Chi C. Wong

Carboxylesterases 1 and 2 Hydrolyze Phospho-Nonsteroidal Anti-Inflammatory Drugs: Relevance to Their Pharmacological Activity

Phospho-nonsteroidal anti-inflammatory drugs (phospho-NSAIDs) are novel NSAID derivatives with improved anticancer activity and reduced side effects in preclinical models. Here, we studied the metabolism of phospho-NSAIDs by carboxylesterases and assessed the impact of carboxylesterases on the anticancer activity of phospho-NSAIDs in vitro and in vivo. The expression of human liver carboxylesterase (CES1) and intestinal carboxylesterase (CES2) in human embryonic kidney 293 cells resulted in the rapid intracellular hydrolysis of phospho-NSAIDs. Kinetic analysis revealed that CES1 is more active in the hydrolysis of phospho-sulindac, phospho-ibuprofen, phospho-naproxen, phospho-indomethacin, and phospho-tyrosol-indomethacin that possessed a bulky acyl moiety, whereas the phospho-aspirins are preferentially hydrolyzed by CES2. Carboxylesterase expression leads to a significant attenuation of the in vitro cytotoxicity of phospho-NSAIDs, suggesting that the integrity of the drug is critical for anticancer activity. Benzil and bis-p-nitrophenyl phosphate (BNPP), two carboxylesterase inhibitors, abrogated the effect of carboxylesterases and resensitized carboxylesterase-expressing cells to the potent cytotoxic effects of phospho-NSAIDs. In mice, coadministration of phospho-sulindac and BNPP partially protected the former from esterase-mediated hydrolysis, and this combination more effectively inhibited the growth of AGS human gastric xenografts in nude mice (57%) compared with phospho-sulindac alone (28%) (p = 0.037). Our results show that carboxylesterase mediates that metabolic inactivation of phospho-NSAIDs, and the inhibition of carboxylesterases improves the efficacy of phospho-NSAIDs in vitro and in vivo.

Preclinical predictors of anticancer drug efficacy: Critical assessment with emphasis on whether nanomolar potency should be required of candidate agents

In the current paradigm of anticancer drug development, candidate compounds are evaluated by testing their in vitro potency against molecular targets relevant to carcinogenesis, their effect on cultured cancer cells, and their ability to inhibit cancer growth in animal models. We discuss the key assumptions inherent in these approaches. In recent years, great emphasis has been placed on selecting for development compounds with nanomolar in vitro potency, expecting that they will be efficacious and safer based on the assumption that they can be used at lower doses (“the nanomolar rule”). However, this rule ignores critical parameters affecting efficacy and toxicity such as physiochemical and absorption, distribution, metabolism and excretion properties, off-target effects, and multitargeting activities. Thus, uncritical application of the nanomolar rule may reject efficacious compounds or select ineffective or toxic compounds. We present examples of efficacious chemotherapeutic (alkylating agents, hormonal agents, antimetabolites, thalidomide, and valproic acid) and chemopreventive (aspirin and sulindac) agents having millimolar potency and compounds with nanomolar potency (cyclooxygenase-2 inhibitors) that, nevertheless, failed or proved to be unsafe. The effect of candidate drugs on animal models of cancer is a better predictor of human drug efficacy; particularly useful are tumor xenografts. Given the cost of failure at clinical stages, it is imperative to keep in mind the limitations of the nanomolar rule and use relevant in vivo models early in drug discovery to prioritize candidates. Although in vivo models will continue having a major role in cancer drug development, more robust approaches that combine high predictive ability with simplicity and low cost should be developed.

Curcumin enhances the lung cancer chemopreventive efficacy of phospho-sulindac by improving its pharmacokinetics.

Phospho-sulindac (PS) is a safe sulindac derivative with promising anticancer efficacy in colon cancer. We evaluated whether its combination with curcumin could enhance the efficacy in the treatment of lung cancer. Curcumin, the principal bioactive component in turmeric, has demonstrated versatile capabilities to modify the therapeutic efficacy of a wide range of anticancer agents. Here, we evaluated the effect of co-administration of curcumin on the anticancer activity of PS in a mouse xenograft model of human lung cancer. Curcumin enhanced the cellular uptake of PS in human lung and colon cancer cell lines. To assess the potential synergism between curcumin and PS in vivo, curcumin was suspended in 10% Tween-80 or formulated in micellar nanoparticles and given to mice by oral gavage prior to the administration of PS. Both formulations of curcumin significantly improved the pharmacokinetic profiles of PS, with the 10% Tween-80 suspension being much more effective than the nanoparticle formation. However, curcumin did not exhibit any significant modification of the metabolite profile of PS. Furthermore, in a mouse subcutaneous xenograft model of human lung cancer, PS (200 mg/kg) in combination with curcumin (500 mg/kg) suspended in 10% Tween-80 (51% inhibition, p<0.05) was significantly more efficacious than PS plus micelle curcumin (30%) or PS (25%) or curcumin alone (no effect). Consistent with the improved pharmacokinetics, the combination treatment group had higher levels of PS and its metabolites in the xenografts compared to PS alone. Our results show that curcumin substantially improves the pharmacokinetics of PS leading to synergistic inhibition of the growth of human lung cancer xenografts, representing a promising drug combination.

Regioselective oxidation of phospho-NSAIDs by human cytochrome P450 and flavin monooxygenase isoforms: implications for their pharmacokinetic properties and safety.

Phospho‐ibuprofen (MDC‐917) and phospho‐sulindac (OXT‐328) are highly effective in cancer and arthritis treatment in preclinical models. Here, we investigated their metabolism by major human cytochrome P450s (CYPs) and flavin monooxygenases (FMOs).

Phospho-ibuprofen (MDC-917) incorporated in nanocarriers: anti-cancer activity in vitro and in vivo.

Phospho‐ibuprofen (P‐I; MDC‐917) inhibits the growth of colon cancer in mice. Here, we investigated the use of nanocarriers to improve its pharmacokinetics (PKs) and anti tumour efficacy.

Pegylation Improves the Pharmacokinetics and Bioavailability of Small-Molecule Drugs Hydrolyzable by Esterases: A Study of Phospho-Ibuprofen

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.

Phospho-aspirin (MDC-22) inhibits breast cancer in preclinical animal models: an effect mediated by EGFR inhibition, p53 acetylation and oxidative stress

BackgroundThe anticancer properties of aspirin are restricted by its gastrointestinal toxicity and its limited efficacy. Therefore, we synthesized phospho-aspirin (PA-2; MDC-22), a novel derivative of aspirin, and evaluated its chemotherapeutic and chemopreventive efficacy in preclinical models of triple negative breast cancer (TNBC).MethodsEfficacy of PA-2 was evaluated in human breast cancer cells in vitro, and in orthotopic and subcutaneous TNBC xenografts in nude mice. Mechanistic studies were also carried out to elucidate the mechanism of action of PA-2.ResultsPA-2 inhibited the growth of TNBC cells in vitro more potently than aspirin. Treatment of established subcutaneous TNBC xenografts (MDA-MB-231 and BT-20) with PA-2 induced a strong growth inhibitory effect, resulting in tumor stasis (79% and 90% inhibition, respectively). PA-2, but not aspirin, significantly prevented the development of orthotopic MDA-MB-231 xenografts (62% inhibition). Mechanistically, PA-2: 1) inhibited the activation of epidermal growth factor receptor (EGFR) and suppressed its downstream signaling cascades, including PI3K/AKT/mTOR and STAT3; 2) induced acetylation of p53 at multiple lysine residues and enhanced its DNA binding activity, leading to cell cycle arrest; and 3) induced oxidative stress by suppressing the thioredoxin system, consequently inhibiting the activation of the redox sensitive transcription factor NF-κB. These molecular alterations were observed in vitro and in vivo, demonstrating their relevance to the anticancer effect of PA-2.ConclusionsOur findings demonstrate that PA-2 possesses potent chemotherapeutic efficacy against TNBC, and is also effective in its chemoprevention, warranting further evaluation as an anticancer agent.

Topical phospho-sulindac (OXT-328) is effective in the treatment of non-melanoma skin cancer

Phospho-sulindac (P-S, OXT-328), a novel sulindac derivative, has shown superior anticancer efficacy and safety compared to sulindac. In this study, we investigated the efficacy of topical P-S hydrogel in the treatment of non-melanoma skin cancer in preclinical models. P-S is a potent inhibitor of A431 epidermoid carcinoma in vitro and achieves this effect by inhibiting cell proliferation and inducing apoptosis. The anticancer efficacy of topical and oral P-S was further evaluated in mice bearing A431 intradermal xenografts. Compared to the controls, topical P-S hydrogel inhibited the A431 xenografts by 70.5% (p<0.01), while oral P-S inhibited it by 43.4% (p<0.05), being significantly less effective than topical P-S (p= 0.017). Topical P-S hydrogel generated significant levels (>500 nmol/g tumor tissue) of intact P-S in the tumors, accounting for 92.5% of the total metabolites in the A431 xenografts. This local delivery of high levels of intact P-S to the A431 xenografts is an important contributor to the potent activity of topical P-S and no local or systemic side effects were noted in the treatment group. Thus, topical P-S is a promising treatment modality against non-melanoma skin cancer and merits further evaluation.

Aerosol administration of phospho-sulindac inhibits lung tumorigenesis

Phospho-sulindac is a sulindac derivative with promising anticancer activity in lung cancer, but its limited metabolic stability presents a major challenge for systemic therapy. We reasoned that inhalation delivery of phospho-sulindac might overcome first-pass metabolism and produce high levels of intact drug in lung tumors. Here, we developed a system for aerosolization of phospho-sulindac and evaluated the antitumor efficacy of inhaled phospho-sulindac in an orthotopic model of human non–small cell lung cancer (A549 cells). We found that administration by inhalation delivered high levels of phospho-sulindac to the lungs and minimized its hydrolysis to less active metabolites. Consequently, inhaled phospho-sulindac (6.5 mg/kg) was highly effective in inhibiting lung tumorigenesis (75%; P < 0.01) and significantly improved the survival of mice bearing orthotopic A549 xenografts. Mechanistically, phospho-sulindac suppressed lung tumorigenesis by (i) inhibiting EGF receptor (EGFR) activation, leading to profound inhibition of Raf/MEK/ERK and PI3K/AKT/mTOR survival cascades; (ii) inducing oxidative stress, which provokes the collapse of mitochondrial membrane potential and mitochondria-dependent cell death; and (iii) inducing autophagic cell death. Our data establish that inhalation delivery of phospho-sulindac is an efficacious approach to the control of lung cancer, which merits further evaluation. Mol Cancer Ther; 12(8); 1417–28. ©2013 AACR.

Phospho-aspirin-2 (MDC-22) inhibits estrogen receptor positive breast cancer growth both in vitro and in vivo by a redox-dependent effect.

Phospho-aspirin (PA-2) is a novel aspirin derivative that exhibits promising anticancer properties and is considerably safer than conventional aspirin. In this study, we investigated the chemotherapeutic efficacy of PA-2 in preclinical models of estrogen receptor positive (ER+) breast cancer and elucidated its mechanism of action. PA-2 inhibited the growth of ER+ cells more potently than aspirin in vitro, and exerted a triple cytokinetic effect that includes induction of apoptosis and cell cycle arrest as well as the inhibition of cell proliferation. PA-2 is highly efficacious in vivo, as treatment of established MCF7 xenografts with PA-2 induced tumor stasis (98.2% inhibition, p<0.01). PA-2 triggered the activation of p53-dependent apoptosis via two distinct mechanisms: 1) acetylation of p53 (at K373), which disrupts its interaction with its transcription repressor MDM2, and 2) translocation of p53 to the mitochondria leading to the dissipation of mitochondrial transmembrane potential (ΔΨm). Consistent with these observations, both the RNAi-mediated knockdown of p53 and forced deactylation via HDAC1 over-expression attenuated the anticancer effect of PA-2 in MCF7 cells. An upstream mediator of the signaling effects of PA-2 is RONS. PA-2 induced oxidative stress in vitro and in mice bearing MCF7 xenografts; its induction effect appears to be tumor-specific. Crucially, administration of N-acetylcysteine, a ROS scavenger, abrogated the effect of PA-2 on p53 acetylation and mitochondria translocation, thus identifying RONS as proximal molecules mediating the anticancer effect of PA-2. In summary, our findings demonstrate that PA-2 is a promising antineoplastic compound against ER+ breast cancer, warranting further evaluation as an anticancer agent.