New therapeutic targets for the treatment of high-risk neuroblastoma.

High-risk neuroblastoma remains a major problem in pediatric oncology, accounting for 15% of childhood cancer deaths. Although incremental improvements in outcome have been achieved with the intensification of conventional chemotherapy agents and the addition of 13-cis-retinoic acid, only one-third of children with high-risk disease are expected to be long-term survivors when treated with current regimens. In addition, the cost of cure can be quite high, as surviving children remain at risk for additional health problems related to long-term toxicities of treatment. Further advances in therapy will require the targeting of tumor cells in a more selective and efficient way so that survival can be improved without substantially increasing toxicity. In this review we summarize ongoing clinical trials and highlight new developments in our understanding of the molecular biology of neuroblastoma, emphasizing potential targets or pathways that may be exploitable therapeutically.

Cell adhesion molecules as targets for therapy of neuroblastoma.

Cell adhesion molecules (CAMs) are glycoproteins expressed on the surface of cell membranes. In normal cells, CAMs participate in a variety of biological processes including cell proliferation, migration and differentiation. In tumor cells, CAMs have been reported to function as oncogenes or tumor suppressors, in signal transduction and as regulators of tumor progression and metastasis. CAMs interact directly with each other as well as with multiple intracellular proteins and extracellular matrix components. Relevant to this review, direct and indirect interactions of CAMs with the extracellular matrix and with the actin cytoskeletal network control cell motility. Cell motility, in turn, regulates the migratory and invasive potential, i.e., the metastatic potential, of tumor cells. The question to be addressed in this review is whether CAM-mediated molecular interactions that regulate metastasis represent potential therapeutic targets for metastatic neuroblastoma.

Neural progenitor cell-mediated delivery of osteoprotegerin limits disease progression in a preclinical model of neuroblastoma bone metastasis.

PURPOSE: Osteoprotegerin (OPG) inhibits osteoclast activation and reduces osteolysis in bone tumors. We hypothesized that tumor-tropic neural progenitor cells (NPCs) engineered to express OPG would reduce neuroblastoma disease burden in the bone. METHODS: Stable expression of green fluorescent protein (NPC-GFP) and OPG (NPC-OPG) was established in human NPCs by lentivirus-mediated transduction. Bone disease was established by intrafemoral injection of luciferase-expressing human neuroblastoma (CHLA-255) cells into 20 SCID mice. Three weeks later, mice began receiving intravenous injection of 2 x 10(6) NPC-OPG or NPC-GFP (control) every 10 days x 3 doses. Disease was monitored with quantitative bioluminescence imaging and x-ray images, which were evaluated on a scale of 0 to 4. These studies were approved by the Institutional Animal Care and Use Committee. RESULTS: Osteoprotegerin treatment in vitro produced no direct toxicity to tumor cells. Coculture of tumor cells with bone marrow significantly increased activation of bone marrow-derived osteoclasts as assessed by tartrate-resistant acid phosphatase staining (156 +/- 10.8 osteoclasts per well) compared to bone marrow culture alone (91.67 +/- 4.7, P = .005). This increase was abrogated by adding OPG-containing media (68.3 +/- 2.8, P = .001). NPC-OPG slowed tumor progression (108-fold increase from pretreatment) compared to mice treated with NPC-GFP (538-fold), as judged by bioluminescence imaging. X-rays subjectively demonstrated less bone disease in NPC-OPG-treated mice (2.27 +/- 0.25) compared to NPC-GFP-treated mice (3.25 +/- 0.22, P = .04). CONCLUSIONS: Neural progenitor cell-mediated delivery of OPG slowed disease progression in a preclinical model of neuroblastoma bone metastasis. The decrease in bone disease was not from direct tumor cell toxicity but likely occurred indirectly through inhibition of osteoclast-directed bone resorption. Thus, targeted delivery of OPG by NPCs may be effective in the treatment of neuroblastoma bone metastasis.

Neural progenitor cell-mediated delivery of interferon beta improves neuroblastoma response to cyclophosphamide.

BACKGROUND: We have shown that continuous systemic delivery of interferon beta (IFN-beta) remodels dysfunctional tumor vasculature, thereby improving tumor perfusion and enhancing delivery and efficacy of chemotherapeutic drugs. We hypothesized that because of their inherent tumor tropism, neural progenitor cells (NPCs) engineered to express IFN-beta could also effect maturation of tumor vasculature without generating high systemic levels of IFN-beta. METHODS: Mice with luciferase-expressing disseminated human neuroblastoma were divided into four groups of equal tumor burden by bioluminescence imaging: (1) untreated controls; (2) NPC-IFN-beta only; (3) cyclophosphamide (CTX) only; and (4) NPC-IFN-beta in combination with CTX. Two million NPC-IFN-beta cells were administered twice, 7 days apart, starting 21 days after tail vein administration of tumor cells. CTX was administered every 6 days for three doses. Mice were killed at 6 weeks, livers and kidneys weighed, and tumor removed for immunohistochemical staining for endothelial cells (CD34), pericytes (alpha-SMA), apoptosis (TUNEL [terminal deoxynucleotidyl transferase dUTP nick-end labeling]), and diI-labeled NPCs. RESULTS: Fluorescent-labeled NPCs confirmed localization of these cells to tumors. The alpha-SMA/CD34 ratio, a marker for vascular maturation, greatly increased in NPC-IFN-beta-treated tumors compared with controls. Bioluminescent signal from luciferase-expressing tumor cells, reflecting tumor burden, was lower with combination therapy than control or either monotherapy, and combination therapy resulted in far less tumor burden by weight in the kidneys and liver. CONCLUSIONS: Targeted delivery of IFN-beta with NPCs produced low circulating levels of IFN-beta, yet the maturing effect on the tumor vasculature and the enhanced efficacy of adjuvant therapy was maintained. Thus, combination therapy of NPC-IFN-beta with CTX warrants further investigation for the treatment of high-risk neuroblastoma patients.

Phase I trial of single-dose temozolomide and continuous administration of o6-benzylguanine in children with brain tumors: a pediatric brain tumor consortium report.

PURPOSE: To estimate the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of escalating doses of temozolomide combined with O(6)-benzylguanine in patients < or =21 years with recurrent brain tumors. EXPERIMENTAL DESIGN: Treatment strata consisted of patients who had previously received no or local radiotherapy (Str1) and patients who had undergone craniospinal radiotherapy or myeloablative chemotherapy (Str2). One-hour i.v. administration of O(6)-benzylguanine at 120 mg/m(2) was followed by 48-h continuous infusion at 30 mg/m(2)/day. Single-dose temozolomide at five dosage levels (267, 355, 472, 628, and 835 mg/m(2)) was given at least 6 h after completion of O(6)-benzylguanine bolus. Treatment was repeated after recovery from toxicities at least 4 weeks apart for a maximum of 12 courses. Dose escalation followed the modified continual reassessment method. Pharmacokinetic analyses of temozolomide and 5-triazeno imidazole carboxamide (MTIC) were done in 28 patients. RESULTS: A total of 44 and 26 eligible patients were enrolled on Str1 and Str2, respectively. Median age at study entry in each stratum was 8.6 and 11.3 years, respectively. Predominant diagnoses were high-grade/brainstem glioma in Str1 and medulloblastoma in Str2. Whereas the estimated MTDs of temozolomide for Str1 and Str2 were 562 and 407 mg/m(2), respectively, the doses recommended for phase II investigations are 472 and 355 mg/m(2), respectively. DLTs were predominantly neutropenia and thrombocytopenia. Three patients with gliomas experienced centrally confirmed partial responses to therapy. Four patients completed all planned therapy. Temozolomide and MTIC exposures were statistically associated with temozolomide dosage. CONCLUSIONS: The current schedule of temozolomide and O(6)-benzylguanine is safe and showed modest activity against recurrent brain tumors in children.

Planarity and constraint of the carbonyl groups in 1,2-diones are determinants for selective inhibition of human carboxylesterase 1.

Carboxylesterases (CE) are ubiquitous enzymes responsible for the detoxification of xenobiotics, including numerous clinically used drugs. Therefore, the selective inhibition of these proteins may prove useful in modulating drug half-life and bioavailability. Recently, we identified 1,2-diones as potent inhibitors of CEs, although little selectivity was observed in the inhibition of either human liver CE (hCE1) or human intestinal CE (hiCE). In this paper, we have further examined the inhibitory properties of ethane-1,2-diones toward these proteins and determined that, when the carbonyl oxygen atoms are cis-coplanar, the compounds demonstrate specificity for hCE1. Conversely, when the dione oxygen atoms are not planar (or are trans-coplanar), the compounds are more potent at hiCE inhibition. These properties have been validated in over 40 1,2-diones that demonstrate inhibitory activity toward at least one of these enzymes. Statistical analysis of the results confirms the correlation (P < 0.001) between the dione dihedral angle and the preferential inhibition of either hiCE or hCE1. Overall, the results presented here define the parameters necessary for small molecule inhibition of human CEs.

Targeting methylguanine-DNA methyltransferase in the treatment of neuroblastoma.

PURPOSE: The combination of temozolomide and irinotecan has preclinical schedule-dependent synergy against neuroblastoma but is not curative for relapsed high-risk patients. We hypothesized that the DNA repair protein methylguanine-DNA methyltransferase (MGMT) is an important resistance factor, and that inactivation of MGMT would sensitize neuroblastoma cells to these agents. EXPERIMENTAL DESIGN: MGMT protein expression was assessed in 74 primary neuroblastoma tumors. Growth inhibition assays were done to determine the IC(50) and the extent of synergy observed with various concentrations of temozolomide, irinotecan, and the MGMT-inactivating agent O(6)-benzylguanine, using cultured syngeneic neuroblastoma cells with either low or high levels of MGMT expression. We then assessed efficacy in a mouse xenograft model of metastatic neuroblastoma. RESULTS: MGMT was expressed by all 74 tumors evaluated. Pretreatment of neuroblastoma cells with O(6)-benzylguanine reduced the IC(50) of temozolomide by 10-fold regardless of level of MGMT expression, and pretreatment with BG followed by temozolomide + irinotecan further reduced the IC(50) in cells with high MGMT expression another 10-fold, to well below clinically achievable concentrations. The combination index was 0.27 to 0.30 for all three drugs in both cell lines, indicating strong synergy. Survival at 100 days for mice with metastatic neuroblastoma was 56% with three-drug treatment, compared with untreated controls (0%, P < 0.001) or temozolomide + irinotecan (10%, P = 0.081). CONCLUSIONS: MGMT is widely expressed in primary neuroblastoma tumors, and is a relevant therapeutic target. Both in vitro and in vivo studies suggest inactivation of MGMT with O(6)-benzylguanine may increase the activity of temozolomide and irinotecan against neuroblastoma.

Selective inhibition of carboxylesterases by isatins, indole-2,3-diones.

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.

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.

Intravascular administration of tumor tropic neural progenitor cells permits targeted delivery of interferon-beta and restricts tumor growth in a murine model of disseminated neuroblastoma.

BACKGROUND: Interferon-beta (IFN-beta) has potent antitumor activity; however, systemic toxicity has limited its clinical use. We investigated the potential of targeted delivery using tumor-tropic neural progenitor cells (NPCs) transduced to express human IFN-beta (hIFN-beta). METHODS: Disseminated neuroblastoma was established in SCID mice by tail vein injection of tumor cells. Fourteen days after tumor cell inoculation, systemic disease was confirmed with bioluminescence imaging (BLI). Mice were then treated by intravenous injection of human F3.C1 NPCs that had been transduced with a replication deficient adenovirus to overexpress hIFN-beta (F3-IFN-beta). Two injections were given: the first at 14 days and the second at 28 days following tumor cell injection. Control mice received NPCs transduced with empty vector adenovirus at the same time points. Progression of disease was monitored using BLI. At sacrifice, organ weights and histology further evaluated tumor burden. RESULTS: After initiation of therapy, BLI demonstrated a significant decrease in the rate of disease progression in mice receiving F3-IFN-beta. At necropsy, control mice had bulky tumor replacing the liver and kidneys, as well as extensive retroperitoneal and mediastinal adenopathy. Impressively, these sites within mice receiving F3-IFN-beta therapy appeared grossly normal with the exception of small nodules within the kidneys of some of the F3-IFN-beta-treated mice. The accumulation of F3.C1 cells within sites of tumor growth was confirmed by fluorescence imaging. Importantly, systemic levels of hIFN-beta in the treated mice remained below detectable levels. CONCLUSIONS: These data indicate that in this model of disseminated neuroblastoma, the tumor-tropic property of F3.C1 NPCs was exploited to target delivery of IFN-beta to disseminated tissue foci, resulting in significant tumor growth delay. The described novel approach for effective IFN-beta therapy may circumvent limitations associated with the systemic toxicity of IFN-beta.

Development of a tumor-selective approach to treat metastatic cancer.

BACKGROUND: Patients diagnosed with metastatic cancer have almost uniformly poor prognoses. The treatments available for patients with disseminated disease are usually not curative and have side effects that limit the therapy that can be given. A treatment that is selectively toxic to tumors would maximize the beneficial effects of therapy and minimize side effects, potentially enabling effective treatment to be administered. METHODS AND FINDINGS: We postulated that the tumor-tropic property of stem cells or progenitor cells could be exploited to selectively deliver a therapeutic gene to metastatic solid tumors, and that expression of an appropriate transgene at tumor loci might mediate cures of metastatic disease. To test this hypothesis, we injected HB1.F3.C1 cells transduced to express an enzyme that efficiently activates the anti-cancer prodrug CPT-11 intravenously into mice bearing disseminated neuroblastoma tumors. The HB1.F3.C1 cells migrated selectively to tumor sites regardless of the size or anatomical location of the tumors. Mice were then treated systemically with CPT-11, and the efficacy of treatment was monitored. Mice treated with the combination of HB1.F3.C1 cells expressing the CPT-11-activating enzyme and this prodrug produced tumor-free survival of 100% of the mice for >6 months (P<0.001 compared to control groups). CONCLUSIONS: The novel and significant finding of this study is that it may be possible to exploit the tumor-tropic property of stem or progenitor cells to mediate effective, tumor-selective therapy for metastatic tumors, for which no tolerated curative treatments are currently available.

Phase II study of oxaliplatin in children with recurrent or refractory medulloblastoma, supratentorial primitive neuroectodermal tumors, and atypical teratoid rhabdoid tumors: a pediatric brain tumor consortium study.

BACKGROUND: An open-label Phase II study of oxaliplatin was conducted to evaluate its safety and efficacy in children with recurrent or refractory medulloblastoma (MB), supratentorial primitive neuroectodermal tumors (SPNET), and atypical teratoid rhabdoid tumor (ATRT). METHODS: Patients were stratified as follows: stratum IA, first recurrence MB with measurable disease; IB, recurrent MB with only cerebral spinal fluid (CSF) positivity or linear leptomeningeal disease (LLD); IC, MB > or =second recurrence; stratum II, recurrent SPNET; stratum III, recurrent ATRT. Patients received oxaliplatin, 130 mg/m(2) intravenously over 2 hours every 3 weeks. The primary objective was to estimate the sustained response rate in stratum 1A. Plasma ultrafiltrate platinum pharmacokinetics were evaluated. RESULTS: A total of 43 patients with a median age of 8.5 years (range, 0.6-18.9 years) were enrolled. In stratum 1A, 2 of 15 had partial responses (PRs, 1 sustained PR). No responses were observed in other strata. The most frequent Grade 3 and 4 toxicities included thrombocytopenia (25.6%), neutropenia (16.3%), leukopenia (12%), increase in serum alanine transaminase (ALT) (7%), vomiting (4.7%), and sensory neuropathy (4.7%). No severe ototoxicity or nephrotoxicity was reported. Plasma ultrafiltrate platinum pharmacokinetic parameters were similar to adults, with a median clearance of 12.2 L/hr (range, 4.4-30 L/hr) and median area under the curve (AUC(0-infinity)) of 9.4 microg/mL/hr (range, 6.2-13.9 microg/mL/hr). CONCLUSIONS: Oxaliplatin was well tolerated in children but has limited activity in children with recurrent CNS embryonal tumors previously treated with platinum compounds.

Pilot study to evaluate MYCN expression as a neuroblastoma cell marker to detect minimal residual disease by RT-PCR.

This pilot study was performed to determine whether MYCN expression warrants further investigation as a tumor marker to detect low levels of residual neuroblastoma (NB). Seven NB cell lines and 30 bone marrow (BM) samples from patients with high-risk NB were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) for MYCN expression, and for the established NB marker tyrosine hydroxylase. MYCN was expressed in all 7 NB cell lines, but not in normal peripheral blood, CD34 cells, or BM. In dilution studies using cell lines with or without DNA amplification of MYCN, 1 NB cell in 10 to 10 nucleated blood cells was detectable by RT-PCR. MYCN was identified in all 21 BM samples in which tumor cells were identified by histologic examination, including 4 samples in which tyrosine hydroxylase was not detected. Additionally, expression of both markers was detected in 5 samples that were negative by histology but presumably contained low levels of tumor cells, consistent with the greater sensitivity of RT-PCR compared with morphologic methods. Detection of MYCN RNA was independent of MYCN DNA amplification status. The selective expression of MYCN in tumor cells, and the sensitivity of detection of MYCN by RT-PCR noted in this and other studies, supports further evaluation of MYCN as a NB marker for molecular detection of minimal residual disease.

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.

Development of an etoposide prodrug for dual prodrug-enzyme antitumor therapy.

Enzyme-prodrug approaches to cancer therapy, theoretically, have the potential to mediate tumor-selective cytotoxicity. However, even if tumor-specific prodrug activation is achieved, enzyme-prodrug systems investigated thus far comprised a single enzyme and a specific prodrug. Although targeted, such systems constitute single-agent therapy, which may be ineffective and/or may promote development of drug resistance. Therefore, a goal of our laboratories was to design and characterize a novel dipiperidinyl derivative of etoposide [1,4'-dipiperidine-1'-carboxylate-etoposide (dp-VP16)] that would act as a prodrug. We envisioned that dp-VP16 would be converted to the active chemotherapeutic agent VP-16 by the same rabbit carboxylesterase (rCE) that we have previously shown to efficiently activate the prodrug irinotecan (CPT-11). This dp-VP16 prodrug might then be used in combination with CPT-11, with both drugs activated by a single enzyme. We evaluated the ability of pure rCE and two human carboxylesterases, hCE1 and hiCE (hCE2), to activate dp-VP16 in vitro, and in neuroblastoma cell lines designed to express/overexpress each enzyme. In SK-N-AS neuroblastoma cell transfectants, expression of rCE or hiCE decreased the IC50 of dp-VP16 as a single agent by 8.3- and 3.4-fold, respectively, in growth inhibition assays. Purified hCE1 did not metabolize dp-VP16 in vitro and did not affect its IC50 in intact cells. The combination indices of sequential exposure to CPT-11 followed by dp-VP16 ranged from approximately 0.4 to 0.6, suggesting that this combination produced greater-than-additive cytotoxicity in neuroblastoma cells expressing rCE. These data provide proof-of-principle that enzyme-prodrug therapy approaches comprised of prodrugs with complementary mechanisms of cytotoxicity that are activated by a single enzyme can be developed.

Inhibition of carboxylesterases by benzil (diphenylethane-1,2-dione) and heterocyclic analogues is dependent upon the aromaticity of the ring and the flexibility of the dione moiety.

Benzil has been identified as a potent selective inhibitor of carboxylesterases (CEs). Essential components of the molecule required for inhibitory activity include the dione moiety and the benzene rings, and substitution within the rings affords increased selectivity toward CEs from different species. Replacement of the benzene rings with heterocyclic substituents increased the K(i) values for the compounds toward three mammalian CEs when using o-nitrophenyl acetate as a substrate. Logarithmic plots of the K(i) values versus the empirical resonance energy, the heat of union of formation energy, or the aromatic stabilization energy determined from molecular orbital calculations for the ring structures yielded linear relationships that allowed prediction of the efficacy of the diones toward CE inhibition. Using these data, we predicted that 2,2'-naphthil would be an excellent inhibitor of mammalian CEs. This was demonstrated to be correct with a K(i) value of 1 nM being observed for a rabbit liver CE. In addition, molecular simulations of the movement of the ring structures around the dione dihedral indicated that the ability of the compounds to inhibit CEs was due, in part, to rotational constraints enforced by the dione moiety. Overall, these studies identify subdomains within the aromatic ethane-1,2-diones, that are responsible for CE inhibition.

Brain tumor oncolysis with replication-conditional herpes simplex virus type 1 expressing the prodrug-activating genes, CYP2B1 and secreted human intestinal carboxylesterase, in combination with cyclophosphamide and irinotecan.

The treatment of malignant glioma is currently ineffective. Oncolytic viruses are being explored as a means to selectively lyse tumor cells in the brain. We have engineered a mutant herpes simplex virus type 1 with deletions in the viral UL39 and gamma(1)34.5 genes and an insertion of the two prodrug activating genes, CYP2B1 and secreted human intestinal carboxylesterase. Each of these can convert the inactive prodrugs, cyclophosphamide and irinotecan (CPT-11), into their active metabolites, respectively. This new oncolytic virus (MGH2) displays increased antitumor efficacy against human glioma cells both in vitro and in vivo when combined with cyclophosphamide and CPT-11. Importantly, cyclophosphamide, CPT-11, or the combination of cyclophosphamide and CPT-11 does not significantly affect oncolytic virus replication. Therefore, MGH2 provides effective multimodal therapy for gliomas in preclinical models when combined with these chemotherapy agents.

Activation and antitumor activity of CPT-11 in plasma esterase-deficient mice.

PURPOSE: To examine the antitumor activity and the pharmacokinetics of CPT-11 (irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin) in a plasma esterase-deficient scid mouse model, bearing human tumor xenografts. EXPERIMENTAL DESIGN: Plasma carboxylesterase (CE)-deficient mice were bred with scid animals to develop a strain that would allow growth of human tumor xenografts. Following xenotransplantation, the effect of the plasma esterase on antitumor activity following CPT-11 administration was assessed. In addition, detailed pharmacokinetic studies examining plasma and biliary disposition of CPT-11 and its metabolites were performed. RESULTS: In mice lacking plasma carboxylesterase, the mean SN-38 systemic exposures were approximately fourfold less than that observed in control animals. Consistent with the pharmacokinetic data, four to fivefold more CPT-11 was required to induce regressions in human Rh30 xenografts grown in esterase-deficient scid mice, as opposed to those grown in scid animals. Additionally, the route of elimination of CPT-11, SN-38, and SN-38 glucuronide (SN-38G) was principally in the bile. CONCLUSIONS: The pharmacokinetic profile for CPT-11 and its metabolites in the esterase-deficient mice more closely reflects that seen in humans. Hence, these mice may represent a more accurate model for antitumor studies with this drug and other agents metabolized by CEs.

Identification and characterization of novel benzil (diphenylethane-1,2-dione) analogues as inhibitors of mammalian carboxylesterases.

Carboxylesterases (CE) are ubiquitous enzymes responsible for the metabolism of xenobiotics. Because the structural and amino acid homology among esterases of different classes, the identification of selective inhibitors of these proteins has proved problematic. Using Telik's target-related affinity profiling (TRAP) technology, we have identified a class of compounds based on benzil (1,2-diphenylethane-1,2-dione) that are potent CE inhibitors, with K(i) values in the low nanomolar range. Benzil and 30 analogues demonstrated selective inhibition of CEs, with no inhibitory activity toward human acetylcholinesterase or butyrylcholinesterase. Analysis of structurally related compounds indicated that the ethane-1,2-dione moiety was essential for enzyme inhibition and that potency was dependent on the presence of, and substitution within, the benzene ring. 3D-QSAR analyses of these benzil analogues for three different mammalian CEs demonstrated excellent correlations of observed versus predicted K(i) (r(2) > 0.91), with cross-validation coefficients (q(2)) of 0.9. Overall, these results suggest that selective inhibitors of CEs with potential for use in clinical applications can be designed.