David A. Cooney

Metabolism and action of amino acid analog anti-cancer agents

The preclinical pharmacology, antitumor activity and toxicity of seven of the more important amino acid analogs, with antineoplastic activity, is discussed in this review. Three of these compounds are antagonists of L-glutamine: acivicin, DON and azaserine; and two are analogs of L-aspartic acid: PALA and L-alanosine. All five of these antimetabolites interrupt cellular nucleotide synthesis and thereby halt the formation of DNA and/or RNA in the tumor cell. The remaining two compounds, buthionine sulfoximine and difluoromethylornithine, are inhibitors of glutathione and polyamine synthesis, respectively, with limited intrinsic antitumor activity; however, because of their powerful biochemical actions and their low systemic toxicities, they are being evaluated as chemotherapeutic adjuncts to or modulators of other more toxic antineoplastic agents.

Initial studies on the mechanism of action of a new oncolytic thiazole nucleoside, 2-β-d-ribofuranosylthiazole-4-carboxamide NSC 286193)

Studies on the mechanism of action of a new oncolytic nucleoside, 2-β-d-ribofuranosylthiazole-4-carboxamide, have been undertaken using P388 murine leukemia cells growing in culture. The title compound was cytotoxic at micromolar levels, but a number of simple substitutions of both the ring and sugar moieties nullified cytotoxicity. Cytofluorimetric analysis revealed that the drug arrests cells in the “S phase” of the cell cycle. At antiproliferative concentrations, the agent inhibited the synthesis of both RNA and DNA. The macromolecular incorporation of preformed pyrimidines, including thymidine, was inhibited by the drug but, among the purines, this effect extended only to members of the adenine family and, in fact, the utilization of guanine and its congeners was reproducibly stimulated. When an examination was made of the ability of a comprehensive series of preformed purines and pyrimidines to overcome the inhibition of thymidine incorporation provoked by exposure to the thiazole nucleoside, the guanines were notably effective, but xanthosine also was shown to be an active antidote. Confirmation that the drug was producing a state of guanine deprivation was provided by high performance liquid chromatography (HPLC) analysis of acid-soluble extracts: a time-dependent fall in the concentrations of GMP and GTP ensued upon exposure to the drug; on the other hand, IMP concentrations increased by ∼15-fold. Pursuant to these findings, an examination was made of the enzymologic steps unique to the biosynthesis of guanine nucleotides in cells exposed to cytotoxic concentrations of the drug. No prominent inhibition of GMP synthetase could be demonstrated in vitro or in culture, but the specific activity of IMP dehydrogenase underwent substantial reductions in both of these cases. HPLC analyses of extracts of cultures exposed to supralethal concentrations of the title compound provided evidence of modest anabolism to the 5′-monophosphate among other products; in vitro a chemically synthesized sample of 2-β-d-ribofuranosylthiazole-4-carboxamide-5′-monophosphate was twenty times more potent than the parent nucleoside in inhibiting IMP dehydrogenase. On kinetic analysis, this inhibition was non-competitive with IMP as the variable substrate.

Pancreatic Islet Cell Tumors Produced by the Combined Action of Streptozotocin and Nicotinamide

Summary Pancreatic islet cell tumors (nesidioblastomas) were produced in 64% (18/28) of the male Holtzman rats treated with both streptozotocin and nicotinamide, while only one tumor was noted in 26 male Holtzman rats treated with streptozotocin alone, and none in rats treated with nicotinamide alone or with the streptozotocin vehicle. The majority of these neoplasms occurred in animals sacrificed 14-18 months after treatment. Hypoglycemia, intensely basophilic cytoplasmic granulation in the islet tumors, and the presence of immunoreactive insulin in tumor extracts indicate that the adenomas secreted insulin.

Tyrphostin induced growth inhibition: correlation with effect on p210bcr-abl autokinase activity in K562 chronic myelogenous leukemia

We have examined a series of tyrosine kinase inhibitors structurally related to erbstatin (tyrphostins) for inhibition of p210bcr-abl autokinase activity in vitro and for growth inhibition of chronic myelogenous leukemia (CML)K562 cells. Of the tyrphostins with IC50 for growth < 50 microM, AG814, AG946, AG952, AG896, AG953, AG956 and AG957 (structurally related to lavendustin A and piceatannol) completely inhibited p210bcr-abl kinase activity in an immune complex kinase assay. Another group of tyrphostins (AG807, AG568, AG763, AG1076, AG490, AG1318, AG556, AG1319, AG555 and AG1111) inhibits growth of K562 cells but not p210bcr-abl tyrosine kinase activity. Of the compounds which inhibit growth and p210bcr-abl tyrosine kinase activity, AG957 inhibits DNA synthesis as early as 2 h (60% inhibition at 20 microM of AG957), a time and concentration of drug where RNA and protein synthesis were not affected. AG957 inhibits p210bcr-abl tyrosine phosphorylation in living cells by 1 h without an inhibition of total protein phosphorylation. Growth inhibition by AG957 was reversible after 4 h of exposure, but irreversible after 24 h. AG957 can be considered as an important lead structure for the development of anti-bcr-abl tyrosine kinase antagonists. These data also raise the possibility that bcr-abl kinase activity is directly linked to maintenance of DNA synthesis in Philadelphia chromosome positive (Ph+) CML cells.

cis-Dichlorodiammineplatinum(II) (NSC-119 875): Preclinical toxicologic evaluation of intravenous injection in dogs, monkeys and mice

cis -Dichlorodiammineplatinum(II), a compound with antineoplastic and antimitotic activity, which inhibited the synthesis of DNA and to a lesser extent of RNA and proteins, was submitted to a preclinical pharmacologic evaluation in 16 dogs, 10 monkeys, and 120 mice. The LD50 following a single dose iv in Swiss mice was 13.4 mg/kg for males and 12.32 mg/kg for females. The minimum lethal dose for the dog was a single iv injection of 2.5 mg/kg or 5 daily consecutive injections of 0.75 mg/kg and for the monkey, 5 daily doses of 2.5 mg/kg. The treatment elicited severe morbidity within 5–17 days and the time of onset was dose related. Toxic signs included severe hemorrhagic enterocolitis, severe hypocellularity of the bone marrow and lymphoid tissues and marked renal lesions (renal tubular necrosis in the dog and nephrosis in the monkey). Dogs also showed occasional pancreatitis and monkeys myocarditis and occasional degeneration of the spermatogenic cells. Renal lesions were the most severe toxic changes in survivors and were manifested by azotemia, hypochloremia, proteinuria, appearance of urinary erythrocytes, leukocytes and decreased 24-hr excretion of lactate dehydrogenase in dogs and by protracted polyuria in monkeys. Occasional hypocalcemia also occurred. In dogs and monkeys that survived the treatment, toxicity regressed entirely within 55–124 days. Treatment affected primarily tissues with a high rate of cell division (intestines, bone marrow, lymphoid tissues and occasionally testes) and tissues known for primary excretion of the compound (intestines and kidneys).

Bleomycin-induced interstitial pneumonia in dogs

The intravenous administration of Bleomycin to 10 dogs at different dosages resulted in varying degrees of interstitial pneumonia in all cases and a lower incidence of nephrosis, foot pad excoriation and ulceration, onychoptosis, and alopecia. Pulmonary changes did not occur as a simple dose-related phenomenon. The lesions required at least 38 days to become apparent and appeared to increase in severity with time. Even at the lowest dose used (0·625 mg/kg body weight) very severe changes were seen 128 days after cessation of therapy. Morphological features of interstitial pneumonia were subpleural localization, focal mesothelial hyperplasia, marked hyperplasia and metaplasia of type II pneumocytes, fetalization of alveoli, and a pleomorphic inflammatory infiltrate. In cross-sections of lung lobes selected for histology approximately 1 to 22% of the parenchyma contained lesions. Involved areas showed marked elastosis, excess of reticular fibres, fibrosis, and increased acid mucopolysaccharides. The administration of Bleomycin produced pulmonary changes similar in many respects to those reported in busulphan-treated patients and desquamative interstitial pneumonia. The finding of interstitial pneumonia and pulmonary fibrosis in dogs treated with low doses over prolonged periods points the need to monitor pulmonary function in humans treated with Bleomycin.

Streptozotocin Diabetes CORRELATION WITH EXTENT OF DEPRESSION OF PANCREATIC ISLET NICOTINAMIDE ADENINE DINUCLEOTIDE

The diabetogenic activity of streptozotocin has been correlated with a reduction in pyridine nucleotide synthesis in the mouse pancreatic islet. To determine the specificity of this reduction for diabetogenicity, a comparative study of streptozotocin, its cytotoxic moiety, 1-methyl-1-nitrosourea, and alloxan was performed. Streptozotocin administered intraperitoneally (i.p.) producd a dose-related reduction in islet NAD which was proportional to the degree of diabetogenicity. A diabetogenic dose, 200 mg/kg, attained a peak plasma N-nitroso intact streptozotocin concentration of 0.224 mumol/ml and reduced the mean islet NAD from a control of 0.78 to 0.15 pmol. At borderline, 150 mg/kg, and nondiabetogenic, 100 mg/kg, doses, plasma concentrations reached 0.161 and 0.136 mumol/ml, and NAD was 0.36 and 0.86 pmol/islet, respectively. 1-Methyl-1-nitrosourea, 100 mg/kg, attained a maximum N-nitroso intact 1-methyl-1-nitrosourea concentration of 0.162 mumol/ml and reduced the mean NAD to 0.58 pmol/islet, and was nondiabetogenic; 200 mg/kg attained a peak plasma concentration of 0.344 mumol/ml and depressed NAD to 0.38 pmol/islet, and was inconsistently diabetogenic. Islet NAD of 0.4 pmol/islet or greater is required for integrity of the beta cell. A diabetogenic dose of alloxan, 500 mg/kg, did not depress NAD, 0.85 pmol/islet, therefore confirming that its mechanism of diabetogenicity differs from that of streptozotocin. In vivo uptake of [methyl-(14)C]streptozotocin by islets was 3.8 times that of [methyl-(14)C]-1-methyl-1-nitrosourea, whereas uptake by the exocrine pancreas favored 1-methyl-1-nitrosourea over streptozotocin 2.4:1. The decreased islet uptake of 1-methyl-1-nitrosourea correlates with the 3.5 times increased molar dosage required to produce islet NAD depression comparable to that of streptozotocin, 150 mg/kg. These studies indicate that the glucose carrier of streptozotocin facilitates uptake of its cytotoxic group, 1-methyl-1-nitrosourea, into islets.

Streptozotocin diabetes—further studies on the mechanism of depression of nicotinamide adenine dinucleotide concentrations in mouse pancreatic islets and liver

Abstract NAD concentrations of individual isolated mouse pancreatic islets were significantly decreased 2 hr after the intravenous administration of a diabetogenic dose of streptozotocin. The decrease in NAD is prevented by pretreatment with nicotinamide. Isolated islets demonstrated a concentration-related reduction in NAD when incubated with streptozotocin in vitro . One hr after the intraperitoneal injection of 14 C-nicotinamide, streptozotocin-treated animals demonstrated a decrease in total radioactive material, and a decreased incorporation into NMN and NAD in islets and liver compared to buffer-treated controls. This was associated with an increase in total radio-active material and labeled nicotinamide in the whole blood of the streptozotocin-treated group. It is proposed that the diabetogenic action of streptozotocin is related to decrease in pancreatic beta cell pyridine nucleotide concentrations resulting from the combination of reduced tissue uptake of precursors and a decrease in synthesis of NAD.

Inhibition of the cardiac mitochondrial calcium pump by adriamycin in vitro

Abstract Adriamycin and daunomycin are clinically useful antineoplastic agents, known for their ability to produce a delayed cardiomyopathy upon chronic administration. Recently it has been suggested that an alteration of myocardial calcium metabolism precedes the cardiomyopathy. This study demonstrates that these cardiotoxic antibiotics inhibit one of the subcellular systems thought to be important in the regulation of calcium metabolism in the heart. In the present communication we report that calcium translocation by cardiac mitochondria is inhibited by these anthracyclines, but that calcium translocation by the cardiac sarcoplasmic reticulum is not inhibited.

Synergistic effect of 5-fluorouracil and N-(phosphonacetyl)-l-aspartate on cell growth and ribonucleic acid synthesis in a human mammary carcinoma

Abstract The biological effects of N-( phosphonacetyl )- l - aspartate (PALA) and 5-fluorouracil (5-FU) were examined singly, and in combination, on the growth of a human mammary carcinoma (MDA) cell line in culture. All combinations of 5-FU (2.5 × 10 −7 to 1.5 × 10 −5 M) and PALA (6.0 × 10 −5 to 3.6 × 10 −3 M) resulted in synergistic inhibition of cell growth as revealed by a 50 per cent isobologram.To examine the biochemical basis for the synergism, measurements of the incorporation of [ 3 H]-5-FU into total non-poly(A)- and poly(A)-RNA, and of the simultaneous incorporation of [ 14 C]deoxyguanosine and [ 3 H]deoxyuridine into DNA, were determined. The combination of 3.7 × 10 −5 M PALA and 1 × 10 −6 M 5-FU produced 65–85 per cent inhibition of cell growth after continuous treatment for 13 days. Treatment of the cells for 3 or 24 hr with the same drug regimen produced approximately a 170 per cent increase in the incorporation of 1 × 10 −6 M [ 3 H]-5-FU into poly(A)RNA in comparison to [ 3 H]-5-FU treatment alone; exposure for 24 hr to 3.7 × 10 −5 M PALA and 1 × 10 −6 M [ 3 H]-5-FU resulted in a 285 per cent increase in the incorporation of [ 3 H]-5-FU into non-poly(A)RNA. The incorporation of either [ 14 C]deoxyguanosine or [ 3 H]deoxyuridine into DNA was not inhibited by this drug regimen; however, the incorporation of [ 3 H]deoxyuridine into DNA was elevated significantly upon 12 or 24 hr of exposure to PALA alone. PALA and 5-FU treatment resulted in a 75 per cent reduction in the concentration of UTP and no change in the concentration of 5-fluorouridine-5′triphosphate 5-FUTP) versus 5-FU treatment alone. Thus, the proportion of 5-FUTP in the total 5FUTP + UTP pool was enhanced more than 3-fold by the combination regimen. These results indicate that the synergistic effect of the combination of PALA and 5-FU on the growth of MDA cells correlates with an increased proportion of 5-FUTP in the pyrimidine nucleotide pool and, consequently, with an enhanced incorporation of 5-FU into RNA, but not with inhibition of DNA synthesis.