Effects of anticancer drugs on folate disposition and excretion.
The nutritional status of cancer patients may be affected due to a variety of factors like poor intake, poor absorption and poor retention. The tumour, by itself or through tumour-associated hormones, may increase the requirements of specific vitamins. Circulating levels of thiamine were found to be low in patients having either bronchogenic carcinoma or carcinoma of the breast. They were found to be depleted further during the treatment with 5-fluorouracil.,
Folic acid deficiency is common in patients with head and neck cancer and also occurs in patients with liver cancer. Low serum folate levels have also been reported in patients with leukaemias,,, lymphomas,, and carcinoma.,
Barford et al, in their studies in normal Wistar rats, showed that methotrexate (MTX) affects the uptake of C and H-Folic acid in a dose-dependent manner.
The folates, on reduction in the body, participate in various single carbon transfer reactions. It was thought to be of interest to see how the folate disposition and excretion are affected by cytotoxic anticancer drugs in tumour-bearing animals.
5-Fluorouracil (5-FU) and cyclophosphamide (CTX) were purchased from Biochem Pharmaceutical Industries, Bombay, India; while methotrexate (MTX) was obtained from Lederle Parenterals Inc; Carolina, Peurto Rico 00630, U.S.A. All drugs were in sterile injection vials, ready for use. Dilutions, when necessary, were made with normal saline.
Tritium labelled folic acid
H-Folic acid (2500 mCi/mmol), labelled non-specifically, was purchased from the Bhabha Atomic Research Centre, Trombay, Bombay, India.
Animals and tumours
C57BL/6 male mice (6-8 weeks old and 20-25 g in weight) were used. Lewis Lung Carcinoma (LL), maintained subcutaneously by serial transplantations, was used for the uptake studies.
C57BI,/6 mice, bearing a 14-day-old LL tumour, were randomized, divided into four groups of six mice each and fed colony diet (Composition: Cracked wheat (70%), cracked Bengal gram (20%), yeast powder (4%), fish meal (5%), shark liver oil (0.25%) and sesame oil (0.75%). The mice in the treated-groups received, as a single injection, one of the following anticancer drugs by i.p. route: 5-FU (250 mg/kg); MTX (12.5 mg/kg) or CTX (180 mg/kg); while those in the control group received the vehicle by the same route. Twenty-four hours after drug treatment, each mouse received 5 µCi of H-folic acid by the oral route and the animals were kept in metabolic cages. Diet was withheld; while water was given ad libitum during the period in metabolic cages.
Urine was collected in two aliquots, 0-6 hours and 6-24 hours and the animals were killed at the end of 24 hours. The liver and tumours were taken out separately into beakers, chopped fine and lyophylised.
The percentage folate uptake and excretion were determined by counting the lyophilised tissue and the urine in a Beckman LS-100 scintillation counter. Fifty µl urine was counted as such in 10 ml of Bray's scintillation mixture, while the lyophylised tissue (about 15-20 mg) was first digested with 0.2 ml of 2N KOH. The volume of urine was non-quenching whereas the quenching due to KOH was corrected by the use of internal standard method.
The per cent folate uptake by the liver was calculated per tissue as a whole. However, the tumour levels were calculated on per g tumour weight basis to account for the variation in the tumour weight due to the drug treatment.
The liver folate uptake in the case of 5-FU treated mice was significantly higher than that in controls (p < 0.001) [Table - 1]. However, the tumour uptake of the 5-FU treated group was not significantly higher than the control [Table - 2]. The urinary excretion of the label in control and 5-FU treated mice was comparable [Table - 3]
In the case of MTX-treated mice, the liver folate uptake of the treated group was significantly lower than the control group (p < 0.001) [Table - 1] The difference in the tumour uptake values of the treated and control groups was not statistically significant [Table - 2] However, an increased urinary excretion of the label was observed in the MTX-treated mice [Table - 3].
As regards the CTX-treated mice, although the liver uptake of the labelled folate was higher in the drug-treated group than that in the controls, it was not statistically significant [Table - 1]. Urinary excretion in CTX-treated mice was slightly lower when compared with that in the control animals [Table - 3].
The levels of labelled folate in the CTX-treated LL tumours, however, were higher, the values being statistically significant (p < 0.025) [Table - 2]
The increased liver folate levels in 5FU treated mice could be explained. on the basis of the mechanism of action of the drug. The formation of a covalently bound ternary complex of 5-FU, 5, 10-methyleneTHF and thymidylate synthetase is necessary for the cytotoxicity of 5-FU., In fact, due to the deprivation of reduced folate by MTX, activity of the combination of MTX followed by 5-FU was found to be less than additive in some schedules.,
Further, it has been shown by in vitro studies using S-180 and HEP-2 cell lines that the sensitivity of the cells is not related to the intracellular levels of 5-FU, but coincides with high levels of folates. Evans et al have shown maximum potentiation of growth inhibition by 5-FU when used in combination with excess folates. Higher intracellular levels of 5, 10-methylene THF are necessary for the pharmacological action of FdUMP with thymidylate synthetase, which can be a limiting factor under certain conditions. Further, potentiation of anticancer activity of 5FU with leucovorin against Friend leukaemia virus could be due to the over coming of this limitation in the metabolic activity.
Machover et al observed that some colorectal tumours, which were previously resistant to 5-FU were rendered sensitive to the drug when used in combination with high doses of folinic acid.
Our studies show a larger amount of the labelled- folate in the liver of mice treated with 5-FU [Table - 1]. Weber, in his studies with different hepatoma cell lines and having different growth rates, has shown a definite link between the increased growth rate and the thymidylate synthetase activity.
The foregoing facts conclusively show the presence of reduced folates as a prerequisite for the anticancer activity of 5FU. The concomitant administration of leucovorin with 5-FU could be avoided here by administering folic acid, as the reducing capacity of the host is not affected by 5-FU. Avoiding the use of leucovorin is also desirable due to its high cost and less stability. Liver is endowed with a high amount of dihydrofolate reductase. Hence, the results suggest a possible use of 5-FU in combination with high doses of folic acid in hepatomas.
5-FU is weakly active against Lewis lung carcinoma. Our studies do not show a significant difference in the tumour uptake values of folic acid in the control and 5-FU treated mice [Table - 2].
As regards CTX, the folate levels of the drug-treated animal tumours were significantly higher than the untreated control tumours [Table - 2]. It would be of interest to see as to why there should be an increased demand for folic acid.
Our results of increased urinary excretion of folic acid [Table - 3] and lower uptake by the liver [Table - 1] in the case of MTX-treated mice are predictable on the basis of literature reports. Folate levels in tumours of MTX-treated and control (non-treated) animals did not show statistically significant difference [Table - 2]