Dianna Green

Phase I studies on chlorozotocin.

A phase I investigation of chlorozotocin, a new‐water soluble chloroethylnitrosourea, was undertaken to define its pharmacologic effects in man. Forty‐three patients received single intravenous doses ranging from 5 to 175 mg/m2 every 6 wk. No signs of toxicity were observed at doses of under 120 mg/m2, but thrombocytopenia occurred at higher doses. The thrombocytopenic nadir appeared to be dose‐dependent and occurred 4 wk after treatment. Platelet transfusions were required in 2 patients who had previously received intensive chemotherapy. No significant leukopenia occurred. A mild reversible and delayed elevation of hepatic transaminases was found in 25% of courses of 120 mg/m2 or more. No renal toxicity was observed and gastrointestinal toxicity was mild. Investigation of clinical pharmacology revealed a rapid triphasic plasma clearance with initial t½s of 3, 15, and 30 min. The concentration of N‐nitroso intact drug at 1 hr was 10% of the initial peak level. Renal excretion accounted for half of the dose. No significant concentration of N‐nitroso intact or radiolabeled drug was detected in the cerebrospinal fluid of 2 patients in whom it was examined. There were objective signs of therapeutic activity in 5 patients, 3 of whom had melanoma. Based on these studies, the recommended dose for phase II investigation of chlorozotocin is 120 mg/m2 every 6 wk.

Chlorozotocin. Mechanism of reduced bone marrow toxicity in mice.

Chlorozotocin is a chloroethyl nitrosourea with a glucose carrier that has curative activity for the murine L1210 leukemia, but is nonmyelosuppressive in mice. To determine the mechanism for this unique property of reduced bone marrow toxicity, comparative studies were conducted with chlorozotocin and CCNU, a myelotoxic chloroethyl nitrosourea. Suspensions of L1210 leukemia and murine bone marrow cells were incubated for 2 h with 0.1 mM [(14)C]-chloroethyl chlorozotocin or CCNU. Chlorozotocin demonstrated a fourfold increased covalent binding of the chloroethyl group to L1210 nuclei when compared to equimolar CCNU. Chlorozotocin alkylation of L1210 cells resulted in the binding of 57 pmol of [(14)C]ethyl group/mg of DNA, which represented a 2.3-fold increased alkylation when compared to CCNU. In marked contrast, the binding of the chloroethyl group to bone marrow nuclei was equivalent for both drugs. In addition, chlorozotocin alkylation of murine bone marrow DNA, 45 pmol of [(14)C]ethyl group/mg of DNA, was equivalent to that of CCNU. The ratio of L1210:bone marrow DNA alkylation was 1.3 for chlorozotocin compared to 0.6 for CCNU. The intracellular carbamoylation of L1210 and bone marrow protein by CCNU was 400- to 600-fold greater than that produced by chlorozotocin. After a 2-h exposure to 0.1, 0.05, or 0.01 mM drug, both chlorozotocin and CCNU produced a reduction in the cloning efficiency of L1210 cells that was dose dependent. However, chlorozotocin was significantly more cytotoxic than CCNU at all three molar concentrations (P < 0.01). Chlorozotocin, 0.1 mM, reduced L1210 DNA synthesis to 1% of control by 48 h, in contrast to 16% with equimolar CCNU (P < 0.01). In mice bearing 10(5) L1210 cells, chlorozotocin produced its optimal antitumor activity (332% increased life span [ILS]) at doses of 48-64 mumol/kg, with >50% indefinite survivors. In contrast, CCNU at the same molar doses resulted in only a 191% ILS; a CCNU dose of 128 mumol/kg was required for comparable optimal L1210 antitumor activity, 413% ILS. On a molar basis, the dose of chlorozotocin that produced optimal in vivo L1210 antitumor activity was one-third to one-half that of CCNU. Chlorozotocin, unlike CCNU, produced no murine bone marrow toxicity at its optimal therapeutic dose. This unique combination of antitumor activity without myelosuppression can be correlated with the advantageous ratio of L1210:bone marrow in vitro DNA alkylation by chlorozotocin (1.3) as compared to equimolar CCNU (0.6).

Correlation of nitrosourea murine bone marrow toxicity with deoxyribonucleic acid alkylation and chromatin binding sites

All of the clinically available nitrosourea antitumor agents produce serious treatment-limiting bone marrow toxicity. A reduction in this toxicity can be achieved by attaching the chloroethylnitrosourea cytotoxic group to C2 (chlorozotocin) or C1 (1-(2-chloroethyl)-3-(beta-D-glucopyranosyl)-1-nitrosourea, GANU) of glucose. Both glucose analogs are less myelotoxic in mice than 1-(2-chloroethyl)-3-cyclohepyl-1-nitrosourea (CCNU) or 1-(4-amino-2-methylpyrimidin-5-yl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), while retaining comparable antitumor activity against the murine L1210 leukemia. To define the nuclear mechanisms for this reduced myelotoxicity, alkylation of L1210 and murine bone marrow DNA was quantitated. With the use of the endonuclease micrococcal nuclease and DNase I, the sites of alkylation within the chromatin substructure were determined. Experiments were performed on L1210 leukemia or bone marrow cells that had been incubated in vitro for 2 hr with 0.1 mM [14C]chloroethyl drug. The quantitative alkylation of DNA by GANU was 1.3-fold greater in L1210, as compared to bone marrow, cells. This ratio of DNA alkylation is comparable to the 1.3 ratio we previously reported for chlorozotocin [L. C. Panasci, D. Green and P. S. Schein, J. clin. Invest. 64, 1103 (1979)]. In contrast, the ratio of alkylation (L1210:bone marrow DNA) for the myelotoxic ACNU was 0.66, similar to 0.59 for CCNU. Nuclease digestion experiments demonstrated that chlorozotocin and GANU preferentially alkylated internucleosomal linker regions of bone marrow chromatin, while nucleosome core particles were the preferred targets of CCNU and ACNU. The reduced myelotoxicity of chlorozotocin and GANU may be correlated with the advantageous ratio of L1210:bone marrow DNA alkylation and preferential alkylation of internucleosomal regions of bone marrow chromatin.

Aspartato(1,2-cyclohexanediamine)platinum(II) complexes: synthesis and characterization; effects of minor impurities on antitumor activity

Abstract Aspartato(1,2-cyclohexanediamine)platinum(II) (ADP), where 1,2-cyclohexanediamine (DAC) is either trans-RR-, trans-SS-, meso-RS-, or a mixture of the three isomers (ADP mixture), has been synthesized and evaluated for antitumor activity. The structures of the complexes have been characterized by various spectroscopic techniques (IR, 1H, 13C, 195Pt and 2D-COSY (1H{1H}) and 2D-HETCOSY (1H{13C}) NMR). Purification and murine antitumor activity of the individual ADP isomers indicate that minor impurities in the ADP mixture have a significant effect on the potency of the platinum complexes.

A phenol technique for extraction of alkylated DNA, RNA, and protein from a single tissue sample

Abstract A procedure for extracting DNA, RNA, and protein from an L1210 leukemia cell sample is described. Sodium deoxycholate and 154 m m NaCl in the presence of phenol extracts cytoplasmic RNA. Sodium deoxycholate, p -aminosalicylate, and 1 m NaCl in the presence of phenol extracts the DNA. The DNA is then freed of RNA with RNase T1 and T2. Cytoplasmic RNA with no detectable DNA and less than 0.02% protein contamination was so obtained. The DNA isolated had less than 3% uracil as compared to thymine and no more than 0.1% protein. The extraction procedures are especially useful for investigating nucleotide alterations in nucleic acids by chemical carcinogens and alkylating agents in tissue samples of only 2 × 10 8 cells. The procedure has also been successfully applied to murine bone marrow cells, human bone marrow cells, HeLa cells, and myeloblasts from a patient with acute myelogenous leukemia.

Synthesis and Characterization of a Series of Water Soluble Amidomalonato-(lR,2R-Cyclohexanediamine)Platinum(II) Complexes

Abstract A new series of water soluble platinum(II) complexes of the type [Pt(DACH)[R-CH(COO)2]], wherein DACH represents IR,2R-cyclohexanediamine and R represents formamido. acetamido, (penta-O-acctyl-gluconyl)amino, and gluconylamino have been synthesized. The modes of binding of amidodicarboxylic acid derivatives in these complexes have been determined by various spectroscopic techniques: 1H, 13 C, and 195PtNMR; 2D-COSY{1H-1H} and 2D-HETCOSY{1H-13C} NMR, MS(FAB), IR and conductivity measurements.

Synthesis and Murzne P388 Antitumor Activity of Uridine Mustard: A Preliminary Report

Abstract 5′-[Bis (2—chloroethyl) amino]—5′-deoxy uridine (uri-dine mustard), compound 5, was synthesized and characterized by its 1H, 13 C, and two-dimensional homonuclear shift correlated (COSY) and two—dimensional heteronuclear correlated NMR spectra. In comparative murine studies, uridine mustard was substantially less leukopenic than the equitherapeutic dose of uracil mustard.