ICANCER RESEARCH 58, 2404-2409. June 1. 1998] Complete Regression of Well-established Tumors Using a Novel Water-soluble Poly(L-Glutamic Acid)-Paclitaxel Conjugate1 Chun Li,2 Dong-Fang Yu, Robert A. Newman, Fernando Cabrai, L. Clifton Stephens, Nancy Hunter, Luka Milas, and Sidney Wallace Department* of Diagnostic Radiology 1C. L. D-F. Y., S. W.J, Clinical Investigation [R. A. N.I. Veterinary Medicine [L C. S.J, ami Experimental Radiation Oncology [N. H.. L. M. ¡,The university of Texas M. D. Anderson Cancer Center, and Department of Pharmacology. The University of Texas Medical School [F. C. /, Houston, Texas 77030 ABSTRACT Despite an intensive search, few water-soluble paclitaxel derivatives have been shown to have a therapeutic index superior to paclitaxel itself. We now report a water-soluble poly(L-glutamic acid)-paclitaxel conjugate (PG-TXL) that produces striking antitumor effects with diminished tox- icity. A single i.v. injection of PG-TXL at its maximum tolerated dose (defined as that dose that produces a maximum 12-15% body weight loss within 2 weeks after a single i.v. injection) equivalent to 60 mg of pacli- taxel/kg and at even a lower dose equivalent to 40 mg of paclitaxel/kg resulted in the disappearance of an established implanted 13762F mam mary adenocarcinoma (mean size, 2000 mm3) in rats. (An equivalent dose of PG-TXL is the amount of conjugate that contains the stated amount of paclitaxel.) Similarly, mice bearing syngeneic OCA-1 ovarian carcinoma (mean size, 500 mm li were tumor-free within 2 weeks after a single i.v. injection of the conjugate at a dose equivalent to 160 mg of paclitaxel/kg. The conjugate has little if any intrinsic tubulin polymerization activity in vitro and is >20 times less potent in supporting the growth of a paclitaxel- dependent CHO mutant cell line. PG-TXL has a prolonged half-life in plasma and greater uptake in tumor as compared with paclitaxel. Fur thermore, only a small amount of total radioactivity from !'(;-[ 'IIITXL was recovered as free [3H]paclitaxel in either the plasma or the tumor tissue within 144 h after drug injection. Histological studies of tumor tissues obtained from mice treated with PG-TXL show fewer apoptotic cells but more extensive tumor necrosis as compared with paclitaxel treatment. These data suggest that in addition to its role as a carrier for selective delivery of paclitaxel to the tumor, PG-TXL exerts distinct pharmacological actions of its own that may contribute to its remarkable antitumor efficacy. INTRODUCTION Paclitaxel (Taxol®) has shown significant antineoplastic activity against various human cancers, including breast and ovarian tumors (1,2). However, a major difficulty in the clinical use of paclitaxel has been its poor water solubility. Previous attempts to prepare water- soluble prodrugs of paclitaxel involved placing low-molecular-weight solubilizing moieties at the C2' or C7 position. These prodrugs are mainly ester derivatives (including succinate, sulfonic acid, and amino acid) and phosphate derivatives (3-7). Although some of these pro- drugs possess adequate aqueous solubility, few have antitumor activ ity comparable to that of the parent drug (3, 4, 7). Several of these derivatives are not suitable for i.v. injection because of their instability in aqueous solution at neutral pH. Recently, Nicolaou et al. (8) reported the synthesis and in vitro biological evaluation of a novel series of prodrugs termed "protaxols." These compounds possess greater aqueous solubility and are converted to paclitaxel as the active drug through an intramolecular hydrolysis mechanism. However, no in vivo data on the antitumor activity of protaxols are yet available. Received 11/12/97; accepted 3/30/98. 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 Supported in part by NIH Grant R29-CA74819-OI. by the University of Texas M. D. Anderson Cancer Center Breast Cancer Research Program, and by the Gianturco Fund. 2 To whom requests for reprints should be addressed, at Department of Diagnostic Radiation. Box 59. M. D. Anderson Cancer Center. 1515 Holcombe Boulevard. Houston. TX 77030. Conjugation of chemotherapeutic agents to macromolecular carri ers is typically performed not only to improve solubility characteris tics of a compound but also to produce desirable pharmacokinetics and to enhance antitumor activity (9-12). Conjugation of paclitaxel to PEG,3 for example, has been reported to result in highly water-soluble prodrugs that are almost completely converted to free paclitaxel upon exposure to aqueous environments. Unfortunately, although the asso ciation with PEG improves the water solubility of paclitaxel, it has not greatly improved the therapeutic index of the PEG-paclitaxel complex compared with free paclitaxel itself (13, 14). With a goal of preparing water-soluble paclitaxel derivatives that possess adequate stability and improved antitumor efficacy, we selected a synthetic polyamino acid, PG (15-17), as a potential carrier for paclitaxel based on the following rationale: (a) PG contains a large number of side chain carboxyl functional groups for drug attachment: (b) PG can be readily degraded by lysosomal enzymes to its nontoxic basic component, L-glutamic acid; and (c) sodium glutamate has been reported to prevent manifes tations of neuropathy induced by paclitaxel, thus enabling higher doses of paclitaxel to be tolerated (18). This report describes the synthesis, characterization, and in vivo antitumor activity of PG-TXL. Attempts are also made to compare the pharmacological properties of PG-TXL with those of paclitaxel. Our findings suggest that in addition to its role as a carrier for selective delivery of paclitaxel to the tumor, PG-TXL also exerts its own pharmacological actions different from those of paclitaxel. MATERIALS AND METHODS Synthesis of PG-TXL. To a solution of 75 mg of PG (A/r 36,200; Sigma Chemical Co., St. Louis. MO) in 1.5 ml of dry /V./V-dimethylformamide were added 20 mg of paclitaxel (Hande Tech. Houston, TX), 15 mg of dicyclo- hexylcarbodiimide, and a trace amount of dimethylaminopyridine. The reac tion was allowed to proceed at room temperature overnight. TLC [silica plate; eluent, CHCl,:methanol (10:1)] showed complete conversion of paclitaxel (R, = 0.55) to the polymer conjugate (/?, = 0). To stop the reaction, the mixture was poured into chloroform. The resulting precipitate was converted to the sodium salt of PG-TXL by dissolving the crude product in NaHCO,. The aqueous solution of PG-TXL was dialyzed against distilled water, filtered, and lyophilized to obtain 88.6 mg of white powder. Paclitaxel content was 21% (w/w, UV method). Yield (conversion to polymer-bound paclitaxel, UV) was 93%. Values for 'H NMR (General Electric model GN 500 spectrometer, 500 MHz, in D2O) were: for aromatic components of paclitaxel. 8= 7.75-7.36 ppm: and for aliphatic components of paclitaxel, S = 6.38 ppm (CI(I-H), 5.97 ppm (C,,-H), 5.63 and 4.78 ppm (C2.-H), 5.55-5.36 ppm (C,.-H and C2-H, m), 5.10 ppm (C5-H), 4.39 ppm (C7-H), 4.10 ppm (C2II-H), 1.97 ppm (OCOCH,), and 1.18-1.20 ppm (C-CH,). Other resonances of paclitaxel were obscured by those of PG. PG resonances at 4.27 ppm (H-a), 2.21 ppm (H-7), and 2.04 ppm (H-ß)were in accordance with the spectrum of pure PG. Characterization of PG-TXL. UV spectra were obtained on a Beckman DU-640 spectrophotometer (Fullerton, CA). The content of paclitaxel conju gated to PG was estimated by UV measurements based on a standard curve generated with known concentrations of paclitaxel in methanol (A = 228 nm). 'The abbreviations used are: PEG, polyethylene glycol; PG, poly(L-glutamic acid); PG-TXL, PG-paclitaxel; MTD. maximum tolerated dose: HPLC. high-performance liquid chromatography; NMR, nuclear magnetic resonance. 2404 Research. on January 25, 2016. © 1998 American Association for Cancer cancerres.aacrjournals.org Downloaded from