THE USE OF LYMPHOCYTE-CULTURES FOR THE IN VESTIGATION ON THE BIOTRANSFORMATION OF DRUGS

In the present paper the rat lymphocytes and mouse myeloma cell cultur e are developed as in vitro test systems and used for the investigatio n on the biotransformation of drugs. The rat lymphocyte culture was es tablished as a suspension culture after the isolation of the cells fro m the spleen of male wistar rats by mechanical disaggregation and dens ity gradient centrifugation with a yield of 107 cells per spleen and a viability of 80%. The addition of the mitogens PHA and Con A. to the culture stimulated the proliferation of the lymphocytes leading to a d oubling of the number of cells comparing with control cultures. Myelom a cells are a permanent cell line of B-lymphocytes. The cultivation wa s caried out as stationary suspension. The marked proliferation of the cells could be increased by addition of Con A. The biochemical proper ties of both kinds of cells are qualitatively comparable. Cytochrome P -450 mediated demethylase activities could be detected, which were 5-1 0fold higher in myeloma cells. The pretreatment with the enzyme induct ors phenobarbitone and 3-methylcholanthrene as well as the addition of the mitogens PHA and Con A increased these turnover rates. Reductive and conjugating activities were not present in the cultures. The estab lished and characterized in vitro systems were applied for the investi gation on the biotransformation of 4 potential drugs. The cardiac effe ctive Trapidil (Rocornal(R)) derivative AR 12463 (5-piperidino-7-[N-pe ntyl-N-(beta-hydroxyethyl)]- amino-s-triazolo[1,5-a]pyrimidine) is tra nsformed in lymphocyte and myeloma cell cultures in two compounds. The se substances revealed as the hydroxypentyl- and the hydroxypyrimidine derivative. Both products are the initiale metabolites for further de gradation reactions in the in vivo biotransformation in the rat. The i mmunostimulator AWD 100-041 (3-(2-mercaptoethyl)quinazoline-2,4-(1 H,3 H)-dione) is metabolized in lymphocyte and myeloma cell culture to th e disulfide of the parent compound. After the incubation of the S-meth yl compound sulfoxidized metabolites originate detectable also in vivo . After the incubation of the anticonvulsant AWD 140-076 (4-chlorophen ylpyrrole-3-morpholino-2-carboxylic acid methyl ester) in the cell cul tures two metabolites are formed in which the oxidative attack takes p lace at the morpholine nitrogen as well as at the pyrrole skeleton. Bo th compounds are the main metabolites in the metabolism in vivo. The b iotransformation of the lipoxygenase inhibitor FLM 5011 (2-Hydroxy-5-m ethyl-laurophenone-oxime) in lymphocyte and myeloma cell cultures is c haracterized by the formation of the omega-hydroxy derivative. This co mpound is the initiale metabolite for the further degradation of the l auryl side chain. All substances were tested in myeloma cells concerni ng their cytotoxicity. The corresponding IC,, values amounted to 4.5 x 10(-6) mol/l for AR 12463, 1.4 x 10(-5) mol/l for AWD 100-041, 1.3 x 10(-4) mol/l for AWD 140-076 and 1.2 x 10(-4) mol/l for FLM 5011, resp ectively. It could not be pointed out a relationship between influenci ng the cell growth and metabolism.