Conjugates of elliptinium acetate with mouse monoclonal anti-alpha-fetoprotein antibodies or Fab fragments: in vitro cytotoxic effects upon human hepatoma cell lines.

Elliptinium acetate (EA) is a new anti-cancer compound displaying cytostatic activity against various malignancies including hepatoma. Using 3 hepatoma cell lines, we compared the in vitro activity of doxorubicin (reference drug), of EA and of conjugates made up with this latter drug and monoclonal antibodies (MAbs). The linkage was performed by a direct oxidation method. Specific immunoconjugates were prepared with an anti-alphafetoprotein (AFP) MAb (AF01) or its Fab fragment (Fab AF01). Non-specific conjugates were obtained with an anti-thyroglobulin MAb (TG01) or its Fab (Fab TG01). Direct membrane injury (51Cr-release), DNA and protein synthesis as well as AFP release were investigated for all compounds. Free EA displayed only weak activity on DNA and protein synthesis, at 10-fold higher molar concentration than doxorubicin. Conjugation of EA with whole AF01 allowed significant potentiation of protein synthesis inhibition without affecting the 3 other tests. In contrast, Fab AF01 x EA conjugates displayed a marked effect in the 4 tests; in particular, this conjugate was at least 100 times more efficient than any other compound when tested in the 51Cr-release test. Neither Fab AF01 nor free EA alone or in combination exhibited such an effect. Fab TG01 x EA conjugate was not directly cytotoxic but potentiated inhibition of DNA and protein synthesis between 2- and 10-fold. The mechanism of the direct cytotoxic effect of anti-AFP Fab x EA conjugate, which has never been described in any other immunodrug model, was investigated.

Polyclonal antibodies to elliptinium acetate: structure-activity relationships and comparison with human anti-elliptinium IgM.

In order to elucidate the immune-mediated hemolytic disease induced in man by elliptinium acetate, a quaternary ammonium compound with antineoplastic activity, polyclonal antibodies directed against this hapten were raised in rabbits. The coupling step between drug and carrier was performed according to a putative human in vivo hapten conjugation mechanism. Structure-activity relationships of the resulting IgG were compared with the epitope site recognized by human anti-elliptinium IgM by using a panel of twelve elliptinium acetate analogues. Although both antibodies were directed principally against the quaternary ammonium ion, a poor correlation between the cross-reactivity indices was obtained. In fact, it appeared that both antibodies recognized specifically the ammonium group plus different regions of the molecule: the indole ring for human antibodies, the N-alkyl group and its vicinity for rabbit ones. The specificity of the obtained rabbit polyclonal antisera is discussed, with regard to the conjugation mechanism of the drug occurring in man.

Benzodiazepine receptors on human blood platelets.

Binding studies conducted on membrane preparation from human platelets using (3H) Ro5-4864 and (3H) diazepam showed specific and saturable binding. Scatchard analysis revealed a single class of binding sites with KD = 10.8 +/- 0.9 nM and Bmax = 775 +/- 105 fmol/mg protein for (3H) Ro5-4864 and KD = 10.5 +/- 1.1 nM and Bmax = 133 +/- 19 fmol/mg for (3H) diazepam. We were unable to detect any GABA binding site on crude membrane preparation, nor did GABA enhance the binding of (3H) Ro5-4864 or (3H) diazepam. This suggests that benzodiazepine receptors are uncoupled to GABA system on human platelets. Ro15-1788, a specific antagonist for "central type" benzodiazepine (BDZ) binding sites was inactive in displacing (3H) Ro5-4864 from membrane receptors, while PK 11195 (a specific ligand for the "peripheral type" receptor) was the most potent of the drugs tested in inhibiting (3H) Ro5-4864 binding. These results indicate that human blood platelets bear "peripheral-type" BDZ receptor. Moreover, we could not detect any (3H) propyl beta carboline specific binding on platelet membranes. Results on benzodiazepine receptors on human circulating lymphocytes are also reported and similarity in pharmacological properties with platelet benzodiazepine receptors is suggested.