8
Preparation of [99mTc(N)PNP] core
2+
[99m
Tc(N)PNP] core was prepared by a two-step process following the procedure reported
2+
in the literature.29,30 The first step involved the preparation of [99mTcN] core which was achieved by
2+
adding freshly eluted 99mTcO (1 mL, 12 mCi) to succinic dihydrazide (5 mg) dissolved in ethanol
-
4
(0.25 mL) and incubating the reaction mixture at room temperature for 30 min after the addition of
00 μL of freshly prepared SnCl solution (1 mg/mL). [99mTcN] intermediate thus prepared was
2+
1
2
characterized by PC studies using a combination of two different solvent systems viz. normal saline
and ethanol:chloroform:toluene:0.5 M ammonium acetate (6:3:3:0.5 v/v). In the second step, freshly
prepared [99mTcN] core (0.5 mL, ~5 mCi) was added with an ethanolic solution (0.4 mL) of PNP2
2+
(0.2 mg) and the reaction mixture was incubated at room temperature for 15 min. The
characterization of [99mTc(N)PNP]2+ core was carried out by RP-HPLC using gradient elution
technique employing 0.1% TFA in water (A) and 0.1 % TFA in acetonitrile (B) as the mobile phase
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(0-4 min 95% A, 4-10 min 95% A to 5% A, 10-25 min 5% A to 25 %, 25-30 min 25% A to 95% A,
30-32 min 95% A). Flow rate of the mobile phase was maintained at 1 mL/min.
Radiolabeling of compound (v) using [99mT(N)PNP] core
2+
Compound (v) was labeled with 99mTc using [99mTc(N)PNP] core following a procedure
2+
which involved the refluxing of compound (v) (1 mg, 1 μmol) with freshly synthesized
[99mTc(N)PNP] core (~5 mCi, 0.9 mL) in a boiling water bath for 30 min. [99mTc(N)PNP]2+-
2+
labeled porphyrin complex was characterized by RP-HPLC using the same gradient system
mentioned earlier.
Radiolabeling of compound (vii) with 99mTc
Radiolabeling of compound (vii) with 99mTc was carried out by following the generalized
procedure usually employed for 99mTc labeling of HYNIC-coupled ligands.31,32 Stock solutions
of SnCl (1 mg/mL) and EDDA (ethylenediamine-N,N’-diacetic acid, 50 mg/mL) were prepared.
2
Compound (vii) (0.5 mg, 0.478 μmol) was dissolved in PBS (phosphate buffered saline, 0.2 mL)
and the resulting solution was transferred to a glass vial containing of tricine (20 mg), EDDA (10
9
9m
-
mg) and SnCl (100 μg) followed by the addition of freshly eluted TcO (1 mL, 12 mCi). The
2
4
reaction mixture was subsequently incubated in a boiling water bath for 30 min. Radiolabeling
yield of 99mTc-labeled HYNIC-coupled porphyrin derivative was determined by RP-HPLC using
the protocol mentioned earlier. Attempts were also made to purify the radiolabeled reaction
mixture in order to improve the percentage radiolabeling yield. Purification was carried out using
®
C-18 Sep-pak cartridges by employing a standard protocol. The cartridge was pre-conditioned
by passing 4 mL of ethanol followed by 2 mL of double distilled water prior to the loading of the
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radiolabeled preparation. Free radiometal was eluted using 600 µL of double distilled water and
subsequently radiolabeled porphyrin was eluted out from the column using 1 mL of ethanol.
Ethanol present in this purified preparation was removed by gentle warming and the preparation
was reconstituted with normal saline before using for biological studies.
Determination of partition coefficient (Log Po/w)
The partition coefficients of 99mTc-labeled porphyrin complexes were determined in
octanol-water system by following the protocol mentioned below. An aliquot of the 99mTc-
labeled porphyrin complex (100 µL) was thoroughly mixed with water (900 µL) and octanol (1
mL) using a vortex mixer and subsequently centrifuged at 3000 rpm for 5 min so as to achieve a
clear separation between the two layers. Equal aliquots (100 µL) were withdrawn from both the
layers and the radioactivity associated with each layer was determined using a well-type NaI(Tl)
scintillation detector. Partition coefficients (Log P ) of 99mTc-labeled porphyrin complexes
o/w
were calculated from these data.
In-vitro serum binding and stability studies
In-vitro serum studies were carried out by following the standardized protocol, which is
briefly mentioned below. Aliquot of 99mTc-labeled porphyrin complex (100 µL) was incubated
with human serum (400 µL) at 37 °C up to 3 h. For determining the serum binding, serum
proteins were precipitated by adding equal volume of acetonitrile to it. The mixture was
subsequently centrifuged at 15000 rpm for 4 min and radioactivity associated with the
supernatant and pellet were counted separately using a NaI(Tl) detector. Percentage serum
binding of 99mTc-labeled porphyrin complexes was calculated from the counts observed in both
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the fractions. For determining the serum stability, aliquots (100 µL) were withdrawn at regular
intervals and the serum proteins were precipitated by adding equal volume of acetonitrile to it.
The mixtures were subsequently centrifuged and the supernatants were analyzed by HPLC using
the protocol mentioned above.
Biodistribution studies
Biological studies, involving pharmacokinetic evaluation and tumor specificity of the
99mTc-labeled porphyrin complexes, were carried out by performing biodistribution studies in
Swiss mice bearing fibrosarcoma tumors. Solid tumors were developed in Swiss mice by
6
implantation of about 10 murine fibrosarcoma cells (~ 100 µL), obtained from the National
Center for Cell Sciences (NCCS, India), subcutaneously on the dorsum of each mouse. The
tumors were allowed to grow until they become ~10 mm in diameter; subsequent to which, the
animals were used for the experiments. Each animal, weighing 20-25 g, was intravenously
injected with ~100 µL of the radiolabeled preparation (~100 µCi, 3.7 MBq) through one of the
lateral tail veins. Biological distribution of both the radiotracers was studied for two different
post-administration time points viz. 1.5 h and 3 h and three animals were used for each time
point. The animals were sacrificed immediately after the designated post-administration time
points through CO asphyxia. Blood samples of the animals were withdrawn by cardiac puncture
2
immediately after sacrifice. Subsequently, the organs/tissues were excised, washed with saline,
dried, weighed in a weighing balance and radioactivity associated with each organ/tissue was
measured using a flat-type NaI(Tl) counter. The percent injected activity (%IA) in various
organs, tissue and tumor was calculated from the above data and expressed as %IA per gram
(%IA/g) of organ/tissue.
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Results
Synthesis of modified porphyrin derivatives
A porphyrin derivative, namely 5-(p-aminopropyleneoxyphenyl)-10,15,20-tris-(p-
carboethoxymethyleneoxyphenyl)-porphyrin (iv) was synthesized and further modified [(v) and
(
vii)] in order radiolabel the derivatives using two different 99mTc cores, e.g. PNP and HYNIC
following a multi-step reaction procedure (Scheme 1 and 2). Various intermediate compounds
and the final products were characterized by standard spectroscopic techniques such as, FT-IR
1
and H-NMR spectroscopy as well as by ESI-Mass spectrometry. Synthesis of compound (ii)
involving the formation of a macrocyclic porphyrin ring was the step having lowest yield owing
to the formation of multiple by-products and repeated column chromatographic purification steps
required for obtaining the porphyrin derivative (ii) in its pure form. Formation of the porphyrin
moiety was confirmed by the presence of an intense Soret band (418 nm) followed by four Q-
bands (515, 552, 594, 650 nm) observed in the UV-Vis spectrum, which is known to be a
3
characteristic of the porphyrin derivatives. Further confirmatory evidences in favour of the
1
formation of compound (ii) were obtained from ESI-MS and H-NMR spectroscopy. The
presence of NMR signal corresponding to two highly shielded protons in the negative region (at -
1
2
.75 ppm) of the H-NMR spectrum as well as obtaining the molecular ion peak in the expected
region of the mass spectrum provided strong evidence in favour of formation of porphyrin ring
structure. Unsymmetrical porphyrin derivative thus synthesized was further derivatized in the
peripheral region of the porphyrin ring via an O-alkylation reaction at the phenolic moiety
resulting the incorporation of a Boc-NH-propyl linker which was subsequently subjected to Boc-
deprotection resulting into the formation of the desired porphyrin derivative (iv). This porphyrin
derivative (iv) was further derivatized following two separate reaction procedures to render it
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suitable for radiolabeling with 99mTc using two different stabilizing chelate structures viz. PNP
and HYNIC. The PNP2 (1-methoxy-2-bis(diphenylphosphino)diethylaminoethane) derivative
and HYNIC used in present study were synthesized separately following the reported
protocols.29,33
Preparation of [99mTcN] and [ Tc(N)PNP] core
2+
99m
2+
[99mTcN]2+ core was synthesized following a reported procedure [29,30] and
characterized by PC using a combination of two sets of solvent systems viz.
ethanol:chloroform:toluene:0.5 M ammonium acetate (6:3:3:0.5 v/v) and normal saline. In the
former solvent system, [99mTcN] intermediate was found to remain near the point of application
+2
(
R = 0-0.2); while in the latter solvent system, it moved towards the solvent front (R = 0.7-1).
f
f
These studies showed that [99mTcN]+2 intermediate was obtained with >95% radiochemical
purity. Synthesized [99mTcN]+2 intermediate was subsequently used for preparation of
[99m
Tc(N)PNP]2+ core which was characterized by RP-HPLC.30 In HPLC chromatogram
free/unlabeled [99mTcN] core was found to be eluted at 3.0 min whereas [ Tc(N)PNP] core
exhibited retention time of 15.0 and 17.5 min (obtained as a doublet). RP-HPLC studies showed
that [99mTc(N)PNP] intermediate could be prepared with >95 % radiochemical purity under the
reaction conditions mentioned earlier.
2+
99m
2+
2+
Preparation and characterization of 99mTc-complexes of (v) and (vii)
Radiolabeling of compound (v) with 99mTc was carried out using [99mTc(N)PNP] core
2+
and the radiolabeled product was characterized by RP-HPLC. 99mTc-labeled porphyrin derivative
(v) was obtained with a radiochemical purity of >95% as determined by RP-HPLC studies where
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99mTc-complex of compound (v) was eluted with a retention time of ~18 min whereas
[99m
Tc(N)PNP] exhibited retention time of 15.0 and 17.5 min under identical conditions (Fig.
2+
1
). On the other hand, compound (vii) containing a HYNIC group attached to one of the
peripheral positions of the porphyrin macrocyle was radiolabeled with 99mTc in presence of
tricine and EDDA as co-ligands. The radiolabeled complex (99mTc-labeled vii) was characterized
by HPLC studies where free 99mTcO eluted from the column at 3.5 min whereas radiolabeled
-
4
complex exhibited retention time of 15 min (Fig. 2). 99mTc-labeled (vii) was obtained with a
radiochemical purity of ~80%, which was further improved to >95% through post-labeling
®
purification using a preconditioned Sep-pak cartridge.
99mTc-labeled porphyrin complexes, prepared either HYNIC or PNP-DTC route, were
obtained with high specific activities. Specific activity of 99mTc(N)PNP-DTC-porphyrin complex
was calculated to be 5 mCi/mg where as the same for 99mTc-HYNIC-porphyrin was found to be
1
4 mCi/mg.
Determination of partition coefficient (LogPo/w)
The partition coefficients of 99mTc-labeled complexes of (v) and (vii) were determined
using octanol-water system and were found to be -0.79±0.03 and -1.86±0.02, respectively. These
values indicate the hydrophilic nature of both the complexes with latter being relatively more
hydrophilic than the former. This may be due to the presence of more polar groups in 99mTc-
HYNIC complex compared to those present in 99mTc(N)PNP complex of the porphyrin
derivatives.
In-vitro serum binding and stability studies
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In-vitro serum binding and stability studies were carried out for 99mTc-complexes of (v)
and (vii) revealed that while 84.7% of the former complex was present in the bound state, only
5
4.7% was found to be bound with serum for the latter one. However, both the radiolabeled
complexes showed excellent serum stability as both the complexes retained >95% radiochemical
purity when incubated in human blood serum till 3 h post-preparation.
Biodistribution studies
Biodistribution studies, carried out in Swiss mice bearing fibrosarcoma tumor, revealed
significant tumor uptake for both the radiolabeled complexes within 1.5 h of administration
(
1.52±0.53 and 3.95±1.42 %IA per g for 99mTc(N)PNP-DTC-porphyrin and 99mTc-HYNIC-
porphyrin, respectively) and retention therein till 3 h post-injection (1.56±0.10 and 3.28±0.27
IA per g for 99mTc(N)PNP-DTC-porphyrin and 99mTc-HYNIC-porphyrin, respectively), up to
%
which the studies were continued (Table 1). However, a careful look at the results of the
biodistribution studies divulges considerable difference in the tumor uptake as well as
distribution pattern of 99mTc-labeled porphyrins prepared through two different routes. A
graphical comparison between blood, liver and tumor uptake values exhibited by both the
complexes at 1.5 h post injection has been shown in Fig. 3. It is noteworthy to mention that
99mTc-porphyrin prepared through HYNIC route showed much higher tumor uptake and better
retention of activity in the tumor compared to those exhibited by 99mTc-porphyrin prepared
through PNP route. On the other hand, 99mTc-porphyrin derivative prepared through PNP route
showed much lower blood uptake (2.41±0.54 and 2.30±0.16 %IA at 1.5 h and 3 h post-
administration, respectively) than that exhibited by 99mTc-labeled porphyrin complex prepared
through HYNIC route (6.63±0.75 and 4.36±0.25 %IA at 1.5 h and 3 h post-administration,
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respectively). This has resulted in almost similar tumor to blood ratios for both the 99mTc-labeled
complexes [0.62±0.08 and 0.67±0.07% IA/g for 99mTc(N)PNP-DTC-porphyrin complex and
0
.53±0.14 and 0.75±0.02% IA/g for 99mTc-HYNIC-porphyrin complex at 1.5 and 3 h post-
administration, respectively] at both the time points (Table 1). The liver uptake of the
99mTc(N)PNP-DTC-porphyrin complex (39.37±3.88 and 34.15±8.25 %IA at 1.5 h and 3 h post-
administration, respectively) was found to be much higher compared to that of 99mTc-HYNIC-
porphyrin complex (19.26±3.48 and 18.45±1.05 %IA at 1.5 h and 3 h post-administration,
respectively) at both the time points and this is in well-correlation with the LogPo/w values
exhibited by the complexes. High liver uptake observed with 99mTc(N)PNP-DTC-porphyrin
could be due to the comparatively higher lipophilicity of the complex which again can be
attributed to the presence of four phenyl groups on PNP2 backbone. Both the complexes
exhibited clearance via hepatobiliary as well as renal pathway, although primary clearance was
through the hepatobiliary route, which is an inherent property exhibited by majority of the
radiolabeled porphyrins.
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Discussion
Porphyrins and their derivatives generated considerable interest and provided
unprecedented opportunities in the development of potential radiopharmaceuticals by virtue of
their ability to mimic essential chemicals in the human body, solubility in serum, rapid wash-out
and inherent ability to localize preferentially in tumor lesions.1-6,34 The possibilities to modify the
chemical structure of porphyrins in order to alter their lipophilicity/hydrophilicity, which
markedly affects the tumor uptake and clearance from the non-target organs, make these
macromolecules an attractive target for developing tumor-specific agents having potential for
either diagnosis or targeted therapy.35
In the quest for developing a radiolabeled porphyrin suitable for diagnostic imaging,
maximum attention was focused towards using 99mTc owing to widespread availability of SPECT
(
single photon emission computed tomography) instrumentation compared with that for PET
positron emission tomography) in most nuclear medicine facilities, the near ideal imaging
(
characteristics of 99mTc via SPECT, its rich and diverse redox chemistry, and its ability to form a
variety of complexes with different porphyrin derivatives.22,23 The ability to attain high specific
activities for targeting low concentration targets through the radiolabeling procedures employing
novel 99mTc-carbonyl, 99mTc-nitrido and 99mTc(III) cores constitute another additional advantage
of using 99mTc for developing radiolabeled porphyrins for diagnostic applications.22
As the tumor accumulation and cell membrane penetration properties of porphyrin
derivatives are strongly dependent on their structures, selection of an appropriate porphyrin is of
prime importance for propitious outcome. In this investigation, we have undertaken the synthesis
of an unsymmetrically substituted porphyrin derivative namely 5-(p-amino-propyloxyphenyl)-
1
0,15,20-tris(carboxymethyleneoxyphenyl)-porphyrin and modified it further to perform radio
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labeling with 99mTc using two different 99mTc-cores viz. 99mTc-HYNIC and 99mTc(N)PNP2 with
an objective to study the effect of different 99mTc-cores on the tumor affinity as well as
pharmacokinetic behavior of the 99mTc-labeled porphyrins.
Among the BFCAs (bi-functional chelating agents) that have been studied for
radiolabeling with 99mTc, HYNIC has shown considerable promise.24 Our interest in using
HYNIC as BFCA for the present work has been primarily due to its excellent labeling efficiency
and the ability to introduce the hydrophilic character in the resulting complex which
consequently can lead to better in-vivo pharmacokinetic behavior of the 99mTc-labeled porphyrin
with respect to undesirable uptake in the non-target organs.22 HYNIC could be linked with
porphyrin derivative via an amide bond between the carboxylic group of the former and free
amine group present in the latter.
For 99mTc-labeling of the porphyrin derivative using 99mTc-nitrido core, the
[99m
Tc(N)PNP]2+ core seems to be an obvious choice, as it offers an unique route for the
preparation of asymmetric 99mTc-complexes owing to the enticing chemical characteristics of the
electrophilic nitrido metal fragment [99mTc(N)PNP] . The structural framework of
2+
[99m
Tc(N)PNP] moiety contains a pseudo tridentate diphosphine ligand (PNP) coordinated to
2+
the 99mTcN group and two labile sites, which complete the pseudo-octahedral environment. The
23
appearance of two peaks in the HPLC chromatogram of [99mTc(N)PNP]2+ moiety is probably
-
due to the presence of weak donor ligands, such as H O, Cl etc. (available in the reaction
2
mixture) which may occupy these two labile positions [Fig. 1(b)]. The [99mTc(N)PNP] metal
fragment is an activated intermediate which selectively reacts with dithiocarbamate derivative of
the porphyrin moiety carrying -donor atoms to form the final complex.23 The resultant
99mTc(N)PNP-DTC-porphyrin complex is relatively lipophilic due to introduction of non-polar
2+
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PNP2 moiety compared to the 99mTc-porphyrin complex formed by using HYNIC as BFCA and
this is evident from the corresponding LogP values exhibited by the radiolabeled porphyrin
complexes.
Owing to its higher lipophilicity, 99mTc(N)PNP-DTC-porphyrin complex was expected to
exhibit superior tumor uptake; although non-specific accumulation was also expected to be
higher. In the same lines, 99mTc-HYNIC-porphyrin complex was expected to yield relatively
lower non-specific accumulation compared to its PNP2 counterpart, along with lower tumor
uptake and retention. However, the results of biodistribution studies in tumor bearing small
animal model revealed that the expected behavior has not exactly been realized. The probable
reason behind the observed results could be the relatively higher blood uptake and longer
retention therein exhibited by the 99mTc-HYNIC-porphyrin complex than that by 99mTc(N)PNP-
DTC-porphyrin complex which might have led to the higher tumor uptake in case of the former
complex. These results of the present study indicate that not only the hydrophilicity/lipophilicity
of the radiolabeled porphyrin, but the other features such as, charge, size and nature of functional
groups present in the coordination sphere could also affect the overall in-vivo performance
exhibited by the same porphyrin when labeled with same radioisotope using different
approaches.
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Conclusions
A porphyrin derivative was derivatized differently in order to radiolabel it with 99mTc by
using two different cores viz. PNP and HYNIC, resulting in the formation of two different 99mTc
complexes of the same porphyrin. Both the 99mTc-labeled porphyrin derivatives were prepared
with >95% radiochemical purity under the optimized radiolabeling conditions and exhibited
adequate in-vitro stability in human serum. However, it was found that selection of the core has a
profound effect on the tumor uptake as well as on the pharmacokinetic behavior of the resultant
99mTc-complexes.
99mTc-porphyrin complex prepared through the PNP route, although
comparatively more lipophilic, exhibited lesser blood uptake and higher liver accumulation
compared to those showed by 99mTc-porphyrin complex, prepared through the HYNIC route. On
the other hand, even though the former complex is more lipophilic in nature, it exhibited
significantly inferior tumor uptake and retention, compared to those shown by the latter. This
observed phenomenon could be attributed to the higher uptake and relatively longer retention of
the 99mTc-porphyrin complex, prepared through the HYNIC route, in the blood which possibly
enhanced the rate of its passive diffusion across the cell membrane in the tumorous tissues
leading to the higher tumor accumulation.
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Acknowledgements
The authors gratefully acknowledge Dr. P.K. Pujari, Associate Director, Radiochemistry
and Isotope Group, Bhabha Atomic Research Centre (BARC) for his constant encouragement
and support. The authors thankfully acknowledge the members of the Radiochemicals Section of
Radiopharmaceuticals Division, BARC for providing 99Mo needed for the construction of
99Mo/99m
Tc generator. The authors thank Shri Umesh Kumar of Radiopharmaceuticals Division
and all the staff members of the Animal House Facility, Radiation Biology and Health Sciences
Division, BARC for the help received during the course of the work reported in the present
manuscript.
Conflict of interest
Bhabha Atomic Research Centre is a constituent unit of Department of Atomic Energy,
Government of India and all research activities of the institute are entirely funded by the
Government of India. The authors declare that there are no competing financial interests.
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