[CANCER RESEARCH 64, 3994 – 4000, June 1, 2004] A Comparative Analysis of Low-Dose Metronomic Cyclophosphamide Reveals Absent or Low-Grade Toxicity on Tissues Highly Sensitive to the Toxic Effects of Maximum Tolerated Dose Regimens Urban Emmenegger, 1 Shan Man, 1 Yuval Shaked, 1 Giulio Francia, 1 John W. Wong, 2 Daniel J. Hicklin, 3 and Robert S. Kerbel 1 1 Molecular and Cellular Biology Research, Sunnybrook and Women’s College Health Sciences Centre, Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada; 2 Department of Anatomic Pathology, Sunnybrook and Women’s College Health Sciences Centre, Toronto-Sunnybrook Regional Cancer Centre, University of Toronto, Toronto, Ontario, Canada; and 3 ImClone Systems, Inc., New York, New York ABSTRACT The survival benefits of traditional maximum tolerated dose (MTD) cytotoxic therapy have been modest for the treatment of most types of metastatic malignancy and, moreover, often come with increased acute and chronic toxicity. Recent studies have demonstrated that the frequent administration of comparatively low doses of cytotoxic agents, with no extended breaks [low-dose metronomic (LDM) chemotherapy], may not only be at least as efficient as MTD therapy but also less toxic. This coincides with an apparent selectivity for “activated” endothelial cells of the tumor vasculature. However, the impact of LDM chemotherapy on the most sensitive target cell populations normally affected by MTD therapy (i.e., bone marrow progenitors, gut mucosa, and hair follicle cells) has not been analyzed in experimental detail. Therefore, we compared effects of LDM and MTD cyclophosphamide (CTX) on bone marrow and gut mucosa. Furthermore, we studied the potential impact of LDM CTX on angiogenesis in the context of wound healing and evidence of organ toxicity. We show absent or moderate hematologic and intestinal toxicity of LDM as opposed to MTD CTX. Of note was the finding of sustained lymphopenia, which is not unexpected given the use of CTX as immuno- suppressive drug. There was no negative impact on wound healing or evidence of organ toxicity. LDM offers clear safety advantages over conventional MTD chemotherapy and therefore would appear to be ideal for long-term combination therapy with targeted antiangiogenic drugs. INTRODUCTION Shortly after the introduction of cytotoxic chemotherapy for the management of neoplastic diseases more than half a century ago, it became evident that frequently observed impressive responses were mostly short lived and that relapsing tumors that initially responded to chemotherapy became drug resistant. Several approaches were devel- oped to try to overcome the development of drug resistance, such as dose escalation of single agents and multidrug combinations, devel- opment of cytotoxic drugs with new modes of action, and by pushing the limits of the maximal tolerated dose (MTD) through improving supportive care measures, growth factor and stem cell support. Al- though some of these approaches were validated recently (e.g., with the successful clinical application of the concept of dose densification in the setting of adjuvant breast cancer treatment; Ref. 1), survival benefits remain modest and often come with the price of increased acute or chronic toxicity (2). The historical emphasis on the concept that more drug is better has resulted in a relative neglect in other important parameters, such as the timing and duration of cytotoxic chemotherapy (3, 4). In this regard, the frequent administration of comparatively low doses of cytotoxic agents, with no extended breaks, sometimes called metronomic chemotherapy (5), may not target tumor cells directly, as is primarily the case for the cyclic MTD approach, but indirectly via inhibiting angiogenesis and vasculogen- esis (2). As such, low-dose metronomic (LDM) chemotherapy may offer several advantages over the MTD approach, as shown by various preclinical studies (e.g., reduced toxicity, treatment response irrespec- tive of the resistance profile of the tumor cell population, and the potential for long-term combination therapy with targeted drugs; Refs. 6, 7). The low toxicity profile of LDM chemotherapy coincides with an apparent selectivity for “activated” endothelial cells of the tumor’s growing neovasculature (8, 9). Several in vitro studies have shown that endothelial cells are much more sensitive, sometimes by one or more orders of magnitude, to exposure of low concentrations of different cytotoxic drugs when compared with many tumor cell lines, fibroblasts of various origin, smooth muscle cells, macrophages, as- trocytes, and mammary and prostate epithelial cells (10 –12, unpub- lished observations). However, no data are yet available regarding the comparative sensitivity of the target cell populations that are normally most sensitive to MTD chemotherapy (i.e., bone marrow progenitors, gut mucosa, and hair follicle cells). Moreover, a thorough preclinical toxicity analysis of LDM therapy beyond the documentation of changes in body weight as a surrogate marker for toxicity is not available despite the fact that LDM chemotherapy is rapidly entering the clinical trial arena (13–17). Therefore, the purpose of the present study was to study the effects of cyclophosphamide (CTX), clinically the most advanced drug in the setting of LDM chemotherapy, on bone marrow function and intesti- nal mucosa (13–15). Given a similar toxicity pattern for successive therapy cycles of MTD CTX, the observation period for MTD exper- iments was 3 weeks (corresponding to one treatment cycle). In the case of LDM CTX, the observation period spanned 42 days for hematologic and intestinal parameters. This period was chosen based on the finding that PC-3 xenograft growth could be controlled com- pletely for at least 42 days with LDM CTX (18). We also studied the potential impact of LDM CTX on wound healing, an angiogenesis- dependent process, and morphologic and/or functional alterations of various organs (in particular, the bladder) in animals exposed to LDM CTX for more prolonged periods. The experiments comparing the effects of LDM versus MTD CTX were performed in non-tumor- bearing C.B-17 severe combined immunodeficient (SCID) and BALB/cJ mice (immunodeficient and immunocompetent mouse strains, respectively, often used for tumor experiments) because the presence of a tumor is known to influence bone marrow function, and treated and control animals also would necessarily have different tumor sizes, making interpretation of the obtained data extremely difficult (19). Our results show an unambiguous advantage with Received 2/18/04; accepted 3/24/04. Grant support: Grants to R. S. Kerbel from NIH (CA-41233) and the National Cancer Institute of Canada (NCIC). R. S. Kerbel holds a Tier I Canada Research Chair in Molecular Medicine. U. Emmenegger is supported by the Swiss National Science Foun- dation and the Swiss Cancer League/Oncosuisse (BIL SKL 1237– 02-2002). Y. Shaked is recipient of a postdoctoral fellowship award from the Canadian Institutes of Health Research (CIHR). 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. Requests for reprints: Robert S. Kerbel, Sunnybrook and Women’s College Health Sciences Centre, Molecular and Cellular Biology, S-217, 2075 Bayview Avenue, Toronto, Ontario, Canada M4N 3M5. Phone: 416-480-5711; Fax: 416-480-5884; E-mail: robert.kerbel@sw.ca. 3994 Research. on July 2, 2015. © 2004 American Association for Cancer cancerres.aacrjournals.org Downloaded from