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In Vitro and in Vivo Enhancement of Ricin-A Chain Immunotoxin Activity by Novel Indolizine Calcium Channel Blockers: Delayed Intracellular Degradation Linked to Lipidosis Induction¹

Laboratoire de Pharmacologie et de Toxicologie Fondamentales, Centre National de la Recherche Scientifique [J-P. J., M. C., C. B., M. A., G. L.], and Laboratoire de Biochimie Medicale, Centre Hospitalier Universitaire Rangueil [T. L.], Toulouse, France; Centre de Recherche Clin-Midy-Sanofi, Montpellier, France [O. T.]; Sanofi-Labaz Research Center, Brussels, Belgium [P. C.]; and Service d'Hématologie, Centre Hospitalier Universitaire Purpan, Toulouse, France [M. A., G. L.]

With regard to increasing the clinical potential of ricin A-chain immunotoxins (RTA-ITs), a novel class of calcium channel blockers, indolizines SR33557 [2-isopropyl-1-((4-(3-N-methyl-N-(3,4-dimethoxy-ß-phenethyl)amino)propyloxy)benzenesulfonyl))indolizine] and SR33287 [isopropyl-2-((1-butylamino-3-propyl)oxy-4-benzoyl)-3-indolizine], were evaluated for their ability to enhance RTA-IT activity in vitro and in vivo. Five µM SR33287 and 5 µM SR33557 were potent enhancers of both anti-Thy 1.2 AT15E RTA-IT (84- and 64-fold, respectively) on T2 cells and anti-CD5 T101 (622- and 538-fold) and T101 F(ab')₂ RTA-IT (34- and 28-fold) on CEM III cells. This was superior to the effect achieved by both 10 µM verapamil and 10 mM NH₄Cl, albeit slightly inferior to that of 50 nM monensin and 5 µM perhexiline.

Murine T2 lymphoma cells bearing the Thy 1.2 antigen were injected i.v. in Thy 1.2 (–) BL. 1.1 mice (median survival time, 17.7 days). Intravenous treatment with 10 µg of AT15E RTA-IT prolonged the survival of mice (median survival time, 26.8 days). When 400 µg of SR33287 were coinjected i.v. with 10 µg of AT15E RTA-IT, mouse survival was further increased, with 5 of 6 mice surviving, disease free, over 42 days.

SR33287 had a significant impact on the intracellular routing of ¹²⁵I-AT15E RTA-IT, which induced a greater than 2-fold increase in intracellular intact AT15E RTA-IT at 90 min. This effect on RTA-IT half-life was distinctly different from that observed with either NH₄Cl or monensin and may be linked to the inhibition of acid lysosomal sphingomyelinase by SR33287, leading to cellular lipidosis.

In conclusion, indolizines appear to be promising agents not only for immunotoxin enhancement but also for increasing the activity of any number of targeted therapeutic agents where modifying either the intracellular routing or increasing the intracellular half-life of the ligand would be beneficial to its cytotoxic activity.

¹ This work was supported in part by Grants ARC 6891 and 6249 from l'Association pour la Recherche sur le Cancer.

² Supported by a grant from la Ligue Nationale Contre le Cancer and in part by the Philippe Foundation, Inc. To whom requests for reprints should be addressed, at Oncology Division, Room M-211, Department of Medicine, Stanford University Medical Center, Stanford, CA 94305-5306.

³ Present address: Laboratory of Molecular Biology, National Cancer Institute, NIH, Bethesda, MD 20892.

Received 8/19/91. Accepted 12/17/91.