Potentiation of BCNU antitumor efficacy by 9-substituted O-6-benzylguanines. Effect of metabolism

Purpose: O-6-Benzylguanine (BG), an O-6-methylguanine-DNA methyltransferase (MGMT) inactivator, potentiates the efficacy of 1,3-bis(2-chloroethyl)-1-n itro-sourea (BCNU) and of other DNA chloroethylating and methylating antica ncer drugs and is currently undergoing clinical trials. O(6-)Benzyl-2'-deox yguanosine (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 i n vivo systems we tested the metabolism, ability to inactivate MGMT, and ef ficacy to potentiate BCNU in vivo of two additional 9-substituted derivativ es of BG, namely O-6-benzyl-9-cyanomethylguanine (CMBG) and O-6-benzylguano sine (BGS). Methods: Metabolism and disposition of these drugs was examined in athymic mice and Sprague-Dawley rats. MGMT suppression was determined i n human medulloblastoma (Daoy) tumor xenografts in athymic mice following t reatment with BGS, dBG, and CMBG and was compared with the loss of resistan ce to BCNU as determined by tumor growth delays. Results: Growth delays at 25 mg/m(2) BCNU and 133 mg/m(2) BG or equimolar doses of CMBG, BGS or dBG w ere 23.0, 2.5, 21.3 days, and 30.4 days, respectively. The above difference s did not correlate with the ED(50)s Of 0.2, 13, 11 mu M, and 2 mu M determ ined for the above compounds, respectively, in cell free extracts. Differen ces in the efficacies of the 9-substituted compounds did correlate, however , with the extent of their metabolic conversion to BG. The maximum concentr ations of BG in blood achieved after the administration of equimolar (250 m u mol/kg) doses of CMBG, BGS and dBG were 10, 30 mu M, and 55 mu M, respect ively. Although such levels were lower than those achieved in circulation b y administration of an equimolar amount of BC, BG levels persisted longer f ollowing treatments with BGS or dBG than after treatment with BG itself. Fo rmation of BG was required for continuous and prolonged (>16 h) suppression of MGMT activity to non-detectable levels (< 5 fmol/mg protein). Conclusio n: Metabolism of BGS and dBG to BG explains the unexpected high efficacy of these compounds in potentiating the antitumor activity of BCNU in the athy mic 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 administra tion, which results in faster and complete suppression of MGMT in Daoy xeno grafts. 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 admin istration 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 thi s compound mainly by pathways other than conversion to BG.