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.

Specificity of isozymes of murine hepatic glutathione S-transferase for the conjugation of glutathione with L-phenylalanine mustard.

Glutathione S-transferase (GST) isozymes play a central role in the protection of cells from cytotoxic chemicals and have a putative role in the intrinsic and acquired resistance of tumors to cytotoxic drugs. We have isolated and purified GST isozymes from mouse liver (M. Warholm et al., Biochemistry, 25: 4119-4125, 1986) and analyzed the metabolic products of the reaction of L-phenylalanine mustard (L-PAM) with glutathione in the presence of GST isozymes, using reverse phase high performance liquid chromatography. At pH 6.5, the spontaneous conjugation of L-PAM and glutathione is suppressed and the major product at 60 min is the monochloro, monohydroxyl derivative of L-PAM. Addition of neither class mu nor class pi isozymes to the reaction has any effect on the metabolism of L-PAM. Only isozymes of the alpha GST class catalyze the conjugation of L-PAM with glutathione. In this case, the major metabolite at 1 h is the monochloro, monoglutathionyl conjugate. Increasing the amount of mu or pi isozyme in the reaction mixture has no effect on the metabolism of L-PAM, whereas increasing the amount of alpha isozyme completely supplants hydrolysis with conjugation. Thus, increased levels of class alpha GST isozyme may represent a specific mechanism whereby cells can acquire resistance to nitrogen mustards.

Prediction of the ability to purge tumor from murine bone marrow using clonogenic assays.

The development of potential purging regimens for autologous bone marrow transplantation has been limited by the inability to predict the antitumor activity of these regimens at doses which will allow engraftment. We describe an in vitro model which estimates the in vivo efficacy of potential purging regimens in mice. The log kill of clonogenic L1210 cells after in vitro incubation with graded doses of 4-hydroperoxycyclophosphamide and vincristine (alone or in combination) was linearly related to the incubation dose of drugs. Clonogenic assays could only directly demonstrate about three logs of cell kill. However, the log linear dose-response allowed the extrapolation of cell kill for doses of drugs whose kill could not be determined directly. The extrapolated cell kill accurately predicted the in vitro activity of the drugs as established by determining the survival of B6D2F1 mice given injections of the drug-treated L1210 cells. Lethally irradiated B6D2F1 mice were given injections of mixtures of syngeneic bone marrow and L1210 cells purged with a combination of 4-hydroperoxycyclophosphamide and vincristine. Combining the results of in vitro granulocyte-macrophage colony-forming unit and clonogenic L1210 sensitivities to this drug combination predicted the survival of mice and, therefore, the effectiveness of the purging regimen.

Noninvasive detection of elevated glutathione levels in MCF-7 cells resistant to 4-hydroperoxycyclophosphamide.

Native human mammary MCF-7 adenocarcinoma cells and a subline displaying resistance to 4-hydroperoxycyclophosphamide, the chemically activated form of cyclophosphamide, were grown as multicellular spheroids or on a collagen sponge matrix and perfused for study by 31P and 13C nuclear magnetic resonance spectroscopy. The natural abundance 13C spectrum of the perfused cells exhibits well-resolved resonances due to the intracellular glutathione (GSH). The resistant cell line shows a higher intensity of the GSH 13C resonances, consistent with the increased GSH concentration determined from biochemical assays of extracts. Treatment of the resistant cell line with buthionine sulfoximine selectively diminishes the intensity of the GSH resonances in the 13C nuclear magnetic resonance spectrum.

Covalent coupling of methotrexate to dextran enhances the penetration of cytotoxicity into a tissue-like matrix.

For antitumor agents introduced directly into the intracranial space, the extent of penetration into tissue, and hence the effectiveness of therapy, depends on the rate of drug elimination from the tissue. To test the hypothesis that slowly eliminated agents would penetrate further through tissues, methotrexate (MTX)-dextran conjugates were produced by covalently linking MTX to dextran through a short-lived ester bond (MTX-ester-dextran; t1/2 approximately 3 days in buffered saline) and a longer-lived amide bond (MTX-amide-dextran; t1/2 > 20 days in buffered saline). The ability of these agents to kill cells and to penetrate through tissue was evaluated using: (a) human brain tumor (H80) cells in a standard format; (b) H80 cells in a novel three-dimensional format that mimics many characteristics of intracranial tumors; and (c) 9L gliosarcoma in the rat brain. Penetration into three-dimensional tissue-like matrices was performed by suspending H80 cells in agarose gels within a hollow fiber that was permeable to MTX but not dextran and injecting MTX or MTX-dextran conjugates into one end of the fiber. The cytotoxicity of MTX-ester-dextran and MTX-amide-dextran against H80 was equivalent to unmodified MTX (50% inhibitory concentration, approximately 0.01 microgram/ml). When released from a biodegradable polyanhydride polymer matrix, MTX and MTX-dextran conjugates retained their ability to inhibit dihydrofolate reductase activity. When MTX or MTX-dextran was diffused into the three-dimensional tumor cell matrix for 10 days, cytotoxic activity penetrated > 2 cm for MTX-amide-dextran and approximately 1 cm for MTX or MTX-ester-dextran; this enhanced penetration correlated with the stability of the MTX-dextran linkage. Intracranial polymeric delivery of MTX or MTX-amide-dextran to rats with intracranial 9L gliosarcoma produced modest but significant increases in survival; conjugation of MTX to dextran appeared to shift the dose-response curve to a lower dosage.

Molecular pharmacology of hepsulfam, NSC 3296801: identification of alkylated nucleosides, alkylation site, and site of DNA cross-linking.

We have determined that hepsulfam, in common with its structural homologue busulfan, alkylates both free guanosine and GMP in DNA at the 7 nitrogen. Mass spectral analysis of the products of the reaction of hepsulfam with guanosine has identified the mono- and bis-alkylated guanosine adducts. UV spectrophotometry and mass spectrometry were used to confirm that alkylation occurred at the 7 nitrogen by following the formation of the formamidopyrimidyl form of the hepsulfam-guanosine adduct at high pH. We have also isolated and identified 1-guanyl,7-hydroxyheptane, 1-guanyl,7-sulfamylheptane, and 1,7-bis(guanyl)heptane from in vitro reaction mixtures of hepsulfam and calf thymus DNA. We have isolated bis-(7-formamidopyrimidyldeoxyguanosinyl)-heptane from an enzymatic digest of DNA treated with hepsulfam. Finally, we have found that hepsulfam forms interstrand cross-links at 5'-GXC-3' sites in model oligonucleotides.

Intrathecal administration of 4-hydroperoxycyclophosphamide in rhesus monkeys.

The preactivated cyclophosphamide analogue, 4-HC, does not require activation by hepatic microsomal enzymes to express its cytotoxic activity and therefore, unlike cyclophosphamide, may be useful for the regional therapy of cancer. In the present study, the pharmacokinetics and toxicology of 4-HC were studied following intraventricular administration of 0.4 mg to rhesus monkeys with chronic indwelling Ommaya reservoirs. 4-HC was measured in cerebrospinal fluid (CSF) and plasma with a high-performance liquid chromatography assay utilizing a fluorometric detector following derivatization with m-aminophenol. The mean peak level of 4-HC in ventricular CSF was 100 microM 5 min after administration. The drug was cleared rapidly and the elimination was monoexponential with a mean half-life of 22 min. The mean clearance from CSF (0.33 ml/min) was 10-fold higher than CSF bulk flow. The drug was distributed throughout the subarachnoid space with lumbar levels approaching ventricular levels by 60 min. Neither acute nor chronic neurotoxicity or systemic toxicity was observed during the 6-wk observation period. Concentrations of 4-HC demonstrated to be cytocidal in vitro against human breast cancer, lymphoid leukemia, and rhabdomyosarcoma were readily achieved in CSF following intraventricular administration. This study demonstrates that intraventricular therapy with 4-HC is feasible and suggests that further study of this approach in the clinical setting should be considered.

Cerebrospinal fluid penetration of active metabolites of cyclophosphamide and ifosfamide in rhesus monkeys.

The penetration of the active metabolites of cyclophosphamide (CP) and ifosfamide (IF) into cerebrospinal fluid (CSF) was determined in rhesus monkeys following an i.v. infusion of 1 gm/m2 of CP and IF. Active metabolites were measured using a high-performance liquid chromatography assay with fluorometric detection following derivatization with m-aminophenol. CSF to blood ratios of the active metabolites of CP and IF were found to be 0.17 and 0.13 following systemic dosing of CP and IF, respectively. The levels achieved in the CSF, however, were equivalent to levels known to be cytocidal to malignant cell lines derived from tumors which metastasize to the central nervous system. Only one animal demonstrated neurotoxicity with IF. CSF levels of active metabolite in this animal were similar to those observed in the other animals.

Hepsulfam sensitivity in human breast cancer cell lines: the role of glutathione and glutathione S-transferase in resistance.

Hepsulfam (NSC 329680, 1,7-heptanediol disulfamate) is an alkylating agent that showed excellent activity against mouse and human mammary carcinoma in preclinical studies. We therefore studied the cytotoxicity of this drug in six human breast cancer cell lines (AdrRMCF7, WTMCF7, Hs578T, MDA-MB-231, T47D, and MDA-MB-468). Clonogenic assays of these cell lines showed a range of sensitivity with the 90% inhibitory concentration ranging from 3.1 microM hepsulfam (MDA-MB-468) to 32.3 microM hepsulfam (AdrRMCF7) after 24-h exposure to the drug. To evaluate possible mechanisms responsible for this observed variation in sensitivity to hepsulfam, we have studied glutathione S-transferase (GST) activity and glutathione (GSH) in these cell lines. Total cytoplasmic GST activity correlated with sensitivity; the most sensitive cell lines had the lowest GST activity, while the two most resistant cell lines, AdrRMCF7 and Hs578T, had the highest GST levels of the six cell lines. Western blot analysis showed that the only detectable isoenzyme was GST-pi. The amount of GST-pi isoform correlated with hepsulfam sensitivity in the three most resistant cell lines and was undetectable in the three most sensitive cell lines. Cellular concentrations of GSH did not correlate with hepsulfam sensitivity. However, GSH depletion with buthionine sulfoximine increased sensitivity to hepsulfam in a dose-dependent fashion in all six cell lines. Evaluation by mass spectrometry revealed that glutathione can form conjugates with hepsulfam. We conclude that the GST/GSH detoxication system plays a role in the sensitivity of these breast cancer cell lines to hepsulfam.

Transforming growth factor-alpha-Pseudomonas exotoxin fusion protein (TGF-alpha-PE38) treatment of subcutaneous and intracranial human glioma and medulloblastoma xenografts in athymic mice.

Epidermal growth factor receptor (EGFR) is amplified or overexpressed in many malignant gliomas and other primary brain tumors but is low or undetectable in normal brain. In the present study, this differential expression has been exploited for targeted brain tumor therapy using a TGF-alpha-Pseudomonas exotoxin recombinant toxin, TGF-alpha-PE38. In vitro experiments demonstrate that the cytotoxicity of this fusion protein is primarily determined by tumor EGFR expression and that TGF-alpha-PE38 cytotoxicity is abolished by pretreatment with excess epidermal growth factor. Treatment with i.p. TGF-alpha-PE38 in nude mice bearing glioblastoma or medulloblastoma s.c. xenografts produced tumor regression and growth delay. For intracranial xenograft implants treated with i.p. TGF-alpha-PE38, significant increases in median survival were noted only for tumors with the highest EGFR expression. However, intracranial tumors treated with a single intratumoral injection of TGF-alpha-PE38 showed increased survival in all xenografts tested. These results indicate that TGF-alpha-PE38 is active against primary human brain tumors ranging from moderate to high EGFR expression. For intracranial tumors, however, the higher survival rates produced by intracranial injection of TGF-alpha-PE38 than by continuous i.p. administration suggest that increased drug clearance or impaired drug delivery reduces the efficacy of systemic TGF-alpha-PE38. Direct delivery of TGF-alpha-PE38 into brain tumors by controlled-release biodegradable polymers or intratumoral implanted catheters, or intrathecal administration into the colony stimulating factor of patients with leptomeningeal metastasis, may represent clinically useful applications of recombinant toxin therapy in tumors with high EGFR expression.

Efficacy of liposomes and hyperthermia in a human tumor xenograft model: importance of triggered drug release.

The tumor drug concentrations, drug distributions, and therapeutic efficacies achieved by three fundamentally different liposomes, nonthermosensitive liposome (NTSL), traditional thermosensitive liposome (TTSL), and low temperature sensitive liposome (LTSL); free doxorubicin (DOX); and saline in combination with hyperthermia (HT) were directly compared in a human tumor xenograft model. NTSL is a nonthermosensitive liposome in the physiological temperature range, TTSL is a traditional thermosensitive liposome that triggers in the range of approximately 42-45 degrees C and releases drug over approximately 30 min, and LTSL is a new low temperature sensitive liposome that triggers in the range of approximately 39-40 degrees C and releases drug in a matter of seconds. Because of the different attributes of the liposomes, it was possible to delineate the relative importance of liposome drug encapsulation, HT cytotoxicity, HT-drug interaction, HT-induced liposomal delivery, and HT-triggered liposomal drug release in achieving antitumor activity. Athymic nude mice bearing the FaDu human tumor xenograft were given a single i.v. dose of 5 mg/kg of DOX (free drug or liposome encapsulated), and the tumors were then heated to either 34 degrees C or 42 degrees C for 1 h at 34 degrees C. All treatment groups were similar, achieving low concentrations of DOX (0-4.5 ng/mg). At 42 degrees C, the LTSL (25.6 ng/mg) achieved the highest DOX concentration (P < 0.04), but all three liposomal formulations (7.3-25.6 ng/mg) were higher than saline or DOX (0-0.7 ng/mg; P < 0.02). LTSL + HT was also the only group that resulted in significant amounts of DNA-bound DOX (silver nitrate-extractable fraction; P < 0.02). Tumor tissue sections were visualized for DOX fluorescence to investigate the local distribution of the drug in the tumor and confirm the relative drug concentrations based on fluorescence intensity. There was relatively little fluorescence seen with treatment groups at 34 degrees C. At 42 degrees C, the LTSL showed the most DOX fluorescence (P < 0.01), and the fluorescence, although not homogeneous, was pervasive throughout the tumor sections. Therapeutic efficacy of treatments was determined from tumor growth time. At 34 degrees C, the only treatment group significantly better than the saline group (9.8 days) was the NTSL group, with a growth time of 20.9 days (P < 0.02). At 42 degrees C, all three liposomal formulations were more efficacious than DOX. LTSL + HT had the longest growth time (51.4 days) and the most number of local controls at 60 days (six of nine tumors). With HT, the DOX concentrations and fluorescence were tightly correlated with tumor growth delay, indicating that adequate (increased) drug delivery can be predictive of therapeutic effect. Overall, the LTSL + HT group showed the largest DOX concentration, the highest and most pervasive DOX fluorescence, and the most antitumor effect. Thus, HT-triggered liposomal drug release may account for the largest differential therapeutic effect and demonstrates the importance of rapid drug release from the drug carriers at the tumor site.

Intraarterial therapy of human glioma xenografts in athymic rats using 4-hydroperoxycyclophosphamide.

The addition of chemotherapy, notably using nitrosoureas, in the treatment of patients with glioblastoma multiforme has resulted in only modest improvements in long-term patient survival over the use of surgical intervention and irradiation alone. Intraarterial (i.a.) chemotherapy offers the potential benefit of increasing tumor drug delivery because of first-pass drug uptake, while minimizing systemic drug levels and toxicity. We have now investigated the i.a. therapy of intracerebral human glioma xenografts in athymic rats with 4-hydroperoxycyclophosphamide (4-HC), a preactivated derivative of cyclophosphamide. Athymic male rats were given intracerebral injections of the human glioma line D-54 MG. On Day 5 after injection, the rats were randomized (n = 8-10) by body weight (mean weight, approximately 300 g). In one set of experiments, each group received either i.v. saline, i.a. saline, 6 mg i.a. 4-HC, 6 mg i.v. 4-HC (6 mg), or 12 mg i.v. 4-HC. Intraarterial 4-HC produced significant increases in median survival (Day 24) compared with i.a. saline controls (140% increase), equivalent doses given i.v. (71% increase), and twice the equivalent dose given i.v. (50% increase) (by Wilcoxon rank sum analysis, P < 0.05 is statistically significant). The i.a. maximum tolerated dose was subsequently determined to be approximately 12.5 mg in non-tumor-bearing rats. Further experiments demonstrated a dose-response increase in survival for i.a. dosages of 6, 9, and 12.5 mg with significant improvement when compared with saline controls and 12.5 mg i.v. Pharmacokinetic experiments also demonstrated a significant first-pass uptake advantage for i.a. (versus i.v.) administered 4-HC. The short plasma half-life and marked antiglioma activity of 4-HC, without the need for hepatic activation, suggest a therapeutic application of this drug in the i.a. treatment of brain tumors.

Nonlinear pharmacokinetics of cyclophosphamide in patients with metastatic breast cancer receiving high-dose chemotherapy followed by autologous bone marrow transplantation.

The pharmacokinetics of cyclophosphamide has been evaluated in 15 patients with metastatic breast cancer undergoing high-dose chemotherapy with alkylating agents followed by autologous bone marrow transplantation. Each patient received two courses of chemotherapy: 4 g/m2 of cyclophosphamide by 90-min infusion prior to peripheral blood progenitor cell collection (the first course) and 6 g/m2 of cyclophosphamide with 800 mg/m2 of thiotepa by 96-h constant infusion before marrow and stem cell reinjection (the second course). In the first course, plasma cyclophosphamide concentration-time data of 9 of 15 patients were fit by a one-compartment model with Michaelis-Menten saturable elimination in parallel with first-order renal elimination. The mean (SD) Vmax and Km values were 1.47 (0.89) microM/min and 575 (347) microM, respectively. The first course data of the remaining six patients were fit using first-order elimination only. In the second drug course, plasma cyclophosphamide disposition curves of 13 of 15 patients demonstrated a decline in concentration following attainment of an initial steady state. The plasma cyclophosphamide disposition data of these patients were fit by a one-compartment pharmacokinetic model, in which the decline of plasma cyclophosphamide concentration after reaching the initial steady state was modeled as being due to an increase in the clearance rate of cyclophosphamide. The mean (SD) initial and final clearance rates were 51 (16) ml/min and 106 (48) ml/min, respectively. Michaelis-Menten elimination was not apparent in the second course because the plasma concentration of cyclophosphamide was much lower. The mean renal clearance rate was 17 ml/min in the first course and 16 ml/min in the second course. Urinary excretion of cyclophosphamide accounted for 17% and 23% of the total dose administered in the first and the second course, respectively. No change in cyclophosphamide clearance rate was apparent in a patient who was taking phenytoin, but a change was present in a patient who was taking phenobarbital. A drug interaction between cyclophosphamide and thiotepa may explain the smaller initial clearance rate for cyclophosphamide during the second drug course.

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.

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.

Establishment of a melphalan-resistant rhabdomyosarcoma xenograft with cross-resistance to vincristine and enhanced sensitivity following buthionine sulfoximine-mediated glutathione depletion.

A melphalan-resistant human rhabdomyosarcoma xenograft, TE-671 MR, was established in athymic mice by serial melphalan treatment of the parent xenograft, TE-671, at the 10% lethal dosage (LD10); significant resistance was evident after ten passages of the tumor. TE-671 MR demonstrated a doubling time of 3.5 days and a latency period to 1000-mm3 tumors of 27.5 days. The glutathione level of TE-671 MR was 2.36 mumol/g tumor, wet weight, 2-fold higher than the parent line. The glutathione S-transferase activity of TE-671 MR was 117.8 mumol/min/mg protein, essentially unchanged from the parent line. Although TE-671 MR demonstrated cross-resistance to vincristine, dot blot analysis did not reveal an elevated expression of mdr1 mRNA in the resistant line. TE-671 MR demonstrated a 9.7-day growth delay following treatment with melphalan at the LD10 (compared to 20.9 days for the parent line). Treatment with L-buthionine-SR-sulfoximine (BSO) resulted in increased sensitivity to melphalan subsequently administered at 50% of the LD10 (melphalan alone, growth delays of 3.7 and 4.6 days in duplicate trials; melphalan plus BSO, growth delays of 7.2 and 9.8 days). Sensitivity to melphalan equal to that of the parent line TE-671 was not achieved, however. Treatment with BSO did not result in significantly enhanced sensitivity to subsequently administered vincristine (50% of the LD10) (vincristine alone, growth delays of 6.8 and 6.9 days in duplicate trials; vincristine plus BSO, growth delays of 10.9 and 7.5 days). These results suggest that generation of melphalan resistance may be associated with development of cross-resistance to vincristine; this resistance may be associated with (although not necessarily mediated by) glutathione elevation; this resistance may be partially overcome by BSO-mediated depletion of glutathione.

Enhanced melphalan cytotoxicity following buthionine sulfoximine-mediated glutathione depletion in a human medulloblastoma xenograft in athymic mice.

The effect and therapeutic consequences of buthionine-(SR)-sulfoximine (BSO)-mediated depletion of glutathione in the human medulloblastoma-derived cell line, TE-671, growing as s.c. xenografts in athymic nude mice were examined. The glutathione content of the s.c. xenografts was 1.11 +/- 0.15 mumol/g (7.79 +/- 1.61 nmol/mg of protein). Administration i.p. to tumor-bearing mice of D,L-BSO (two doses at 12-h intervals; 5 mmol/kg) depleted the glutathione content of the xenografts to 25.7% of control. Administration of a 30 mM solution of L-BSO in drinking water for 96 h depleted the glutathione content to 17.4% of control. Depletion of glutathione with these regimens resulted in a significant increase in the s.c. tumor growth delay over that produced by melphalan alone: 17.2 days versus 12.6 days for D,L-BSO (i.p.) plus melphalan versus melphalan and 22.9 days versus 16.6 days for L-BSO (p.o.) plus melphalan versus melphalan. These studies demonstrate the increased cytotoxicity of melphalan resulting from BSO-mediated depletion of glutathione in human medulloblastoma and support further efforts to modulate the chemosensitivity and radiosensitivity of this tumor by modulation of glutathione.

Intrathecal 4-hydroperoxycyclophosphamide: neurotoxicity, cerebrospinal fluid pharmacokinetics, and antitumor activity in a rabbit model of VX2 leptomeningeal carcinomatosis.

Dissemination of tumor to the leptomeninges and cerebrospinal fluid represents a common pattern of metastasis for many cancers; however, few chemotherapeutic agents are available for intrathecal (i.t.) use and treatment results are often poor. We studied the neurotoxicity and pharmacokinetics of i.t. 4-hydroperoxycyclophosphamide (4-HC) in the rabbit and the activity of i.t. 4-HC in a VX2 rabbit model of leptomeningeal carcinomatosis to evaluate the potential use of 4-HC in the treatment of leptomeningeal tumors. Toxicity studies examined 4-HC doses ranging from 0.5 to 6.0 mumol administered by intraventricular injection weekly for 4 to 8 weeks. Clinical or histological neurotoxicity was not observed in rabbits treated with < 1.0 mumol 4-HC for 4 weeks. Clinical toxicity, characterized by lethargy, weight loss, seizures, or death, was apparent at doses > 2.0 mumol. Vasculitis of superficial arteries was observed in rabbits treated with > 1.0 mumol 4-HC. In cerebrospinal fluid pharmacokinetic studies, the mean drug half-life after intraventricular or intralumbar administration was 24.3 and 18.2 min. Regional inequities in drug exposure were apparent as area under the clearance curve values for cerebrospinal fluid distant from the injection site were lower than those of proximate sites (P < 0.001). Weekly intraventricular treatment of VX2 leptomeningeal tumor-bearing rabbits with 0.5 or 1.0 mumol of 4-HC resulted in an increased life span of 22.5 and 35%, respectively. These results indicate that i.t. 4-HC, at doses lower than those producing neurotoxicity in the rabbit, is effective treatment for VX2 leptomeningeal carcinomatosis.

The effect of an amino acid-lowering diet on the rate of melphalan entry into brain and xenotransplanted glioma.

Melphalan (L-phenylalanine mustard, L-PAM, alkeran; molecular weight, 305,000) is transported across tumor cell membranes and the blood-brain barrier by the large neutral amino acid (LNAA) transport system. Normally, plasma LNAA levels are high enough and the affinity low enough that this system does not transport much melphalan into the brain. However, plasma amino acids can be reduced by fasting and protein-free diet. We used this method to reduce competition and to increase melphalan transport into brain tumors. In nude mice fasted for 12 h and then fed a protein-free diet for 2 and 6 h, mean plasma LNAA levels were 46% and 42% of control values. Nude mice with xenotransplanted D-54MG human gliomas were used to study tissue distribution and uptake kinetics of [3H]melphalan in a control group and a diet group (after a 12-h fast and 2 h of a 0% protein diet). The K1 (blood-to-tissue transfer constant) of melphalan, determined by graphical analysis and by nonlinear fitting to a 2-compartment model, was higher in the diet group in all tumor regions except the necrotic center of subcutaneous tumors; the increase was significant in the tumor periphery of brain and s.c. tumors. The ratio of K1s (diet to control) varied from 1.2 to 1.3 in brain tumors, 1.9 to 2.1 in subcutaneous tumors, and 1.8 to 3.1 in tumor-free brain. The apparent [3H]melphalan distribution space was significantly higher in the tumor periphery of both brain and subcutaneous tumors of the 15- and 30-min diet group. We also measured blood-brain barrier transport of [alpha-14C]aminoisobutyric acid and blood flow (with [131I]iodoantipyrine): the K1 of [alpha-14C]aminoisobutyric acid was 28.1 +/- 6.6 (SE) in brain tumors and 24.3 +/- 8.9 microliters/g/min in subcutaneous tumors. Blood flow was 58.2 --> 3.9 in brain tumors and 5.2 +/- 0.4 ml/100 g/min in subcutaneous tumors. Fasting, when combined with a protein-free diet, reduces plasma amino acid levels and thereby reduces competition between melphalan and LNAAs. This may increase the amount of melphalan that can enter a brain tumor without increasing the administered drug dose and suggests a therapeutic manipulation that can be used to increase the delivery of melphalan.