Studies on the antitumour action of a synthetic immunoadjuvant - maltosyl palmitate

Abstract

Eight different mono- and di-saccharides were converted to sugar acyl palmitates by esterification with palmitoyl chloride and of these maltosyl palmitate showed the highest adjuvant effect on humoral antibody production in rabbits against sheep red blood cells. Several maltosyl palmitates were produced as indicated by TLC. They constituted a variation in the number of palmitic acid residues per maltose molecule, and they formed aggregates with one another. The major product, band IV, bas an Rf of 0.46 and a ratio approximating four palmitate residues per maltose molecule. This glycolipid exhibited no acute toxicity as shown by the limulus lysate assay, the chick embryo lethality test and high dose administration in Swiss mice (10 mg) and Fischer rats (1 mg). Preliminary studies showed that no chronic toxicity was observed upon administration of 50 mg of the material, three times per week to Fischer rats as shown by evaluation of food and water intake, weight gain, haematological analyses, biochemical analyses of serum and urine, and the histological examination of various tissues and organs. Also, there was no decrease in survival times as a result of this treatment carried out over several months in three species of mice (AKR/J, A/J, C3H/HeJ). The TD50, originally obtained, of MAT cells was found to be 1.6 X 10 (exposant 3) at 4 weeks post-inoculation which was reduced upon storage at -80 °C and after several passages in Fischer rats to 10 (exposant 2). The best glycolipid treatment was seen to be that administered on the day of tumour cell inoculation and continued every third day thereafter. This showed the least number of tumour takes and the smallest increase in tumour size. A dose of 10 mg of glycolipid was seen to be the most effective dose in three tumour-host Systems (MAT-Fischer rat, C12TSV5S-Syrian hamster, B16-C57BL/J mice). The glycolipid also showed good antitumor activity against two other tumour-host Systems: NH (Sprague- Dawley rats) and L26 (BALB/c mice) tumours. Labelled glycolipid localised in organs of immunological importance in the Fischer rat, e.g. spleen, bone marrow, liver as well as in the tumour after s.c. administration to tumour-bearing animals. Maltosyl palmitate showed the highest mitogenic activity for Fischer rat spleen cells as compared to palmitates derived from other sugars. This product also showed increased mitogenic activity of the spleen cells of Swiss, AKR/J and BALB/c mice but not for those of the Syrian hamster or the C57BL/J mouse. Macrophage activation by glycolipid in vitro showed increased cytotoxicity against MAT cells. The in vitro cytotoxicity of macrophages and spleen cells of Fischer rats and Syrian hamsters was increased against MAT or C12TSV5S cells, respectively, when injected in vivo with tumour cells and the glycol lipid, administered separately. Immunisation with neuraminidase-treated, reduced and acetylated or irradiated MAT cells was unsuccessful. No protection was afforded by such treatment against subsequent exposure of the Fischer rats to the MAT. Combination of glycolipid treatment with two LPS preparations resulted in synergy of the effects of the two immunoadjuvants as seen by decreased tumour size and a reduced tumour growth rate. Intralesional inoculation of oil-in-PBS emulsions of glycolipid and/or BCG into large MAT tumours resulted in the greatest increase in survival on treatment with glycolipid followed in efficacity by treatment with BCG or a combination of the two. In the case of tumours of a small size, the combination of the two immunoadjuvants was seen to be the most effective in increasing survival, the next best survival was seen by treatment with glycolipid alone. The effectiveness of this glycolipid, band IV as an antitumour immunoadjuvant was verified by comparison with other known immunopotentiators, all of which showed delayed tumour appearance and some tumour growth inhibition. Of these, glycolipid was judged to be the best followed by levamisole, BCG, pyran copolymer and Corynebacterium parvum in order of decreasing efficacy. Différent maltosyl palmitate bands showed differing activities - bands II, IV and VI were mitogenic for Fischer rat spleen cells; bands IV and VI, injected in vivo along with sheep red blood cells, showed increased PFC response of splenic lymphocytes as compared to animals injected with sheep red blood cells alone. Band IV showed the best antitumour activity as seen by reduced tumour takes and decreased tumour size; band VI was effective but less so than band IV; band III, which showed low mitogenicity, showed some antitumour activity though not at the level of either bands IV or VI. A commercially available glycolipid sorbitol monopalmitate also showed good mitogenicity, increased PFC response and an antitumour activity equal to that of band IV and was seen to be better than sorbitol substituted with other fatty acids. Maltosyl palmitate proved to be a useful adjunct to surgical removal of tumours in Syrian hamsters and Fischer rats in 2 out of 3 experiments and 5 out of 6 experiments, respectively, as seen by reduced tumour resurgence and reduced growth of the recurrent tumours. The best result was achieved by the administration of glycolipid twice prior to surgical excision and twice weekly subsequently. When glycolipid was used in conjunction with chemotherapy, the combination with bleomycin gave the best results. Preliminary results are presented for the identification of tumour antigens, separated by lEF, of tumour cell membrane polypeptides, their isolation and testing of their capacity to elicit inhibiton of macrophage migration obtained for tumour-sensitized animals. An age-dependent résistance to MAT cell growth was seen in Fischer rats. There was increased résistance in 40-day, 8-month and 14-month old animals and increased susceptibility to tumour growth in 2-month and 10- to 12-month old rats.