Ewing sarcoma (EWS) is a tumor of the bone and soft-tissue that primarily affects adolescents and young adults. With current therapies, 70% of patients with localized disease survive, but patients with metastatic or recurrent disease have a poor outcome. We found that EWS cell lines are defective in DNA break repair and are sensitive to PARP inhibitors (PARPis). PARPi-induced cytotoxicity in EWS cells was 10- to 1,000-fold higher after administration of the DNA-damaging agents irinotecan or temozolomide. We developed an orthotopic EWS mouse model and performed pharmacokinetic and pharmacodynamic studies using 3 different PARPis that are in clinical development for pediatric cancer. Irinotecan administered on a low-dose, protracted schedule previously optimized for pediatric patients was an effective DNA-damaging agent when combined with PARPis; it was also better tolerated than combinations with temozolomide. Combining PARPis with irinotecan and temozolomide gave complete and durable responses in more than 80% of the mice.

Both replication-incompetent and replication-selective adenoviruses are being developed for the treatment of cancer and other diseases. Concerns have been raised about the safety of intra-vascular adenovirus administration following a patient death on a clinical trial with a replication-defective adenovirus. In addition, the feasibility of vascular delivery to distant tumors has been questioned. dl1520 (ONYX-015) is a replication-selective adenovirus that has previously shown safety and antitumoral activity following intratumoral injection. This is the first report of intra-vascular administration with a genetically engineered, replication-selective virus. A phase I dose-escalation trial was performed inpatients with liver-predominant gastrointestinal carcinoma (n = 11 total; primarily colorectal). dl1520 was infused into the hepatic artery at doses of 2 × 10 8 -2 × 10 12 particles for

Carboxylesterases (CES) have important roles in pesticide and drug metabolism, and contribute to the clearance of ester-containing xenobiotics in mammals. Tissues with the highest levels of CES expression are the liver and small intestine. In addition to xenobiotics, CES also harness their broad substrate specificity to hydrolyze endobiotics, such as cholesteryl esters and triacylglycerols. Here we determined if two human CES isoforms, CES1 and CES2, hydrolyze the endocannabinoids 2-arachidonoylglycerol (2AG) and anandamide (AEA), and two prostaglandin glyceryl esters (PG-Gs), which are formed by COX-mediated oxygenation of 2AG. We show that recombinant CES1 and CES2 efficiently hydrolyze 2AG to arachidonic acid (AA), but not amide-containing AEA. Steady-state kinetic parameters for CES1- and CES2-mediated 2AG hydrolysis were, respectively: kcat, 59 and 43min−1; Km, 49 and 46μM; kcat/Km, 1.2 and 0.93μM−1 min−1. kcat/Km values are comparable to published values for rat monoacylglycerol lipase (MAGL)-catalyzed 2AG hydrolysis. Furthermore, we show that CES1 and CES2 also efficiently hydrolyze PGE2-G and PGF2α-G. In addition, when cultured human THP1 macrophages were treated with exogenous 2AG or PG-G (10μM, 1h), significant quantities of AA or PGs were detected in the culture medium; however, the ability of macrophages to metabolize these compounds was inhibited (60-80%) following treatment with paraoxon, the toxic metabolite of the insecticide parathion. Incubation of THP1 cell lysates with small-molecule inhibitors targeting CES1 (thieno[3,2-e][1]benzothiophene-4,5-dione or JZL184) significantly reduced lipid glyceryl ester hydrolase activities (40-50% for 2AG and 80-95% for PG-Gs). Immunodepletion of CES1 also markedly reduced 2AG and PG-G hydrolase activities. These results suggested that CES1 is in part responsible for the hydrolysis of 2AG and PG-Gs in THP1 cells, although it did not rule out a role for other hydrolases, especially with regard to 2AG metabolism since a substantial portion of its hydrolysis was not inactivated by the inhibitors. An enzyme (Mr 31-32kDa) of unknown function was detected by serine hydrolase activity profiling of THP1 cells and may be a candidate. Finally, the amounts of in situ generated 2AG and PG-Gs in macrophages were enhanced by treating the cells with bioactive metabolites of OP insecticides. Collectively, the results suggest that in addition to MAGL and fatty-acid amide hydrolase (FAAH), which have both been documented to terminate endocannabinoid signaling, CES may also have a role. Furthermore, since PG-Gs have been shown to possess biological activities in their own right, CES may represent an important enzyme class that regulates their in vivo levels.

Cholesteryl esters are hydrolyzed by cholesteryl ester hydrolase (CEH) yielding free cholesterol for export from macrophages. Hence, CEH has an important regulatory role in macrophage reverse cholesterol transport (RCT). CEH and human carboxylesterase 1 (CES1) appear to be the same enzyme. CES1 is inhibited by oxons, the bioactive metabolites of organophosphate (OP) pesticides. Here, we show that CES1 protein is robustly expressed in human THP-1 monocytes/macrophages and its biochemical activity inhibited following treatment of cell lysates and intact cells with chlorpyrifos oxon, paraoxon, or methyl paraoxon (with nanomolar IC 50 values) or after immunodepletion of CES1 protein. CES1 protein expression in cells is unaffected by 24-h paraoxon treatment, suggesting the reduced hydrolytic activity is due to covalent inhibition of CES1 by oxons and not down-regulation of expression. Most significantly, treatment of cholesterol-loaded macrophages with either paraoxon (a non-specific CES inhibitor) or benzil (a specific CES inhibitor) caused enhanced retention of intracellular cholesteryl esters and a "foamy" phenotype, consistent with reduced cholesteryl ester mobilization. Thus, exposure to OP pesticides, which results in the inhibition of CES1, may also inhibit macrophage RCT, an important process in the regression of atherosclerosis.

The activation of the anticancer prodrug CPT-11, to its active metabolite SN-38, is primarily mediated by carboxylesterases (CE). In humans, three CEs have been identified, of which human liver CE (hCE1; CES1) and human intestinal CE (hiCE; CES2) demonstrate significant ability to hydrolyze the drug. However, while the kinetic parameters of CPT-11 hydrolysis have been measured, the actual contribution of each enzyme to activate the drug in biological samples has not been addressed. Hence, we have used a combination of specific CE inhibition and conventional chromatographic techniques to determine the amounts, and hydrolytic activity, of CEs present within human liver, kidney, intestinal and lung specimens. These studies confirm that hiCE demonstrates the most efficient kinetic parameters for CPT-11 activation, however, due to the high levels of hCE1 that are expressed in liver, the latter enzyme can contribute up to 50% of the total of drug hydrolysis in this tissue. Conversely, in human duodenum, jejunum, ileum and kidney, where hCE1 expression is very low, greater than 99% of the conversion of CPT-11 to SN-38 was mediated by hiCE. Furthermore, analysis of lung microsomal extracts indicated that CPT-11 activation was more proficient in samples obtained from smokers. Overall, our studies demonstrate that hCE1 plays a significant role in CPT-11 hydrolysis even though it is up to 100-fold less efficient at drug activation than hiCE, and that drug activation in the intestine and kidney are likely major contributors to SN-38 production in vivo.

Carboxylesterases (CE) are ubiquitous enzymes thought to be responsible for the metabolism and detoxification of xenobiotics. Numerous clinically used drugs including Demerol, lidocaine, capecitabine, and CPT-11 are hydrolyzed by these enzymes. Hence, the identification and application of selective CE inhibitors may prove useful in modulating the metabolism of esterified drugs in vivo. Having recently identified benzil (diphenylethane-1,2-dione) as a potent selective inhibitor of CEs, we sought to evaluate the inhibitory activity of related 1,2-diones toward these enzymes. Biochemical assays and kinetic studies demonstrated that isatins (indole-2,3-diones), containing hydrophobic groups attached at a variety of positions within these molecules, could act as potent, specific CE inhibitors. Interestingly, the inhibitory potency of the isatin compounds was related to their hydrophobicity, such that compounds with clogP values of <1.25 were ineffective at enzyme inhibition. Conversely, analogs demonstrating clogP values>5 routinely yielded Ki values in the nM range. Furthermore, excellent 3D QSAR correlates were obtained for two human CEs, hCE1 and hiCE. While the isatin analogues were generally less effective at CE inhibition than the benzils, the former may represent valid lead compounds for the development of inhibitors for use in modulating drug metabolism in vivo.

CPT-11 is a potent antitumor agent that is activated by carboxylesterases (CE) and intracellular expression of CEs that can activate the drug results in increased cytotoxicity to the drug. As activation of (1-piperidino)-1-piperidino]carbonyloxycamptothecin) by human CEs is relatively inefficient, we have developed enzyme/prodrug therapy approaches based on the CE/CPT-11 combination using a rabbit liver CE (rCE). However, the in vivo application of this technology may be hampered by the development of an immune response to rCE. Therefore, we have developed a mutant human CE (hCE1m6), based on the human liver CE hCE1, that can activate CPT-11 approximately 70-fold more efficiently than the wild-type protein and can be expressed at high levels in mammalian cells. Indeed, adenoviral-mediated delivery of hCE1m6 with human tumor cells resulted in up to a 670-fold reduction in the IC 50 value for CPT-11, as compared to cells transduced with vector control virus. Furthermore, xenograft studies with human tumors expressing hCE1m6 confirm the ability of this enzyme to activate CPT-11 in vivo and induce antitumor activity. We propose that this enzyme should likely be less immunogenic than rCE and would be suitable for the in vivo application of CE/CPT-11 enzyme/prodrug therapy.

Carboxylesterases (CE) are members of the esterase family of enzymes, and as their name suggests, they are responsible for the hydrolysis of carboxylesters into the corresponding alcohol and carboxylic acid. To date, no endogenous CE substrates have been identified and as such, these proteins are thought to act as a mechanism to detoxify ester-containing xenobiotics. As a consequence, they are expressed in tissues that might be exposed to such agents (lung and gut epithelia, liver, kidney, etc.). CEs demonstrate very broad substrate specificities and can hydrolyze compounds as diverse as cocaine, oseltamivir (Tamiflu), permethrin and irinotecan. In addition, these enzymes are irreversibly inhibited by organophosphates such as Sarin and Tabun. In this overview, we will compare and contrast the two human enzymes that have been characterized, and evaluate the biology of the interaction of these proteins with organophosphates (principally nerve agents).