Efficacy of 5-fluorouracil in combination with methoxyphenyl maleamic acid in murine tumors.
Combination chemotherapy studies were carried out in vivo against sarcoma 180 (ascites)(S180) and Ehrlich (ascites) carcinoma (EAC) tumours using cytotoxic drugs and methoxyphenyl maleamic acid (MPMA), an intermediate in the synthesis of pyrrolidine-nitrogen-mustards. Preliminary data have suggested that the combination of 5-fluorouracil (5-FU) and methoxyphenyl maleamic acid (MPMA) was more active than 5-FU used singly against EAC tumour. The possible therapeutic potential of this combination was further investigated in EAC tumour.
Anticancer drugs have well known therapeutic limitations, which have continued to stimulate the search for new agents with enhanced therapeutic efficacy. Considerable efforts have been directed towards the generation of unique compounds with biological characteristics unshared by related anti-proliferative drugs especially those in clinical use. Although, most of them have been reported to be effective agents available for the treatment of human cancers, they have been the subject of intense and extensive research directed towards understanding the mechanisms of therapeutic and cytotoxic effects.
Earlier studies recognized that the growth of both normal and neoplastic cells is affected by intracellular levels of chemotherapeutic agents. Differences in cell kinetics, pharmacokinetics and membrane transport are significant to permit pharmacological manipulations to find vastly improved selective toxicity for cancer cells. Modification of drug activity, however, involves the use of modulating agents that may offer selective protection against toxicity of normal tissue without compromising anti-tumour activity.
In our earlier study, we observed that methoxyphenyl maleamic acid (MPMA) potentiated the activity of cyclophosphamide against P388 leukaemia. This finding raised the possibility that MPMA may be useful in combination with cytotoxic drugs in other rodent tumours. To test this possibility, we investigated the ability of NIPMA against S180 (ascites) and Ehrlich (ascites) carcinoma when used in combination with cytotoxic drugs.
We now report the results of our study in this paper.
Methoxyphenyl maleamic acid (MPMA) s obtained as an intermediate during the synthesis of pyrrolidinedione-nitrogen- mustards,. Anticancer drugs were purchased from the local market and their LD10 doses were used. Test solutions were freshly prepared either in suspension form (0.2% Carboxymethyl cellulose) or in normal saline.
Sarcoma 180 (ascites) (S180):
Swiss (Webster) mice of either sex (weighing between 20-24 g) were used. Tumour cells were harvested from the peritoneal cavity of animals with a 7-day-old S180 (ascites) tumour under aseptic conditions. Each recipient mouse received 2 x 10 cells by the intraperitoneal route,.
Ehrlich (ascites) carcinoma (EAC):
Swiss (Webster) mice of either sex (weighing 20-25 g) were used. Tumour cells were harvested from the peritoneal cavity of animals with a 7-day-old EAC tumour under aseptic conditions. Each recipient mouse received 5 x 10 cells by the intraperitoneal route.
The animals were randomized in groups of six. They were fed normal colony diet. (Composition: Cracked wheat (70%), cracked Bengal gram (20%), yeast power (4%), fish-meal (5%), shark liver oil (0.25%) and sesame oil (0.75%) and were given water ad libitum.
Treatment protocol and evaluation:
The administration of MPMA singly or in combination with conventional anticancer drugs was initiated (day 1) and continued on days 5 and 9. The controls received the vehicle (0.2% carboxyinethyl cellulose (w/v) or distilled water/normal saline) by the same route.
The body weights of both control and treated animals were recorded on days 1 and 5 to determine toxicity, if any. The median survival time (MST) of animals treated with either anticancer drug alone or with MPMA and anticancer drug administered simultaneously at the same dosages were compared with each other. The efficacy of co-administration was determined on the basis of increase in the survival time of treated group (T) as compared to that of control group (C) using the following expression.
Median Survival Time (days) of treated animals (T)
--------------------------------------------------------------- x 100
Median Survival Time (days) of treated animals (C)
T/C ? 125 indicates anti-tumour activity.
Modification factor (MF) was determined from the MST of combined treatment group divided by the MST of known cytotoxic drug treated group.
The statistical significance of differences between the combined treatment group and the anticancer drug-treated group was calculated by Student's T test and expressed as p values.
The effects of the combinations of cytotoxic drugs and MPMA were studied in two murine tumour models. The combinations were not effective against S180 (ascitic) tumour. However, the combination of 5-FU and MPMA was of promising therapeutic value, particularly in EAC tumour. The addition of MPMA resulted in an almost two- fold increased efficacy of 5-FU. Combinations of other cytotoxic drugs with MPMA did not show any additional therapeutic benefits (Table 1).
A significant consequence of MPMA in combination with 5-FU was that the survival period was not only markedly, increased but it persisted as such through out the treatment period resulting in cures of some tumour bearing animals. These results were further confirmed by using different treatment schedules of 5-FU and MPMA which employed fractionated doses of both 5-FU and MPMA (Table 2). Starting with the dose of 100 mg/kg, the combined activity started diminishing gradually. The lower doses of MPMA were not effective.
The effect of combined treatment on a well-grown EAC tumour was also studied. Injections were given i.p. on days 5, 9 and 13. In this, the inability of MPMA to prevent growth inhibition was observed. It may be mentioned here that the addition of MPMA to 5-FU did not increase host toxicity as judged by data on body weight loss in the combined treatment group.
The biologic and pharmacologic basis of combination chemotherapy has been widely studied in laboratory animals. From a clinical point of view, the combination of several anti-neoplastic drugs is aimed towards (i) increasing therapeutic synergism by exploiting different mechanisms of action with subsequent improvement of therapeutic activity, (ii) preventing or delaying the emergence of resistant cell clones through the mechanism of action of drugs used, (iii) increasing patient's tolerance to the toxic effects of the drugs by properly varying their dosages and (iv) making use of differing pharmacologic characteristics of various compounds to achieve rapid and complete regression without toxicity to the host.
A number of investigators have examined the synergism of pharmacological compounds and therapeutic agents both in vitro and in vivo to enhance tumour eradication. Our previous study has shown that the combination of MPMA with certain anticancer drugs has enhanced their cytotoxic effectiveness . The present study has shown that the combination of 5-FU with MPMA has resulted in an increased anti-tumour effect of 5-FU against EAC tumour as compared to the 5FU administered alone. Although MPMA, of its own, did not possess anti-tumour activity, the observed interaction between MPMA and 5-FU appears to be a synergistic effect rather than the additive one.
A study of various doses of 5-FU, administered concomitantly with MPMA, confirmed the efficacy of this combination in the treatment of EAC tumour (Table 2). This combination, however, did not appear to be of much therapeutic value in further regimens used. An increased life expectancy could not be obtained after MPMA administered with higher doses of 5-FU in an advanced EAC tumour. The lower cytotoxic activity of other combinations might be ascribed to a reduced cellular uptake of these drugs as compared to 5-FU or to intracellular degradation or repair mechanisms.
The present data indicate that MPMA acts as a biochemical modulator for the intracellular accumulation of 5- FU. Thus modulatory effect may be due to a higher rate of phosphorylation rather than the decreased one, hence a greater rate of 5-FU phosphorylation and 5-FU accumulation. It would also contribute to an increased incorporation of 5-FU nucleotide into RNA. This possibility is currently under investigation.
An equally significant implication from our study is that lesser amounts of 5-FU may be required when co-administered with MPMA. This combined approach has produced a larger percentage of cure in mice bearing EAC tumour than a method which uses only a single cytotoxic drug. The apparent lack of toxicity makes it an attractive drug combination for possible use in the modulation of anti-tumour activity of 5-FU in vivo, if appropriate tissue concentrations of the drug could be achieved.
The 5-FU uptake data will indicate that MPMA will probably increase the net cellular content of 5-FU in malignant cells in vivo only if 5-FU is present in adequate amounts outside the cell. In order to achieve greater influx than efflux, the time and method of in vivo 5FU administration should be aimed to maximize the amount of 5-FU outside the malignant cells. It may be mentioned here that since specific structural determinants are important for enhancing the activity of particular cytotoxic drug(s), different intermediates could be synthesized and tested for their potentiating properties.
The authors wish to thank Mr. MG Nirmalkar for his technical assistance and Ms. Premlata, M Kotenkar for typing the manuscript.