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

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 reticulocytes, 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 substrates 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.

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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 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 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 lidinophosphonium hexafluorophosphate (20.8 mg, 40 Amol). that convert pro-MMP-2 to active MMP-2. Unfortunately, Doxorubicin hydrochloride (18.6 mg, 32 Amol, Aldrich, mouse tumor models do not accurately reflect the cell types Milwaukee, WI) was added as a suspension in dimethyl- observed in human tumors because both xenografts and formamide (0.1 mL) followed by diisopropylethylamine transgenic tumor models are composed primarily of (0.0139 mL, 80 Amol). The reaction was stirred for 2 hours. rapidly growing tumor cells with relatively few stromal Solvent was removed under vacuo. The sample was cells. dissolved in H2O:CH3CN and purified using a Dynamax This study discovered MMP-activated doxorubicin pro- C18 reverse-phase column (41.4 250 mm, Varian, Palo drugs that had a greater therapeutic index than doxorubi- Alto, CA) with a linear gradient from 30% to 50% cin using HT1080 cells as a model system. In the absence of acetonitrile, 0.05% ammonium acetate over 20 minutes information about enzyme activity levels, prodrugs were with a flow rate of 45 mL/min. Fractions were pooled and identified that were efficiently cleaved by MMP-2, -9, and freeze dried to afford the purified peptide-doxorubicin -14 because these enzymes were frequently overexpressed conjugate (ES m/e calculated for C48H63N5O16 [M-H] , in human tumors, affected tumor progression in preclinical 965.06; found, 964.6). Caution: Doxorubicin is a highly toxic models, and were very active in vitro. Because these MMPs compound. Appropriate measures should be taken to avoid may have insufficient activity for prodrug activation in vivo, exposure to the compound. prodrug design was further constrained so that prodrugs Enzyme Assays were likely be activated by other MMPs, with the exception Doxorubicin prodrugs were incubated with MMP-2, -9, of MMP-11 (12), based on the known MMP substrate or -14 or neprilysin to compare the competency of these specificities (13). HT1080 cells were used as a model system conjugates as substrates for these enzymes. Recombinant

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MMP-2, -9, and -14 were purified from HT1080 fibrosarco- intrinsic fluorescence as above. To determine the amount ma cells, insect cells, and Escherichia coli, respectively, as of metabolism that was due to MMPs, cultures were described previously (12, 16, 17). MMP-2 and -9 were incubated with 0.15 mmol/L marimastat, a broad-spec- activated with 4-aminophenylmercuric acetate for 2 hours trum MMP inhibitor (21). This concentration of marima- at 37jC. Unreacted 4-aminophenylmercuric acetate was stat was sufficient to inhibit MMPs based on in vitro removed using Bio-Spin 6 columns (Bio-Rad, Hercules, assays and the conversion of pro-MMP-2 to MMP-2 in CA). Neprilysin was purified from rat kidney as described HT1080 cultures (data not shown). To determine the (18). Conjugates (1 Amol/L) were incubated with either 10 fraction of metabolism due to neprilysin, cultures were nmol/L MMP-2, 2 nmol/L MMP-9, or 5 nmol/L MMP-14 incubated 100 nmol/L phosphoramidon. in 50 mmol/L HEPES (pH 7.5), 10 mmol/L CaCl2, 0.1% ProdrugMetabolisminMice Brij-35 at 37jC. At various times up to 60 minutes, an HT1080 tumors were transplanted into naive mice using aliquot of the reaction was removed and injected into a trocar needles. Dry conjugate powders were dissolved Novapak C18 reverse-phase high-performance liquid chro- in N,N-dimethylacetamide and diluted with 5% dextrose matography column (Waters, Milford, MA). The reaction to yield a final dosing solution of 10% N,N-dimethylace- mixture was eluted with a 27% to 63% acetonitrile gra- tamide, 4.5% dextrose. Mice were then given the desired dient containing 0.1% trifluoroacetic acid. Doxorubicin- dose using 0.1 mL dosing solution. Drugs were given i.v. containing compounds were detected and quantified using into the tail vein. At the indicated times, three mice per f the intrinsic fluorescence of doxorubicin (excitation wave- time point were anesthetized with CO2. Blood ( 1 mL) length, 480 nm; emission wavelength, 580 nm). Alternately, was collected by cardiac puncture in a syringe containing conjugates (1 Amol/L) were incubated with 10 nmol/L 0.1 mL sodium citrate to prevent coagulation and cells were neprilysin in 50 mmol/L Tris-HCl (pH 7.5), 10 mmol/L removed by centrifugation. The resulting plasma was CaCl2, 0.1% Brij-35 and processed as above. The substrate transferred to another tube and frozen in liquid nitrogen. peak area was used to determine the kcat/Km for each Tumor and heart tissue were removed, frozen in liquid j conjugate under first-order conditions (substrate << Km): nitrogen, and stored at 80 C. For analysis, tissues were thawed on ice, weighed, and minced with scissors, and kcat=Km ¼ lnðS0=StÞ=ðt ðEÞÞ cold, citrated mouse plasma was added (Cocalico Biolog- ical, Reamstown, PA). Plasma was added at 5 mL/g for where S0 is the initial substrate concentration, St is the tumor tissue and 10 mL/g for heart tissue. Slurries were substrate concentration at various times, t is time, and E is homogenized for 1 minute and 0.5 mL was transferred to enzyme concentration (19). an Eppendorf tube. Silver nitrate (0.1 mL, 33%) was ProdrugMetabolisminCulturedHT1080Cells added to the homogenate to improve the recovery of To measure the rate of prodrug metabolism in cell doxorubicin and the mixture was vortexed briefly. Aceto- culture, actively growing HT1080 fibrosarcoma cells nitrile (0.05 mL) was then added, vortexed briefly, and (American Type Culture Collection, Manassas, VA) were mixed for 15 minutes. This mixture was centrifuged for plated at 60% confluence. Two hours after plating, the cell 5 minutes. The supernatant was transferred to another medium (DMEM, 10% fetal bovine serum) was removed tube, dried under a nitrogen stream, and stored at 80jC. and replaced with DMEM containing 0.1% bovine serum Doxorubicin-containing compounds were separated by albumin and 40 nmol/L phorbol 12-myristate 13-acetate. reverse-phase high-performance liquid chromatography Phorbol 12-myristate 13-acetate was added to increase the as above and quantified using a doxorubicin standard expression of MMP-9 and conversion of pro-MMP-2 to curve. Control experiments showed that this method active MMP-2 (20). After 16 hours, doxorubicin prodrug extracted 80% doxorubicin-containing compounds and that (500 nmol/L) was added to the medium. Aliquots of metabolism of doxorubicin-containing compounds during medium (0.1 mL) were removed at the indicated times, analysis was negligible. 0.3 mL acetonitrile was added to each aliquot, and the Inhibition ofHT1080 XenograftGrowth sample was mixed and then centrifuged for 5 minutes. HT1080 tumors were transplanted into naive mice. After Solvent was evaporated from clarified supernatant (0.3 mL) 5 to 7 days, tumors had reached a median size of 75 to 100 using a nitrogen stream. Samples were then vortexed with mm3. Mice were then grouped into cohorts of 10 mice such 0.06 mL acetonitrile. Buffer A [0.06 mL; 14 mmol/L that each cohort had the same median tumor size and a sodium phosphate, 0.5 mmol/L triethylamine (pH 4.2)] similar tumor size distribution. Treatment was then was then added, vortexed, and centrifuged. Supernatant initiated as described in Results and tumors were measured (0.06 mL) was mixed with 0.06 mL buffer A and 0.1 mL every 3 days with calipers. was injected onto a Novapak C18 column at a flow rate of Reticulocyte Measurements 1 mL/min and eluted with a linear gradient from Three mice per condition were anesthetized with CO2 75% buffer A, 25% acetonitrile to 50% buffer A, 50% 3 days after the last treatment because control experiments acetonitrile over 12 minutes. Control experiments showed showed that this time corresponded to the nadir of the that doxorubicin and doxorubicin conjugates were recov- reticulocyte decrease. Blood (0.5 mL) was collected by ered with f80% yield using this method. Doxorubicin- cardiac puncture in a syringe containing EDTA to prevent containing compounds were detected based on their coagulation. RBC and reticulocyte concentrations were

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determined the same day using an Advia 120 Hematology of compounds to humans. Although the necessary solubil- Analyzer. ity would depend on clinical usage, a target solubility of Lethal Dose Determination 1 mg/mL was chosen to guide compound design. Increasing doses of drugs were given i.v. to five mice per Prodrug Cleavage by MMP-2, -9, and -14 In vitro dose. Animals were observed for 30 days for signs of severe To determine the effect of prime-side length on MMP toxicity and euthanized if required according to Dupont cleavage rate, peptides of various lengths were attached to Pharmaceuticals Animal Care and Use Committee guide- doxorubicin and the catalytic efficiencies for cleavage by lines. MMP-2, -9, and -14 were determined (Table 1). For this analysis, simple collagen-like peptide sequences (PLGfL) Results were used because most MMPs cleave peptides containing Design ofMMP-Activated Doxorubicin Prodrugs this sequence (reviewed in ref. 13). A conjugate with the one prime-side residue, compound 1, was not detectably Doxorubicin prodrugs were formed by linking the cleaved by MMP-2 or -9. A conjugate with two prime-side COOH terminus of a peptide to the amino group of residues, compound 2, was efficiently cleaved by MMP-9, doxorubicin (Fig. 1). The resulting prodrug is not cytotoxic modestly cleaved by MMP-2, and poorly cleaved by MMP- because the peptide prevents the prodrug from entering 14. Conjugates with three prime-side residues, compound cells (22). MMP cleavage occurs in the middle of the 3, or four prime-side residues, compound 4, were cleaved illustrated hexapeptide generating a tripeptide doxorubicin efficiently by MMP-2, -9, and -14. The absolute k K conjugate (Fig. 1). The tripeptide doxorubicin conjugate cat/ m must be further cleaved by extracellular proteases to form values for efficiently cleaved conjugates, such as com- Leu-Dox before doxorubicin-containing compounds can pounds 3 and 4, were close to values for the most efficient k K efficiently penetrate cells. Leu-Dox may be converted to MMP substrates. In particular, cat/ m values for the best reported fluorogenic peptide substrates for MMP-2, -9, doxorubicin either inside or outside cells. Leucine was 1 1 chosen as the COOH-terminal residue because Leu-Dox and -14 are 629, 87, and 1,590 mmol/L s , respectively was more efficiently converted to doxorubicin than other (24–26). Metabolism ofConjugates in Cultured HT1080 Cells amino acid doxorubicin conjugates (23) and leucine allowed efficient MMP-2, -9, and -14 cleavage (data not The metabolism rate of conjugates with two or more shown). The peptide was capped to prevent aminopepti- prime-side residues in cultured HT1080 cells was deter- dase degradation and increase solubility. Sufficient aque- mined because in vitro enzymatic digestions showed that ous solubility was required to allow the i.v. administration these conjugates were substrates for MMP-2, -9, or -14. For this analysis, conjugates were incubated with cultured HT1080 cells, aliquots of culture supernatant were removed after various times, and the composition of doxorubicin-containing compounds was determined. Using compound 2 as an example, the parental conjugates disappeared linearly with time for several hours and the major metabolite was L-Dox (Fig. 2A). Other metabolites, suchasLL-Dox,whichwouldbeformedbyMMP cleavage of compound 2, were only detected in trace amounts. To determine why these expected metabolites were not detected at higher levels, compounds were cleaved by MMP-2 in vitro and then added to cultured HT1080 cells. Analysis of these culture supernatants showed that post-MMP cleavage metabolites were rapidly converted to L-Dox (data not shown). The kinetics of conversion of these post-MMP cleavage metabolites to L- Dox were consistent with the trace amounts of these metabolites detected following incubation of the parental compounds. To allow comparisons between compounds, the fraction processed was defined as the fraction of compounds present in the culture supernatant that could result from MMP cleavage. The fraction processed per hour was calculated by fitting a straight line to the fraction processed values measured during the first 6 hours after Figure 1. Structure of doxorubicin (A) and compound 6, a representative incubation of the compound with cultured HT1080 cells. prodrug, with expected enzymatic cleavage products (B). The MMP Although this method is considered the most rigorous, f cleavage site is indicated by . Residues on the NH2-terminal side of the cleavage site are designated P1, P2, etc., whereas residues on the COOH- very similar values would be obtained by simply terminal side of the cleavage site are designated P1V,P2V, etc. See Results considering the formation of L-Dox. Analysis of com- for further description. pounds 2 to 4 showed a dramatic effect of prime-side

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Table 1. In vitro properties of selected prodrugs

1 1 Code Structure kcat/Km (mmol/L s ) Solubility (mg/mL) MMP-2 MMP-9 MMP-14 Neprilysin

1 Ac-PLG-L-Dox <1 <1 ND ND ND 2 Ac-PLG-LL-Dox 18 130 4 22 0.004 3 Peg-PLG-LYL-Dox 150 310 >250 13 0.02 4 Ac-PLG-LYAL-Dox 190 300 130 >120 0.0002 5 Ac-PLG-HofOrnL-Dox 150 48 >250 <0.1 0.3 6 Ac-E-PCitG-HofYL-Dox 19 43 42 <1 2.1

Abbreviations: Peg, polyethylene glycol; Hof, homophenylalanine; Orn, ornithine; Cit, citrulline; ND, not determined.

length on processing in HT1080 cultures; the fraction were detectably degraded in the absence of cultured processed per hour was 0.02, 0.065, and 0.3 for these HT1080 cells (data not shown). compounds that contained 2, 3, or 4 prime-side residues, To determine the amount of the metabolism that resulted respectively (Fig. 2B). None of the compounds in Fig. 2B from MMP cleavage, reactions were conducted with marimastat, a broad-spectrum MMP inhibitor. This analysis showed that most of the cleavage of compounds 2 and 4 were not due to MMPs (Fig. 2B). In the case of compound 3, approximately one half of the metabolism resulted from MMPs. Similar results were obtained with other small- molecule MMP inhibitors (data not shown). To identify the putative non-MMP-metabolizing enzyme, the effect of other protease inhibitors on compound 4 metabolism was tested. These studies revealed that 100 nmol/L phosphoramidon greatly reduced the metabolism of compound 4 (Fig. 2C). At this concentration, phosphoramidon is specific for neprilysin, a cell surface protease (27). Other data supported the hypothesis that neprilysin was the major non-MMP-metabolizing enzyme for conjugates in cultured HT1080 cells. First, HT1080 cells expressed neprilysin based on Western blotting (data not shown). Second, the non-MMP metabolism rate of conjugates correlated well with the in vitro catalytic efficiency of neprilysin cleavage (Table 1; Fig. 2B; data not shown). Third, other cell lines that expressed neprilysin had non- MMP metabolism rates that correlated with their levels of neprilysin expression (data not shown). Because neprilysin is expressed outside tumor tissue with high levels in kidneys (27) and would, therefore, lead to nontumor activation of prodrugs, compounds were identified that were good substrates for MMP-2, -9, and -14 but were poor substrates for neprilysin. The data for two such compounds, compounds 5 and 6, are summa- rized in Table 1. In the case of compound 5, MMP selectivity over neprilysin was first achieved by substi- tuting ornithine for tyrosine in the P2V position. The ornithine substitution had the added benefit of Figure 2. Metabolism rates of selected prodrugs by cultured HT1080 increasing solubility to 0.3 mg/mL. MMP selectivity over cells. A, amount of L-Dox and doxorubicin formed from compound 2 in cultured HT1080 cells. Amounts are represented as the fraction of all neprilysin with good solubility was later obtained in doxorubicin-containing compounds detected. The total amount of doxo- compounds, such as compound 6, which did not contain rubicin-containing compounds differed by <5% at different times. B, non- any positively charged residues. Consistent with the MMP and MMP processing of several conjugates in cultured HT1080 cells. neprilysin hypothesis, the MMP-selective compounds, The fraction of metabolism due to MMPs was determined in reactions with 150 nmol/L marimastat. C, effect of phosphoramidon (Phos.)and compounds 5 and 6, were primarily metabolized by marimastat on compound 4 processing in cultured HT1080 cells. MMPs in cultured HT1080 cells (Fig. 2B).

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Analysis of conjugate metabolism in cultured HT1080 cells also showed that there was little correlation between the enzymatic cleavage of conjugates by MMP-2, -9, or -14 and the cleavage of conjugates by cultured HT1080 cells (Table 1; Fig. 2B; data not shown). Potential reasons for this lack of correlation will be discussed later. Compound 5 Preferentially Accumulates Doxorubicin inTumor Xenografts Relative to HeartTissue To determine if the metabolism of conjugates by cultured HT1080 cells was sufficient to lead to the preferential accumulation of doxorubicin in tumor tissue, mice with HT1080 xenografts were injected with 1.4 Amol/kg compound 5, L-Dox, or doxorubicin. At various times after injection, tumor and heart tissue were analyzed for doxorubicin levels. Heart tissue was chosen as a reference tissue because there were published data comparing tumor and heart levels of doxorubicin and L-Dox. Both doxorubicin and L-Dox administration resulted in more doxorubicin in heart tissue than HT1080 xenografts (Fig. 3A and B), consistent with published results (28–31). In contrast, compound 5 administration led to more doxorubicin in HT080 tissue than heart (Fig. 3C). One way to quantify these differences is to integrate the area under the doxorubicin time- concentration curves. This integration showed that compound 5 had a 15-fold better tumor/heart doxorubicin ratio than doxorubicin (Table 2). This analysis also showed that the tumor doxorubicin levels from compound 5 were approximately one third those from doxorubicin at equimolar doses. To gain further insight into compound 5 metabolism, the concentration-time profiles for compound 5 and its metabolites in plasma, tumor, and heart were determined. As in cultured HT1080 cells, the major doxorubicin- containing compounds in the tissues following administration of compound 5 were compound 5, L-Dox, and doxorubicin (Fig. 4). Compound 5 disappeared rapidly from the plasma compartment. Whereas L-Dox levels were measurable in plasma, doxorubicin levels in plasma were below detectable limits. In heart tissue, L-Dox levels Figure 3. Doxorubicin deposition in HT1080 xenografts and heart tissue resulting from administration of 1.4 Amol/kg doxorubicin (A), L-Dox (B), or were also less than the levels of compound 5 (Fig. 4B). compound 5 (C). Bars, SE. Unlike plasma, heart tissue accumulated doxorubicin presumably by metabolism of L-Dox. Unlike plasma and heart, L-Dox levels in HT1080 tissue exceeded compound 5 levels. Because neither compound 5 nor L-Dox preferen- In particular, mice became flushed due to redistribution of tially accumulated in HT1080 tissue (Fig. 4; data not blood to the extremities. This redistribution also led to a shown), these findings provide strong evidence that rapid decrease in blood pressure and death within minutes compound 5 was preferentially metabolized in HT1080 after compound injection. Administration of an equivalent tissue relative to heart and plasma. As a final test for amount of doxorubicin did not cause detectable flushing or tumor-specific metabolism, conjugates that could not be acute toxicity. cleaved by MMP-2, -9, or -14 were injected into mice. These Several factors supported the hypothesis that conjugates conjugates did not lead to detectable levels of doxorubicin caused mast cell degranulation leading to the release of in HT1080 xenografts (data not shown). histamine and other vasoactive peptides. First, the Some Conjugates Cause AcuteToxicity with Adminis- flushing and reduced blood pressure were consistent with tration at High Doses mast cell degranulation. Second, preadministration of When compound 5 was given at higher doses that were terfenadine, an antihistamine, partially prevented acute compatible with xenograft efficacy studies, such as toxicity. Third, acute toxicity was prevented by adminis- 14 mmol/kg, an unexpected acute toxicity was observed. tration of a nonlethal dose of compound 5 showing

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Table 2. Doxorubicin deposition in HT1080 xenografts and heart respectively, were cured. At these doxorubicin doses, mice tissue lost an average of 10% body weight, which was operationally defined as the maximum tolerated dose. Compound Doxorubicin AUC Tumor/heart ratio 0-24 Mice treated with compound 6 had no detectable weight (nmol g1 h1) loss. HT1080 Heart Absolute Normalized The observed tumor growth inhibition of compound 6 was in reasonable agreement with that expected from the Doxorubicin 3.1 8.2 0.38 1 levels of doxorubicin deposited in tumors. In particular, L-Dox 0.58 1.1 0.53 1.4 14 Amol/kg compound 6 deposited approximately one half 5 1.3 0.24 5.6 15 the doxorubicin concentration in tumors as 14 Amol/kg doxorubicin. If the doxorubicin deposited by compound 6 Abbreviation: Doxorubicin AUC0-24, area of the doxorubicin concentration- was equipotent with that deposited by doxorubicin, then time curve from 0 to 24 hours. 14 Amol/kg compound 6 q4dx3 should cause a similar growth inhibition to 7 Amol/kg doxorubicin q4dx3 and 14 Amol/kg compound 6 qdx10 should be more effective than desensitization consistent with mast cell degranulation. 14 Amol/kg doxorubicin q4dx3. As shown in Fig. 5, tumor Fourth, doxorubicin was reported to cause mast cell degranulation in mice, rats, and dogs when given at very high doses (32–34). Based on these data, we hypothesize that some non-doxorubicin structures cooperate with doxorubicin to cause mast cell degranulation. Identification of Soluble, Selective Conjugates without AcuteToxicity Based on these findings, compounds were screened for those that were specific for MMP cleavage in vitro and cultured cells yet lacked the acute toxicity observed with compound 5. This screening effort revealed that all compounds with positively charged residues caused acute toxicity at 14 Amol/kg. In contrast, compounds, such as compound 6, did not cause acute toxicity at doses up to 112 Amol/kg, the maximum dose that could be given because of compound solubility limitations. Like compound 5, compound 6 was selectively cleaved by MMP-2, -9, and -14, relative to neprilysin (Table 1), was primarily metabolized by MMPs in cultured HT1080 cells (Fig. 2B), and had good aqueous solubility (Table 1). Furthermore, compound 6 led to the selective deposition of doxorubicin in HT1080 xenografts, relative to heart tissue, with a tumor/heart doxorubicin ratio of 12 and doxorubicin HT1080 levels approximately one half those from an equimolar dose of doxorubicin at 14 Amol/kg. Compound 6 Is More Efficacious Than Doxorubicin with LessToxicity To determine if the doxorubicin deposited by conjugates was effective at reducing tumor growth, mice were implanted with HT1080 xenografts, grouped into cohorts of 10 mice with a similar median tumor size and tumor size distribution, and treated with doxorubicin or compound 6. These studies showed that 14 Amol/kg compound 6 q4dx3 was slightly less effective than 7 Amol/kg doxorubicin q4dx3, whereas 14 Amol/kg compound 6 qdx10 was more effective than the maximum tolerated levels of doxorubicin (Fig. 5). In particular, 8 of 10 mice treated with the higher dose of compound 6 were cured of their HT1080 xenografts as defined by lack of detectable tumors 75 days after implantation. In contrast, only 2 of 10 and 0 of 10 mice treated with Figure 4. Compound 5 is rapidly metabolized to L-Dox and doxorubicin in doxorubicin at 14 Amol/kg q4dx3 and 6 Amol/kg qdx10, HT1080 xenografts (C) and heart tissue (B) but not plasma (A). Bars, SE.

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doxorubicin deposited by compound 6 was effective at reducing the growth of HT1080 xenografts, whereas the reduced doxorubicin deposition in nontumor tissues led to an increased maximum tolerated dose and reduced marrow toxicity. The results of this study with MMP-activated doxorubicin prodrugs are similar to those from previous studies with extracellular enzyme-activated doxorubicin prodrugs. In particular, doxorubicin prodrugs activated by prostate- specific antigen (36, 37), neprilysin (38), and plasmin (39, 40) have been studied in mice. For the prostate-specific antigen–activated prodrug L-377,202, the doxorubicin concentration in a prostate cell xenograft was increased 2-fold and the heart doxorubicin concentration was reduced 2-fold leading to a 4-fold increased tumor/heart doxorubicin ratio (41). For the neprilysin-activated prodrug CPI-0004Na, tumor doxorubicin concentrations were increased 2-fold and heart doxorubicin concentrations Figure 5. Compound 6 inhibits the growth of HT1080 xenografts better were decreased 10-fold, leading to a 20-fold increase in than doxorubicin. See legend for doses and schedules. Day 0, time of tumor implantation. Arrows, beginning and end of treatment. A tumor the doxorubicin tumor/heart ratio (38). In both of these volume of 1 mm3 was assigned to tumors that were undetectable. cases, the prodrugs had a higher maximum tolerated dose than doxorubicin and inhibited xenograft growth better than doxorubicin (37–39). Very similar results with growth inhibition by compound 6 is consistent with these respect to improvements in the maximum tolerated dose predictions. and inhibition of xenograft growth were obtained with a Mice treated with compound 6 did not show any overt different prostate-specific antigen–activated prodrug (36) signs of toxicity, including weight loss or changes in coat and two plasmin-activated prodrugs (40). texture. To investigate compound 6 toxicity in detail, mice Although preclinical studies with tumor-activated pro- were given increasing doses of either compound 6 or drugs show that it is possible to increase the therapeutic doxorubicin. This experiment showed that the LD50 for index of doxorubicin in mice, it is unclear whether the doxorubicin was f42 Amol/kg, whereas administration of benefits observed in mice will translate to humans. The first compound 6 at 112 Amol/kg did not cause any detectable human trial with L-377,202 were recently completed and adverse effects. As a measure of marrow toxicity, retic- suggest that some of the improvements observed in ulocytes were quantified. Reticulocytes are short-lived mice will be translated to humans (42). In the case of precursors of RBC in blood that are easily quantified (35). MMP-activated prodrugs, this issue is particularly difficult Doxorubicin-treated mice showed a dramatic decrease in reticulocytes with an ED50 of 2.5 Amol/kg (Fig. 6). In contrast, mice given 14 Amol/kg compound 6 qdx10 had reticulocyte levels (average, 5.6% RBC; SD, 3.3% RBC) that were not statistically different from vehicle-treated mice (average, 3.0% RBC; SD, 1.2% RBC). Taken together, these results show that compound 6 is more effective than doxorubicin at reducing the growth of HT1080 xenografts with significantly less toxicity than doxorubicin.

Discussion In this study, MMP-activated doxorubicin prodrugs were discovered that had a higher therapeutic index than doxorubicin when HT1080 cells were used as a preclinical model. To achieve this increased therapeutic index, prodrugs were identified that were efficiently cleaved in vitro by MMP-2, -9, and -14, poorly cleaved by neprilysin, an off-target metalloprotease, and lacked positively charged residues to avoid acute toxicity. Such conjugates were specifically metabolized by MMPs in Figure 6. Effect of doxorubicin dose on reticulocyte counts. Mice were cultured HT1080 cells and preferentially deposited doxo- given the indicated dose of doxorubicin daily for 10 d. Three days after the rubicin in HT1080 xenografts relative to heart. The last dose, reticulocyte counts were determined. Bars, SD.

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to address because mouse tumors are poor models for were not saturating for metabolism in HT1080 cultures human tumors with respect to MMP expression and (data not shown), differences in the assay conditions activity. In particular, MMPs are frequently expressed between the in vitro assays and the cell-based assays could and activated by different cell types within human tumors. yield divergent results. In this case, the differences would For instance, MMP-2 is primarily expressed by stromal be specific for MMPs because the neprilysin in vitro and fibroblasts, yet pro-MMP-2 is activated by MMP-14 that is in vivo rates were well correlated. Clearly, additional work present on tumor cells. Because xenografts and mouse will be needed to resolve this issue. transgenic tumor models contain relatively few stromal An acute toxicity similar to that with compound 5 was fibroblasts, it is difficult to mimic the MMP activity of reported with h-Ala-Leu-Ala-Leu-Dox (44). Interestingly, human tumors in mice. To deal with this complexity, our capping of the positively charged NH2 terminus in this studies used HT1080 cells, a fibrosarcoma cell line. By conjugate with a succininc group also eliminated the acute choosing a fibroblast cell tumor line that expressed MMP-1, toxicity of this conjugate. These investigators, however, -2, -3, -7, -9, -14, -15, -16, and -18 (14, 15), we hoped to better attributed this improvement to decreased aggregation of represent the MMP composition in human tumors than the prodrugs. with more conventional epithelial tumor cell lines. Al- The use of MMPs to activate prodrugs should be though HT1080 cells are a rational choice, inhibition of applicable to other cytotoxics. For instance, other inves- HT1080 growth does not remove many of the unknowns in tigators have made paclitaxel and vinblastine prodrugs. translating these preclinical findings into the clinic. Although the peptide sequence identified with the doxo- If MMP-activated doxorubicin prodrugs have an in- rubicin prodrug is a good starting point for these other creased therapeutic index relative to doxorubicin in cytotoxics, it is likely that additional optimization will be humans, then there are two major ways to exploit this required because the cytotoxic moiety affects the enzymatic increased therapeutic index. Ideally, one would like to utilization of the resulting conjugate. It is also likely that increase the efficacy of doxorubicin by increasing the tumor other cytotoxic prodrugs will not have the acute toxicity concentration of doxorubicin with prodrug administration. due to mast cell degranulation that was observed with the Although such a strategy clearly works in HT1080 doxorubicin prodrugs. In particular, the positively charged xenografts, it is unclear whether this strategy will be peptides apparently increase the amount of histamine successful in human tumors. In particular, several studies release that can result from doxorubicin alone under some have investigated the use of high-dose chemotherapy circumstances. where autologous hematopoietic progenitor cell transplan- In a prodrug that requires multiple steps for activation, tation is used to allow chemotherapeutic doses that would like the MMP-activated doxorubicin prodrugs, the initial otherwise be myeloablative. Whereas overall survival was activation step should have the slowest rate and subse- similar between high-dose chemotherapy and conventional quent steps should be rapid compared with the initial step chemotherapy in four trials for patients with metastatic for optimal prodrug performance. The MMP-activated breast cancer, six of seven trials found increased disease- prodrugs described here do not possess this optimal free survival using high-dose chemotherapy (reviewed in property. In particular, the removal of the prime-side ref. 43). Although the apparent lack of increase survival is residues following MMP cleavage appears rapid, relative to discouraging, these studies may not have had sufficient MMP cleavage, except for the conversion of L-Dox to patients to detect meaningful differences. For this and other doxorubicin. This conclusion is supported by the low reasons, high-dose chemotherapy trials are ongoing. concentrations of intermediates with multiple prime-side Alternatively, an increased therapeutic index of doxorubi- residues and the high concentration of L-Dox observed in cin prodrugs could be used to decrease the toxicity from cultured cells and tumor tissue. Additionally, L-Dox doxorubicin therapy. Such decreased toxicity could allow concentrations in HT1080 xenografts at times soon after the use of other therapies that would not have been injection exceeded doxorubicin concentrations at later possible due to toxicities from doxorubicin. times. Taken together, these data show that a significant The reason for the lack of correlation between the in vitro fraction of the L-Dox that is formed in the HT1080 enzymatic efficiencies for conjugate cleavage and the xenografts diffuses outside the tumor before conversion metabolism rates in cultured HT1080 cells is unclear. to doxorubicin. If this loss could be reduced, then the Possibly, the major metabolizing MMP in cultured HT1080 potency, and potentially therapeutic index, of prodrugs cells is not MMP-2, -9, or -14. Consistent with this would be increased. Because the enzymatic conversion of hypothesis, HT1080 cells express several MMPs other than L-Dox to doxorubicin appears relatively slow, it would be MMP-2, -9, and -14, including MMP-1, -3, -7, -15, -16, and -18 interesting to investigate nonenzymatic mechanisms to (14, 15). Alternately, the conditions used for in vitro assays eliminate the final residue, such as the self-immolating may not accurately reflect the in vivo activities of the MMPs. linkers described previously (45). The enzymatic assays use soluble MMPs in detergent- containing solutions with substrate concentrations well below the Km and the cell-based assays use membrane- Acknowledgments bound MMPs with conjugates dissolved in 0.1% bovine We thank Bob Grafstrom, Kurt Auger, Simon Robinson, and Peter Ho for serum albumin, DMEM. Although conjugate concentrations advice and encouragement and Jim Duan for the gift of marimastat.

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References 25. Bickett D, Green M, Berman J, et al. A high throughput fluorogenic substrate for interstitial collagenase (MMP-1) and gelatinase (MMP-9). 1. Hardman J, Limbird L, Gilman A. The pharmacologic basis of Anal Biochem 1993;212:58 – 64. therapeutics. 10th ed. New York: McGraw-Hill; 2001. 26. Mucha A, Cuniasse P, Kannan R, et al. Membrane type-1 matrix 2. Gerwitz D. A critical evaluation of the mechanisms of action proposed metalloproteinase and stromelysin-3 cleave more efficiently synthetic for the antitumor effects of the anthracycline antibiotics Adriamycin and substrates containing unusual amino acids in their P1V positions. J Biol daunorubicin. Biochem Pharmacol 1999;57:727 – 41. Chem 1998;273:2763 – 8. 3. Woessner J, Nagase H. Matrix metalloproteinases and TIMPs. 1st ed. 27. Turner A. Neprilysin. In: Barrett A, Rawlings N, Woessner J, editors. New York: Oxford University Press; 2000. Handbook of proteolytic enzymes. San Diego: Academic Press; 1998. 4. Strongin A, Collier I, Bannikov G, et al. Mechanism of cell surface p. 1080 – 5. activation of 72-kDa type IV collagenase. Isolation of the activated form of 28. Broggini M, Colombo T, Martini A, Donnelli M. Studies on the the membrane metalloprotease. J Biol Chem 1995;270:5331 – 8. comparative distribution and biliary excretion of doxorubicin and 4V- 5. McCawley L, Matrisian L. Matrix metalloproteinases: they’re not just epi-doxorubicin in mice and rats. Cancer Treat Rep 1980;64:897 – 904. for matrix anymore. Curr Opin Cell Biol 2001;13:534 – 40. 29. Formelli F, Carsana R, Pollini C. Pharmacokinetics of 4V-deoxy-4V- 6. Egeblad M, Werb Z. New functions for the matrix metalloproteinases in iodo-doxorubicin in plasma and tissues of tumor-bearing mice compared cancer progression. Nat Rev Cancer 2002;2:161 – 74. to doxorubicin. Cancer Res 1987;47:5401 – 6. 7. H-YHa, Moon H-B, Nam M-S, et al. Overexpression of membrane-type 30. van der Vijgh W, Maessen P, Pinedo H. Comparative metabolism and matrix metalloproteinase-1 gene induces mammary gland abnormalities pharmacokinetics of doxorubicin and 4V-epidoxorubicin in plasma, heart, and adenocarcinoma in transgenic mice. Cancer Res 2001;61:984 – 90. and tumor of tumor-bearing mice. Cancer Chemother Pharmacol 1990; 8. Itoh T, Tanioka M, Yoshida H, et al. Reduced angiogenesis and tumor 26:9 – 12. progression in gelatinase A-deficient mice. Cancer Res 1998;58:1048 – 51. 31. de Jong J, Klein I, Bast A, van der Vijgh W. Analysis and 9. Itoh T. Experimental metastasis is suppressed in MMP-9-deficient pharmacokinetics of N-L-leucyldoxorubicin and metabolites in tissues of mice. Clin Exp Metastasis 1999;17:177 – 81. tumor-bearing BALB/c mice. Cancer Chemother Pharmacol 1992;31: 156 – 60. 10. Jones J, Glynn P, Walker R. Expression of MMP-2 and MMP-9, their inhibitors, and the activators MT1-MMP in primary breast carcinomas. 32. Decorti G, Bartoli Klugman F, Candusio L, Baldini L. Characterization J Pathol 1998;189:161 – 8. of histamine secretion induced by anthracyclines in rat peritoneal mast cells. Biochem Pharmacol 1986;35:1939 – 42. 11. Elenbaas B, Weinberg R. Heterotypic signaling between epithelial tumor cells and fibroblasts in carcinoma formation. Exp Cell Res 2001; 33. Decorti G, Bartoli Klugman F, Candusio L, et al. Uptake of Adriamycin 264:169 – 84. by rat and mouse mast cells and correlation with histamine release. Cancer Res 1989;49:1921 – 6. 12. Pan W, Arnone M, Kendall M, et al. Identification of peptide substrates for human MMP-11 (stromelysin-3) using phage display. J Biol 34. Bristow M, Sageman W, Scott R, et al. Acute and chronic Chem 2003;278:27820 – 7. cardiovascular effects of doxorubicin in the dog: the cardiovascular pharmacology of drug-induced histamine release. J Cardiovasc Pharmacol 13. Nagase H, Fields G. Human matrix metalloproteinase specificity 1980;2:487 – 515. studies using collagen sequence-based synthetic peptides. Biopolymers 1996;40:399 – 416. 35. Burkitt H, Young B, Heath J. Wheater’s functional histology. In: Wheater’s functional histology. 3rd ed. Churchill Livingstone; 1993. p. 44. 14. Giambernardi T, Grant G, Taylor G, et al. Overview of matrix metalloproteinase expression in cultured human cells. Matrix Biol 1997; 36. Khan S, Denmeade S. In vivo activity of a PSA-activated doxorubicin 16:483 – 96. prodrug against PSA-producing human prostate cancer xenografts. Prostate 2000;45:80 – 3. 15. Grant G, Giambernardi T, Grant A, Klebe R. Overview of expression of matrix metalloproteinases (MMP-17, MMP-18, MMP-20) in cultured 37. DeFeo-Jones D, Garsky V, Wong B, et al. A peptide-doxorubicin human cells. Matrix Biol 1999;18:145 – 8. ‘‘prodrug’’ activated by prostate-specific antigen selectively kills prostate 16. Sang A, Bodden M, Windsor L. Activation of human progelatinase A tumor cells positive for prostate-specific antigen in vivo. Nat Med 2000; by collagenase and matrilysin: activation by procollagenase by matrilysin. 6:1248 – 52. J Protein Chem 1996;15:243 – 53. 38. Dubois V, Dasnois L, Lebtahi K, et al. CPI-0004NA, a new 17. George H, Marchand P, Murphy K, et al. Recombinant human 92- extracellularly tumor-activated prodrug of doxorubicin: in vivo toxicity, kilodalton type V collagenase/gelatinase from baculovirus infected insect activity, and tissue distribution confirm tumor cell selectivity. Cancer Res cells: expression, purification, and characterization. Protein Expr Purif 1997; 2002;62:2327 – 31. 10:154– 61. 39. Pan C, Cardarelli P, Nieder M, et al. CD10 is a key enzyme involved in 18. Sonnenberg J, Sakane Y, Jeng A, et al. Identification of protease the activation of tumor-activated prodrug CPI-0004Na and novel ana- logues: implications for the design of novel peptide prodrugs for the 3.4.24.11 as the major atrial natriuretic factor degrading enzyme in the rat + kidney. Peptides 1988;9:173 – 80. therapy of CD10 tumors. Cancer Res 2003;63:5526 – 31. 19. Copeland R. Enzymes: a practical introduction to structure, mecha- 40. Devy L, de Groot F, Balcher S, et al. Plasmin-activated doxorubicin nism, and data analysis. 2nd ed. New York: John Wiley & Sons, Inc.; 2000. prodrugs containing a spacer reduce tumor growth and angiogenesis without systemic toxicity. FASEB J 2004;18:565 – 7. 20. Stanton H, Gavrilovic J, Atkinson S, et al. The activation of proMMP- 2 (gelatinase A) by HT1080 fibrosarcoma cells is promoted by culture on a 41. Wong B, DeFeo-Jones D, Jones R, et al. PSA-specific and non- fibronectin substrate and is concomitant with an increase in processing of PSA-specific conversion of a PSA-targeted peptide conjugate of doxoru- MT1-MMP (MMP-14) to a 45 kDa form. J Cell Sci 1998;111:2789 – 98. bicin to its active metabolites. Drug Metab Dispos 2001;29:313 – 8. 21. Rasmussen H, McCann P. Matrix metalloproteinase inhibition as a 42. DiPaola R, Rinehart J, Nemunatis J, et al. Characterization of a novel novel anticancer strategy: a review with special focus on batimastat and prostate-specific antigen-activated peptide-doxorubicin conjugate in marimastat. Pharmacol Ther 1997;75:69 – 75. patients with prostate cancer. J Clin Oncol 2002;20:1874 – 9. 22. Trouet A, Passioukov A, Van derpoorten K, et al. Extracellularly 43. Tartarone A, Romano G, Galasso R, et al. Should we continue to tumor-activated prodrugs for the selective chemotherapy of cancer: study high-dose chemotherapy in metastatic breast cancer patients? A application to doxorubicin and preliminary in vitro and in vivo studies. critical review of the published data. Bone Marrow Transplant 2003; Cancer Res 2001;61:2843 – 6. 31:525 – 30. 23. Baurain R, Masquelier M, Deprez-de Campeneere D, Trouet A. Amino 44. Fernandez A, Van derpoorten K, Dasnois L, et al. N-succinyl-(h- acid and dipeptide derivatives of daunorubicin. 2. Cellular pharmacology alanyl-L-leucyl-L-alanyl-L-leucyl)doxorubicin: an extracellularly tumor-acti- and antitumor activity on L1210 leukemic cells in vitro and in vivo. J Med vated prodrug devoid of intravenous acute toxicity. J Med Chem 2001;44: Chem 1980;23:1171 – 4. 3750 – 3. 24. Knight C, Willenbrock F, Murphy G. A novel coumarin-labelled peptide 45. Niculescu-Duvaz I, Niculescu-Duvaz D, Friedlos F, et al. Self- for sensitive continuous assays of the matrix metalloproteinases. FEBS immolative anthracycline prodrugs for suicide gene therapy. J Med Chem Lett 1992;296:263 – 6. 1999;42:2485 – 9.

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