IN VITRO AND IN VIVO BIOLOGIC EVALUATION OF LONG-CIRCULATING BIODEGRADABLE DRUG CARRIERS LOADED WITH THE PURE ANTIESTROGEN RU 58668 Thibault AMELLER, Ve ´ronique MARSAUD, Philippe LEGRAND, Ruxandra GREF and Jack-Michel RENOIR * UMR CNRS 8612, Pharmacologie Cellulaire et Mole ´culaire, Cha ˆtenay-Malabry, France We have developed a parenteral delivery system for the administration of the highly promising pure antiestrogen RU 58668 (RU). Two types of nanoparticles (NP) made of biode- gradable copolymers and coated with polyethylene-glycol (PEG) chains were prepared: nanospheres (NS) (diameter, 110 nm) and nanocapsules (NC) with an oily core (diame- ter, 250 nm). The amount of RU incorporated into NS and NC was 33 vs. 5 g RU/mg of polymer, respectively. Coating with PEG chains prolonged the antiestrogenic po- tency of RU, as shown by a prolonged antiuterotrophic activ- ity of encapsulated RU into PEG-poly(D,L lactic acid) (PLA) NS, as compared to that of conventional nonpegylated NS. In mice bearing MCF-7 estrogen-dependent tumors, free RU injected at 4.3 mg/kg/week by i.v. route slightly decreased the estradiol-promoted (0.5 mg/kg/week) tumor growth while RU-loaded PEG-PLA NS injected at the same dose strongly reduced it. Analysis of cell cycle parameters in tumors treated with RU indicated that RU-loaded PEG-PLA NS in- jected at 4.3 mg/kg/week in MCF-7 tumors decreased cyclin D 1 and cyclin E simultaneously, and increased p27. The anti- tumoral activity of RU encapsulated within pegylated NC was stronger than that of RU entrapped with pegylated NS loaded at an equivalent dose. Indeed, the former decreased the tumor size in nude mice transplanted with the estrogen receptor-positive but estrogen-independent MCF-7/Ras breast cancer cells at a concentration 2.5 times lower than that of the latter (0.4 mg/kg/week compared to 1 mg/kg/ week). Empty PEG-PLA NS and NC were devoid of an- tiuterotrophic and antitumoral activities. Altogether, these results suggest that the incorporation of the pure antiestro- gen RU into long-circulating NP could represent a novel antiestrogen drug delivery system for the parenteral route. © 2003 Wiley-Liss, Inc. Key words: breast cancer; antiestrogen; drug delivery; nanoparticles Hormone therapy consists of the abrogation of signals carried by natural hormones in order to block their biologic effects. This type of therapy has been used as a treatment for hormone-dependent cancers for more than 30 years. Worldwide, close to 1 million women per year are diagnosed with breast cancer. 1 Of all breast cancer patients, about one-third respond to hormone therapy, and this response is unfortunately only transient with a median dura- tion of about 18 months. 2 Endocrine therapy of hormone-depen- dent breast cancer consists of a variety of both medical and surgical ablative treatment modalities aiming at suppressing the mitogenic action of endogenous estradiol. 3 The classic mechanism by which estradiol exerts its effects is through binding to its intranuclear receptor (estradiol receptor (ER)) and the activation of target genes that promote cell cycle progression and tumor growth in breast cancer. A number of molecules displaying partial or total antagonistic activity have been synthesized. 3 However, the recent discovery of a second ER, ER, 4 has considerably complicated our understanding of the mechanism of action of estradiol and also of the pharmacology of these antiestrogenic molecules. 5 Tamoxifen (Nolvadex), delivered orally at 40 mg/day, is the most frequently used antiestrogen at the moment. Tamoxifen and its active metab- olite 4-hydroxy-tamoxifen are mixed antagonists/agonists whose activity is determined by the nature of the promoter to which ER binds, the nature of the ER ( or ) predominantly present in the tissue and the cellular content in terms of the coactivator/coinhibi- tor ratio. 5 For example, tamoxifen acts as a strong antagonist in the breast, but behaves as a full agonist in the uterus where it can induce carcinomas. 3 Thus, tamoxifen appears as an archetypal selective estrogen receptor modulator (SERM), with several unde- sirable side effects (hot flashes, induction of uterine cancers), but also some beneficial effects in the skeleton (inhibitory of bone resorption) as well as in the cardiovascular and central nervous systems (decrease of the incidence of coronary heart disease, improvement of cognitive function and delay of the onset of Alzheimer’s disease, respectively (see MacGregor and Jordan 3 and references cited therein). Unfortunately, tamoxifen resistance al- ways occurs, and in that case, a blockade of E 2 synthesis by aromatase inhibitors is sometimes of benefit (see Simpson et al. 6 for a review). In other cases, there is no response to aromatase inhibitors. Thus, despite encouraging improvements in breast can- cer treatment, prognosis of metatastic disease is dramatic, stressing the need of new drugs and new administration strategies. Among the antiestrogenic compounds that have been synthesized, the 7-estradiol derivative ICI 182,780 7 and the 11-estradiol deriv- ative RU 58668 (RU) 8 have been shown to inhibit tamoxifen resistance. 9 –11 Both are considered as pure antiestrogens, com- pounds that exert their activity even in the absence of E 2 . ICI 182,780 (Faslodex or Fulvestrant) has recently obtained the Marketing Authorization Application from the FDA. 12 However, in mice bearing human breast cancer tumors, RU act as an inhibitor of tumor progression, while ICI 182,780 11,13 behaves as a cyto- static drug. Therefore, RU appears as a promising antihormone for breast cancer treatment, and for this reason, we decided to work with RU rather than the ICI molecule. Nevertheless, the administration of a pure antiestrogen will have detrimental effects on skeleton and other tissues where estradiol has great benefits, if it is widely distributed throughout the body. RU also has poor oral bioavailability. 8 Taking into account that Abbreviations: E 2 , estradiol; ER, estradiol receptor; ERE, estrogen re- sponsive element; IC 50 , 50% inhibitory concentration; LUC, luciferase; NC, nanocapsules; NP, nanoparticles; NS, nanospheres; PEG, polyethyl- ene-glycol; PLA, poly(D,L lactic acid); RES, reticulo endothelial system; RU, RU 58668 (11-[4 –[5-[(4, 4, 5, 5, 5, pentafluoropentyl)sulfonyl]pen- tyloxy]phenyl]-estra-1, 3, 5 (10)-triene-3,17-diol); SERD, selective estro- gen receptor downregulator; SERM, selective estrogen receptor modulator. Grant sponsor: Association pour la Recherche contre le Cancer; Grant numbers: 9863 and 5970; Grant sponsor: Centre National de la Recherche Scientifique; Grant sponsor: Paris XI University; Grant sponsor: Minist` ere de la Recherche et de la Technologie; Grant sponsor: Ligue Nationale contre le Cancer; Grant sponsor: Ernst Schering Foundation. *Correspondence to: UMR CNRS 8612, Pharmacologie Cellulaire et Mol´ eculaire, 5 rue Jean-Baptiste Cl´ ement, 92296 Chˆ atenay-Malabry, France. Fax: + 33-(0)1-46-83-58-32. E-mail: Michel.Renoir@cep.u-psud.fr Received 28 November 2002; Revised 13 March 2003; Accepted 31 March 2003 DOI 10.1002/ijc.11248 Int. J. Cancer: 106, 446 – 454 (2003) © 2003 Wiley-Liss, Inc. Publication of the International Union Against Cancer