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.

Potentiation of the chemotherapeutic effect of 5-fluorouracil by combination with guanosine 5'-monophosphate.

The chemotherapeutic effect of the 5-fluorouracil (5-FU)-guanosine 5'-monophosphate (GMP) combination in various mouse tumor systems was compared with that of 5-FU monotherapy. Antitumor activity of 5-FU against L-1210 leukemia was potentiated without increasing its toxicity to the host when GMP at 30-100 mg/kg/day was injected simultaneously with 5-FU. Any time interval between the administrations of 5-FU and GMP diminished the increase in survival. Moreover, the combination of 5-FU and GMP at 100 mg/kg/day produced marked antitumor effects in the P-388 leukemia, ascites sarcoma 180, and Ehrlich ascites carcinoma systems. GMP also potentiated the antitumor activity of 5-FU in solid tumor systems (adenocarcinoma 755 and Lewis lung carcinoma) when given by intravenous injection, but not intraperitoneal injection. The therapeutic effect of 5-FU on various murine tumors was markedly potentiated by GMP at 100 mg/kg/day or less without increasing the toxicity to the host.

Effect of pyrimidines, purines and their nucleosides on antitumor activity of 5-fluorouracil against L-1210 leukemia.

The chemotherapeutic action of 5-fluorouracil (5-FU) monotherapy on L-1210 leukemia in mice was compared with combinations of pyrimidines (uracil, uridine, deoxyuridine, cytosine, cytidine, deoxycytidine, thymine and thymidine) or purines (adenine, adenosine, deoxyadenosine, guanine, guanosine, deoxyguanosine and inosine) with 5-FU. The antitumor activity of 5-FU was enhanced by coadministration of uracil, thymine or guanosine, but the toxicity of the first two compounds was also enhanced. Only when 5-FU was administered with guanosine, was not only the antitumor activity but also the therapeutic ratio potentiated without increasing its toxicity. A time interval between the administration of 5-FU and guanosine diminished the survival effect. Therefore, the 5-FU-guanosine combination produced its optimal chemotherapeutic effect by simultaneous injection. The potentiation of antitumor effect of 5-FU by guanosine was prevented completely by cytidine or uridine, and partially by deoxyuridine, adenosine or deoxyadenosine.

Antitumor activity of 1-acyloxymethyl derivatives of 5-fluorouracil against L1210 leukemia.

Antitumor activity of 15 1-acyloxymethyl derivatives of 5-fluorouracil was examined by both intraperitoneal injection and oral administration in L1210 leukemia system. Therapeutic ratio for dodecanoyloxymethyl derivative by intraperitoneal injection was 23 which was greater than that for 5-fluorouracil (11). On the other hand, undecanoyloxymethyl derivative of 5-fluorouracil showed the highest therapeutic ratio (5.8) when administered orally which was greater than that for 5-fluorouracil (1.9) and 1-(2-tetrahydrofuryl)-5-fluorouracil (1.0).

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.

Antitumor activity of alkyldisulfide derivatives of 6 mercaptopurines against L-1210 leukemia.

Antitumor activity of twenty seven 6-alkyl disulfide derivatives of 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) were examined in the system of murine L-1210 leukemia. When given by intraperitoneal administration, maximum increase in life span produced by iso-pentyl (75%) and heptyl (68%) disulfides of 6-MP and sec-butyl (83%), pentyl (78%), and naphthyryl (90%) disulfides of 6-TG were higher than that of parent compounds (6-MP: 53%, 6-TG: 64%). Compounds with higher therapeutic ratio than respective parent compound were decyl and naphthyryl disulfide derivatives of 6-MP and almost all the derivatives of 6-TG tested (propyl, butyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, decyl, benzyl, and naphthyryl disulfides of 6-TG). Among them, decyl derivatives of both 6-MP and 6-TG showed the highest therapeutic ratio as high as 50 and 48, while those of parent compounds were 6.2 and 5.0, respectively. The improvement of therapeutic effect by the modification to decyl disulfide of 6-MP was demonstrated inthe L-1210 system, but it was not found in the Sarcoma-180 system reported previously. By oral administration, these derivatives were active against the leukemia but they were not superior to the parent compounds.

Excretion of 1-hexylcarbamoyl-5-fluorouracil in urine of mice.

Excretion and metabolites in urine and feces of mice after oral administration of l-hexylcarbamoyl-5-fluorouracil-6-14C (14C-HCFU) or 5-fluorouracil-6-14C (14C-FU) were examined. After oral administration, about 90% of 14C-HCFU was excreted in urine within 48 h but not in feces. Major radioactive compounds in urine were 1-(3-carboxypropylcarbamoyl)-5-fluorouracil (CPRFU), FU, 5, 6-dihydro-5-fluorouracil (DHFU) and alpha-fluoro-beta-alanine (FBAL). Excretion ratio of these metabolites within 48 h was 13.5, 32.5, 20.2 and 23.9%, respectively, CPRFU and FU were rapidly excreted in urine after 14C-HCFU administration followed by DHFU and FBAL. Excretion of the latter two degradation products of FU was slower. On the other hand, major metabolites in urine after 14C-FU administration were DHFU and FBAL, but intact FU was slightly excreted. Excretion ratio of FU, DHFU and FBAL within 48 h was 4.8, 58.1 and 28.2%, respectively.

Antitumor activity of 1-alkoxycarbonylalkylcarbamoyl-5-fluorouracil derivatives by oral administration.

Antitumor activity of seven 5-fluorouracil derivatives having carbamoyl linkage with amino acid was examined against L-1210 leukemia, adenocarcinoma 755, ascites sarcoma 180, Ehrlich ascites carcinoma and Lewis lung carcinoma by oral administration. These compounds showed more than 30% increase in life-span (ILS) against L-1210 at optimal doses when given by oral administration. Therapeutic ratios (ILSmax/ILS30) of 1-methoxycarbonylmethylcarbamoyl and 1-(1-ethoxycarbonyl-3-methylthiopropylcarbamoyl) derivatives of 5-fluorouracil in L-1210 system were 4.8 and 4.7, respectively. 1-Methoxycarbonylmethylcarbamoyl and 1-(2-ethoxycarbonylethylcarbamoyl) derivatives of 5-fluorouracil inhibited completely the growth of adenocarcinoma 755 when given orally, but only 1-methoxycarbonylmethylcarbamoyl derivative inhibited 99 and 98% of the growth of ascites sarcoma 180 and Ehrlich ascites carcinoma, respectively. The latter compound increased the life-span to 48% at optimal dose in Lewis lung carcinoma system.

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.

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.

Structure-antitumor activity relationship of purin-6-yl alkyl disulfides.

Antitumor activity and toxicity to host of newly synthesized disulfide derivatives of 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) were examined in murine ascites sarcoma-180 system (total packed cell volume method) by parenteral administration. The compounds tested were 6-alkyl disulfides (carbon number of alkyl group: 2, 3, 4, 5, 6, 7, 8, 10, and 14), 6-branched-alkyl disulfides (iso-propyl, sec-butyl, and tert-butyl), and 6-aralkyl disulfide (naphthyryl). Most disulfide derivatives of 6-MP and 6-TG showed higher antitumor activity (lower ED50) and higher toxicity to host (lower LD50) than parent compounds, but ratios of increase in activity and toxicity were different with each other. The compounds with higher chemotherapeutic index (LD50/ED50) than parent compounds were alpha-naphthyryl (8.7) disulfide in a series of 6-MP (7.5) derivatives; and sec-butyl (27), tert-butyl (24), octyl (23), decyl (26), and alpha-naphthyryl (28) disulfides in a series of 6-TG (14) derivatives. These 6-TG derivatives were promising for antitumor agents.

Metabolic fate of 1-hexylcarbamoyl-5-fluorouracil after oral administration in mice.

1. The metabolic fate of a new antitumour agent, 1-hexylcarbamoyl-5-fluoro[6-14C]uracil (14C-HCFU) was compared with that of 5-fluoro[6-14C]uracil (14C-FU) after oral administration to mice. 2. 1-(5-Hydroxyhexylcarbamoyl)-5-fluorouracil (5-hydroxy-HCFU) and 1-(5-oxohexylcarbamoyl)-5-fluorouracil (5-keto-HCFU) were found as major intermediate metabolites of 14C-HCFU and were produced by omega-1 oxidation. 3. FU was detected in plasma 180 min after oral administration of 14C-HCFU, whereas unchanged FU disappeared within 60 min after 14C-FU. 4. 14C-HCFU and resulting FU were retained in tissues for a long period after oral administration, while administered 14C-FU was rapidly degraded.

Effect of 1-hexylcarbamoyl-5-fluorouracil on spontaneous mammary adenocarcinoma of mice.

The antitumor activity of 1-hexylcarbamoyl-5-fluorouracil (HCFU) in various schedules of long-term oral administration was examined in spontaneous mammary adenocarcinoma of SHN mice, an autochthonous tumor system. In the control group, the average time to local recurrence and average longevity after surgical intervention were 21 and 48 days, respectively. Oral administration of HCFU at 200 approximately 300 mg/kg/day, 3 times a week for 5 consecutive days every 2 or 3 weeks was markedly effective against the adenocarcinoma. The optimal schedule was 20 administrations of HCFU at 300 mg/kg/day, 3 times a week. The average time to local recurrence after the operation was increased to 200% and average postoperative survival was also prolonged to 150%. Growth of the tumors was slower and lung metastases at autopsy were found to be suppressed by HCFU. The effect of HCFU in delaying local recurrence and prolonging longevity was slightly affected by the schedule of administration.

Antitumor activity of metabolites of 1-hexylcarbamoyl-5-fluorouracil and related compounds against L1210 leukemia in vivo and L5178Y lymphoma cells in vitro.

Antitumor activity of metabolites of 1-hexylcarbamoyl-5-fluorouracil (HCFU) and related compounds was examined in vivo and in vitro. Carboxypentyl and carboxypropyl carbamoyl derivatives of 5-fluorouracil (FU) were moderately active against L1210 by oral administration but less active by intraperitoneal administration. However, 5-hydroxy- and 5-oxo-hexylcarbamoyl derivatives of FU were markedly or moderately active against the leukemia by both oral and intraperitoneal administrations. Therapeutic ratios for the metabolites were less than that for HCFU by both oral and intraperitoneal administrations. Metabolites of HCFU had growth inhibitory activity against L5178Y in vitro similar to alkylcarbamoyl derivatives of FU. Activity of metabolites was lower than that of FU and higher than that of HCFU in vitro. It means that HCFU is converted into FU through active intermediate metabolites in vivo.

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.