Anticancer Activity of an Imageable Curcuminoid 1-[2-Aminoethyl-(6-hydrazinopyridine-3-carbamidyl)-3,5-bis-(2-fluorobenzylidene)-4-piperidone (EFAH)

Article abstract of DOI:10.1111/j.1747-0285.2011.01271.x

3,5-Bis(2-fluorobenzylidine)-4-piperidone or EF24 is a potent anticancer derivative of curcumin. Using an amine derivative of EF24, we synthesized a hydrazinonicotinic acid conjugate, EFAH, for Tc-99m radiolabelling and single photon emission tomography imaging. The aqueous solubility of EFAH (3.5mg/mL) was significantly more than that of EF24 (1.2mg/mL); the octanol/water partition coefficient of EFAH was estimated at log P=0.33. As an antiproliferative agent, EFAH was as effective as EF24 in suppressing the proliferation of H441, MiaPaCa-2 and Panc-1 cells. Daily intraperitoneal injection of EFAH (5μg) for 3weeks in mice carrying xenografts of Panc-1 pancreatic cancer showed a mean tumour volume reduction of 79%; the tumour weight decreased by 82% in the treated group. For imaging and biodistribution, EFAH was labelled with Tc-99m (98% RCY) and intravenously administered in rats. Approximately 23.7% and 14.3% of injected dose accumulated in liver and intestine, respectively, suggesting that EFAH is mostly eliminated by hepatobiliary route. The results indicate that HYNIC modification of EF24 for Tc-99m radiolabelling does not affect its antiproliferative efficacy. For the first time, a visual biodisposition of EF24 in a live animal model has been demonstrated. Such knowledge could be of benefit in developing therapeutic curcuminoids, such as EF24.

Full text of DOI:10.1111/j.1747-0285.2011.01271.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01271.x
Chem Biol Drug Des 2012; 79: 194–201
Research Article
Anticancer Activity of an Imageable Curcuminoid
1-[2-Aminoethyl-(6-hydrazinopyridine-3-
carbamidyl)-3,5-bis-(2-fluorobenzylidene)-4-
piperidone (EFAH)
imaging can provide such enabling technologies where the ability
to image the distribution of drug may offer an important tool in
real-time management of therapy. Molecularly targeted drugs are
especially amenable to this strategy. Arguably, as drug distribution
may be heterogeneous even in histologically identical tumours,
image-based knowledge of drug accumulation in cancer tissue may
help in predicating the drug effect. The focus in our laboratory has
been to add imaging dimension to the development of anticancer
drugs by synthesizing related compounds that could be easily moni-
tored by non-invasive imaging. The role of such technology in has-
tening anticancer drug development is acknowledged in a Critical
Path Initiative by the FDA and NCI (1–3). Imaging provides both
structural and functional information under physiologic conditions
and eliminates the sampling errors inherent in other methods (4). It
is also possible to carry out longitudinal studies in the same subject
to collect time-point-specific data. We describe results of our study
on the development of a novel imageable curcuminoid.
Pallavi Lagisetty, Dharmalingam
Subramaniam, Kaustuv Sahoo, Shrikant
Anant and Vibhudutta Awasthi*
Department of Pharmaceutical Sciences and Small Animal Imaging
Facility, University of Oklahoma Health Science Center, 1110 N,
Stonewall Avenue, Oklahoma City, OK 73117, USA
*Corresponding author: Vibhudutta Awasthi, vawasthi@ouhsc.edu
3,5-Bis(2-fluorobenzylidine)-4-piperidone or EF24 is
a potent anticancer derivative of curcumin. Using
an amine derivative of EF24, we synthesized a hy-
drazinonicotinic acid conjugate, EFAH, for Tc-99m
radiolabelling and single photon emission tomogra-
phy imaging. The aqueous solubility of EFAH
(3.5 mg ⁄ mL) was significantly more than that of
EF24 (1.2 mg ⁄ mL); the octanol ⁄ water partition coef-
ficient of EFAH was estimated at log P = 0.33. As an
antiproliferative agent, EFAH was as effective as
EF24 in suppressing the proliferation of H441, Mia-
PaCa-2 and Panc-1 cells. Daily intraperitoneal injec-
tion of EFAH (5 lg) for 3 weeks in mice carrying
xenografts of Panc-1 pancreatic cancer showed a
mean tumour volume reduction of 79%; the tumour
weight decreased by 82% in the treated group. For
imaging and biodistribution, EFAH was labelled
with Tc-99m (98% RCY) and intravenously adminis-
tered in rats. Approximately 23.7% and 14.3% of
injected dose accumulated in liver and intestine,
respectively, suggesting that EFAH is mostly elimi-
nated by hepatobiliary route. The results indicate
that HYNIC modification of EF24 for Tc-99m radi-
olabelling does not affect its antiproliferative effi-
cacy. For the first time, a visual biodisposition of
EF24 in a live animal model has been demonstrated.
Such knowledge could be of benefit in developing
therapeutic curcuminoids, such as EF24.
Curcumin is an unsaturated polyphenolic b-ketone present in the
rhizome of Indian dietary spice turmeric (Curcuma longa L.). Several
investigations have demonstrated that curcumin is chemopreventive
in all three stages of carcinogenesis – initiation, progression and
promotion (5–9). The pharmacological safety and efficacy of curcu-
min make it an attractive therapeutic molecule, but the poor bio-
availability after oral administration remains a major barrier (10).
The problem of bioavailability has been attributed to its lack of
water solubility, inefficient absorption and rapid metabolism (11). It
is therefore of interest to modify the core structure of curcumin
with a goal of enhanced bioavailability and potency. Synthetic ana-
logues of curcumin with enhanced cytotoxicity and improved physi-
cochemical properties are now emerging (12–14). One of these
compounds is 3,5-bis(2-fluorobenzylidine)-4-piperidone (also known
as EF24) which has been shown to possess potent anticancer activ-
ity, both in vitro and in vivo (15).
Key words: cancer, curcuminoid, EF24, imaging, single photon emis-
sion tomography, Tc-99m
In this study, we report the synthesis of a modified EF24 derivative,
1-[2-aminoethyl-(6-hydrazinopyridine-3-carbamidyl)-3,5-bis-(2-fluorob-
enzylidene)-4-piperidone (EFAH). EFAH could be efficiently labelled
with a gamma ray-emitting Tc-99m radionuclide. We demonstrate
that the modification does not affect the antiproliferative activity of
3,5-bis(benzylidene)-4-piperidone series in cancer cells, both in vitro
and in vivo. Finally, we show that the Tc-99m-labelled analogue of
the compound provides quantitative information about biodistribu-
tion from single photon emission tomography (SPECT) in a rat
Received 24 May 2011, revised 21 September 2011 and accepted for
publication 16 November 2011
There is a growing understanding among cancer researchers about
the utility of visual, temporal and quantitative information pertain-
ing to the delivery of drug in tumour tissue. In most instances,
194

Imageable Curcuminoid EFAH
model. To our knowledge, this is the first image-based non-invasive
investigation of EF24.
eluted with 10% methanol in methylene chloride. The fractions con-
taining the pure compound were collected and concentrated to dry-
ness to obtain the compound 2 as a yellow solid (252 mg, 53%
yield, m.p. 151–152 ꢀC).
Methods and Materials
All reagents were obtained from commercial sources and were used
N’-(5-{2-[3,5-Bis-(2-fluorobenzylidene)-4-oxo-
piperidin-1-yl]-ethylcarbamoyl}-pyridin-2-yl)-
hydrazinecarboxylic acid tert-butyl ester (3)
Succunimidyl-6-boc-hydrazinonicotinic acid was synthesized in-house
following a reported method (16). Succunimidyl-6-boc-hydrazinoni-
cotinic acid (360 mg, 1.03 mmol) was added to compound 2
(280 mg, 0.79 mmol) in DMF containing diisopropyl ethylamine
(650 lL, 3.95 mmol). The reaction mixture was stirred at room tem-
perature for 48 h and monitored by TLC where a new spot
appeared with polarity in between the reactants. After completion
of the reaction, the solvent was evaporated to dryness, and the
pure conjugate was separated by column chromatography on silica
(200–300 mesh) with 10% methanol in chloroform. The fractions
containing the desired compound were collected and dried to yield
compound 3 as yellow foamy solid (290 mg, 62% yield, m.p. 123–
125 ꢀC). Rf (10% methanol in chloroform) = 0.40.
1
directly without further purification. H NMR spectra and 13C NMR
spectra were recorded at 300 and 75 MHz on Varian VX-300 (Varian
Inc., Palo Alto, CA, USA). The spectra were referenced to the resid-
ual protonated solvents. Abbreviations such as s, d, t, m, br and dd
used in the description of NMR spectra denote singlet, doublet,
triplet, multiplet, broad, and double doublet respectively. The chemi-
cal shifts and coupling constants were reported in d parts per mil-
lion (p.p.m.) and hertz (Hz), respectively. The mass spectra were
recorded by Finnigon MAT LCQ mass spectrometer (San Jose, CA,
USA). The NMR and mass spectroscopy data for the synthesized
compounds are provided in the supplemental document (Appendix
S1). The reverse-phase high-performance liquid chromatography (RP-
HPLC) was performed with Beckman Model 126 pump, 166 absor-
bance detector, Bioscan Model B-FC-300 radioisotope detector. HPLC
solvents consisted of water and acetonitrile with 0.1% trifluoroace-
tic acid. Radionuclide Tc-99m, as pertechnetate, was obtained from
OUHSC Nuclear Pharmacy (Oklahoma City, OK, USA). All intermedi-
ate and final products were monitored by thin layer chromatography
(TLC) on 250-lm silica plates. Where applicable, the compounds
were purified by column chromatography using 200–300 mesh silica
gel columns. Melting points were recorded on an Electrothermal
Mel-Temp melting point apparatus (Thermo Scientific, Waltham,
MA, USA). The reported melting points (ꢀC) are uncorrected.
N-{2-[3,5-Bis-(2-fluorobenzylidene)-4-oxo-piperidin-
1-yl]-ethyl}-6-hydrazinonicotinamide (4)
To deprotect compound 3, hydrochloric acid (55 lL of 11 N) was
added to a solution of compound 3 in 5 mL of methylene chloride
(120 mg, 0.20 mmol). The reaction mixture was stirred at room tem-
perature for 5 h, and a slower moving spot was obtained in TLC.
The reaction mixture was evaporated to dryness, and compound 4
was isolated as yellow solid. Methylene chloride and ether wash
was given to the solid to remove the impurities (86 mg, 87% yield,
m.p. 222–224ꢀC). Rf (30% MeOH in Chloroform) = 0.10.
3,5-Bis-(2-fluorobenzylidene)-4-piperidone (1)
Hydrochloric Acid gas (generated in situ) was bubbled into a solu-
tion of 4-piperidone hydrochloride monohydrate (3 g, 19.5 mmol) in
glacial acetic acid (80 mL) until a clear solution was obtained
(about 15 min). To the reaction mixture, 2-fluorobenzaldehyde (6 mL,
56.5 mmol) was added and the mixture was left at room tempera-
ture for 48 h. The crystals formed were filtered on a Buchner fun-
nel, washed with absolute ethanol (50 mL) and ether (50 mL), and
followed by drying to obtain 1 as yellow crystalline solid (5.71 g,
94% yield, m.p. 185–186 ꢀC). Free base of 1 was generated by
treating the acetate salt with 10% potassium carbonate solution.
Partition coefficient
The partition coefficient (KO ⁄ W) was determined in triplicate in octa-
nol ⁄ water system. The initial oil and water phases were prepared
as phases saturated in the other solvent. About 20 mL of octanol
was mixed with 20 mL deionized water in a screw-capped glass
bottle and agitated overnight at 25 ꢀC. The mixture was allowed to
settle for 6 h to separate water-rich octanol phase (oil phase), from
octanol-rich water phase (water phase). About 3.0 mg of drug was
dissolved in 2 mL of oil phase in a screw-capped glass tube, and
2 mL of water phase was added. The tubes were agitated over-
night followed by a 6 h equilibrium period. EFAH was spectrophoto-
metrically analysed in both the phases and estimated against a
calibration curve. KO ⁄ W was calculated as a ratio between drug in
oil and water phases.
1-(2-Aminoethyl)-3,5-bis-(2-fluorobenzylidene)-4-
piperidone (2)
2-Bromoethylamine hydrobromide (204 mg, 1.00 mmol), compound 1
(311 mg, 1.00 mmol), cesium carbonate (325 mg, 1.00 mmol) and
potassium iodide (166 mg, 1.00 mmol) were taken together in a
flask containing DMF (5 mL). The reaction mixture was heated at
80 ꢀC for 18 h. The progress of the reaction was monitored by an
appearance of a new spot in silica TLC. After the reaction was
complete, the reaction mixture was filtered and the solvent was
dried. The residue was dissolved in chloroform and washed with
saturated sodium chloride solution followed by water. The organic
phase was separated, dried over anhydrous sodium sulphate and
concentrated to obtain a crude yellow solid. The crude compound
was passed through a silica column, and the title compound was
Tc-99m-labelled N-{2-[3,5-Bis-(2-
fluorobenzylidene)-4-oxo-piperidin-1-yl]-ethyl}-6-
hydrazinonicotinamide (5)
About 300 lL of tricine solution (100 mg ⁄ mL in water) was added
to a solution of compound 4 (30 lg) in 30 lL water. Na99mTcO4
(2 mCi in 0.5 mL of saline) and 10 lL SnCl2 solution (1 mg ⁄ mL in
Chem Biol Drug Des 2012; 79: 194–201
195

Lagisetty et al.
1 N HCl) were added to the reaction mixture. The reaction mixture
was incubated at room temperature for 15 min. Quantitative radi-
olabelling (>95%) was obtained within 15 min of incubation at room
temperature. The labelling was followed by Whatman paper chro-
matography in acetone where free pertechnetate had an Rf of 0.8
and the labelled compound had an Rf of 0.1. Upon radio-RP-HPLC
using a gradient of acetonitrile (20–100%) at 1.5 mL ⁄ min at
254 nm, compound 5 eluted at 9.2 min without any contamination
of free pertechnetate (2.5 min).
tional Animal Care Committee of the University of Oklahoma Health
Sciences Center.
Biodistribution of Tc-99m-EFAH
Female athymic nude rats (125–150 g) were obtained from Harlan
Laboratories (Indianapolis, IN, USA), and housed in a controlled
environment with 12 h day ⁄ night cycle. The animals were allowed
to acclimatize at least 1 week before inoculation of lung adenocar-
cinoma H441 cells. On the day of tumour implantation, the animals
were anaesthetized with 2–3% isoflurane in oxygen stream. About
0.1 mL H441 cell suspension in phosphate-buffered saline (100 mil-
lion cells ⁄ mL) was subcutaneously injected in the left dorsal thigh
region. The animals were returned to their cages, and the tumour
was allowed to grow till a palpable tumour was visible in majority
of the animals.
Stability of Tc-99m label against plasma
challenge
The stability of Tc-99m labelling of EFAH was tested by challenging Tc-
99m-EFAH with plasma at 37 ꢀC for up to 24 h. Briefly, about 300 lL of
human plasma was incubated with 148 MBq (200 lL) of Tc-99m-EFAH.
After indicated times, a 10-lL aliquot of supernatant was injected into
a reverse-phase HPLC column (C-18, 10 lm Sonoma) eluted under a 20-
min gradient of acetonitrile (20–100%) at 1.5 mL⁄ min.
We investigated the distribution of Tc-99m-EFAH by imaging in a
NanoSPECT machine (Bioscan, Washington, DC, USA). The rats
were anaesthetized using 2–3% isoflurane in oxygen stream and
placed inside the detector. The body temperature was maintained
at 37 € 1 ꢀC by Minerve anaesthesia system (Bioscan). Approxi-
mately 500 lCi of Tc-99m-EFAH (ꢀ0.2 mL) was intravenously
injected in the tail vein. Planar whole-body images were acquired
at various times over 24 h. After the final imaging session at 24 h,
the rats were subjected to a biodistribution study. Briefly, the ani-
mals were euthanized by an intraperitoneal overdose of a euthana-
sia solution (Euthasol, Virbac Corporation, Fort Worth, TX, USA).
Various organs were excised, washed with saline, weighed, and
appropriate tissue samples were counted in an automated gamma
counter (Perkin-Elmer, Boston, MA, USA). Total blood volume, bone
and muscle mass were estimated as 5.7%, 10% and 40% of body
weight, respectively. A diluted sample of injected Tc-99-EFAH served
as a standard for comparison.
Cell culture and drug treatment
Human lung adenocarcinoma cell line NCI-H441 (ATCC Number:
HTB-174) was obtained from the American Type Culture Collection
(Manassas, VA, USA). H441 cells were maintained at 37 ꢀC with
5% CO2 in McCoy's 5A Medium (Invitrogen, Carlsbad, CA, USA)
supplemented with 5% heat-inactivated foetal bovine serum (FBS)
and gentamicin (Gibco Laboratories, Grand Island, NY, USA). Pancre-
atic cancer cells Panc-1 and MiaPaCa-2 were maintained in RPMI
1640 1· (Catalogue number 11 875) in 10% heat-inactivated FBS;
penicillin at 100 U and streptomycin at 100 lg ⁄ mL of medium were
added as antibiotics.
To evaluate the antiproliferative activity of synthesized compounds,
the cells were seeded in a 96-well flat bottom tissue culture plates
at a density of 5 · 103 cells per well. The cells were allowed to
attach and grow overnight. The test compounds were dissolved in
dimethyl sulfoxide (DMSO) and added to cells in the culture medium
supplemented with 5% FBS. The DMSO concentration was main-
tained at 0.1% per well. The control wells received equivalent vol-
ume of DMSO without any drugs. The cells were allowed to remain
in the treatment medium for 24, 48 and 72 h.
Mouse model of xenograft tumour
Panc-1 cells (ATCC) were grown in RPMI 1640, containing 10%
heat-inactivated foetal bovine serum (Sigma Chemical Co., St.
Louis, MO, USA) and 1% antibiotic–antimycotic solution (Media-
tech, Inc.) at 37 ꢀC in a humidified atmosphere of 5% CO2. Five-
week-old male athymic nude mice (The Jackson Laboratory, Bar
Harbor, ME, USA) were maintained with water and standard
mouse chow ad libidum. The animals were injected with 1 · 106
cells in the left and right flank and allowed to form xenografts.
EFAH was intraperitoneally administered daily (5 lg ⁄ injection) for
3 weeks until killing. Tumours were measured weekly with a Ver-
nier caliper, and the tumour volumes were calculated according to
the formula length · width · depth · 0.5236. At the end of the
treatment, the animals were killed, and the tumours were
removed and weighed.
Cell proliferation
The total number of cells after treatment was estimated by hexosa-
minidase assay (17). Briefly, the medium was removed and
hexosaminidase substrate solution in citrate buffer pH 5 (7.5 mM),
p-nitrophenol-N-acetyl-beta-D-glucosaminidase (Calbiochem, San Diego,
CA, USA), was added at 60 lL per well. The plate was incubated
at 37 ꢀC in 100% humidity for 30 min, before stopping the reaction
by adding 90 lL of 50 mM glycine containing 5 mM of EDTA (pH
10.4); absorbance was measured at 405 nm.
Data analysis
The in vitro biological data were analysed for significance of differ-
ence at p < 0.05 using PRISM 5.0 (GraphPad Software, Inc., La Jolla,
CA, USA). The in vivo biodistribution data were analysed for pre-
sentation as per cent injected dose per gram tissue as well as
Animal studies
The animal experiments were performed according to the NIH Ani-
mal Use and Care Guidelines and were approved by the Institu-
196
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Imageable Curcuminoid EFAH
accumulation per organ. All data were corrected for the decay of
Tc-99m radioactivity (physical decay T1 ⁄ 2 = 6 h).
was found to possess more aqueous solubility (1.2 versus
3.5 mg ⁄ mL, respectively). This may be of significance in the
design of aqueous parenteral formulation, as well as in altering
the oral bioavailability. According to the Lipinski's rule, drugs
with octanol ⁄ water partition coefficient (log P) <5.0 possess more
drug-likeness than the compounds with larger log P values (24).
We report that the log P value of EFAH in octanol ⁄ water system
is 0.33.
Results and Discussion
Despite recent advances in molecular and tumour biology in can-
cer and the introduction of several new agents, inclusive of
molecularly targeted therapies, the disease continues to be a con-
temporary challenge. Chemotherapy with anticancer drugs is the
mainstay for the treatment of cancer. Antineoplastic agents are
cytotoxic by design and can cause severe systemic adverse effects
in therapeutic doses. Curcumin and its synthetic analogues have
shown some selectivity towards cancer cells. For instance, it has
been shown that curcumin is antiproliferative in cancerous cells
without being toxic to normal cells (18). We also showed recently
that a congener of curcuminoid EF24 is not toxic to lung fibro-
blasts, but suppresses growth of cancer cells (19,20). To enable
labelling of EF24 with imageable radionuclide, we modified 3,5-
bis-(2-fluorobenzylidene)-4-piperidone to carry HYNIC moiety and
investigated whether the intrinsic anticancer activity of the com-
pound is preserved after the modification. The synthesis of EF24
was accomplished by acid-catalysed Claisen-Schmidt condensation
of 4-piperidone and 2-fluoro benzaldehyde, and has been reported
elsewhere (21,22).
When compound 4 was labelled with Tc-99m in the presence of tri-
cine and stannous chloride, over 98% of labelling yield was
obtained. Because of the ideal nuclear characteristics, Tc-99m
(T1 ⁄ 2 = 6 h, 140 KeV) remains the most commonly used radionuclide
for SPECT. It easily forms a stable complex with hydrazinonicotina-
mide. The labelled compound 5 was characterized by HPLC
(Figure 2). The retention time of Tc-99m-labelled EFAH was mea-
sured as 9.25 min. When challenged with human plasma, there
was negligible decomplexation of Tc-99m radioactivity for up to
24 h of incubation (Figure 2).
In vitro anticancer activity of EFAH
A significant concern in modifying drugs for radiolabelling purposes
is the tendency of modified drug to lose activity and ⁄ or potency.
Therefore, we evaluated EFAH in multiple cells lines and compared
its antiproliferative activity with that of EF24. The experimental data
demonstrated that EFAH was as effective as EF24 in H441, Panc-1
and MiaPaCa-2 cell lines. Cell proliferation was studied in cancer
cells in culture by estimating the decrease in the activity of lyso-
somal enzyme hexosaminidase (Figure 3). It is clear that EFAH pos-
sessed significant antiproliferative activity in all the three cells
tested, and the potency was comparable with that shown by EF24.
The results suggest that EFAH can be further developed as an im-
ageable anticancer compound.
Synthesis and Tc-99m radiolabelling
In our previous studies, we have shown that modification of
piperidinyl nitrogen in EF24 does not adversely affect its activity
(23). Here, we modified piperidinyl nitrogen with bromoethylamine
to obtain compound 2 (82% yield). Upon a reaction of N-hy-
droxysuccinimidyl-6-boc-hydrazino nicotinamide with the free pri-
mary amine in 2, compound 3 containing a hydrazinonicotinoyl
(HYNIC) moiety was obtained, which on deprotection afforded
compound 4 (Figure 1). Compared to the precursor EF24, EFAH
Biodistribution and imaging of Tc-99m-EFAH
The importance of imaging in cancer drug development is increas-
ing with advances in instrumentation, chemistry and imaging tech-
nologies (25). The objective behind this strategy has been to
enable monitoring of the adequacy and selectivity of drug accumu-
lation in pathologic tissue. A textbook example may be the pre-
therapy elucidation of ibritumomab distribution by imaging before
initiating radioimmunotherapy of non-Hodgkin's lymphoma with
Y-90-labelled ibritumomab or Zevalin (26). In the presented work,
we report a similar approach to the development of synthetic
curcuminoids.
We followed up our in vitro study by investigating in vivo biodis-
position of Tc-99m-EFAH in
a rat model of xenograft lung
tumour. Figure 4 shows the accumulation of radioactivity in vari-
ous organs after 24 h of injection. The accompanying whole-body
images are shown in Figure 5, and they confirm the overall
results obtained from biodistribution data. In the sequential
images through 24 h, the radiolabelled compound appeared to
travel from liver to intestine and into the faeces. No other organ
except liver and intestines accumulated significant radioactivity,
suggesting that its clearance depended on hepatobiliary route.
Figure 1: Synthesis of EFAH. The reactions conditions were as
follows: (i) bromoethylamine, KI, Cs2CO3, DMF, 80 ꢀC, 5 h; (ii) HY-
NIC, DMF, DIPEA, room temperature, 24 h; (iii) DCM, HCl, room tem-
perature, 2 h, and (iv) tricine, Na99mTcO4, Stannous chloride, room
temperature, 15 min.
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Lagisetty et al.
Figure 2: The stability of Tc-
99m-EFAH was analysed by
reverse-phase radioHPLC using a
20-min acetonitrile gradient (20–
100%). The labelled compound
was incubated with human plasma
at 37 ꢀC, and the appearance of
free Tc-99m-pertechnetate peak at
2.5–2.75 min (white arrow) was
monitored over time. Labelled com-
pound eluted at just <10 min (black
arrow).
A
B
C
Figure 3: EFAH inhibits proliferation of (A) lung adenocarcinoma H441 cells, and pancreatic cancer cells (B) MiaPaCa-2 and (C) Panc-1.
The cells were treated with 1 and 10 lM EFAH for 24–72 h, and the cell proliferation was measured by estimating hexosaminidase activity
as described in the text.
As expected, the tumour tissue accumulated measurable, but lit-
tle radiolabelled compound. The same observation was made in
the scintigraphic images. Previously, Ravindranath and Chandr-
asekhara (27) reported that the majority of tritiated (H-3) curcu-
min administered in rats eliminated in faeces. The in vivo
behaviour of Tc-99m-EFAH is suggestive of a stable complex of
Tc-99m. When the rat urine collected after 30 min of injection
was injected in HPLC column, no free Tc-99m radioactivity was
found (data not shown).
Efficacy of EFAH as an antiproliferative agent
To test whether EFAH is active as an antiproliferative agent in
vivo, we administered EFAH in a mouse model of xenograft
Panc-1 tumours. The xenograft tumours were allowed to grow to
a size of approximately 500 mm3, following which EFAH was
intraperitoneally administered on daily basis for 3 weeks. We
observed a remarkable reduction in tumour size after treatment
with EFAH. EFAH inhibited the growth of the tumours xenografts
(Figure 6), whereas the control tumours grew during the treat-
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Imageable Curcuminoid EFAH
A
B
Figure 4: Biodistribution of Tc-
99m-EFAH in nude rats carrying
H441 xenograft tumours. (A) Per
cent injected dose per gram of tis-
sue and (B) per cent injected dose
per organ. The rats were intrave-
nously injected with about 500 lCi
of Tc-99m-EFAH and euthanized
after 24 h for biodistribution.
ment period, reaching a size of over 2000 mm3. Panc-1 tumours
in the EFAH-treated animals were smaller throughout, measuring
approximately 434 mm3 at the end of the treatment period. The
excised tumours from the EFAH-treated mice averaged 0.45 g,
whereas those from the control group weighed about 2.5 g (Fig-
ure 6B).
Although the mechanism of action of EF24 is not clearly under-
stood, evidences suggest that it causes apoptosis via a redox-
dependent mechanism (28,29). EF24 and its analogues containing
dienone moiety are capable of serving as Michael acceptors. In cis-
platin-resistant ovarian cancer cells, EF24 was found to cause apop-
totic cell death by induction of PTEN and inhibition of AKT activities
(30). In a more elaborate in vivo study by Subramaniam, et al., (15)
EF24 was found to induce caspase-mediated apoptosis in HCT-116
colon cancer xenografts. At the same time, marked reduction in
AKT activity, as well as decreased cyclooxygenase-2, interleukin-8,
and vascular endothelial growth factor mRNA, and protein expres-
sion was reported (15). All these studies show that EF24-induced
apoptosis is accompanied by G2-M cell cycle arrest. Further, EF24
has been shown to suppress NF-kB signalling pathway through a
direct action on IjB kinase in lung, breast, ovarian and cervical can-
cer cells (31). NF-kB plays an important role in oncogenesis, and
there is significant evidence to hypothesize that the anticancer
activity of curcuminoids may be mediated by inhibition of NF-kB
Figure 5: Gamma camera images of rats injected with Tc-99m-
EFAH: rats injected with approximately 500 lCi of Tc-99m-EFAH
were imaged in a NanoSPECT system. A dynamic image acquisi-
tion of Tc-99m-EFAH kinetics in the first 30 min of intravenous
administration (upper panel) was followed by static images (at
0.5, 2, 4 and 24 h) showing accumulation of Tc-99m-EFAH in liver
and gut.
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Lagisetty et al.
Acknowledgments
A
The authors are thankful to the OUHSC-Nuclear Pharmacy for sup-
plying Tc-99m-pertechnetate radioactivity. The authors also acknowl-
edge the funding from National Cancer Institute (R03 CA143614-01).
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Additional Supporting Information may be found in the online ver-
sion of this article:
Appendix S1. NMR and mass spectroscopy data.
Please note: Wiley-Blackwell is not responsible for the content or
functionality of any supporting materials supplied by the authors.
Any queries (other than missing material) should be directed to the
corresponding author for the article.M
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