Metabolic fate on 1-hexylcarbamoyl-5-fluorouracil and 5-fluorouracil in mice bearing ascites sarcoma 180.

Patterns of metabolism and disposition in plasma of tumor-bearing mice after oral administration of [6(-14)C]1-hexylcarbamoyl-5-fluorouracil ([6(-14)C]HCFU) resembled those in plasma of normal mice, but elimination of [6(-14)C]HCFU and 5-fluorouracil (FUra) was slower in tumor-bearing mice. The level of 1-(5-hydroxyhexylcarbamoyl)-5-fluorouracil (HHCFU) was lower in tumor-bearing mice. Also detected in plasma were [6(-14)C]HCFU, HHCFU, 1-(3-carboxypropylcarbamoyl)-5-fluorouracil, FUra, 5,6-dihydro-5-fluorouracil, and alpha-fluoro-beta-alanine. FUra originating from [6(-14)C]HCFU was retained over 6 hours, whereas intact FUra after [6(-14)C]FUra administration disappeared within 2 hours. The pattern of metabolism in ascitic fluid was similar to that in plasma after [6(-14)C]HCFU and [6(-14)C]FUra administration, but FUra was retained for a longer period in ascitic fluid. In sarcoma 180 cells, the maximum concentration of total radioactivity was observed 1 or 2 hours after [6(-14)C]FUra or [6(-14)C]HCFU administration, respectively, and the level of intact HCFU was very low. The principal metabolites were nucleotides that were maintained for a long period after administration of both compounds. The pattern of other metabolites after [6(-14)C]HCFU administration was also similar to that after [6(-14)C]FUra administration.

Murine lymphoma L5178Y cells resistant to purine antagonists: differences in cross-resistance to thioguanine-platinum(II) and selenoguanine-platinum(II).

To determine whether the antitumor activities of thioguanine-platinum(II) [TG-Pt(II)] and selenoguanine-platinum(II) [SeG-Pt(II)] are due to direct actions of these compounds or to the actions of their hydrolysis products, studies were made on a purine antagonist-resistant, murine lymphoma L5178Y/MP subline that lacked the anabolic enzyme hypoxanthine-guanine phosphoribosyltransferase necessary for tumor inhibition. The L5178Y/MP subline proved to be highly resistant to both TG-Pt(II) and thioguanine; the resistance ratios to the two compounds were almost identical. The subline showed high resistance to selenoguanine, but the cross-resistance to SeG-Pt(II) was negligible. Whether the compounds exhibit the delayed cytotoxicity characteristic of purine antagonists was also investigated. Delayed cytotoxicity was demonstrated for TG-Pt(II) as well as for thioguanine and other purine antagonists but not for SeG-Pt(II) or cis-dichlorodiammineplatinum(II). Experiments on cross-resistance and delayed cytotoxicity showed differences in the cytotoxicities of TG-Pt(II) and SeG-Pt(II): TG-Pt(II) exerted its activity through its hydrolysis product thioguanine, whereas SeG-Pt(II) compound was cytotoxic itself.

Relationship between antitumor effect and metabolites of 5-fluorouracil in combination treatment with 5-fluorouracil and guanosine in ascites sarcoma 180 tumor system.

The antitumor activity of 5-fluorouracil (FUra) against ascites Sarcoma 180 was significantly enhanced by coadministration of guanosine, and slightly by adenosine, but not by cytidine or uridine. In advanced ascites Sarcoma 180, guanosine also enhanced the action of FUra, but adenosine, uridine, and cytidine did not. The potentiation of antitumor activity by guanosine was reversed by addition of cytidine. The antitumor activity of FUra was significantly potentiated when guanosine was administered either 0 to 15 min before or 5 min after FUra. Changes in metabolites of FUra after potentiation by guanosine were investigated. Total radioactivity in the plasma was significantly decreased 10 min after the combined administration of [6-14C]FUra (3 mg/kg i.p.) and guanosine (100 mg/kg i.p.) in comparison with that of [6-14C]FUra alone and was slightly decreased by coadministration of [6-14C]FUra and adenosine. Conversely, it was significantly increased by uridine or cytidine. The decrease in total radioactivity in the plasma caused by guanosine was completely reversed by addition of cytidine. FUra, 5-fluorouridine, alpha-fluoro-beta-ureidopropionic acid, and alpha-fluoro-beta-alanine were found in the plasma. Intact FUra accounted for about 55% of the total radioactivity. The proportion of metabolites of [6-14C]FUra was not changed by coadministration of [6-14C]FUra and guanosine, adenosine, or cytidine, but the proportion of FUrd was increased by uridine. In the ascitic fluid, the total radioactivity derived from [6-14C]FUra was decreased by its combined administration with guanosine, and it was reversed by addition of cytidine. This pattern was similar to that in the plasma. The main FUra compound was intact FUra itself (90%), and 5-fluorouridine accounted for 1% of the total radioactivity in the ascitic fluid. On the other hand, total radioactivity of [6-14C]FUra in the tumor cells was significantly and slightly increased by guanosine and adenosine, respectively. Total radioactivity after [6-14C]FUra in combination with uridine or cytidine was less than that after [6-14C]FUra alone. Incorporation of [6-14C]FUra into RNA was increased about 3.7 times by its combination with guanosine in comparison with FUra alone, and it was increased 2.0, 0.6, and 0.7 times by adenosine, uridine, and cytidine, respectively. Moreover, FUra-nucleotides were significantly increased by guanosine. The increased radioactivity in RNA and FUra-nucleotides of tumor cells caused by guanosine was completely reversed by cytidine. These changes in incorporation into tumor cells were comparable to those in antitumor activity against ascites Sarcoma 180. The potentiation of antitumor activity of FUra by guanosine was considered to be due to an increase in incorporation of FUra into FUra-nucleotides and RNA in the tumor cells.

Antitumor activity of 1,2-diaminocyclohexane--platinum complexes against sarcoma-180 ascites form.

Platinum complexes derived from three isomers of 1,2-diaminocyclohexane have been synthesized and their antitumor activities were evaluated against ascites Sarcoma-180. All the platinum complexes had high antitumor activity. Platinum complexes derived from cis-1,2-diaminocyclohexane were more effective than those derived from trans-l-and trans-d-1,2-diaminocyclohexane. Among the platinum complexes tested, oxalato(cis-1,2-diminocyclohexane)platinum had a remarkably high therapeutic index. Modification of the nonleaving group as well as that of the leaving group is important in order to find better antitumor platinum complexes.

Mechanism of cytotoxicity of 5-fluorouracil: distinction between the irreversible cytotoxic effect of 5-fluorouridine and the reversible cytotoxic effect of 5-fluoro-2'-deoxyuridine on murine lymphoma L5178Y cells in culture.

To determine the relative importance of the DNA and RNA effects in the cytotoxicity of 5-fluorouracil, we determined the effects of brief exposure to 5-fluorouridine and 5-fluoro-2'-deoxyuridine, compared with 5-fluorouracil, on the subsequent growth of murine lymphoma L 5178 Y cells during prolonged incubations following the removal of the drug from the culture medium. 5-Fluoro-2'-deoxyuridine markedly inhibited cell proliferation by continuous exposure. However, its inhibitory effect by exposure even at 1,000 muM for 1 h easily disappeared within 1 week after removal of the drug from the culture medium. This result indicates that the cytotoxic effect of 5-fluoro-2'-deoxyuridine is reversible on cell proliferation. On the other hand, the exposure to 5-fluorouridine, even at the low concentration of 1.5 muM for 1 h, caused a substantial and irreversible inhibition on cell proliferation. The cytotoxic effect of 5-fluorouracil was also an irreversible action similar to that of 5-fluorouridine.

A compound possessing antitumor and interferon-inducing activities derived from the common antigen (OEP) of Pseudomonas aeruginosa.

OEP, a component consisting mainly of protein with small amounts of lipids and sugars, has been isolated from the autolysate of Pseudomonas aeruginosa and purified by physicochemical methods. It possesses remarkable biological properties, showing antitumor and interferon-inducing activities. As regards the antitumor activity of the sample, the ED50 value against ascites sarcoma-180 was 1 microgram/kg mouse/day, and its interferon-inducing activity amounted to 15 units at a concentration of 0.01 microgram/ml. Both activities increased after protease digestion, reaching about ten times those of the sample which had not undergone digestion. The protease-treated OEP contained 17% protein, 14.5% total sugars, 31% lipids, 12.5% hexosamine, 3.8% KDO, and 2.7% phosphorus. Neutral sugars consisted of 12.4% rhamnose, 2.7% mannose, 66.9% glucose, and other unidentified material. Total lipids derived from OEP consisted of 65% loosely-bound and 35% covalently-bound lipids; the former contained C14:10, C16:0, C16:1, C18:0, and C15:1 acids and the latter, beta-OH C10:0, C12:0, alpha-OH C12:0, beta-OH C12:0, C16:0, and C16:1 acids. The antitumor and interferon-inducing activities of OEP remained after the removal of loosely-bound lipids from OEP.

Pharmacokinetics of 1-alkylcarbamoyl-5-fluorouracils in plasma and ascites fluid after oral administration in mice.

Pharmacokinetics of 1-alkylcarbamoyl-5-fluorouracils was examined in mice bearing sarcoma 180. The alkylcarbamoyl derivatives were absorbed rapidly as intact form through the gastrointestinal tract and distributed into ascites fluid. Concentration-x-time (C-x-t) values of 5-fluorouracil formed in plasma and ascites fluid decreased in order by extension of the carbon chain of the alkyl moiety. C-x-t value of 5-fluorouracil formed in ascites fluid after hexylcarbamoyl derivative was higher than that in plasma. Antitumor activity of the compounds was correlated with both maximum concentration (Cmax) and C-x-t values of 5-fluorouracil formed and Cmax of total (intact form plus 5-fluorouracil formed) in ascites fluid (P < 0.01), and with C-x-t values in ascites fluid and Cmax and C-x-t values of 5-fluorouracil formed in plasma (P < 0.05). Alkylcarbamoyl structure was valuable for rapid absorption through the gastrointestinal tract and blood-ascites barrier and for maintenance of 5-fluorouracil level in plasma and ascites fluid.

Antitumor activity of 1-alkylcarbamoyl derivatives of 5-fluorouracil in a variety of mouse tumors.

The antitumor properties of 1-alkylcarbamoyl derivatives of 5-fluorouracil were examined in various mouse tumor systems to select promising compounds for clinical use. Almost all alkylcarbamoyl derivatives were active against various tumors when given by oral administration. Among them, 1-methyl, 1-ethyl, 1-isopropyl, 1-hexyl and 1-octyl carbamoyl derivatives of 5-fluorouracil were moderately or markedly active in six mouse tumor systems tested. However, 1-methyl, 1-ethyl, and 1-isopropyl carbamoyl derivatives were toxic to mice, though not lethal. As a result, 1-hexyl and 1-octyl carbamoyl derivatives were selected as the best candidates for antitumor agents in further study.

Antitumor activity of 3',5'-diesters of 5-fluoro-2'-deoxyuridine against murine leukemia L1210 cells.

Antitumor activity of several 3',5'-diesters of 5-fluoro-2'-deoxyuridine (FUdR) against L1210 leukemia cells following intraperitoneal administration was examined. Esters of FUdR with aromatic acid or aliphatic acid of longer chain length were markedly active. Their activities, with respect to ILS30, were as much as 100 times that of unesterified FUdR. 3',5'-ditoluoyl FUdR also had an improved therapeutic effect: its therapeutic ratio was increased to 8.1, as against 2.0 for FUdR. On the other hand, 3',5'-diesters of FUdR with aliphatic acid of shorter chain length do not appear to be as active as FUdR. The relationship between the antitumor activity and plasma levels has also been examined. After 3',5'-diacetyl FUdR, which is one of the drug group showing low cytotoxicity, the plasma concentration rapidly decreased to an unmeasurable level 3 h after dosing. This tendency is similar to that shown in FUdR. On the other hand, with 3',5'-dipalmitoyl FUdR and 3',5'-dibenzoyl FUdR, each of which has a marked antitumor effect, plasma concentrations decreased slowly and were maintained for as long as 48 h after dosing. The results show that the cytotoxicity of diesters of FUdR is correlated with the duration of a high plasma level of FUdR.

Antitumor activity of alkylesters of 1-beta-D-ribofuranosyl-5-fluorouracil-5'-phosphate against murine lymphoma L5178Y resistant to 1-beta-D-ribofuranosyl-5-fluorouracil.

A 1-beta-D-ribofuranosyl-5-fluorouracil-resistant clone, designated L5178Y/FUR, was established in this experiment. This is one of the colonies derived from a subculture which acquired resistance to 1-beta-D-ribofuranosyl-5-fluorouracil by passaging the L5178Y cells through four sucessive episodes of culture in Fischers' medium containing 1-beta-D-ribofuranosyl-5-fluorouracil; each analog treatment was followed by recovery intervals. In this subline, IC99 values for 1-beta-D-ribofuranosyl-5-fluorouracil was 0.57 muM, which was 24 times as great as in the parent line (0.0024 muM), and this subline showed a cross resistance to 5-fluorouracil. New alkylesters of 1-beta-D-ribofuranosyl-5-fluorouracil 5'-phosphate (FUMP-alkylesters) were synthesized and their antitumor activity was examined against 1-beta-D-5-fluorouracil-sensitive and -resistant L5178Y sublines. Only hexadecylester of 1-beta-D-ribofuranosyl-5-fluorouracil 5'-phosphate was effective, not only to 1-beta-D-ribofuranosyl-5-fluorouracil line but also to 1-beta-D-ribofuranolsyl-5-fluorouracil-resistant subline. Other compounds showed cross resistant in the 1-beta-D-ribofuranosyl-5-fluorouracil-resistant cells.

The difference in mechanism of action of 5-fluorouracil and its nucleosides in L5178y cells.

The mechanism of antitumor activity of 5-fluorouracil (FU) was studied in mouse leukemia L5178Y cells in vitro. FU increased labeled-thymidine incorporation into acid-insoluble fraction and inhibited labeled-deoxycytidine incorporation as did 5-fluorouridine (FUR) and 5-fluoro-2'-deoxyuridine (FUdR). FU and FUR inhibited labeled-uridine incorporation but FUdR did not. For reversal method at the equieffective concentration of FU, FUR or FUdR, antiproliferating effects of FU and FUR were partially reversed by thymidine and deoxyuridine though FUdR toxicity was completely abolished by both compounds. These results demonstrate that FU and FUR affect not only thymidylate synthesis as a consequence of the conversion to deoxymononucleotide, but also the site concerning functioning RNA synthesis in L5178Y cells, and the FUdR is a specific inhibitor of thymidylate synthesis.

Isolation and characterization of the murine lymphoma L5178Y cell line highly resistant to 5-fluorouracil.

5-Fluorouracil-resistant cell line designated as L5178Y/FU was established in this experiment. This is one of the colonies derived from the subculture which acquired resistance to 5-fluorouracil by passing the L5178Y cells through ten successive episodes of culture in Fischer's medium containing 5-fluorouracil, in which each 5-fluorouracil treatment was followed by recovery intervals. The resistance of this line is about 80-fold that of the IC99 of 5-fluorouracil, and also shows a cross resistance to both 5-fluorouridine and 5-fluoro-2'-deoxyuridine. 5-Fluoro-2'-deoxyuridine resistant subline designated as L5178Y/FUdR was also isolated from the subculture which acquired resistance to 5-fluoro-2'-deoxyuridine by using the same selection procedure. The resistance of this line is about 650-fold that of the IC99 of 5-fluoro-2'-deoxyuridine, and shows a partial cross-resistance to 5-fluorouridine, but not to 5-fluorouracil.

Inhibition of phosphoribosylation of 5-fluorouracil by purines.

The mechanism of the reversal of 5-fluorouracil cytotoxicity in L5178Y cells by hypoxanthine, adenine and inosine was examined in a cell-free system. A crude extract of the cells possessed high hypoxanthine and adenine phosphoribosyltransferase and purine nucleoside phosphorylase activities. Hypoxanthine (2 mM), adenine (5 mM) and inosine (5 mM) inhibited the nucleotide formation from 5-fluorouracil at 0.2 mM 5-phosphoribosyl 1-pyrophosphate (PRPP). However, at a higher concentration of PRPP (2.5 mM), the inhibition of hypoxanthine was not found. It suggests that the inhibition of 5-fluorouracil metabolism is due to a deficiency of PRPP induced by phosphoribosylation of hypoxanthine and adenine.

Effect of centrophenoxine on cyclophosphamide concentration in blood.

In order to clarify the mechanism of potentiation of cyclophosphamide activity by centrophenoxine, blood concentration of total and activated cyclophosphamide was examined. Blood concentration of cyclophosphamide increased by the compound and biological half-life of activated cyclophosphamide was markedly increased to 30 min from 18 min in intraperitoneal administration. At the same time, concentration of active form in ascites fluid was also increased and biological half-life of the active form was increased to 50 min from 18 min. Similar increase in blood level of active form was also shown by p-chlorophenoxyacetic acid and probenecid, but concentrations at early stages after injection was not increased. As a result, potentiation of cyclophosphamide activity by centrophenoxine was found to be due to maintenance of active form in both blood and ascites fluid at higher levels than those in the control.

Antineoplastic effect of orally administered 1-alkyl carbamoyl derivatives of 5-fluorouracil on sc implanted Lewis lung carcinoma and B16 melanoma.

The antitumor activity of 1-alkyl carbamoyl derivatives of 5-fluorouracil against Lewis lung carcinoma and B16 melanoma by long-term oral administration was examined. The 1-hexyl and 1-phenethyl carbamoyl-5-fluorouracil derviatives were markedly active against early Lewis lung carcinoma among the derivatives tested. These compounds were not markedly active against advanced Lewis lung carcinoma but did show acceptable activity. Increases in lifespan in mice with early Lewis lung carcinoma at optimal doses of 1-hexyl and 1-phenethyl carbamoxyl-5-fluorouracil were 98% and 78% respectively. In advanced Lewis lung carcinoma, the 1-hexyl derivative was active by either intermittent or daily administration, but the 1-phenethyl derivative was active only by daily administration. Lung metastases were not inhibited by optimal doses of the 1-hexyl derivative but were completely inhibited by the 1-phenethyl derivative. The 1-hexyl derivative was also active against B16 melanoma and the increase in lifespan at optimal doses was 27%. As a result, 1-hexyl carbamoyl-5-fluorouracil was found to be the most active derivative against early Lewis lung carcinoma and B16 melanoma. However, 1-phenethyl carbamoyl-5-fluorouracil was the most active derivative against advanced Lewis lung carcinoma by daily administration and this compound completely inhibited lung metastases, while 5-fluorouracil and cyclophosphamide did not inhibit lung metastases.