Some biological properties of the impure dichloride salt of tetrakis[p-(dimethylamino)phenyl]ethylene and a pinacolone.

The impure dichloride salt of tetrakis[p-(dimethylamino)phenyl]ethylene and a pinacolone that is a substituted acetophenone show several biological properties, one of which is activity against lymphosarcoma in mice. The involvement, if any, of free radicals in the biological properties of these substances is discussed.

Effect of liver disease on pharmacokinetics and toxicity of 9-beta-D-arabinofuranosyladenine-5-phosphate.

Plasma levels of 9-beta-D-arabinofuranosyladenine-5'-phosphate (ara-AMP) and its metabolites 9-beta-D-arabinofuranosyladenine (ara-A and 9-beta-D-arabinofuranosylhypoxanthine (ara-Hx) were determined by high-performance liquid chromatography in four patients with chronic active hepatitis positive for hepatitis B surface antigen and eight patients with severe herpesvirus infections and normal liver function. Ara-AMP was given intravenously over a 30-min period in doses ranging from 10 to 30 mg of ara-A equivalent/kg per day. The metabolism of ara-AMP did not differ significantly between the two patient groups. Ara-AMP was quickly converted to ara-A, which was rapidly deaminated to ara-Hx. The mean half-lives of ara-AMP, ara-A, and ara-Hx were 0.14 hr, 0.17 hr, and 3.5 hr, respectively. Thus, ara-AMP is rapidly metabolized and does not act as a depot form of ara-A. Patients with chronic active hepatitis demonstrated increased bone marrow sensitivity to ara-AMP and a musculoskeletal pain syndrome not observed in patients treated for herpesvirus infections.

KLN205--a murine lung carcinoma cell line.

KLN205 cells, a cloned cell line established from the Nettesheim lung carcinoma, grow in various synthetic media such as MEM, Fisher's or Roswell Park Memorial Institute Medium (RPMI) with the addition of 5 to 20% fetal bovine serum (FBS), calf-serum (CS) or horse serum (HS). They grow optimally in minimum Eagle's medium plus nonessential amino acids (NEAA) plus 5 to 10% FBS or HS. The cells are transplantable to DBA/2, BDF1, AKD2F1, and BALB/c, but not to C3H/He or ICR mice. The growth curves, plating efficiency, ultrastructural characteristics, modal number of chromosomes and transplantability to mice of various strains are almost the same for early and late passage of cells passaged in vitro. These parameters for 16th and 36th passages were: doubling time, 31 and 33 hr; plating efficiency, 12.4 +/- 1.2 and 14.6 +/- 2.6%; modal number of chromosomes, 73 and 76; lung colony formation in DBA/2, 50 and 45.9/mouse; and subcutaneous tumor diameter 24.5 and 27.4 mm, respectively. Only the numbers of lung colonies formed in BDF1 mice were different: 24.4/mouse with 16th passage cells, and 10.2/mouse with 36th passage cells. The results suggest that KLN205 is a relatively stable cultured cell line through 36 passages. As was expected, immunosuppression by higher concentrations of triaminolone acetonide (TA) enhanced lung colony formation in BDF1 mice. On the other hand, a low concentration of TA inhibited lung colony formation in DBA/2 mice, which was unexpected. These results suggest that KLN205 offers a model for investigations on metastases to lungs as well as chemotherapy for lung carcinoma.

In vivo effects in mice produced by a 3'-branched homologue of alpha-2'-deoxythioguanosine.

Studies with a 3'-branched chain homolog (alpha-3'-BCTGdR) of 2'-deoxythioguanosine (alpha-TGdR) showed that it did not prolong the survival of mice bearing the Mecca lymphosarcoma. Host toxicity was quite profound and resembled that seen with 6-thioguanine (6-TG). Evidence was obtained that this nucleoside derivative was not appreciably converted to 6-TG in the mouse. Mice treated with toxic doses of 6-TG or alpha-3'-BCTGdR were found to have very similar pathological changes. The granulocytes were eliminated from the peripheral blood, bone marrow was acellular, and some more limited damage was seen in the intestinal crypts. Experiments with radiosulfur-labeled drugs demonstrated that alpha-3'-BCTGdR was incorporated into the DNA of mouse bone marrow, predominantly in the chain-terminating position, with the result that shorter chains of DNA accumulated. The new homolog, unlike alpha-TGdR, was phosphorylated in bone marrow as well as in tumor, and incorporated well into the DNA both of bone marrow and of the neoplastic cells. In devising other homologs attention must be given to the specificity of the kinases, i.e., to whether phosphorylation is superior in tumor cells or in the growing normal cells.

Improved antitumor effects in 3'-branched homologues of 2'-deoxythioguanosine. Synthesis and evaluation of thioguanine nucleosides of 2,3-dideoxy-3-(hydroxymethyl)-D-erythro-pentofuranose.

The 3-(hydroxymethyl) branched homologue of 2-deoxyribofuranose was synthesized from the corresponding branched ribofuranose 2-O-(S-methyl dithiocarbonate) with tributyltin hydride in the first direct, one-step deoxygenation at C-2 of a ribofuranose. Nucleoside coupling afforded the corresponding 3'-branched 2'-deoxyribonucleosides of thioguanine. The alpha- and beta-nucleosides were equally inhibitory to growth of WI-L2 human lymphoblastoid cells, were phosphorylated and incorporated into the DNA of Mecca lymphosarcoma in mice to the same degree, and were more effective in these tests than the parent analogue alpha-2'-deoxythioguanosine. These results indicate that the hydroxy functions at the 3' and 5' positions of 2'-deoxynucleosides are interchangeable and that acceptance by the that the furanose ring oxygen and 2'-methylene are corresponding interchangeable, and that acceptance by the enzymes is improved if primary hydroxyls are provided at both the 3' and 5' positions.

Pharmacology and toxicology of alpha-2'-deoxythioguanosine in cancer patients.

alpha-2'-Deoxythioguanosine (alpha-TGdR) was administered as a single dose to 13 cancer patients in 18 experiments at dose levels of 150--1500 mg/m2 and as a daily dose to 22 patients in 42 experiments at dose levels of 100--4000 mg/m2/day X 5 days. No significant toxicity was observed. Blood levels and rates of excretion were determined with radiosulfur-labeled alpha-TGdR. Approximately 80% of the dose was excreted in the urine in 24 hours, initially as unchanged alpha-TGdR and increasingly as metabolites. Metabolites appear to be nucleosides and do not include 6-thioguanine, 6-thioxanthine, or 6-thiouric acid to any measurable extent. Small amounts of the alpha-TGdR in blood samples were bound to albumin and to erythrocyte membranes. Blood plasma levels of alpha-TGdR at the highest doses were in the range of 200--300 micrometer, declining in 24 hours to 67--124 micrometer.

Resistance to 9-beta-D-arabinofuranosyladenine in murine tumor cells.

Two lines of the 6C3HED (Gardner lymphosarcoma), 6C3HED-LeP and 6C3HED-ADL, were studied. The former is exquisitely sensitive to 9-beta-D-arabinofuranosyladenine (ara-A) and the latter is resistant. Cytological examinations and strain specificity tests indicated that they are both 6C3HED. DNA synthesis in the sensitive line was found to be more sensitive to ara-A in whole-cell incubations than it was in the resistant line. In cell-free extracts, the DNA synthesis of the sensitive line showed greater inhibition by 9-beta-D-arabinofuranosyladenine 5'-triphosphate. Lower ability to form 9-beta-D-arabinofuranosyladenine 5'-triphosphate or to allow access to the intracellular space was eliminated as an explanation for the resistance. Cells from an ara-A-resistant human leukemia were tested, and the DNA synthesis of the cells, in either whole cells or cell-free extract, was unaffected by ara-A or 9-beta-D-arabinofuranosyladenine 5'-triphosphate, respectively. This suggests that resistance has emerged by reason of change in the DNA polymerase(s) and that the finding may be important in the clinical use of ara-A.

Growth characteristics and drug responses of a murine lung carcinoma in vitro and in vivo.

Cells obtained from the Nettesheim lung carcinoma of DBA/2 mice, a heterogenous population grown s.c., were cultured as monolayers. These cells were serially subcultured and cloned twice, and a clone was selected for further study. This clone produced malignant tumors at the injected site when injected s.c. into male DBA/2 or C57BL/L x DBA/2 F1 mice. Referred as KLN205, this cell line had the highest rate of lung colony formation on i.v. injection. It was subcultured for over 15 generations, and its cytological characteristics were investigated. The s.c. and lung colony growth were examined histologically. The effects of treatment with two antimetabolite drugs, arabinosyl-6-mercaptopurine (NSC 406021) and 6-selenoguanosine (NSC 137679) were determined in culture and in vivo. The former was relatively ineffective; the latter was very effective both in vivo and in vitro. Several drugs used clinically for the treatment of lung cancer were also tested. This established and characterized cell line is proposed as a potential model for testing other chemotherapeutic treatments.

Enhancement of the antitumor activity of arabinofuranosyladenine of 2'-deoxycoformycin.

The 6C3HED lymphosarcoma, a tumor cell line very sensitive to 9-beta-D-arabinofuranosyladenine (ara-A), and 6C3HED/ara-A, a line resistant to ara-A, were studied. Both were responsive to 9-beta-D-arabinofuranosylcytosine (ara-C). Two lines of cells. L1210 and L1210/ara-C, are both resistant to ara-A and have very high levels of the deaminase that inactivates ara-A. When an effective inhibitor of the deaminase, 2'-deoxycoformycin, was combined with ara-A in the treatment of mice bearing L1210 or L1210/ara-C tumors, both became responsive to ara-A. Studies are reported on the extent of effects of 2'-deoxycoformycin at several dose levels and the duration of its effect in tumor cells and normal tissues. Single doses produce essentially complete inhibition of the deaminase, and little recovery was seen before 24 hr. However, DNA synthesis in normal tissues recovered more quickly. It is suggested that ara-A and ara-C, the former as a new derivative (9-beta-D-arabinofuranosyladenine 5'-phosphate) and possibly combined with 2'-deoxycoformycin, be regarded as potentially alternative drugs for the treatment of neoplasms.

9-beta-D-arabinofuranosyladenine 5'-phosphate metabolism and excretion in humans.

9-beta-D-Arabinofuranosyladenine (ara-A) was converted chemically to the 9-beta-D-arabinofuranosyladenine 5'-phosphate (ara-A-5'-P) and administered i.v. to four cancer patients in seven experiments. Urinary excretion and plasma levels of radioactivity were monitored for 24 hr in each case. Radioactivity present as unchanged ara-A-5'-P, ara-A, and the deamination product of ara-A, 9-beta-D-arabinofuranosylhypoxanthine, was determined. Excretion was, as in earlier studies with ara-A, given i.v., largely as 6-beta-D-arabinofuranosylhypoxanthine. However, in contrast to the 88 to 97% excretion of ara-A and products in 24 hr when ara-A was given by i.v. push, excretion was 41.47 to 79.1% in 24 hr when ara-A-5'-P was given. With the exception of one experiment at a low dose, where plasma ara-A levels were significant for 6 hr, the plasma levels of ara-A were sustained at significant levels for 24 hr after a single dose of ara-A-5'-P. The doses of ara-A-5'-P given were well tolerated by the four patients. Indications are that this derivative provides important advantages (solubility and sustained blood levels) over ara-A.

Inhibition of DNA chain growth by alpha-2'-deoxythioguanosine.

Mecca lymphosarcoma cells were incubated with (35-S)-alpha-2'-deoxythioguanosine for 8 hr and DNA was analyzed in alkaline sucrose gradients. 35-S radioactivity was found exclusively in a low-molecular-weight fraction. Pulse-chase experiments showed that 35-S-containing DNA fragments formed during the pulse were not incorporated into high-molecular-weight DNA following the chase. These results, together with the previous observation that (35-S)-alpha-2'-deoxythioguanosine was found predominantly in the terminal nucleoside position of DNA chains, suggested that alpha-2'deoxythioguanosine, once incorporated, terminates chain elongation.