A synthetic compound (506), 0(1-6) D-glucosamine disaccharide 1,4'-bisphosphate, which is acylated at * Corresponding author. (08:K27, Re-mutant) in both effective dose and degree of stimulation. However, compound 406, like the biosynthetic lipid A precursor, was far less active in characteristic endotoxic properties such as pyrogenicity and the activity to prepare the local Shwartzman reaction, though the compound had as high a lethal toxicity as F515 lipid A for mice loaded with galactosamine (5). Galanos et al. (6) have reported essentially the same results in experiments with the same synthetic preparations. The marked differences noted between the synthetic compound, 406, and the natural product, F515 lipid A, regarding biological activities that more or less characterize endotoxic lipopolysaccharide (LPS) and its lipid A moiety have been assumed to be attributable to the following fact: all four acyl groups bound to the amino groups at C-2 and C-2' positions 225

Summary Nebulisation chemotherapy, a chemotherapeutic method for the treatment of lung cancer that involves the administration of anticancer agents through the inhalation of nebulised aerosols, has been found to be highly effective (Tatsumura et al., 1983a,b). We confirmed that 5-FU administered by this method accumulates in the trachea, bronchi and regional lymph nodes of patients treated before surgery, along with 5-FU metabolites, FUR and FUdR, indicating that 5-FU is directly incorporated and metabolised in the respiratory tract. Parallel result were obtained using mongrel dogs. The 5-FU levels in other organs, such as the heart and liver, were found to be extremely low. Only a trace of 5-FU was found in the serum of both the patients and the dogs. We further investigated the anti-tumour effect of this therapy in ten selected patients and observed a satisfactory anti-tumour response of 60.0%. These results, along with our previous finding that the retention time of isotope tracers inhaled as aerosol is considerably longer in tumour tissues than in normal parts (Tatsumura et al., 1983a) al., 1983a,b). The present study further supports the therapeutic value of this method and presents some data which help explain how this therapy works. Materials and methodsAdministration and analysis of 5-FU in mongrel dogs The concentrations of 5-FU and its metabolites, FUR and FUdR, in the tissues and sera were measured using a recently developed, high-performance liquid chromatographic (HPLC) method (Masuike et al., 1985).Eighteen mongrel dogs weighing 15.5-16.5 kg were used in the experiment. All dogs were anaesthetised with ketamine hydrochloride (Ketalar) (10-20 mg kg-') and atropine sulfate (0.03-0.04 mg kg-'). They were then intubated, mechanically ventilated, and given supplementary oxygen. Anaesthesia was maintained by an intravenous administration of pentobarbital (5-10 mg kg-'). The inhalant was prepared by mixing 5-FU (50 mg kg-') with expectorant, was nebulised by an ultrasonic nebuliser, and was then sent into the respiratory apparatus. Oxygen was supplied to the circuit at a rate of 2 1/min and ventilation was carried out at 100-150 ml min-' at 20 times/min. This circuit carried the aerosol to the bronchial trees at the alveolar level.Cardiac arrest was induced by an intravenous injection of KCI after each procedure. Tissue samples were collected after

The structure/activity relationship of lipid A, a bioactive center of endotoxic lipopolysaccharides, in the activation of the clotting enzyme cascade of a horseshoe crab amoebocyte lysate (Limulus activity) and the complement system in human serum, was examined using synthetic lipids A and related compounds. Regarding Limulus activity, a newly developed colorimetric method, which utilizes a mixture of recombined clotting factors and a chromogenic substance, was much more sensitive for detecting changes in the chemical structure of test compounds than the conventional gelation method using the amoebocyte whole lysate. (pl-6)-~-Glocosamine disaccharide bisphosphates, which had neither 3-hydroxyacyl nor 3-acyloxyacyl groups, and acylglucosamine phosphates, which in structure correspond or are analogous to the non-reducing or reducing moieties of lipids A and biosynthetic disaccharide lipid A precursors showed only negligible activity in the colorimetric tests, but they exhibited a distinct though much weaker gelation activity than the parent disaccharide molecules. The assay results obtained by the colorimetric Limulus test correlate better with the pyrogenicity of the test synthetic compounds than those given by the gelation method, although the dependence of pyrogenicity on chemical structure is greater. The presence of 3-hydroxyacyl groups on the bisphosphorylated (j1-6)-~-glucosamine disaccharide backbone is the prerequisite for effective activation of the clotting enzyme cascade of horseshoe crab amoebocyte lysate, while the presence of an adequate number (one or two) of 3-acyloxyacyl groups on the disaccharide bisphosphate backbone is needed for full pyrogenicity.Complement activation, on the other hand, showed structural requirements quite different from those for the colorimetric Limulus activity and the pyrogenicity. The disaccharide compounds that had only non-hydroxylated acyl groups, acylated glucosamine phosphates that had the structure of the non-reducing portion of lipids A and biosynthetic disaccharide precursors, which were scarcely active in the colorimetric Limulus test, caused complement activation comparable to or sometimes stronger than that of the parent disaccharide molecules. Acylglucosamine phosphates, corresponding in structure to the reducing moiety of disaccharide compounds, however, showed little activity.A series of compounds corresponding in structure to the lipid A portions of endotoxic lipopolysaccharides of Enterobacteriaceae, their analogs and their structural components have been synthesized [l -71. Endotoxic and immunobiological activities of these synthetic compounds have been extensively studied in comparison with those of reference lipopolysaccharides and lipid A preparations of bacterial origin by a few groups including ours [8-231. A synthetic counterpart (LA-1 5-PP) of lipid A from an Escherichiu coli Re mutant exhibited full endotoxic and immunobiological activities identical to those of a reference bacterial product [lo, 15,191. The bioactivities of a synthetic compound...

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