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 LD 10 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.

Chemotherapeutic agents that alkylate the O6 position of guanine in DNA such as BCNU,31-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, fotemustine,dacarbazine, streptozotocin, procarbazine, and TMZ are used primarily to treat brain cancer, melanoma, lymphoma, and gastrointestinal cancers. The effectiveness of these agents, however, is limited by AGT (1), a protein that repairs O6-alkylguanine adducts and is up-regulated in several tumors during progression (2, 3, 4, 5). Furthermore, selection of resistant AGT phenotypic populations after treatment with alkylating agents seems to be the reason for the recurrence of tumors of even a more resistant phenotype (6). Tumor resistance to DNA alkylation could be theoretically reversed with AGT pseudosubstrates that react with and inactivate the protein. The principle that depletion of AGT enhances the antitumor chemotherapeutic potency of DNA alkylating drugs has been tested and well documented experimentally using several O6-benzyl (7, 8, 9) and 4-bromothenyl (10) derivatives of guanine, and clinical trials are under way to test such compounds against brain tumors. Despite considerable advances in this field, methodologies to sensitize tumors by depleting AGT and the selection of the appropriate chemotherapeutic agent to be combined with AGT depleting drugs are still under evaluation. BG, the prototype AGT inhibitor, is nonspecific, and it also sensitizes normal tissue to alkylating agents,thus increasing their toxicity, mutagenicity, and carcinogenicity,which are limits to its utility to achieve the desirable therapeutic effect (11, 12, 13). To overcome the nonspecificity of BG,strategies for the protection of sensitive normal tissue have been considered. The transduction of AGT mutant forms that are resistant to BG-mediated depletion of AGT in hematopoietic bone marrow cells or other cell populations that might be poisoned by the combination of BG and alkylating agents is an interesting approach to avoid unacceptable myelosuppression in patients (14, 15, 16, 17, 18), but complications often associated with gene therapy could be avoided with the discovery of more tumor-selective AGT inactivators. Although progress in this direction has been slow, there are numerous reports indicating that the efficacy of chemotherapy could be improved by either modifying the route of administration of BG (19) or by using alternative AGT inhibitors (20, 21). An AGT inhibitor that has not only resulted in impressive tumor growth delays in combination with BCNU but also in eradicating the AGT-efficient, nitrosourea-resistant Daoy tumor xenografts is the deoxyribonucleoside analogue of BG, dBG (9). The effectiveness of this compound, which may be attributed to its water solubility, its systemic availability and persistence in circulation, along with its metabolic conversion to more potent AGT inhibitors (21), warrants further comparisons of BG with additional 9-substituted, water-soluble BG derivatives.

An additional important issue in combining DNA alkylating agents with AGT inhibitors is whether to include such inhibitors in the treatment of tumors with no or low AGT content, especially because such a combination limits the dose of the alkylating agent. Dose is important for several reasons, including the fact that the alkylating agent itself might quench low levels of AGT. A case in point is TMZ, which at a dose of 100 mg/kg eliminates all of the AGT activity in tumors having moderate AGT levels for a prolonged time period (11). In addition, the inverse correlation between AGT levels and effectiveness of BCNU against central nervous system tumors (22, 23)suggest that there may be no benefit in treating AGT-deficient tumors with AGT inhibitors. Determining the threshold of AGT activity that could be overcome by alkylating agents without the use of AGT inactivators may be complicated by tumor heterogeneity in AGT levels in some human tumors. However, such heterogeneity has not been demonstrated conclusively in gliomas, and therefore, conclusions derived from xenograft experiments may also apply in patients with such malignancies.

In this report, we address the question of whether AGT depletion prior to the administration of BCNU or TMZ enhances the response of typical glial tumors having low (<50 fmol/mg protein) to moderate (75 fmol/mg protein) levels of AGT activity. It is expected that AGT inhibitors will not enhance the efficacy of TMZ against tumors with modest AGT levels when this agent is administered at its maximum effective dose. On the other hand, the markedly higher toxicity of BCNU as compared with TMZ is expected to render the combination of AGT inhibitors and BCNU more effective than BCNU itself in the treatment of tumors having modest levels of AGT activity, such as those usually found in the central nervous system.

Chemicals.

BG and dBG were synthesized and purified according to methods published previously (24, 25). BCNU was purchased from Bristol-Myers Squibb (Princeton, NJ), and TMZ was donated by Schering-Plough, Inc.(Madison, NJ).

Animals.

BALB/c nu/nu athymic mice, 4 weeks of age, were purchased from Harlan Labs (San Diego, CA). Mice were maintained under barrier conditions and given sterilized food (Harlan Teklad laboratory diet)and water.

Tumor Lines.

The tumor lines D-54, SWB39, SWB40, SWB77 (human glioblastomas), and SWB61 (human anaplastic oligodendroglioma) were chosen for their lack of genomic instability and effective MMR repair.4They were grown in 5% fetal bovine serum (Life Technologies, Inc.,Gaithersburg, MD) in Eagle’s MEM (Life Technologies, Inc.)supplemented with lysine, valine, methionine, and leucine (100μ m each), nonessential amino acids (1:100 dilution of stock from Life Technologies, Inc.), 1 mm sodium pyruvate,1 μm α- hydroxocobalamin, 10 μm folic acid, and 0.2 mg/ml gentamicin. s.c. tumors grew upon injection of 3–4 million cells/animal. Tumor xenografts D-54, SWB39, SWB40, SWB77, and SWB61 had AGT activities of <10, 10, 45, 75, and 16 fmol/mg protein,respectively, as determined by the biochemical assay (26). Respective mitotic indices in xenografts were 5.5, 8.2, 12.1, 5.7, and 12.9 m/high-powered field. Extensive necrosis was observed in growing xenografts SWB40 and SWB61, which is in agreement with their rapid growth patterns.

Drug Treatment.

All treatments were administered i.p. BG and dBG were dissolved in 40%PEG 400:60% PBS. The pH of the solvent was corrected to 7.0 with sodium bicarbonate before the addition of the drug. BCNU was administered in 5% ethanol in water from a stock solution of 20 mg/ml of anhydrous ethanol. Depending on the dose of BCNU, the injected ethanol carrier varied from 10 to 15% ethanol. TMZ was dissolved in 100% DMSO. AGT inhibitors were administered at volumes of 30 ml/m2 and alkylating drugs at 20 ml/m2. Drug doses were calculated as mg/m2 using the formula: meters (m) = weight(g)2/3 × K ×10−4, where K is 10.5 for mice (27). In animals of 20 ± 2 g used in this study, the weight (kg) of the animal is ∼2.6 times the area surface(m2).

Tumor Implantation and Treatment.

Two hundred μl containing 3 × 106 cells of D-54, SWB39, SWB40, SWB77, and SWB61 tumor cells in 5% serum media were injected at the left flank of athymic mice, 6 weeks of age,weighing between 18 and 22 g. Visible tumors appeared in most of the animals within 3 weeks after implantation. The tumors were subsequently measured in two perpendicular dimensions, and their volumes were estimated using the formula (α2 ×β)/2, where α is the shorter and β the longer of the two dimensions. Treatment was administered to animals with tumors ranging between 150 and 200 mm3. Tumors were measured every other day until their volumes exceeded five times the volume of the tumor at treatment. The data were analyzed using the Wilcoxon rank sum test, comparing the time from treatment to five times treatment volume in individual animals in each of the groups. Growth delay was the difference between the median time to five times treatment volume in the treatment group minus the median time to five times treatment volume in the control group. The number of tumor regressions (number of tumors measuring smaller than the volume on the treatment day)occurring in each group was also determined. Groups were compared with the two-tailed Fisher’s exact test. Two control groups received 40%PEG:60% PBS, followed by 10% ethanol:water in 2 h or 40%PEG:60% PBS and BCNU in 10% ethanol. Unless otherwise indicated,10–12 animals were used in each experimental group.

Toxicity of BCNU and Temozolomide.

The toxicity of BCNU, TMZ, and their combinations with BG and dBG were determined in the Balb/c athymic mouse (Table 1). The LD10 for BCNU is between 70 and 75 mg/m2 when the drug was delivered i.p. Treatments with doses of 70 mg/m2 induced a temporary loss of weight of ∼10% and no deaths. All deaths at higher doses occurred within 72 h from treatment. The AGT inhibitors BG and dBG sensitized animals to BCNU, and their use necessitated a decrease of the dose of the chemotherapeutic drug to 30 and 35 mg/m2, respectively. The combination of dBG and BCNU at 250 and 35 mg/m2, respectively, was the maximum tolerated exposure to this drug combination, inducing a 12%weight loss but no deaths. Increasing the dBG dose to 300 mg/m2 depleted the intestinal AGT in mice and sensitized the animals to the toxicity of BCNU, even at doses <35 mg/m2. At 180 mg/m2 BG, a dose that is equimolar to 250 mg/m2 dBG, 35 mg/m2 BCNU induced a 40% incidence of death and a weight loss 2-fold greater than that induced by an equimolar doses of dBG and 35 mg/m2 BCNU. Animal deaths were prevented by reducing the dose of BCNU to 30 mg/m2. Therefore, administration of equimolar doses of BG and dBG required treatment with 30 and 35 mg/m2 BCNU, respectively, to achieve the same extent of weight loss and no deaths (equitoxic treatments). Comparisons between BG and dBG at equimolar doses with slight variation in the BCNU dose to achieve comparable toxicity is justified because such treatments achieve the best possible tumor response for the combination of these two AGT inactivators with BCNU without causing any animal deaths.

TMZ was tolerated well by athymic mice at a single dose of 400 mg/m2 with only 1 of 10 animals dying 5 days after treatment. At 600 mg/m2, 5 of 10 animals died acutely (in <24 h), whereas an additional 2 died within 27 days from treatment. No deaths were seen in animals treated with doses <400 mg/m2, and toxicity was manifested only as mild weight loss. Acute toxicities of TMZ and BCNU, based on weight loss,were comparable at maximum tolerated doses of 300 and 70 mg/m2, respectively. As it was the case with BCNU, the toxicity of TMZ was also potentiated by administration of AGT inhibitors 2 h prior to treatment. Equimolar doses of BG or dBG in 40% PEG of 180 and 250 mg/m2, respectively,caused acute animal deaths when combined with TMZ at doses >200 mg/m2. Sporadic deaths observed between 5 and 12 days after treatment with dBG (250 mg/m2)or BG (180 mg/m2) and TMZ at 200 mg/m2 necessitated further reduction of the TMZ dose to 180 mg/m2.

Treatment Efficacy of BCNU against Glial Tumors.

The efficacy of BCNU against glial tumors was tested using the D-54,SWB39, SWB61, SWB40, and SWB77 xenografts growing s.c. in nude mice. The median time and range for these tumors to quintuple in size was 8 (7, 8, 9, 10, 11), 11 (9, 10, 11, 12), 12 (7, 8, 9, 10, 11, 12, 13, 14, 15, 16),11 (9, 10, 11, 12, 13, 14, 15), and 11 (9, 10, 11, 12, 13) days,respectively. The effect of BCNU at doses ranging between 35 and 75 mg/m2 on growth delay of these xenografts and the toxicity of BCNU at these doses are shown on Table 2. A dose of 35 mg/m2 BCNU was ineffective against all tumor xenografts tested, including those with negligible levels of AGT activity (D- 54, SWB61, and SWB39). However, SWB61 SWB39, and D-54 tumor xenografts, with AGT activity <20 fmol/mg protein, responded to a single BCNU treatment at doses of 65–70 mg/m2,whereas those with higher AGT activities did not respond (SWB77) or responded weakly (SWB40). Further escalation of the dose induced animal deaths, as expected from the toxicity data without further delaying tumor growth in the survivors.

Treatment Efficacy of BCNU and dBG.

Table 3 shows the effect of depletion of AGT prior to BCNU treatment. Depletion of AGT was accomplished with dBG, which has been shown previously to suppress AGT activity to <2% of its baseline for a period lasting for at least 16 h after its administration (21). BCNU was administered at 2 h after 250 mg/m2 dBG,which is approximately the molar equivalent to a dose of 180 mg/m2 BG. Escalation of the dBG dose above this level did not cause toxicity by itself but resulted in unacceptable toxicity by BCNU with no increase in tumor growth delay. The increased toxicity of BCNU with higher doses of dBG is likely attributable to depletion of intestinal AGT (9) and the exposing of the brush epithelium to cross-linking DNA damage. The toxicity of the optimal dBG/BCNU combination, as determined from acute loss of weight, was similar to that achieved for the maximum tolerated dose of BCNU alone. However, the tumor growth delay was markedly enhanced in glioblastomas expressing AGT activity at levels >45 fmol/mg protein. Although the increase in tumor growth delay induced by dBG (250 mg/m2) and BCNU (35 mg/m2) observed in tumors with low AGT activity(<20 fmol/mg protein) was not statistically significant as compared with an equitoxic dose of BCNU alone (70 mg/m2),there was a definite benefit (P < 0.025) as compared with 35 mg/m2 BCNU alone (Tables 2 and 3).

Comparison between BG and dBG in Enhancing the Efficacy of BCNU.

A direct comparison between BG and dBG in enhancing the efficacy of BCNU against the AGT-efficient SWB77 tumor xenografts is shown in Fig. 1. Treatments with either dBG-BCNU (at 250 and 35 mg/m2) or BG-BCNU (at 180 and 30 mg/m2) were isotoxic to a treatment with 70 mg/m2 BCNU alone (Tables 2 and 4). Tumor delays with either drug combination were markedly greater than those observed when the same tumors were treated with the full dose (70 mg/m2 of BCNU alone, but dBG was more effective than BG (P < 0.001) in enhancing the efficacy of BCNU. A statistically significant difference (P < 0.001)between BG and dBG was also determined when these two compounds were used in combination with BCNU against SWB40 tumor xenografts (Table 4). It was noted that dBG-BCNU caused similar delays in SWB77 and SWB40,but BG increased the efficacy of BCNU, more notably in SWB40 than in SWB77. There was a small but statistically significant(P < 0.05) advantage in using the reduced dose of BCNU and AGT inhibitors rather than the maximum tolerated dose of BCNU without AGT inhibition (70 mg/m2) against the SWB61 tumor xenografts, which have marginal AGT activity, but not against D-54, which has negligible AGT levels.

Treatment Efficacy of TMZ.

Three tumor xenografts, SWB61, SWB40 and SWB77, were treated with TMZ at single i.p. doses of 300 mg/m2 and at 200 mg/m2 (Table 5). Although none of the implanted tumors were eradicated by TMZ treatment alone, the growth of SWB61 and SWB40 was retarded for >50 days. TMZ induced a moderate response in SWB77, where tumor growth delay was ∼20 days. There were no statistical differences in the tumor response between the doses of 200 and 300 mg/m2 TMZ in any of the tumors treated, despite evident greater toxicity of the latter dose. Further escalation of the dose to 600 mg/m2 against SWB61 did not delay growth but had a rather profound effect in toxicity. It was concluded that the maximum therapeutic effect of TMZ (single-dose i.p.) was achieved at 200–300 mg/m2.

Treatment Efficacy of TMZ and dBG or BG.

AGT inhibitors enhanced the efficacy of TMZ alone against the AGT-positive SWB77 xenografts compared with the efficacy of TMZ alone at its maximum tolerated dose of 300 mg/m2. At equimolar doses, BG was less effective than dBG in enhancing TMZ antitumor activity against SWB77, although the two treatments produced equal toxicity in the animals (Table 6). A significant advantage in using lower levels of TMZ in combination with BG or dBG rather than greater doses of TMZ alone was also seen in SWB40 tumor xenografts. However, with SWB40, the difference between BG and dBG in enhancing the efficacy of TMZ (Fig. 2; Table 6) was not statistically significant. There was no enhancement of TMZ efficacy against SWB61 by BG or dBG (data not shown).

The goals of this study were to test the importance of low to modest (10–75 fmol/mg protein) levels of AGT activity in the resistance of gliomas to both methylating and chloroethylating agents and to determine the threshold of AGT activity required to completely block the chemotherapeutic effectiveness of BCNU and of TMZ. Furthermore, this study aimed to determine the benefit, if any, of using AGT inactivators in combination with either TMZ or BCNU against brain neoplasms with low or nondetectable AGT activity and whether the selectivity of the combination of AGT inhibitors and alkylating drugs against tumors outweighs the imposed toxicity of such combinations on normal tissue in the mouse model.

DNA repair of the damage induced by alkylating agents occurs with the aid of several systems (28), such as direct reversal of O6-alkylguanine, nucleotide excision repair, MMR, base excision repair, error-free and error-prone tolerance during DNA replication, and recombinational repair. Although BCNU is toxic via the formation of cross-links, DNA excision repair seems to be of minor importance as compared with O6-alkylguanine reversal (29). However, BCNU ultimately bridges the N1 of G and N3 of C residues via the intermediate formation of O6-N1cycloethyl derivative of guanine yielding cross-links that are not subject to repair by either excision or by AGT (30). Inhibition of the O6-2-chloroethyl reversal for a period of at least 18 h, which is required for the cross-links to be formed, is therefore necessary to preserve the cytotoxic effect of BCNU. TMZ, on the other hand, exerts its toxicity by invoking the response of the MMR system via the recognition of O6-methylguanine containing odd bp by the MSH2, MLH1, and PMS2 complexed to MSH3 or MSH6. As a result of the different mechanisms by which BCNU and TMZ exert their toxicities, an inverse correlation between BCNU toxicity and AGT levels is expected with all tumors, whereas such a correlation is expected with TMZ only with tumors that have at least a functional MMR and a rapid rate of turnover. The tumors used in this communication express similar levels of MLH1, MSH2, and PMS2, which migrate to the nucleus after exposure to TMZ and show no apparent instability after TMZ treatment, as determined by microsatellite assays.4 The above indicate the presence of functional MMR systems (31). Therefore, the resistance of SWB77 and to a lesser extent that of SWB40 to TMZ was attributable, at least partially, to AGT. The poorer response of SWB77 to TMZ, as compared with that of SWB40 and SWB61, even after the depletion of the AGT activity, indicates that SWB77 may resist alkylating damage by mechanisms not yet identified. Different responses of SWB77 on one hand and SWB40 or SWB61 on the other, in cell cycle responses to DNA damage that could determine the efficiency of death via the action of MMR-related mechanisms, could also account for the resistance of SWB77 to TMZ (32).

Gliomas with low levels of AGT (<20 fmol/mg protein), such as SWB61 and SWB39, were minimally responsive to BCNU, and their resistance to this agent was not improved by pretreatment with AGT inhibitors,indicating the presence of yet unknown mechanisms of resistance. In this regard, SWB39 and SWB61 differ from SWB40 and SWB77, which are resistant to BCNU mainly because of their moderate levels of AGT. SWB39, SWB61, D-54, and even the AGT-effective tumor SWB40 were responsive to TMZ alone in the absence of AGT inhibition, which indicates that AGT activity <45 fmol/mg protein did not significantly contribute to the resistance of these tumors to TMZ. Furthermore, the high efficacy of TMZ against SWB61 suggests that mechanisms of resistance other than AGT, which contribute to the weak response of these tumors to BCNU, can be overcome by TMZ. These data indicate that the threshold of AGT activity required for resistance to TMZ is markedly higher than that determined for BCNU. These experiments also demonstrate that the maximum benefit of the combination of AGT inhibitors and BCNU requires fine adjustments of the doses of these two agents.

In mice, the doses of BG and dBG required for maximum efficacy were 180 and 250 mg/m2, respectively. Doses of BG and dBG can be lowered to 150 and 200 mg/m2 with only a mild decline in the enhancement of antitumor efficacy of the alkylating agents. Doses of 300 dBG induced unacceptable depletion of AGT from normal mouse tissues (21) without improving the suppression of AGT from tumors, and therefore, such doses rendered these tissues extremely sensitive to the alkylating drug, causing severe weight loss and deaths to the treated animals. Overdosing with AGT inhibitors did not improve the tumor response in the survivors,indicating that BG and dBG cause maximal AGT depletion in the tumor when administered at 150–180 and 200–250 mg/m2,respectively.

One of the important findings of this study is that the potential of other AGT inhibitors to enhance the efficacy of chemotherapy with DNA alkylating drugs and theoretically to surpass BG, the prototype AGT inhibitor currently undergoing clinical trials. It has been shown previously that dBG, which has a small but significant advantage to suppress tumor AGT as compared with O6-benzyl-9-cyanomethylguanine and O6-benzylguanosine, is also more effective in enhancing the efficacy of BCNU against human medulloblastoma (Daoy)tumors (21). Comparisons between these 9-substituted derivatives of BG have shown that small variations in both the extent and also the duration of suppression could translate into marked differences in the response of tumor to BCNU chemotherapy. Careful screening of additional compounds should therefore allow the discovery of AGT inactivators that are more effective than those presently at hand. In this report, we demonstrate that dBG is significantly more effective than BG in enhancing the efficacy of BCNU against two AGT-efficient glioblastomas. This could be attributed to the more prolonged exposure of tumors to AGT inhibitors derived from dBG than from BG metabolism (21), causing a more protracted AGT suppression. Comparison of BG and 8-oxo-BG levels in plasma and cerebrospinal fluid of primates given systemic BG and dBG,respectively, may suggest a greater potency of BG over dBG (33). However, the comparisons were made at equal doses and not at equimolar or equitoxic doses that would have allowed direct comparisons between that report and the data presented here. Our results with TMZ contrast with those reported earlier showing marginal benefit on TMZ efficacy by BG in single-administration regimens (7, 34). This is probably because of the optimization of the chemotherapeutic regimens and fine adjustment of the TMZ and BG or dBG doses in this study.

Overall, we have shown that for glial tumors with AGT activity <45 fmol/mg protein, there is no significant benefit in using an AGT inhibitor and a reduced dose of BCNU as compared with an isotoxic full dose of BCNU. However, tumors with AGT of 45 fmol/mg protein or greater activity are unresponsive to BCNU alone and require pretreatment with an AGT inhibitor to respond to BCNU treatment. It is interesting that tumors, which are resistant to BCNU because of their AGT content, are more responsive to treatment combining BCNU and AGT inhibitors than tumors with low AGT activity and responsive to BCNU alone. This indicates that the latter may rely on mechanisms of resistance other than the AGT. Unlike BCNU, the performance of TMZ is not as dependent on tumor AGT at the AGT levels tested. Consistent improvement of tumor delay because of AGT inhibition prior to TMZ treatment is observed with SWB77. The similar efficacy of TMZ against SWB61, SWB39, and SWB40 suggests that the (different) mechanisms of resistance these tumors may relay on are easily overcome by a single dose of TMZ. On the other hand, the failure to obtain the same response with SWB77 as with SWB61 and SWB40, even under AGT inhibition conditions, indicates that the efficacy of TMZ may also be limited by yet uncharacterized mechanisms of resistance in some tumors. The tumors tested here are neither rich in AGT nor have MMR defects, which may confer resistance to TMZ.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

        
1

Supported by National Cancer Institute Grants CA 57725 and CA 78561 and the National Cancer Institute, Department of Health and Human Services, under contract with ABL and The Children’s Brain Tumor Foundation of the Southwest.

                
3

The abbreviations used are: BCNU,1,3-bis(2-chloroethyl)-1-nitrosourea; AGT, O6-alkylguanine-DNA alkyltransferase; BG, O6-benzylguanine; dBG, O6-benzyl-2′-deoxyguanosine; MMR, mismatch repair; TMZ, temozolomide; PEG, polyethyleneglycol.

        
4

D. B. Bocangel, S. Mitra, and D. M. Kokkinakis, unpublished observations.

Table 1

Toxicity of carmustine (BCNU) and TMZ: Effect of BG and dBG on potentiating the toxicity of BCNU and TMZ in athymic mice

Alkylating drugaDoseb mg/m2AGT inhibitoraDoseb mg/m2Weight loss % of controlDeathsc
BCNU 65 none  05 ± 2d 0 /10e 
BCNU 70 none  10 ± 3 0 /10 
BCNU 75 none  15 ± 3 3 /10 
BCNU 85 none  24 ± 4 6 /10 
BCNU 35 BG 200 22 ± 6 5 /10 
BCNU 35 BG 180 23 ± 3 4 /10 
BCNU 30 BG 200 14 ± 4 1 /10 
BCNU 30 BG 180 10 ± 2 0 /10 
BCNU 35 dBG 300 21 ± 4 7 /10 
BCNU 35 dBG 250 12 ± 3 0 /10 
BCNU 35 dBG 200 10 ± 2 0 /10 
TMZ 600 none  21 ± 4 5 /10 
TMZ 400 none  16 ± 4 1 /10 
TMZ 200 none  05 ± 2 0 /10 
TMZ 400 dBG 250 15 ± 3 4 /10 
TMZ 200 dBG 250 12 ± 3 1 /10 
TMZ 180 dBG 250 03 ± 1 0 /10 
TMZ 180 BG 180 03 ± 2 0 /10 
TMZ 200 BG 180 06 ± 3 2 /10 
Alkylating drugaDoseb mg/m2AGT inhibitoraDoseb mg/m2Weight loss % of controlDeathsc
BCNU 65 none  05 ± 2d 0 /10e 
BCNU 70 none  10 ± 3 0 /10 
BCNU 75 none  15 ± 3 3 /10 
BCNU 85 none  24 ± 4 6 /10 
BCNU 35 BG 200 22 ± 6 5 /10 
BCNU 35 BG 180 23 ± 3 4 /10 
BCNU 30 BG 200 14 ± 4 1 /10 
BCNU 30 BG 180 10 ± 2 0 /10 
BCNU 35 dBG 300 21 ± 4 7 /10 
BCNU 35 dBG 250 12 ± 3 0 /10 
BCNU 35 dBG 200 10 ± 2 0 /10 
TMZ 600 none  21 ± 4 5 /10 
TMZ 400 none  16 ± 4 1 /10 
TMZ 200 none  05 ± 2 0 /10 
TMZ 400 dBG 250 15 ± 3 4 /10 
TMZ 200 dBG 250 12 ± 3 1 /10 
TMZ 180 dBG 250 03 ± 1 0 /10 
TMZ 180 BG 180 03 ± 2 0 /10 
TMZ 200 BG 180 06 ± 3 2 /10 
a

BCNU stock 10 mg/ml in 100%ethanol was diluted to appropriate concentrations with 5% ethanol. TMZ was dissolved in DMSO.

b

dBG and BG were dissolved in 40%PEG in PBS. BCNU and TMZ were injected i.p. (injection volume,0.15–0.2 ml/animal) 2 h after treatment with i.p. dBG, BG, or vehicle (injection volume, 0.2–0.3 ml/animal).

c

Number of deaths per number of animals treated.

d

Mean ± SD.

e

Animals were monitored for 20 days from initiation of treatment.

Table 2

Response of human glial tumor xenografts in athymic mice to a single i.p. treatment with BCNU: Effect of dose

TumorBCNUa mg/m2No. of animalsNo. of deathsWeight loss Mean %No. of regressionsMedian to 5× treatment volumeT-Cb Days
D-54 35 10 
SWB61 35 12 
SWB39 35 10 −1 
SWB40 35 11 
SWB77 35 10 −1 
D-54 65       
SWB61 65 10 21 9c 
SWB39 65 10 22 11c 
SWB40 65 13 16 4c 
SWB77 65 10 12 
D-54 70 12 10 26 18c 
SWB61 70 20 8c 
SWB39 70 21 10c 
SWB40 70 19 8c 
SWB77 70 14 
D-54 75 12 23 27 19c 
TumorBCNUa mg/m2No. of animalsNo. of deathsWeight loss Mean %No. of regressionsMedian to 5× treatment volumeT-Cb Days
D-54 35 10 
SWB61 35 12 
SWB39 35 10 −1 
SWB40 35 11 
SWB77 35 10 −1 
D-54 65       
SWB61 65 10 21 9c 
SWB39 65 10 22 11c 
SWB40 65 13 16 4c 
SWB77 65 10 12 
D-54 70 12 10 26 18c 
SWB61 70 20 8c 
SWB39 70 21 10c 
SWB40 70 19 8c 
SWB77 70 14 
D-54 75 12 23 27 19c 
a

BCNU stock 10 mg/ml in 100%ethanol was diluted to appropriate concentrations with 5% ethanol. BCNU was injected i.p. (injection volume, 0.15–0.2 ml/animal).

b

Tumor growth delay as compared with untreated controls. Animals were killed when tumor exceeded 1200 mm3.

c

Statistically significant(P < 0.05) as compared with untreated controls or to those treated with 35 mg/m2 BCNU.

Table 3

Enhancement of the efficacy of BCNU with dBG against human glial tumor xenografts in athymic mice

TumorBCNUa mg/m2dBG mg/m2No. of animalNo. of deathsWeight loss Mean %No. of regressionsMedian to 5× treatment volumeT-Cb Days
D-54 35 300 12 29 17 
 35 250 12 12 24 16 
 30 250 12 10 21 13 
SWB61 35 250 12 10 26 14 
 35 200 12 25 13 
SWB39 35 250 12 22 11 
SWB40 35 250 13 10 11 40 29 
SWB77 35 250 12 12 49 38 
SWB77 35 250 14 12 14 41 30 
TumorBCNUa mg/m2dBG mg/m2No. of animalNo. of deathsWeight loss Mean %No. of regressionsMedian to 5× treatment volumeT-Cb Days
D-54 35 300 12 29 17 
 35 250 12 12 24 16 
 30 250 12 10 21 13 
SWB61 35 250 12 10 26 14 
 35 200 12 25 13 
SWB39 35 250 12 22 11 
SWB40 35 250 13 10 11 40 29 
SWB77 35 250 12 12 49 38 
SWB77 35 250 14 12 14 41 30 
a

BCNU stock 10 mg/ml in 100%ethanol was diluted to appropriate concentrations with 5% ethanol. dBG was dissolved in 40% PEG in PBS. BCNU was injected i.p. (injection volume, 0.15–0.2 ml/animal) 2 h after treatment with i.p. dBG(injection volume, 0.2–0.3 ml/animal).

b

T-C tumor growth delay. All values were statistically significant as compared with untreated controls.

Fig. 1.

Treatment of s.c. SWB77 human glioblastoma xenografts in athymic mice with BG or dBG and BCNU. The treatments were administered i.p. when tumors had reached volumes of 150–200 mm3. ○, vehicle plus BCNU 35 mg/m2 at 2-h intervals; ▾, BG 180 mg/m2 plus 30 mg/m2BCNU; •, dBG 250 mg/m2 plus 35 mg/m2BCNU. Vehicle for dBG and BG was 40% PEG 400 in PBS. BCNU was administered as a solution in 10% ethanol.

Fig. 1.

Treatment of s.c. SWB77 human glioblastoma xenografts in athymic mice with BG or dBG and BCNU. The treatments were administered i.p. when tumors had reached volumes of 150–200 mm3. ○, vehicle plus BCNU 35 mg/m2 at 2-h intervals; ▾, BG 180 mg/m2 plus 30 mg/m2BCNU; •, dBG 250 mg/m2 plus 35 mg/m2BCNU. Vehicle for dBG and BG was 40% PEG 400 in PBS. BCNU was administered as a solution in 10% ethanol.

Close modal
Table 4

Comparison of the enhancement of efficacy of BCNU by BG and dBG against SWB77, SWB40, SWB61, and D-54 human glial tumor xenografts in athymic mice

TumorBCNUa mg/m2dBG or BG mg/m2No. of animalsNo. of deathsWeight loss Mean %No. of regressionsMedian to 5× treatment volumeT-Cb Days
SWB77 35 250 (dBG) 14 12 14 41 30 
 30 180 (BG) 12 11 22 11 
SWB40 35 250 (dBG) 13 10 11 40 29 
 30 180 (BG) 12 12 11 31 20 
SWB61 35 250 (dBG) 12 10 26 14 
 30 180 (BG) 12 11 10 24 12 
D-54 35 250 (dBG) 12 12 24 16 
 30 180 (BG) 12 10 18 10 
TumorBCNUa mg/m2dBG or BG mg/m2No. of animalsNo. of deathsWeight loss Mean %No. of regressionsMedian to 5× treatment volumeT-Cb Days
SWB77 35 250 (dBG) 14 12 14 41 30 
 30 180 (BG) 12 11 22 11 
SWB40 35 250 (dBG) 13 10 11 40 29 
 30 180 (BG) 12 12 11 31 20 
SWB61 35 250 (dBG) 12 10 26 14 
 30 180 (BG) 12 11 10 24 12 
D-54 35 250 (dBG) 12 12 24 16 
 30 180 (BG) 12 10 18 10 
a

BCNU stock 10 mg/ml in 100%ethanol was diluted to appropriate concentrations with 5% ethanol. dBG was dissolved in 40% PEG in PBS. BCNU was injected i.p. (injection volume, 0.15–0.2 ml/animal) 2 h after treatment with i.p. dBG(injection volume, 0.2–0.3 ml/animal).

b

T-C tumor growth delay as compared with untreated controls. All values were statistically significant as compared with untreated controls.

Table 5

Response of human glial tumor xenografts in athymic mice to a single i.p. treatment with TMZ

TumorTMZa mg/m2No. of animalsNo. of deathsWeight loss Mean %No. of regressionsMedian to 5× treatment volumeT-Cb Days
SWB77 300 10 10 34 23 
SWB77 200 10 10 30 19 
SWB40 300 10 10 10 66 53 
SWB40 200 10 10 59 46 
SWB61 600 12 10 10 61 54 
SWB61 300 10 10 63 56 
SWB61 200 10 10 61 54 
TumorTMZa mg/m2No. of animalsNo. of deathsWeight loss Mean %No. of regressionsMedian to 5× treatment volumeT-Cb Days
SWB77 300 10 10 34 23 
SWB77 200 10 10 30 19 
SWB40 300 10 10 10 66 53 
SWB40 200 10 10 59 46 
SWB61 600 12 10 10 61 54 
SWB61 300 10 10 63 56 
SWB61 200 10 10 61 54 
a

TMZ was dissolved in DMSO and injected i.p. (injection volume, 0.15–0.2 ml/animal).

b

T-C tumor growth delay as compared with untreated controls. All values were statistically significant as compared with untreated controls.

Table 6

Response of human glial tumor xenografts in athymic mice to a single treatment of BG or dBG, followed by a single i.p. treatment with TMZ

TumorTMZa mg/m2AGT inhibitor mg/m2No. of animalsNo. of deathsWeight loss Mean %No. of regressionsMedian to 5× treatment volumeT-Cb Days
SWB77 180 180 (BG) 12 12 43 31 
SWB77 180 250 (dBG) 12 10 12 61 49 
SWB77 200  12 10 28 16 
SWB40 180 180 (BG) 12 10 12 68 56 
SWB40 180 250 (dBG) 12 11 12 75 63 
SWB40 200  12 12 53 41 
SWB40 300  12 12 67 55 
TumorTMZa mg/m2AGT inhibitor mg/m2No. of animalsNo. of deathsWeight loss Mean %No. of regressionsMedian to 5× treatment volumeT-Cb Days
SWB77 180 180 (BG) 12 12 43 31 
SWB77 180 250 (dBG) 12 10 12 61 49 
SWB77 200  12 10 28 16 
SWB40 180 180 (BG) 12 10 12 68 56 
SWB40 180 250 (dBG) 12 11 12 75 63 
SWB40 200  12 12 53 41 
SWB40 300  12 12 67 55 
a

TMZ was dissolved in DMSO. dBG and BG were dissolved in 40% PEG in PBS. TMZ was injected i.p.(injection volume, 0.15–0.2 ml/animal) 2 h after treatment with i.p. dBG (injection volume, 0.2–0.3 ml/animal).

b

T-C tumor growth delay as compared with untreated controls. All values were statistically significant as compared with untreated controls.

Fig. 2.

Treatment of s.c. SWB40 human glioblastoma xenografts in athymic mice with BG or dBG and TMZ. The treatments were administered i.p. when tumors had reached volumes of 150–200 mm3. •, 40% PEG in PBS followed with 20 ml/m2 DMSO 2 h later; ○, dBG 250 mg/m2followed by 20 ml/m2 DMSO 2 h later; □, 40% PEG 400 in PBS vehicle followed in 2 h with 200 mg/m2temozolomide in DMSO; ▪, 40% PEG 400 in PBS vehicle followed in 2 h with 300 mg/m2 temozolomide in DMSO; ▿ BG 180 mg/m2 plus 200 mg/m2 temozolomide; ▾, dBG 250 mg/m2 plus 200 mg/m2 TMZ in DMSO. Vehicle for dBG and BG was 40% PEG 400 in PBS.

Fig. 2.

Treatment of s.c. SWB40 human glioblastoma xenografts in athymic mice with BG or dBG and TMZ. The treatments were administered i.p. when tumors had reached volumes of 150–200 mm3. •, 40% PEG in PBS followed with 20 ml/m2 DMSO 2 h later; ○, dBG 250 mg/m2followed by 20 ml/m2 DMSO 2 h later; □, 40% PEG 400 in PBS vehicle followed in 2 h with 200 mg/m2temozolomide in DMSO; ▪, 40% PEG 400 in PBS vehicle followed in 2 h with 300 mg/m2 temozolomide in DMSO; ▿ BG 180 mg/m2 plus 200 mg/m2 temozolomide; ▾, dBG 250 mg/m2 plus 200 mg/m2 TMZ in DMSO. Vehicle for dBG and BG was 40% PEG 400 in PBS.

Close modal

We acknowledge Jacquelynn Wick for assistance in the completion of xenograft experiments.

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