High-dose oral tamoxifen, a potential multidrug-resistance-reversal agent: phase I trial in combination with vinblastine.

BACKGROUND: P-glycoprotein mediates resistance to natural-product anti-neoplastic agents like vinblastine through an active transport process resulting in reduced intracellular concentration of these agents. The triphenylethylene antiestrogen tamoxifen and its major metabolite N-desmethyltamoxifen at concentrations of 4-6 microM enhance the intracellular concentration of natural-product antineoplastics and augment the cytotoxicity of such drugs three-fold to 10-fold in a variety of human and murine cell lines. PURPOSE: On the basis of these preclinical findings, we conducted a phase I clinical trial of high-dose, oral tamoxifen administered in conjunction with a 5-day continuous infusion of vinblastine. METHODS: We studied 53 patients with advanced epithelial tumors. Tamoxifen was given orally as a loading dose on day 1, followed by two doses a day on days 2-13. Vinblastine was given as a 120-hour continuous infusion (1.5 mg/m2 per day) on days 9-13 of each tamoxifen course. The starting dose of tamoxifen was 40 mg/m2 administered twice a day following a loading dose of 150 mg/m2. The maximum dose was 260 mg/m2 twice a day following a loading dose of 680 mg/m2. Treatment cycles were repeated every 28 days. RESULTS: The dose-limiting toxic effects of tamoxifen were neurologic and began within 3-5 days after the start of treatment. They consisted of tremor, hyperreflexia, dysmetria, unsteady gait, and dizziness. One patient experienced a grand mal seizure 24 hours after the last tamoxifen dose. Toxic effects were rapidly reversible. Asymptomatic prolongation of the QT interval on electrocardiogram occurred at doses of tamoxifen of 80 mg/m2 or higher given twice a day. No coagulation or ophthalmologic abnormalities occurred. Tamoxifen did not enhance the toxicity of vinblastine. Mean plasma concentrations of tamoxifen or N-desmethyltamoxifen at 260 mg/m2 tamoxifen given twice a day for 13 days were 6.04 and 6.56 microM, respectively. There was no relationship between plasma antiestrogen content and the development of neurotoxic effects. CONCLUSIONS: Tamoxifen at 150 mg/m2 given twice a day following a loading dose of 400 mg/m2 results in plasma levels of tamoxifen and N-desmethyltamoxifen of 4 and 6 microM, respectively, without dose-limiting toxicity. We recommend this dose for phase II trials of tamoxifen to modulate P-glycoprotein-mediated drug resistance. IMPLICATIONS: Our study demonstrates that high-dose tamoxifen can be safely administered and that plasma concentrations that may inhibit P-glycoprotein function can be achieved.

Enhancement of nitrosourea activity in medulloblastoma and glioblastoma multiforme.

BACKGROUND: Although chemotherapy offers promise of increased survival for children with medulloblastoma and glioblastoma multiforme, drug resistance occurs frequently, resulting in tumor progression and death. Resistance to nitrosoureas and methylating agents, which damage DNA, can be mediated by a DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGAT). Depletion of this protein with alkylguanines or methylating agents, however, restores tumor cell sensitivity to the cytotoxicity of chloroethylnitrosoureas (e.g., carmustine [BCNU]). PURPOSE: This study was designed to determine whether resistance to the activity of nitrosourea (the drug BCNU) in BCNU-resistant human medulloblastoma (D341 Med) and human glioblastoma multiforme (D-456 MG) can be reversed by the methylating agent streptozocin and the O6-substituted guanines O6-methylguanine and O6-benzylguanine. METHODS: Xenografts were grown subcutaneously in athymic BALB/c mice. BCNU was administered as a single intraperitoneal injection at doses of 100 mg/m2, 75 mg/m2, or 38 mg/m2--i.e., 1.0, 0.75, or 0.38, respectively, of the dose lethal to 10% of treated animals (LD10). Mice were treated intraperitoneally with a single dose of O6-benzylguanine or O6-methylguanine (240 mg/m2) or with streptozocin (600 mg/m2) daily for 4 days. Response was assessed by tumor growth delay and tumor regression. AGAT activity in the xenografts was measured at 1 and 6 hours after pretreatment, at the time tumors were excised. RESULTS: Pretreatment with O6-benzylguanine, O6-methylguanine, or streptozocin reduced AGAT activity to 4%, 25%, and 95% of control values, respectively, in D341 Med and 0%, 0%, and 25% of control values, respectively, in D-456 MG 1 hour after injection. After 6 hours, levels changed to 7%, 61%, and 116% of control values in D341 Med and 0%, 79%, and 21% of control values in D-456 MG, respectively. Both D341 Med and D-456 MG xenografts were completely resistant to BCNU at its LD10. Pretreatment with O6-benzylguanine increased BCNU sensitivity in both types of xenograft. In contrast, treatment with BCNU plus O6-methylguanine or streptozocin did not produce growth delays substantially different from those produced by BCNU alone, reflecting the more efficient depletion of AGAT by O6-benzylguanine. Following therapy with BCNU plus O6-benzylguanine at 0.38 LD10, tumor regressions were seen in eight of 10 D341 Med and in all 10 D-456 MG xenografts. CONCLUSION: We recommend comprehensive clinical toxicologic evaluation of combination therapy with O6-benzylguanine plus BCNU, which would allow subsequent design of phase I clinical trials.

Increased melphalan activity in intracranial human medulloblastoma and glioma xenografts following buthionine sulfoximine-mediated glutathione depletion.

In previous studies we demonstrated that administration of buthionine sulfoximine (BSO) to athymic BALB/c mice bearing intracranial human glioma xenografts resulted in highly selective depletion of glutathione in neoplastic tissue with minimal effects on contralateral normal brain tissue. In the present study we treated athymic BALB/c mice bearing intracranial human glioma (D-54 MG) or medulloblastoma (TE-671) xenografts with melphalan alone or BSO followed by melphalan. Administration of BSO depleted intracellular glutathione to 7.5% of the control level. BSO plus melphalan resulted in a significant increase in median survival over that produced by melphalan alone: 45.3% versus 26.4% in TE-671 and 69% versus 27.6% in D-54 MG. These studies justify further efforts to modulate chemotherapeutic and radiotherapeutic interventions of primary malignant brain tumors by depletion of glutathione.

Chemotherapy of subcutaneous and intracranial human medulloblastoma xenografts in athymic nude mice.

The continuous human medulloblastoma cell line TE-671 was grown as s.c. and intracranial xenografts in athymic nude mice. Tumor-bearing animals were treated with chemotherapeutic agents at the 10% lethal dose; s.c. xenografts were sensitive to melphalan, 1-(2-chloroethyl)-3-(2,6-dioxo-1-piperidyl)-1-nitrosourea, and 5-azacytidine. No consistent response could be demonstrated to 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-monophosphate, and no response to methylglyoxal bis(guanyl hydrazone), N-trifluoroacetyl adriamycin-14-valerate, or to 1-beta-D-arabinofuranosylcytosine was observed. Melphalan produced a significant (P = less than or equal to 0.007) increase in the median survival of mice bearing intracranial xenografts, whereas no response was seen to 1-(2-chloroethyl)-3-(2,6-dioxo-1-piperidyl)-1-nitrosourea or 5-azacytidine. This model will allow analysis of the chemotherapeutic profile of human medulloblastoma, and provides a means to differentiate cellular sensitivity and resistance from drug access to the intracranial site.

Sister chromatid exchange induction in patients with anaplastic gliomas undergoing treatment with radiation plus diaziquone or 1,3-bis(2-chloroethyl)-1-nitrosourea.

Diaziquone (AZQ) (NSC 182986), a lipid-soluble benzoquinone derivative, is presently being tested in a Phase III clinical trial to determine its efficacy in patients with anaplastic gliomas compared to the more standard 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) treatment following whole-brain irradiation. These patients on single-drug chemotherapy allowed us to evaluate the effects of each agent on sister chromatid exchange (SCE) induction in vivo. Eight weeks following the final radiation treatment, patients were randomly assigned to one of two groups: (a) 200 mg BCNU/m2, i.v., every 8 weeks; of (b) 15 mg AZQ/m2/day, i.v., for 3 consecutive days, every 4 weeks. Blood (5-10 ml) was drawn by venipuncture before treatment, within 10 h after treatment, and for two BCNU-treated patients at various other times. Peripheral blood lymphocytes were cultured by standard techniques for analysis of SCE. Eight weeks after irradiation but before chemotherapy, the mean SCE frequency in the patients' peripheral blood lymphocytes was 9.6 SCEs/metaphase. Following treatment with AZQ or BCNU, the baseline SCE frequency was increased more than 2-fold or 3-fold, respectively. Two months after BCNU treatment, there was less than a 25% reduction in SCE levels compared to samples taken and cultured within 10 h after treatment. These data show that lesions leading to SCE in human peripheral blood lymphocytes are relatively longlived, and that on a mg/m2 basis, AZQ is a more potent inducer of SCE in vivo than is BCNU.

Chemotherapy and radiation therapy of human medulloblastoma in athymic nude mice.

The human medulloblastoma cell line TE-671 was grown s.c. and intracranially in athymic nude mice. Tumor-bearing animals treated with chemotherapeutic agents or radiation were compared to untreated tumor-bearing controls. Tumors growing s.c. were sensitive to cyclophosphamide and vincristine with growth delays in duplicate trials of 15.8/16.5 and 12.9/15.0 days, respectively. These tumors were minimally responsive to the 2,5-bis(1-aziridinyl-3,6-dioxodiethyl ester of 1,4-cyclohexadiene-1,4-dicarbamic acid (NSC 182986) and cis-diamminedichloroplatinum II and unresponsive to methotrexate, 2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido(2,3-d)pyrimidine (NSC 351521), 1,3-bis(2-chloroethyl)-1-nitrosourea (NSC 409962), and procarbazine. Radiation therapy with 2500 or 1500 rads as a single fraction produced a marked response, with growth delays of 39.5 and 21.1 days, respectively. Cyclophosphamide produced a significant (p less than 0.0005) increase in the median survival of mice with intracranial tumors. Vincristine produced a minimal increase in the median survival while no response was seen to the 2,5-bis(1-aziridinyl-3,6-dioxodiethyl ester of (1,4-cyclohexadiene-1,4-dicarbamic acid at the dose level and schedule tested. This model system will allow further analysis of the therapeutic sensitivity of human medulloblastoma to other agents or combined-modality regimens.

Treatment of human glioma and medulloblastoma tumor lines in athymic mice with diaziquone and diaziquone-based drug combinations.

We used diaziquone (NSC 182986) alone and in combination with other antineoplastic drugs to treat six human glioma and one human medulloblastoma tumor lines growing s.c. in athymic mice. Pharmacokinetic studies of diaziquone in the plasma of athymic mice indicated rapid clearance with a half-life of approximately 11.5 min. Diaziquone produced significant growth delays in at least one experiment using each of seven different tumor lines, and it produced consistent and significant delays in five of the seven. There was no obvious difference between a single dose and a dose administered once daily for 5 days, and tumor regressions to a volume smaller than that at treatment were uncommon in any of the single-drug experiments. Using our most extensively characterized human glioma line, D-54 MG, we found striking enhancement of the therapeutic effect by using nontoxic combinations of either diaziquone and carmustine (1,3-bis(2-chloroethyl)-1-nitrosourea, NSC 409962) or diaziquone and procarbazine (NSC 77213). These combinations produced significant increases in the median growth delay, significant increases in the number of tumor regressions, and some instances in which no palpable tumors were present 100 days after treatment. In contrast, in experiments using diaziquone -based chemotherapy combinations with either cyclophosphamide, cis-platinum, or vincristine, there was only slight enhancement of the therapeutic effect. These results, using human glioma and medulloblastoma tumor lines in athymic mice, suggest a broad range of activity of diaziquone against primary nervous system tumors and enhancement of its therapeutic effect with either 1,3-bis(2-chloroethyl)-1-nitrosourea or procarbazine. If Phase II and Phase III clinical trials corroborate these findings, the value of the nude mouse system for the evaluation of new therapeutic approaches to brain neoplasms would be further confirmed.

In vitro and in vivo inhibition of glioblastoma and neuroblastoma with MDL101731, a novel ribonucleoside diphosphate reductase inhibitor.

We examined the effects of MDL101731, a novel ribonucleoside reductase inhibitor, against human glioblastomas and neuroblastoma, both in vitro and in xenograft models, to determine its activity against malignant brain tumors. MDL101731 produced a concentration-dependent inhibition of both glioblastoma cell lines (HS683 and J889H) and neuroblastoma (SK-N-MC) in nanomolar concentrations (IC50, 30-90 nM). s.c. xenografts of human glioblastoma (D54) in athymic mice increased to five times their initial volume at a median of 7.4 days in control animals, while tumor regression occurred in 12 of 12 animals treated with MDL101731 (100 mg/kg, i.p., two times/week) during 22 days of treatment (P < 0.0001). Intracerebral implants of D54 carried a median survival of 20 days in control animals, whereas animals receiving MDL101731 (100 mg/kg, i.p., two times/week, days 10-35) had a median survival of 46.5 days (P < 0.0001). Intracerebral xenografts of SK-N-MC in athymic mice resulted in a median survival of 23 days in control animals and 26 days in animals treated with carmustine (1,3-bis(2-chloroethyl)-1-nitrosourea 20 mg/kg/week, i.v. x 2; difference not significant). There was 90% survival in animals treated with MDL101731 (200 mg/kg, i.v., two times/week, days 7-35) up to 90 days after implant. These studies indicate that MDL101731 has potent antiproliferative activity against human malignant brain tumors.

In vitro versus in vivo correlations of chemosensitivity of human medulloblastoma.

An in vitro clonogenic assay was used to test the chemosensitivity of the human medulloblastoma cell line TE-671. Dose-response relationships for reduction in colony formation were generated for cyclophosphamide, vincristine, Adriamycin, 1,3-bis(2-chloroethyl)-1-nitrosourea (NSC 409962), and 1,4-cyclohexadiene-1,4-dicarbamic acid, 2,5-bis(1-aziridinyl)-3,6-dioxo-diethylester (NSC 182986); and the in vitro drug dose at which there is a 75% reduction in the number of colonies in comparison to controls (ID75S) were derived from these data. Methotrexate produced no colony reduction at any dose tested up to 1000 micrograms/ml. The in vitro results were compared to growth delays in s.c. TE-671 xenografts in athymic mice treated with the same agents. Agents with an ID75 less than assumed in vivo plasma drug concentrations were all active in vivo, whereas two of the three agents with an ID75 greater than assumed in vivo plasma drug concentrations demonstrated no in vivo activity. These results suggest that for these agents, the relationship between the ID75 of the drug and its in vivo concentration allows in vitro clonogenic assay results to agree with in vivo growth delay responses.

Treatment of human brain tumor xenografts with O6-benzyl-2'-deoxyguanosine and BCNU.

O6-Methylguanine-DNA methyltransferase (MGMT), a constitutively expressed DNA repair protein, removes alkyl groups from the O6-position of guanine in DNA. Tumor cells with high MGMT activity are resistant to nitrosoureas and other agents that form toxic O6-alkyl adducts. O6-Benzylguanine (BG) inactivates the MGMT protein and thereby enhances the sensitivity of tumor cells to alkylating drugs. However, the therapeutic potential of BG is limited by its poor solubility and its nonspecific inactivation of MGMT in normal tissues as well as in tumor tissues. Consequently, BG analogues are being developed to identify agents that have more favorable pharmacological characteristics. We evaluated O6-benzyl-2'-deoxyguanosine (dBG), the 2'-deoxyribonucleoside analogue of BG, for its ability to inhibit MGMT and to potentiate 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in a MGMT-positive human brain tumor xenograft, Daoy. When given i.p. 1 h before BCNU (25 mg/m2) to animals bearing s.c. tumors, dBG (134 mg/m2) produced a growth delay of 24.7 days, compared to 21.6 days after treatment with an equimolar dose of BG (90 mg/m2) plus BCNU and -0.6 days after treatment with BCNU alone. The combination of dBG + BCNU also increased the survival of animals bearing intracranial tumors by 65%. By increasing the dose of dBG to 300 mg/m2 (the maximum dose that could be delivered i.p. in a standard treatment volume), the growth delay of s.c. tumors increased from -0.1 days with BCNU alone to 39.3 days. dBG suppressed both tumor and liver MGMT activity to less than 1.5% of baseline, and dBG + BCNU induced extensive perivascular apoptosis. Because dBG is a 10-fold less potent MGMT inhibitor than BG in HT-29 cell extracts, these results illustrate the capacity of BG analogues to potentiate BCNU toxicity, despite less in vitro activity than the parent compound, and emphasize the importance of in vivo evaluation of BG analogues.

Activity of temozolomide in the treatment of central nervous system tumor xenografts.

The activity of 8-carbamoyl-3-methylimidazo[5,1-d]-1,2,3,5-tetrazin- 4(3H)-one (temozolomide) in the treatment of a panel of xenografts derived from ependymoma, medulloblastoma, and childhood and adult high-grade glioma was evaluated in athymic nude mice bearing s.c. and intracranial tumors. Temozolomide administered daily for a total of five doses demonstrated marked activity against a panel of Mer+ xenografts despite marginal to moderate activity of 1,3-bis(2-chloroethyl)-1-nitrosourea. The growth delays produced by temozolomide in these xenografts were 1.8-7.5-fold greater than those produced by procarbazine. Although temozolomide demonstrated marginal activity against the Mer+ cell line D341 Med when a 5-day schedule was used, a high-dose 1-day schedule resulted in moderate activity. Temozolomide produced increases in median survival of 1285% (adult glioma D-54 MG), 323% (childhood glioma D-456 MG), and 68% (ependymoma D612 EP). Pretreatment of mice with O6-benzylguanine increased temozolomide-induced mortality, requiring reduction of the dosage from 1200 to 750 mg/m2 on the single-day regimen. O6-Benzylguanine pretreatment of mice bearing Mer+ D341 Med increased the growth delay of temozolomide, in duplicate experiments, from -3.1 to 4.8 and 1.1 to 4.9 days. These studies suggest that temozolomide may be active in the treatment of a broad spectrum of central nervous system cancers, including Mer+ tumors resistant to 1,3-bis(2-chloroethyl)-1-nitrosourea.

Elevated DNA polymerase alpha, DNA polymerase beta, and DNA topoisomerase II in a melphalan-resistant rhabdomyosarcoma xenograft that is cross-resistant to nitrosoureas and topotecan.

Previous investigations have revealed that the human TE-671 MR human rhabdomyosarcoma xenograft selected in vivo for melphalan resistance (M. C. Rosenberg, et al., Cancer Res., 49: 6917-6922, 1989) is cross-resistant to a wide variety of alkylating agents and to bleomycin, but is collaterally sensitive to etoposide. Although glutathione levels were noted to be elevated in TE-671 MR compared to the melphalan-sensitive parental TE-671 xenograft, treatment with buthionine sulfoximine to deplete glutathione levels did not fully restore melphalan sensitivity in the TE-671 MR xenograft. The present studies were undertaken to search for additional mechanisms of resistance in the TE-671 MR xenograft. Drug sensitivity testing performed at the dose of agents that was lethal to 10% of the animals revealed that the TE-671 MR xenograft maintained resistance to the bifunctional cross-linking agent 1,3-bis(2-chloroethyl)-1-nitrosourea and was cross-resistant to the topoisomerase I poison topotecan. Treatment with buthionine sulfoximine did not sensitize the TE-671 MR xenograft to 1,3-bis(2-chloroethyl)-1-nitrosourea. Further, even though O6-alkylguanine-DNA alkyltransferase levels were high in both the TE-671 and TE-671 MR xenografts, depletion of O6-alkylguanine-DNA alkyltransferase activity by treatment with O6-benzylguanine substantially sensitized the TE-671 xenografts but not the TE-671 MR xenografts, suggesting an additional mechanism of resistance. Measurement of additional enzyme activities that might be involved in DNA repair revealed significant elevations in DNA polymerase alpha (46 +/- 8 (SD) units/mg protein in TE-671, 69 +/- 6 units/mg protein in TE-671 MR, P < 0.05) and DNA polymerase beta (0.43 +/- 0.01 units/mg protein in TE-671, 0.78 +/- 0.12 units/mg protein in TE-671 MR, P < 0.05) but not DNA polymerase delta or total DNA ligase. Examination of topoisomerases by activity assays and Western blotting revealed a 2-fold increase in topoisomerase II and a 2-fold decrease in topoisomerase I in the TE-671 MR xenograft compared to the parental xenograft, apparently explaining the collateral sensitivity to etoposide and cross-resistance to topotecan. These results suggest that TE-671 MR xenografts contain multiple changes in activities of DNA repair-related proteins and other nuclear proteins that could contribute to alkylating agent resistance.

Therapeutic analysis of melphalan-resistant human rhabdomyosarcoma xenograft TE-671 MR.

Investigations with the melphalan-resistant human rhabdomyosarcoma xenograft TE-671 MR were carried out to identify patterns of cross-resistance and collateral sensitivity and to define the mechanism(s) mediating melphalan resistance. TE-671 MR was cross-resistant to thio-TEPA, mitomycin, vincristine, and cisplatin, and partially resistant to chlorambucil and cyclophosphamide. TE-671 MR and the parent line TE-671 were both resistant to 1,3-bis(2-chloroethyl)-nitrosourea and expressed similar levels of O6-alkylguanine-DNA alkyltransferase. TE-671 MR retained full sensitivity to actinomycin D and demonstrated enhanced sensitivity to VP-16 compared to TE-671. Treatment of TE-671 MR with melphalan plus VP-16 resulted in greater than additive growth delays. The frequency of hypoxic regions was similar in TE-671 MR and TE-671, respectively. Measurement of tumor-to-plasma levels at 180 min following i.p. administration of melphalan at 0.5 of the 10% lethal dosage showed mean tumor-to-plasma ratios of 3.81 in TE-671 MR and 7.38 in TE-671, respectively. The lower drug levels in TE-671 MR may be contributing to the resistance to melphalan and thus indicate the need for further studies to define the reasons for these differences in tumor drug level.

Experimental chemotherapy of human medulloblastoma cell lines and transplantable xenografts with bifunctional alkylating agents.

A series of bifunctional alkylators were tested against the genotypically and phenotypically heterogeneous continuous human medulloblastoma cell lines, TE-671, Daoy, and D283 Med in vitro and against TE-671 and Daoy growing as s.c. and intracranial xenografts in athymic mice. Drugs tested included melphalan, cyclophosphamide, iphosphamide, phenylketocyclophosphamide, thiotepa, 1,3-bis(2-chloroethyl)-1-nitrosourea (in vivo), and busulfan (in vivo). Melphalan and phenylketocyclophosphamide were the most active agents in vitro with drug doses at which there is a 90% reduction in the number of colonies in comparison to controls of 2.13, 5.29, and 4.72 microM for melphalan and 4.60, 5.01, and 4.34 microM for phenylketocyclophosphamide against TE-671, D283 Med, and Daoy, respectively. Melphalan, cyclophosphamide, iphosphamide, phenylketocyclophosphamide, and thiotepa produced significant growth delays against s.c. TE-671 and Daoy xenografts, while no activity could be demonstrated for 1,3-bis(2-chloroethyl)-1-nitrosourea or busulfan. Melphalan, cyclophosphamide, iphosphamide, and thiotepa also produced significant increases in median survival in mice bearing intracranial TE-671 and Daoy xenografts. These results extend our previous studies demonstrating the antitumor activity of nitrogen and phosphoramide mustard-based bifunctional alkylating agents in the treatment of human medulloblastoma continuous cell lines and transplantable xenografts, and support the continued use of these agents in clinical trials.

Experimental chemotherapy of human medulloblastoma with classical alkylators.

Seven classical alkylators were tested for activity against the continuous human medulloblastoma cell line TE-671 grown in vitro and as s.c. and intracranial xenografts in athymic mice. Drugs tested included melphalan, cyclophosphamide (4-hydroperoxycyclophosphamide in vitro), iphosphamide (4-hydroperoxyiphosphamide in vitro), phenylketocyclo-phosphamide, phenylketoiphosphamide, Asta Z 7557, and thiotriethyl-enephosphoramide. All agents were active, with melphalan demonstrating the most activity in vitro and in vivo. Comparative studies of cyclophosphamide and phenylketocyclophosphamide revealed partition coefficients (log P) of 0.73 and greater than 1.69, respectively, and cyclophosphamide exhibited greater cytotoxic activity in post- (equitoxic) drug administration murine plasma. Hematological toxicity was limited to leukopenia/neutropenia for both of these agents. These studies suggest that the classical alkylators may have a role in the treatment of medulloblastoma and provide a means to further analyze their therapeutic potential.

Modulation of O6-alkylguanine-DNA alkyltransferase-mediated carmustine resistance using streptozotocin: a phase I trial.

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) resistance may be mediated by repair of chloroethylated guanine before stable cross-linking occurs. Guanine adducts may be repaired by the enzyme O6-alkylguanine-DNA alkyltransferase (O6-AGAT). Such repair irreversibly inactivates O6-AGAT. Streptozotocin (STZ) forms adducts at the O6 position of guanine; repair of these adducts consumes O6-AGAT. In vivo STZ potentiates BCNU cytotoxicity. The purpose of this trial was to determine the maximum tolerated dose of BCNU that can be administered together with STZ. The STZ dose was 500 mg/m2/day for 4 days and was not escalated. BCNU was given 4 h after the third dose of STZ at a starting dose of 75 mg/m2. A total of 43 patients were entered in the study. There were 4 dose escalations, reaching a maximum tolerated BCNU dose of 175 mg/m2. At this dose, thrombocytopenia was the dose-limiting toxicity (one patient, 25-49 x 10(9)/liter; 2 patients, less than 25 x 10(9)/liter); neutropenia was less severe (2 patients, 2.0-3.9 x 10(9)/liter, 1 patient, 1.0-1.9 x 10(9)/liter). Two other commonly seen toxicities were elevations in the serum alkaline phosphatase and mild elevations in the serum creatinine. Peripheral blood lymphocyte O6-AGAT levels decreased from a mean of 212 fmol/mg protein pretherapy to 8.2 fmol/mg protein on day 3 prior to BCNU (P = 0.03). Three partial responses were seen. There were no therapy-related fatalities, and toxicity was easily managed. This study established that 150 mg of BCNU can be administered safely together with STZ, 500 mg/m2/day for 4 days. Additional studies are required to determine whether O6-AGAT-mediated BCNU resistance is suppressed.

Synergy between methionine stress and chemotherapy in the treatment of brain tumor xenografts in athymic mice.

This study describes a novel approach to the treatment of brain tumors with the combination of recombinant L-methionine-alpha-deamino-gamma-lyase and chemotherapeutic regimens that are currently used against such tumors. The growth of Daoy, SWB77, and D-54 xenografts in athymic mice was arrested after the depletion of mouse plasma methionine (MET) with a combination of a MET- and choline-free diet and recombinant L-methionine-alpha-deamino-gamma-lyase. The treated tumor-bearing mice were rescued from the toxic effects of MET withdrawal with daily i.p. homocystine. This regimen suppressed plasma MET to levels below 5 microM for several days, with no treatment-related deaths. MET depletion for 10-12 days induced mitotic and cell cycle arrest, apoptotic death, and widespread necrosis in tumors but did not prevent tumor regrowth after cessation of the regimen. However, when a single dose of 35 mg/m(2) of N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU), which was otherwise ineffective as a single therapy in any of the tumors tested, was given at the end of the MET depletion regimen, a more than 80-day growth delay was observed for Daoy and D-54, whereas the growth of SWB77 was delayed by 20 days. MET-depleting regimens also trebled the efficacy of temozolomide (TMZ) against SWB77 when TMZ was given to animals as a single dose of 180 mg/m(2) at the end of a 10-day period of MET depletion. The enhanced responses of both Daoy and SWB77 to DNA alkylating agents such as BCNU and TMZ could be attributed to the down-regulation of O(6)-methylguanine-DNA methyltransferase activity. However, the synergy of MET depletion and BCNU observed with D-54 tumors, which do not express measurable O(6)-methylguanine-DNA methyltransferase protein, is probably mediated by a different mechanism. MET depletion specifically sensitizes tumors to alkylating agents and does not significantly lower the toxicity of either BCNU or TMZ for the host. In this regard, the combination approach of MET depletion and genotoxic chemotherapy demonstrates significant promise for clinical evaluation.

Melphalan transport, glutathione levels, and glutathione-S-transferase activity in human medulloblastoma.

Melphalan transport, glutathione levels, and glutathione-S-transferase activity were measured in two continuous human medulloblastoma cell lines and transplantable xenografts in athymic nude mice, TE-671 and Daoy. In vitro mean glutathione levels were 10.06 nmol/10(6) cells in TE-671 and 2.96 nmol/10(6) cells in Daoy. In vitro mean glutathione-S-transferase values were 91.52 nmol/min/mg protein in TE-671 and 50.31 nmol/min/mg protein in Daoy. Transport studies revealed kinetic parameters of Km = 108.3 microM, Vmax = 363.1 pmol/10(6) cells/min in TE-671 and Km = 111.7 microM, Vmax = 180.6 pmol/10(6) cells/min in Daoy. Melphalan transport was inhibited by both DL-alpha-2-aminobicyclo[2.2.1]heptane-2- carboxylic acid and sodium ion depletion in TE-671 and Daoy cells in vitro, indicating that both systems of amino acid transport are functional in these medulloblastoma lines. In vivo s.c. xenograft glutathione values were lower (7.79 nmol/mg protein) in TE-671 than in Daoy (13.68 nmol/mg protein). The mean plasma concentration in mice given a 10% lethal dose (71.3 mg/m2) of melphalan i.p. was 50.3 microM at 10 min, with the half-life of 29.9 min. At this dose, s.c. xenograft levels were 2- to 3-fold higher in TE-671 than in Daoy tumors for the 3-h period measured. These studies demonstrate transport parameters confirming facilitated transport of melphalan in human medulloblastoma, a mean murine plasma melphalan concentration (following treatment with melphalan) above the in vitro drug dose at which there is a 90% reduction in the number of colonies in comparison to controls for TE-671 and Daoy for 2 h, and glutathione and glutathione-S-transferase levels in the same range previously reported in other melphalan-sensitive and melphalan-resistant human tumors. Future work with spontaneous and acquired melphalan-resistant human medulloblastoma cell lines and xenografts will define the role of these mechanisms in mediating drug resistance.

Activity of intrathecal 4-hydroperoxycyclophosphamide in a nude rat model of human neoplastic meningitis.

Neoplastic meningitis can result from leptomeningeal dissemination of a variety of cancers. We now report the development of animal models of human neoplastic meningitis and activity of intrathecal 4-hydroperoxycyclophosphamide (4-HC) against the human rhabdomyosarcoma cell line TE-671 and the human glioma cell line D-54 MG grown in the subarachnoid space of athymic rats. The injection of 5 x 10(5) TE-671 or D-54 MG cells resulted in leptomeningeal tumor growth from the base of the brain to the cauda equina. Daily weights and neurological examinations revealed progressive neurological deficits and weight loss, with death occurring between Days 21 and 27 for TE-671 and Days 14 and 26 for D-54 MG. 4-HC toxicity in non-tumor-bearing rats was assessed at dose levels of 2.0, 10.0, 15.0, and 20.0 mM, with clinical and histological evidence of neurotoxicity observed at the 2 highest dose levels. Intrathecal treatment with 4-HC on Day 8 following injection of TE-671 resulted in an increase in median survival of 20% (P = 0.04) at 1.0 mM 4-HC and 41% (P less than 0.001) at 2.5 mM 4-HC. Intrathecal treatment with 4-HC (2.5 mM) on Day 5 following injection of D-54 MG resulted in an increase in median survival of 23% (P = 0.009). These studies show the usefulness of the athymic rat model of human neoplastic meningitis and demonstrate the efficacy in vivo of intrathecally administered 4-HC against a human glioma and a human rhabdomyosarcoma cell line and the lack of toxicity at therapeutic levels of 4-HC in normal athymic rats.

Methylator resistance mediated by mismatch repair deficiency in a glioblastoma multiforme xenograft.

A methylator-resistant human glioblastoma multiforme xenograft, D-245 MG (PR), in athymic nude mice was established by serially treating the parent xenograft D-245 MG with procarbazine. D-245 MG xenografts were sensitive to procarbazine, temozolomide, N-methyl-N-nitrosourea, 1,3-bis(2-chloroethyl)-1-nitrosourea, 9-aminocamptothecin, topotecan, CPT-11, cyclophosphamide, and busulfan. D-245 MG (PR) xenografts were resistant to procarbazine, temozolomide, N-methyl-N-nitrosourea, and busulfan, but they were sensitive to the other agents. Both D-245 MG and D-245 MG (PR) xenografts displayed no O6-alkylguanine-DNA alkyltransferase activity, and their levels of glutathione and glutathione-S-transferase were similar. D-245 MG xenografts expressed the human mismatch repair proteins hMSH2 and hMLH1, whereas D-245 MG (PR) expressed hMLH1 but not hMSH2.