Doses Of BCNU Research Articles

Phase 1 trial of O6‐benzylguanine and BCNU in children with CNS tumors: A Children's Oncology Group study

Efficacy of nitrosoureas is limited by host repair of drug-induced alkylation. O(6)-benzylguanine (O(6)-BG), an inhibitor of host alkylation repair, and BCNU were studied in children with refractory/untreatable central nervous system tumors to determine dose-limiting toxicities (DLTs) and maximum tolerated dose (MTD) of BCNU administered following O(6)-BG. O(6)-BG (120 mg/m(2) IV over 1 hr) was followed by BCNU (IV over 1 hr). Cohorts of three to six patients were treated with escalating doses of BCNU. Courses were repeated every 6 weeks. Patients in Stage 1 were accrued irrespective of prior treatment. Once the MTD was exceeded, Stage II accrual was limited to less heavily pretreated patients (</= two prior chemotherapy regimens, no prior central axis radiation, no prior bone marrow transplant, and no bone marrow involvement). Twelve patients in Stage I and 13 in Stage II (less heavily pretreated patients only) were evaluable for toxicity. The MTD of BCNU administered with O(6)-BG (120 mg/m(2) IV) was 58 mg/m(2) in less-heavily pretreated patients. Myelosuppression, which was cumulative in some patients receiving multiple cycles of therapy, was the predominate DLT. Twenty-four patients were evaluable for response: after two courses of therapy, 6 had stable disease, 17 had progressive disease, and 1 patient had a minor response but progressed after four courses of therapy. Based on lack of activity of this combination in adult phase II studies, no further testing of O(6)-BG plus BCNU in children is planned. Strategies to decrease hematopoeitic toxicity of BCNU plus O(6)-BG are required.

Tumorigenesis of Chemotherapeutic Drug-Resistant Cancer Stem-Like Cells in Brain Glioma

Glioblastoma multiforme (GBM) is the most frequently occurring brain cancer. Although the existence of cancer stem cells (CSCs) in GBM has been established, there is little evidence to explain the link between CSCs and chemoresistance. In this study, we developed a dissociated cell system of human GBM cells, A172 and established GBM2 cells, that have shown resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). After exposure to a lethal dose of BCNU, the small population of GBM cancer cells survived and proliferated, as opposed to direct inhibition of the apoptosis and activation of the proliferation signal. Also, these cells contained subpopulations of stem-like cells, expressing CD133, CD117, CD90, CD71, and CD45 cell-surface markers, and had the capacity for multipotency. Moreover, we observed that BCNU-resistant subpopulations derived from GBM cancer cells can be grown to tumors when transplanted into severe combined immunodeficient (SCID) mouse brain. These results demonstrated that BCNU-resistant subpopulations derived from GBM have cancer stem-like cell properties. These findings provide further evidence that CSCs in GBM display chemotherapeutic drug resistance. Hopefully, it will be possible to improve the therapeutic outcome of GBM, leading to better anticancer strategies.

CXCR4 Inhibition Synergizes with Cytotoxic Chemotherapy in Gliomas

The chemokine receptor CXCR4 is expressed in many different cancers. In malignant brain tumors, CXCR4 signaling has been implicated in tumor growth, survival, and migration, and pharmacologic inhibition of CXCR4 results in decreased tumor growth in preclinical models. To understand how CXCR4 inhibitors may be incorporated into clinical therapy, we examined determinants of responsiveness to CXCR4 inhibition. Because optimal use of CXCR4 inhibition will likely be a part of multimodality therapy, we also investigated the efficacy of CXCR4 inhibition combined with conventional cytotoxic chemotherapy. CXCR4 protein levels and responsiveness to the CXCR4 inhibitor AMD3100 were determined in a panel of glioblastoma multiforme cell lines. The effects of AMD3100, alone or in combination with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), on cell growth were determined for several of these cell lines in vitro. We used an orthotopic model of glioblastoma multiforme to evaluate the antitumor efficacy of AMD3100 combined with BCNU in vivo. The level of CXCR4 protein expression in glioblastoma multiforme cells predicts the dose at which there is a response to AMD3100; cells that express higher levels of CXCR4 protein require higher doses for equivalent response. In all cell lines tested, treatment of glioblastoma multiforme cells with BCNU followed by AMD3100 results in synergistic antitumor efficacy in vitro. This synergy can also be seen in an orthotopic glioblastoma multiforme model. Treatment with subtherapeutic doses of BCNU in combination with AMD3100 results in tumor regression in vivo, and this reflects both increased apoptosis and decreased proliferation following combination therapy. These studies support testing CXCR4 inhibitors in patients with glioblastoma multiforme and establish that inhibition of CXCR4 synergizes with conventional cytotoxic therapies in a clinically relevant combinatorial strategy.

Long-term outcome of high-dose chemotherapy and autologous stem cell transplantation in relapsed/refractory Hodgkin’s disease: A cohort of 199 pts from Royal Marsden Hospital

7622 Background: The purpose was to assess prognostic factors and outcome of pts with relapsed/refractory Hodgkin disease (HD) who have received high-dose chemotherapy and autologous stem cell transplant (ASCT). Methods: The retrospective study was approved by Research Ethics Committee. Primary end-point was overall (OS) and progression free survival (PFS). OS was defined as death from any cause measured from date of transplant and PFS defined as disease progression or death from any cause. Data on 199 pts who received ASCT between 1985–6/05 was reviewed. Results: Median time from 1st treatment(tx) to ASCT was 2.5 y (0.5–27.3). Demography at ASCT:61% Stage IV, median age 31y(18–69); median prior tx regimens 3 (1–7); median Hasenclever index 3 (0–6); 150 pts had responding disease (53 CR, 97 PR), 49 pts had stable/untested relapse/refractory disease. 62% pts received MBE as conditioning tx and bone marrow was source of stem cells in 57%. Post-ASCT, 61% (122/199) pts attained CR with an overall response (CR+PR) of 85%. 12 pts had non-relapse mortality;10 died before 1990 of interstitial pneumonitis (with higher dose BCNU). Of 122 pts attaining CR, 27 relapsed;3 after attaining CR for &gt;5y and 1 after attaining CR for&gt;10-y. 5-y risk of relapse in patients attaining CR was 21%. Median OS/PFS from ASCT was 9y/2.6y. 105 pts are alive at median follow-up of 10.3y. 5-y OS/PFS was 52/45% and 10-y OS/PFS was 49.5/41% for whole group. 10-y OS/PFS for pts in CR vs PR vs rest at ASCT was 72/66% vs 55/44% vs 11/5%. 20(10%) pts developed 2nd cancer (7 secondary AML/MDS). Probability of developing 2nd cancer at 10y is 6.9% (95%CI 3.6–13%) and 19.8% (95%CI 12–32%) at 20-y. Conclusions: These data provide the longest followup reported for patients receiving ASCT for relapsed/refractory HD. In addition to previously described prognostic factors, our data shows that Hasenclever index &lt;3 influences outcome favorably and attaining CR at ASCT leads to a significantly better outcome. [Table: see text] No significant financial relationships to disclose.

Hydrogen peroxide and redox modulation sensitize primary mouse hepatocytes to TNF-induced apoptosis

Tumor necrosis factor α (TNF) plays an important role in mediating hepatocyte injury in various liver pathologies. TNF treatment alone does not cause the death of primary cultured hepatocytes, suggesting other factors are necessary to mediate TNF-induced injury. In this work the question of whether reactive oxygen species can sensitize primary cultured hepatocytes to TNF-induced apoptosis and necrosis was investigated. Sublethal levels of H 2O 2, either as bolus doses or steady-state levels generated by glucose oxidase, were found to sensitize cultured hepatocytes to TNF-induced apoptosis. High levels of H 2O 2 also triggered necrosis in hepatocytes regardless of whether TNF was present. Similarly, antimycin, a complex III inhibitor that increases reactive oxygen species generation from mitochondria, sensitized hepatocytes to TNF-induced apoptosis at low doses but caused necrosis at high doses. Redox changes seem to be important in sensitizing primary hepatocytes, because diamide, a thiol-oxidizing agent, and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of GSSG reductase, also increased TNF-induced apoptosis in cultured primary hepatocytes at sublethal doses. High doses of diamide and BCNU predominantly triggered necrotic cell death. Agents that sensitized hepatocytes to TNF-induced apoptosis—H 2O 2, antimycin, diamide, BCNU—all caused a dramatic fall in the GSH/GSSG ratio. These redox alterations were found to inhibit TNF-induced IκB-α phosphorylation and NF-κB translocation to the nucleus, thus presumably inhibiting expression of genes necessary to inhibit the cytotoxic effects of TNF. Taken together, these results suggest that oxidation of the intracellular environment of hepatocytes by reactive oxygen species or redox-modulating agents interferes with NF-κB signaling pathways to sensitize hepatocytes to TNF-induced apoptosis. The TNF-induced apoptosis seems to occur only in a certain redox range—in which redox changes can inhibit NF-κB activity but not completely inhibit caspase activity. The implication for liver disease is that concomitant TNF exposure and reactive oxygen species, either extrinsically generated (e.g., nonparenchymal or inflammatory cells) or intrinsically generated in hepatocytes (e.g., mitochondria), may act in concert to promote apoptosis and liver injury.

Over-representation of specific regions of chromosome 22 in cells from human glioma correlate with resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea

BackgroundGlioblastoma multiforme is the most malignant form of brain tumor. Despite treatment including surgical resection, adjuvant chemotherapy, and radiation, these tumors typically recur. The recurrent tumor is often resistant to further therapy with the same agent, suggesting that the surviving cells that repopulate the tumor mass have an intrinsic genetic advantage. We previously demonstrated that cells selected for resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) are near-diploid, with over-representation of part or all of chromosomes 7 and 22. While cells from untreated gliomas often have over-representation of chromosome 7, chromosome 22 is typically under-represented.MethodsWe have analyzed cells from primary and recurrent tumors from the same patient before and after in vitro selection for resistance to clinically relevant doses of BCNU. Karyotypic analyses were done to demonstrate the genetic makeup of these cells, and fluorescent in situ hybridization analyses have defined the region(s) of chromosome 22 retained in these BCNU-resistant cells.ResultsKaryotypic analyses demonstrated that cells selected for BCNU resistance were near-diploid with over-representation of chromosomes 7 and 22. In cells where whole copies of chromosome 22 were not identified, numerous fragments of this chromosome were retained and inserted into several marker and derivative chromosomes. Fluorescent in situ hybridization analyses using whole chromosome paints confirmed this finding. Additional FISH analysis using bacterial artificial chromosome probes spanning the length of chromosome 22 have allowed us to map the over-represented region to 22q12.3–13.32.ConclusionCells selected for BCNU resistance either in vivo or in vitro retain sequences mapped to chromosome 22. The specific over-representation of sequences mapped to 22q12.3–13.32 suggest the presence of a DNA sequence important to BCNU survival and/or resistance located in this region of chromosome 22.

737. Efficient In Vivo Selection and Chemoprotection Following Drug Resistance Gene Therapy in a Nonmyeloablative Allogeneic Transplantation Setting in the Dog

We have previously demonstrated successful in vivo selection and chemoprotection in dogs that received myeloablative allogeneic stem cell transplantation with gene-modified cells. Here we wished to investigate whether I vivo selection, chemoprotection, and modulation of donor chimerism could also be achieved after nonmyeloablative transplantation which should be less toxic and thus more appropriate for older patients and patients with genetic diseases. We have used a lentivirus vector encoding the P140K mutant of methylguanine-DNA methyltransferase (MGMT-P140K) to transduce DLA matched littermate CD34+ cells. The use of a lentivirus vector allowed us to keep the ex vivo/transduction time short, less than 24 hours which improves engraftment in the allogeneic setting. Three dogs received a nonmyeloablative dose of total body irradiation (TBI) ranging from 200 cGy to 400 cGy before infusion of the gene-modified CD34+ cells and early post-transplant chemotherapy to facilitate engraftment. All three dogs had stable gene marking and donor chimerism, before receiving a course of treatment with O6BG/BCNU. One to three treatments led to a sustained increase in gene marking in all dogs with maximum levels achieved of >75%. Furthermore, the transduced cells conferred chemoprotection relative to control animals and prevented severe neutropenia even with ascending doses of BCNU. Our initial analysis of retrovirus integration with LAM-PCR suggest that before and after in vivo selection of chemoprotected cells multiple clones contribute to hematopoiesis and no side effects or malignancies have been observed.Our results suggest that drug resistance gene therapy and subsequent in vivo selection is feasible and safe in the nonmyeloablative transplantation setting. We are continuing to modify the pre-transplant immunosuppression and post-transplant in vivo selection to facilitate engraftment of gene-modified cells. In nonmyeloablative allogeneic transplantation for leukemia where post-transplant relapse remains a substantial complication, the application of this model could reduce the toxicity of chemotherapy treatment and allow for more intensive treatment regimens. In addition this strategy could be used to increase the percentage of gene-marked donor cells in patients with genetic diseases and low donor chimerism. These studies and future studies in the nonmyeloablative setting are required to assess the long-term safety of chemo-protective gene therapy. We have previously demonstrated successful in vivo selection and chemoprotection in dogs that received myeloablative allogeneic stem cell transplantation with gene-modified cells. Here we wished to investigate whether I vivo selection, chemoprotection, and modulation of donor chimerism could also be achieved after nonmyeloablative transplantation which should be less toxic and thus more appropriate for older patients and patients with genetic diseases. We have used a lentivirus vector encoding the P140K mutant of methylguanine-DNA methyltransferase (MGMT-P140K) to transduce DLA matched littermate CD34+ cells. The use of a lentivirus vector allowed us to keep the ex vivo/transduction time short, less than 24 hours which improves engraftment in the allogeneic setting. Three dogs received a nonmyeloablative dose of total body irradiation (TBI) ranging from 200 cGy to 400 cGy before infusion of the gene-modified CD34+ cells and early post-transplant chemotherapy to facilitate engraftment. All three dogs had stable gene marking and donor chimerism, before receiving a course of treatment with O6BG/BCNU. One to three treatments led to a sustained increase in gene marking in all dogs with maximum levels achieved of >75%. Furthermore, the transduced cells conferred chemoprotection relative to control animals and prevented severe neutropenia even with ascending doses of BCNU. Our initial analysis of retrovirus integration with LAM-PCR suggest that before and after in vivo selection of chemoprotected cells multiple clones contribute to hematopoiesis and no side effects or malignancies have been observed. Our results suggest that drug resistance gene therapy and subsequent in vivo selection is feasible and safe in the nonmyeloablative transplantation setting. We are continuing to modify the pre-transplant immunosuppression and post-transplant in vivo selection to facilitate engraftment of gene-modified cells. In nonmyeloablative allogeneic transplantation for leukemia where post-transplant relapse remains a substantial complication, the application of this model could reduce the toxicity of chemotherapy treatment and allow for more intensive treatment regimens. In addition this strategy could be used to increase the percentage of gene-marked donor cells in patients with genetic diseases and low donor chimerism. These studies and future studies in the nonmyeloablative setting are required to assess the long-term safety of chemo-protective gene therapy.

Proton and sodium MRI assessment of emerging tumor chemotherapeutic resistance.

The ultimate goal of any cancer therapy is to target the elimination of neoplastic cells. Although newer therapeutic strategies are in constant development, therapeutic assessment has been hampered by the inability to assess, rapidly and quantitatively, efficacy in vivo. Diffusion imaging and, more recently, sodium MRI have demonstrated their distinct abilities to detect therapy-induced alterations in tumor cellularity, which has been demonstrated to be indicative of therapeutic efficacy. More importantly, both imaging modalities detect tumor response much earlier than traditional methodologies that rely on macroscopic volumetric changes. In this study, the correlation between tumor sodium and diffusion was further tested to demonstrate the sensitivity of sodium imaging to gauge tumor response to therapy by using a 9L rat gliosarcoma treated with varying doses of BCNU [1,3-bis(2-chloroethyl)-1-nitrosourea]. This orthotopic model has been demonstrated to display variability in response to BCNU therapy where initial insult has been shown to lead to drug-resistance. In brief, a single 26.6 mg/kg BCNU dose yielded dramatic responses in both diffusion and sodium MRI. However, a second equivalent BCNU dose yielded a much smaller change in diffusion and sodium, suggesting a drop in tumor sensitivity to BCNU. The MRI responses of animals treated with 13.3 mg/kg BCNU were much lower and similar responses were observed after the initial and secondary applications of BCNU. Furthermore, these results were further validated using volumetric measurements of the tumor and also ex vivo determination of tumor sensitivity to BCNU. Overall, these experiments demonstrate the sensitivity and applicability of sodium and diffusion MRI as tools for dynamic assessment of tumor response to therapy.

Gene expression profile induced by BCNU in human glioma cell lines with differential MGMT expression

Chemotherapy with the alkylating agent BCNU (1,3-bis (2-chloroethyl)-1-nitrosourea) is the most commonly used chemotherapeutic agent for gliomas. However, the usefulness of this agent is limited because tumor cell resistance to BCNU is frequently found in clinical brain tumor therapy. The O6-methylguanine-DNA methyltransferase protein (MGMT) reverses alkylation at the O6 position of guanine and we have reported the role of MGMT in the response of brain tumors to alkylating agents. However, the different mechanisms underlying the patterns related to MGMT remain unclear. To better understand the molecular mechanism by which BCNU exerts its effect in glioma cell lines according MGMT expression, we used microarray technology to interrogate 3800 known genes and determine the gene expression profiles altered by BCNU treatment. Our results showed that treatment with BCNU alters the expression of a diverse group of genes in a time-dependent manner. A subset of gene changes was found common in both glioma cell lines and other subset is specific of each cell line. After 24 h of BCNU treatment, up-regulation of transcription factors involved in the nucleation of both RNA polymerase II and III transcription initiation complexes was reported. Interestingly, BCNU promoted the expression of actin-dependent regulators of chromatin. Similar effects were found with higher BCNU doses in MGMT+ cell line showing a similar mechanism that in MGMT-deficient cell with standard doses. Our data suggest that human glioma cell lines treated with BCNU, independently of MGMT expression, show changes in the expression of cell cycle and survival-related genes interfering the transcription mechanisms and the chromatin regulation.

A phase I trial with extended cohort of gemcitabine and vinorelbine followed by autologous peripheral blood stem cell transplantation for recurrent or refractory Hodgkin’s lymphoma

6651 Background: High dose chemotherapy and autologous stem cell transplantation (ASCT) is the most effective treatment for recurrent or refractory Hodgkin’s lymphoma (HL). Disease recurrence is still a major cause of treatment failure. Augmented BCNU-regimens are reported to have good outcomes but greater pulmonary toxicity. A novel transplant regimen incorporating gemcitabine and vinorelbine, active drugs in HL different from alkylating agents, was tested to establish MTD of gemcitabine and to assess safety at the MTD. Methods: In this phase I extended cohort study, dose escalation with gemcitabine in combination with vinorelbine followed by high dose BCNU, etoposide (VP-16), and cyclophosphamide (CY) and ASCT. 18 patients with recurrent or refractory HL were enrolled. 7 patients had dose escalation before determining the MTD of gemcitabine at 1250 mg/m2 on the basis of elevated liver transaminases and a symptom complex of fever, HA, and skin toxicity. 11 additional patients were studied at the MTD to establish safety. The regimen consisted of gemcitabine 1250 mg/m2 CI and vinorelbine 30 mg/m2 on day-13 and day-8, BCNU 10 mg/kg on day-6, VP-16 60 mg/kg on day-4, CY 100 mg/kg on day-2. Regimen-related toxicity was the primary endpoint. FFP and EFS were secondary endpoints. Results: 5 patients had no risk features; 13 had high risk features (stage IV disease at relapse, failure to achieve min disease, or B sxs at relapse). Median f/u time was 231 days (44 to 791d). Gr 1–2 skin rash was seen but with complete resolution. Transient fever and liver transaminase elevations also fully recovered. All patients engrafted. Remarkable was reduction in BCNU-induced pneumonitis in only 2 of 18 patients (11%), as compared to our standard regimen incidence rate of 35%. Est FFP and EFS at 1 year in low risk patients was 100% and for high risk patients, 73%(CI:47–99%) Conclusions: This novel transplant regimen has decreased incidence of pulmonary toxicity compared with augmented BCNU-regimens and provides a reduced toxicity platform for further development of post-transplant immunotherapy to improve transplant outcome. No significant financial relationships to disclose.

Safety and Efficacy of CBV Followed by Autologous PBSC Transplant in Children with Lymphoma after Failed Induction or First Relapse - a Children's Oncology Group Study.

Purpose: To determine the safety and efficacy of high dose cyclophosphamide, BCNU and etoposide (CBV) chemotherapy followed by autologous non-purged PBSCT in children with Hodgkin's (HD) and non-Hodgkin's lymphoma (NHL) who have either failed primary induction therapy or have experienced a first relapse.Methods: At study entry, patients received 2–4 courses of reinduction therapy as determined by the local institution. When a CR or PR was achieved, G-CSF mobilized PBSCs were harvested and patients underwent autologous PBSCT after a preparative therapy of cyclophosphamide 6000 mg/m2, BCNU 450–300 mg/m2, and etoposide 2400 mg/m2. Patients were to receive a set dose of 5x106 CD34 cells/kg. Patients were followed for disease status and toxicities. 69 patients (38 HD, 31 NHL) were entered onto study between April 1998 and June 2002. Survival statistics were calculated as Kaplan Meier estimates.Results: 41/69 (27/38 HD, 14/31 NHL) achieved a CR/PR at the conclusion of reinduction; 14 failed due to PD; 7 had SD; 7 were inevaluable. 38 patients (27 HD, 11 NHL) proceeded to autologous PBSCT. Overall 28/38 (20 HD, 8 NHL) of the transplanted patients survive and 21/28 (15 HD, 6 NHL) are progression free. The survival rates at 12 months and 24 months were 88%(±6) (HD 92%(±6), NHL 78%(±14)) and 73%(±10) (HD 76%(±11), NHL 65%(±22)), respectively. The rates of PFS at 12 months and 24 months were 65%(±8)(HD 72%(±9); NHL 46%(±17)) and 53%(±11) (HD 56%(±12); NHL 46%(±19)), respectively. 10 patients died after PBSCT. The causes of death were PD (N=5), infection (N=4), and toxicity (N=1). The first 17 patients received BCNU 450 mg/m2 and 8 of these experienced grade 3 or 4 pulmonary or renal toxicity. The dose was thus dropped to 300 mg/m2 and an additional 21 patients underwent PBSCT and 3 of these patients developed grade 3 or 4 pulmonary or renal toxicity. The incidence of grade 3 or 4 pulmonary of renal toxicity at 300mg/m2 was significantly lower than that at 450 mg/m2 (p=0.04, Fisher's exact test).Conclusions: For children with primary resistant or first relapsed lymphoma who achieve a CR or PR after reinduction, CBV preparative therapy followed by PBSCT for children with primarily resistant or relapsed lymphoma results in an acceptable survival but a lower PFS due to a high relapse rate following transplant. A BCNU dose of 300 mg/m2, but not a dose of 450 mg/m2 is well tolerated by these heavily treated patients.

Evaluation of in vitro and in vivo antitumor activity of BCNU-loaded PLGA wafer against 9L gliosarcoma

The purpose of the present study was to develop implantable BCNU-loaded poly( d, l-lactide-co-glycolide) (PLGA) wafer for the controlled release of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and to evaluate its in vitro and in vivo antitumor activity. The release rate of BCNU from PLGA wafer increased with the increase of BCNU amount loaded and the release was continued until 7 days. In vitro and in vivo antitumor activity of BCNU-loaded PLGA wafer was investigated using in vitro cytotoxicity against 9L gliosarcoma cells and a subcutaneous (s.c.) solid tumor model of 9L gliosarcoma, respectively. The wafers containing BCNU showed more effective cytotoxicity than BCNU powder due to its short half-life and inhibited the proliferation of 9L gliosarcoma cells. BCNU-loaded PLGA wafer delayed the growth of the tumors significantly and increasing the dose of BCNU in the wafer resulted in a substantial regression of the tumor. These results of antitumor activity of BCNU-loaded PLGA wafer demonstrate the feasibility of the wafers for clinical application.

Evaluation of safety and tolerance of escalating doses of RSR13 administered with a fixed dose of BCNU every six weeks in patients with recurrent malignant glioma: Results of the phase I NABTT 9806 clinical trial

1533 Background: RSR13 is an allosteric modifier of hemoglobin which has been shown to increase tumor oxygen delivery, with potential to improve cytotoxic therapy. Previous studies in glioblastoma have demonstrated that the agent is tolerable at 100 mg/kg when delivered daily prior to radiotherapy, and survival was reported in excess of one year. Preclinical studies have shown enhancement of the response to certain chemotherapeuticagents as well. This protocol is assessing the safety and tolerance of escalating doses of RSR13 when administered prior to BCNU in patients with recurrent high grade glioma, as well as maximum tolerated dose and pharmacokinetics of RSR13 when administered with BCNU. Methods: RSR13, in escalating doses of 25 mg/kg, to a maximum of 100 mg/ kg, in cohorts of six patients each, was administered over 30 minutes prior to infusion of BCNU 200 mg., every six weeks, for a maximum of six cycles. Only nonhematologic toxicities are assessed in determining the dose limiting toxicity (DLT) of the combination of agents. Results: A total of 33 patients have been accrued, 14 females, 19 males. Two grade 3/4 hematologic toxicities occurred in the first cohort of six patients at 25 mg/kg, therefore this cohort was expanded to 12. One non-hematologic DLT was observed in this cohort (generalized weakness) and in the 50 mg/kg cohort, a DLT of pulmonary edema was observed. There were no DLTs in the 75 mg/kg cohort. Symptomatic hypoxia was observed as a significant adverse event in two patients in the 100 mg/kg cohort which is still enrolling. Conclusions: Hematologic toxicity is manageable in this drug combination. The maximum tolerated dose of RSR13 with BCNU is projected to be 75 or 100 mg/kg. No significant financial relationships to disclose.

Evaluation of safety and tolerance of escalating doses of RSR13 administered with a fixed dose of BCNU every six weeks in patients with recurrent malignant glioma: Results of the phase I NABTT 9806 clinical trial

1533 Background: RSR13 is an allosteric modifier of hemoglobin which has been shown to increase tumor oxygen delivery, with potential to improve cytotoxic therapy. Previous studies in glioblastoma have demonstrated that the agent is tolerable at 100 mg/kg when delivered daily prior to radiotherapy, and survival was reported in excess of one year. Preclinical studies have shown enhancement of the response to certain chemotherapeuticagents as well. This protocol is assessing the safety and tolerance of escalating doses of RSR13 when administered prior to BCNU in patients with recurrent high grade glioma, as well as maximum tolerated dose and pharmacokinetics of RSR13 when administered with BCNU. Methods: RSR13, in escalating doses of 25 mg/kg, to a maximum of 100 mg/ kg, in cohorts of six patients each, was administered over 30 minutes prior to infusion of BCNU 200 mg., every six weeks, for a maximum of six cycles. Only nonhematologic toxicities are assessed in determining the dose limiting toxicity (DLT) o...

913. MGMT (P140K) Allows for Safe, Efficient, and Sustained In Vivo Selection and Chemoprotection in a Large Animal Model

In vivo selection of genetically modified cells has the potential to improve autologous and allogeneic stem cell gene therapy and transplantation. We have previously reported successful in vivo selection of MGMT overexpressing cells in a clinically relevant canine model of allogeneic and autologous stem cell transplantation. Here we describe long-term stable in vivo selection and chemoprotection using the MGMT system. CD34-enriched donor marrow cells were transduced with an RD114-pseudotype retroviral vector encoding P140K and eGFP. Transduced cells were infused into two myeloablated DLA-identical allogeneic recipients or two autologous recipients. After stable engraftment four dogs were treated with various regimens of O6BG and BCNU or O6BG and temozolomide. Initially, the dose of 5 mg/kg O6BG and 0.4 mg/kg BCNU increased marking, but also caused dramatic myelosuppression. After blood cell counts recovered an identical dose of O6BG and BCNU achieved additional selection with no myelosuppression demonstrating marrow protection. The pronounced myelosuppression initially observed in the first dog was alleviated in the second dog by using slowly escalating doses of BCNU with more frequent treatments before further dose escalation. As an alternate selection strategy temozolomide was used in place of BCNU in the two autologous dogs. Initially, low doses of temozolomide followed by dose escalation of up to 600 mg/m2 and 700 mg/m2 has shown efficient in vivo selection without myelosuppression. The chemoprotection of bone marrow in all dogs appears to be very durable and protects against myelosuppression at chemotherapy doses that lead to pronounced cytopenia in control animals. Gene marking in hematopoietic repopulating cells was assessed by flow cytometry and real-time PCR. In vivo selection was observed in all lineages with granulocyte marking of up to 89% with O6BG and BCNU and 98% with O6BG and temozolomide. The marking is very durable with follow up after selection to 20 months. Morphological analysis of marrow aspirates 12 months and 7 months after selection showed no evidence of myelodysplasia. In an attempt to characterize the clones contributing to long-term hematopoiesis linear-amplification mediated PCR was carried out on peripheral blood DNA. Integration analysis has shown dogs are polyclonal before and after selection with no trend towards oligoclonal or monoclonal states. To probe the absolute number of provirus in clones contributing to long-term hematopoiesis real-time PCR analysis was carried out on peripheral blood DNA. The provirus copy number was less than two copies per cell. Our data demonstrate that less than two provirus copies are sufficient to protect clones from chemotherapy. In summary our data from a clinically relevant canine model demonstrate that MGMT(P140K)-transduced cells can be safely, efficiently and durably selected with a follow up of up to 20 months, and that the high marking levels attained by in vivo selection provide effective marrow protection. Finally, using P140K gene-marked stem cell transplants in conjunction with either BCNU or temozolomide for treatment of solid tumors would alleviate the dose limiting myelosuppression often seen with such treatments.

1006. Preliminary Results of a Phase I Trial Using Retroviral Gene Transfer of G156A MGMT To Protect Hematopoiesis during BG and BCNU Therapy of Advanced Malignancies

We have extensively studied the G156A MGMT mutation which confers O6alkylguanine DNA alkyltransferase (AGT) that is resistant to O6benzylguanine (BG). In a Phase I clinical trial at our institution, BG was shown to inhibit tumor AGT at 120mg/m2 and the maximum tolerated dose of BCNU was 40 mg/m2. In the laboratory, we have shown that the retrovirus MFG-G156A-MGMT confers BG and BCNU resistance ex vivo into human CD34+ cells. We have also reported that G156A-MGMT transduced murine hematopoietic progenitors repopulate the bone marrow and can be enriched up to 1000-fold after 2 cycles of BG and BCNU. These studies demonstrate the strong capacity of this model to confer BG and BCNU resistance and a selection advantage in vivo. Based on these data, we initiated a clinical trial using gene transfer into autologous CD34+ cells in patients with advanced malignancies. Patients undergo CD34+ mobilization with G-CSF and GM-CSF. CD34+ enriched cells (CliniMACS, Miltenyi) are transduced with MFG-G156A MGMT (produced in PG13 cells by the NGVL, K. Cornetta, Director) in the presence of the fibronectin fragment CH-296 (provided by Takara Bio Inc) and the cytokine combination SCF, Tpo, and Flt-3 ligand for 86 hours. The cell culture is harvested on day 4 and the entire culture is re-infused into the patient who received BG and BCNU 48 hours prior. Patients receive subsequent cycles of BG and BCNU every 6 weeks with analysis of marrow and blood cells for evidence of gene transfer prior to each cycle. In the first three patients, the frequency of gene transfer measured by proviral containing CFU in the post transduction culture was 20 ± 7% and 6.3 ± 3 % of the cells expressed G156A-AGT by flow cytometry. An average of 4.4 ± 2 x 107viable mononuclear cells with a CD34 purity of 93 ± 5% were infused. In one patient, 10% of bone marrow CFU showed presence of the provirus 5 weeks after infusion but prior to the second cycle of BG and BCNU. In this same patient, insertion site analysis by LAM-PCR showed a similar banding pattern in both the lymphocyte-monocyte and granulocyte fractions, indicating multilineage reconsitituion by a single transduced stem cell. Our preliminary results demonstrate efficient gene transfer into cytokine stimulated CD34+ cells using the MFG-G156A-MGMT vector and these cells can be infused without toxicity. Given the strong degree of selection observed in pre-clinical models, we anticipate enrichment for transduced cells following each cycle of chemotherapy.

Hammerhead ribozyme-mediated sensitization of human tumor cells after treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea.

O(6)-Methylguanine DNA methyltransferase (MGMT) protects tumor cells from the cytotoxic effects of DNA-alkylating agents such as 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU). To improve the therapeutic index of BCNU, biochemical strategies to inhibit MGMT temporarily by systemic administration of small molecules, such as O(6)-benzylguanine, have been developed and are showing promise in clinical trials. In this study, an alternative molecular strategy for modulating BCNU resistance was explored using hammerhead ribozymes (Rz) designed to degrade the long-lived MGMT mRNA. We had previously identified several ribozymes capable of decreasing MGMT levels in HeLa cells. Using colony formation assays, the BCNU-induced cell kill was shown to be increased by 1 to 3 logs in the HeLa/Rz clones compared with wild-type HeLa cells at a BCNU dose of 100 microM. In the current study, 10 randomly selected clones of Rz161, 212, and a reconstructed Rz178/212 were assayed for MGMT activity, MGMT mRNA, and sensitivity to BCNU. The 30 clones exhibited almost identical results in the three assays, i.e., nearly undetectable MGMT activity, greatly diminished MGMT mRNA, and comparable sensitivity to BCNU using the 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) viability assay. The effects of catalytically inactive ribozymes carrying a single point mutation were compared with their active counterparts in vitro and in stably transfected clones to determine whether antisense inhibition was a contributor to the inhibition of MGMT activity we observed. Collectively, these results suggest that the hammerhead ribozymes characterized in this study will be excellent candidates for future gene therapy approaches targeting MGMT.

High-dose BCNU followed by autologous hematopoietic stem cell transplantation in supratentorial high-grade malignant gliomas: a retrospective analysis of 114 patients.

Conventional treatment of high-grade glioma includes maximal surgical resection followed by external radiation therapy. Despite this treatment, the prognosis for patients is poor. High doses of chemotherapy might be another way to increase the response rate and median survival. Increasing doses of BCNU might be more effective, but also provokes unacceptable myelotoxicity. This dose-limiting toxicity can be circumvented by using autologous blood stem cell rescue. We report our experience of high-dose BCNU followed by transplantation of autologous hematopoietic stem cells in 114 patients with high-grade gliomas. Of the 114 gliomas, 78 were glioblastoma multiforme (GM) (68%), 24 anaplastic astrocytomas (AA) (21%), and 12 anaplastic oligodendrogliomas (OD) (11%). Complete surgical resection was performed for 22 patients (18 GM and 4 AA). The median age was 44 years (range 17-65). A total of 84 patients received autologous hematopoietic stem cells from bone marrow harvest, while 30 patients received granulocyte colony-stimulating factor followed by apheresis and received peripheral blood progenitor cells (PBPC). High dose of BCNU (800 mg/m(2)) was given at least 1 month after neurosurgery. Bone marrow or PBPC was transplanted 48-72 h after chemotherapy. Radiotherapy was started approximately 40 days after transplantation to a total of 60 Gy. Median follow-up was 89 months (19-163). The overall survival (OS) was, respectively, 12 months for GM, 37 months for OD and 81 months for AA. Histological type appeared to be the main discriminating factor, with a worse prognosis for GM. Within the GM population, age, completeness of surgery, and response appeared to be one important prognostic factors. The AA and OD populations were small to reliably assess prognostic factors. On multivariate analysis, the main prognostic factors were histologic type, quality of surgery, and age (P<0.005). Five of 114 patients had lethal complications from the procedure. Four of these patients had a Karnovsky performance score (KPS) of 60%. The protocol thus appears to be feasible but patients should be selected for KPS more than 70%. We observed long-term survivors, although the OS and the time to treatment failure seem to be comparable to that described for other treatment. Additional pilot studies are unlikely to reveal more than a modest benefit from this procedure and therefore a randomized study should be performed.

Efficacy of BCNU and paclitaxel loaded subcutaneous implants in the interstitial chemotherapy of U-87 MG human glioblastoma xenografts

Nude mice were challenged with human U-87 MG glioblastoma tumors to assess the efficacy of different cytostatics and different application protocols. While the intraperitoneal application of BCNU solutions (3 times 20 mg BCNU/kg) had no effect on tumor growth, the application of polymer matrices made of a physical mixture of poly(1,3-bis[carboxyphenoxpropane]-co-sebacic acid) 20:80 with poly( d, l-lactic-co-glycolic acid) loaded with 0.25 mg BCNU, slowed down the growth of tumors significantly. When the animals were treated with implants carrying 0.25 mg BCNU they responded to the treatment whether the tumor had been inoculated recently (9 days ago) or whether it was fully established (after 20 days). After its sensitivity was proven, the xenograft model was used to further investigate the efficacy of anticancer drugs and some treatment regimens using polymer implants. Thus the tumor model allowed to discriminate between the efficacy of different doses of BCNU. Only implants loaded with 0.75 or 1 mg of BCNU led to a substantial suppression of tumor growth over approximately 2 months. While BCNU was only able to suppress the growth of the tumor, the combination of BCNU with paclitaxel led to a complete remission in some animals. These preliminary results suggest that combinations of cytostatics might improve local chemotherapy of malignant glioma substantially. Based on our data it will be worthwhile to investigate implants that release drugs such as BCNU and paclitaxel closer. Amongst other factors we will try to elucidate the effect of repetitive doses of drugs using programmable implants.

Synergistic effect of genistein and BCNU on growth inhibition and cytotoxicity of glioblastoma cells.

Recent experiments have shown that dietary soy isoflavones such as genistein can significantly suppress invasiveness and growth of a number of human malignancies. This study examined whether genistein, at a concentration typical of plasma levels following soy diet intake, in combination with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU, carmustine) exhibited an additive or synergistic inhibitory effect on the growth of glioma cells. The human glioblastoma multiforme (GBM) cell line U87 and the rodent C6 glioma were treated with genistein at 4 microM, combined with BCNU (0-50 microM). Monolayer cell growth and cytotoxicity, as measured by colonigenic survival in soft agarose, were then compared in control and drug-treated cultures. Presence of apoptosis, using the DNA ladder assay and laser scanning cytometry (LSC), was investigated in all cell lines at those concentrations where an enhancement of antiproliferative effect of BCNU in presence of genistein was observed. A 32-41% increase in monolayer growth inhibition and a 28-42% increase in colony cytotoxicity in the U87 cell line were observed when genistein (4 microM) was added to BCNU in the 0-10 microM dose range. In the C6 cell line, a 30-36% increase in monolayer growth inhibition and a 39-54% increase in colony cytotoxicity were observed with the BCNU dose range of 0-50 microM. All experiments showed a significant increase in growth inhibition and a decrease in colonogenic survival (P < 0.05). We were unable to detect apoptosis in any of the lines when genistein was combined with BCNU. These results indicate that genistein at typical adult dietary plasma levels can significantly enhance the antiproliferative and cytotoxic action of BCNU. The implication for treatment of GBM may be a reduction in the chemotherapeutic dose recommendations of these agents and subsequently a decrease in the risk of treatment sequelae for these patients.