Potential antiglioma activity of 9-hydroxy-2-N-methylellipticine as determined by pharmacological and human tumor clonogenic cell studies.

The antiglioma activity of elliptinium (HME) was investigated in a human glioma clonogenic cell assay. Early passage cells of three human glioma cell lines (SF126, SF375, and SF407) were exposed to HME at the clinically achievable dose of 3 microM for 3 h. At this HME concentration, clonogenic cell survival was reduced by more than 3 logs in SF126 and SF375, and by 0.8 logs in SF407. A study of the kinetics of cell kill showed that whereas at moderate (less than or equal to 1.5 microM) HME doses cell kill increased with treatment time up to a maximum at approximately 3 h, cytotoxicity was more dose than time dependent at higher doses. Flash treatment of SF375 cells with 3 microM HME resulted in more than 2 logs clonogenic cell kill. Using high-pressure liquid chromatography, we investigated the in vitro decay kinetics of HME under our in vitro drug treatment conditions and observed a very rapid, protein nondependent 40% drop in HME concentration which was dose dependent and was probably due to HME adsorption on the surface of tissue culture plasticware. Subsequent decay of the drug was very slow, with a decay rate constant of 0.022/h and a half-life of 298 h. In order to determine whether HME crosses the blood-brain barrier, we measured the rat brain capillary permeability coefficient, P, of [3H]HME and [14C]HME. The mean P value of 2.2 X 10(-6) cm/s +/- 16% (SD) suggests that HME crosses the blood-brain barrier (t 1/2 = 46 min) consistent with its molecular size and octanol-water partition coefficient.

Central nervous system toxicity and cerebrospinal fluid pharmacokinetics of intraventricular 3-[(4-amino-2-methyl-5-pyrimidinyl)ethyl]-1-(2-chloroethyl)-1-nitro soureas and other nitrosoureas in beagles.

The central nervous system toxicity and cerebrospinal fluid (CSF) pharmacokinetics of 3-[(4-amino-2-methyl-5-pyrimidinyl)ethyl]-1-(2-chloroethyl)-1- nitrosoureas, a (ACNU) were determined in beagles and compared to those for three other nitrosoureas, 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea, 1,3-bis(2-chloroethyl)-1-nitrosourea, and chlorozotocin. Of the four drugs, ACNU was tolerated best and at doses of 0.2 to 0.8 mg/week for 8 consecutive weeks. We found that the average half-time for CSF elimination of ACNU was 18 min (range, 12 to 38 min). This value exceeded the known rate of ACNU decomposition in aqueous solution (28 to 29 min), implying that the disappearance of ACNU from CSF was due to hydrolytic decomposition and cellular entry and/or transcapillary loss across central nervous system capillaries. The drug exposure integral (C X t) of ACNU in the CSF after a "toxic dose low" of 0.8 mg in the dogs would achieve the equivalent of in vitro cell kills in excess of 3 logs for rat 9L and human glioma 126 cells. As a potential therapeutic agent for meningeal neoplasia, the major limiting factor may be that the CSF elimination of ACNU is rapid compared to its equilibration time from ventricle to spinal- and cerebral convexity-subarachnoid space. Based on these results, we have instituted clinical Phase I trials of intra-CSF ACNU.

Levels of N7-(2-hydroxyethyl)guanine as a molecular dosimeter of drug delivery to human brain tumors.

The level of N7-(2-hydroxyethyl)guanine (N7-HOEtG), one of the DNA alkylation products formed by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) treatment, was measured in human brain tumor samples by high performance liquid chromatography with electrochemical detection. The tumors from 6 recurrent chemotherapy-naive patients with recurrent glioblastoma multiforme were analyzed as controls. The mean level of N7-HOEtG in DNA of these specimens was 0.42 pmol/mg DNA. Samples were also obtained from a patient with a recurrent glioblastoma multiforme after direct intratumoral therapy with BCNU in ethanol (DTI-015). The levels of N7-HOEtG in the samples distal, medial, and adjacent to the site of injection were 0.8, 2.6, and 369.5 pmol/mg DNA, respectively. Comparison of the level of N7-HOEtG detected in the distal sample after injection with BCNU in ethanol with the mean level of the untreated samples indicated that it was not sufficiently different to be ruled out as a chance occurrence. Comparison of the levels of N7-HOEtG in the medial and adjacent brain tumor samples with the mean level of the control samples showed values that were approximately 6- and 879-fold higher. These results demonstrate that intratumoral administration of BCNU in ethanol produces significant levels of DNA alkylation and suggest that DNA adduct measurements provide a unique molecular dosimeter to evaluate delivery of alkylating agents to brain tumors.

5-Azacytidine hydrolysis kinetics measured by high-pressure liquid chromatography and 13C-NMR spectroscopy.

Hydrolysis of 5-azacytidine, an experimental anticancer drug, in aqueous buffers was measured using a high-pressure liquid chromatographic (HPLC) procedure and a 13C-NMR method. The former utilized a 17.5-micron Aminex A-6 strong cation-exchanger column eluted with 0.4 M, pH 4.6 ammonium formate buffer at a flow rate of 0.4 ml/min. The hydrolysis sequence as well as the existence of a labile intermediate, N-formylguanylribosylurea, was unequivocally established using 6-13C-5-azacytidine and NMR spectral techniques. A reversible ring opening step to the N-formylguanylribosylurea with an equilibrium constant of 0.58 +/- 0.03 between pH 5.6 and 8.5, followed by an irreversible formation of guanylribosylurea, was found by HPLC. The data confirm previous assumptions on the hydrolytic kinetics. The pH dependency of hydrolysis was examined, and the hydrolysis profile gave a normal V shape with the most stable pH at 7.0. Rather stable intravenous dosage forms can be formulated.

Induction of lacI mutations in Big Blue Rat-2 cells treated with 1-(2-hydroxyethyl)-1-nitrosourea: a model system for the analysis of mutagenic potential of the hydroxyethyl adducts produced by 1,3-bis (2-chloroethyl)-1-nitrosourea.

We have investigated the genotoxic effects of 1-(2-hydroxyethyl)-1-nitrosourea (HENU). We have chosen this agent because of its demonstrated ability to produce N7-(2-hydroxyethyl) guanine (N7-HOEtG) and O(6)-(2-hydroxyethyl) 2'-deoxyguanosine (O(6)-HOEtdG); two of the DNA alkylation products produced by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU). For these studies, we have used the Big Blue Rat-2 cell line that contains a lambda/lacI shuttle vector. Treatment of these cells with HENU produced a dose dependent increase in the levels of N7-HOEtG and O(6)-HOEtdG as quantified by HPLC with electrochemical detection. Treatment of Big Blue Rat-2 cells with either 0, 1 or 5mM HENU resulted in mutation frequencies of 7.2+/-2.2x10(-5), 45.2+/-2.9x10(-5) and 120.3+/-24.4x10(-5), respectively. Comparison of the mutation frequencies demonstrates that 1 and 5mM HENU treatments have increased the mutation frequency by 6- and 16-fold, respectively. This increase in mutation frequency was statistically significant (P<0.001). Sequence analysis of HENU-induced mutations have revealed primarily G:C-->A:T transitions (52%) and a significant number of A:T-->T:A transversions (16%). We propose that the observed G:C-->A:T transitions are produced by the DNA alkylation product O(6)-HOEtdG. These results suggest that the formation of O(6)-HOEtdG by BCNU treatment contributes to its observed mutagenic properties.

Inhibition of microsomal lipid peroxidation by naphthoquinones: structure-activity relationships and possible mechanisms of action.

Menadione (2-methyl-1,4-naphthoquinone) is a remarkably potent inhibitor of microsomal lipid peroxidation, effective at submicromolar concentrations. Its possible mechanism of action and the relationship between naphthoquinone structure and antioxidant activity were the topics of this investigation. In the microsomal lipid-peroxidizing system dependent on NADPH and ferric pyrophosphate, menadione, at concentrations of 50 microM or higher virtually eliminated the accumulation of malondialdehyde and lipid hydroperoxides. In the NADPH-independent, cumene hydroperoxide-dependent system, menadione was also an effective antioxidant, but only in the presence of reducing equivalents. These and other observations indicate that a reduced form of menadione, either the hydroquinone or semiquinone, is the active antioxidant, and suggest that it may trap hydroperoxy radicals, alkoxy radicals, or other free radicals involved in propagating lipid peroxidation. Moreover, these results show that electron diversion per se cannot account for the antioxidant effects of menadione. A comparison of the antioxidant activities of eight 1,4-naphthoquinones indicated that methyl substitution of C-2, lack of steric hindrance at C-3 or C-5, and (in the case of weak acids) a relatively high pKa are favorable structural features associated with strong antioxidant activity.

Carbon-13 nuclear magnetic resonance spectroscopy. Structure of the anticoagulant warfarin and related compounds in solution.

Carbon-13 nuclear magnetic resonance spectra have been obtained for the coumarin-related compounds warfarin and phenprocoumon. The spectral assignments indicate that warfarin exists as a mixture of cyclic hemiketal diastereomers in dimethyl sulfoxide solution. The sodium salt of warfarin exists as the ringopen form in water solution.