Lucy M. Rose

Metabolism and Metabolic Actions of 6-Methylpurine and 2-Fluoroadenine in Human Cells

Activation of purine nucleoside analogs by Escherichia coli purine nucleoside phosphorylase (PNP) is being evaluated as a suicide gene therapy strategy for the treatment of cancer. Because the mechanisms of action of two toxic purine bases, 6-methylpurine (MeP) and 2-fluoroadenine (F-Ade), that are generated by this approach are poorly understood, mechanistic studies were initiated to learn how these compounds differ from agents that are being used currently. The concentration of F-Ade, MeP, or 5-fluorouracil required to inhibit CEM cell growth by 50% after a 4-hr incubation was 0.15, 9, or 120 microM, respectively. F-Ade and MeP were also toxic to quiescent MRC-5, CEM, and Balb 3T3 cells. Treatment of CEM, MRC-5, or Balb 3T3 cells with either F-Ade or MeP resulted in the inhibition of protein, RNA, and DNA syntheses. CEM cells converted F-Ade and MeP to F-ATP and MeP-ribonucleoside triphosphate (MeP-R-TP), respectively. The half-life for disappearance of HeP-ribonucleoside triphosphate from CEM cells was approximately 48 hr, whereas the half-lives of F-ATP and ATP were approximately 5 hr. Both MeP and F-Ade were incorporated into the RNA and DNA of CEM cells. These studies indicated that the mechanisms of action of F-Ade and MeP were quite different from those of other anticancer agents, and suggested that the generation of these agents in tumor cells by E. coli PNP could result in significant advantages over those generated by either herpes simplex virus thymidine kinase or E. coli cytosine deaminase. These advantages include a novel mechanism of action resulting in toxicity to nonproliferating and proliferating tumor cells and the high potency of these agents during short-term treatment.

Metabolism and Metabolic Effects of Halopurine Nucleosides in Tumor Cells in Culture

Abstract Within a series of halo derivatives of adenosine, deoxyadenosine and arabinosyladenine attempts have been made to correlate structure with cytotoxicity, substrate activity for adenosine deaminase and nucleoside kinases, and efficacy of the corresponding nucleotides against target enzymes.

Metabolism of 4′-thio-β-d-arabinofuranosylcytosine in CEM cells

Because of the excellent in vivo activity of 4′-thio-β-d-arabinofuranosylcytosine (T-araC) against a variety of human solid tumors, we have studied its metabolism in CEM cells to determine how the biochemical pharmacology of this compound differs from that of β-d-arabinofuranosylcytosine (araC). Although there were many quantitative differences in the metabolism of T-araC and araC, the basic mechanism of action of T-araC was similar to that of araC: it was phosphorylated to T-araC-5′-triphosphate (T-araCTP) and inhibited DNA synthesis. The major differences between these two compounds were: (i) T-araC was phosphorylated to active metabolites at 1% the rate of araC; (ii) T-araCTP was 10- to 20-fold more potent as an inhibitor of DNA synthesis than was the 5′-triphosphate of araC (araCTP); (iii) the half-life of T-araCTP was twice that of araCTP; (iv) the catalytic efficiency of T-araC with cytidine deaminase was 10% that of araC; and (v) the 5′-monophosphate of araC was a better substrate for deoxycytidine 5′-monophosphate deaminase than was the 5′-monophosphate of T-araC. Of these differences in the metabolism of these two compounds, we propose that the prolonged retention of T-araCTP is a major factor contributing to the activity of T-araC against solid tumors. The data in this study represent another example of how relatively small structural changes in nucleoside analogs can profoundly affect the biochemical activity.

The Synthesis and Biological Activity of Certain 4′-Thionucleosides

Abstract Results are presented on the synthesis and biological activity of several types of 4′-thionucleosides as potential anticancer agents. Detailed studies on the mechanism of action of 4′-thiothymidine are also presented.

Biochemical aspects of chemotherapy of mouse colon carcinoma. Fluoropyrimidines and pyrazofurin

Fluorouracil was metabolized to nucleotides and incorporated into RNA of mouse colon carcinomas and normal tissues. No significant difference was observed in three mouse colon tumors, but the extent of incorporation of the analog into RNA of normal colon tissue was lower than that in colon tumors. Fluorouracil phosphoribosyltransferase activity, although low, was observed to be about five times higher in mouse colon carcinomas than it was in normal colon tissue. Fluorouracil and 5‐fluoro‐2′‐deoxyuridine, as anticipated, inhibited the incorporation of [6‐3H]‐2′‐deoxyuridine into DNA of colon carcinomas and normal tissues. Colon and spleen tissue made a more rapid recovery of capacity for DNA synthesis than did colon tumors. In normal tissues examined the recovery from inhibition of DNA synthesis by fluorodeoxyuridine appeared to be more rapid than was recovery from fluorouracil inhibition. Effects of pyrazofurin, an inhibitor of orotidylic acid decarboxylase, on pyrimidine synthesis in mouse colon carcinomas and in normal tissues was analyzed by means of high‐pressure liquid chromatography. Consequences of pyrazofurin‐induced inhibition of pyrimidine biosynthesis in mouse colon carcinomas were evident in decreased pools of pyrimidine ribonucleotides. Orotidylic acid did not accumulate behind this block but elevated levels of orotic acid and orotidine were observed in acid‐soluble extracts. The maximum reduction in uracil ribonucleotide pools was observed 24 hours after pyrazofurin treatment. Recovery of uracil ribonucleotide pools was evident within 48 hours and was complete 72 hours after treatment. The maximum levels of orotic acid and orotidine in colon carcinomas were attained 24 hours after treatment; these levels remained elevated above control levels for 72 hours after pyrazofurin treatment. The pool of uracil ribonucleotides was not depressed in colon and spleen tissue from pyrazofurin‐treated animals; nevertheless, pronounced elevation of orotic acid and orotidine levels in these normal tissues was observed. These results reveal differences in effects of pyrazofurin on pyrimidine ribonucleotide pools in mouse colon carcinoma and in colon tissue. These differences may be due in part to availability or extent of utilization of exogenous pyrimidines or precursors of pyrimidines. Such differences may be exploited by means of scheduled combination chemotherapy with inhibitors of pyrimidine synthesis and pyrimidine analogs.

Disposition in mice of 7-hydroxystaurosporine, a protein kinase inhibitor with antitumor activity

UCN-01, a hydroxylated derivative of staurosporine, was selected for study because of its promising antitumor activity. For mice dosed intravenously, subcutaneously, or by oral gavage with this compound, the maximum tolerated doses (MTD) were 20, 10, and >100 mg/kg, respectively. UCN-01 was stable in mouse and dog plasma, but in human plasma it was converted to a metabolite in a process not inhibited by standard protease and esterase inhibitors. Following n intravenous dose of 10 mg/kg UCN-01, the half-lives for the initial (t1/2α) and terminal (t1/2β) exponential phases of elimination were 10 and 85 min, respectively; the area under the plasma concentration-time curve (AUC value) was 117 μg min ml−1. In mice dosed by oral gavage with 10 mg/kg, the calculated value for the half-life of the elimination phase was 150 min. The AUC value was 15 μg min ml−1, giving a value for bioavailability of 13%. After subcutaneous dosing with 10 mg/kg, the calculated values for half-lives for the distribution and elimination phases were 23 and 130 min, respectively; the AUC value was 113 μg min ml−1. Since this value is equivalent to that obtained for intravenous dosing, administration of UCN-01 by the subcutaneous route may be an alternative to intravenous dosing in preclinical and clinical trials.

Alterations in nucleotide pools induced by 3-deazaadenosine and related compounds role of adenylate deaminase

3-Deazaadenine, 3-deazaadenosine, and the carbocyclic analog of 3-deazaadenosine produced similar effects on nucleotide pools of L1210 cells in culture: each caused an increase in IMP and a decrease in adenine nucleotides and had no effect on nucleotides of uracil and cytosine. Concentrations of 50-100 microM were required to produce these effects. Although 3-deazaadenosine and carbocyclic 3-deazaadenosine are known to be potent inhibitors of adenosylhomocysteine hydrolase, the effects on nucleotide pools apparently are not mediated via this inhibition because they are also produced by the base, 3-deazaadenine, and because the concentrations required are higher than those required to inhibit the hydrolase. Cells grown in the presence of 3-deazaadenine or 3-deazaadenosine contained phosphates of 3-deazaadenosine (the mono- and triphosphates were isolated); from cells grown in the presence of the carbocyclic analog of 3-deazaadenosine, the monophosphate was isolated, but evidence for the presence of the triphosphate was not obtained. A cell-free supernatant fraction from L1210 cells supplemented with ATP catalyzed the formation of monophosphates from 3-deazaadenosine or carbocyclic 3-deazaadenosine, and a cell-free supernatant fraction supplemented with 5-phosphoribosyl 1-pyrophosphate (PRPP) catalyzed the formation of 3-deaza-AMP from 3-deazaadenine. Adenosine kinase apparently was not solely responsible for the phosphorylation of the nucleosides because a cell line that lacked this enzyme converted 3-deazaadenosine to phosphates. No evidence was obtained that the effects on nucleotide pools resulted from a block of the IMP-AMP conversion, but the results could be rationalized as a consequence of increased AMP deaminase activity. This explanation is supported by two observations: (a) coformycin, an inhibitor of AMP deaminase, prevented the effects on nucleotide pools, and (b) 3-deazaadenine decreased the conversion of carbocyclic adenosine to carbocyclic ATP and increased its conversion to carbocyclic GTP. The latter conversion requires the action of AMP deaminase and the observed effects can be rationalized by a nucleoside analog-mediated increase in AMP deaminase activity. Because these effects on nucleotide pools are produced only by concentrations higher than those required to inhibit adenosylhomocysteine hydrolase, they may not contribute significantly to the biological effects of 3-deazaadenosine or carbocyclic 3-deazaadenosine.(ABSTRACT TRUNCATED AT 400 WORDS)

Metabolism of O6-Propyl and N6-Propyl-carbovir in CEM Cells

Abstract The metabolism of O6-propyl-carbovir and N6-propyl-carbovir, two selective inhibitors of HIV replication, has been evaluated in CEM cells. Both compounds were phosphorylated in intact cells to carbovir-5′-triphosphate. The metabolism of these two agents was inhibited by deoxycoformycin and mycophenolic acid, but not erythro-9-(2-hydroxy-3-nonyl)adenine. No evidence of the 5′-triphosphate of either compound was detected in CEM cells.

Identification of 7-(2-hydroxyethyl)guanine as a product of alkylation of calf thymus DNA with Clomesone

Evidence at the molecular level is presented in support of alkylation of O6-guanine moieties of DNA as the mechanism of cytotoxicity of Clomesone to HT-29 cells and consists in the isolation and identification of a product resulting from alkylation of calf thymus DNA with Clomesone, followed by depurination to yield 7-(2-hydroxyethyl)guanine, whose formation is reasonably explained by O6-guanine chloroethylation followed by intramolecular alkylation at N7 of guanine and subsequent hydrolysis to the hydroxyethylguanine.

Metabolism and mechanism of action of some new purine antimetabolites.

Abstract Studies on 8-aza-6-thioinosine (azaMPR) have revealed a very complex picture of its chemistry and metabolism. Under physiologic conditions, azaMPR rearranges at a significant rate to N-β- d -ribofuranosyl[1,2,3]thiadiazolo [5,4-d] pyrimidine-7-amine (TPR). This ribosylamine (TPR) is, in turn, unstable under these conditions undergoing isomerization, via opening of the ribofuranose ring, to give the four possible ribosylamines — the α- and β-ribofuranoses and the α- and β-ribopyranoses and, after a longer period, loss of the sugar moiety giving the biologically inactive [1,2,3]thiadiazolo[5,4-d]pyrimidin-7-amine. An examination of extracts of cells treated with azaMPR showed the presence of three nucleotides — 8-aza-6-thioinosinic acid and N-α- and β- d -ribofuranosyl[1,2,3]thiadiazolo [5,4-d] pyrimidin-7-amine 5′-phosphates (TPRP and α-TPRP). TPR, which is more cytotoxic than azaMPR, gives rise in treated cells to TPRP and its α-anomer only. AzaMPR and TPR are both excellent substrates for adenosine kinase and selectively reduce guanine nucleotide pools and inhibit the synthesis of RNA and DNA without affecting protein synthesis. It is possible that TPRP is the biologically active metabolite, but this has not been established. 8-Aza-S-methyl-6-thioinosine and 8-aza-O6-methylinosine are also highly cytotoxic nucleosides, as are the corresponding ethyl compounds. The thio compounds, poor substrates for adenosine deaminase and excellent substrates for adenosine kinase, owe their biologic activity to their conversion to the alkylthio ribonucleotides. On the other hand, the O6-alkyl compounds are substrates for both the deaminase and the kinase, and conversion to 8-azainosinic acid probably contributes to their activity. The conversion of 8-aza-O6-methylguanosine to 8-azaguanosine is entirely responsible for its activity since it is a good substrate for the deaminase, is not a substrate for the kinase, and its activity is blocked by 2′-deoxycoformycin, a potent inhibitor of the deaminase. 2-Amino-6-chloro-1-deazapurine (ACD), a highly cytotoxic purine analog with activity against leukemia L1210, is converted to its ribonucleotide in cells, presumably by hypoxanthine-guanine phosphoribosyl transferase. Since the ribonucleoside of ACD is inactive, it would appear not to be a substrate for either purine nucleoside phosphorylase or any cellular kinase. The mechanism of action of ACD ribonucleotide is currently under investigation.