ATase Depletion Research Articles

Phase I trial of temozolomide, thalidimide, and lomustine in patients with metastatic melanoma in the brain.

8571 Background: Temozolomide (TMZ) and Lomustine (CCNU) are alkylating and active cytotoxic agents for metastatic melanoma (MM). The DNA repair enzyme O6-alkylguinine-DNA alkyltransferase (ATase), is the major cause of drug resistance for alkylating agents. Depletion of ATase by continuous dosing of TMZ may improve the anti-tumor activity of alkylating agents. This Phase I study is designed to establish a safe and tolerated regimen of oral cytotoxic agents, TMZ and CCNU in combination with a cytostatic agent, thalidomide (TLD), in patients with MM in the brain. Methods: Patients (pts) with stage IV MM in the brain with or without extracranial metastasis were entered successively onto 3 treatment cohorts: level 1, CCNU 30 mg/m2on day 1; level 2, CCNU 45 mg/m2on day 1; level 3, CCNU 60 mg/m2 on day 1. CCNU was administered at a flat dose of 30 mg/m2 on day 29 for all cohorts. TMZ was administered at 75mg/m2/d x 6 weeks followed by a 2-week break. TLD was administered at 200 mg/m2/d to the first 3 patient in level 1, then 200 mg/d in all other patients. Safety was assessed at weeks 2, 4, 6, 8 and every 4 weeks thereafter; tumor response was evaluated every 8 weeks. Results: Seventeen pts were enrolled, 7 pts each in levels 1 and 2, and 3 pts in level 3. . The MTD was determined to be dose level 2: CCNU 45 mg/m2on day 1. Three pts had a deep venous thrombosis. The most common adverse events were fatigue, constipation and myelosuppression. Of 16 pts assessable for disease response: 2 experienced a partial response, 2 stable disease and 2 mixed response. Disease response in brain and visceral organs were observed in dose levels 1 and 2. Conclusions: The combination therapy of TMZ, TLD and CCNU is safe and well tolerated in patients with MM in the brain. Clinical activity was seen at both levels 1 and 2. Further study of this oral drug combination therapy in these patients with extremely poor prognosis and lack of effective therapy is warranted.

Lomeguatrib, a Potent Inhibitor of O6-Alkylguanine-DNA-Alkyltransferase: Phase I Safety, Pharmacodynamic, and Pharmacokinetic Trial and Evaluation in Combination with Temozolomide in Patients with Advanced Solid Tumors

A major mechanism of resistance to temozolomide involves the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (ATase). The main aims of this phase I trial were to determine an ATase-depleting dose (ADD) of lomeguatrib, a potent pseudosubstrate inhibitor, and to define a suitable dose of temozolomide to be used in combination with lomeguatrib in patients with advanced cancer. Lomeguatrib was administered at dose levels of 10 to 40 mg/m2 days 1 to 5, as a single agent, and also in combination with temozolomide. Once the ADD of lomeguatrib was identified, the dose of temozolomide in combination was increased, in successive patient cohorts, from 50 to 175 mg/m2 on days 1 to 5 of a 28-day cycle to define the maximal tolerated dose and dose-limiting toxicity of the combination. Thirty-eight patients with advanced solid tumors were enrolled. More than 95% ATase depletion within 4 hours of the first dose was achieved in peripheral blood mononuclear cells at lomeguatrib doses of > or =10 mg/m2/d i.v. or > or =20 mg/m2/d orally, and tumor biopsies showed > or =92% ATase depletion. At the ADD of lomeguatrib i.v., the maximal tolerated dose of temozolomide in combination was 150 mg/m2 days 1 to 5. The dose limiting toxicity of the combination of lomeguatrib and temozolomide was myelosuppression. The toxicity of lomeguatrib alone was minimal. In 23 patients with measurable disease, one complete response was seen and 12 patients had stable disease for at least 3 months. This first administration of lomeguatrib to man successfully established an oral ADD of lomeguatrib and identified a combination regimen with temozolomide suitable for future clinical evaluation.

Pharmacokinetic, biochemical and clinical effects of dimethyltriazenoimidazole-4-carboxamide-bischloroethylnitrosourea combination therapy in patients with advanced breast cancer.

We assessed whether split dosing with the methylating agent DTIC is an effective strategy for inactivating the DNA repair protein O6-alkylguanine DNA-ATase in order to decrease tumour resistance to BCNU. ATase levels in PBMCs were used as a surrogate for tumour ATase depletion to determine whether this correlated with either the pharmacokinetics of DTIC and its major metabolite AIC or other clinical sequelae. Two 1 hr infusions of DTIC (400 mg/m(2)) 4 hr apart followed another 4 hr later by BCNU (75 mg/m(2)) were administered every 6 weeks in 7 patients with heavily pretreated advanced breast cancer. The extent and kinetics of ATase depletion and recovery in PBMCs varied not only between patients but also between cycles in the same patient. Serial FNAs showed heterogeneity in tumour ATase expression but no clear pattern of change in ATase activity. DTIC and AIC exhibited biphasic clearance from the blood, consistent with a 2-compartment pharmacokinetic model. The AUC of AIC was strongly correlated with the percentage decrease in PBMC ATase levels. There were no clinical responses, and toxicity in neutrophils and platelets was marked. Split-dose DTIC therefore does not appear to be a clinically effective approach to overcome O(6)-alkylating agent resistance in advanced breast cancer.

O6-(4-bromothenyl)guanine improves the therapeutic index of temozolomide against A375M melanoma xenografts

Tumour resistance to methylating agents is linked to expression of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (ATase). There is considerable interest in improving the efficacy of O6-alkylating chemotherapy by prior depletion of ATase. We have tested the ability of a modified guanine base, O6-(4-bromothenyl)guanine (4BTG), to inactivate ATase and to enhance the anti-tumour effect of temozolomide in an animal model system. A375M human melanoma xenografts were established in the flanks of nude mice. ATase depletion after a single dose of 4BTG or O6-BG (20 mg/kg i.p.) was determined over a 24 hr period. Subsequently, we tested the effect of 4BTG (20 mg/kg i.p. daily) and/or temozolomide (80–175 mg/kg i.p. daily) over a 5-day schedule on tumour growth. 4BTG was an effective inactivator of ATase in tumour, producing complete depletion within 2 hr of dosing. Furthermore, it enhanced the tumour growth delay achieved with temozolomide, increasing the tumour quintupling time by 8.7 days (95% confidence interval 6.1–11.3 days, p < 0.0001). Whilst the delay in tumour growth was indistinguishable from that observed with O6-benzylguanine (O6-BG) and temozolomide, the 4BTG combination resulted in considerably less toxicity (0/9 vs. 2/9 deaths; 6.84% weight loss vs. 9.48%, p = 0.019). 4BTG is a potent inactivator of ATase and enhances the therapeutic ratio of temozolomide in this model system to a greater extent than O6-BG. Int. J. Cancer 85:248–252, 2000. ©2000 Wiley-Liss, Inc.

O(6)-(4-bromothenyl)guanine improves the therapeutic index of temozolomide against A375M melanoma xenografts.

Tumour resistance to methylating agents is linked to expression of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (ATase). There is considerable interest in improving the efficacy of O(6)-alkylating chemotherapy by prior depletion of ATase. We have tested the ability of a modified guanine base, O(6)-(4-bromothenyl)guanine (4BTG), to inactivate ATase and to enhance the anti-tumour effect of temozolomide in an animal model system. A375M human melanoma xenografts were established in the flanks of nude mice. ATase depletion after a single dose of 4BTG or O(6)-BG (20 mg/kg i.p.) was determined over a 24 hr period. Subsequently, we tested the effect of 4BTG (20 mg/kg i.p. daily) and/or temozolomide (80-175 mg/kg i.p. daily) over a 5-day schedule on tumour growth. 4BTG was an effective inactivator of ATase in tumour, producing complete depletion within 2 hr of dosing. Furthermore, it enhanced the tumour growth delay achieved with temozolomide, increasing the tumour quintupling time by 8.7 days (95% confidence interval 6.1-11.3 days, p < 0.0001). Whilst the delay in tumour growth was indistinguishable from that observed with O(6)-benzylguanine (O(6)-BG) and temozolomide, the 4BTG combination resulted in considerably less toxicity (0/9 vs. 2/9 deaths; 6.84% weight loss vs. 9.48%, p = 0.019). 4BTG is a potent inactivator of ATase and enhances the therapeutic ratio of temozolomide in this model system to a greater extent than O(6)-BG.

Four-hourly scheduling of temozolomide improves tumour growth delay but not therapeutic index in A375M melanoma xenografts

To establish whether temozolomide is more effective against A375M human melanoma xenografts if given every 4 h rather than every 24 h, in order to exploit depletion of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (ATase) by prior doses of the drug. ATase depletion in A375M human melanoma xenografts was determined over 24 h after a single dose of temozolomide. The effect of different drug schedules (all of total dose 500 mg/kg) in delaying the growth of the xenografts was tested, and ATase depletion and DNA methylation damage assessed in tumour and normal tissue. Maximal depletion of ATase in tumour, to 2.52 +/- 0.23% of pretreatment levels, occurred 4-8 h after a single 100 mg/kg i.p. dose of temozolomide, with 23.0% recovery of protein levels at 24 h. Scheduling of temozolomide every 4 h increased tumour growth delay (33.6 +/- 1.39 days with temozolomide 100 mg/kg 4-hourly x versus 23.2 +/- 1.43 days with temozolomide 100 mg/kg once daily x 5; P < 0.0001) at the expense of increased toxicity (17.4 +/- 1.55% animal weight loss versus 10.6 +/- 1.27%. respectively). Temozolomide every 4 h did not increase ATase depletion compared with the 5-day schedule, but resulted in greater DNA 06-guanine methylation (29.0% more in tumour, 20.8% in liver and 56.0% in brain, comparing areas under the methylation-time curve). The 4-hourly schedule of temozolomide delayed tumour growth significantly more than the once-daily and 12-hourly schedules, probably as a result of greater DNA damage inflicted, but also increased toxicity. It remains to be seen if this regimen confers a net benefit over the standard schedule.

HMLH1 expression and cellular responses of ovarian tumour cells to treatment with cytotoxic anticancer agents.

Loss of expression of the hMLH1 and hPMS2 subunits of the MutL alpha-mismatch repair complex is a frequent event (9/10) in independent cisplatin resistant derivatives of a human ovarian carcinoma cell line. However, only hMLH1 mRNA is decreased in these MutL alpha-deficient lines. No alterations in the levels of the hMSH2 and hMSH6 (GTBP) subunits of the MutS alpha-complex are observed. An increase in the proportion of ovarian tumours negative for the hMLH1 subunit is observed in samples taken at second look laparotomy after chemotherapy (36%: 4/11), compared to untreated tumours (10%: 4/39). No significant difference is observed for hMSH2, hMSH6 or hPMS2. Furthermore, cisplatin and doxorubicin-resistant ovarian lines deficient in hMLH1 expression are cross-resistant to 6-thioguanine and the methylating agent N-methyl-N-nitrosourea (MNU). Depletion of O6-alkylguanine-DNA-alkyltransferase (ATase) activity confers only limited increased sensitivity to MNU. Thus the mismatch repair deficient lines retain DNA damage tolerance even after ATase depletion. The hMLH1 deficient lines also lose ability to engage G1 and G2 cell cycle arrest after cisplatin damage. Together these data suggest that loss of hMLH1 expression may be a high frequency event following exposure of ovarian tumour cells to cisplatin and may be critically involved in the development of drug resistance. Thus, the hMLH1 status of these cells appears to be highly correlated with the ability to engage cell death and cell cycle arrest after DNA damage induced by cisplatin.

Formation and loss of O6-methyldeoxyguanosine in human leucocyte DNA following sequential DTIC and fotemustine chemotherapy

There is increasing evidence to indicate that O6-methyldeoxyguanosine (O6-MedG) formation in DNA is a critical cytotoxic event following exposure to certain anti-tumour alkylating agents and that the DNA repair protein O6-alkylguanine-DNA alkyltransferase (ATase) can confer resistance to these agents. We recently demonstrated a wide inter-individual variation in the depletion and subsequent regeneration of ATase in human peripheral blood lymphocytes following sequential DTIC (400 mg m-2) and fotemustine (100 mg m-2) treatment, with the nadir ATase activity occurring approximately 4 h after DTIC administration. We have now measured the formation and loss of O6-methyldeoxyguanosine (O6-MedG) in the DNA of peripheral leucocytes of eight patients receiving this treatment regimen. O6-MedG could be detected within 1 h and maximal levels occurred approximately 3-5 h after DTIC administration. Following the first treatment cycle, considerable inter-individual variation was observed in the peak O6-MedG levels, with values ranging from 0.71 to 14.3 mumol of O6-MedG per mol of dG (6.41 +/- 5.53, mean +/- s.d.). Inter- and intra-individual variation in the extent of O6-MedG formation was also seen in patients receiving additional treatment cycles. This may be a consequence of inter-patient differences in the capacity for metabolism of DTIC to release a methylating intermediate and could be one of the determinants of clinical response. Both the pretreatment ATase levels and the extent of ATase depletion were inversely correlated with the amount of O6-MedG formed in leucocyte DNA when expressed either as peak levels (r = -0.59 and -0.75 respectively) or as the area under the concentration-time curve (r = -0.72 and -0.73 respectively). One complete and one partial clinical response were seen, and these occurred in the two patients with the highest O6-MedG levels in the peripheral leucocyte DNA, although the true significance of this observation has yet to be established.

Sequential administration of varying doses of dacarbazine and fotemustine in advanced malignant melanoma

There is increasing experimental evidence to suggest that expression of O6-alkylguanine-DNA-alkyltransferase (ATase) is a major factor in resistance to dacarbazine (DTIC). We recently demonstrated a progressive ATase depletion in human peripheral lymphocytes with nadir levels occurring at 4-6 h after DTIC administration (Lee et al., 1991). Therefore in an attempt to improve the clinical response rate of DTIC, fotemustine was administered 4 h after DTIC administration; since in the case of fotemustine, ATase removes the chloroethyl lesions from the O6-position of guanine, thereby preventing the formation of the cytotoxic cross-links. Sixty patients with widely metastatic melanoma received DTIC at 400, 500 or 800 mg m-2 followed by fotemustine (100 mg m-1) at 4 h after DTIC administration. Treatment was repeated every 28 days with a total of 169 cycles of chemotherapy administered; 75, 57 and 37 treatment cycles with 400, 500 and 800 mg m-2 DTIC groups respectively. Eighteen of the 60 patients responded (with three complete response); response rates were linearly related to dose, being 24%, 30% and 40% in patients receiving 400, 500 and 800 mg m-2 of DTIC respectively and the overall response rate was 30%. Median survival was 3.6 months (range, 1-15 months) with no statistically significant difference between the different DTIC treatment groups (P = 0.67). Nine patients are alive at 5 to 26 months (median 10 months); three patients with no tumour and five patients with stable disease. A statistically significant relationship was seen between the development of severe haematological toxicity (WHO > or = 3) with increasing dosage of DTIC and significant subclinical pulmonary damage was seen in 11 patients where the lung function was monitored during the course of treatment. In conclusion, it appears that with this small group of patients, escalation of DTIC dosage might not significantly affect response rates but does increase haematological toxicity. The present study provides a framework for other studies in an attempt to modulate ATase-mediated drug resistance in tumour tissues but the associated toxicity will need careful monitoring.

Dosage and cycle effects of dacarbazine (DTIC) and fotemustine on O6-alkylguanine-DNA alkyltransferase in human peripheral blood mononuclear cells

There is increasing experimental evidence to suggest that endogenous expression of O6-alkylguanine-DNA-alkyltransferase (ATase) is a major factor in cellular resistance to certain chemotherapeutic agents including dacarbazine (DTIC). We have recently shown wide interindividual variation in the depletion and subsequent regeneration of ATase in peripheral blood mononuclear cells (PMCs) following DTIC and this has now been extended to ascertain whether or not depletion is related to dosage of DTIC used and repeated treatment cycles of chemotherapy. ATase levels were measured in three groups of 25 patients (pts) up to 24 h after receiving DTIC at 400 mg m-2, 500 mg m-2 or 800 mg m-2. Each group also received fotemustine (100 mg m-2), 4 h after DTIC. The lowest extent of ATase depletion (highest nadir ATase) was seen in patients receiving 400 mg m-2. The mean nadir ATase, expressed as a percentage of pre-treatment ATase, was respectively 56.3%, 26.4% and 23.9% for 400 mg m-2, 500 mg m-2 and 800 mg m-2. The median nadir of ATase activity for pts receiving 800 mg m-2 pts was at 4-6 h and for pts given lower doses it was at 2-3 h. In addition, repeated measures analysis of variance of observations before chemotherapy, then at 2, 3, 4, 6 and 18 h after chemotherapy provides some evidence that ATase was depleted to a lesser extent after cycle 1 than after subsequent cycles (P = 0.025). It also provides evidence that the change in ATase activity over time varied with dose and cycle. The findings can be interpreted on the basis of a dosage-dependent metabolism of DTIC to an agent capable of methylation of DNA and subsequent depletion of PMC ATase: with higher DTIC doses, the extent of ATase depletion may be limited by the pharmacokinetics of DTIC metabolism. PMC ATase was measured in another group of 8 pts at various times after receiving only fotemustine (100 mg m-2) and in contrast to DTIC, no ATase depletion was seen suggesting that insufficient concentrations of fotemustine and/or its metabolites were available to react with DNA to produce a depletion of PMC ATase activity.

Cyclophosphamide decreases O6-alkylguanine-DNA alkyltransferase activity in peripheral lymphocytes of patients undergoing bone marrow transplantation.

O6-alkylguanine-DNA-alkyltransferase (ATase) levels were measured in extracts of peripheral blood lymphocytes taken at various times during chemotherapy from 19 patients with various haematological malignancies. Seven patients with advanced Hodgkin's disease received preparative treatment consisting of cyclophosphamide (1.5 g m-2, daily) administered on days 1 to 4 and BCNU (600 mg m-2) on day 5 prior to autologous bone marrow rescue (ABMR) delivered on day 7. Treatment in the remaining 12 patients consisted of cyclophosphamide (1.8 g m-2, daily) given on days 1 and 2 followed at day 4 with total body irradiation (TBI) administered in six fractions over the subsequent 3 days to a total dose of 1200 cGy prior to bone marrow transplantation. In the Hodgkin's group, significant decreases in ATase activity were seen during the cyclophosphamide treatment, and the median ATase nadir was 32% (range 0% to 57%) of pretreatment levels following 4 days of cyclophosphamide. In one patient, no ATase activity was detectable following the 4th cyclophosphamide treatment. ATase activities decreased further after BCNU administration to a median of 19% (range 0% to 32%) of pretreatment levels. Extensive cyclophosphamide-induced reduction of lymphocyte ATase levels was also seen in the other group of 12 patients treated with cyclophosphamide/TBI: postcyclophosphamide median ATase nadir was 35% (range 12% to 78%) of the pretreatment levels. No ATase depletion was seen when cyclophosphamide (up to 10 mM) was incubated for 2 h with pure recombinant human ATase in vitro whereas ATase activity was reduced by 90% on preincubation with 100 microns acrolein or with greater than 1 mM phosphoramide mustard. This suggests that a cyclophosphamide-induced decrease in ATase levels in human peripheral lymphocytes in vivo may be due to depletion mediated by the production of intracellular acrolein. Since ATase appears to be a principal mechanism in cellular resistance to the cytotoxic effects of BCNU and related alkylating agents, these observations suggest that a cyclophosphamide-induced reduction in ATase activity may be an additional factor in the effectiveness of the combined sequential therapy.

In vivo depletion of O6-alkylguanine-DNA-alkyltransferase in lymphocytes and melanoma of patients treated with CB 10-277, a new DTIC analogue

There is increasing evidence to suggest that alkylation of guanine residues in DNA at the O6 position is the critical cytotoxic event following treatment with dacarbazine (DTIC) and related drugs and that endogenous O6-alkylguanine-DNA alkyltransferase (ATase) gene expression may be a major factor in resistance to such agents. 1-p-Carboxyl-3,3-dimethylphenyltriazene (CB10-277) was recently selected for clinical evaluation as a DTIC analogue with improved solubility, stability and (possibly) metabolic activation. Serial ATase levels were measured in peripheral blood lymphocytes of nine patients and in biopsied melanoma samples of two patients undergoing treatment with 24-h continuous infusion of CB10-277 (12 g/m2). Wide individual variations in pre-treatment levels as well as in the post-treatment depletion of lymphocyte ATase were seen. Progressive depletion of lymphocyte ATase was seen during continuous infusion of CB10-277 in all patients. Complete suppression of lymphocyte ATase activity occurred in two patients whose pre-treatment ATase levels were low. Immediately following completion of the CB10-277 infusion, the median ATase activity was 17% of pre-treatment levels (range, 0-67%). At 24 h after the end of the infusion, no recovery of lymphocyte ATase activity was observed in six patients, but significant recovery to 50%, 100% and 102% of pre-treatment activity occurred in the other three. In three patients who returned for subsequent cycles of chemotherapy at 4 weeks after the first dose, pre-treatment ATase levels showed a 3- to 4-fold increase relative to the original pre-treatment values. A significant correlation was found between the extent of ATase depletion and the initial lymphocyte ATase levels (r = 0.725, P < 0.05). Haematological toxicity developed in two patients and was associated with low pre-treatment ATase activity. Depletion of tumour ATase activity was noted in these patients, with residual activity amounting to 8% and 11% of pre-treatment levels, respectively, in the biopsies melanoma tissues. These results indicate extensive metabolism of CB10-277 to a methylating agent capable of mediating alkylation of DNA and subsequent depletion of lymphocyte and tumour ATase levels and further indicate that the effects on lymphocytes may reflect effects on the target tumour.