[CANCER RESEARCH 43, 5699-5706, December 1983] Comparative Cytotoxic and Cytokinetic Effects of the Epipodophyllotoxins 4'-Demethylepipodophyllotoxin-9-(4,6-O-2-ethylidene-#-D-glucopyranoside) and 4'-Demethylepipodophyllotoxin-9-(4,6-O-2-thenylidene-/5-D- glucopyranoside) and Their Metabolites on Human Leukemic Lymphoblasts1 Lois W. Dow,2 Joseph A. Sinkule,3 A. Thomas Look, Agnes Horvath, and William E. Evans Division of Hematology-Oncology [L. W. D., A. T. L, A. H.], and the Pharmacokinetics and Pharmacodynamics Section [J. A. S., W. E. £.],St. Jude Children's Research Hospital, Memphis, Tennessee 38101 ABSTRACT We compared the effects of the epipodophyllotoxins 4'-de- methylepipodophyllotoxin-9-(4,6-O-2-ethylidene-l8-D-glucopyran- oside) (VP-16-213) and 4'-demethylepipodophyllotoxin-9-(4,6-O- 2-thenylidene-0-D-glucopyranoside) (VM-26) and several of their derivatives on cell cycle progression and viability of human leukemic lymphoblasts (CCRF-CEM). The c/s-(picro)-lactone de rivatives, like both VP-16-213 and VM-26, produced G2-phase arrest and cytotoxicity, but only at concentrations 100 times greater than required with the parent compounds. The epiagly- cone derivative showed potent cytotoxicity: at 100 to 250 nM, it reduced cloning efficiency by 50%, an effect requiring 25 to 40 nM VM-26 and 340 to 425 nM VP-16-213. In contrast to VM-26 and VP-16-213, the epiaglycone arrested cells in M, consistent with evidence that it, like podophyllotoxin, is an inhibitor of microtubule assembly. At concentrations resulting in 50% cell kill and an increase of cells in M, however, the epiaglycone produced little change in the proportion of cells in Gìor early S phase, while podophyllotoxin produced a shift of cells to mid- and late S. The hydroxy acid derivatives, although found in detectable quantities in patients' urine and serum, were inactive in vitro. Structural differences among the compounds account for their different biochemical and cell kinetic effects and, hence, different cytotoxic activities. Because the epiaglycone is a potent compound and combines activities of both the podophyllotoxins and 4'-demethyl derivatives, further studies of its cytotoxicity are indicated. INTRODUCTION The epipodophyllotoxins VM-264 and VP-16-213 are active against a variety of human tumors including acute lymphoblastic leukemia, acute nonlymphocytic leukemia, lymphomas, small-cell carcinoma of the lung, and germ cell tumors (1, 25, 30). Both 'Supported by Leukemia Program Project Grant CA 20180 and Childhood Cancer Center Support (Core) Grant CA 21765 from the National Cancer Institute and by ALSAC. 1To whom requests for reprints should be addressed, at 332 North Lauderdale, Memphis, Tenn. 38101. 3 Recipient of Grant RR-05584-18 from the Division of Research Resources, NIH. 4The abbreviations used are: VM-26, 4'-demethylepipodophyllotoxin-9-(4,6-O- 2-thenylidene-/S-D-glucopyranoside); VP-16-213, 4'-demethylepipodophyllotoxin-9- (4,6-O-2-ethylidene-/3-D-glucopyranoside); DMSO, dimethyl sulfoxide; HPLC, high- pressure liquid chromatography; TLC, thin-layer chromatography; NMR, nuclear magnetic resonance; FBS, fetal bovine serum. Received January 10,1983; accepted August 24,1983. agents are cell cycle phase-specific and cause an accumulation of cells in late S or G2 phase at concentrations below 1 to 2 UM (8, 12, 17, 23). Pharmacological studies have shown that their actions result in single-strand DNA breaks, inhibition of nucleo- side uptake, and inhibition of NADH-linked cellular respiration (11,21,22); related podophyllotoxin compounds appear to inhibit microtubule assembly and nucleoside uptake only (19). Despite extensive investigation, the precise biochemical mechanisms responsible for cytotoxicity of the epipodophyllotoxins are not clear. Studies by Creaven (4) indicate that VM-26 and VP-16-213 are metabolized extensively in humans. Less than 22% of a standard VM-26 dose and less than 67% of a standard VP-16- 213 dose are eliminated as unchanged drug in the urine. Greater than 50% of the radioactivity recovered in plasma at 48 hr after administration of tritiated VM-26 or VP-16-213 is in the form of metabolites; pharmacokinetic data suggest that as much as 86% of a single dose of VM-26 is metabolized. Similar studies indicate extensive metabolism of VP-16-213 as well. Cytotoxicity studies of drug metabolites have been limited. For example, Ozols ef al. (24) found that the adriamycinol and Adriamycin aglycone derivatives of Adriamycin were toxic to primary human ovarian cancer cells of 3 patients. Since the parent VM-26 and VP-16-213 compounds are metabolized ex tensively, the biological activity of their derivatives should help explain the mechanism of epipodophyllotoxin cytotoxicity. Al though studies of the parent compounds have been reported, data concerning the cytotoxic and cytokinetic effects of the potential metabolites are not available. In this paper, we describe the cytotoxic and cell kinetic effects of the c/s-(picro)-lactone, hydroxy acid, and epiaglycone derivatives of VM-26 and VP-16- 213 on the human lymphoblastic leukemia cell line CCRF-CEM (hereafter called CEM), in comparison with effects of the parent epipodophyllotoxins and podophyllotoxin. MATERIALS AND METHODS Drugs, Chemicals, and Equipment. VM-26 (NSC 122819) and VP- 16-213 (NSC 141540) were gifts from Bristol Laboratories (Syracuse, N. Y.). The potential metabolites of VM-26 and VP-16-213 were synthesized as described below or provided by Dr. H. Stähelin(Sandoz Ltd., Basel, Switzerland). Podophyllotoxin, trimethylsilane, deuterated DMSO, deu- terochloroform, and potassium bromide were purchased from Sigma Chemical Co. (St. Louis, Mo.). HPLC-grade methanol, DMSO, acetoni- trile, ethyl acetate, and chloroform were obtained from Burdick and Jackson Laboratories (Muskegon, Mich.). TLC plates included analytical silica gel (0.25-mm layer) commercially available from Supelco (Bellefonte, DECEMBER 1983 5699 Research. on January 22, 2022. © 1983 American Association for Cancer cancerres.aacrjournals.org Downloaded from