George Mattheolabakis, Chi C Wong, Yu Sun, Carol A Amella...

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Pegylation improves the pharmacokinetics and bioavailability of small-molecule drugs

hydrolysable by esterases: A study of phospho-ibuprofen

George Mattheolabakis, Chi C Wong, Yu Sun, Carol A Amella, Robert Richards, Panayiotis P.

Constantinides, Basil Rigas

Department of Medicine, Stony Brook University (G.M., C.C.W., Y.S., C.A.A., R.R., B.R.) and

Medicon Pharmaceuticals (P.C., B.R.), Stony Brook, NY, USA

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on July 21, 2014 as DOI: 10.1124/jpet.114.217208

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Running Title: Pegylation prevents phospho-ibuprofen hydrolysis

Correspondence

Basil Rigas

Department of Medicine

HSC, T17-080

Stony Brook University

Stony Brook, NY 11794-8173, USA

Tel: 631-444-9538; Fax: 631-444-9553

E-mail: [email protected]

Number of Text Pages: 21

Number of Tables: 1

Number of Figures: 4

Number of Words:

• Abstract: 243

• Introduction: 483

• Discussion: 776

Abbreviations

CES = carboxylesterases

DCM = dichloromethane

NSAIDs = Nonsteroidal anti-inflammatory drugs

PI = phospho-ibuprofen

PI-PEG = phospho-ibuprofen – polyethylene glycol

PK = pharmacokinetic


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ABSTRACT

Esterase hydrolysis of drugs can accelerate their elimination thereby limiting their efficacy. Poly-

ethylene glycol covalently attached on drugs (pegylation) is known to improve the efficiency of

many drugs. Using as a test agent the novel phospho-ibuprofen (PI), we examined whether

pegylation of PI could abrogate its hydrolytic degradation by esterases; PI, known to inhibit

colon cancer growth, has a carboxylic ester hydrolysable by carboxylesterases (CES). We

covalently attached PEG-2000 to PI (PI-PEG) and studied its stability by exposing it to cells

overexpressing CES and by administering it to mice. We also evaluated PI-PEG’s anticancer

efficacy in human colon cancer xenografts and in Apcmin/+ mice. PI-PEG was stable in the

presence of cells overexpressing CES1 or CES2, while PI was extensively hydrolyzed (90.2 ±

0.7 %, 14.3 ± 1.1 %, mean ± SEM). In mice, PI was nearly completely hydrolyzed. Intraveous

administration of PI-PEG resulted in significant levels in blood and in colon cancer xenografts

(xenograft values in parentheses): AUC0-24h = 2,351 (2,621) (nmole/g)xh; Cmax = 1,965 (886)

nmole/g; Tmax = 0.08 (2) h. The blood levels of ibuprofen, its main hydrolytic product, were

minimal. Compared to controls, PI-PEG inhibited the growth of the xenografts by 74.8%

(p<0.01), and reduced intestinal tumor multiplicity in Apcmin/+ mice by 73.1% (p<0.01)

prolonging their survival (100% vs. 55.1% of controls; p=0.013).Pegylation protects PI from

esterase hydrolysis and improves its pharmacokinetics. In preclinical models of colon cancer, PI-

PEG is a safe and efficacious agent that merits further evaluation.


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1. INTRODUCTION

Carboxylesterases (CES) are a multigene family of mammalian enzymes widely

distributed throughout the body that catalyze the hydrolysis of esters, amides, thioesters, and

carbamates. Humans have two carboxylesterases, hCE1 and hCE2. Both are expressed in the

liver, but the intestine expresses only hCE2. CES represent a first-line defense against

internalized molecules, a function crucial to the survival of an organism. However, the function

of CES may be detrimental to drug development, if preservation of the integrity of a drug is

required for its function. This has been the case with phospho-ibuprofen (PI, MDC-917), which

consists of ibuprofen covalently attached to a diethylphosphate group via a butyl spacer moiety

(Mattheolabakis et al., 2012a). The spacer and ibuprofen are bound through a carboxylic ester

bond, which we have shown to be subject to hydrolysis by CES 1 and 2. Since PI is not a pro-

drug, we sought to preserve the molecule intact in vivo by circumventing the ability of CES to

hydrolyze it. We explored whether pegylating PI would serve that purpose.

Pegylation, the covalent attachment of one or more molecules of polyethylene glycol

(PEG) to a target molecule, is an efficient method used to improve the pharmacokinetic (PK)

properties and therapeutic efficacy of both low- and high- molecular weight compounds.

Pegylation of drugs improves their hydrophilicity and other physiochemical properties, enhances

their bioavailability, stability and circulation half-life in vivo, and reduces their potential toxicity

(Greenwald et al., 2003; Hong et al., 2010). Pegylated compounds have shown promising

efficacy in humans. For example, PEG-camptothecin (PROTHECAN®), an ester-based drug, is

in phase 2 clinical trials (Greenwald et al., 2003; Scott et al., 2009); PEG-paclitaxel is currently

in phase 1 trials (Choi and Jo, 2004); and PEG-interferon is currently used for treatment of

hepatitis C (Hoofnagle and Seeff, 2006). We reasoned that in the case of PI, pegylation will have


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two desirable effects. First, it may enhance its solubility because of PEG’s hydrophilicity;

second, it may protect it from CES due to PEG’s sheer size making it a poor substrate for CES.

PI is a promising candidate anticancer agent. In preclinical models it has exhibited

significant efficacy against colon and breast cancer (Mattheolabakis et al., 2012a; Sun et al.,

2012). It is of interest that in both applications the efficacy of PI against cancer was enhanced by

formulating it in a nanocarrier that likely protected it from CES. Our recent PK study revealed

that the bioavailability of PI in mice is low (<5%) independently of the administration route, a

finding ascribed to limited aqueous solubility, hydrolysis by esterases, and rapid clearance from

the systemic circulation (Xie et al., 2011).

Here, we report the synthesis of pegylated PI (PI-PEG), consisting of mPEG2000

attached to PI at its phosphate moiety (Fig. 1); mPEG2000 is a neutral, biocompatible and

biodegradable methoxy-poly(ethylene oxide). Pegylation of PI rendered it resistant to esterase

hydrolysis, resulting in improved pharmacokinetic (PK) properties and enhanced biodistribution.


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2. MATERIALS AND METHODS

Ibuprofen was obtained from TCI America (Portland, OR). Methoxy-polyethylene glycol

MW=2,000 (mPEG2000), 1,4-butanodiol, and all other reagents were purchased from Sigma-

Aldrich (St. Louis, MO). Human colon cancer cell lines were purchased from American Type

Culture Collection (ATCC, Manassas, VA) and cultured following the recommendations of

ATCC.

2.1. PI-PEG synthesis

A modified H-phosphate reaction was used to synthesize PI-PEG (Tirosh et al., 1997).

Nuclear magnetic resonance (NMR) spectra were recorded in CDCl3 solution at 400 Mhz using a

Varian Instrument (Santa Clara, CA). The reaction proceeded in two stages as depicted in Fig.1.

2.1.1. Synthesis of 4-hydroxybutyl 2-(4-isobutylphenyl)acetate 3

Ibuprofen 1, (5 g, 0.024 mol) was dissolved with 4-dimethylaminopyridine (0.59 g, 0.004

mol), dicyclohexylazodicarboxylate (DCC, 6.5 g, 0.031 mol) and pyridine (1.91 g, 0.024 mol)

in 80 ml of dry dichloromethane (DCM). The reaction was allowed to proceed for 30 min at

room temperature. This mixture was slowly added to a suspension of 1,4-butanodiol 2 (13 g,

0.144 mol) in 140 ml of dry DCM and allowed to react overnight with continuous stirring. The

resulting solution was filtered, washed with 10% K2CO3, water and brine, dried over magnesium

sulfate and evaporated under reduced pressure. The residue was subjected to silica gel

chromatography and eluted with a gradient of hexane-ethyl acetate to obtain 3.


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2.1.2. Synthesis of 4-(hydroxy(mPEG)phosphoryloxy butyl 2-(4-isobutylphenyl) propanoate 5

1.6 ml of PCl3 was added to a solution of imidazole (3.89 g, 0.056 mol) in 180 ml of dry

DCM under stirring over an ice-water bath. This was followed by the addition of 8.2 ml of

triethylamine in 20 ml DCM. After 15 minutes, 20 ml of 3 dissolved in DCM was added

dropwise over 30 min. The reaction continued for an additional 40 min before being quenched by

adding 100 ml of water-pyridine (1:4 v/v). After 15 min, 300 ml of chloroform were added and

the organic layer was washed twice with 100 ml of water, dried over MgSO4 and evaporated

under reduced pressure. The residue was dissolved in 50 ml of DCM. Lyophilized mPEG2000

(3.5 g) and pivaloyl chloride (0.35 g, 0.0029 mol) were added to the reaction mixture. After 10

minutes of stirring, the organic solvent was evaporated to dryness under reduced pressure. A

solution of 0.8 g I2 in 15 ml of water-pyridine (1:1 v/v) was added in order to oxidize the H-

phosphate. Oxidation took place for 10 min and was stopped by the addition of 100 ml 5%

aqueous Na2S2O3 solution. PI-PEG 5 was extracted from the aqueous medium with 100 ml of

chloroform and 100 ml of DCM. The organic layers were combined, dried over MgSO4 and

evaporated under reduced pressure. PI-PEG 5 was purified by repeated cycles of acetone

precipitation at -80oC.

2.2. HPLC analysis

PI-PEG was quantified using an HPLC Waters Alliance 2695 equipped with a Waters

2998 photodiode array detector (220 nm, Waters, Milford, MA) and a Thermo BDS Hypersil

C18 column (150 X 4.6 mm, particle size 3 μm, Thermo Fisher Scientific, Waltham, MA). The

mobile phase consisted of a gradient between buffer A (H2O, acetonitrile, trifluoroacetic acid

94.9:5:0.1 v/v/v) and buffer B (acetonitrile). Using various concentrations of PI-PEG in


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acetonitrile we determined that the range of linear responses of our HPLC method was between

10 and 400 μM.

2.3. In situ hydrolysis by CES1 and CES2

HEK293 cells were seeded into poly-L-lysine coated 24-well plates at a density of 2.0 x

105 cells/per well. After 24 h, HEK293 cells were transfected with the carboxylesterases 1

(CES1) or carboxylesterases 2 (CES2) plasmids, or empty pCMV-XL6 vector with

Lipofectamine 2000. Briefly, the transfection complexes were formed in Opti-MEM (Invitrogen,

Carlsbad, CA) and then added to cells after incubation for 20 min. Over-expression of CES1 and

CES2 was confirmed by RT-PCR. Hydrolysis assays were performed 22-24 h after transfection.

The media were aspirated and replaced with complete RPMI media containing 100 µM PI-PEG

or PI. After 1 h incubation, the cells were washed once with complete media, and collected in

200 µl lysis solution (50% ethanol). Extraction was performed by sonication for 5 min followed

by addition of 400 µl of ethanol. The samples were centrifuged at 17,000 x g for 10 min and

analyzed by HPLC.

2.4. Acute toxicity studies

All animal studies were approved by the Institutional Animal Care and Use Committee at

Stony Brook University. PI-PEG dissolved in water was injected intraperitoneally (i.p.) with

escalating single doses of 400, 800, 1,600, 3,200 and 4,000 mg/kg that corresponds to the

equivalent dose of 66.7, 113.3, 266.7, 533,3 and 666,7 mg/kg of PI, respectively, (2 mice per

dose) into female CD-1 mice (Harlan, Indiana, IN) with 3 days intervals between injections for 3

subsequent injections. Mice were monitored daily for body weight changes, overall health


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condition, and signs of toxicity. At the end of the study, mice were euthanized and necropsies

were performed.

2.5. PK and drug distribution studies in mice bearing colon cancer xenografts

A single dose of an aqueous solution of PI-PEG, 2,400 mg/kg (equimolar to 400 mg/kg

of PI), was injected i.p. or intravenously (i.v.) into female athymic nude mice (Harlan, Indiana,

IN) bearing SW-480 human colon cancer xenografts. Groups of 2 mice were sacrificed at various

time points following drug administration. Blood was collected through heart puncture and drug

was extracted with two volumes of acetonitrile. Various organs and the xenografts from the

animals were also collected, homogenized and sonicated in PBS, and extracted with two volumes

of acetonitrile. After centrifugation for 10 min at 5,000 g, drug levels were measured in the

supernatants by HPLC, to determine the area under the curve (AUC0-24h), the maximum

concentration, the time of the maximum time and drug’s circulation half-time. As a control,

equimolar amounts of PI were administered i.p. or i.v. .

2.6. Efficacy studies in mice

Human colon cancer xenografts in mice: SW-480 human colon cancer cells (2×106 cells

suspended in 100 μl PBS) were implanted subcutaneously (s.c.) into both flanks of 5-6 week old

female athymic nude mice. When the average tumor volume reached ∼100 mm3, mice were

given i.p. vehicle (water) or PI-PEG 4,000 mg/kg daily x5d/week. Tumor dimensions were

measured using a caliper, and tumor volumes were calculated using the following formula: L ×

W × (L + W/2) × 0.56. On day 17, animals were euthanized and tumors were harvested. We

performed hematoxylin-and-eosin (H&E) staining on lung, liver and kidneys sections to evaluate

their histological characteristics and determine any abnormalities.


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Efficacy in Apcmin/+ mice: Six-week-old C57BL/6J Apcmin/+ mice (The Jackson

Laboratories, Bar Harbor, ME; n = 9/group) were treated daily by oral gavage as follows: vehicle

(water); PI 400 mg/kg; PI-PEG 2,400 mg/kg; and PEG 2,000 mg/kg (equimolar to PI-PEG). At

16 weeks of age, the animals were sacrificed, their small intestines and colons were removed,

opened longitudinally and tumors were counted using a magnifying lens. Kaplan-Meier survival

estimates were calculated for each group and compared using the log-rank test. Animal weights

were compared across time using linear mixed models with group included as a between subjects

effect and time as a within-subjects factor. The best-fitting variance-covariance structure was

chosen using information criteria.

3. RESULTS

3.1. Synthesis of PI-PEG

PI-PEG was synthesized as depicted in Fig. 1 and characterized by 1H NMR, 31P NMR

and HPLC. The 1H NMR (Supplemental Figure) shows an excess of PEG molecules (peak at 3.6

ppm) present in the final product compared to ibuprofen alone (benzyl peaks at 7.05 and 7.2

ppm). In all samples, unreacted PEG was <10% of the total on a molar basis. The HPLC

chromatogram showed a single peak at 4.8 min representing PI-PEG (Fig 2). Because the UV

absorption of the PI-PEG originates from the ibuprofen part of the molecule, we concluded that

our samples contained <0.5% of other ibuprofen by-products.

3.2. PI-PEG is resistant to carboxylesterase-mediated hydrolysis in vitro

We have previously shown that intact phospho-NSAIDs are much more potent than their

hydrolyzed products (Wong et al., 2012). However, phospho-NSAIDs are highly susceptible to


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degradation by carboxylesterases in vivo at the carboxyl ester bond. Thus, the stability of PI-PEG

is critical for optimal efficacy. We evaluated the metabolic stability of PI-PEG in CES1- and

CES2-overexpessing cells and compared it to PI, the non-pegylated counterpart. In CES1- and

CES2-overexpressing cells, PI undergoes rapid degradation with 90.2 ± 0.7 % (mean ± SEM for

this and all subsequent values) and 14.3 ± 1.1 % of the total drug hydrolyzed, respectively, after

1 h incubation. In contrast, PI-PEG is highly stable in the presence of carboxylesterase, as we

could not detect any apparent hydrolysis in either CES1- or CES2- expressing cells. Hence,

pegylation significantly protected the carboxyl-ester moiety in PI-PEG from carboxylesterase-

mediated hydrolysis, suggesting that this chemical modification can significantly improve the

metabolic stability in vivo.

3.3. PK and biodistribution of PI-PEG

We studied the PK properties of PI-PEG and PI after a single i.v. or i.p. administration to

mice; these compounds were given at equimolar doses (2400 mg/kg and 400 mg/kg,

respectively). In contrast to PI that was essentially completely hydrolyzed, PI-PEG remained

predominantly intact in vivo, regardless of its route of administration.

As shown in Fig. 3 and Table 1, i.v. administration of PI-PEG led to similar AUC0-24h

values in both blood and tumor xenografts (2,351 and 2,621 nmole/g*h, respectively). As

expected for this route of administration , the blood Cmax was higher (2.2 fold) than that of

tumors but the Tmax in blood (5 min) preceded considerably that of tumors (2 h), indicating an

active and prolonged drug uptake process by the tumors. Both the blood and tumor drug levels

showed a similar decay, leveling off to near zero after 4 h. Of note, ibuprofen, a hydrolysis

product of PI-PEG was detected in both blood and tumors. However, the levels of ibuprofen

were small indicating a low level PI-PEG hydrolysis. Based on AUC0-24h values, ibuprofen in


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blood was 4% of PI-PEG (99 vs. 2,351 nmole/g*h, respectively) and in tumor 11% (293 vs.

2,621 nmole/g*h, respectively), confirming the marked resistance of PI-PEG to in vivo

hydrolysis. In fact, given the similar Tmax of ibuprofen in blood and tumors (2 h), it is likely that

the low-level degradation of PI-PEG occurs in tumors.

In contrast to PI-PEG and in agreement with our previous report (Xie et al., 2011), when

administered i.v., PI was almost completely hydrolyzed. Even at its maximum tolerated dose

(100 mg/kg), PI exhibited low levels in the blood (AUC0-24h 2.6 nmole/g*h) and was

undetectable in the tumors, being rapidly hydrolyzed to release quantitatively its parent

compound, ibuprofen.

PI-PEG is encountered at distinctly high levels in kidneys that exceed those of all other

organs (Fig. 3C). The Cmax of PI-PEG in kidneys is 5-fold higher than that of stomach, the organ

with the next highest Cmax (4,884 vs. 1,003 μM). The higher levels of PI-PEG in the kidneys

(increased AUC0-24h, Suppl. Table) indicate that the kidneys are an important elimination

pathway for the hydrophilic PI-PEG.

When PI-PEG was given i.v. at doses as high as 4,000 mg/kg that corresponds to the

equivalent dose of 666.7 mg/kg of PI, there was no evidence of toxicity. Further increase in its

dose was precluded by the high viscosity of the i.v. solution. Thus we could not determine acute

or sub-chronic maximum tolerated dose of PI-PEG. In contrast, the MTD of non-pegylated PI

was 100 mg/kg thus, pegylation of PI results in at least 7-fold increase in its MTD in mice. These

findings indicate that pegylation of PI is associated with a significant increase in its safety.

The PK and biodistribution parameters of PI-PEG after its i.p. and i.v. administration are

summarized in Table 1, Fig. 3 and Supplementary Fig. 2. PI-PEG levels after i.p. administration

increased rapidly and blood Cmax=922 µM, Tmax=30 min, and AUC0-24h =2,044 nmole/g*h. In the


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tumors, the PI-PEG levels peaked at 1 h with Cmax=245 nmole/g and AUC0-24h =1,248 nmole/g*h

(Table 1). We also detected ibuprofen in the plasma and tumors, albeit at much lower levels

compared to PI (Fig. 3, middle panels).

Administration by i.v. injection resulted in a 3.6-fold higher peak drug level and 2.1-fold

higher total drug exposure in the tumors and resulted in reduced drug accumulation in other

organs (based on their respective Cmax and AUC0-24h values) compared to i.p. injection. Overall,

i.v. administration enhanced the delivery of PI-PEG to tumors compared to the i.p. route.

Compared to i.v. administration, when PI-PEG was given i.p. it generated higher AUC0-24h in the

lungs (9.7 fold), liver (3 fold), the spleen (12.8 fold), and the heart (5.3 fold). However, i.v.

administration in mice is limited by the viscosity of the PI-PEG solution, especially when high

drug concentrations are used (3,000 to 4,000 mg/kg). Thus, we continued our studies using i.p.

administration.

3.4. Animal toxicity studies

We evaluated the in vivo toxicity of PI-PEG in mice applying standard protocols for acute

toxicity studies. Mice treated with PI-PEG up to 4,000 mg/kg were healthy and without any body

weight loss. In particular, there were no abnormalities in the major organs upon gross and

histological examination. In addition, all mice treated with PI-PEG on a long-term basis for

efficacy studies showed no evidence of toxicity (Fig. 4).

3.5. PI-PEG is efficacious in the treatment and prevention of colon cancer

We evaluated the chemotherapeutic potential of PI-PEG in a subcutaneous xenograft

model of SW-480 human colon cancer cells. When the tumors reached ~100 mm3 volume, mice

were treated with PI-PEG (4,000 mg/kg/d, i.p., 5 times per wk) for 17 days. As shown in Fig. 4,


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PI-PEG suppressed tumor growth. This effect was statistically significant, beginning at 7 days

after the initiation of treatment and continuing until the end of the study (day 7 p<0.03; day 10 to

17, p<0.01). Compared to the vehicle control, PI-PEG reduced tumor volume by 74.8% (p<0.01,

Fig. 4).

Next, we evaluated the chemopreventive potential of PI-PEG in the Apcmin/+ mouse

model. Equimolar doses of PI (400 mg/kg/d), PEG (2,000 mg/kg/d) and PI-PEG (2,400 mg/kg/d)

were administered to Apcmin/+ mice by oral gavage once daily for 10 weeks. Both PI and PI-PEG

decreased the total number of tumors in the small intestine by 77.2% and 73.1%, respectively, as

compared to the control group (p<0.01 for both compared to control; no significance between PI

and PI-PEG groups). PI-PEG and PI reduced colon tumor multiplicity by 91.1% (p<0.01) and

64.3% (p<0.02, no significance for PI vs. PI-PEG), respectively. PEG (mPEG2000) reduced the

number of colon tumors by 40.5% (p>0.1 compared to control; Figure 4). Based on the log-rank

tests, survival among both PI and PI-PEG treated animals (100% for both) was significantly

greater (p=0.013) compared to survival for the control or PEG groups (55.6% and 71.4%

respectively, Fig. 4). In both studies, PI-PEG was well tolerated with no weight loss during

treatment.


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4. DISCUSSION

Pegylation has emerged as an efficacious approach to overcoming inherent limitations of

candidate drugs such as low aqueous solubility, rapid metabolism and elimination, low

bioavailability and toxicity. Indeed, several clinically used products are based on this approach

(Harris and Chess, 2003; Mattheolabakis et al., 2012b). Here, we demonstrate that pegylation of

a new class of small molecules can be used to circumvent the hydrolytic activity of the

ubiquitous carboxylesterases that inactivates otherwise efficacious compounds.

The test compound used to demonstrate the feasibility of this approach was PI, a member

of the promising phospho-NSAIDs (Xie et al., 2011). The pegylation of PI was achieved through

a three-step process starting with ibuprofen. The final product had a purity of over 90%; the low

levels of PEG in PI-PEG were of no biological significance, e.g., PEG even at doses equimolar

to PI-PEG failed to affect tumor multiplicity.

The pegylation of PI had a major impact on its physicochemical properties, interaction

with CESs and pharmacokinetic profile. The lipophilic PI (logP = 5.22) became readily soluble

in aqueous solutions, including blood and other body fluids. In addition, PI-PEG resisted CES

hydrolysis both in vitro and in vivo. In sharp contrast to PI that was hydrolyzed by both isoforms

of CES (nearly completely by CES1), PI-PEG remained essentially intact when exposed to cell

lines overexpressing CES1 and CES2. A similar pattern was observed in vivo as well. PI is

known to be completely hydrolyzed by mice due to the action of their carboxylesterases

independently of the administration route (Wong et al., 2012).

A likely explanation of the resistance of PI-PEG to CES hydrolysis is that the PEG

moiety through steric hindrance rendered the PI-PEG molecule inaccessible to the catalytic site

of CES. PEG is not only 5-fold larger than PI based on molecular weight, but can also behave as


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a random coil in an aqueous solvent. Thus it is conceivable that the PEG tail sterically prevents

the enzymatic hydrolysis of the new molecule, which, as a result, remains intact. Since we have

shown that all phospho-NSAIDs synthesized to date are subject to CES degradation (unpublished

data), their pegylation may offer a significant pharmacological advantage for this class of drugs.

The two critical changes in the properties of PI brought about by its pegylation

(hydrophilicity and resistance to enzymatic hydrolysis) had a direct effect on its PK and

biodistribution. The levels of PI-PEG in the circulation of mice were very high (Cmax >1,000

µM) in contrast to the essentially undetectable levels of PI (Xie et al., 2011). PI-PEG survived its

exposure to CES which led to high tumor drug levels, where it was present consistently and

independently of its administration route. It is of interest that while the i.p. route delivered

similar amounts of PI-PEG to the blood (practically identical AUC0-24h values), i.v.

administration led to almost double tumor drug levels. Since the i.v. route led to more than

double Cmax values of PI-PEG, it is conceivable that tumor drug uptake is dependent on the

blood-tumor drug gradient.

The biodistribution of PI-PEG likely reflects its enhanced hydrophilicity. The organ with

the highest levels of PI-PEG is the kidney (5 fold higher than the next highest). Thus is possible

that PI-PEG is predominantly eliminated through the kidney.

PI-PEG showed considerable efficacy against colon cancer in animal models and

inhibited tumorigenesis when employed as either a chemopreventive or a chemotherapeutic

agent. In fact, PI-PEG showed roughly the same efficacy in both applications (~75%).

Remarkably, the effect of PI-PEG in the colon in terms of tumor multiplicity was clearly superior

to that of PI (>90% reduction vs. 64%). Even though the difference between the two compounds


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failed to reach statistical significance (probably due to our modest sample size), the trend in

favor of PI-PEG is clear.

In addition to efficacy, the safety of anticancer agents is of extreme importance,

especially for chemoprevention, when they are administered for prolonged periods of time to

healthy subjects. NSAIDs have as strong a potential to serve as chemopreventive agents

(Johnson et al., 2010), but their toxicity, mostly gastrointestinal and renal, will be limiting in

that context, especially since toxic levels can become cumulative with prolonged use (Rainsford,

1999). PI-PEG appears to be a very safe agent, as evidenced by the absence of gastrointestinal

and other organ toxicity in mice as presented in this study.

In summary, our preclinical data show that pegylation is promising as an approach that

can enhance the pharmacological properties of compounds like PI and structurally similar

phospho-NSAIDs that are subject to enzymatic degradation. Furthermore, PI-PEG is a safe and

efficacious anticancer agent, exhibiting the essential pharmacologic and safety properties

required of a successful candidate anticancer agent.


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Authorship contributions

Participated in research design: Mattheolabakis, Rigas

Conducted experiments: Mattheolabakis, Wong, Sun

Performed data analysis: Mattheolabakis, Rigas

Wrote or contributed to the writing of the manuscript: Mattheolabakis, Wong, Amella, Richards,

Constantinides, Rigas


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Footnotes Section

Statement of conflicts of interest: Dr. Rigas has an equity position in Medicon

Pharmaceuticals, Inc. and Dr. Constantinides is a consultant for the company.

This work was supported in part by an assistance award (W81XWH-10-1-0873) from the US Army Medical Research Acquisition Activity


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FIGURE LEGENDS

Figure 1: Synthesis of PI-PEG. The synthesis was carried out using the H-phosphate strategy as

described in Methods. Briefly, Ibuprofen 1 reacted with excess 1,4-butanodiol 2 according to a

DCC coupling method to obtain 3, which reacted with PCl3 and mPEG2000 to produce the final

product PI-PEG 5.

Figure 2: HPLC chromatogram of PI-PEG. The prominent peak with retention time 4.8 min

originates from PI-PEG. The minor peaks at 6.1 min and 8.5 min originate from ibuprofen

byproducts, and represent < 0.5% of all detected compounds.

Figure 3: PK and biodistribution of PI-PEG. Blood and tumor levels of PI-PEG (A) and its

metabolite, ibuprofen (B), after i.v. or i.p. administration, determined by HPLC as in Methods.

The biodistribution of PI-PEG after i.v. administration (C), and of ibuprofen, its metabolite (D).

S.I., small intestine.

Figure 4: PI-PEG inhibits colon cancer in vivo. A. PI-PEG administered by i.p inhibits the

growth of SW-480 human colon cancer cell xenografts in nude mice. Statistically significant

inhibition started on day 11. B. PI-PEG administered orally to Apcmin/+ mice suppressed tumor

multiplicity in the entire gastrointestinal (GI) tract and also in the colon presented separately. It

also improved the survival rate compared to controls (p<0.05). C. Hematoxylin and eosin

staining of xenograft tissue sections from animals treated with PI-PEG and sacrificed on day 17

of treatment: 1- lung, 2- liver, and 3- kidney.


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TABLES

Table 1: PK parameters of PI-PEG after i.v. and i.p. injection

Delivery Route

Cmax

(nmole/g)

Tmax

(h)

AUC0-24h

(nmole/g x h)

t1/2

(h)

Intravenously

Blood 1,965 0.08 2,351 0.69

Tumor 886 2 2,621 2.44

Intraperitoneally

Blood 922 0.5 2,044 2.54

Tumor 245 1 1,248 2.87


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OH

O

+HOOH DMAP, Pyridine

DCC, DCM

O

O

OH

1PCL3

CH2Cl2-TEA

(i) (ii)

H2O

O

O

O P

O

H

OH(i) PV-Cl, pyridine

(ii) CH3O-PEG-OH

(iii) I2, H20

O

O

O PO-

O

O-PEG-OCH3

2

3

45

Figure 1


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Figure 2

0.4

0.3

0.2

0.1

0.0

-0.1

0 5 10

Retention time (min)

UV Absorption (AU)


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Figure 3

0

1200

2400

0 8 16 24

Intraperitoneal

Intravenous

Blood (nmole/g)

0

1200

2400

0 8 16 24

Tumor (nmole/g)

Time (h)

0

60

120

0 8 16 24

Intraperitoneal

Intravenous

PI-PEG

Ibuprofen

0

60

120

0 8 16 24

Blood (nmole/g)

Tumor (nmole/g)

A

B

0

2500

5000

S.I. Lung Liver Stomach Spleen Kidney Heart Tumor

5 min 30 min

1h 2h

4h

0

100

200

PI-PEG (nmole/g)

Ibuprofen (nmole/g)

C

D


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Figure 4

0

40

80

Control PI-PEG PI PEG

G.I. tract

0

50

100

0 5 10

Survival (%)

Treatment (wks)

Control

PEG

PI

PI-PEG

0

400

800

0 5 10 15 20

Tumor volume (mm3)

Treatment (days)

Control

PI-PEG

* *

0

1

2

Control PI-PEG PI PEG

Colon * *

** *

Numberof tumors at sacrifice

A

B

A B C1 2 3

C


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