Franz A. Schmid

Further evaluation of bimolane and analogs as potential antitumor agents

Bimolane has been shown to have good antitumor activity and on an equitoxic basis its activity is usually better than the chemically related razoxane. Resistance patterns of these two piperazinediones were similar. They exhibited cross-resistance to each other but little or no cross-resistance to most other clinically used drugs tested. Partial resistance was observed, however, between the piperazinediones and vincristine, daunorubicin and doxorubicin. The antitumor activities of three new analogs were compared with bimolane, ICRF-154 and razoxane against four rodent tumors. In general, bimolane was most effective.

[13N]Ammonia and l-[amide-13N]glutamine metabolism in glutaminase-sensitive and glutaminase-resistant murine tumors

The short-term metabolic fate of labeled nitrogen derived from [13N]ammonia or from L-[amide-13N]glutamine was determined in murine tumors known to be resistant (Ridgeway Osteogenic Sarcoma (ROS] or sensitive (Sarcoma-180 (S-180)) to glutaminase therapy. At 5 min after intraperitoneal injection of [13N]ammonia or of L-[amide-13N]glutamine, only about 0.7% of the label recovered in both tumors was in protein and nucleic acid. After [13N]ammonia administration, most of the label (over 80%) was in a metabolized form; a large portion of this metabolized label (50-57%) was in the urea fraction with a smaller amount in glutamine (37-42%). The major short-term fate of label derived from L-[amide-13N]glutamine was incorporation into components of the urea cycle with smaller amounts in the acidic metabolites and in acidic amino acids. No labeled urea was found during in vitro studies in which S-180 tumor slices were incubated with [13N]ammonia, suggesting that the [13N]urea formed in the tumor in the in vivo experiments was not due to de novo synthesis through carbamyl phosphate in the tumor. Both tumors exhibited very low glutamine synthetase activity. Following glutaminase treatment, glutamine synthetase and gamma-glutamyltransferase activities, while remaining low, increased in the resistant tumor but not in the sensitive tumor; this increase may be related to the insensitivity of the ROS tumor toward glutaminase treatment.

L1210 Leukemia hybrids isolated after fusion of alkylating agent-resistant sublines

The two resistant lines, L1210/CPA (cyclophosphamide) and L1210/MeCCNU (1-(2-chloroethyl)-3-(trans-4-methyl-cyclohexyl)-1-nitrosourea) were used, each of which is not cross-resistant to the drug to which the other line is resistant. Their resistance was used as markers as well as the basis for selection of the hybrids. For the production of hybrids five in-vivo or in-vitro schedules were employed. The in-vitro methods produced six successful hybrid lines, but the in-vivo schedules produced none. Resistance to both CPA and MeCCNU was expressed dominantly in the hybrids. The hybrids had chromosome modes ranging from 68 to 78. This study shows that CPA and MeCCNU can be used both as markers and as selective agents, and that CPA and MeCCNU resistance in L1210 leukemia are dominantly expressed in the hybrid.

OCCURRENCE OF DEAMINATED MONOPHOSPHATE NUCLEOTIDE IN EXTRACELLULAR SITES AFTER GIVING ARABINOFURANOSYLCYTOSINE OR DEOXYCYTIDINE TO MICE

Contrary to the widely held belief that cells are impermeable to nucleotides. Ortiz and coworkers have provided evidence that certain nucleotides can penetrate mammalian cells. They have shown that arabinofuranosylcytosine-5-monophosphate (ara-CMP) is about one quarter as effective as ara-C in inhibiting multiplication or in killing mouse fibroblast cells. They also demonstrated that D-arabonofuranosyladenine-5-monophosphate (ara-AMP) is a considerably more potent inhibitor of cell multiplication than is the parent nucleoside. Cohen and Plunkett further demonstrated that 3H,32P-ara-AMP enters the cell intact during a four-hour incubation at 5% of the initial rate of penetration of ara-A. In recent studies of the metabolism of arabinofuranosylcytosine (ara-C) and of deoxycytidine (CdR) in vioo, we have obtained evidence that certain nucleotides may be able to escape from cells and circulate widely. Tritium-labeled ara-C or CdR was injected subcutaneously in BD2Fl mice or in similar mice five days after intraperitoneal inoculation of lo6 L1210 leukemic cells. One hour later the animals were killed. The acid-soluble radioactivity in different tissues was fractionated with Ag 1 x 8 anion exchange columns into nucleoside and mono-, di-, and triphosphate nucleotide fractions as described in TABLE I . Normal (N) or leukemic (L) mice at five days after transplantation of L1210 received S.C. injections of ara-C-3H, 8.97 nmole, 0.1

Cryofragility and other membrane characteristics of solid mouse tumors

i/g and were killed one hour later. Tissues were homogenized in cold 10% PCA. PCA was removed as KC104 after addition of 2N KOH. The supernatant was applied to a chromaflex column containing 0.7 x 4 cm of Ag 1 x 8 anionic exchange resin (200-400 mesh, in chloride form, purchased from Bio-Rad Labs). The nucleoside and mono-, di-, and triphosphate nucleotides fractions were eluted with 10 ml of water, 0.15 M, 0.3 M, and 1 M N H 4 H C 0 3 , respectively. An aliquot of each extract was used for determination of radioactivity in Packard Tri-Carb liquid scintillation spectrophotometer. Different fractions from certain of the tissues were selected for determination of their content of ara-C and ara-U. These fractions were evaporated at 80-90 C under reduced pressure to remove water and to decompose N H 4 H C 0 3 . Residues from nucleoside fractions were dissolved in 0.5 ml water. and aliquots were used for descending paper chromatography (Whatman no. 3) in isopropanol: conc. HCI: HzO. 68 : 17 : 14.4

Antitumor Activity of Cytoxan

Abstract The membrane fragility of Ridgway osteogenic sarcoma (ROS) was compared with that of other solid tumors in an attempt to explain its sensitivity to chemotherapeutic agents. The sensitivity of ROS to freezing, hypotonicity, and to the mechanical insult of homogenization was determined by bioassay. The results were compared with those obtained with an ROS subline resistant to cyclophosphamide (ROS/CPA), with melanoma B16 and the recently induced sarcoma S1976, the latter two being relatively drug-resistant tumors. Representative values of the viability as expressed by percent of tumor takes were: (a) after storage at −78 °C for 24 hr in Earles balanced salt solution without DMSO, for ROS 0%, ROS/CPA 11%, B16 82%, and S1976 100%; (b) after incubation at 37 °C for 2 hr in distilled water, for ROS 0%, ROS/CPA 0%, B16 42%, and S1976 80%; (c) after mechanical dispersion in a tissue grinder, for ROS 17%, ROS/CPA 28%, B16 100%, and S1976 100%. Relevant therapeutic indices for ROS, B16, and S1976 were: for single doses of cyclophosphamide 12, 3.1, 3.5, melphalan 3.5, 1.5, 1.5, medphalan 4.2, 1.5, 1.2 and for 6 daily doses of 5-fluorouracil 1.5,