Lyudmila Tsurkan

Tumor-Targeted Enzyme/Prodrug Therapy Mediates Long-term Disease-Free Survival of Mice Bearing Disseminated Neuroblastoma

Neural stem cells and progenitor cells migrate selectively to tumor loci in vivo. We exploited the tumor-tropic properties of HB1.F3.C1 cells, an immortalized cell line derived from human fetal telencephalon, to deliver the cDNA encoding a secreted form of rabbit carboxylesterase (rCE) to disseminated neuroblastoma tumors in mice. This enzyme activates the prodrug CPT-11 more efficiently than do human enzymes. Mice bearing multiple tumors were treated with rCE-expressing HB1.F3.C1 cells and schedules of administration of CPT-11 that produced levels of active drug (SN-38) tolerated by patients. Both HB1.F3.C1 cells and CPT-11 were given i.v. None of the untreated mice and 30% of mice that received only CPT-11 survived long term. In contrast, 90% of mice treated with rCE-expressing HB1.F3.C1 cells and 15 mg/kg CPT-11 survived for 1 year without detectable tumors. Plasma carboxylesterase activity and SN-38 levels in mice receiving both rCE-expressing HB1.F3.C1 cells (HB1.F3.C1/AdCMVrCE) and CPT-11 were comparable with those in mice receiving CPT-11 only. These data support the hypothesis that the antitumor effect of the described neural stem/progenitor cell-directed enzyme prodrug therapy (NDEPT) is mediated by production of high concentrations of active drug selectively at tumor sites, thereby maximizing the antitumor effect of CPT-11. NDEPT approaches merit further investigation as effective, targeted therapy for metastatic tumors. We propose that the described approach may have greatest use for eradicating minimum residual disease.

Targeting Oxidative Stress in Embryonal Rhabdomyosarcoma

Rhabdomyosarcoma is a soft-tissue sarcoma with molecular and cellular features of developing skeletal muscle. Rhabdomyosarcoma has two major histologic subtypes, embryonal and alveolar, each with distinct clinical, molecular, and genetic features. Genomic analysis shows that embryonal tumors have more structural and copy number variations than alveolar tumors. Mutations in the RAS/NF1 pathway are significantly associated with intermediate- and high-risk embryonal rhabdomyosarcomas (ERMS). In contrast, alveolar rhabdomyosarcomas (ARMS) have fewer genetic lesions overall and no known recurrently mutated cancer consensus genes. To identify therapeutics for ERMS, we developed and characterized orthotopic xenografts of tumors that were sequenced in our study. High-throughput screening of primary cultures derived from those xenografts identified oxidative stress as a pathway of therapeutic relevance for ERMS.

Organ-specific carboxylesterase profiling identifies the small intestine and kidney as major contributors of activation of the anticancer prodrug CPT-11

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.

Targeting the DNA Repair Pathway in Ewing Sarcoma

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 three 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.

An improved human carboxylesterase for enzyme/prodrug therapy with CPT-11

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 CPT-11 (irinotecan-7-ethyl-10-[4-(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 IC50 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.

Biochemical and molecular analysis of carboxylesterase-mediated hydrolysis of cocaine and heroin

Background and purpose:  Carboxylesterases (CEs) metabolize a wide range of xenobiotic substrates including heroin, cocaine, meperidine and the anticancer agent CPT‐11. In this study, we have purified to homogeneity human liver and intestinal CEs and compared their ability with hydrolyse heroin, cocaine and CPT‐11.

ATP-dependent Mitochondrial Porphyrin Importer ABCB6 Protects against Phenylhydrazine Toxicity

Background: The role of the mitochondrial ABC transporter, Abcb6, in vivo is unknown. Results: Abcb6-null mice are incapable of ATP-dependent import of mitochondrial porphyrins. Despite compensatory changes in the porphyrin pathway, Abcb6-null mice are less viable after a porphyrin-inducing stress. Conclusion: Abcb6 absence abolished ATP-dependent mitochondrial porphyrin uptake and deregulated porphyrin pathway genes. Significance: Disrupted Abcb6 function may produce porphyria after certain stresses. Abcb6 is a mammalian mitochondrial ATP-binding cassette (ABC) transporter that regulates de novo porphyrin synthesis. In previous studies, haploinsufficient (Abcb6+/−) embryonic stem cells showed impaired porphyrin synthesis. Unexpectedly, Abcb6−/− mice derived from these stem cells appeared phenotypically normal. We hypothesized that other ATP-dependent and/or -independent mechanisms conserve porphyrins. Here, we demonstrate that Abcb6−/− mice lack mitochondrial ATP-driven import of coproporphyrin III. Gene expression analysis revealed that loss of Abcb6 results in up-regulation of compensatory porphyrin and iron pathways, associated with elevated protoporphyrin IX (PPIX). Phenylhydrazine-induced stress caused higher mortality in Abcb6−/− mice, possibly because of sustained elevation of PPIX and an inability to convert PPIX to heme despite elevated ferrochelatase levels. Therefore, Abcb6 is the sole ATP-dependent porphyrin importer, and loss of Abcb6 produces up-regulation of heme and iron pathways necessary for normal development. However, under extreme demand for porphyrins (e.g. phenylhydrazine stress), these adaptations appear inadequate, which suggests that under these conditions Abcb6 is important for optimal survival.

Analysis of Mammalian Carboxylesterase Inhibition by Trifluoromethylketone-Containing Compounds

Carboxylesterases (CE) are ubiquitous enzymes that hydrolyze numerous ester-containing xenobiotics, including complex molecules, such as the anticancer drugs irinotecan (CPT-11) and capecitabine and the pyrethroid insecticides. Because of the role of CEs in the metabolism of many exogenous and endogenous ester-containing compounds, a number of studies have examined the inhibition of this class of enzymes. Trifluoromethylketone-containing (TFK) compounds have been identified as potent CE inhibitors. In this article, we present inhibition constants for 21 compounds, including a series of sulfanyl, sulfinyl, and sulfonyl TFKs with three mammalian CEs, as well as human acetyl- and butyrylcholinesterase. To examine the nature of the slow tight-binding inhibitor/enzyme interaction, assays were performed using either a 5-min or a 24-h preincubation period. Results showed that the length of the preincubation interval significantly affects the inhibition constants on a structurally dependent basis. The TFK-containing compounds were generally potent inhibitors of mammalian CEs, with Ki values as low as 0.3 nM observed. In most cases, thioether-containing compounds were more potent inhibitors then their sulfinyl or sulfonyl analogs. QSAR analyses demonstrated excellent observed versus predicted values correlations (r2 ranging from 0.908–0.948), with cross-correlation coefficients (q2) of ∼0.9. In addition, pseudoreceptor models for the TKF analogs were very similar to structures and models previously obtained using benzil- or sulfonamide-based CE inhibitors. These studies indicate that more potent, selective CE inhibitors, containing long alkyl or aromatic groups attached to the thioether chemotype in TFKs, can be developed for use in in vivo enzyme inhibition.

Inhibition of human carboxylesterases hCE1 and hiCE by cholinesterase inhibitors

Carboxylesterases (CEs) are ubiquitously expressed proteins that are responsible for the detoxification of xenobiotics. They tend to be expressed in tissues likely to be exposed to such agents (e.g., lung and gut epithelia, liver) and can hydrolyze numerous agents, including many clinically used drugs. Due to the considerable structural similarity between cholinesterases (ChE) and CEs, we have assessed the ability of a series of ChE inhibitors to modulate the activity of the human liver (hCE1) and the human intestinal CE (hiCE) isoforms. We observed inhibition of hCE1 and hiCE by carbamate-containing small molecules, including those used for the treatment of Alzheimers disease. For example, rivastigmine resulted in greater than 95% inhibition of hiCE that was irreversible under the conditions used. Hence, the administration of esterified drugs, in combination with these carbamates, may inadvertently result in decreased hydrolysis of the former, thereby limiting their efficacy. Therefore drug:drug interactions should be carefully evaluated in individuals receiving ChE inhibitors.

Intracellular inhibition of carboxylesterases by benzil: modulation of CPT-11 cytotoxicity

Carboxylesterases are ubiquitous proteins responsible for the detoxification of xenobiotics. However, these enzymes also activate prodrugs, such as the anticancer agents capecitabine and CPT-11. As a consequence, overexpression of carboxylesterases within tumor cells sensitizes these cells to CPT-11. We have recently identified two classes of carboxylesterase inhibitors based on either a benzil (diphenylethane-1,2-dione) or a benzene sulfonamide scaffold and showed that these compounds inhibit carboxylesterases with Kis in the low nanomolar range. Because both classes of inhibitors show reversible enzyme inhibition, conventional in vitro biochemical assays would not accurately reflect the in situ levels of carboxylesterase activity or inhibition. Therefore, we have developed a novel assay for the determination of intracellular carboxylesterase activity using 4-methylumbelliferone as a substrate. These studies show that benzil and a dimethylbenzil analogue efficiently enter cells and inhibit human intestinal carboxylesterase and rabbit liver carboxylesterase intracellularly. This inhibition results in reduced cytotoxicity to CPT-11 due to the lack of carboxylesterase-mediated conversion of the prodrug to SN-38. These results suggest that intracellular modulation of carboxylesterase activity with benzil or its analogues may be applied to minimize the toxicity of normal cells to CPT-11. [Mol Cancer Ther 2006;5(9):2281–8]