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Biomedical subjects

M E Dolan

Publications and source records attributed to M E Dolan.

At least 55 records · Page 3Linked to original sources
PubMed1997

Intraarterial O6-benzylguanine enables the specific therapy of nitrosourea-resistant intracranial human glioma xenografts in athymic rats with 1,3-bis(2-chloroethyl)-1-nitrosourea.

The prognosis for patients with malignant gliomas continues to be dismal. The high degree of resistance of gliomas to nitrosourea-based chemotherapy is one major factor in poor treatment outcome. The identification of O6-alkylguanine-DNA alkyltransferase (AGAT) as a major determinant of nitrosourea resistance has resulted in the development of several agents to inactivate this repair protein and counteract tumor cell resistance. However, a major problem in preclinical trials has been the marked nitrosourea dose limitations imposed by the prior administration of AGAT-depleting agents. We investigated the AGAT depletion and selective enhancement of BCNU activity of intraarterial (i.a.) O6-benzylguanine (O6BG) in the human malignant glioma xenograft D-456 MG growing intracranially (i.e.) in athymic rats. Whereas i.a. O6BG at 2.5 mg/kg produced 100% inhibition of D-456 MG AGAT i.e. activity 8 h after administration, intraperitoneal (i.p.) O6BG at this dose produced only 40% inhibition, requiring dose escalation to 10 mg/kg to produce 100% AGAT depletion. Prior administration of i.p. O6BG (10 mg/kg) and i.a. O6BG (2.5 mg/kg) limited maximum tolerated intravenous (i.v.) BCNU doses (37.5 mg/kg when given alone) to 6.25 and 25 mg/kg, respectively. Higher doses of BCNU alone or in combination with O6BG produced histopathologic evidence of cerebral and hepatic toxicity. Therapy experiments revealed a significantly improved median survival for rats treated with O6BG i.a. (2.5 mg/kg) plus BCNU i.v. (25 mg/kg, days 61 and 59 in duplicate experiments) compared with saline (day 21. P = 0.001). O6BG i.a. or i.p. (days 22 and 23, P = 0.001), BCNU i.v. (37.5 mg/kg, day 29, P = 0.001), and O6BG i.p. (10 mg/kg), plus BCNU i.v. (6.25 mg/kg, day 37, P < 0.001). Therefore, O6BG i.a., by virtue of rapid AGAT depletion and selective uptake into i.c. tumors, offers significant potential for regional chemomodulation of AGAT-mediated nitrosourea resistance in malignant human gliomas with concomitant reduction of systemic toxicity.

Alkyl and Aryl Transferases↗
PubMed1997

O6-benzylguanine and its role in chemotherapy.

The presence of the DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT) in tumor cells is a significant source of resistance to chemotherapeutic alkylnitrosoureas and alkyltriazenes. O6-Benzylguanine provides a means to effectively inactivate the AGT protein and increase the chemotherapeutic effectiveness of chloroethylating and methylating agents in vitro and in human tumor xenograft models. Phase I clinical trials of the combination of O6-benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea are ongoing. Efforts directed at overcoming potential enhanced hematopoietic toxicity and mutagenicity have included the use of gene therapy to express an alkyltransferase gene in the relevant marrow stem cells. Altered AGT proteins resistant to O6-benzylguanine generated from point mutations in the mammalian alkyltransferase gene have been expressed in animal models using retroviral transduction techniques. It is anticipated that the successful application of this approach in humans may provide a means to increase the therapeutic index of O6-benzylguanine and 1, 3-bis(2-chloroethyl)-1-nitrosourea.

Animals↗
PubMed1996

Intracellular Localization and intercellular heterogeneity of the human DNA repair protein O(6)-methylguanine-DNA methyltransferase.

O(6)-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that removes alkyl adducts from DNA and may be important in tumor resistance to alkylation chemotherapy. MGMT was visualized in human cells and tumor tissues with monoclonal antibodies against MGMT and immunofluorescence microscopy, and fluorescent signals were quantified by digital image analysis. MGMT was found both in the cytoplasm and the nucleus, and in either locale the protein reacts with alkylated DNA bases and becomes inactivated and lost from the cell. Cell lines in culture and xenografts showed a broad normal distribution of nuclear MGMT levels, but human brain tumors often showed a skewed distribution, with a significant fraction of cells with high levels of MGMT. O(6)-Benzylguanine, a suicide substrate inactivator for MGMT activity, reduced MGMT in human cells and in a mouse xenograft to levels undetectable by antibody assay 1 h post-treatment. In melanoma specimens taken from a patient 3 h post-treatment with temozolomide, MGMT levels were reduced by 70%. This quantitative immunofluorescence assay can be used to monitor MGMT and it depletion in human tumors to improve the use of alkylating agents in cancer chemotherapy.

Animals↗
PubMed1996

Peripheral blood mononuclear cell dihydropyrimidine dehydrogenase activity in volunteers with and without diabetes mellitus.

It has been reported that cancer patients with diabetes mellitus receiving a continuous infusion of 5-fluorouracil (5-FU) have more toxicity and higher plasma 5-FU levels that patients without diabetes mellitus. Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-FU. DPD activity in peripheral blood mononuclear cells has been reported to correlate inversely with 5-FU plasma levels in patients. We therefore undertook a study to compare the activity of DPD in peripheral blood mononuclear cells of human subjects with and without diabetes mellitus. The study groups comprised 43 volunteers with and 39 without diabetes mellitus, and peripheral blood mononuclear cell DPD activity was assayed on samples obtained between 8 a.m. and 11 a.m. DPD activity was not decreased in diabetic subjects. There was no relationship between DPD activity and gender body mass index, or race. There was a modest correlation between DPD activity and age (r=0.19, P =0.08). We conclude that increases in 5-FU-related toxicities in diabetics must be related to factors other than peripheral blood mononuclear cell DPD activity.

Adult↗
PubMed1996

Pharmacodynamics of fluorouracil-based induction chemotherapy in advanced head and neck cancer.

PURPOSE AND METHODS: To optimize the biochemical modulation of fluorouracil (5-FU), we administered the pure I-stereoisomer of leucovorin (LV) as a 132-hour continuous intravenous infusion (CIV) with cisplatin 100 mg/m2, 5-FU 640 mg/m2/d as a 120-hour CIV, and interferon alfa-2b (IFN) at 2 MU/m2/d for 6 days for three cycles (I-PFL-IFN). Pharmacologic parameters included morning (AM) and afternoon (PM) plasma concentrations of 5-FU, LV and its active metabolite 5-methyl tetrahydrofolate (MTHF), and dihydropyrimidine dehydrogenase (DPD) activity in peripheral mononuclear cells. RESULTS: Eighty-nine patients were treated (86 stage IV). Neutropenia and mucositis were the most common toxicities. Sixty-six percent achieved a complete remission (CR). There was a trend for higher PM versus AM 5-FU concentrations (median, 1.64 v 1.51 mumoles/L; P = .08), but not for LV plus MTHF (P = .66). The mean +/- SD DPD activity was 0.21 +/- 0.14 nmol/min/mg and did not correlate with plasma concentrations of 5-FU or LV plus MTHF or clinical toxicities. Higher PM 5-FU concentrations correlated with worse leukopenia (P = .04) and severity of mucositis (P = .04). PM 5-FU concentration was higher in women than in men (P = .07), with no apparent difference in severity of toxicities. The maximum 5-FU concentration was higher in CR than non-CR patients (median, 2.01 v 1.54 mumoles/L; P = .02) and higher in women than men who achieved a CR (median, 2.77 v 1.91 mumoles/L; P = .03). No correlation of CR with dose-intensity was found. CONCLUSION: L-PFL-IFN is active in stage IV head and neck cancer. 5-FU concentration is a significant predictor of toxicity. In women, optimization of response outcome requires a higher 5-FU concentration. Individualized 5-FU dosing to obtain higher 5-FU plasma concentrations may be indicated.

Adolescent↗
PubMed1996

Pharmacokinetics and metabolism in rats of 2,4-diamino-6-benzyloxy-5- nitrosopyrimidine, an inactivator of O6-alkylguanine-DNA alkyltransferase.

Inactivation of the human DNA repair protein, O6-alkylguanine-DNA-alkyltransferase (AGT), by exposure to O6-benzylguanine leads to a dramatic enhancement in the cytotoxic response of cells to chemotherapeutic alkylnitrosoureas. Benzylated pyrimidines identified as more potent inactivators than O6-benzylguanine in vitro include 2,4-diamino-6-benzyloxy-5-nitrosopyrimidine (5-nitroso-BP) and 2,4-diamino-6-benzyloxy-5-nitropyrimidine (5-nitro-BP). In efforts to determine the clinical usefulness of these benzylated pyrimidines, we examined the metabolism and pharmacokinetics of 5-nitroso-BP in Sprague-Dawley rats, together with its potency as an AGT inactivator in mice. The mean plasma half-life, clearance, and volume of distribution of 5-nitroso-BP in rats were, respectively, 3.8 min, 22 liters/hr/kg, and 2.1 liters/kg. Two metabolites were identified in rat plasma (i.e. 5-nitro-BP and 2,4,5-triamino-6-benzyloxypyrimidine) after intravenous administration of 5-nitroso-BP in rat. Reduction of 5-nitroso-BP (100 microM) occurred primarily in cytosol and was inhibited (> 95%) by 1 mM menadione. Dicumarol (100 microM), a DT-diaphorase inhibitor, did not significantly inhibit this reaction. This indicated a possible role of a dicumarol-resistant quinone reductase. At higher substrate and protein concentration, NADPH-dependent oxidation of 5-nitroso-BP to 5-nitro-BP primarily occurred in microsomes and was completely inhibited by 1-aminobenzotriazole (1 mM), a P450 inhibitor. Unfortunately, neither 5-nitroso-BP nor 5-nitro-BP was as effective as O6-benzylguanine at depleting AGT activity in mouse liver or spleen. At 1 hr after injection of 15 mg/kg O6-benzylguanine, 5-nitroso-BP, or 5-nitro-BP, AGT levels in liver fell to 1%, 66%, and 71% basal activity, respectively. Rapid cytosolic reduction of 5-nitroso-BP may explain the lack of potency of the pyrimidines in vivo.

Animals↗
PubMed1995

Human liver oxidative metabolism of O6-benzylguanine.

The oxidation of O6-benzylguanine, an inactivator of O6-alkylguanine-DNA alkyltransferase, was examined using human liver cytosol, microsomes, and several P450 isoforms. Incubation of O6-benzylguanine with human liver cytosol resulted in the formation of O6-benzyl-8-oxoguanine, which was inhibited by menadione, a potent inhibitor of aldehyde oxidase. Inhibition by allopurinol, a xanthine oxidase inhibitor, was less dramatic. Oxidation of O6-benzylguanine also occurred with pooled human liver microsomes and was inhibited by both furafylline and troleandomycin, selective inhibitors of CYP1A2 and CYP3A4, respectively. Human P450s CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2E1, and CYP3A4 expressed in Hep G2 hepatoma cells using vaccinia virus vectors were incubated with 10 or 200 microM O6-benzylguanine. At 10 microM, O6-benzylguanine was oxidized primarily by CYP1A2 and to a lesser extent by CYP3A4. However, an appreciable increase in CYP3A4 contribution was noted at 200 microM. CYP1A2 exhibited a more than 200-fold higher relative catalytic activity (Vmax/Km) compared with CYP3A4. Therefore, at therapeutically relevant concentrations of O6-benzylguanine, CYP1A2 could be primarily involved in its oxidation since it shows a much lower Km value (1.3 microM) than CYP3A4 (52.2 microM) and cytosol (81.5 microM). However, one would expect interindividual variation in the extent of oxidation of O6-benzylguanine depending on the levels of aldehyde oxidase, CYP1A2, and CYP3A4.

Carcinoma, Hepatocellular↗
PubMed1995

Increased killing of prostate, breast, colon, and lung tumor cells by the combination of inactivators of O6-alkylguanine-DNA alkyltransferase and N,N'-bis(2-chloroethyl)-N-nitrosourea.

The ability of a number of compounds that act as inactivators of O6-alkylguanine-DNA alkyltransferase (AGT) to sensitize human tumor cell lines to the effects of N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU) were examined. The AGT inactivators tested included O6-benzylguanine (BG) and its 8-aza-, 8-bromo-, 8-methyl-, 8-oxo, and 8-amino-derivatives and O6-[p-(hydroxymethyl)benzyl]guanine. All of these compounds except the 8-amino-derivative were active in greatly increasing the killing of HT29 colon, Du145 prostate, MCF-7 breast and A549 lung tumor cells by BCNU. Their activities were comparable to those of BG. Two pyrimidines, 2,4-diamino-6-benzyloxy-5-nitrosopyrimidine and 2,4-diamino-6-benzyloxy-5-nitropyrimidine, were found to be considerably more potent than BG in enhancing BCNU-induced cell killing. The addition of a steroid group to the 9-position of BG forming either O6-benzyl-9-[3-oxo-4-androsten-17 beta-yloxycarbonyl)methyl]guanine or O6-benzyl-9-[3-oxo-5 alpha-androstan-17 beta-yloxycarbonyl)methyl]guanine also produced compounds effective in enhancing the cytotoxicity of BCNU when added at 10 microM. These results indicate that a range of potent compounds with potentially different pharmacokinetics is available to test the hypothesis that inactivation of AGT overcomes the resistance of many tumor cells to nitrosoureas.

Antineoplastic Agents, Alkylating↗
PubMed1995

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.

Animals↗
PubMed1995

8-Substituted O6-benzylguanine, substituted 6(4)-(benzyloxy)pyrimidine, and related derivatives as inactivators of human O6-alkylguanine-DNA alkyltransferase.

Several 8-substituted O6-benzylguanines, 2- and/or 8-substituted 6-(benzyloxy)purines, substituted 6(4)-(benzyloxy)pyrimidines, and a 6-(benzyloxy)-s-triazine were tested for their ability to inactivate the human DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT, alkyltransferase). Two types of compounds were identified as being significantly more effective than O6-benzylguanine (the prototype low molecular weight inactivator) at inactivating AGT in human HT29 colon tumor cell extracts. These were 8-substituted O6-benzylguanines bearing electron-withdrawing groups at the 8-position (e.g. 8-aza-O6-benzylguanine and O6-benzyl-8-bromoguanine) and 5-substituted 2,4-diamino-6-(benzyloxy)pyrimidines bearing electron-withdrawing groups at the 5-position (e.g. 2,4-diamino-6-(benzyloxy)-5-nitroso- and 2,4-diamino-6-(benzyloxy)-5-nitropyrimidine). The latter derivatives were also more effective than O6-benzylguanine at inactivating AGT in intact HT29 colon tumor cells. Provided these types of purines and pyrimidines do not exhibit undesirable toxicity, they may be superior to O6-benzylguanine as chemotherapeutic adjuvants for enhancing the effectiveness of antitumor drugs for which the mechanism of action involves modification of the O6-position of DNA guanine residues.

Cells, Cultured↗
PubMed1995

Pharmacokinetics of O6-benzylguanine in rats and its metabolism by rat liver microsomes.

O6-Benzylguanine is an effective inhibitor of the DNA repair protein, O6-alkylguanine-DNA alkyltransferase, and enhances the effectiveness of 1,3-bis(2-chloroethyl)-1-nitrosourea in cells in culture and animal tumor models. To prepare O6-benzylguanine for clinical trials and to determine the availability and disposition of O6-benzyl-7,8-dihydro-8-oxoguanine (O6-benzyl-8-oxoguanine), its major metabolite, pharmacokinetic parameters of these compounds were investigated in male Sprague-Dawley rats. Noncompartmental pharmacokinetic parameters were determined following intravenous administration of O6-benzylguanine or O6-benzyl-8-oxoguanine in rats. Half-life, clearance, and volume of distribution were respectively, 1.6 hr, 160 ml/hr/kg, and 405 ml/kg for O6-benzylguanine, and 1.2 hr, 312 ml/hr/kg, and 507 ml/kg for O6-benzyl-8-oxoguanine. At least 37% of O6-benzylguanine was converted to O6-benzyl-8-oxoguanine after administration of O6-benzylguanine. Renal excretion accounted for 8 and 62% of the administered O6-benzylguanine and O6-benzyl-8-oxoguanine, respectively. Administration of phenobarbital to rats before O6-benzylguanine resulted in a 17- to 19-fold increase in the amount of oxidized product in the urine. Kinetic constants, KM and Vmax were estimated as 19.6 microM and 0.02 nmol/min/mg protein and 13.4 microM and 0.96 nmol/min/mg protein, for uninduced and induced rat liver microsomes, respectively. The use of inhibitors of cytosolic enzymes, xanthine oxidase, and aldehyde oxidase indicated that aldehyde oxidase is primarily involved in the cytosolic oxidation of O6-benzylguanine.

Animals↗
PubMed1995

Pharmacokinetics and pharmacodynamics of oltipraz as a chemopreventive agent.

Oral oltipraz, as a single-dose treatment, was evaluated as a chemopreventive agent in 31 normal subjects. In a subset of subjects, the relationship between plasma oltipraz concentrations and the induction of lymphocyte glutathione (GSH) and glutathione S-transferase (GST) enzyme levels was evaluated. Pharmacokinetic analysis revealed nonlinear disposition of oltipraz with disproportionate 40-fold increase and 9.5-fold decrease in peak plasma concentrations (Cmax) and p.o. clearance, respectively, over the dose range of 100-500 mg. There was no correlation between the oltipraz dose and the absorption rate or the time to reach Cmax. Since oltipraz undergoes extensive metabolism, saturable first-pass elimination could be one of the sources of nonlinearity. Pharmacodynamic evaluation was conducted based on the percentage of elevation of GSH and GST levels over baseline in lymphocytes of subjects receiving 100 mg and 125 mg oltipraz. Induction was observed in both dose groups with a time lag between the maximum concentrations of oltipraz and that of GSH or GST. We also observed a linear correlation between oltipraz Cmax and the corresponding GSH and GST elevations. Subjects with higher Cmax values showed a greater increase over baseline in the GSH and GST levels. Mild toxicities were observed at all dose levels. The most common were flatulence, hunger, fatigue, and headache. These preliminary results indicate that oltipraz may be effective in inducing GSH and GST, an enzyme capable of carcinogen elimination.

Administration, Oral↗
PubMed1994

Anti-neoplastic activity of sequenced administration of O6-benzylguanine, streptozotocin, and 1,3-bis(2-chloroethyl)-1-nitrosourea in vitro and in vivo.

The purpose of this study was to evaluate the anti-tumor activity of sequenced administration of O6-benzylguanine (BG), streptozotocin (STZ), and 1,3-bis(2-chloroethyl)-1- nitrosourea (BCNU) in vitro and in vivo. We measured the recovery of O6-methylguanine DNA methyltransferase (MGMT) and BCNU cytotoxicity in the human glioma SF767 cell line, and anti-tumor activity against xenografts following exposure to BG, STZ or the combination of BG + STZ combined with BCNU. In SF767 cells, the combination of BG (10 microM) + STZ (0.05 mM) produced sustained inhibition of MGMT activity for at least 24 hr, and a greater potentiation of BCNU cytotoxicity than either agent alone. The combined treatment of BG + STZ increased BCNU-induced cell kill by 0.5 to 1.0 log over BG or STZ alone. The maximally tolerated doses of the combination of BG + STZ + BCNU administered to nude mice i.p. were the following: BG (80 mg/kg), STZ (100 mg/kg), and BCNU (15 mg/kg). Utilizing these doses of BG and STZ, the depletion and repletion profile of MGMT activity in SF767 xenografts was measured. STZ at 100 mg/kg did not affect xenograft MGMT activity. Subsequent to BG treatment, xenograft MGMT activity was inactivated completely for 12 hr, and the tumors gradually recovered approximately 40% of control activity by 24 hr. The combination of BG + STZ produced sustained inhibition of MGMT activity for 24 hr in the xenografts with complete recovery of MGMT activity by 48 hr. Administration of the combination of BG + BCNU to nude mice bearing SF767 tumor resulted in significant inhibition of tumor growth for 23 days. However, the addition of STZ to this combination provided no greater anti-tumor activity than that observed with BG + BCNU. The three-drug combination of BG, STZ, and BCNU produced no more than 2.4 to 13.0% weight loss with occasional lethal toxicity. Collectively, these data suggest that prolonged depletion of MGMT might be required for optimal reversal of BCNU resistance both in vitro and in vivo.

Animals↗
PubMed1994

Metabolism of O6-benzylguanine, an inactivator of O6-alkylguanine-DNA alkyltransferase.

O6-Benzylguanine effectively inactivates the DNA repair protein, O6-alkylguanine-DNA alkyltransferase, leading to an increase in the therapeutic index of 1,3-bis(2-chloroethyl)-1-nitrosourea in nude mouse xenograft studies. To investigate the fate of this inactivator in mammalian systems, we examined its biodistribution and metabolism following i.p. administration of 8-[3H]-O6-benzylguanine to male Sprague-Dawley rats and BALB/c mice. Following administration to rats, there were significantly higher levels of radioactivity in liver than in lung, spleen, kidney, small intestine, and esophagus for up to 24 h. Major urinary metabolites were identified as O6-benzyl-7,8-dihydro-8-oxoguanine, N2-acetyl-O6-benzylguanine, and N2-acetyl-O6-benzyl-7,8-dihydro-8-oxoguanine. Debenzylated metabolites included guanine, 7,8-dihydro-8-oxoguanine, and N2-acetylguanine. In contrast to rat metabolism, acetylated derivatives were not found in mouse urine. However, O6-benzyl-7,8-dihydro-8-oxoguanine was a major metabolite in the mouse. O6-Benzyl-7,8-dihydro-8-oxoguanine was a very effective O6-alkylguanine-DNA alkyltransferase inactivator and exhibited a 50% effective dose in HT29 cell extracts of 0.3 microM compared to 0.2 microM for O6-benzylguanine. The O6-alkylguanine-DNA alkyltransferase depleting activity of N2-acetyl-O6-benzylguanine and N2-acetyl-O6-benzyl-7,8-dihydro-8-oxoguanine were, respectively, 120- and 325-fold lower than O6-benzylguanine in HT29 cell-free extracts.

Animals↗
PubMed1994

Effect of 1,19-bis(ethylamino)-5,10,15-triazanonadecane on human tumor xenografts.

The polyamine analogue 1,19-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4), 5 mg/kg i.p., was given twice daily on days 0-3 and 7-10 (cycle 1) to nude mice with human malignant gliomas (SF-767 and U-87 MG), lung adenocarcinoma (A549), and colon carcinomas (HCT116 and HT29). A second cycle of drug was given to mice with SF-767 and A549 tumors on days 42-45 and 49-52. The maximum animal weight loss varied between 4 and 12%, which was observed 10-15 days following the initiation of treatment, but no overt toxic reactions were noted. The SF-767 brain tumors were extremely responsive to BE-4-4-4-4 alone (3 of 8 complete regressions after 2 cycles); however, the growth of the U-87 MG brain tumor was only slightly inhibited by BE-4-4-4-4 treatment. There was significant inhibition of tumor growth after treatment with one cycle of BE-4-4-4-4 in animals carrying the A549, HCT116, and HT29 tumors. At day 73, the growth of the A549 tumor was inhibited by 78 and 89% following one or two cycles of BE-4-4-4-4, respectively. The mitotic index of A549 tumors was 18 times greater in control mice than in those treated with BE-4-4-4-4 for one or two cycles 99 days after initiation of treatment. 1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) was given to mice carrying the U-87 MG or A549 tumors on day 4 (cycle 1) and day 46 (cycle 2) in the maximal tolerated dose of 50 mg/kg for BCNU alone and 40 mg/kg for BCNU plus BE-4-4-4-4. BCNU alone significantly inhibited the growth of U-87 MG tumors but not the growth of A549 tumors. Treatment with the combination of BCNU and BE-4-4-4-4 was significantly better than BCNU alone for A549 tumors and better than BE-4-4-4-4 alone for U87 tumors. However, in both animal groups treated with the combination, there was a significant weight loss, which was not observed for animals treated with either agent alone. These data suggest a role for BE-4-4-4-4 in the treatment of brain, lung, and colon tumors.

Animals↗
PubMed1994

Extended depletion of O6-methylguanine-DNA methyltransferase activity following O6-benzyl-2'-deoxyguanosine or O6-benzylguanine combined with streptozotocin treatment enhances 1,3-bis(2-chloroethyl)-1-nitrosourea cytotoxicity.

We have recently suggested that optimal reversal of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) resistance might require complete inactivation of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) for at least 24 h following BCNU administration (22). This study was undertaken to further evaluate the functional importance of the regeneration rate of MGMT activity following O6-benzylguanine (BG), O6-benzyl-2'-deoxyguanosine (dBG), and streptozotocin (STZ) in determining the potentiation of BCNU cytotoxicity in the highly resistant colon carcinoma cell line HT-29. To this end, we measured the enhancement of BCNU cytotoxicity utilizing regimens which provided complete inhibition, with partial or complete recovery of MGMT activity by 24 h. We were able to modulate the recovery rate of MGMT activity following BG or dBG administration by repeated washing of cells with complete medium. Subsequent to equally inhibitory doses of BG (100 microM) or dBG (1.0 mM) treatment without washing, MGMT activity was completely inactivated for 24 h. However, MGMT activity recovered to control levels by 24 h when cells were treated with BG or dBG and washed 4 times with complete medium. This recovery was completely inhibited for 24 h by combining BG or dBG with 2.5 mM STZ. These differential repletion profiles produced disparate potentiation of BCNU cytotoxicity. The regimens which produced complete inactivation of MGMT for 24 h produced the greatest enhancement of BCNU cytotoxicity. BG or dBG (without a wash) potentiated BCNU cytotoxicity by approximately 3 logs of synergistic cell kill. When the recovery rate of MGMT activity was markedly enhanced via washing of cells, BG-BCNU or dBG-BCNU produced less than 1 log of synergistic cell kill. The addition of STZ to BG or dBG inhibited this temporal recovery for 24 h and potentiated BCNU cytotoxicity by approximately 4 logs. These data further demonstrate that extended depletion of MGMT is required for optimal reversal of BCNU resistance. Because a three-drug combination of BG-STZ-BCNU or dBG-STZ-BCNU consistently produced greater cytotoxicity than any two-drug regimen, clinical testing of these combinations is warranted. Additionally, our data suggest that the design of clinical regimens targeting the inactivation of MGMT and the reversal of BCNU resistance should consider the functional importance of extended depletion of MGMT in order to increase the possibility of antitumor responses.

Antineoplastic Agents↗
PubMed1994

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.

Alkyl and Aryl Transferases↗