Enhancement of fluorinated pyrimidine-induced cytotoxicity by leucovorin in human colorectal carcinoma cell lines.

Reduced folates have been shown to increase the cytotoxicity of 5-fluorouracil (5-FU) by stabilizing the 5-fluoro-2'-deoxyuridine-5'-monophosphate-thymidylate synthase complex, thus increasing the block in the DNA synthetic pathway. Using an in vitro colorimetric [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cytotoxicity assay, we tested the effects of 5-FU and 5-fluoro-2'-deoxyuridine (FdUrd) with and without leucovorin (LV) on a panel of 11 human colorectal carcinoma cell lines. The effect of LV on 5-FU and FdUrd was quantitatively similar. A clinically achievable level of LV (20 microM) increased the cytotoxicity in all three replicate experiments in 10 of the 11 cell lines (P less than .05, binomial test). LV alone at a concentration of 20 microM had no effect on cell survival. In three cell lines, 50% inhibition of growth occurred at a clinically achievable area under the curve of 5-FU alone. With the addition of LV, one additional cell line showed 50% growth inhibition at a clinically achievable level of 5-FU. Hence large clinical trials may be necessary to detect a significant improvement in survival as a result of adding LV to the fluorinated pyrimidines.

Analysis of gene expression in regenerating rat liver by hybridization of nuclear and cytoplasmic RNA with DNA.

To determine whether massive gene activation occurs in rat liver following partial hepatectomy, DNA-RNA hybridization-saturation and RNA depletion experiments were performed. RNA was extracted from whole cells, nuclei, post-mitochondrial extracts, and polysomes obtained from livers of normal, sham-operated, and partially hepatectomized rats. The purified RNA was labeled with [3H]dimethyl sulfate in vitro and hybridized with nuclera DNA under conditions in which only repetitive sequence transcripts form hybrids with DNA. For comparative purposes, experiments were also performed with nuclear RNA labeled with [32P3phosphoric acid in vivo. The following observations were made: (a) for whole-cell RNA the saturation levels obtained in the hybrization reaction are the same regardless of the source of RNA USED (NORMAL, SHAM-OPERATED, OR PARTIALLY HEPATECTOMIZED RATS); (B) NO DIFFERENCES IN THE SATURATION LEVELS WERE FOUND WHEN LIVER NUCLEAR RNA from these three groups of animals were used; (c) the concentration of nuclear RNA from 6-hr regenerating liver necessary to saturate the DNA is slightly higher than that of nuclear RNA obtained from normal rat liver; (d) cytoplasmic RNA from 6-hr regenerating liver saturates the DNA at a much lower concentration than that required for RNA from normal or sham-operated rats. Our results suggest that for repetitive sequence transcripts, massive "derepression" of the genome does not occur at the early stages of liver regeneration. The alterations detected reflect primarily changes in RNA concentrations rather than qualitative alterations in gene expression. Increased transport of repetitive sequence transcripts from nucleus to cytoplasm appears to take place in regenerating liver.

Effects of leucovorin on idoxuridine cytotoxicity and DNA incorporation.

Leucovorin (LV) increased the growth inhibition produced by iododeoxyuridine (IdUrd), a halogenated analogue of thymidine, in a murine tumor cell line (L1210) and three human tumor cell lines (HL-60, HT-29, and MCF-7). This increased growth inhibition was associated with increased incorporation of IdUrd into DNA. Consistent with previous reports, IdUrd (as iododeoxyuridine monophosphate) inhibited thymidylate synthase (TS) and was dehalogenated intracellularly by TS to generate thymidine nucleotides. In all four cell lines, LV decreased the dehalogenation of IdUrd, producing a 3-fold increase in the labeled iododeoxyuridine triphosphate/dTTP ratios in cytoplasm and labeled IdUrd/thymidine in DNA derived from tritiated IdUrd. In intact L1210 cells, apparent TS activity was inhibited 50% by 3 microM IdUrd alone and 75% by the combination of 3 microM IdUrd and 20 microM LV. Apparent TS activity was unchanged with 20 microM LV alone. In all cell lines except HL-60, the ratios of labeled iododeoxyuridine triphosphate/dTTP derived from tritiated IdUrd were 3-fold lower than the labeled IdUrd/thymidine ratios in DNA. This observation suggests that replicative DNA polymerases preferentially incorporate iododeoxyuridine triphosphate into DNA compared to the endogenous substrate dTTP. This preferential incorporation was independent of the effect of LV. These novel findings suggested that a potential mechanism for the effects of LV on IdUrd was increased inhibition of TS analogous to the interaction between fluoropyrimidines and LV. Enzyme inhibition studies using L1210 cell extracts showed that iododeoxyuridine monophosphate was a weak inhibitor of TS (Ki greater than 10 microM) when compared to 5-fluorodeoxyuridine monophosphate (Ki less than 10 nM). Despite the major differences in potency of these two halogenated pyrimidines, LV appears to modulate the activity of IdUrd as well as 5-fluorodeoxyuridine. LV may provide a clinically useful approach to improve the radiosensitizing and/or cytotoxic properties of IdUrd.

Pharmacokinetics of the hypoxic radiosensitizers misonidazole and demethylmisonidazole after intraperitoneal administration in humans.

The hypoxic radiosensitizers misonidazole or demethylmisonidazole were administered i.p. in a 2-liter volume to 6 patients affected by advanced ovarian carcinoma, and the pharmacokinetic course of the two drugs was studied. The clearance of misonidazole and demethylmisonidazole from the peritoneal fluid was 19.1 and 12.4 ml/min, respectively. At 3 hr after drug administration, both radiosensitizers had peritoneal fluid concentrations more than 8 times larger than in the plasma. The concentration x time exposure in the peritoneal fluid was 3.2 times larger than in plasma for misonidazole and 7.6 times for demethylmisonidazole. The advantage of i.p. delivery compared with systemic delivery decreases with distance from the peritoneal surface, but the advantage may be maintained for up to 1 mm or 100 cell layers. These differences between the two routes of administration provide a rational basis for the expectation that a substantial increase of the therapeutic benefits of misonidazole and demethylmisonidazole in potentiating radiation therapy or chemotherapy can be expected in treating tumors confined to the i.p. space.

Plasma pharmacokinetics of adriamycin and adriamycinol: implications for the design of in vitro experiments and treatment protocols.

The plasma pharmacokinetics of Adriamycin and adriamycinol following a 15-min infusion of 75 mg/sq m of Adriamycin were studied in ten patients previously untreated with Adriamycin. The disappearance kinetics of Adriamycin could adequately be described by a biexponential equation with an initial half-life of 8-min and a terminal half-life of 30 hr. The major drug exposure (area under the concentration-time curve) occurs during the terminal phase where drug concentrations are generally less than 10(-7) M (0.05 micrograms/ml). An improvement in the high-performance liquid chromatography sensitivity facilitated the determination of the terminal phase. The plasma kinetics of adriamycinol, the major and only known active metabolite of Adriamycin, show a rapid initial increase in plasma concentration followed by a slow decline which parallels that of Adriamycin during the terminal phase. The relative drug exposure of adriamycinol to Adriamycin was approximately 50%. The relationship between the measured plasma drug levels and free drug available for distribution into tissues was studied by comparing the plasma binding characteristics of Adriamycin and adriamycinol. A constant 20 to 25% of the total plasma concentrations of both Adriamycin and adriamycinol was freely diffusible over the whole range of observed concentrations, 20 nM to 2 microM. Thus, the free drug exposure (area under the concentration-time curve) of tumor and host tissues in vivo can be determined from these plasma measurements, since the free drug exposures in plasma and in extracellular fluid are equivalent. These results can also serve as a guide for the design of clinically relevant in vitro studies of Adriamycin and adriamycinol. The pharmacokinetic parameters determined in this study have been used to simulate plasma concentration-time courses for a variety of Adriamycin treatment schedules. Alternatives are suggested which reduce peak plasma Adriamycin concentration while antitumor area under the concentration-time curve is maintained.

Phase I and clinical pharmacology study of intravenous flavone acetic acid (NSC 347512).

We have conducted a Phase I and pharmacological study of flavone acetic acid, one of a series of novel flavonoids. The drug was administered i.v. weekly for 4 weeks, with a 2-week rest and then repeated. Flavone acetic acid was given initially in a 1-h infusion, but at the 3900-mg/m2 dose level, the infusion time was lengthened to 3 h. A total of 31 patients were treated with 9 different dose levels, ranging from 330 to 6400 mg/m2. Dose-limiting toxicity was acute hypotension that began after about one-third of each drug dose had been infused and rarely lasted more than a few minutes after the infusion was discontinued. In addition, subjective fatigue and asthenia causing unacceptable patient discomfort was dose limiting. A significant side effect noted that was not dose limiting was diarrhea during the infusion. This drug exhibited nonlinear pharmacokinetic behavior. Plasma levels exceeded 300 micrograms/ml during the infusion at the maximally tolerated dose. After the infusion ended the principal half-life was about 2 h. In 24-h urine collections 27% of the flavone acetic acid dose was recovered as intact drug and an additional 37% was recovered as a metabolite. The maximally tolerated dose determined in this study is 6400 mg/m2 given i.v. over 3 h.

Carbamazepine and its -10,11-epoxide metabolite in plasma and CSF. Relationship to antidepressant response.

Levels of carbamazepine and its -10,11-epoxide metabolite were measured in plasma and CSF of affectively ill patients treated only with carbamazepine for an average of 33 days at an average dosage of 1,055 mg/day. The CSF levels of carbamazepine were 2.06 micrograms/mL (ie, 31% of plasma levels, which equaled 6.55 micrograms/mL); CSF -10,11-epoxide concentrations averaged 0.91 micrograms/mL in 18 subjects (63% of those found in plasma). Carbamazepine levels in plasma or CSF were not related to degree of antidepressant or antimanic response. In contrast, concentrations of the -10,11-epoxide metabolite were correlated with the degree of antidepressant response. This preliminary study suggests the possibility that the -10,11-epoxide metabolite of carbamazepine may be related to the degree of clinical efficacy in affectively ill patients and may thus possess active psychotropic properties in man in addition to its reported anticonvulsant effects in animals.

Enzymatic defense against radiation damage in mice. Effect of selenium and vitamin E depletion.

Radiation effects are mediated in part by the generation of oxygen-derived free radicals and hydrogen peroxide. Membrane polyunsaturated fatty acids are important biological targets of these toxic molecules which cause lipid peroxidation. Radiation damage to DNA is also known to result in base hydroperoxides, especially thymidine hydroperoxide. Glutathione (GSH) is known to inhibit lipid peroxidation both chemically and through its interaction with the selenium-dependent glutathione peroxidase (GSH-Px). Although cytosolic GSH-Px can metabolize organic lipid peroxides in solution, it cannot metabolize phospholipid peroxides in micelles. This may be due to the interference of phase differences between the aqueous cytosol and the membrane, or the result of steric hindrance. Recent studies have suggested the presence of a membrane-bound GSH-dependent peroxidase system. We examined the cytosolic versus membrane-associated GSH-Px, in various tissues of mice on a selenium and vitamin E deficient diet, and found significant differences among organs in the distribution of enzyme activity in these two subcellular fractions. The effect of single high-dose whole body irradiation did not appear to be related to the activity of these enzymes.

Mechanism of hydrolysis of halogenated nitrosoureas.

The hydrolysis kinetics of halogenated nitrosoureas were investigated using chlorozotocin as a model. Evidence is presented to show that the hydrolytic reaction of halogenated nitrosoureas between pH 3 and 8 is a summation of spontaneous water and hydroxide-ion-catalyzed reactions; the later reaction is the sum of two parallel reactions. The relative contribuiton of each reaction changes with pH and results in different product distributions. Dianionic phosphate (HPO42-) increased the amount of free chloride-ion production without significantly altering the hydrolysis rate. A mechanism is proposed to explain this behavior. The role of hydrolytic decomposition products produced at physiological pH on the biological activity of nitrosoureas is discussed.