1-hexylcarbamoyl-5-fluorouracil is more cytostatic than 5-fluorouracil against human tumors in vitro

The sensitivity of HeLa cells and 15 human tumors, including eight gastric cancers, five colorectal cancers and two lung cancers to 1-hexylcarbamoyl-5-fluorouracil (HCFU) was compared with that to 5-fluorouracil (5-FU) in vitro. HeLa cells were doubly sensitive to HCFU, as compared to 5-FU. After the HeLa cells had been treated with 5-FU or HCFU at 77 μM for 1–5 h, the intracellular levels of 5-FU and HCFU were determined, using gas chromatographic-mass spectrometric methods. The level of HCFU plus 5-FU in the HCFU-treated cells was twice as high as the level of 5-FU in the 5-FU-treated cells. The sensitivity to HCFU in 15 tumor tissues varied with the tissue; however, all tissues tested were more sensitive to HCFU than to 5-FU, assessed using the succinate dehydrogenase inhibition test. These results suggest that the hexylcarbamoyl structure facilitates the rapid uptake of HCFU through the cell membrane. HCFU may prove to be more effective for treating each individual patient with a malignant lesion.     

Anti-tumor effects of carmofur on human 5-FU resistant cells]

As a part of studies to clarify the efficacy mechanism of carmofur (HCFU), a 5-FU analog, the degree of cross-resistance to HCFU of cell lines of cultured human colon cancer DLD-1 and a stomach cancer NUGC-3, both of which had acquired resistance to 5-FU, was studied in vitro and in vivo. In vitro, both resistant cell lines showed no or little cross-resistance to HCFU under a short-term drug exposure, and very low cross-resistance to HCFU, as compared to resistance to 5-FU, under a long-term drug exposure. In therapeutic experiments using nude mice bearing parent or subcutaneously transplanted 5-FU-resistant DLD-1 cells, HCFU exerted almost the same growth-inhibitory effects on both tumors. These results suggest that HCFU itself may have its own anti-tumor mechanism, different from that of 5-FU.     

Enhancement of antitumor effects of 1-hexylcarbamoyl-5-fluorouracil combined with hyperthermia on Ehrlich ascites tumor in vivo and Nakahara-Fukuoka sarcoma cell in vitro

1-Hexylcarbamoyl-5-fluorouracil (HCFU) was found to be hydrolyzed to 5-fluorouracil (5-FU) depending on temperature. In this study, the antitumor effects of HCFU with hyperthermia were studied on Ehrlich ascites in tumor-bearing CD-1 mice and Nakahara-Fukuoka sarcoma cells. Concurrently, the change of 5-FU concentration in the Ehrlich tumor was also observed. In vivo, Ehrlich cells were transplanted in the foot pads of mice. HCFU (80 mg/kg) and 5-FU (40 mg/kg) (same molarity) were administered orally on the 3rd, 6th, and 9th days after transplantation and simultaneously hyperthermia (43.5 degrees C, 45 min) was induced. The antitumor effect was evaluated by the tumor volume. On the 9th day after transplantation, HCFU (150 mg/kg) and 5-FU (75 mg/kg) were administered with or without hyperthermia (43.5 degrees C, 45 min) and the tumor tissues were taken for 5-FU concentration analysis. In addition, Nakahara-Fukuoka sarcoma cells were incubated (42 degrees C various times) with our without HCFU and 5-FU (both 1 x 10(-4) M) in vitro. The cytotoxicity was evaluated by colony forming assay. The most effective condition was found to be the combination of HCFU with hyperthermia in vitro and in vivo. The highest 5-FU concentration was observed by the treatment of HCFU combined with hyperthermia in vivo. Hence, HCFU could be clinically useful in thermochemotherapy.     

Comparative clinical study on 5-FU concentrations for oral HCFU and i.v. 5-FU

Using the cross-over method, the same patients were administered continuous intravenous injections of 5-FU and HCFU, an oral derivative of 5-FU widely used for breast and colon cancer in Japan. The pharmacokinetics of 5-FU in blood of both drugs were then compared. The AUC of the 5-FU concentration in blood of the HCFU 100 mg group tended to be higher than that in the 5-FU 250 mg group and lower than that in the 5-FU 500 mg group. There was, however, no statistically significant difference between the HCFU and 5-FU 250 mg groups (p = 0.3274), or between the HCFU and 5-FU 500 mg groups (p = 0.1921).     

Serum concentration of HCFU and 5-FU after oral administration of HCFU in postoperative digestive cancer patients]

In an attempt to establish the optimum dose of HCFU, the effect of the presence of stomach on the absorption of HCFU and both the rise and maintenance of blood HCFU levels was evaluated in patients with gastric cancer (total gastrectomy group) and with colorectal cancer (non-gastrectomy group). The blood concentrations of HCFU fractions and 5-FU were determined in terms of pharmacokinetic parameters in these groups. The Tmax was significantly different between the two groups, with HCFU fraction and 5-FU levels that were significantly higher in the total gastrectomy group 30 minutes after administration. No differences were found in Cmax or AUC. There were no significant differences in Cmax or AUC among the various subgroups given different doses of HCFU (100, 150 and 200 mg) in either group, although dose-dependency was observed. Similar results were obtained in crossover tests. The 5-FU remained at an effective concentration of 0.05 microgram/ml, 4 hours after a single dose of 100 mg HCFU.     

Sensitivity test for 5-fluorouracil and its analogues, 1-(2-tetrahydrofuryl)-5-fluorouracil, uracil/1-(2-tetrahydrofuryl)-5-fluorouracil (4:1) and 1-hexylcarbamoyl-5-fluorouracil, using the subrenal capsule assay

The chemosensitivity of 20 human neoplastic tissues including 13 gastric and 7 colorectal cancers was tested using 5-fluorouracil (5-FU) and its analogues: 1-(2-tetrahydrofuryl)-5-FU (FT), uracil/FT (UFT) and 1-hexylcarbamoyl-5-FU (HCFU), and the in vivo subrenal capsule (SRC) assay. The relative variation of tumor size (delta TS/TSo) was calculated as follows: delta TS/TS0 = (TS6-TS0/TS0) x 100%, where TS6 was the tumor size on day 6 and TS0 on day 0, and the chemosensitivity was considered to be sensitive when delta TS/TS0 in the treated group was decreased to below -10%. The mean tumor size was -10.9 +/- (SD) 10.9% for 5-FU, -12.3 +/- 17.1% for FT, -18.4 +/- 15.8% for UFT and -17.9 +/- 15.4% for HCFU. The decrease of tumor size was marked when exposed to UFT (p less than 0.01) or HCFU (p less than 0.02), compared with that to 5-FU. Positive correlations were noted between the tumor sizes of 5-FU and its analogues (5-FU vs. FT, r = 0.851; 5-FU vs. UFT, r = 0.746; 5-FU vs. HCFU, r = 0.685). In 9 tissues resistant to 5-FU, 2 (22%) were sensitive to FT, 4 (44%) to UFT, 5 (56%) to HCFU and 7 tissues (78%) to at least one of these analogues. These results suggest that the SRC assay is useful for predicting the effective drug among 5-FU and 5-FU analogues, for individual patients with cancer.     

Human colorectal carcinoma is more sensitive to HCFU than 5-FU and tegafur in in vitro and in vivo drug sensitivity tests

The sensitivity of human colorectal cancer to 5-fluorouracil (5-FU) and its derivatives: 1-(tetrahydro-2-furyl)-5-FU (tegafur) and 1-hexylcarbamoyl-5-FU (HCFU) was determined by in vitro succinate dehydrogenase inhibition (SDI) test and in vivo subrenal capsule (SRC) assay. Using the SDI test with 25 colorectal cancer specimens, the succinate dehydrogenase (SD) activity decreased to 73.6 +/- 17.8% for 5-FU-treated cells and 37.2 +/- 17.0% for HCFU-treated cells, compared to that of control cells. The chemosensitivity-positive rates were 16% for 5-FU and 68% for HCFU. Using the SRC assay with 7 colorectal cancer specimens, the relative variation of tumor size, which was calculated by delta TS/TS0 = (TS6-TS0)/TS0 X 100 (%), decreased to -12.6 +/- 10.1% for 5-FU, -14.9 +/- 12.4% for tegafur and -23.9 +/- 14.2% for HCFU, and the inhibition of tumor growth following exposure to HCFU was evident. The chemosensitivity-positive rates were 49% for 5-FU, 57% for tegafur and 71% for HCFU. Our results show that HCFU is more effective to colorectal cancer than 5-FU and FT-207, and the chemosensitivity test of HCFU will be useful in determining the effective drug for the treatment of colorectal cancer.     

5-FU and HCFU concentrations in serum and tissues in hepatocellular carcinoma after HCFU oral administration

Before proceeding with a resection of a hepatocellular carcinoma (HCC) in patients, the concentrations of 5-FU and 1-Hexylcarbamoyl-5-fluorouracil (HCFU) have been measured in the serum, the liver, and in the cancer tissue after an oral administration of HCFU (300 mg). The maximum 5-FU value was found to be 3.45 +/- 3.21 micrograms/ml (n = 22) at 2 hours, and this value decreased linearly. The concentrations of 5-FU in the liver tissues were 0.02 +/- 0.01 micrograms/g (n = 16) and the concentrations of 5-FU in cancer tissues were 0.03 +/- 0.01 micrograms/g (n = 16). There were no statistical differences found between cirrhotic patients and non-cirrhotic patients. These results indicate that the concentrations of HCFU in the serum and tissues were not influenced by liver damage.     

HCFU and 5-fluorouracil levels in the blood and tissue of hepatocellular carcinoma after oral administration of HCFU

HCFU was orally administered to 14 patients with hepatocellular-carcinoma, (including 11 patients with liver cirrhosis) and evaluated of HCFU and 5 fluorouracil (5-FU) levels. Blood and tissue 6-8 hr. after oral administration. The concentration of 5-FU in tissue was almost in the effective levels. In addition, the 5-FU level in the tissue of hepatocellular carcinoma tended to be higher than in non cancerous portion of the liver. 5-FU tissue concentration was not correlated with various laboratory data for the liver function (K-ICG, T. bil, GOT, GPT, etc.) From these results, it is suggested that HCFU is a useful anticancer agent for hepatocellular carcinoma especially for the cases accompanied liver cirrhosis.     

Effects of HCFU and TNP 470 on liver metastasis of BALB/c retroperitoneal sarcoma (LMFS)

Combination therapy using HCFU and TNP 470, which inhibits angiogenesis, was examined for effectiveness against the footpad injection model of LMFS tumor. This LMFS, a retroperitoneal sarcoma of BALB/c mice, proliferated at the inoculation site (100% take) and all mice operated on after day 15 had spontaneous metastatic nodules in the liver. Mice with the LMFS tumor were given HCFU and 5-FU (5 days/week), and/or TNP 470 (3 days/week) from day 3 for 3 weeks and sacrified at day 28 under anesthesia. Seven of 10 mice receiving 5-FU and TNP 470 died from the side effects of the drugs. Mean tumor weight and liver metastatic nodules were determined and compared with a control group. The results were as follows: HCFU group: 94.6%, 11.8%, 5 FU group: 73.9%, 28.8%, TNP 470 group: 67.6%, 44%, HCFU and TNP 470 group: 33.3%, 6.4%. Mice with LMFS were given HCFU and/or TNP 470 from day 3 for 4 weeks. The foot on the injected side was amputated on day 15, and the animals were sacrified on day 35. Liver metastatic nodules compared with those of the operation (OP) group as follows: OP + HCFU group: 14.4%, OP + TNP group: 39.1%, and OP + HCFU + TNP 470 group: 5.4%. Histologically, 5 of 5 mice of the OP group, 3 of 5 of the OP + HCFU group, 4 of 5 of the OP + TNP 470 group and 1 of 5 of the OP + HCFU + TNP 470 group had liver metastases. These results show that while either HCFU or TNP 470 is effective by itself, a combination of these drugs is more effective against liver metastasis.     

Combined effects of interferon alpha-A/D with fluoropyrimidine derivatives in the subrenal capsule assay

The combined effects of interferon alpha-A/D (IFN alpha-A/D) with 5-fluorouracil and fluoropyrimidine derivatives such as 1-(2-tetrahydrofuryl)-5-fluorouracil (tegafur), UFT, 1-hexylcarbamoyl-5-fluorouracil (HCFU) and 5'-deoxy-5-fluorouracil (5'-DFUR), were examined by 4-day subrenal capsule assay. Four human tumor xenografts serially transplanted in athymic mice, H-111, SH-10 established from gastric cancers, CH-4 and CH-5 from colon cancers, were used. In this experiment, the adequate dose of each agent was estimated as 50 mg/kg for 5-FU, 473 mg/kg for tegafur, 433 mg/kg for UFT, 50 mg/kg for HCFU, 185 mg/kg for 5'-DFUR and 1 x 10(5) IU for IFN alpha-A/D, respectively. When synergistic, additive and subadditive effects were defined as positive combined effects, all combinations produced positive combined effects against H-111 and CH-5, while negative ones were observed for all combinations against CH-4. The combinations of 5-FU, HCFU and 5'-DFUR with IFN alpha-A/D produced synergistic effects against SH-10. These results indicate that the combination therapy of 5-FU and fluoropyrimidine derivatives with IFN alpha-A/D would be useful.     

A case of leukoencephalopathy caused by HCFU

After a hysterectomy, a bilateral salpingo-oophorectomy, two courses of intra-peritoneal chemotherapy of Cisplatinum, Carmofur (HCFU, 600 mg/day [per os]) were given a patient who had ovarian granulosa cell tumor (malignant, stage Iai). Dizziness and loss of consciousness developed about 60 days after administration of HCFU, and leucoencephalopathy was diagnosed. A CT revealed a diffuse low density area in the white matter of the cerebrum. Myelin Basic Protein in the spinal fluid was found to amount to 9.8 mg/dl, which in norm. person is less than 4.0 mg/dl. Also, it showed parallel changes with the course of the clinical findings. HCFU easily dissolves to fat and changes to 5-FU without enzymes in the liver cell. Further HCFU also passes through Blood Brain Barrier to Produce 5-FU and its derivatives, in which the alpha-Fluoro-beta-Alanine is thought to be the culprit that brings on leucoencephalopathy. Even so, HCFU should be dosed when needed in spite of this risk. Other 5-FU modifiers also have been reported to produce the same effect in several cases.     

Influence of immunopotentiators on the activation of 5-FU derivatives

As it has been shown that various kinds of immunopotentiators inhibit the hepatic microsomal drug-metabolizing enzymes and reduce the activation of masked compounds such as tegafur, we investigated the influence of immunopotentiators (OK-432 and PSK) on the activation of carmofur (HCFU), which is known to be transformed spontaneously in to the tumor-active substance, 5-fluorouracil (5-FU), in vivo, and compared the results to those obtained for tegafur. After oral administration of tegafur, the area under the plasma concentration curves of 5-FU in rats pretreated with OK-432 and PSK were 72% and 79% of those in untreated rats, while in the case of HCFU almost similar plasma concentration curves for 5-FU were obtained in both treated and untreated rats. These results show that the activation of HCFU to 5-FU is not influenced by treatment with these immunopotentiators. Furthermore, our clinical investigation showed that the survival curves of cancer patients given combination therapy of tegafur with OK-432 tended to be lower than those given a single therapy of tegafur, supporting the above mentioned basic results.     

Effects of urokinase on the transfer of 1-hexylcarbamoyl-5-fluorouracil (HCFU) into the blood, bile and pancreatic juice

We studied effects of urokinase (Uronase) on the transfer of an oral anticancer agent, 1-hexylcarbamoyl-5-fluorouracil (HCFU) into blood, bile and pancreatic juice in 5 patients in whom pancreaticoduodenectomy has been performed for cancer of the periampullary region, and who had been simultaneously provided with drainage of the bile duct and pancreatic duct. Following oral administration of 500 mg of HCFU, HCFU and 5-FU concentrations in blood reached a peak at 2 hours, those in bile at 4 hours, and those in pancreatic juice at 4 to 6 hours. The administration of 24,000 IU of urokinase in combination with HCFU resulted in increased HCFU and 5-FU concentrations in blood, bile and pancreatic juice--the HCFU concentration in bile increased to 3 times and that in pancreatic juice, to about 5 times the level in the some counterparts in urokinase--untreated patients. These changes seemed to have resulted from the acceleration by urokinase of distribution of the anticancer agent into the organs.     

Inhibition of HCFU absorption after resection for gastric cancer--application of hydroxyaluminium gel]

HCFU (carmofur; Mifurol) is an 5-FU analog. The maximum blood concentration of HCFU in HCFU fraction (Cmax) after gastric resection was higher than before resection. Hot sensation and pollakiuria, characteristic side effects of HCFU, are dependent on concentration of HCFU fraction in blood. Therefore, it is considered that the frequency of occurrence of side effects after gastric resection is high. For that reason, we thought that if absorption of HCFU could be reduced, fewer side effects would result. We focused on the fact that drugs which include aluminium gel may decrease absorption in combined drugs, and thought it would be possible to delay absorption in HCFU by using them. We studied the HCFU concentration in the HCFU fraction and 5-FU concentration in blood, respectively, in two cases: 1) single oral administration of HCFU 100 mg and 2) coadministration with hydroxyaluminium gel (ALG) 10 ml in the whole or partial resection of gastric cancers for 8 patients. We found that the concentration of HCFU in the HCFU fraction 2 hours after its administration decreased significantly: 3.24 +/- 1.78 (single administration), 1.37 +/- 0.91 (coadministration with ALG) (p = 0.023). HCFU concentration in the HCFU fraction seemed to decrease for coadministration with ALG in the area under the time-blood concentration curve (AUG) (p = 0.071). The 5-FU concentration did not seem to decrease in either case. From these results, the coadministration of HCFU with ALG seems to be effective for the inhibition of adverse drug reaction after the resection of gastric cancers.     

Cerebrospinal fluid distributions of systemically administered fluorinated pyrimidines

Transfer of systemically administered fluorinated pyrimidines (Tegafur, TAC-278, HCFU and FD-1) to cerebrospinal fluid was studied in 7 patients primary brain tumors. Seven patients had had irradiation and also had V-P shunt operation for hydrocephalus 5-FU concentration in CSF was extremely high in FD-1 and TAC-278 administration, but not in Tegafur and HCFU administration. In addition, Tegafur and HCFU did not reveal any cumulative effects of 5-FU in CSF by continuous prolonged systemic administration. The facts suggest strongly the usefullness of the agents in the treatment of intracranial neoplasms, which have high CSF concentrations. However, intermediate metabolites of 5-FU in CSF are different from those in systemic pathway, and FD-1 and TAC-278 produce CNS toxicities. Therefore, further extensive studies are necessary to utilize these agents for the treatment of intracranial neoplasms.     

Randomized controlled trial of the efficacy of adjuvant immunochemotherapy and adjuvant chemotherapy for colorectal cancer, using different combinations of the intracutaneous streptococcal preparation OK-432 and the oral pyrimidines 1-hexylcarbamoyl-5-fluorouracil and uracil/tegafur

Background We investigated the efficacy and safety of adjuvant immunochemotherapy and adjuvant chemotherapy for colorectal cancer, using different combinations of the intracutaneous streptococcal preparation OK-432 and the oral pyrimidines 1-hexylcarbamoyl-5-fluorouracil (carmofur, HCFU) and uracil/tegafur (UFT).Methods Patients with stage II, III, or IV (Dukes B, C) colorectal cancer were enrolled and randomly assigned to one of three groups: an immunochemotherapy group (mitomycin C [MMC] + 5-fluorouracil [5-FU] + HCFU + OK-432), a chemotherapy group (MMC + 5-FU + HCFU), and a control group (surgery alone) for those with colon cancer (study 1); and an immunochemotherapy group (MMC + 5-FU + UFT + OK-432), a chemotherapy group (MMC + 5-FU + UFT), and a control group (surgery alone) for those with rectal cancer (study 2).Results A total of 760 patients with colon cancer and 669 patients with rectal cancer were entered into this randomized clinical trial (RCT). The incidence of side-effects was in the order of: immunochemotherapy group chemotherapy group control group in both the cohort of patients with colon cancer and the cohort with rectal cancer. In particular, the frequency of leucopenia and skin disorders was significantly higher than control groups. There were no severe adverse events such as death related to the adjuvant therapy. In both the colon cancer and rectal cancer cohorts, no significant difference in the 5-year survival rate and disease-free survival rate was noted among the three groups.Conclusion The results of an RCT demonstrated that the combination of MMC + 5-FU + HCFU + OK-432 for colon cancer and that of MMC + 5-FU + UFT + OK-432 for rectal cancer could not prolong the survival of patients with surgically resected colorectal cancer, but that both combinations were well tolerated as adjuvant therapy.     

Subrenal capsule assay using nude mice as a predictor of the response of the gastric cancer to chemotherapy

Feasibility of utilizing human gastric cancers as first transplant generation xenografts in nude mice for determining tumor sensitivity to chemotherapeutic agents was demonstrated by applying subrenal capsule (SRC) assay. A total of 55 human gastric tumors from patients were tested in this assay. Mitomycin-C (MMC) and hexycarbamyl-5-FU (HCFU, 5-FU derivative) were selected for the treatment of these patients after surgery and also for this assay as first transplant. Evaluable rate of MMC in this assay was 92.7% and that of HCFU was 90.9%. Sensitivity of tumors to MMC was 25% and to HCFU was 32%. Correlation between response to chemotherapy of human tumors in patients and in nude mice was 78.6%. These results indicate that this assay could predict effective drugs for patients with gastric cancer.     

Pharmacological and pharmacodynamic aspects of cancer chemotherapy, with special reference to 5-fluorouracil and its derivatives

5-FU analogs being investigated in Japan are; 5-FU, FT, FD-1, HCFU, UFT, TAC-278, 5'-DFUR, FF-705, and TK-117. They exert antitumor activity via 5-FU which is an intermediate metabolite of those derivatives. In this paper, UFT, TAC-278, and 5'-DFUR, were particularly investigated from the point of view of pharmacokinetics of those drugs (or 5-FU) in the blood, normal and tumor tissues of patients with gastric cancer. As a result, it is concluded that observation of the sequential changes of 5-FU level in various tissues of humans is potential in designing cancer chemotherapy.     

A multicenter randomized study comparing 5-fluorouracil continuous infusion (ci) plus 1-hexylcarbamoyl-5-fluorouracil and 5-FU ci alone in colorectal cancer

To verify the effectiveness of oral 1-hexylcarbamoyl-5-fluorouracil (HCFU) in improving the surgical cure rate in advanced colorectal cancer, a multicenter randomized comparative study was conducted. A total of 429 patients who had had curative resection for stage II and III colorectal cancer were randomly assigned to a study group receiving a 14-day course of 5-FU continuous infusion (320 mg/m2/day) followed by oral HCFU for a year (300 mg/day), or to the control group receiving a 14-day course of 5-FU continuous infusion alone. In terms of background factors, no significant differences were found between the 214 patients in the study group and the 215 in the control group. Adverse reactions during the treatment were more frequently seen in the study group. But with few exceptions, the toxicities were mild and the compliance was acceptable. The 5-year overall survival rate of the study group was similar to that of the control group. The 5-year disease-free survival rate of the study group was better than that of the control group in the patients with colon cancer (hazard ratio=1.87; 95% confidence interval 1.03-3.38; p=0.037). However, this benefit was not seen in the patients with rectal cancer. A significant improvement in the disease-free survival rate was demonstrated through the addition of HCFU to 5-FU continuous infusion for the patients with colon cancer. The usefulness of oral fluoropyrimidine as an adjuvant for curative surgery for colon cancer was further warranted.