Esterase hydrolysis of drugs can accelerate their elimination, thereby limiting their efficacy. Polyethylene glycol (PEG) covalently attached to 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 hydrolyzable by carboxylesterases (CES). We covalently attached mPEG-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, whereas PI was extensively hydrolyzed (90.2 ± 0.7%, 14.3 ± 1.1%, mean ± S.E.M.). In mice, PI was nearly completely hydrolyzed. Intravenous administration of PI-PEG resulted in significant levels in blood and in colon cancer xenografts (xenograft values in parentheses): area under the curve for 0–24 hours = 2351 (2621) (nmol/g) × h; Cmax = 1965 (886) nmol/g; Tmax = 0.08 (2) hour. The blood levels of ibuprofen, its main hydrolytic product, were minimal. Compared with 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% versus 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.
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. Because PI is not a prodrug, 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; Enzon, Inc., Piscataway, NJ), 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 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 mPEG-2000 attached to PI at its phosphate moiety (Fig. 1). mPEG-2000 is a neutral, biocompatible, and biodegradable methoxy-poly(ethylene oxide). Pegylation of PI rendered it resistant to esterase hydrolysis, resulting in improved PK properties and enhanced biodistribution.
Materials and Methods
Ibuprofen was obtained from TCI America (Portland, OR). Methoxy-polyethylene glycol mol. wt. = 2000 (mPEG-2000), 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 (Manassas, VA) and cultured following the manufacturer’s recommendations.
A modified H-phosphate reaction was used to synthesize PI-PEG (Tirosh et al., 1997). 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.
Synthesis of 4-Hydroxybutyl 2-(4-Isobutylphenyl) Acetate (Compound 3).
Ibuprofen, compound 1 (5 g, 0.024 mol) was dissolved with 4-dimethylaminopyridine (0.59 g, 0.004 mol), dicyclohexylazodicarboxylate (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 minutes at room temperature. This mixture was slowly added to a suspension of 1,4-butanodiol, compound 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 compound 3.
Synthesis of 4-Hydroxy(mPEG)Phosphoryloxy Butyl 2-(4-Isobutylphenyl) Propanoate (Compound 5).
PCl3 (1.6 ml) 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 compound 3 dissolved in DCM was added dropwise over 30 minutes. The reaction continued for an additional 40 minutes before being quenched by adding 100 ml of water-pyridine (1:4 v/v). After 15 minutes, 300 ml of chloroform was 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 mPEG-2000 (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 to oxidize the H-phosphate. Oxidation took place for 10 minutes and was stopped by the addition of 100 ml 5% aqueous Na2S2O3 solution. PI-PEG compound 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 compound 5 was purified by repeated cycles of acetone precipitation at −80°C.
High-Performance Liquid Chromatography Analysis
PI-PEG was quantified using a high-performance liquid chromatography (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 × 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 acetonitrile we determined that the range of linear responses of our HPLC method was between 10 and 400 μM.
In Situ Hydrolysis by CES1 and CES2
Human embryonic kidney 293 cells were seeded into poly-l-lysine–coated 24-well plates at a density of 2.0 × 105 cells/per well. After 24 hours, human embryonic kidney 293 cells were transfected with the CES1 or 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 minutes. Overexpression of CES1 and CES2 was confirmed by reverse-transcription polymerase chain reaction. Hydrolysis assays were performed 22–24 hours after transfection. The media were aspirated and replaced with complete RPMI media containing 100 µM PI-PEG or PI. After 1-hour 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 minutes followed by addition of 400 µl of ethanol. The samples were centrifuged at 17,000g for 10 minutes and analyzed by HPLC.
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 with escalating single doses of 400, 800, 1600, 3200, and 4000 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, Indianapolis, IN) with 3-day intervals between injections for three subsequent injections. Mice were monitored daily for body weight changes, overall health condition, and signs of toxicity. At the end of the study, mice were euthanized and necropsies were performed.
PK and Drug Distribution Studies in Mice Bearing Colon Cancer Xenografts
A single dose of an aqueous solution of PI-PEG, 2400 mg/kg (equimolar to 400 mg/kg PI), was injected intraperitoneally or intravenously into female athymic nude mice (Harlan) bearing SW-480 human colon cancer xenografts. Groups of 2 mice were killed at various time points after 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 phosphate-buffered saline and extracted with two volumes of acetonitrile. After centrifugation for 10 minutes at 5000g, drug levels were measured in the supernatants by HPLC to determine the area under the curve (AUC0-24 hours), the maximum concentration (Cmax), the time of the maximum concentration (Tmax), and drug’s circulation half-time. As a control, equimolar amounts of PI were administered intraperitoneally or intravenously.
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 into both flanks of 5- to 6-week-old female athymic nude mice. When the average tumor volume reached ∼100 mm3, mice were given intraperitoneal vehicle (water) or PI-PEG 4000 mg/kg daily × 5 days/wk. 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 staining on lung, liver, and kidneys sections to evaluate their histologic characteristics and determine any abnormalities.
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), 400 mg/kg PI, 2400 mg/kg PI-PEG, and 2000 mg/kg PEG (equimolar to PI-PEG). At 16 weeks of age, the animals were killed, their small intestines and colons were removed and 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.
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 Fig. 1) shows an excess of PEG molecules (peak at 3.6 ppm) present in the final product compared with 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 minutes 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.
PI-PEG Is Resistant to Carboxylesterase-Mediated Hydrolysis In Vitro.
We previously showed that intact phospho–nonsteroidal anti-inflammatory drugs (NSAIDs) are much more potent than their hydrolyzed products (Wong et al., 2012). However, phospho-NSAIDs are highly susceptible to 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-overexpressing cells and compared it to PI, the nonpegylated counterpart. In CES1- and CES2-overexpressing cells, PI undergoes rapid degradation with 90.2 ± 0.7% (mean ± S.E.M. for this and all subsequent values) and 14.3 ± 1.1% of the total drug hydrolyzed, respectively, after 1 hour 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.
PK and Biodistribution of PI-PEG.
We studied the PK properties of PI-PEG and PI after a single intravenous or intraperitoneal administration to mice; these compounds were given at equimolar doses (2400 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, intravenous administration of PI-PEG led to similar AUC0-24 hours values in both blood and tumor xenografts (2351 and 2621 nmol/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 minutes) preceded considerably that of tumors (2 hours), 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 hours. Notably, 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 AUC0-24 hours values, ibuprofen in blood was 4% of PI-PEG (99 versus 2351 nmol/g × h, respectively) and in tumor 11% (293 versus 2621 nmol/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 hours), 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 intravenously, PI was almost completely hydrolyzed. Even at its maximum tolerated dose (100 mg/kg), PI exhibited low levels in the blood (AUC0-24 hours 2.6 nmol/g × h) and was undetectable in the tumors, being rapidly hydrolyzed to release quantitatively its parent compound, ibuprofen.
PI-PEG is encountered in kidneys at distinctly high levels that exceed those of all other organs (Fig. 3C). The Cmax of PI-PEG in kidneys is 5-fold higher than that of the stomach, the organ with the next highest Cmax (4884 versus 1003 μM). The higher levels of PI-PEG in the kidneys (increased AUC0-24 hours; Supplemental Table 1) indicate that the kidneys are an important elimination pathway for the hydrophilic PI-PEG.
When PI-PEG was given intravenously at doses as high as 4000 mg/kg that correspond to the equivalent dose of 666.7 mg/kg PI, there was no evidence of toxicity. Further increase in its dose was precluded by the high viscosity of the intravenous solution. Thus we could not determine acute or subchronic maximum tolerated dose of PI-PEG. In contrast, the maximum tolerated dose of nonpegylated PI was 100 mg/kg; thus, pegylation of PI results in at least a 7-fold increase in its maximum tolerated dose 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 intraperitoneal and intravenous administration are summarized in Fig. 3, Supplemental Fig. 2, and Table 1. PI-PEG levels after intraperitoneal administration increased rapidly and blood Cmax = 922 µM, Tmax = 30 minutes, and AUC0-24 hours = 2044 nmol/g × h. In the tumors, the PI-PEG levels peaked at 1 hour with Cmax = 245 nmol/g and AUC0-24 hours = 1248 nmol/g × h (Table 1). We also detected ibuprofen in the plasma and tumors, albeit at much lower levels compared with PI (Fig. 3B).
Administration by intravenous 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 AUC0-24 hours values) compared with intraperitoneal injection. Overall, intravenous administration enhanced the delivery of PI-PEG to tumors compared with the intraperitoneal route. Compared with intravenous administration, when PI-PEG was given intraperitoneally it generated higher AUC0-24 hours in the lungs (9.7-fold), liver (3-fold), the spleen (12.8-fold), and the heart (5.3-fold). However, intravenous administration in mice is limited by the viscosity of the PI-PEG solution, especially when high drug concentrations are used (3000 to 4000 mg/kg). Thus, we continued our studies using intraperitoneal administration.
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 4000 mg/kg were healthy and without any body weight loss. In particular, there were no abnormalities in the major organs upon gross and histologic examination. In addition, all mice treated with PI-PEG on a long-term basis for efficacy studies showed no evidence of toxicity (Fig. 4).
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 (4000 mg/kg per day i.p., 5 times per week) for 17 days. As shown in Fig. 4, 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; days 10 to 17, P < 0.01). Compared with 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 per day), PEG (2000 mg/kg per day), and PI-PEG (2400 mg/kg per day) 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, compared with the control group (P < 0.01 for both compared with 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 versus PI-PEG), respectively. PEG (mPEG-2000) reduced the number of colon tumors by 40.5% (P > 0.1 compared with control; Fig. 4). On the basis of the log-rank tests, survival among both PI- and PI-PEG–treated animals (100% for both) was significantly greater (P = 0.013) compared with 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.
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 inactivate 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 biologic 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, independent 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 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. Because we showed that all phospho-NSAIDs synthesized to date are subject to CES degradation (C. C. Wong, K. W. Cheng, I. Papayannis, G. Mattheolabakis, L. Huang, G. Xie, N. Ouyang, and B. Rigas, 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 > 1000 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 although the intraperitoneal route delivered similar amounts of PI-PEG to the blood (practically identical AUC0-24 hours values), intravenous administration led to almost double tumor drug levels. Because the intravenous 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, it 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 versus 64%). Although the difference between the two compounds failed to reach statistical significance (probably because of 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 because toxic levels can become cumulative with prolonged use (Rainsford, 1999). PI-PEG apears 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.
Participated in research design: Mattheolabakis, Constantinides, Rigas.
Conducted experiments: Mattheolabakis, Wong, Sun.
Performed data analysis: Mattheolabakis, Amella, Richards, Rigas.
Wrote or contributed to the writing of the manuscript: Mattheolabakis, Wong, Amella, Richards, Constantinides, Rigas.
- Received June 2, 2014.
- Accepted July 18, 2014.
This work was supported in part by an assistance award [W81XWH-10-1-0873] from the US Army Medical Research Acquisition Activity. B.R. has an equity position in Medicon Pharmaceuticals, Inc. P.P.C. is a consultant for Medicon Pharmaceuticals, Inc.
- area under the curve
- high-performance liquid chromatography
- nonsteroidal anti-inflammatory drugs
- polyethylene glycol
- phospho-ibuprofen–polyethylene glycol
- Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics