A novel ibuprofen derivative with anti-lung cancer properties: Synthesis, formulation, pharmacokinetic and efficacy studies

Article abstract of DOI:10.1016/j.ijpharm.2014.10.019

Phospho-non-steroidal anti-inflammatory drugs (phospho-NSAIDs) are a novel class of NSAID derivatives with potent antitumor activity. However, phospho-NSAIDs have limited stability in vivo due to their rapid hydrolysis by carboxylesterases at their carboxylic ester link. Here, we synthesized phospho-ibuprofen amide (PIA), a metabolically stable analog of phospho-ibuprofen, formulated it in nanocarriers, and evaluated its pharmacokinetics and anticancer efficacy in pre-clinical models of human lung cancer. PIA was 10-fold more potent than ibuprofen in suppressing the growth of human non-small-cell lung cancer (NSCLC) cell lines, an effect mediated by favorably altering cytokinetics and inducing oxidative stress. Pharmacokinetic studies in rats revealed that liposome-encapsulated PIA exhibited remarkable resistance to hydrolysis by carboxylesterases, remaining largely intact in the systemic circulation, and demonstrated selective distribution to the lungs. The antitumor activity of liposomal PIA was evaluated in a metastatic model of human NSCLC in mice. Liposomal PIA strongly inhibited lung tumorigenesis (>95%) and was significantly (p < 0.05) more efficacious than ibuprofen. We observed a significant induction of urinary 8-iso-prostaglandin F_2α in vivo, which indicates that ROS stress probably plays an important role in mediating the antitumor efficacy of PIA. Our findings suggest that liposomal PIA is a potent agent in the treatment of lung cancer and merits further evaluation.

Full text of DOI:10.1016/j.ijpharm.2014.10.019

International Journal of Pharmaceutics 477 (2014) 236–243
Contents lists available at ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
A novel ibuprofen derivative with anti-lung cancer properties:
Synthesis, formulation, pharmacokinetic and efficacy studies
Ka-Wing Cheng a,1, Ting Nie a,1, Nengtai Ouyang a,b, Ninche Alston a, Chi C. Wong a,c
,
George Mattheolabakis a, Ioannis Papayannis a, Liqun Huang a, Basil Rigas a,
*
a
Division of Cancer Prevention, Department of Medicine, Stony Brook University, Stony Brook, NY, USA
Medicon Pharmaceuticals, Inc., Stony Brook, NY, USA
b
c
Department of Medicine and Therapeutics, Chinese University of Hong Kong, Hong Kong
A R T I C L E I N F O
A B S T R A C T
Phospho-non-steroidal anti-inflammatory drugs (phospho-NSAIDs) are
Article history:
Received 9 May 2014
a
novel class of NSAID
derivatives with potent antitumor activity. However, phospho-NSAIDs have limited stability in vivo
due to their rapid hydrolysis by carboxylesterases at their carboxylic ester link. Here, we synthesized
phospho-ibuprofen amide (PIA), a metabolically stable analog of phospho-ibuprofen, formulated it in
nanocarriers, and evaluated its pharmacokinetics and anticancer efficacy in pre-clinical models of human
lung cancer. PIA was 10-fold more potent than ibuprofen in suppressing the growth of human non-small-
cell lung cancer (NSCLC) cell lines, an effect mediated by favorably altering cytokinetics and inducing
oxidative stress. Pharmacokinetic studies in rats revealed that liposome-encapsulated PIA exhibited
remarkable resistance to hydrolysis by carboxylesterases, remaining largely intact in the systemic
circulation, and demonstrated selective distribution to the lungs. The antitumor activity of liposomal PIA
was evaluated in a metastatic model of human NSCLC in mice. Liposomal PIA strongly inhibited lung
tumorigenesis (>95%) and was significantly (p < 0.05) more efficacious than ibuprofen. We observed a
Received in revised form 22 July 2014
Accepted 7 October 2014
Available online 11 October 2014
Keywords:
Lung cancer
Liposome
Phospho-ibuprofen amide
Ibuprofen
Xenografts
significant induction of urinary 8-iso-prostaglandin F2 in vivo, which indicates that ROS stress probably
a
plays an important role in mediating the antitumor efficacy of PIA. Our findings suggest that liposomal
PIA is a potent agent in the treatment of lung cancer and merits further evaluation.
ã 2014 Elsevier B.V. All rights reserved.
1. Introduction
renal toxicity (Henry and McGettigan, 2003; Wolfe et al., 1999;
Yoshikawa and Naito, 2011). Our group has developed novel
Lung cancer is the leading cause of cancer related deaths in both
males and females in the United States of America, accounting for
approximately 30% of all deaths due to cancer (Jemal et al., 2010).
Epidemiological studies have shown that daily administration of
derivatives of NSAIDs, such as phospho-ibuprofen (PI), by
covalently attaching to NSAIDs a diethyl-phosphate moiety via a
spacer molecule.
PIhasdemonstratedenhancedanticancerefficacyover ibuprofen
in inhibiting the growth of human colon cancer cell lines in vitro and
in human colon cancer xenografts in mice, without any significant
gastrointestinal toxicity (Xie et al., 2011). However, its efficacy in
vivo is suboptimal due to its poor pharmacokinetic properties.
Specifically, rapid hydrolysis by blood esterases and its poor
solubility render the bioavailability of intact PI below 5% (Wong
et al., 2012). Herein, we have synthesized phospho-ibuprofen-
amide (PIA), a metabolically stable amide analog of PI, formulated it
in liposomes, and evaluated the pharmacokinetics and anticancer
efficacy of liposome-encapsulated PIA.
ibuprofen,
a non-steroidal anti-inflammatory drug (NSAID)
reduces the risk of lung cancer (Harris et al., 2007). As with most
NSAIDs, however, long term use of ibuprofen is associated with
significant side effects, most prominently gastrointestinal and
Abbreviations: DCF-DA, 2,7-dichlorofluorescein diacetate; 8-iso-PGF2 , 8-iso-
a
prostaglandin F2 ; NSAIDs, Non-steroidal anti-inflammatory drugs; NSCLC, Non-
a
small-cell lung cancer; PIA, Phospho-ibuprofen amide; ROS, Reactive oxygen
species.
* Corresponding author at: Division of Cancer Prevention, HSC, T-17 Room 080,
Stony Brook, New York, USA. 11794-8173. Tel.: +1 631 444 9538; fax: +1 631 444
9553.
Incorporation of drugs in nanocarriers, such as liposomes
(Flenniken et al., 2006; Gabizon, 2001), dendrimers (Lee et al.,
2005) and polymeric micelles (Blanco et al., 2009; Sutton et al.,
2007), is a promising approach to overcome the limitations of
E-mail address: basil.rigas@stonybrook.edu (B. Rigas).
These authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.ijpharm.2014.10.019
0378-5173/ã 2014 Elsevier B.V. All rights reserved.

K.-W. Cheng et al. / International Journal of Pharmaceutics 477 (2014) 236–243
237
many anticancer drugs related to poor water solubility and
metabolic instability (Alexis et al., 2010; Peer et al., 2007). Recent
studies have shown that bioavailability and efficacy of poorly
soluble lipophilic anticancer agents can be improved by incorpo-
was purified by silica gel open column chromatography using
hexane:ethyl acetate (60:40, v/v) as the eluant. Fractions were
collected and their profiles were checked by high-performance
liquid chromatography (HPLC). The pure fractions were combined
and evaporated to give a slightly yellow liquid with a percent yield
ration in b-lapachone micelles (Blanco et al., 2010) and liposomes
(Mattheolabakis et al., 2012). The increased blood solubility of
these modified nanoparticles and the subsequent prolonged
circulation time decrease the dose needed to achieve the
equivalent pharmacological effect of the free compound. Our
group has demonstrated the successful use of liposomes to
improve the bioavailability and efficacy of PI (Nie et al., 2012).
Given the structural similarity between PI and PIA, we hypothe-
sized that the efficacy of PIA could be significantly improved using
liposome as a delivery vehicle.
Herein, we describe the synthesis and liposomal formulation of
PIA, a novel derivative of ibuprofen and report on its pharmacoki-
netic properties and efficacy in suppressing lung cancer in pre-
clinical models.
of 85%. 1H NMR (CDCl3):
d 0.9 (s, 6H), 1.2 (m, 6H), 1.3 (d, 3H), 1.6 (m,
4H), 1.8 (m, 5H), 2.5 (d, 2H), 3.6 (m, 1H), 4.05 (m, 8H), 5.5 (s, 1H), 7.1
(d, 2H), and 7.2 (d, 2H).
2.3. Preparation of liposome-encapsulated PIA
Liposomes were prepared as previously described (Nie et al.,
2012). Briefly, Soy-PC, DSPE–PEG (1,2-distearoyl-sn-glycero-3-
phosphoethanolamine-N-[amino(polyethylene glycol)-2000]; am-
monium salt) and PIA were dissolved in chloroform. The solution
was evaporated to a thin film, rehydrated with phosphate buffered
saline (PBS) and gradually extruded through double polycarbonate
membranes of 0.08- or 0.02-mm pore size (Waters, Milford, MA)
using an extruder device (Lipex Biomembranes, Vancouver, BC,
Canada). The final concentration of each component was 37 mg/mL
Soy-PC, 7.4 mg/mL DSPE–PEG and 20 mg/mL PIA. The non-
encapsulated PIA was separated from liposomes by extensive
dialysis against saline. The liposomes were freeze-dried in a
LABCONCO freeze drier (LABCONCO Co., Kansas, Missouri) under
the following conditions: ꢁ40 ꢂC, 50 mTorr. The amounts of PIA
incorporated into liposomes were determined by HPLC. The molar
ratios between soy PC, DSPE–PEG and PIA were determined by 1H
NMR spectroscopic analysis of freeze-dried liposomal PIA.
2. Material and methods
2.1. Reagents
All reagents and solvents were of ACS grade. 1H NMR spectra
were recorded on a Varian 400 spectrometer. Samples prepared for
NMR analysis were dissolved in CDCl3. Chemical shifts are reported
in ppm relative to TMS. Electron ionization mass spectra were
recorded on a Thermo Scientific DSQ (II) mass spectrometer.
Thin-layer chromatography (TLC) was performed on silica gel
sheets (Tiedel-deHaën, Sleeze, Germany) containing a fluorescent
indicator. Flash column chromatographic separations were carried
out using 60 Å silica gel (TSI Chemical Company, Cambridge, MA).
All experiments dealing with moisture- or air-sensitive com-
pounds were conducted under dry nitrogen. Proliferating cell
nuclear antigen (PCNA) antibody was from Cell Signaling (Danvers,
MA). Trx-1 and Trx-R antibodies were from Abcam (Cambridge,
MA).
2.4. Characterization of liposomal PIA
Size distribution of PIA-encapsulated liposomes was measured
by dynamic light scattering, using Zeta Plus with the BI-MAS
option (Brookhaven Instruments Co., Holtsville, NY). All measure-
ments were performed in triplicate at 25 ꢂC at a measurement
angle of 90ꢂ. The morphology of liposomes was examined by
transmission electron microscopy (TEM) with negative staining.
Briefly, the samples were prepared by wetting a carbon-coated grid
with a small drop of a diluted liposome or micelle solution. Upon
drying, they were stained with 1% uranyl acetate and 2%
phosphotungstic acid, air-dried at room temperature and viewed
under a FEI BioTwinG2 electron microscope (FEI, Hillsboro, OR).
2.2. Synthesis of PIA
2.2.1. Synthesis of N-(4-hydroxy-butyl)-2-(4-isobutyl-phenyl)-
propionamide
Ibuprofen (0.228 g, 1 mmol), 3-amino propanol (0.138 mL,
1.5 mmol) and enzotriazole-N,N,N0,N0-tetramethyl-uronium-hexa-
fluoro-phosphate (HBTU, 0.57 g, 1.5 mmol) were dissolved in 5 mL
of DMF containing diisopropylethylamine (DIPEA, 0.17 mL,
1 mmol). The reaction mixture was stirred at room temperature
for 4 h. The reaction was monitored by TLC. The remnant was
dissolved in ethyl acetate, and then washed sequentially with 1 M
HCl, saturated NaHCO3, distilled water, and brine, and dried over
anhydrous sodium sulfate (Na2SO4). After the solvent was
removed, the crude product was purified by flash column
chromatography to give N-(4-hydroxy-butyl)-2-(4-isobutyl-phe-
nyl)-propionamide as a white solid with a 95% yield. 1H NMR
2.5. Cell culture
A549, H23 and H358 human non-small-cell lung cancer (NSCLC)
cell lines were purchased from American Type Culture Collection
(ATCC, Manassas, VA). A549 was cultured in F12K medium and
H23 and H358 in RPMI medium, as recommended by ATCC.
2.6. Cytokinetic analyses
Cell viability was measured with the MTT assay (Roche
Diagnostics, Indianapolis, IN). For cell cycle analysis, cells were
fixed using cold 70% ethanol and stained with PI following
standard protocols prior to flow cytometric analysis. Apoptosis and
necrosis were assessed by staining cells with Annexin V-FITC and
propidium iodide (PI) and analyzing them by flow cytometry
following standard protocols (Kozoni et al., 2000).
(CDCl3):
d 0.9 (s, 6H),1.3 (d, 3H), 1.6 (m, 4H), 1.8 (m, 5H), 2.5 (d, 2H),
3.6 (m, 1H), 4.05 (m, 4H), 5.5 (s, 1H), 7.1 (d, 2H), and 7.2 (d, 2H).
2.2.2. Synthesis of phosphoric acid diethyl ester 4-[2-(4-isobutyl-
phenyl)-propionylamino]-butyl ester
Under nitrogen, diethylchlorophosphate (0.43 g, 1.25 mmol)
was added drop-wise to a solution of alcohol (1e, 0.299 g, 1 mmol)
in dichloromethane (10 mL) containing diisopropylethylamine
(0.17 mL, 1 mmol) and 4-(dimethylamino) pyridine (6 mg,
0.05 mmol). The reaction mixture was stirred overnight. The
reaction solution was washed with water (2 ꢀ 25 mL), dried over
anhydrous Na2SO4, filtered and concentrated. The crude residue
2.7. Oxidative stress assay
Reactive oxygen and nitrogen species (RONS) levels in cultured
cells were determined using the general RONS probe dichlorodi-
hydrofluorescein diacetate (DCF-DA). A549, H23 or H358 cells
treated with PIA for 1.5 h were loaded with DCF-DA, and

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K.-W. Cheng et al. / International Journal of Pharmaceutics 477 (2014) 236–243
fluorescence intensity was analyzed on a FACScaliber following
standard protocols (BD Bioscience).
3. Results
The effect of PIA or liposomal PIA on the redox state of the mice
bearing orthotopic lung tumors was assessed by measuring the
levels of urinary 8-iso-prostaglandin F2 (8-iso-PGF2 ) using an
3.1. Preparation and characterization of liposomal PIA
Liposomal PIA was prepared as described in Section 2. PIA is
more hydrophobic compared to ibuprofen (octanol–water parti-
tion coefficient, Log P = 4.78 vs. 3.75), and thus could be more
efficiently incorporated in liposomes. The average hydrodynamic
radius of PIA-encapsulated liposomes was 220 nm. Negative-
staining TEM (Fig. 1A) shows that the liposomes were homoge-
nous, unilamellar and round vesicles with average diameter in
accordance with the hydrodynamic diameter. The lipid to drug
loading ratio of liposomal PIA was determined by 1H NMR
spectroscopy to be 1:1. In contrast, ibuprofen was virtually
incompatible with the liposome carrier as evidenced by a 1:0 lipid
to drug ratio. As shown in Fig. 1B, cellular uptake of PIA was much
higher than that of ibuprofen (18 ꢃ 1.2 vs. 0.1 ꢃ0.05 nmol/mg
protein, mean ꢃ SEM for this and all subsequent values), and
liposomal formulation of PIA further enhanced its already superior
uptake by over 4-fold.
a
a
ELISA kit (Oxford Biomedical Research, MI, USA).
2.8. Pharmacokinetic and tissue distribution studies
A single dose of PIA 200 mg/kg suspended in corn oil was given
to mice by i.p. injection. Liposomal PIA was administered to rats
with a single i.v. dose of 200 mg/kg. After treatment, the animals
were sacrificed at designated time points, and blood was collected
and immediately centrifuged to obtain the plasma. Acetonitrile
(2ꢀ the volume of plasma) was added to the plasma samples,
mixed and centrifuged at 13,000 ꢀ g for 15 min. The supernatants
were analyzed by HPLC.
For analysis of tissue distribution, PIA was administered to mice
by i.p. injection and liposomal PIA by i.v. injection, and the animals
were euthanized 1 h post treatment. Organs were excised and
snap-frozen in liquid nitrogen and stored at ꢁ80 ꢂC until analysis.
Accurately weighed amounts of the organs were homogenized
individually in PBS (pH 7.4), sonicated, and extracted with
acetonitrile. Subsequent steps for drug level determination were
the same as those applied to the plasma samples.
3.2. PIA inhibits the growth of NSCLC human lung cancer cells
To evaluate the effect of the chemical modification of ibuprofen
on its anticancer activity, we determined the 24-h IC50 values of PIA
and ibuprofen in A549, H23 and H358 NSCLC cell lines (Fig. 1C). PIA
demonstrated a substantially enhanced cytotoxicity (7- to >14-fold)
compared to ibuprofen in the three cell lines. The potent growth
2.9. In vivo efficacy in lung cancer xenografts
All animal experiments were performed with the approval of the
Institutional Animal Care and Use Committee, State University of
NewYork atStonyBrook.Femaleathymicnudemice(6–7weeks old)
were purchased from Harlan (Harlan Industries, Indianapolis, IN).
Afteracclimation for 1week, 6 ꢀ106 A549 cellswere injectedi.v. into
the tail vein of the animals. Two weeks post implantation when the
tumors were established in the lungs (monitored by fluorescence
imaging; Maestro, Wobum, MA), the mice were randomized into the
vehicle and PIA treatment groups (n = 6). PIA was suspended in corn
oil and given to the animals by i.p. injection at a dose of 200 mg/kg,
once per day, five days per week for 6 weeks.
To evaluate whether liposomal incorporation of PIA could
improve its anticancer efficacy and whether PIA is significantly
better than its parent NSAID ibuprofen, we performed another
study using the following treatment groups: vehicle, ibuprofen and
liposomal PIA, which were administered to the animals by tail vein
injection once a week at 200 mg/kg.
inhibitoryeffectofPIAresultsfromitscytokineticeffect.At1.5 ꢀ IC50
,
PIA significantly induced apoptosis in the cell lines by 2- to 5-fold
relativetocontrol(Fig.2A).Equimolarconcentrationofibuprofendid
not appreciably induce apoptosis. Even at a concentration equal to
4 times that of PIA, ibuprofen only moderately induced apoptosis by
<2-fold. PIA alsoinhibitedcell cycle progression by arresting it atthe
G1 phase(Fig. 2B). The G1 phasecell populationincreasedfrom62.8%
to 77.9% in A549 cells, 45.0–56.0% in H23 cells, and 51.0–62.1% in
H358 cells after 24-h treatment with 0.75 ꢀ IC50 PIA. Ibuprofen, at
4 times the concentration of PIA, exhibited a comparable G1 phase
arresting effect in the H23 and H358 cell lines, but failed to do so in
the A549 cells.
3.3. PIA inhibits the growth of lung cancer xenografts
To assess the in vivo efficacy of PIA against NSCLC, we treated
athymic nude mice bearing orthotopic GFP-A549 xenografts (n = 6)
with vehicle control or PIA administered i.p. Relative fluorescence
intensity of GFP and lung weight were used to assess the antitumor
efficacy of PIA (Fig. 3). The average tumor volume of the PIA-
treated mice was 74% lower (p < 0.001) than that of the control
group assessed by GFP luminosity at sacrifice. In addition, the
average tumor weight of the PIA-treated group was 67% lower than
that of the control. The average lung weight from another group of
mice not implanted with cancer cells or subjected to vehicle or PIA
treatment served as the normal control. Noticeably, the lung
weight of the vehicle-treated group was 5.7-fold higher than that
of normal mice. PIA i.p. treatment maintained lung weight of the
mice near the normal levels. PIA also appeared to prevent health
deterioration caused by the disease. By week 8, the average body
weight of the PIA treatment group was essentially the same as that
at baseline, whereas the vehicle control group had lost ꢄ30% of
their body weights (Fig. 3B).
At sacrifice, lungs and tumors were excised from the animals
and their images were taken on the Maestro imaging machine.
Immediately following this step, the lungs (including the
orthotopic xenografts) and other organs/tissues were snap-frozen
in liquid nitrogen or preserved in formalin for subsequent analysis.
2.10. Immunohistochemistry
Tissue samples were fixed in 10% buffered formalin and
embedded in paraffin, and 4 mm-thick sections were placed on
slides. The tissue sections were dewaxed and rehydrated according
to standard protocols. The expression of PCNA, Trx-1 and Trx-R was
determined by immunohistochemistry (IHC) staining following
standard protocols. Scoring was performed according to the
method reported in our previous study (Huang et al., 2011).
2.11. Statistical analyses
To test our hypothesis that PIA is metabolically stable in vivo and
that intact PIA is the pharmacological active compound, we
analyzed its pharmacokinetic profile in the plasma and its tissue
distribution. In agreement with our expectations, intact PIA was
the dominant compound detected in the blood and organs (Fig. 3C
Data are expressed as mean ꢃ SEM. Results were analyzed using
the Student’s t-test. P values <0.05 were considered statistically
significant.

K.-W. Cheng et al. / International Journal of Pharmaceutics 477 (2014) 236–243
239
Fig. 1. Phospho-ibuprofen amide (PIA) is a more potent anticancer agent than ibuprofen in vitro. (A) PIA was formed by conjugation of ibuprofen to a diethyl phosphate group
through an amide bond. Left: Ibuprofen and PIA. Right: Transmission electron microscopic image of liposomal PIA. (B) PIA is more hydrophobic than ibuprofen. A549 cells were
treated with PIA or ibuprofen. Drug uptake was determined by HPLC and expressed as nmol/mg protein. PIA, but not ibuprofen, could be encapsulated in liposomes with a 1:1
lipid:drug ratio. (C) Cytotoxicity of ibuprofen and PIA in non-small-cell lung cancer cell lines. IC50 values were determined by the MTT assay and summarized in the table.
Fig. 2. The effect of PIA and ibuprofen on cytokinetics in NSCLC cell lines. Cells were treated with PIA or ibuprofen. (A) Apoptosis was determined by flow cytometry in cells
stained with Annexin-V (abscissa) and PI (ordinate). PIA 1 ꢀ IC50 strongly induced apoptosis in A549 (upper), H23 (middle) and H358 (bottom) cells. Ibuprofen (4ꢀ the
concentration of PIA) only moderately induced apoptosis. (B) Cell cycle analysis was performed on PI stained cells by flow cytometry. Percentages of cells in G1, S and
G2 phases are shown in the histograms.

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K.-W. Cheng et al. / International Journal of Pharmaceutics 477 (2014) 236–243
Fig. 3. Xenograft inhibitory effect and biodistribution of PIA in mice. GFP-transfected A549 cells (5 ꢀ106) were injected into athymic nude mice through a tail vein. Treatment
with vehicle or PIA (i.p. 200 mg/kg/day, 6 days/week) was started after two weeks when orthotopic tumors were established (monitored by in vivo imaging; Maestro, Wobum,
MA). (A) PIA significantly (74%, p < 0.001) inhibited the growth of orthotopic lung cancer xenografts. Upper: Representative images of the lungs (cancerous and non-cancerous
tissues) of mice from the vehicle and PIA-treated group at sacrifice, respectively. Lower left: GFP luminosity of the lungs of the vehicle vs. the PIA-treatment group. Lower right:
Average lung weight of the vehicle and PIA-treatment group. (B) Change in body weights of the mice during the study period. (C) A representative HPLC chromatogram of a
blood sample from a PIA-treated mouse and the UV-absorption spectrum of PIA. (D) Pharmacokinetic profiles of PIA in mice. Mice were treated with a single dose of PIA (i.p.
200 mg/kg) and euthanized at designated time points. Levels of PIA in the blood (left) and different organs (right) were determined by HPLC.
and D). The plasma level of PIA peaked 1 h post drug treatment,
gradually decreased toward later time points and became
undetectable after 4 h. These data suggest that the novel PIA is
highly resistant to hydrolysis by carboxylesterases, in contrast to
phospho-ibuprofen (Nie et al., 2012). It was also found that PIA
administered by i.p. injection was predominantly subjected to
liver/kidney metabolism/excretion, with only a small percentage
(<5%) being delivered to the lungs.
(p < 0.001) relative to vehicle control (Fig. 4B). In addition, the
average lung weight of the PIA-treated group was 80% lower than
that of the control. At the equivalent dose, ibuprofen was much less
effective,asindicatedbyonly55%reductioninGFPfluorescenceand
20% reduction in lung weight compared to the control group. Of
note, liposomal PIA was administered only once a week, which
equated only one-sixth of the total drug dose of free PIA used in the
other efficacy study. Thus, liposomal formulation proved to
profoundly enhance the efficacy of PIA in inhibiting the growth
of A549 lung cancer xenografts. IHC staining of paraffin-embedded
lung cancer xenograft sections with PCNA showed that liposomal
PIA significantly (p < 0.05) decreased the percentage of actively
proliferating cells compared to controls (Fig. 4C).
3.4. Liposomal incorporation targets PIA to the lungs and enhances its
anticancer activity
To explore the feasibility to selectively deliver PIA to the lungs
by encapsulating it in liposomes, we evaluated the pharmacoki-
netics and tissue distribution of liposomal PIA in rats after a single
i.v. administration of the drug (equivalent to 200 mg/kg PIA). In the
plasma (Fig. 4A), intact PIA was the predominant form which
3.5. PIA induces oxidative stress in vivo and in vitro
Our previous study (Sun et al., 2012) showed that phospho-
ibuprofen induces oxidative stress in breast cancer cell lines by
suppressing the thioredoxin (Trx) system, which plays an
important role in mediating its anticancer activity. Hence, we
analyzed the change of the status of oxidative stress in mice
bearing A549 xenografts after treatment with PIA. The results
showed that PIA given i.p. and liposomal PIA given i.v. both
significantly (p < 0.01) induced oxidative stress in vivo, as reflected
peaked (Cmax = 5 mM) at 30 min, gradually decreased and was
undetectable after 2.5 h. Tissue distribution analysis showed that
liposomal PIA preferentially delivered PIA to the lungs, reaching
2.3 nmol/mg protein, which was significantly higher than the
levels in other organs.
To assess whether the selective delivery of liposomal PIA to the
lungs could be translated into enhanced anticancer activity, we
tested the effect of i.v. liposomal PIA (200 mg/kg, once/week) on the
growth oforthotopicA549 xenografts. We alsocompareditseffectto
that of ibuprofen. Remarkably, fluorescence imaging showed that
liposomal PIA inhibited the growth of A549 xenografts by 95%
by the much higher levels of urinary 8-iso-PGF2 in the PIA-treated
a
group relative to control (Fig. 5A). Immunohistochemical staining
of the orthotopic xenograft sections showed that PIA significantly

K.-W. Cheng et al. / International Journal of Pharmaceutics 477 (2014) 236–243
241
Fig. 4. Biodistribution of liposomal PIA and its xenograft inhibitory effect in vivo. (A) Pharmacokinetic profiles of liposomal PIA in rats. Rats were treated with a single dose of
liposomal PIA (i.v. 200 mg/kg) and euthanized at designated time points. Levels of PIA in the blood (upper) and different organs (lower) were determined by HPLC. (B) GFP-
transfected A549 cells (5 ꢀ106) were injected into athymic nude mice through a tail vein. Treatment with vehicle, ibuprofen or PIA (i.v. 200 mg/kg/day, once/week) was
started after two weeks when orthotopic tumors were established. Upper: Representative images of the lungs from the vehicle, ibuprofen, and liposomal PIA treatment group
at sacrifice, respectively. Lower: Percent GFP luminosity and weight of the lungs (cancerous and non-cancerous tissues) from the ibuprofen and liposomal PIA treatment
groups, relative to the vehicle control. (C) Immunostaining of paraffin-embedded tissue sections from the orthotopic A549 xenografts. Results are expressed as percentage
positively stained cells. Left: Representative images of the PCNA-stained tissue sections from the vehicle and PIA-treated groups. . Right: Histogram showing percent PCNA-
positive cells of the tissue sections from the vehicle and PIA treatment groups.
suppressed the expression of Trx-1 and Trx reductase (Fig. 5B),
which likely contributed to the induction of oxidative stress in vivo.
In vitro, treatment of A549 cells with PIA at 1.5 ꢀ IC50 for 1.5 h
significantly induced the level of intracellular ROS by 4.9-fold
(Fig. 5C). Further analysis revealed that PIA treatment also
increased the levels of ROS in the H23 and H358 NSCLC cell lines
by 17% and 65%, respectively. The equimolar doses of ibuprofen
failed to induce ROS in these cell lines. Even at doses equal to 3–
4 times those of PIA, ibuprofen was only able to induce ROS by 40%,
3%, and 8% in A549, H23 and H358 cells, respectively. These data
suggest that the induction of oxidative stress may play an
important role in mediating the activity of PIA against human
NSCLC in pre-clinical models.
suppressed the growth of orthotopic A549 lung tumors (75%
inhibition) in immunodeficient mice, and this effect was further
enhanced (95% inhibition) by its formulation in liposomes. The
parent NSAID ibuprofen was much less efficacious, inhibiting
tumor growth by about half (55%). These results indicate that, PIA,
especially liposome-incorporated PIA, is a potent inhibitor of lung
cancer.
Our data suggest the induction of oxidative stress as an
important mechanism of cell death caused by PIA. This effect was
noted both in cultured cells, by using the ROS probe DCF-DA, and in
animals, by assaying urinary levels of 8-iso-PGF2 , a widely used
a
marker of oxidative stress in vivo (Milne et al., 2005). This finding is
in agreement with previous studies (Huang et al., 2010; Mackenzie
et al., 2010, 2011), which showed that ROS induction is a pivotal
event in the anticancer effect of phospho-NSAIDs. Cancer cells are
often in a heightened state of oxidative stress (Diehn et al., 2009).
Hence, they are vulnerable to excess ROS (Fruehauf and Meyskens,
2007), and the induction of ROS may underlie the potent effect of
PIA in suppressing tumor growth.
A critical contributing factor to the potent anticancer activity of
PIA is its metabolic stability. PIA is an amide derivative of phospho-
ibuprofen. Phospho-ibuprofen, whilst being considerably more
cytotoxic toward cancer cells than ibuprofen, displayed only
moderately improved efficacy in vivo. In vivo, phospho-NSAIDs are
susceptible to hydrolysis at their carboxylic ester bond by
carboxylesterases in the liver, intestine or plasma (Nie et al.,
2012). Phospho-ibuprofen, for example, is rapidly hydrolyzed by
carboxylesterase 1 to give the considerably less active ibuprofen
4. Discussion
The present study establishes that PIA is a novel agent with
significantly improved anticancer activity over its parent NSAID,
ibuprofen. The anticancer efficacy of PIA is mediated by (i) its
potent cytokinetic effect on lung cancer cells; and (ii) its
remarkable metabolic stability against esterase-mediated inacti-
vation. Furthermore, incorporation of PIA into liposomes signifi-
cantly enhanced its antitumor efficacy by improving its
pharmacokinetics and targeting it to the lungs.
PIA is a potent inhibitor of lung cancer cell growth in vitro (ꢄ10-
fold more potent than ibuprofen). PIA exerts a potent cytokinetic
effect, which is a result of induction of apoptosis, inhibition of cell
proliferation and of cell cycle progression (G0/G1). In vivo, PIA

242
K.-W. Cheng et al. / International Journal of Pharmaceutics 477 (2014) 236–243
Fig. 5. PIA induces oxidative stress in vivo and in vitro. (A) Urinary 8-iso-prostaglandin F2 (8-iso-PGF2 ) levels of athymic nude mice bearing A549 xenografts treated with
a
a
vehicle or PIA. Left: PIA, i.p. 200 mg/kg/day, 6 days/week for 6 weeks. Right: i.v. 200 mg/kg/day, once/week for 6 weeks. (B) Immunostaining of paraffin-embedded tissue
sections from the orthotopic A549 xenografts. Upper left: Representative images of Trx-1 stained tissue sections. Lower left: Representative images of Trx-R stained tissue
sections. Right: Histograms showing IHC scores of the tissue sections from the different treatment groups. (C) PIA induced ROS in A549, H23 and H358 cells more potently than
ibuprofen. Cells were treated with PIA 1.5 ꢀ IC50 or ibuprofen (4ꢀ the concentration of PIA) for 1.5 h, stained with DCF-DA and analyzed by flow cytometry. Left: Histograms
showing the induction of ROS by PIA vs. ibuprofen in different NSCLC cell lines. Percentages of ROS induction by PIA and by ibuprofen are summarized in the table.
(Wong et al., 2012). PIA, on the other hand, exhibits remarkable
metabolic stability, as manifested in its resistance to hydrolysis by
carboxylesterases. As a consequence, PIA evades carboxylesterase-
mediated inactivation and remains predominantly intact in the
circulation of mice, thereby improving the delivery of the potent,
intact drug to lung tumors.
The phospho-modification has an important impact on the
physicochemical properties of NSAIDs. PIA is significantly more
hydrophobic than ibuprofen and is only sparsely soluble in water.
This property allows PIA to permeate cell membranes readily
(confirmed by the 180-fold enhanced cellular uptake), but it limits
bioavailability and prevents the use of i.v. administration due to the
high risk of drug aggregation in the circulation. Although i.p.
administration of PIA demonstrated much better bioavailability
than the oral route (data not shown), it did not result in preferential
drug delivery to the lungs. Therefore, the therapeutic efficacy
achieved with i.p. delivery was sub-optimal.
other organs. The authors thus proposed liposome to be a
promising carrier to target the anticancer drug to the lungs for
lung cancer treatment. Because of the targeted delivery, i.v.
administration of liposomal PIA only required one-sixth of the
dosage of free PIA given by i.p. over the study period to achieve the
nearly complete tumor elimination. In addition, liposomes
facilitate tumor targeting via the enhanced permeability and
retention (EPR) effect whereby they extravasate through the
“leaky” vasculature of tumors (Maeda et al., 2000). All these factors
may contribute to the potent efficacy and enhanced therapeutic
index of liposomal PIA in the inhibition of lung tumor growth.
In conclusion, our findings suggest that PIA is a promising agent
against human NSCLC and that liposomes enhance its intrinsic
anticancer effect, greatly improving its therapeutic index.
References
The anticancer efficacy of PIA can be augmented through
incorporation into liposomes. Nanocarriers, such as liposomes, are
well-established vehicles for the solubilization of hydrophobic
drugs and for improving pharmacokinetic properties. We have
demonstrated highly efficient loading of PIA in liposomes, with a
drug:lipid ratio of 1:1. The incorporation of PIA into liposomes
increased its already superior ability to penetrate cells compared to
ibuprofen in vitro. In vivo biodistribution studies indicate that
liposomal PIA was preferentially delivered to the lungs, resulting in
higher levels than those in other organs. A recent study (Wei et al.,
2014) reported the capability of liposomes to alter the biodis-
tribution of paclitaxel, manifested in the significantly enhanced
delivery of the drug to the lungs by a factor of 14–20 compared to
Alexis, F., Pridgen, E.M., Langer, R., Farokhzad, O.C., 2010. Nanoparticle technologies
for cancer therapy. Handb. Exp. Pharmacol. 55–86.
Blanco, E., Kessinger, C.W., Sumer, B.D., Gao, J., 2009. Multifunctional micellar
nanomedicine for cancer therapy. Exp. Biol. Med. (Maywood) 234, 123–131.
Blanco, E., Bey, E.A., Khemtong, C., Yang, S.G., Setti-Guthi, J., Chen, H., Kessinger, C.W.,
Carnevale, K.A., Bornmann, W.G., Boothman, D.A., Gao, J., 2010. Beta-lapachone
micellar nanotherapeutics for non-small cell lung cancer therapy. Cancer Res.
70, 3896–3904.
Diehn, M., Cho, R.W., Lobo, N.A., Kalisky, T., Dorie, M.J., Kulp, A.N., Qian, D., Lam, J.S.,
Ailles, L.E., Wong, M., Joshua, B., Kaplan, M.J., Wapnir, I., Dirbas, F.M., Somlo, G.,
Garberoglio, C., Paz, B., Shen, J., Lau, S.K., Quake, S.R., Brown, J.M., Weissman, I.L.,
Clarke, M.F., 2009. Association of reactive oxygen species levels and radio-
resistance in cancer stem cells. Nature 458, 780–783.
Flenniken, M.L., Willits, D.A., Harmsen, A.L., Liepold, L.O., Harmsen, A.G., Young, M.J.,
Douglas, T., 2006. Melanoma and lymphocyte cell-specific targeting incorpo-
rated into a heat shock protein cage architecture. Chem. Biol. 13, 161–170.

K.-W. Cheng et al. / International Journal of Pharmaceutics 477 (2014) 236–243
243
Fruehauf, J.P., Meyskens Jr., F.L., 2007. Reactive oxygen species: a breath of life or
death? Clin. Cancer Res. 13, 789–794.
Mattheolabakis, G., Nie, T., Constantinides, P.P., Rigas, B., 2012. Sterically stabilized
liposomes incorporating the novel anticancer agent phospho-ibuprofen (MDC-
917): preparation, characterization, and in vitro/in vivo evaluation. Pharm. Res.
29, 1435–1443.
Gabizon, A.A., 2001. Pegylated liposomal doxorubicin: metamorphosis of an old
drug into a new form of chemotherapy. Cancer Invest. 19, 424–436.
Harris, R.E., Beebe-Donk, J., Alshafie, G.A., 2007. Reduced risk of human lung cancer
by selective cyclooxygenase 2 (COX-2) blockade: results of a case control study.
Int. J. Biol. Sci. 3, 328–334.
Milne, G.L., Musiek, E.S., Morrow, J.D., 2005. F2-isoprostanes as markers of oxidative
stress in vivo: an overview. Biomarkers 10, S10–S23.
Nie, T., Wong, C.C., Alston, N., Aro, P., Constantinides, P.P., Rigas, B., 2012. Phospho-
ibuprofen (MDC-917) incorporated in nanocarriers: anti-cancer activity in vitro
and in vivo. Br. J. Pharmacol. 166, 991–1001.
Henry, D., McGettigan, P., 2003. Epidemiology overview of gastrointestinal and
renal toxicity of NSAIDs. Int. J. Clin. Pract. Suppl. 43–49.
Huang, L., Zhu, C., Sun, Y., Xie, G., Mackenzie, G.G., Qiao, G., Komninou, D., Rigas, B.,
2010. Phospho-sulindac (OXT-922) inhibits the growth of human colon cancer
cell lines: a redox/polyamine-dependent effect. Carcinogenesis 31, 1982–1990.
Huang, L., Mackenzie, G.G., Sun, Y., Ouyang, N., Xie, G., Vrankova, K., Komninou, D.,
Rigas, B., 2011. Chemotherapeutic properties of phospho-nonsteroidal anti-
inflammatory drugs, a new class of anticancer compounds. Cancer Res. 71,
7617–7627.
Peer, D., Karp, J.M., Hong, S., Farokhzad, O.C., Margalit, R., Langer, R., 2007.
Nanocarriers as an emerging platform for cancer therapy. Nat. Nanotechnol. 2,
751–760.
Sun, Y., Rowehl, L.M., Huang, L., Mackenzie, G.G., Vrankova, K., Komninou, D., Rigas,
B., 2012. Phospho-ibuprofen (MDC-917) suppresses breast cancer growth: an
effect controlled by the thioredoxin system. Breast Cancer Res. 14, R20.
Sutton, D., Nasongkla, N., Blanco, E., Gao, J., 2007. Functionalized micellar systems
for cancer targeted drug delivery. Pharm. Res. 24, 1029–1046.
Wei, Y., Xue, Z., Ye, Y., Huang, Y., Zhao, L., 2014. Paclitaxel targeting to lungs by way of
liposomes prepared by the effervescent dispersion technique. Arch. Pharm. Res.
37, 728–737.
Jemal, A., Siegel, R., Xu, J., Ward, E., 2010. Cancer statistics: 2010. CA Cancer J. Clin. 60,
277–300.
Kozoni, V., Tsioulias, G., Shiff, S., Rigas, B., 2000. The effect of lithocholic acid on
proliferation and apoptosis during the early stages of colon carcinogenesis:
differential effect on apoptosis in the presence of
Carcinogenesis 21, 999–1005.
Lee, C.C., MacKay, J.A., Frechet, J.M., Szoka, F.C., 2005. Designing dendrimers for
biological applications. Nat. Biotechnol. 23, 1517–1526.
a
colon carcinogen.
Wolfe, M.M., Lichtenstein, D.R., Singh, G., 1999. Gastrointestinal toxicity of
nonsteroidal antiinflammatory drugs. N. Engl. J. Med. 340, 1888–1899.
Wong, C.C., Cheng, K.W., Xie, G., Zhou, D., Zhu, C.H., Constantinides, P.P., Rigas, B.,
2012. Carboxylesterases
1 and 2 hydrolyze phospho-nonsteroidal anti-
Mackenzie, G.G., Sun, Y., Huang, L., Xie, G., Ouyang, N., Gupta, R.C., Johnson, F.,
Komninou, D., Kopelovich, L., Rigas, B., 2010. Phospho-sulindac (OXT-328), a
novel sulindac derivative, is safe and effective in colon cancer prevention in
mice. Gastroenterology 139, 1320–1332.
Mackenzie, G.G., Ouyang, N., Xie, G., Vrankova, K., Huang, L., Sun, Y., Komninou, D.,
Kopelovich, L., Rigas, B., 2011. Phospho-sulindac (OXT-328) combined with
difluoromethylornithine prevents colon cancer in mice. Cancer Prevent. Res. 4,
1052–1060.
inflammatory drugs: relevance to their pharmacological activity. J. Pharmacol.
Exp. Ther. 340, 422–432.
Xie, G., Sun, Y., Nie, T., Mackenzie, G.G., Huang, L., Kopelovich, L., Komninou, D.,
Rigas, B., 2011. Phospho-ibuprofen (MDC-917) is a novel agent against colon
cancer: efficacy, metabolism, and pharmacokinetics in mouse models. J.
Pharmacol. Exp. Ther. 337, 876–886.
Yoshikawa, T., Naito, Y., 2011. Pathogenesis of NSAIDs-induced gastrointestinal
ulcers. Nihon rinsho 69, 995–1002.
Maeda, H., Wu, J., Sawa, T., Matsumura, Y., Hori, K., 2000. Tumor vascular
permeability and the EPR effect in macromolecular therapeutics: a review. J.
Control Release 65, 271–284.