Phase I pharmacokinetic study of intraperitoneal etoposide.

The synergistic interaction of etoposide with cisplatin in certain tumors prompted an evaluation of its potential role in the i.p. treatment of ovarian cancer and other intraabdominal malignancies. We conducted a Phase I evaluation of etoposide as a single agent to determine the maximum tolerated dose i.p., to describe dose-limiting and other toxic effects, and to examine the pharmacokinetics of etoposide in this setting. Etoposide was diluted in 2 liters of normal saline, and instilled i.p. over 10 to 25 min following maximal drainage of ascites. The dwelling time was 4 h, followed by peritoneal drainage. Twenty-two patients received 56 courses at doses which ranged from 100 to 800 mg/m2. The median age was 49, the median performance status was 1, and 18 patients had received prior chemotherapy, with or without radiation. The principal acute toxicity was abdominal pain in 10 patients; this was usually accompanied by signs of peritoneal irritation and was always responsive to nonsteroidal antiinflammatory medications. The major toxicity was dose-related neutropenia; Grade 3 or 4 toxicity affected five of six patients at 800 mg/m2. Thrombocytopenia, nausea and vomiting, and alopecia were also observed. The recommended dose for further study is 700 mg/m2. The pharmacokinetics of etoposide in plasma and peritoneal fluid was measured in 19 patients. Peritoneal levels over the 4-h dwelling time declined monoexponentially with a harmonic mean half-life of 3.5 h (range, 1.9 to 7.8). Plasma levels rose to a peak at 2.9 +/- 1.7 (SD) h and then declined exponentially with a harmonic mean terminal half-life of 7.7 h (range, 4.2 to 15.6). The plasma area under the concentration-time curve increased linearly with respect to dose. The relative pharmacological advantage (ratio of peritoneal to plasma area under concentration-time curve) for i.p. administration was measured as 2.8 and was independent of dose. Based on the high plasma protein binding of etoposide (94%) and the minimal protein binding in the fluid instilled i.p., the ratio of the areas under the concentration-time curves of free drug is estimated to be 4%. These results illustrate that tumor confined to the peritoneal cavity would be exposed to substantially higher free (diffusible) drug concentrations following i.p. than following i.v. administration and support the further evaluation of etoposide by this route.

Direct in vitro photoaffinity labeling of DNA with daunorubicin, adriamycin, and rubidazone.

Irradiation by daylight fluorescent lamps, of a physical complex of duplex DNA with either daunorubicin, adriamycin, or rubidazone produced covalent adducts to DNA. The photoincorporated drug could not be extracted by phenol extraction which removed 99% of the non-photolyzed drug from its physical complex with DNA. The photoadduct was also stable to dialysis, Mg2+ addition, column chromatography, and thermal denaturation and alkali treatment of the DNA. The photoinduced adduct was proportional to the amount of incident irradiation, and the amount of DNA present, and as much as 30--45% of the drug which was physically associated could be photoincorporated. The drugs were not incorporated extensively into single-stranded DNA which lacks the ability to bind these antitumor agents by intercalation. Although the photochemical mechanisms of photoaffinity labeling DNA with these antineoplastic agents are unknown, this approach may prove to be useful for trageting their cellular sites of actions.

Modification of the carcinogenic process in colorectal cancer by endogenous and exogenous factors: effect of colestipol hydrochloride on tumors induced by dimethylhydrazine.

The effect of the bile acid sequestrant, colestipol hydrochloride, on the incidence of dimethylhydrazine-induced tumors of the large intestine was determined in male Swiss mice. The subcutaneous administration of dimethylhydrazine (15 mg/kg) produced tumors in approximately 89% of the animals with an average of 1.70 tumors per tumor-bearing animal. When carcinogen-treated animals received dietary colestipol (0.52%, w/w) from 4 weeks prior to the first injection of dimethylhydrazine until the time of death, there was an increase in the number of tumors per tumor-bearing animal to 2.23. In an attempt to understand the nature of this enhancement, animals were administered dietary colestipol at different times in relation to the administration of the carcinogen. The number of tumors per tumor-bearing animal for the different protocols was: post-initiation colestipol exposure, 1.70; colestipol exposure concomitant with dimethylhydrazine, 1.41; pre-initiation colestipol exposure, 2.23. Thus, colestipol appeared to function both as an anticarcinogen and as a promoter (pre-initiation). Since colestipol has the capacity to bind a number of chemical agents, the different biological effects probably reflect the multifactorial nature of colorectal cancer with the end result dependent on the balance between opposing factors. The selective administration of colestipol in relation to carcinogen administration may prove useful in elucidating the various factors involved in the etiology of this disease.

Amelioration of doxorubicin-induced skin necrosis in mice by butylated hydroxytoluene.

The effect of butylated hydroxytoluene (BHT) on doxorubicin (Adriamycin)-induced skin ulcers was investigated in mice. The skin lesions produced by a single intradermal (ID) injection of doxorubicin (0.05 mg; 1 mg/ml) reached maximum size between 5 and 10 days after injection of ADR. Different concentrations of BHT were administered by different routes and at different times in relation to the injection of doxorubicin. The most effective dose of BHT was 4 mg/animal. The topical application of BHT immediately following doxorubicin injection reduced the area of the ulcer by 57%; the immediate ID injection of BHT reduced the size of the ulcer by 84%. Additional studies are required to determine whether BHT will be a clinically useful modifier of the toxicity associated with doxorubicin extravasation in cancer patients.

Effect of hyperthermia and doxorubicin on nucleoid sedimentation and poly (ADP-ribose) polymerase activity in L1210 cells.

We report on the individual and combined effects of doxorubicin (DOX) and hyperthermia (HYP) on nucleoid sedimentation and poly (ADP-ribose) polymerase (PARP) activity of L1210 cells. The effects of HYP and DOX on nucleoid sedimentation (increased sedimentation) were similar and correlated with cell viability. No correlation of PARP activity with cell toxicity was evident; the activity of PARP was inhibited by HYP (42 degrees C; 1-3 h) and stimulated by DOX (1-10 microM; 30 min). The HYP-induced inhibition of PARP was actually ameliorated by simultaneous exposure to DOX. Although separate studies have previously suggested that chromatin alterations or the inhibition of PARP might play a role in the effect of HYP, the correlation of nucleoid changes (rather than PARP activity) with cell viability emphasizes the contribution of the former. Furthermore, the results suggest that the nucleoid technique may prove useful in screening potential treatment modalities.

2,4-Diamino-6-(bis-2-chloroethyl)aminomethyl pteridine. A new potent anticancer drug.

2,4-Diamino-6-(bis-2-chloroethyl)aminomethyl pteridine has been synthesized and found to be highly potent against L-1210 mouse leukemia lymphoblasts. A single dose of 5 mg/kg injected 24 h after the tumor inoculation increased the life-span of 50% of mice to more than 200%, while the other 50% of animals were cured.

Mechanism of butylated hydroxytoluene-associated modification of diethylnitrosamine-induced squamous stomach carcinoma.

The metabolism of butylated hydroxytoluene (BHT) and the effect of BHT on the metabolism of diethylnitrosamine (DEN) was studied in male and female BALB/c mice to further understanding of the selective protection of BHT on the incidence of DEN-induced squamous-stomach carcinomas in female (but not in male) mice. Following intragastric administration of [14C]BHT, the antioxidant was covalently bound to tissue macromolecules. The relative distribution of this bound BHT varied with time; 8 hr after [14C]BHT administration, most of the covalently bound BHT was associated with the protein components; at 96 hr the nucleic acid components bound more BHT than did the protein components. Animals pretreated with BHT and given [14C]DEN intragastrically had lower blood levels of radioactivity and eliminated a larger percentage of DEN and/or its metabolites in the urine and as carbon dioxide than animals given [14C]DEN alone. The binding of DEN and/or its metabolites to cellular macromolecules of the squamous stomach of female animals was decreased following pretreatment with BHT. However, the BHT-associated decrease in DEN binding was also observed in the squamous stomach of male animals and in the liver of both sexes, although the tumour incidence in these target organs for DEN carcinogenesis is not modified by BHT. These results suggest that the BHT-associated decrease in the binding of DEN to DNA is of a generalized rather than a selective nature, and may be insufficient to account for the protective effect of BHT. Two parameters that were found to parallel the susceptibility of DEN target tissues to the anticarcinogenic effects of BHT were the relative degree of inhibition of DEN bound to RNA species and the relative amount of BHT bound to DNA. Thus the anticarcinogenic properties of BHT may be more complex than an induction of enzymes that detoxify the carcinogen and/or an inhibition of enzymes that activate the carcinogen with a resulting decrease in the quantity of carcinogen available for electrophilic reactions.

Studies on nitrosamine metabolism. III. Comparison of the subcellular distribution of radioactivity in tissues of RFM and BALB/c mice following administration of [14C]diethylnitrosamine.

The tissue distribution of radioactivity in male RFM and BALB/c mice was investigated following a single dose of [14C]-labeled diethylnitrosamine (DEN). The radiolabel was associated with all the subcellular fractions, and the majority of the radioactivity was located in the cytosol fraction. The majority of the radiolabel was usually eliminated from the fractions within 16-24 hrs after [14C]DEN administration.

Association of nitrosamine-derived radioactivity with nuclear and mitochondrial DNA in mice.

In order to better understand the carcinogenic process, a comparative study was undertaken of the association of nitrosamine-derived radioactivity with nuclear and mitochondrial DNA isolated from tumor-susceptible and non-tumor-susceptible tissues of two strains of mice with different susceptibilities to tumor induction by dimethylnitrosamine (DMN) or diethylnitrosamine (DEN). A single dose of either [14C]-DMN or [14C]DEN was administered by intragastric intubation to young adult male RFM or BALB/c mice. The DNA isolated from the mitochondrial fraction of tumor-susceptible tissues bound several times (10 to 90) more nitrosamine than the nuclear DNA. These results suggest that mitochondrial DNA may be a target in the carcinogenic process induced by nitrosamine compounds.

Effect of monocyclic derivatives on DNA repair in human lymphocytes.

Several monocyclic compounds, widely used industrially, were observed to inhibit semiconservative DNA synthesis and DNA repair synthesis in human peripheral lymphocytes. The compounds were: anisole, rho-cresol, butylated hydroxytoluene, butylated hydroxyanisole, 2-tert-butyl-4-methylphenol,2,6-di-tert-butyl-4-nitrosophenol and rho-methoxyphenol.

Effect of adriamycin and hyperthermia on the sedimentation of nucleoids from L1210 cells.

We report on the individual and combined effects of adriamycin (ADR) and hyperthermia (HYP) on the sedimentation behavior of L1210 mouse leukemia cell nucleoids in neutral sucrose gradients. Nucleoid sedimentation profiles obtained from cells incubated with ADR (1-10 microM; 30 min; 37 degrees C) exhibited an increased sedimentation rate associated with an increased protein content of these subnuclear units. Exposure of cells to HYP (1-3 h; 42 degrees C) produced similar results. Simultaneous exposure of L1210 cells to conditions of HYP and ADR which resulted in minimal changes in nucleoid sedimentation when used singly, produced an enhanced effect. A similar enhancement was observed with other intercalating antineoplastic agents believed to exert their effect, at least partially, via free radicals (daunorubicin, amsacrine, bisantrene, mitoxantrone). However, enhancement with HYP was not observed with (a) the classic intercalating agent, ethidium bromide; (b) non-intercalating DNA-breaking agents (bleomycin, lithocholic acid, etoposide); (c) inhibitors of poly (ADP-ribose) polymerase (m-methoxybenzamide, benzamide); or (d) non-intercalating antineoplastic agents capable of causing free radical formation (bleomycin). The results suggest that DNA intercalating agents capable of initiating free radical processes may show an enhanced toxicity with simultaneous HYP treatment, and that the nucleoid assay may be a means of screening agents with these biological properties for potential clinical usefulness in combination with HYP.

Purification and properties of phosphoglucomutase from Fleischmann's yeast.

1. A procedure has been described for the purification of the major isozyme of yeast phosphoglucomutase of highest known specific activity. 2. The native enzyme has a molecular weight of about 65400 and was found to be homogeneous as judged by sucrose density gradient centrifugation, gel filtration, electrophoresis on acrylamide gel and ultracentrifugal analysis. In the presence of denaturing agents such as guanidine hydrochloride or sodium dodecyl sulfate, the enzyme dissociated into 32000-molecular-weight subunits. 3. As isolated, the enzyme has one mole of phosphate bound per mole of enzyme. Preparations incubated with 1.0 mM EDTA in 10 mM citrate buffer, pH 5.5 and dialysed against 10 mM metal-free citrate buffer, pH 5.5, contain no intrinsically bound Zn2+ and were enzymically inactive but fully active in the presence of 5 mM Mg2+ and 84% as active with 0.5 mM Zn2+. Simultaneous presence of both ions at these concentrations did not enhance activity. Enzyme was completely and irreversibly inactivated by preincubation with Be2+. Inactive enzyme had one mole of Be2+ bound per mole of enzyme. 4. Enzyme exhibited "ping-pong" kinetics rather than "random sequential". Km values for glucose 1-phosphate and for glucose 1,6-bisphosphate were calculated to be 2.34 times 10(-5) M and 2.24 times 10(-6) M, respectively. Rate of enzyme phosphate turnover was studied with rapid-mixing technique. The rates of 32P release from 32P-labeled enzyme and its appearance as glucose 6-[32P]phosphate were comparable and remained unaffected by addition of glucose 1,6-bisphosphate.

Resolution of malignant small bowel obstruction after intraperitoneal etoposide therapy.

In the case we have reported, abdominal carcinomatosis with malignant small bowel obstruction (due to metastatic lung adenocarcinoma) developed despite ten cycles of cisplatinvinblastine and eight months of treatment with interferon and interleukin-2. Etoposide (VP-16) administered intraperitoneally as part of a phase I trial produced dramatic relief of the obstruction.

Protective effect of butylated hydroxytoluene on adriamycin-induced skin necrosis in the rat.

The effect of butylated hydroxytoluene (BHT) on adriamycin (ADR)-induced skin ulcers was investigated using rats. The application of BHT reduced the size of the skin ulcers by the following amounts: topical application (20%); intradermal injection (50%); repeated applications of BHT (82%). Additional studies are required to determine if BHT will be a modifier of ADR extravasation in cancer patients.

Development of lung metastases after curative intraperitoneal chemotherapy in a rat colon cancer model.

A model of colon peritoneal carcinomatosis was developed by injecting 5 x 10(7) viable tumor cells intraperitoneally into Fisher 344 rats. All 40 control rats developed bulky abdominal tumor with ascites and died of peritoneal carcinomatosis and bowel obstruction (median survival 5 weeks). One day after tumor implantation, treatment group rats received a single intraperitoneal injection of single agent or combination chemotherapy. The most active intraperitoneal single agents were 5-fluorouracil, cisplatin, and etoposide. The most active combination was 5-fluorouracil and cisplatin. Combination chemotherapy produced a significant increase in median, 10-week, and 20-week survival (vs control and single agent). Six of 11 (55%) rats treated with intraperitoneal combination chemotherapy dying between 10-20 weeks died of lung metastasis with cure of intraperitoneal tumor. The increased ability of intraperitoneal combination chemotherapy to cure intraperitoneal disease was offset by the development of lung metastasis.

Radioactive components in the acid-soluble fraction of mouse liver cytosol after dimethylnitrosamine[methyl-14C] administration.

The acid-soluble components of mouse-liver cytosol, prepared at time intervals after intragastric administration of a single carcinogenic dose of 14C-dimethylnitrosamine, were separated by column chromatography. The columns were calibrated with known in vitro metabolites of the nitrosamine, and the elution profiles were compared with those of the mouse-liver system. The results suggest that the 14C-methyl label is transferred to many of the same compounds as identified in vitro, but that numerous other labeled compounds are also present. The possible significance of these metabolites to the pathogenic processes induced by dimethylnitrosamine has yet to be determined.

Resistance to adriamycin cytotoxicity among respiratory-deficient mutants in yeast.

Saccharomyces cell uptake of Adriamycin and the ensuing cytotoxic response were found to be dependent upon the ionic strength of the medium used for drug treatment. A given concentration of Adriamycin which inhibited growth in complete medium ws found to be significantly cytotoxic when administered in water. Many survivors after Adriamycin treatment in water were found to be respiratory-deficient petite mutants containing mitochondrial deoxyribonucleic acid mutations. Petite mutants arising after Adriamycin treatment were not induced but selected from the preexisting population of spontaneously derived petite mutants (normal frequency, 2%) due to an increased resistance of these mutants to killing by Adriamycin as compared with normal respiratory-sufficient cells. The responses to Adriamycin in mitochondrial deoxyribonucleic acid respiratory-deficient mutants (rho-, rho degrees, mit-) with different impaired mitochondrial functions was studied. All were similarly more resistant to killing by Adriamycin than wild-type cells. The common deficiency shared by these mutants, i.e., nonfunctioning electron transport, may play a role in protecting these mutants from Adriamycin cytotoxicity. In addition, normal cells grown on glycerol, requiring aerobic respiration for carbon source utilization were more susceptible to killing by Adriamycin than cells grown on glucose. These studies suggest that a mitochndrial function in yeast may interact with Adriamycin to potentiate a cell cytotoxic mechanism of the drug.