Synthesis, radiolabeling and evaluation of a new positively charged 99mTc-labeled fatty acid derivative for myocardial imaging

Article abstract of DOI:10.1002/jlcr.1836

¹²³I-labeled fatty acids and ¹⁸F-FDG are used as metabolic markers for detecting myocardial abnormalities. However, a ^99mTc-based molecule may find wider application. In the present work, a new ^99mTc-labeled, un

Full text of DOI:10.1002/jlcr.1836

Research Article
Received 28 June 2010,
Revised 31 August 2010,
Accepted 31 August 2010 Published online 30 November 2010 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1836
Synthesis, radiolabeling and evaluation of a
new positively charged Tc-labeled fatty
99m
acid derivative for myocardial imaging
Anupam Mathur,a Madhava B. Mallia,b Haladhar D. Sarma,c
Ã
Sharmila Banerjee,b and Meera Venkateshb
123I-labeled fatty acids and 18F-FDG are used as metabolic markers for detecting myocardial abnormalities. However,
99mTc-based molecule may find wider application. In the present work, a new 99mTc-labeled, uni-positively charged,
a
16-carbon fatty acid has been prepared and evaluated in normal Swiss mice. The results are then compared with the neutral
analogue reported earlier.
A 16-cysteinyl hexadecanoic acid conjugate was synthesized in a six-step synthetic procedure starting with
16-bromohexadecanoic acid. The ligand upon incubation with [99mTcN(PNP6)]21 core formed the required positively
charged complex in ꢀ85% yield. The complex, which was obtained as a mixture of syn-anti isomers, was purified by HPLC
and the major fraction was used for in vivo studies in Swiss mice. The biodistribution studies in Swiss mice showed initial
uptake similar to 125I-IPPA followed by rapid clearance from the myocardium till 10 min p.i. Thereafter, the rate of clearance
was significantly decreased, an observation reported earlier for positively charged fatty acid complexes. In terms of
absolute uptake, the positively charged complex performed better than the neutral analogue reported earlier. The
positively charged fatty acid complexes, prepared using [99mTcN(PNP)]21 core, seems to be better candidates for the
development of myocardial metabolic tracers than their neutral counterparts.
Keywords: [99mTcN(PNP)]21 core; myocardial imaging; fatty acid; [125I]-IPPA; PNP ligand
washout kinetics, which reflect fatty acid metabolism, with
ischemic areas showing low uptake and delayed washout.3
The 123I-labeled fatty acids are based on cyclotron produced
Introduction
Fatty acids are the major source of energy, contributing nearly
radioisotope, 123I. Although number of cyclotrons is increasing
every year, cyclotron-based isotopes are currently not widely
available in all parts of the world. Hence, molecules labeled with
99mTc, which is a more widely available isotope, are preferable.
Several reports are available in the literature on the synthesis
and evaluation of 99mTc-labeled fatty acid derivatives based on
various metal cores albeit with suboptimal properties.8–18
Among the different ways of labeling molecules with 99mTc,
the use of [99mTcN(PNP)]21 core has shown excellent in vivo
characteristics suitable for myocardial imaging.19,20 This core
forms pseudo-octahedral complexes, where the two cis-positions
in the square basal plane are being occupied by phosphorus
ꢀ90% of the energy needs, in normal myocardium under
fasting condition.1 They are transported across the lipid
membrane either by passive diffusion or via protein transpor-
ter.2 Once inside the myocardium, either they get metabolized
via the b-oxidation pathway releasing energy required for the
normal functioning of the heart or get stored in the form of
triglycerides.3
An ischemic myocardium exhibits change in its normal
metabolic pathway using fatty acids. The viable ischemic
myocardial cells shift to anaerobic metabolic pathway using
glucose for their energy needs and survival. It has been seen
even after establishing normal oxygenation levels in previously
ischemic cells, the cells show predominant glucose metabolism
(ischemic memory) before reverting to normal state. Thus,
radiolabeled fatty acids provide important information for early
identification of cardiac abnormalities in high-risk patients.1,4,5
The currently used radiolabeled fatty acids are 123I-labeled
aRadiopharmaceuticals Program, Board of Radiation and Isotope Technology
(BRIT), Mumbai 400705, India
bRadiopharmaceuticals Division, Bhabha Atomic Research Centre (BARC),
Trombay, Mumbai 400085, India
Iodophenylpentadecanoic acid
(
123I-IPPA) and beta-methyl
iodophenyl pentadecanoic acid (123I-BMIPP).1,6,7 Both are meta-
bolic substrates but 123I-IPPA is rapidly metabolized, resulting in
rapid clearance from the heart, whereas 123I-BMIPP being a
branched chain fatty acid does not readily undergo b-oxidation
and is retained. Thus, 123I-IPPA imaging involves study of
cRadiation Biology and Health Science Division, Bhabha Atomic Research Centre
(BARC), Trombay, Mumbai 400085, India
*Correspondence to: Meera Venkatesh, Radiopharmaceuticals Division, Bhabha
Atomic Research Centre, Trombay, Mumbai 400085, India.
E-mail: meerav@barc.gov.in
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A. Mathur et al.
atoms of the long chain PNP ligand and the other two cis- (0.1 mL) was added. The reaction mixture was then refluxed
positions occupied by the p-donor bidentate ligand having overnight and cooled. Excess ethanol was removed under
donor groups such as SS/SO/SN. Thus, the amino acid cysteine is vacuum and ice-cold water (20 mL) was added to the oily
a useful bi-functional chelator, where a bio-molecule, like fatty residue. The aqueous layer was extracted with chloroform
acid, can be attached either at –COOH or –NH2 group, and the (3 Â 10 mL). The chloroform extracts were pooled, dried over
other two groups, SH and NH2/COOH, can be used for labeling anhydrous sodium sulphate, filtered and evaporated to obtain
with [99mTcN(PNP)]21 core.21 The overall charge of the complex the target compound. Yield: 97% (ꢀ525 mg). IR (neat, cmÀ1
)
depends on the groups in cysteine that are coordinated to the 2919 (s); 2849 (m); 1740 (s).
[
99mTcN(PNP)]21 core, with the overall charge being positive, if
–SH and –NH2 group of cysteine are involved in coordination, or Ethyl 16-phthalimidohexadecanoate (2)
neutral, if –SH and –COOH groups are coordinated to the
99mTcN(PNP)]21 core.
The compound 1 (500 mg, 1.37 mmol) and phthalimide (203 mg,
1.37 mmol) were dissolved in acetonitrile (15 mL) and anhydrous
potassium carbonate (209 mg, 1.5 mmol) was added. The
reaction mixture was then refluxed overnight. The progress of
the reaction was monitored by TLC in ethyl acetate:hexane
(1:9 v/v) mixture. Upon completion of the reaction, the reaction
mixture was cooled, filtered and solvent was removed under
vacuum to obtain the crude product. The target compound was
obtained by silica gel chromatography (10% ethyl acetate: 90%
chloroform). Yield ꢀ87% (511 mg). Rf = 0.8 (ethylacetate/hexane,
1:9 v/v). IR (neat, cmÀ1) 2919 (s); 2849 (m); 1740 (s); 1704 (s);
[
The desired characteristics of a 99mTc-labeled fatty acid are
high uptake in the normal myocardium with retention long
enough to carry out SPECT imaging and rapid clearance from
background tissue/organs such as blood, liver and lungs.5 The
presence of PNP ligand in the complex while increasing the lipo-
philicity of the complex, and thus the initial uptake in the myo-
cardium, also helps in fast clearance of the activity from the
non-target organs due to its hydrolysable ether linkages.
Recently, a series of neutral and positively charged fatty acid
derivatives labeled with
[
99mTcN(PNP)]21 core have been
1
1464 (m); 1406 (m); 1174 (m); 1064 (m); 714 (s). H-NMR (CDCl3,
prepared and evaluated in biological systems.8 Our group
working on similar complexes has earlier reported the syn-
thesis and bio-evaluation of a neutral complex of a long chain
16-carbon fatty acid derivative.9 In the present work, a positively
charged structural analogue of the previously reported
16-carbon fatty acid derivative was synthesized, radiolabeled
and evaluated in normal Swiss mice. The results are compared
with the neutral analogue as well as 125I-IPPA reported earlier.9
d ppm) 7.790–7.853 (C6H4C-, 2H, m); 7.672–7.733 (C6H4C-, 2H, m);
4.057–4.164 (-COOCH2CH3, 2H, q, J = 7.2 Hz); 3.626–3.698
(-CH2CH2N(CO)2-, 2H, t, J = 7 Hz); 2.235–2.309 (-CH2CH2COOEt,
2H, t, J = 7.4 Hz); 1.622 (-CH2CH2COOEt and -CH2CH2N(CO)2-,
4H, m); 1.206–1.277 [(CH2)11 and -COOCH2CH3, 25H, m].
Ethyl 16-aminohexadecanoate (3)
To compound 2 (300 mg, 0.69 mmol) dissolved in ethanol
(15 mL), 80% hydrazine hydrate (219 mL, 3.49 mmol) was added
and the reaction mixture refluxed for 3 h. Thereafter, the
reaction mixture was brought to room temperature and excess
hydrazine hydrate and ethanol were removed under vacuum.
The precipitate obtained was re-dissolved in ethanol (15 mL) and
treated with 2 N HCl (5 mL). The reaction mixture was again
refluxed overnight, cooled and then excess solvent was
removed under vacuum to obtain a white powder. The crude
product obtained was again refluxed overnight in excess
ethanol and concentrated H2SO4 to esterify the fatty acid
hydrolyzed in the previous step. The reaction mixture was
cooled to obtain a white precipitate of phthalhydrazide, which
was removed by filtration. The filtrate was then evaporated
under vacuum to obtain an oily residue, which was dissolved in
cold water and the pH was brought above 7 with 5% sodium
bicarbonate solution. The alkaline solution was extracted with
chloroform (3 Â 10 mL) and the pooled extracts were dried over
anhydrous sodium sulphate. The chloroform was removed to
give the pure product in quantitative yield (ꢀ230 mg). IR (neat,
cmÀ1) 3197 (w); 2916 (s); 2848 (m); 1738 (s); 1577 (m); 1518 (s);
Experimental
The 16-bromo hexadecanoic acid, triethyl silane, succinic
dihydrazide and stannous chloride were obtained from Aldrich,
USA. N-Boc S-trityl cysteine, N-ethyl, N’-(3-dimethylamino)
carbodiimide hydrochloride (EDCI) and potassium carbonate
were purchased from Fluka, Germany. Hydrazine hydrate (80%)
¨
was purchased from Riedel-de-Haen, Germany. Phthalimide was
obtained from Loba Chemicals, India. All other reagents used
were of analytical grade. Sodium pertechnetate (Na99mTcO4) was
eluted with normal saline just before use from a 99Mo-99mTc gel
generator, supplied by Board of Radiation and Isotope
Technology, India. Bis[(diethoxypropylphosphanyl) ethyl]ethoxy
ethylamine (PNP6) was obtained as a gift from Prof. Adriano
Duatti, University of Ferrara, Italy. Silica gel plates (Silica Gel 60
F254) were obtained from Merck, India. The HPLC of the prepared
complex was carried out on a JASCO PU 2080 Plus dual pump
HPLC system, Japan, with a JASCO 2075 Plus tunable absorption
detector and Gina Star radiometric detector system, using a
C18 reversed phase HiQ Sil (5 mm, 4 Â 250 mm) column. The
IR spectra of all the compounds were recorded on a JASCO
FT-IT/420 spectrophotometer, Japan. The 1H-NMR spectra
were recorded either on 200 or 300 MHz Bruker Spectro-
photometer, USA.
1
1463 (m); 1182 (s); 721 (m). H-NMR (CDCl3, d ppm) 4.056–4.162
(–COOCH2CH3, 2H, q, J = 7 Hz); 3.000 (–CH2CH2NH2, 2H, m);
2.234–2.308 (–CH2CH2COOEt, 2H, t, J = 7.4 Hz); 1.895
(–CH2CH2NH2 and –CH2CH2NH2, 4H, m); 1.602 (–CH2CH2COOEt,
2H, m); 1.239–1.274 [(CH2)11 and –COOCH2CH3, 25H, m].
Synthesis
Ethyl 16-(N-Boc, S-trityl cysteinyl)hexadecanoate (4)
Ethyl 16-bromohexadecanoate (1)
The compound 3 (150 mg, 0.5 mmol) and N-Boc, S-trityl cysteine
The 16-bromo hexadecanoic acid (500 mg, 1.5 mmol) was (233 mg, 0.5 mmol) were dissolved in dry dichloromethane
dissolved in ethanol (20 mL) and concentrated sulphuric acid (15 mL) and cooled to 01C with continuous stirring. To the
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A. Mathur et al.
cooled reaction mixture, EDCI (106 mg, 0.55 mmol) was added
and stirred for 1 h after which the reaction was continued
overnight at room temperature. The progress of the reaction
was monitored by TLC. On completion of the reaction, the
reaction mixture was washed with water (3 Â 10 mL) and
the organic phase was dried over anhydrous sodium sulphate.
The crude product was obtained after removing dichloro-
methane and purified using silica gel column chromatography
(ethyl acetate:chloroform, 5:95 v/v) to obtain the desired product
4. Yield ꢀ72% (267 mg). Rf = 0.6 (ethyl acetate/chloroform, 5:95
v/v). IR (neat, cmÀ1) 3316 (b); 3057 (w); 2975 (w); 2925 (s); 2853
(s); 1713 (bs); 1681 (m); 1488 (m); 1366 (m); 1168 (s); 1032 (w);
Quality control
HPLC
The radiochemical purity of the [99mTcN(PNP)]12 core prepared
as well as the complex was assessed by HPLC using a C18
reversed phase column. Water (A) and methanol (B) were used
as the mobile phase and the following gradient elution
technique was adopted for the separation (0 min 50% A,
15 min 0% A, 50 min 0% A). Flow rate was maintained at
1 mL/min. About 25 mL of the test solution was injected into the
column and elution was monitored by observing the radio-
activity profile.
1
743 (s); 700 (s). H-NMR (CDCl3, d ppm) 7.221–7.436 [(C6H5)3C–,
The purification of the complex was carried out in the same
analytical column used for characterization. The 99mTc-pertech-
netate with high radioactive concentration (50 mCi/mL) was
used for the radiolabeling. The methanolic fraction containing
the pure complex obtained upon HPLC purification was
evaporated under vacuum and reformulated in 2 mL of 10%
ethanolic solution. About 500 mCi of the pure radiolabeled fatty
acid could be obtained by this method, which was sufficient to
carry out further studies.
15H, m]; 5.996 (–CHCONHCH2–, 1H, s); 4.825 (–NHCHCH2S–, 1H,
m); 4.057–4.164 (–COOCH2CH3, 2H, q, J = 6.9Hz); 3.817
(–NHCHCH2S–, 1H, m); 3.142–3.190 (–CH2CH2NHCO–, 2H, m);
2.67–2.74 (–CHCHAHBS–, 1H, m); 2.45–2.53 (–CHCHAHBS–, 1H, m);
2.265–2.314 (–CH2CH2COOEt, 2H, t, J = 7.4Hz); 1.617
(–CH2CH2COOEt and –CH2CH2NHCO–, 4H, m); 1.415 [(CH3)3C–,
9H, s]; 1.15–1.35 [(CH2)11 and –COOCH2CH3, 25H, m].
16-(N-Boc, S-trityl cysteinyl) hexadecanoic acid (5)
The compound 4 (60 mg, 0.08 mmol) dissolved in methanol
(320 mL) was treated with KOH solution (1 M, 160 mL, 0.16 mmol).
The reaction mixture was stirred at room temperature for 48 h.
The progress of the reaction was monitored by TLC. Upon
completion of the reaction, methanol was removed under
vacuum, 5 mL of water was added and the pH of the reaction
mixture was adjusted to 3 using 2 N HCl. The white precipitate of
the target compound obtained was filtered and dried under
vacuum. Yield ꢀ80% (57 mg). Rf = 0.2 (ethyl acetate/chloroform,
Partition coefficient (Log Po=w
)
The HPLC purified labeled compound (100 mL) was mixed with
water (0.9 mL) and octanol (1 mL) on a vortex mixer for about
1 min and then centrifuged for 5 min to effect the separation of
the two layers. Equal aliquots of the two layers were withdrawn
and measured for the radioactivity. The readings thus obtained
were used to calculate the Log Po/w value of the complex.
1:9 v/v). IR (neat, cmÀ1) 3313 (b); 3057 (w); 2924 (s); 2852 (s); Stability studies
1685 (s); 1656 (s); 1530 (b); 1491 (m); 1444 (m); 1366 (m); 1248
1
Cysteine challenge: For cysteine challenge studies, purified fatty
(w); 1167 (s); 1033 (w); 742 (s); 699 (s). H-NMR (CDCl3, d ppm)
acid complex (50 mL, 10 mCi), 10 mM cysteine solution (50 mL) and
saline (400 mL) were mixed in a 5 mL vial and incubated at 371C
for 30 min. Thereafter, the sample was analyzed by TLC
(EtOH:CHCl3:benzene:0.5 M ammonium acetate (1.5:2:1.5:0.5)
for possible degradation of the original complex (Fatty acid
complex: Rf = 0.1–0.2).
Serum stability: To assess the stability of the fatty acid complex
in human serum, purified fatty acid complex (50 mL, 10 mCi) was
incubated with human serum (450 mL) at 371C for 30 min.
7.209–7.430 [(C6H5)3C–, 15H, m]; 6.178 (–CHCONHCH2–, 1H, s);
5.092 (–NHCHCH2S–, 1H, m); 3.849 (–NHCHCH2S–, 1H, m);
3.083–3.171 (–CH2CH2NHCO–, 2H, m); 2.667 (–CHCHAHBS–, 1H,
m); 2.523 (–CHCHAHBS–, 1H, m); 2.321–2.345 (–CH2CH2COOH,
2H, t, J = 7.2 Hz); 1.620 (–CH2CH2COOH and –CH2CH2NHCO–, 4H,
m); 1.405 [(CH3)3C–, 9H, s]; 1.259 [(CH2)11, 22H, s].
16-Cysteinyl hexadecanoic acid (6)
The compound 5 (40 mg, 0.06 mmol) was stirred with trifluoro- Thereafter, the serum proteins were precipitated by addition of
acetic acid (2 mL) for 2 h at room temperature. To the yellow ethanol (500 mL), the solution was centrifuged and the super-
solution, triethyl silane was added drop-wise until it becomes natant was analyzed by TLC to determine the stability of the
colorless and stirring was continued for another 15 min. Upon complex in serum.
removal of the solvent, the target compound was obtained as a
sticky white solid, which was used as such for radiolabeling.
In vivo evaluation studies
All procedures performed herein were in accordance with the
national laws pertaining to the conduct of animal experiments.
Radiolabeling
In a typical labeling procedure, succinic dihydrazide (5 mg),
stannous chloride (0.1 mg) and ethanol (250 mL) were taken in a
vial to which freshly eluted Na99mTcO4 (50 mCi, 750 mL) was
added. Upon keeping the reaction mixture at room temperature
Normal Swiss mice (20–25 g body weight) were used for the
in vivo distribution assays of the prepared fatty acid complexes.
All the mice involved in the study were kept under fasting for
6–7 h prior to the experiment, although water was given ad
libitum. The HPLC purified radiolabeled preparation (100 mL,
20 mCi) was administered intravenously through tail vein of each
animal. Individual sets of animals (n = 3) were utilized for
studying the biodistribution at different time points (2, 5, 10
and 30 min). The animals were sacrificed immediately at the end
of the respective time point and the relevant organs and tissue
for 20 min
[
99mTcN]21 intermediate was formed. To this
intermediate, PNP6 ligand (ꢀ2.5 mg) and fatty acid cysteine
conjugate (5 mg), each dissolved in nitrogen purged ethanol
(250 mL), were added simultaneously and the reaction mixture
heated at 901C for 30 min. Thereafter, the reaction mixture was
cooled and then characterized by HPLC.
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A. Mathur et al.
were excised for measurement of associated activity. The organs ligand to the [99mTcN]21 core sterically orients P donor groups
were weighed and the activity associated with each was (good p-acceptor) to occupy the basal cis-positions in a square
measured in a flat-bed type NaI(Tl) counter with suitable energy pyramidal complex leaving the other two cis-positions, occupied
window for 99mTc (140 keV710%). For the sake of comparison, by labile groups, available for coordination with a suitable
the activity retained in each organ/tissue was expressed as a p-donor bi-dentate ligand. The fatty acid cysteine ligand now
percent value of the injected dose per gram (% ID/g).
occupies this position forming a unipositively charged [212]
asymmetric pseudo-octahedral complex. The fatty acid complex
prepared herein is similar to the complexes reported earlier.21
Therefore, it is logical to presume the present complex also to
have similar structure.
Results and discussion
The scheme followed for the synthesis of cysteine conjugated
fatty acid derivative is shown in Figure 1. The target compound
6 was synthesized following a six-step synthetic procedure. The
first step involved the protection of the carboxylic group of
bromo fatty acid followed by conversion of the halide to amine
via the Gabriel phthalimide synthesis. The free amino group of
16-amino hexanoic ester derivative was then conjugated with
acid group of N-Boc, S-Trt cysteine using EDCI to obtain
compound 4. The compound 4 was then de-esterified using
methanolic KOH to generate the free acid 5. All the inter-
mediates were characterized using FT-IR and 1H-NMR. Finally,
The prepared complex was characterized by HPLC. The
asymmetric centre in cysteine residue leads to
a final
[
99mTcN(PNP6)]-fatty acid complex as a mixture of syn and
anti-isomers.22 Consequently, two peaks were observed at
25 min (64.8%) and 26.6 min (19.7%) respectively (Figure 3).
The stereochemistry of the isomers can be ascertained only after
preparing the complex in macroscopic level. The major fraction
was isolated by HPLC and used for carrying out the stability and
in vivo biological studies.
The labeled complex did not show any significant degree of
trans-chelation upon challenging with cysteine. However, the
complex was observed to degrade in human serum (about 10%
over a period of 30 min). The Log Po/w value of the present fatty
acid complex was found to be 1.2, which was much lower
compared to the neutral analogue (1.8)9 and 125I-IPPA (1.7).
Figure 4 shows the myocardial uptake and clearance pattern
of the complex under evaluation. The results obtained with
125I-IPPA9 as well as earlier reported neutral analogue9 are also
shown for comparison. It could be observed that initial uptake
shown by the present complex (9.8872.99% ID/g) in the myo-
cardium at 2 min post injection (p.i.) is similar to that of 125I-IPPA.
However, initially accumulated activity was not retained as
the target compound
6 was obtained by simultaneous
deprotection of Boc- and trityl-groups using trifluoroacetic
acid/triethyl silane combination and used as such without
further characterization.
The [99mTcN]21 core forms exceptionally inert complexes
when p-donor (N and SÀ atoms from cysteine) as well as p-
acceptor (two P atoms from PNP6) ligands are present together
in the complex.21 The radiolabeling of the prepared fatty acid
derivative was carried out as shown in Figure 2. The formation of
[
[
99mTcN(PNP)]-fatty acid complex involved prior preparation of
99mTcN]21 core followed by simultaneous addition of PNP6
ligand and fatty acid derivative. The addition of long chain PNP6
Figure 1. Synthesis of 16-cysteinyl hexadecanoic acid.
Figure 2. Synthesis of [99mTcN(PNP)]-fatty acid complex.
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A. Mathur et al.
Figure 4. Myocardial uptake pattern of charged fatty acid complex compared with
125I-IPPA and neutral fatty acid complex.
mitochondria. And that such
a situation will complicate
the interpretation of the results. Though, in the present study,
no attempt was made to determine the extent of such
interaction, there are few points which indicate that mitochon-
drial retention of the present cationic complex is insignificant, if
not completely absent. The retention of activity in the myo-
cardium through the Coulombic attraction with the mitochon-
dria is often characterized by steady retention of activity for
prolonged period as can be seen in the case of the cationic
perfusion agents, viz. Sestamibi and Tetrofosmin. However, the
clearance pattern of the cationic fatty acid complex from the
myocardium, evaluated in the present study, showed similarity
to neutral fatty acid complex rather than the cationic perfusion
agents.
Variation of heart/blood, heart/lung and heart/liver ratio
with time observed with positively charged fatty acid complex,
125I-IPPA and neutral fatty acid complex is shown in Figure 5. It
can be seen that there is no significant difference in heart/lung
and heart/liver ratio among the three different radiolabeled
compounds. The 125I-IPPA, however, showed slightly better
heart/blood ratio compared to the other two complexes.
The clearance of activity from different organs exhibited by
the three radiolabeled compounds is shown in Figure 6. For the
positively charged fatty acid complex injected activity cleared
mostly through hepatobiliary route. Similar observation could
be made for the neutral analogue as well. Increase in activity in
intestine with time indicates fast clearance of activity from liver.
This is due to the hydrolysis of the ether groups of PNP ligand in
the liver, which facilitates easy clearance of the activity.23 Also, a
different mechanism based on P-glycoproteins (Pgp) or multi-
drug resistance-associated protein (MDR)-Pgp has been sug-
gested to be the probable mechanism for rapid elimination of
Figure 3. HPLC chromatograms of (a) [99mTcN(PNP)] core, (b) [99mTcN(PNP)]-fatty
acid complex and (c) Purified [99mTcN(PNP)]-fatty acid complex.
indicated by the subsequent clearance from the myocardium.
This probably is an indication that the [99mTcN(PNP)]-cysteine
moiety is not functioning like the 125I-iodophenyl ring in 125I-
IPPA. Although clearance of activity was observed from the
myocardium, however, it was not uniform. The initial rapid
clearance phase up to 10 min p.i. was followed by slow phase
with 1.3970.36% ID/g remaining after 30 min p.i. It could be
noted that activity retained at the end of 30 min p.i. is around
50% of the activity observed at 5 min p.i. This is similar to
the observation made by Cazzola et al.8 The positive charge on
the complex may lead to the retention of activity in the
[
99mTcN(PNP)] lipophilic compounds from the tissues.24 Contrary
to this it could be observed that in the case of 125I-IPPA
clearance of activity from the liver is very slow, which resulted in
low heart/liver ratio.
Conclusions
A new 16-carbon fatty acid cysteine derivative was synthesized
and labeled with [99mTcN(PNP)]21 core in good yields. The
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A. Mathur et al.
Figure 5. Comparison of (a) Heart/Blood, (b) Heart/Lung and (c)Heart/Liver
ratios of charged fatty acid complex with 125I-IPPA and neutral fatty acid
complex.
Figure 6. Comparison of clearance pattern of (a) charged fatty acid complex,
(b) 125I-IPPA and (c) neutral fatty acid complex from non-target organs.
biodistribution studies in Swiss mice showed high myocardial analogue. Positively charged fatty acid derivatives, prepared
extraction similar to the 125I-labeled standard agent IPPA at using [99mTcN(PNP)]21 core, seems to be better candidates for
2 min p.i., however, with poor retention subsequently. The the development of myocardial metabolic tracers than their
complex showed better uptake characteristics than the neutral neutral counterparts.
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A. Mathur et al.
Acknowledgements
[11] M. Walther, C. M. Jung, R. Bergmann, J. Pietzsch, K. Rode,
K. Fahmy, P. Mirtschink, S. Stehr, A. Heintz, G. Wunderlich,
W. Kraus, H. J. Pietzsch, J. Kropp, A. Deussen, H. Spies,
Bioconjugate Chem. 2007, 18, 216.
[12] T. Uehara, T. Uemura, S. Hirabayashi, S. Adachi, K. Odaka,
H. Akizawa, Y. Magata, T. Irie, Y. Arano, J. Med. Chem. 2007,
50, 543.
The authors are thankful to Prof. Adriano Duatti for providing
PNP6 ligand. The support and encouragement of Dr
V. Venugopal, Director, Radiochemistry and Isotope group,
BARC is gratefully acknowledged.
[13] A. C. Heintz, C. M. Jung, S. N. Stehr, P. Mirtschink, M. Walther,
J. Pietzsch, R. Bergmann, H. J. Pietzsch, H. Spies, G. Wunderlich,
J. Kropp, A. Deussen, Nucl. Med. Commun. 2007, 28, 637.
[14] Y. Magata, T. Kawaguchi, M. Ukon, N. Yamamura, T. Uehara,
K. Ogawa, Y. Arano, T. Temma, T. Mukai, E. Tadamura, H. Saji,
Bioconjugate Chem. 2004, 15, 389.
References
[1] K. Yoshinaga, N. Tamaki, Curr. Opin. Biotech. 2007, 18, 52.
[2] K. Milger, T. Herrmann, C. Becker, D. Gotthardt, J. Zickwolf,
R. Ehehalt, P. A. Watkins, W. Stremmel, J. Fu¨llekrug, J. Cell Sci.
2006, 119, 4678.
[15] T. Chu, Y. Zhang, X. Liu, Y. Wang, S. Hu, X. Wang, Appl. Radiat.
Isotop. 2004, 60, 845.
[16] G. S. Jr. Jones, D. R. Elmaleh, H. W. Strass, A. J. Fichman, Bioorg.
Med. Chem. Lett. 1996, 6(20), 2399.
[3] N. Tamaki, K. Morita, Y. Kuge, E. Tsukamoto, J. Nucl. Med. 2000,
41, 1525–1534.
[17] G. S. Jr. Jones, D. R. Elmaleh, H. W. Strass, A. J. Fichman, Nucl. Med.
Biol. 1994, 21(1), 117.
[4] V. Dilsizian, T. M. Bateman, S.R. Bergmann, R. D. Prez,
M. Y. Magram, A. E. Goodbody, J. W. Babich, J. E. Udelson,
Circulation 2005, 112, 2169.
[5] C. J. Pastore, J. W. Babich, J. E. Udelson, J. Nucl. Cardiol. 2007, 14,
S153.
[18] R. H. Mach, H. F. Kung, P. Jungwiwattanaporn, Y. Guo, Nucl. Med.
Biol. 1991, 18(2), 215.
[19] Y. S. Kim, J. Wang, A. Broisat, D. K. Glover, S. Liu, J. Nucl. Cardiol.
2008, 15, 535.
[20] A. Boschi, L. Uccelli, C. Bolzati, A. Duatti, N. Sabba, E. Moretti, G. D.
Domenico, G. Zavattini, F. Refosco, M. Giganti, J. Nucl. Med. 2003,
44, 806.
[6] F. F. Jr Knapp, K. R. Ambrose, M. M. Goodman, Eur. J. Nucl. Med.
1986, 12, S539.
[7] F. F. Jr. Knapp, J. Kropp, Eur. J. Nucl. Med. 1995, 22, 361.
[8] E. Cazzola, E. Benini, M. Pasquali, P. Mirtschink, M. Walther,
H. J. Pietzsch, L. Uccelli, A. Boschi, C. Bolzati, A. Duatti,
Bioconjugate Chem. 2008, 19, 450.
[9] A. Mathur, S. Subramanian, M. B. Mallia, S. Banerjee, G. Samuel,
H. D. Sarma, M. Venkatesh, Bioorg. Med. Chem. 2008, 16, 7927.
[10] P. Mirtschink, S. N. Stehr, H. J. Pietzsch, R. Bergmann, J. Pietzsch,
G. Wunderlich, A. C. Heintz, J. Kropp, H. Spies, W. Kraus,
A. Deussen, M. Walther, Bioconjugate Chem. 2008, 19, 389.
[21] A. Boschi, C. Bolzati, E. Benini, E. Malago, L. Uccelli, A. Duatti,
A. Piffanelli, F. Refosco, F. Tisato, Bioconjugate Chem. 2001, 12,
1035.
[22] C. Bolzati, A. Mahmood, E. Malago, L. Uccelli, A. Boschi, A. G.
Jones, F. Refosco, A. Duatti, F. Tisato, Bioconjugate Chem. 2003,
14, 1279.
[23] E. C. Lisic, M. J. Heeg, E. Deutsch, Nucl. Med. Biol. 1999, 26, 563.
[24] C. Bolzati, M. C. Ceccato, S. Agostini, S. Tokunaga, D. Casara, G.
Bandoli, J. Nucl. Med. 2008, 49, 1336.
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