[CANCER RESEARCH 59, 4898 – 4905, October 1, 1999] Potent Topoisomerase I Inhibition by Novel Silatecans Eliminates Glioma Proliferation in Vitro and in Vivo 1 Ian F. Pollack, 2 Melanie Erff, David Bom, Thomas G. Burke, J. Thompson Strode, and Dennis P. Curran Department of Neurosurgery, Children’s Hospital of Pittsburgh and University of Pittsburgh Cancer Institute Brain Tumor Center [I. F. P., M. E.], University of Pittsburgh School of Medicine [I. F. P., M. E.], and Department of Chemistry, University of Pittsburgh [I. F. P., M. E., D. B., D. P. C.], Pittsburgh, Pennsylvania 15213-2583, and Experimental Therapeutics Program, Markey Cancer Center, University of Kentucky Colleges of Pharmacy and Medicine, Lexington, Kentucky 40506-0286 [T. G. B., J. T. S.] ABSTRACT Although topoisomerase inhibitors, such as camptothecin and topotecan, have been widely used in the treatment of nonglial tumors, their application for gliomas has been limited by poor efficacy relative to toxicity that may in part reflect limited bioavailability and blood stability of these agents. How- ever, the potential promise of this class of agents has fostered efforts to develop new, more potent, and less toxic inhibitors that may be clinically relevant. Using a cascade radical annulation route to the camptothecin fam- ily, we developed a series of novel camptothecin analogues, 7-silylcamptoth- ecins (“silatecans”), that exhibited potent inhibition of topoisomerase I, dra- matically improved blood stability, and sufficient lipophilicity to favor blood- brain barrier transit. We explored the efficacy of a series of these agents against a panel of five high-grade glioma cell lines to identify a promising compound for further preclinical testing. One of the most active agents in our systems (DB67) inhibited tumor growth in vitro with an ED 50 ranging be- tween 2 and 40 ng/ml, at least 10-fold more potent than the effects observed with topotecan, and at least comparable with those of SN-38, the active metabolite of CPT-11. Because DB67 also exhibited the highest human blood stability of any of the agents examined, this agent was then selected for in vivo studies. A dose-escalation study of this agent in a nude mouse U87 glioma model system demonstrated a concentration-dependent effect, with tumor growth inhibition at day 28 postimplantation (the day control animals began to require sacrifice because of large tumor size) of 61  7% and 73  3% after administration of DB67 doses of 3 and 10 mg/kg/day, respectively, for 5 days beginning on postimplantation day 7. Animals that continued treatment with 10 mg/kg/day in three additional 21-day cycles all remained progression free after >90 days of follow-up but later developed enlarging tumors after treatment was stopped. However, a slightly higher dose (30 mg/kg/day) induced complete tumor regression after only two cycles in all study animals and was effective even if treatment was delayed until large, bulky tumors had developed. Application of the 30 mg/kg/day dose to treat established intracra- nial glioma xenografts led to long-term (>90 day) survival in six of six animals, whereas all controls died of progressive disease (P < 0.00001). No apparent toxicity was encountered in any of the treated animals. In summary, the present studies indicate that silatecans may hold significant promise for the treatment of high-grade gliomas and provide a rationale for proceeding with further preclinical evaluation of their efficacy and safety versus com- mercially available camptothecin derivatives. INTRODUCTION High-grade gliomas present a major therapeutic challenge (1– 4) because these poorly circumscribed, biologically aggressive lesions typically are refractory to surgery, radiotherapy, and conventional chemotherapy. Accordingly, there is a strong rationale for examining the efficacy of novel chemotherapeutic strategies as a means for enhancing disease control. Topo 3 I inhibitors are a class of agents that interfere with DNA “unwinding” during DNA replication and RNA transcription and stabilize DNA-topo I complexes through noncovalent interactions to yield enzyme-linked DNA single-strand breaks. Prolonged exposure to these agents in replicating cells produces lethal double-strand DNA breaks that can trigger the induction of programmed cell death (5). In this way, these agents function by subverting the normal topo I enzyme to “poison,” and ultimately induce cytotoxicity in, rapidly dividing tumor cells (6), which typically have high levels of topo I activity (7–9). Topo I inhibitors have shown significant promise as antineoplastic agents in in vitro studies. Unfortunately, early topo I inhibitors, such as camptothecin (10, 11), suffered from poor solubil- ity, limited bioavailability secondary to albumin binding, and rapid hydrolysis at physiological pH of the lactone ring to an inactive open form (6, 11). Subsequently, a number of camptothecin derivatives, such as topotecan and irinotecan (CPT-11), were developed that exhibited somewhat improved aqueous solubility and bioavailability (12, 13). Topotecan has shown substantial promise in preclinical and initial clinical studies with several tumor types (6, 11, 14, 15), but this activity has yet to be duplicated in patients with brain tumors (16 –19), despite the apparent cerebrospinal fluid penetrance of this agent (20). In one study that used a 24-h infusion of 5.5–7.5 mg/m 2 every 21 days, no activity was observed in nine high-grade, non- brainstem gliomas or in 14 brainstem gliomas (16). Other studies of topotecan in recurrent central nervous system tumors have also observed little if any activity, even using high doses (3.5 mg/m 2 / day administered in 5-day courses; Refs. 16 –19). One potential shortcoming of topotecan that may account for its limited clinical activity against brain tumors is its rapid hydrolysis to the inactive open-ring form (13, 21, 22), which may limit the ability of thera- peutically relevant concentrations of the active drug to persist within the tumor for a sufficient duration to induce cytotoxicity. CPT-11, which is also undergoing early clinical testing (12, 23– 26), is actually a largely inactive prodrug that is metabolized to form SN-38 (27); significant interpatient differences in metabolism may account for wide variability in the accumulation of active and inactive drug metabolites and hence both toxicity and efficacy (23, 28, 29). Recognizing the potential promise but apparent limitations of the available topo I inhibitors, we undertook synthesis of a series of novel camptothecin derivatives, using a cascade radical annulation route to the campothecin family. To enhance lipophilicity (and the potential for transfer across the blood-brain barrier) as well as potentially inhibit hydrolysis and albumin binding and increase blood stability, a series of 7-silyl-modified campothecins (silatecans) were developed. These agents exhibit potent inhibition of topo I (30, 31), dramatically improved blood stability, and profound inhibition of glioma growth in vitro, with some derivatives exhibiting more than 10-fold greater Received 10/30/98; accepted 1/25/99. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by Grants NS01810 and NS37704 (to I. F. P.), CA63653 (to T. G. B.), GM31678 (to D. P. C.), American Cancer Society Research Grant Awards DHP-138 and RPG-95– 059-03-CDD (to T. G. B.), and the Lucille Markey Trust Grant (to T. G. B.). 2 To whom requests for reprints should be addressed, at Department of Neurosurgery, Children’s Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA 15213. Phone: (412) 692-5881; Fax: (412) 692-5921; E-mail: Pollaci@chplink.chp.edu. 3 The abbreviations used are: topo, topoisomerase; MTS, 3-(4,5-dimethylthiazol-2-yl)- 5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PMS, phenazine metho- sulfate; HPLC, high-performance liquid chromatography; TEA, triethylamine; DB67, 7-tert-butyldimethylsilyl-10-hydroxycamptothecin. 4898 Research. on November 30, 2015. © 1999 American Association for Cancer cancerres.aacrjournals.org Downloaded from