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Matrix Metalloproteinase–Activated Doxorubicin Prodrugs Inhibit HT1080 Xenograft Growth Better Than Doxorubicin with Less Toxicity

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Matrix Metalloproteinase–Activated Doxorubicin Prodrugs Inhibit HT1080 Xenograft Growth Better Than Doxorubicin with Less Toxicity 751 Matrix metalloproteinase–activated doxorubicin prodrugs inhibit HT1080 xenograft growth better than doxorubicin with less toxicity Charles F. Albright, Nilsa Graciani, Wei Han, MMP-selective conjugates were given to mice with Eddy Yue, Ross Stein, Zhihong Lai, HT1080 xenografts and the distribution of doxorubicin Melody Diamond, Randine Dowling, was determined. These studies showed that MMP- Lisa Grimminger, Shu-Yun Zhang, selective conjugates were preferentially metabolized in Davette Behrens, Amy Musselman, HT1080 xenografts, relative to heart and plasma, leading Robert Bruckner, Mingzhu Zhang, Xiang Jiang, to 10-fold increases in the tumor/heart ratio of doxorubi- Daniel Hu, Anne Higley, Susan DiMeo, cin. The doxorubicin deposited by a MMP-selective Maria Rafalski, Sandya Mandlekar, Bruce Car, prodrug, compound 6, was more effective than doxorubi- Swamy Yeleswaram, Andrew Stern, cin at reducing HT1080 xenograft growth. In particular, Robert A. Copeland, Andrew Combs, compound 6 cured 8 of 10 mice with HT1080 xenografts Steve P. Seitz, George L. Trainor, Rebecca Taub, at doses below the maximum tolerated dose, whereas Pearl Huang, and Allen Oliff doxorubicin cured 2 of 20 mice at its maximum tolerated dose. Compound 6 was less toxic than doxorubicin at this Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, Connecticut efficacious dose because mice treated with compound 6 had no detectable changes in body weight or reticulo- cytes, a marker for marrow toxicity. Hence, MMP- activated doxorubicin prodrugs have a much higher Abstract therapeutic index than doxorubicin using HT1080 xeno- Matrix metalloproteinase (MMP)–activated prodrugs were grafts as a preclinical model. [Mol Cancer Ther 2005;4(5): formed by coupling MMP-cleavable peptides to doxorubicin. 751–60] The resulting conjugates were excellent in vitro sub- strates for MMP-2, -9, and -14. HT1080, a fibrosarcoma cell line, was used as a model system to test these Introduction prodrugs because these cells, like tumor stromal fibro- Doxorubicin is an anthracycline natural product that is blasts, expressed several MMPs. In cultured HT1080 frequently used to treat breast cancer, liver cancer, soft- cells, simple MMP-cleavable peptides were primarily tissue sarcomas, and non-Hodgkin’s lymphoma (reviewed metabolized by neprilysin, a membrane-bound metallopro- in ref. 1). Doxorubicin kills tumor cells by causing teinase. MMP-selective metabolism in cultured HT1080 topoisomerase II–stimulated DNA strand breaks and cells was obtained by designing conjugates that were potentially other mechanisms (reviewed in ref. 2). Like good MMP substrates but poor neprilysin substrates. To other cytotoxic drugs, doxorubicin doses are limited by determine how conjugates were metabolized in animals, unwanted toxicity to nontumor tissues. Myelosuppression is the dose-limiting toxicity for doxorubicin, although stomatitis, mucositis, and alopecia are also frequently Received 1/7/05; revised 2/9/05; accepted 3/2/05. observed. In addition to these typical chemotherapeutic The costs of publication of this article were defrayed in part by the toxicities, doxorubicin causes cardiomyopathy that payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to depends on the cumulative dose of doxorubicin. indicate this fact. Matrix metalloproteinases (MMP) are a family of >20 Note: N. Graciani is currently at Wyeth Pharmaceutical, 500 Arcola Road, enzymes (reviewed in ref. 3). MMPs are synthesized as Collegeville, PA 19426. E. Yue and S. Yeleswaram are currently at inactive proenzymes that become activated by proteolysis. Incyte Pharmaceuticals, Route 141 and Henry Clay Drive, Wilmington, DE 19880-0400. R. Stein is currently at the Department of Neurology, For instance, the secreted enzyme pro-MMP-2 is proteo- Harvard Medical School, Cambridge, MA 02139. Z. Lai, M. Diamond, lyzed and activated by the membrane-anchored MMP-14 S-Y. Zhang, R.A. Copeland, P. Huang, and A. Oliff are currently at (4). Once activated, MMPs can cleave a variety of GlaxoSmithKline, Collegeville, PA 19426. L. Grimminger is currently at Johnson & Johnson, Malvern, PA 19355. A. Musselman is currently at extracellular matrix proteins, such as collagen, laminin, Merck Research Laboratories, Merck & Co., West Point, PA 19486. fibronectin, and elastin, which are potentially important M. Zhang is currently at Neurocrine Biosciences, Inc., 1055 Science Center Drive, San Diego, CA 92130. A. Stern is currently at Ensemble for tumor angiogenesis, invasion, and metastasis. Addi- Discovery Corp., 99 Erie Street, Cambridge, MA 02139. R. Taub is tionally, MMPs cleave a variety of other proteins, such currently at Roche Pharmaceuticals, Nutley, NJ 07110. as growth factor receptors and cell adhesion molecules, Requests for reprints: Charles F. Albright, Bristol-Myers Squibb which may be important for tumor growth and survival Pharmaceutical Research Institute, 5 Research Parkway, Wallingford, CT 06492. Phone: 203-677-5937; Fax: 203-677-7569. (reviewed in refs. 5, 6). MMPs can be inactivated by tissue E-mail: [email protected] inhibitors of MMPs, a family of four proteins, which bind Copyright C 2005 American Association for Cancer Research. tightly to MMPs. Mol Cancer Ther 2005;4(5). May 2005 Downloaded from mct.aacrjournals.org on September 29, 2021. © 2005 American Association for Cancer Research. 752 MMP-Activated Doxorubicin Prodrugs Increasing the therapeutic index of doxorubicin could because these cells grow as xenografts, are sensitive to benefit cancer treatment by allowing greater concentrations doxorubicin, and express several different MMPs (14, 15). of doxorubicin in tumors and thereby potentially greater Furthermore, HT1080 cells are derived from a fibrosarcoma tumor growth inhibition. Alternatively, increasing the and were, therefore, hoped to mimic stromal fibroblasts, a therapeutic index of doxorubicin could allow reduced major constituent of many human tumors. Using this toxicities and thereby permit the use of other cytotoxics that strategy, prodrugs were identified that were excellent would be prevented because of overlapping toxicities. One in vitro substrates for MMP-2, -9, and -14. Optimization of way to improve the therapeutic index of doxorubicin is to conjugate structure led to prodrugs that were selectively create prodrugs that are selectively activated in the tumor metabolized by MMPs in HT1080 cultures and preferen- environment. MMPs are attractive enzymes to activate tially deposited doxorubicin in HT1080 xenografts relative prodrugs in the tumor environment because MMPs are to heart tissue. The preferential doxorubicin deposition led intimately connected with tumorigenesis. In particular, to an improved therapeutic index, relative to doxorubicin, preclinical studies implicate MMPs in tumor progression as shown by improved HT1080 xenograft growth inhibition (reviewed in ref. 6). For instance, overexpression of MMP- with reduced toxicity. 14 in mouse mammary tissue causes mammary tumors (7), whereas loss of MMP-2 or -9 reduced the lung colonization by B16-LBL6 melanoma cells and Lewis lung carcinoma Materials and Methods cells (8, 9). Additionally, MMP expression in human Compound Synthesis tumors frequently correlates with disease progression All peptides were synthesized following the same (reviewed in ref. 6). For instance, one study found MMP- protocol. An example synthesis of compound 1 is provided. 2 and -14 expression in 90% of breast carcinomas and a The peptide acid was synthesized on the solid phase strong correlation between tumor cell membrane staining starting with commercially available Fmoc-Leu-Wang resin and lymph node metastases (10). Although there are strong (0.40 g, 0.25 mmol, Advance Chemtech, Louisville, KY). connections between MMPs and tumor progression, pub- The synthesis was done on an ABI 433A peptide lished studies do not determine the absolute or relative synthesizer (Applied Biosystems, Foster City, CA) using MMP activity in the tumor environment. Such information standard Fmoc protocols. The completed peptide on resin is clearly critical to the design of MMP-activated prodrugs was N-acetylated with acetic anhydride. The desired because the MMP enzyme activity must be sufficient to peptide was cleaved from the resin with 90% trifluoroacetic activate efficacious levels of prodrug. acid in water for 2 hours. After solvent removal, the Preclinical testing of MMP-activated prodrugs is com- peptide was dissolved in H2O:CH3CN and freeze dried. plicated by differences between mouse and human tumors. Product was confirmed by mass spectrometry (ES m/e À Many human tumors, including breast cancers, are com- calculated for C21H36N4O6 [M-H] , 439.54; found, 439.3). posed of roughly equal numbers of tumor cells and stromal Analytic high-performance liquid chromatography on a cells, including stromal fibroblasts, inflammatory cells, and Metachem Monochrome C18 reverse-phase column (50 Â endothelial cells (reviewed in ref. 11). The interplay of these 4.6 mm, MetaChem Technologies, Torrance, CA) showed cell types is thought to be crucial for MMP activation in that crude peptide to be 85% pure. To this intermediate (19.9 mg, different cell types express and activate specific MMPs 40 Amol) dissolved in dimethylformamide (0.2 mL) in a (reviewed in ref. 6). For instance, stromal fibroblasts small amber vial was added benzotriazol-1-yl-oxytripyrro- express MMP-2, yet tumor cells are the source of MMP-14
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