Mutagenesis by O(6)-methyl-, O(6)-ethyl-, and O(6)-benzylguanine and O(4)-methylthymine in human cells: effects of O(6)-alkylguanine-DNA alkyltransferase and mismatch repair.

Double-stranded and gapped shuttle vectors were used to study mutagenesis in human cells by O(6)-methyl (m(6)G)-, O(6)-ethyl (e(6)G)-, and O(6)-benzylguanine (b(6)G), and O(4)-methylthymine (m(4)T) when these bases were incorporated site-specifically in the ATG initiation codon of a lacZ' gene. Vectors were transfected into either human kidney cells (293) or colon tumor cells (SO) or into mismatch repair defective human colon tumor cells (H6 and LoVo). Cellular O(6)-alkylguanine-DNA alkyltransferase (alkyltransferase) was optionally inactivated by treating cells with O(6)-benzylguanine prior to transfection. In alkyltransferase competent cells, the mutagenicity of all the modified bases was substantially higher in gapped plasmids than in double-stranded plasmids. Alkyltransferase inactivation increased mutagenesis by the three O(6)-substituted guanines in both double-stranded and gapped plasmids but did not affect m(4)T mutagenesis. In the absence of alkyltransferase, mutagenesis by m(6)G and to a lesser extent e(6)G in double-stranded vectors was higher in the mismatch repair defective H6 and LoVo cells than in SO or 293 cells indicating that e(6)G as well as m(6)G were subject to mismatch repair processing in these cells. The level of mutagenesis by m(4)T and b(6)G was not affected by mismatch repair status. When incorporated in gapped plasmids and in the absence of alkyltransferase, the order of mutagenicity for the modified bases was m(4)T > e(6)G congruent with m(6)G > b(6)G. The O(6)-substituted guanines primarily produced G-->A transitions while m(4)T primarily produced T-->C transitions. However, m(4)T also produced a significant number of T-->A transversion mutations in addition to T-->C transitions in mismatch repair deficient LoVo cells.

Thresholds of O6-alkylguanine-DNA alkyltransferase which confer significant resistance of human glial tumor xenografts to treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea or temozolomide.

Bis-2-chloroethylnitrosourea (BCNU) or temozolomide (TMZ) were tested alone or in combination with the AGT inhibitors O6-benzyl-2'-deoxyguanosine (dBG) or O6-benzylguanine (BG) against human glial tumor xenografts growing s.c. in athymic mice. Four glioblastoma (SWB77, SWB40, SWB39, and D-54) and one anaplastic oligodendroglioma (SWB61) xenografts having O6-alkylguanine-DNA alkyltransferase (AGT) activities of 75, 45, 10, < 10, and 16 fmol/mg protein, respectively, were used. BCNU at 35 mg/m2 was ineffective against these tumors, although 70 mg/m2 (LD10, 75 mg/m2) produced a marked tumor growth delay (T-C) in D54 but had no effect against SWB40 or SWB77. Coadministration of BG or dBG and BCNU necessitated reduction of the BCNU dose to a maximum of 30 and 35 mg/m2, respectively, because of increased toxicity. Optimized treatment with dBG (250 mg/m2) and BCNU (35 mg/m2) resulted in T-Cs of 30, 29, 11, 16, and 14 days for SWB77, SWB40, SWB39, D-54 and SWB61, respectively. These delays were more pronounced than those induced with optimized, isotoxic treatments with BG (180 mg/m2) and BCNU (30 mg/m2). In comparison to BCNU, TMZ was less toxic, with an LD10 of 400 mg/m2. TMZ (300 mg/m2) was more effective than BCNU against SWB77, SWB40, and SWB61, inducing T-Cs of 23, 53, and 56 days, respectively. BG and dBG enhanced the toxicity of TMZ in athymic mice by decreasing the LD10 from 400 to 200 mg/m2. TMZ (180 mg/m2) with either BG (180 mg/m2) or dBG (250 mg/m2) resulted in T-Cs of 31 and 49 days in SWB77, respectively, as compared with 16 days for TMZ (180 mg/m2) alone. In SWB40, the combination of TMZ with dBG, but not with BG, was significantly more effective than the maximum tolerated dose of TMZ (300 mg/m2) alone. The combination of TMZ with AGT inactivators had no benefit, as compared with TMZ alone, against xenografts with marginal AGT activity. In conclusion, at equimolar doses dBG was less toxic than BG in athymic mice when combined with either BCNU or TMZ. In this regard, BCNU or TMZ can be used at higher doses in combination with dBG than with BG. This study further demonstrates that there is a significant benefit of depleting AGT with nonspecific AGT inhibitors prior to treatment with either BCNU or TMZ in tumors having AGT activity >45 fmol/mg protein.

Debenzylation of O(6)-benzyl-8-oxoguanine in human liver: implications for O(6)-benzylguanine metabolism.

O(6)-Benzylguanine (BG) effectively inactivates the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase, and enhances the effectiveness of 1,3-bis(2-chloroethyl)-1-nitrosourea in cells in culture and tumor-bearing animals. BG is presently in phase II clinical trials. In humans, BG is converted to O(6)-benzyl-8-oxoguanine (8-oxoBG), a longer-lived, yet equally potent inactivator. We have isolated and identified the debenzylated product, 8-oxoguanine, in plasma and urine of patients following administration of BG. The purpose of this work was to determine the human liver enzymes responsible for the debenzylation of 8-oxoBG. Therefore, 8-oxoBG was incubated with human liver microsomes and cytosol, and the concentration of 8-oxoguanine was determined. No appreciable product was formed in the cytosol; however, increasing amounts of 8-oxoguanine were formed with increasing concentrations of pooled human liver microsomes. The amount of 8-oxoguanine formed increased with time and substrate concentration. Co-incubation of human liver microsomes with 8-oxoBG and various cytochrome P450 isoform-selective inhibitors suggested the possible involvement of CYP1A2, 2E1, and/or 2A6 in this reaction. Incubation of 8-oxoBG with baculovirus cDNA-overexpressed CYP1A2, 2E1, 2A6, and 3A4 demonstrated that formation of 8-oxoguanine was due mainly to CYP1A2. Debenzylation of 8-oxoBG complied with Michaelis-Menten kinetics with K(m) and V(max) values of 35.9 microM and 0.59 pmol/min/pmol of CYP1A2, respectively. CYP1A2 appears to be mainly responsible for the debenzylation of 8-oxoBG in human liver.

Inactivation of human O(6)-alkylguanine-DNA alkyltransferase by modified oligodeoxyribonucleotides containing O(6)-benzylguanine.

Inactivation of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT) enhances tumor cell killing by therapeutic alkylating agents. O(6)-Benzylguanine (b(6)G) can inactivate AGT and is currently in clinical trials to enhance therapy. Short oligodeoxyribonucleotides containing b(6)G are much more effective inactivators, but their use for therapeutic purposes is likely to be compromised by metabolic instability. We have therefore examined the ability to inactivate AGT of an 11-mer oligodeoxyribonucleotide containing b(6)G (11-mpBG) when modified with terminal methylphosphonate linkages to protect it from nucleases. This modification did not reduce the ability to serve as a substrate/inactivator for AGT, and 11-mpBG had an ED(50) value of 1.3 nM, more than 300-fold lower than that for b(6)G. A similar oligodeoxyribonucleotide containing O(6)-methylguanine (m(6)G) was also found to be a good substrate (ED(50) value of 10 nM), but the benzylated form was repaired more rapidly and preferentially. When added to HT29 cell cultures, 5 microM 11-mpBG was able to cause a prolonged inactivation of cellular AGT for at least 72 h and to greatly sensitize the cells to killing by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). The 11-mpMG was ineffective at up to 20 microM, suggesting that the benzyl group allows better uptake into the cell. However, even with 11-mpBG, the 1000-fold decrease in potency toward AGT in HT29 cells compared to that toward the protein in vitro suggests that uptake may be a limiting factor. These results suggest that oligodeoxyribonucleotides such as 11-mpBG may prove to be useful drugs for potentiation of alkylating agent chemotherapy if uptake can be improved.

Inactivation of O6-alkylguanine-DNA alkyltransferase by 8-substituted O6-benzylguanine analogs in mice.

PURPOSE: The purpose of this study was to determine the usefulness of various 8-substituted O6-benzylguanine (BG) analogs as modulators of the DNA repair protein. O6-alkylguanine-DNA alkyltransferase (AGT). More specifically, the degree of inactivation of AGT in mouse brain, liver, kidney and tumor by O6-benzyl-8-oxoguanine (8-oxoBG), 8-aza-O6-benzylguanine (8-azaBG), O6-benzyl-8-bromoguanine (8-bromoBG) and O6-benzyl-8-trifluoromethylguanine (8-tfmBG) was compared to inactivation by BG, a modulator in phase II clinical trials. BG is converted rapidly to 8-oxoBG in rodents, monkeys and humans. It was reasoned that 8-substituted analogs of BG would exhibit different pharmacological properties compared to BG which could influence tissue bioavailability and, thus, the extent of AGT inactivation in vivo. We compared the tissue distribution of these agents and AGT activity following administration of the 8-substituted analogs. MATERIALS AND METHODS: At various time points up to 24 h after i.p. administration of the BG analogs, tissues (i.e. brain, liver, kidney), A549 lung tumor xenografts (i.p.) or D456 brain tumor xenografts (i.c.) were harvested from athymic nude mice for AGT analysis. AGT activity was quantified in tissue extracts using a biochemical assay with [3H]methylated DNA as a substrate. In addition, concentrations of BG and 8-oxoBG were determined by HPLC with fluorescence detection in mouse tissues following administration of drug. RESULTS: Each of the 8-substituted analogs of BG demonstrated variable AGT inactivation capabilities that were comparable to or better than those of BG especially in kidney and brain tissues. There was a more pronounced depletion of AGT inactivation in brain and D456 brain tumor xenografts following administration of BG compared to 8-oxoBG that could be explained by a much greater concentration of AGT-inactivating drug (BG plus the metabolite 8-oxoBG for mice treated with BG versus 8-oxoBG for mice treated with 8-oxoBG) present in these tissues. The AUCs for brain, kidney and liver were 3.2, 6.9 and 1 1.8 times greater for BG than for 8-oxoBG. CONCLUSIONS: 8-substituted analogs of BG possess unique AGT-inactivation profiles in vivo that are different from that of BG. The AGT-inhibitory activities of BG and its major metabolite, 8-oxoBG, are related to tissue disposition of both drugs.

Phase I trial of carmustine plus O6-benzylguanine for patients with recurrent or progressive malignant glioma.

PURPOSE: The major mechanism of resistance to alkylnitrosourea therapy involves the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT), which removes chloroethylation or methylation damage from the O(6) position of guanine. O(6)-benzylguanine (O(6)-BG) is an AGT substrate that inhibits AGT by suicide inactivation. We conducted a phase I trial of carmustine (BCNU) plus O(6)-BG to define the toxicity and maximum-tolerated dose (MTD) of BCNU in conjunction with the preadministration of O(6)-BG with recurrent or progressive malignant glioma. PATIENTS AND METHODS: Patients were treated with O(6)-BG at a dose of 100 mg/m(2) followed 1 hour later by BCNU. Cohorts of three to six patients were treated with escalating doses of BCNU, and patients were observed for at least 6 weeks before being considered assessable for toxicity. Plasma samples were collected and analyzed for O(6)-BG, 8-oxo-O(6)-BG, and 8-oxoguanine concentration. RESULTS: Twenty-three patients were treated (22 with glioblastoma multiforme and one with anaplastic astrocytoma). Four dose levels of BCNU (13.5, 27, 40, and 55 mg/m(2)) were evaluated, with the highest dose level being complicated by grade 3 or 4 thrombocytopenia and neutropenia. O(6)-BG rapidly disappeared from plasma (elimination half-life = 0. 54 +/- 0.14 hours) and was converted to a longer-lived metabolite, 8-oxo-O(6)-BG (elimination half-life = 5.6 +/- 2.7 hours) and further to 8-oxoguanine. There was no detectable O(6)-BG 5 hours after the start of the O(6)-BG infusion; however, 8-oxo-O(6)-BG and 8-oxoguanine concentrations were detected 25 hours after O(6)-BG infusion. The mean area under the concentration-time curve (AUC) of 8-oxo-O(6)-BG was 17.5 times greater than the mean AUC for O(6)-BG. CONCLUSION: These results indicate that the MTD of BCNU when given in combination with O(6)-BG at a dose of 100 mg/m(2) is 40 mg/m(2) administered at 6-week intervals. This study provides the foundation for a phase II trial of O(6)-BG plus BCNU in nitrosourea-resistant malignant glioma.

Plasma and cerebrospinal fluid pharmacokinetics of O6-benzylguanine and analogues in nonhuman primates.

O6-Benzylguanine (BG) is a potent, specific inactivator of the DNA repair protein, O6-alkylguanine-DNA alkyltransferase, that enhances the sensitivity of tumor cell lines and tumor xenografts to chloroethylnitrosoureas. To search for BG analogues with greater penetration into the cerebrospinal fluid (CSF), we evaluated plasma and CSF pharmacokinetics of BG, 8-aza-O6-benzylguanine (8-azaBG), O6-benzyl-8-bromoguanine (8-BrBG), O6-benzyl-8-oxoguanine (8-oxoBG), O6-benzyl-8-trifluoromethylguanine (8-tfmBG), and O6-benzyl-2'-deoxyguanosine (B2dG) after i.v. administration of 200 mg/m2 of drug through an indwelling Ommaya reservoir in a nonhuman primate model. BG and its analogues were quantified in plasma and CSF using reverse-phase high-performance liquid chromatography assays. The plasma clearances of the four 8-substituted BG analogues were similar (0.04-0.06 l/h/kg), but half-lives ranged from <2 to >24 h. BG was converted to 8-oxoBG, an equally potent O6-alkylguanine-DNA alkyltransferase inactivator, and the elimination of 8-oxoBG was much slower than that of BG. As a result, the plasma area under the curve of 8-oxoBG was 3.5-fold greater than that of BG. B2dG was metabolized to BG and 8-oxoBG, but this pathway accounted for only 20% of B2dG elimination. The CSF penetration percentages (based on the ratio of AUC(CSF): AUCplasma) for BG, 8-azaBG, 8-oxoBG, 8-tfmBG, 8-BrBG, and B2dG were 3.2, 0.18, 4.1, 1.4, <0.3, and 2.0%, respectively. The CSF penetration of BG and its active metabolite 8-oxoBG is greater than the penetration of 8-azaBG, 8-BrBG, 8-tfmBG, and B2dG.

O6-benzylguanine-mediated enhancement of nitrosourea activity in Mer- central nervous system tumor xenografts--implications for clinical trials.

PURPOSE: To evaluate the role of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) plus O6-benzylguanine (O6-BG) in the treatment of both Mer+ and Mer- tumors. METHODS: The effect of pretreatment with O6-BG on the activity of BCNU against Mer- human central nervous tumor xenografts D-54 MG and D-245 MG was evaluated in athymic nude mice. RESULTS: BCNU (1.0 LD10; dose lethal to 10% of treated animals) produced growth delays of 8.9 days and 7.5 days and tumor regressions in six of ten and one of nine animals against D-54 MG, which was derived from a human malignant glioma xenograft. Dose reduction of BCNU to 0.38 LD10 eliminated antitumor activity. The combination of BCNU (0.38 LD10) plus O6-BG produced growth delays of 8.8 days and 7.9 days, with tumor regressions in four of ten and two of nine animals, respectively. BCNU (1.0 LD10) produced a growth delay of 49.8 days and ten of ten tumor regressions against D-245 MG, which was derived from a glioblastoma multiforme. BCNU (0.38 LD10) produced a growth delay of 19.4 days, with nine of ten tumor regressions. The combination of BCNU (0.38 LD10) plus O6-BG produced a growth delay of 65.7 days and seven of eight tumor regressions. CONCLUSION: These results suggest that the combination of BCNU plus O6-BG may be a rational intervention for both Mer+ as well as Mer- tumors.

Potentiation of BCNU antitumor efficacy by 9-substituted O6-benzylguanines. Effect of metabolism.

PURPOSE: O6-Benzylguanine (BG), an O6-methylguanine-DNA methyltransferase (MGMT) inactivator, potentiates the efficacy of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and of other DNA chloroethylating and methylating anticancer drugs and is currently undergoing clinical trials. O6-Benzyl-2'-deoxyguanosine (dBG), a less effective MGMT inactivator than BG in vitro, is at least as effective as BG in combination with BCNU against tumor xenografts in athymic mice. In order to identify the mechanism of dBG activation in in vivo systems we tested the metabolism, ability to inactivate MGMT, and efficacy to potentiate BCNU in vivo of two additional 9-substituted derivatives of BG, namely O6-benzyl-9-cyanomethylguanine (CMBG) and O6-benzylguanosine (BGS). METHODS: Metabolism and disposition of these drugs was examined in athymic mice and Sprague-Dawley rats. MGMT suppression was determined in human medulloblastoma (Daoy) tumor xenografts in athymic mice following treatment with BGS, dBG, and CMBG and was compared with the loss of resistance to BCNU as determined by tumor growth delays. RESULTS: Growth delays at 25 mg/m2 BCNU and 133 mg/m2 BG or equimolar doses of CMBG, BGS or dBG were 23.0, 2.5, 21.3 days. and 30.4 days, respectively. The above differences did not correlate with the ED50S of 0.2, 13, 11 microM, and 2 microM determined for the above compounds, respectively, in cell free extracts. Differences in the efficacies of the 9-substituted compounds did correlate, however, with the extent of their metabolic conversion to BG. The maximum concentrations of BG in blood achieved after the administration of equimolar (250 micromol/kg) doses of CMBG, BGS and dBG were 10, 30 microM, and 55 microM, respectively. Although such levels were lower than those achieved in circulation by administration of an equimolar amount of BG, BG levels persisted longer following treatments with BGS or dBG than after treatment with BG itself. Formation of BG was required for continuous and prolonged (> 16 h) suppression of MGMT activity to non-detectable levels (< 5 fmol/mg protein). CONCLUSION: Metabolism of BGS and dBG to BG explains the unexpected high efficacy of these compounds in potentiating the antitumor activity of BCNU in the athymic mouse model. The faster and more effective suppression of tumor MGMT by dBG and its greater efficacy, as compared with BGS, also correlates with a more rapid accumulation of BG in blood after dBG than after BGS administration, which results in faster and complete suppression of MGMT in Daov xenografts. Thus, metabolism of dBG and BGS to BG appears to be the determining factor for continuous and prolonged suppression of MGMT activity, and that near complete suppression of such activity during and following BCNU administration is required for the higher efficacy of treatments. Similarly, the failure of CMBG to suppress tumor MGMT to the same extent as BGS, in spite of their similar ED50 values, could be attributed to the metabolism of this compound mainly by pathways other than conversion to BG.

Eradication of human medulloblastoma tumor xenografts with a combination of O6-benzyl-2'-deoxyguanosine and 1,3-bis(2-chloroethyl)1-nitrosourea.

O6-Benzyl-2'-deoxyguanosine (dBG), a water-soluble inhibitor of O6-methylguanine-DNA methyltransferase (MGMT), potentiates the efficacy of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) against MGMT-positive, BCNU-resistant Daoy human medulloblastoma tumor xenografts in athymic mice (S. C. Schold et al., Cancer Res., 56: 2076-2081, 1996). Such potentiation was comparable to that observed for O6-benzylguanine, the prototype MGMT inhibitor that is currently undergoing clinical trials. In this study, we optimized the therapeutic effect of the dBG and BCNU combination against brain tumor xenografts without inducing substantial toxicity in the host by adjusting the doses of both compounds. dBG was escalated from 133 mg/m2 to 200 and 300 mg/m2, whereas corresponding doses of BCNU were reduced from 25 mg/m2 to 17 and 11 mg/m2, respectively. The growth delays of 30.2, 38.4, and 22.3 days, respectively, observed for the above regimens suggest that the optimal drug combination is not achieved with maximum doses of dBG. In fact, the highest doses of dBG (300 mg/m2) contributed to more frequent BCNU-related toxicities, despite the reduced BCNU dosage, and a reduction of the therapeutic effect. Toxicity was related to the depletion of MGMT activity in the gut of host mice and was manifested by edema, inflammation, and hemorrhage in the bowel wall by subsequent BCNU administration. With additional dosage adjustments, we found that tumor suppression of >90 days without toxicity was observed at 200 mg/m2 dBG and 23 mg/m2 BCNU. At these doses, tumors were eradicated (regressed to an undetectable size for >90 days) in 8 of 12 animals. Thus, dBG is the first of the MGMT inhibitors to show a curative effect in combination with BCNU against a human central nervous system tumor xenograft in athymic mice.

Inhibition of O(6)-methylguanine-DNA methyltransferase increases azoxymethane-induced colonic tumors in rats.

Azoxymethane (AOM) causes O(6)-methylguanine adduct formation which leads to G-->A transitions. Their repair is carried out by O(6)-methylguanine-DNA methyltransferase (MGMT). To evaluate the importance of this repair event in AOM-induced carcinogenesis, we examined the effect of O(6)-benzylguanine (BG), a potent inhibitor of MGMT, on colonic tumor development. Rats were treated weekly for 2 weeks at 0 and 24 h with BG (60 mg/kg body wt i.p.) or vehicle (40% polyethylene glycol, PEG-400), followed 2 h after the first dose of BG with AOM (15 mg/kg body wt) or vehicle (saline) i.p. Rats were killed 35 weeks later and tumors harvested and DNA extracted. In the AOM-treated groups, BG caused a significant increase in tumor incidence with tumors in 65.9%, versus 30.8% in the AOM/PEG-treated group (P < 0.05). In the BG/AOM group there was also a significant increase in tumor multiplicity, with 2.3 tumors/tumor-bearing rat, versus 1.6 tumors/tumor- bearing rat in the AOM/PEG group (P < 0.05). Since O(6)-methylguanine adducts can cause activating mutations in the K-ras and beta-catenin genes, we examined the effects of BG on these mutations. In the BG group there were seven mutations in codon 12 or 13 of exon 1 of the K-ras gene in 51 tumors examined, compared with no K-ras mutations in 17 tumors analyzed in the AOM/PEG group (P = 0.12). In the BG/AOM group there were 10 mutations in exon 3 of the beta-catenin gene among 48 tumors evaluated, compared with six mutations in 16 tumors analyzed in the PEG/AOM group (P = 0.16). In summary, MGMT inhibition increases AOM-induced colonic tumor incidence and multiplicity in rats.

Modulation of cyclophosphamide activity by O6-alkylguanine-DNA alkyltransferase.

PURPOSE: The human medulloblastoma cell line D283 Med (4-HCR), a line resistant to 4-hydroperoxycyclophosphamide (4-HC), displays enhanced repair of DNA interstrand crosslinks induced by phosphoramide mustard. D283 Med (4-HCR) cells are cross-resistant to 1,3-bis(2-chloroethyl)- -nitrosourea, but partial sensitivity is restored after elevated levels of O6-alkylguanine-DNA alkyltransferase (AGT) are depleted by O6-benzylguanine (O6-BG). Studies were conducted to define the activity of 4-HC and 4-hydroperoxydidechlorocyclophosphamide against D283 Med (4-HCR) after AGT is depleted by O6-BG. METHODS: Limiting dilution and xenograft studies were conducted to define the activity of 4-HC and 4-hydroperoxydidechlorocyclophosphamide with or without O6-BG. RESULTS: The activity of 4-HC and 4-hydroperoxydidechlorocyclophosphamide against D283 Med (4-HCR) was increased after AGT depletion by O6-BG preincubation. Similar studies with Chinese hamster ovary cells, with or without stable transfection with a plasmid expressing the human AGT protein, revealed that the AGT-expressing cells were significantly less sensitive to 4-HC and 4-hydroperoxydidechlorocyclophosphamide. Reaction of DNA with 4-HC, phosphoramide mustard, or acrolein revealed that only 4-HC and acrolein caused a decrease in AGT levels. CONCLUSIONS: We propose that a small but potentially significant part of the cellular toxicity of cyclophosphamide in these cells is due to acrolein, and that this toxicity is abrogated by removal of the acrolein adduct from DNA by AGT.

Phase I trial of O6-benzylguanine for patients undergoing surgery for malignant glioma.

PURPOSE: The major mechanism of resistance to alkylnitrosourea therapy is the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT), which removes chlorethylation or methylation damage from the O6-position of guanine. O6-benzylguanine (O6-BG) is an AGT substrate that inhibits AGT by suicide inactivation. We conducted a phase I trial to define the presurgical dose required for depletion of tumor AGT activity in patients with malignant glioma. MATERIALS AND METHODS: Patients were to be treated 18 hours before craniotomy with intravenous doses that ranged between 40 and 100 mg/m2 given over 1 hour. Resected tumor was snap-frozen in liquid nitrogen and AGT activity analyzed by high-pressure liquid chromatography (HPLC). Up to 13 patients were treated at a specific dose of O6-BG, with a target end point of > or = 11 of 13 patients with undetectable tumor AGT levels (< 10 fmol/mg protein). RESULTS: Thirty patients with malignant gliomas were enrolled, with 11 of 11 patients treated at 100 mg/m2 O6-BG demonstrating tumor AGT levels less than 10 fmol/mg protein. No toxicity was noted in any patient treated. CONCLUSION: These results indicate that 100 mg/m2 of O6-BG can maintain tumor AGT levels less than 10 fmol/mg protein for at least 18 hours after treatment, a time interval in which bis(2-chloroethyl)nitrosourea (BCNU)-induced chloroethyl adducts are fully converted into interstrand cross-links. A 100-mg/m2 dose of O6-BG will be used in combination with BCNU in another phase I trial designed to determine the maximal-tolerated dose of BCNU.

Reaction of O6-benzylguanine-resistant mutants of human O6-alkylguanine-DNA alkyltransferase with O6-benzylguanine in oligodeoxyribonucleotides.

Inactivation of the human DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT), by O6-benzylguanine renders tumor cells susceptible to killing by alkylating agents. AGT mutants resistant to O6-benzylguanine can be made by converting Pro140 to an alanine (P140A) or Gly156 to an alanine (G156A). These mutations had a much smaller effect on the reaction with O6-benzylguanine when it was incorporated into a short single-stranded oligodeoxyribonucleotide. Such oligodeoxyribonucleotides could form the basis for the design of improved AGT inhibitors. AGT and mutants P140A and G156A preferentially reacted with O6-benzylguanine when incubated with a mixture of two 16-mer oligodeoxyribonucleotides, one containing O6-benzylguanine and the other, O6-methylguanine. When the 6 amino acids located in positions 159-164 in AGT were replaced by the equivalent sequence from the Escherichia coli Ada-C protein (mutant AGT/6ada) the preference for benzyl repair was eliminated. Further mutation incorporating the P140A change into AGT/6ada giving mutant P140A/6ada led to a protein that resembled Ada-C in preference for the repair of methyl groups, but P140A/6ada did not differ from P140A in reaction with the free base O6-benzylguanine. Changes in the AGT active site pocket can therefore affect the preference for repair of O6-benzyl or -methyl groups when present in an oligodeoxyribonucleotide without altering the reaction with free O6-benzylguanine.

Effect of ionic state of 2'-deoxyguanosine and solvent on its aralkylation by benzyl bromide.

To extend studies of the aralkylation of nucleic acid components under a variety of solvent conditions, we determined product distributions from the reactions of benzyl bromide with 2'-deoxyguanosine and the anion of 2'-deoxyguanosine in 2,2, 2-trifluoroethanol (TFE) and compared these distributions with those from the reaction of the anion with benzyl bromide in N, N-dimethylacetamide (DMA). 7-Benzylguanine was the only benzylated product detected in the reaction with the neutral nucleoside in TFE. In striking contrast, the reaction of the anion of 2'-deoxyguanosine with benzyl bromide in TFE produced N2-benzyl-2'-deoxyguanosine in significant yield and with high selectivity. The reaction of the anion of 2'-deoxyguanosine with benzyl bromide in DMA produced products derived only from reaction at the 1- and/or 7-position of the nucleoside. The weakly nucleophilic but protic polar solvent TFE and the iminolate tautomeric form of the 2'-deoxyguanosine anion appear to be essential for benzylation at the exocyclic N2-position.

O6-alkylguanine-DNA alkyltransferase inactivation by ester prodrugs of O6-benzylguanine derivatives and their rate of hydrolysis by cellular esterases.

To modulate the bioavailability and perhaps improve the tumor cell selectivity of O6-alkylguanine-DNA alkyltransferase (AGT) inactivators, pivaloyloxymethyl ester derivatives of O6-benzylguanine (BG) were synthesized and tested as AGT inactivators and as substrates for cellular esterases. The potential prodrugs examined were the 7- and 9-pivaloyloxymethyl derivatives of O6-benzylguanine (7- and 9-esterBG), and of 8-aza-O6-benzylguanine (8-aza-7-esterBG and 8-aza-9-esterBG) and the 9-pivaloyloxymethyl derivative of 8-bromo-O6-benzylguanine (8-bromo-9-esterBG). The benzylated purines were all potent inactivators of the pure AGT and of the AGT activity in HT29 cells and cell extracts. Each ester was at least 75 times less potent than the corresponding benzylated purine against the pure human AGT. In contrast, the activities of esters and their respective benzylated purine were similar in crude cell extracts and in intact cells. The increase in potency of esters in cellular extracts could be explained by a conversion of the respective prodrug to the more potent benzylated purine in the presence of cellular esterases. The apparent catalytic activity (Vmax/Km) of liver microsomal esterase for 8-azaBG ester prodrugs was 70-130 times greater than for BG prodrugs and 10-20 times greater than for 8-bromo-9-esterBG. Tumor cell hydrolysis of the esters varied considerably as a function of cell type and prodrug structure. These data suggest that these or related prodrugs may be advantageous for selective AGT inactivation in certain tumor types.

Comparison of mutagenesis by O6-methyl- and O6-ethylguanine and O4-methylthymine in Escherichia coli using double-stranded and gapped plasmids.

To compare mutagenesis by O6-methylguanine (m6G), O4-methylthymine (m4T) and O6-ethylguanine (e6G), and assess their genotoxicity in Escherichia coli, double-stranded and gapped plasmids were constructed containing a single m6G, e6G or m4T in the initiation codon (ATG) of a lacZ' gene. Modified base induced mutations were scored by the loss of lacZ' activity on X-gal-containing media resulting in formation of white or sectored (mutant) rather than blue (non-mutant) colonies. Genotoxicity experiments with gapped plasmids containing the modified bases indicated that m4T produced a greater number of bacterial colonies than m6G or e6G. m4T was more mutagenic (45% mutant colonies) than m6G (6%) or e6G (11%) in repair competent (w.t.) E. coli when incorporated in double-stranded plasmids. In gapped plasmids, m4T produced 99% mutant colonies (as was observed previously for e6G) in both w.t. E. coli or E. coli deficient in both O6-alkylguanine-DNA alkyltransferases as well as methylation-directed mismatch repair (ada(-)-ogt(-)-mutS[-]). m6G in gapped plasmids produced 62% mutant colonies in w.t. E. coli, but this percentage increased to 94% in the ada(-)-ogt(-)-mutS(-) strain. In double-stranded plasmids both m4T and m6G produced very similar distributions of mutant and non-mutant colonies in the ada(-)-ogt(-)-mutS(-) strain. These observations led to the conclusion that differences in the mutagenicity of m6G and m4T in w.t. E. coli were a result of preferential repair of m6G compared to m4T by alkyltransferase and mismatch repair mechanisms, and did not reflect differences in their respective coding efficiency or their inherent obstructiveness to DNA synthesis as was observed with e6G. The combination of alkyltransferase and mismatch repair was concluded to be primarily responsible for the apparent genotoxicity of m6G compared to m4T in double-stranded plasmids.

Effect of DNA on the inactivation of O6-alkylguanine-DNA alkyltransferase by 9-substituted O6-benzylguanine derivatives.

Studies were carried out on the inactivation of pure human O6-alkylguanine-DNA alkyltransferase by 9-substituted O6-benzylguanine derivatives in the presence and absence of DNA. The addition of DNA increased the rate of inactivation of the alkyltransferase by O6-benzylguanine and its 9-methyl derivative but had little effect on the rate of inactivation by the 9-cyanomethyl derivative. In contrast, when O6-benzylguanine derivatives with larger 9-substituents such as ribose, 2'-deoxyribose, dihydrotestosterone, or 2-hydroxy-3-(isopropoxy)propyl were used, the addition of DNA was strongly inhibitory to the inactivation. In the case of O6-benzylguanine, O6-benzylguanosine, and O6-benzyl-2'-deoxyguanosine, these results were confirmed by directly measuring the rate of formation by the alkyltransferase of guanine, guanosine, or 2'-deoxyguanosine, respectively. The data indicated that the presence of DNA activated the alkyltransferase, rendering it more reactive with O6-benzylguanine or O6-benzyl-9-methylguanine, but that DNA interferes with the binding of inhibitors with larger 9-substituents, presumably by competing for the same binding site. Since these inactivators readily inactivate alkyltransferase in cells, the amount of cellular alkyltransferase bound to DNA must be small or readily exchangeable with the free form.

Repair of O6-benzylguanine by the Escherichia coli Ada and Ogt and the human O6-alkylguanine-DNA alkyltransferases.

O6-Methylguanine is removed from DNA via the transfer of the methyl group to a cysteine acceptor site present in the DNA repair protein O6-alkylguanine-DNA alkyltransferase. The human alkyltransferase is inactivated by the free base O6-benzylguanine, raising the possibility that substantially larger alkyl groups could also be accepted as substrates. However, the Escherichia coli alkyltransferase, Ada-C, is not inactivated by O6-benzylguanine. The Ada-C protein was rendered capable of reaction by the incorporation of two site-directed mutations converting Ala316 to a proline (A316P) and Trp336 to alanine (W336A) or glycine (W336G). These changes increase the space at the active site of the protein where Cys321 is buried and thus permit access of the O6-benzylguanine inhibitor. Reaction of the mutant A316P/W336A-Ada-C with O6-benzylguanine was greatly stimulated by the presence of DNA, providing strong support for the concept that binding of DNA to the Ada-C protein activates the protein. The Ada-C protein was able to repair O6-benzylguanine in a 16-mer oligodeoxyribonucleotide. However, the rate of repair was very slow, whereas the E. coli Ogt, the human alkyltransferase, and the mutant A316P/W336A-Ada-C alkyltransferases reacted very rapidly with this 16-mer substrate and preferentially repaired it when incubated with a mixture of the methylated and benzylated 16-mers. These results show that benzyl groups are better substrates than methyl groups for alkyltransferases provided that steric factors do not prevent binding of the substrate in the correct orientation for alkyl group transfer.

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.