Synthesis and Biological Evaluation of EC72: A New Folate-Targeted

Bioconjugate Chem. 2005, 16, 803−811

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Synthesis and Biological Evaluation of EC72: A New Folate-Targeted Chemotherapeutic Christopher P. Leamon,* Joseph A. Reddy, Iontcho R. Vlahov, Marilynn Vetzel, Nikki Parker, Jeffrey S. Nicoson, Le-Cun Xu, and Elaine Westrick Endocyte, Inc., 3000 Kent Avenue, West Lafayette, Indiana 47906. Received December 6, 2004; Revised Manuscript Received April 14, 2005

A novel folate conjugate of mitomycin C, herein referred to as EC72, was designed and evaluated for biological activity against FR-positive cells and tumors. EC72 was produced by coupling folic acid-γcysteine to 7-N-modified MMC via a disulfide bond. This water soluble conjugate was found to retain high affinity for FR-positive cells, and it produced dose responsive activity in vitro against a panel of folate receptor (FR)-positive cell lines. EC72’s activity was considered to be targeted and specific for the FR since (i) excess folic acid blocked biological activity, and (ii) FR-negative cell lines were unresponsive to this drug. Initial in vivo tests confirmed EC72’s activity in both syngeneic and xenograft models, and this activity occurred in the apparent absence of gross or pathological toxicity. These results are significant, since daily dosing of EC72 for more than 30 consecutive days yielded no evidence of myelosuppression or toxicity to major organs, including the FR-positive kidneys. The latter observation supports published data, indicating that the apically oriented kidney proximal tubule FRs function to salvage folates prior to their excretion and to return these molecules back into systemic circulation. Overall, EC72’s performance in vitro and in vivo warrants further preclinical study before this novel targeted chemotherapeutic is considered for clinical investigation.

INTRODUCTION

Notwithstanding the noteworthy advances in its treatment, cancer persists as the second largest cause of death in America, with one in four deaths being caused by this disease (1). Chemotherapy remains the primary method of treatment for metastasized or disseminated cancers. Traditionally, chemotherapy regimens have been designed to administer the untargeted drug intermittently (e.g. once every three to six weeks) at its maximum tolerated dose followed by a recovery period. This approach has been used for over 50 years with dismal improvements in patient cure rates. One reason for this apparent failure is that this method of treatment is plagued by toxicity resulting from the nondiscriminate uptake of these poisons into proliferating cells of both normal and neoplastic tissue. Second, the kinetics of tumor tissue recovery is much faster than the kinetics of normal tissue recovery, thus making tumor elimination highly unlikely in response to infrequent drug administration. In theory, the normal tissue toxicity manifested by any drug should be decreased or eliminated if the pharmacophore were specifically targeted to the diseased cells. This alternative approach might also enable a more aggressive metronomic-style of dosing, which would be expected to produce better therapeutic outcomes because the tumor would never have the opportunity to “recover”. As such, tumor-specific targeting of drugs remains one of the most important goals in cancer treatment today. The vitamin folic acid is a ligand capable of targeting covalently attached bioactive agents quite specifically to folate receptor (FR)-positive tumors. The FR, which is a well-known tumor associated protein, can actively internalize bound folates via endocytosis (2, 3). It has been * To whom correspondence should be addressed. Phone (765) 463-7175; fax (765) 463-9271; e-mail [email protected].

detected at very high levels in >90% of ovarian and other gynecological cancers, as well as at high to moderate levels in kidney, brain, lung, and breast carcinomas (414). Not surprisingly, the concept of “folate-drug targeting” has been effectively exemplified over the past 14 years using payloads as diverse as small radiopharmaceutical agents to large DNA-containing formulations (see (15) for a comprehensive overview of this technology). Recent efforts from a small molecule research project led to the creation of a folate-mitomycin C (MMC) conjugate. This novel FR-targetable chemotherapeutic, named EC72, was constructed using disulfide linker technology to afford molecular separation of MMC from folate once this conjugate enters the endosomes of FRpositive cancer cells. As reported below, EC72 was found to produce encouraging in vitro and in vivo therapeutic activity in multiple FR-positive models, and these data warrant further preclinical study of this new investigational agent. EXPERIMENTAL PROCEDURES

Materials. N10-Trifluoroacetylpteroic acid was purchased from Eprova AG, Schaffhausen, Switzerland. Peptide synthesis reagents were purchased from NovaBiochem (La Jolla, CA) and Bachem (San Carlos, CA). Folate-free RPMI media (FFRPMI) and PBS were obtained from Gibco, Grand Island, NY. 3H-Thymidine and N1a-(3H-methyl)mitomycin K were purchased from Moravek Biochemicals, Brea, CA. Synthesis, Purification, and Analytical Characterization of EC72 and 3H-Methyl-EC72. Construction of EC72 began with the synthesis of the thiol containing folate linker, Pte-γGlu-Cys-OH, using a preloaded FmocCys(Trt) Wang resin and standard solid-phase techniques. The linker was purified on a preparative RPHPLC system connected to a Novapak HR C18 19 × 300

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mm column. A gradient method was used, starting with 99% mobile phase A (10 mM ammonium acetate buffer, pH 5.5) and 1% mobile phase B (acetonitrile) and reaching 50% B within 30 min at a flow rate of 15 mL/min. Fractions corresponding to the main peak (UV monitoring at 280 nm) were combined, and the acetonitrile was removed under reduced pressure. The rest was subjected to freeze-drying for 48 h to give the folate linker at >94% purity based on analytical HPLC (RT ) 10 min) on NovaPak C18 3.9 × 150 mm column and using a gradient of solvents, starting with 99% mobile phase A (1 mM phosphate buffer, pH 6.5) and 1% mobile phase B (acetonitrile) and reaching 50% B within 30 min at a flow rate of 1.0 mL/min. 1H NMR (300 MHz, DMSO-d6): 1.87-2.93 (m,1H), 1.98-2.04 (m,1H), 2.10-2.15 (m, 2H), 2.54-2.68 (dd, J ) 13.5 Hz, J ) 5.6 Hz, 1H), 2.71-2.82 (dd, J ) 13.5 Hz, J ) 4.7 Hz, 1H), 4.05-4.08 (dd, J ) 5.6 Hz, J ) 4.7 Hz, 1H), 4.15-4.20 (dd, J ) 8.2 Hz, J ) 4.4 Hz, 1H), 6.61-6.63 (d, J ) 8.7 Hz, 2H), 7.60-7.62 (d, J ) 8.7 Hz, 2H), 8.61 (s, 1H). The other reaction partner, pyridyldithioethylmitomycin C, was prepared by treating the commercially available 2-pyridyldithioethylamine with the vinylogous methyl ester in the quinone moiety of mitomycin A (16). The resulting 2-pyridyldithiol derivative was used for sulfenylation of the thiol group in Pte-γGlu-Cys-OH to provide EC72 (Pte-γGlu-Cys-SS-MMC). In brief, 100 mg (0.21 mmol) of Pte-γGlu-Cys-OH were mixed with 4.0 mL of HPLC-grade water, and the resulting suspension was bubbled with argon. While purging, the mixture was treated with 0.1 N sodium bicarbonate until the pH reached 7.0, at which time the solution turned clear yellow. One hundred twenty milligrams (0.24 mmol) of pyridyldithioethylmitomycin C dissolved in 3.0 mL of methanol was then added to the reaction vessel. The reaction mixture was stirred under argon for 1 h, after which methanol and some of the water were removed under reduced pressure. The resulting dark green suspension was filtered through a 0.45 µm filter to give a dark green solution; the sample was then loaded onto a preparative RP-HPLC system connected to a Novapak HR C18 19 × 300 mm column. A gradient method was used starting with 99% mobile phase A (10 mM ammonium acetate buffer, pH 6.3) and 1% mobile phase B (acetonitrile) and reaching 50% B within 30 min at a flow rate of 15 mL/min. Fractions corresponding to the main peak (UV monitoring at 280 nm) were combined, and the acetonitrile was removed under reduced pressure. The remaining material was subjected to freeze-drying for 48 h to yield 68 mg of final EC72 conjugate. EC72’s analytical HPLC profile revealed 93% purity (tR ) 16.5 min) on a NovaPak C18 3.9 × 150 mm column, using a gradient of solvents starting with 99% mobile phase A (1 mM phosphate buffer, pH 6.5) and 1% mobile phase B (acetonitrile) while reaching 50% B within 30 min at a flow rate of 1.0 mL/min. The structure of EC72 was characterized by 1H NMR (500 MHz, D2O): δ 1.73 (s, 3H), 2.07-2.13 (m,1H), 2.28-2.32 (m,1H), 2.46-2.53 (m, 2H), 2,56-2.61 (m, 1H), 2.64-2.73 (m,1H), 2.90-2.96 (dd, J ) 14.0 Hz, J ) 8.3 Hz, 1H), 2.97-2.99 (m, 2H), 3.083.11 (dd, J ) 14.0 Hz, J ) 1.7 Hz, 1H), 3.25 (s, 3H), 3.463.49 (dd, J ) 10.6 Hz, J ) 4.4 Hz, 1H), 3.50-3.64 (m, 3H), 4.07-4.10 (d, J ) 13.3 Hz, 1H), 4.16-4.21 (t, J ) 10.6 Hz, 1H), 4.38-4.43 (m, 2H), 4.50 (s, 2H), 6.68-6.70 (d, J ) 8.7 Hz, 2H), 7.62-76.4 (d, J ) 8.7 Hz, 2H), 8.66 (s, 1H) and ES MS: (m-H)- 935.6, (m+H)+ 937.4. The synthesis of 3H-methyl-EC72 was performed as described above except that N1a-(3H-methyl)mitomycin K (Moravek Biochemicals) was substituted for mitomycin

Leamon et al.

A. The reaction mixture was condensed to 0.5 mL under reduced pressure, and the residue was purified by HPLC using the aforementioned conditions. The eluates containing 3H-methyl-EC72 were collected and condensed to 1 mL under reduced pressure. The radiochemical purity was 98.5%, and the specific activity was 3.2 Ci/mmol. Cell Culture. Cells were grown continuously as a monolayer using folate-free RPMI medium (FFRPMI) containing 10% heat-inactivated fetal calf serum (HIFCS) at 37 °C in a 5% CO2/95% air-humidified atmosphere with no antibiotics. The HIFCS contained its normal complement of endogenous folates which enabled the cells to sustain growth in this more physiologically relevant medium (3). All cell experiments were performed using FFRPMI containing 10% HIFCS (FFRPMI/HIFCS) as the growth medium, except where indicated. Folate Receptor Assay. A folate receptor (FR) assay was performed as previously described by Parker et al. (14), and all sample preparation procedures were performed at 4 °C. Briefly, tissue samples were homogenized in homogenization buffer (10 mM Tris, pH 8.0, 0.02 mg/ mL each of leupeptin and aprotinin; 1 mL buffer per 50 mg of tissue). Membrane pellets were collected by centrifugation (40 000g for 60 min. at 4 °C) and resuspended in solubilization buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 25 mM n-octyl-β-D-glucopyranoside, 5 mM EDTA, and 0.02% sodium azide). Insoluble material was removed by another centrifugation, and the total protein concentration of the supernatants was determined by the BCA method (Pierce Chemical Co.). Each sample was diluted to 0.25 mg/mL in solubilization buffer, and the solubilized FRs were concentrated by passing through Microcon-30 microconcentrators (30K molecular weight cutoff; Millipore Corporation). After a brief acid wash step to release endogenous folates, samples were neutralized and then allowed to bind 3H-folic acid (120 nM 3H-folic acid (Amersham) in 10 mM Na2PO4, 1.8 mM KH2PO4, pH 7.4, containing 500 mM NaCl, 2.7 mM KCl, and 25 mM n-octyl-β-D-glucopyranoside) or 3H-folic acid competed with 120 µM unlabeled folic acid. The concentrators were washed three times with a detergent-based buffer (50 mM n-octyl-β-D-glucopyranoside, 0.7 M NaCl in PBS, pH 7.4). After the final wash, the retentates containing the solubilized FRs were recovered with PBS containing 4% Triton-X 100. The samples were then counted in a liquid scintillation counter (Packard Bioscience Co.). CPM values were converted to picomole of FR using the reagent’s specific activity, and the final results were normalized with respect to the sample protein content. Relative Affinity Assay. The relative affinity of various folate derivatives was determined according to the method described by Westerhoff et al. (17) with slight modification. Briefly, FR-positive KB cells were suspended into FFRPMI/HIFCS following exposure to 0.25% trypsin in phosphate-buffered saline (PBS) at room temperature for 3 min. Following a 5 min 800g spin and one PBS wash, the final cell pellet was suspended in FFRPMI (no serum). Cells were incubated for 15 min on ice with 100 nM of 3H-folic acid in the absence and presence of increasing concentrations of folate-containing test articles. Samples were centrifuged at 10 000g for 5 min, cell pellets were suspended in buffer, transferred to individual vials containing 5 mL of scintillation cocktail, and then counted for radioactivity. Negative control tubes contained only the 3H-folic acid in FFRPMI (no competitor). Positive control tubes contained a final concentration of 1 mM folic acid, and CPMs measured in these samples (representing nonspecific binding of label) were subtracted from all samples. Notably, relative

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Biological Evaluation of EC72

affinities were defined as the inverse molar ratio of compound required to displace 50% of 3H-folic acid bound to FR on KB cells, and the relative affinity of folic acid for the FR was set to 1. Kinetics and Specificity of EC72 Activity. KB cells were seeded in 12-well Falcon plates and allowed to form adherent monolayers (∼50% confluent) overnight in FFRPMI/HIFCS. Thirty minutes prior to the addition of EC72, spent medium was aspirated from all wells and replaced with fresh FFRPMI. Note, designated wells received media containing 100 µM folic acid, and cells within the latter wells were used to determine the targeting specificity, since cytotoxic activity produced in the presence of excess folic acid (enables competition for FR binding) would signify the portion of the total activity that was unrelated to FR-specific delivery. Following a single rinse with 1 mL of media, cells were exposed to 100 nM of EC72 in 1 mL of FFRPMI/HIFCS for only 10 min at 37 °C (pulse). Cells were then washed three times with media and chased for 22, 46, or 70 h. Spent media was aspirated from all wells and replaced with fresh media containing 5 µCi/mL 3H-thymidine. Following a 2 h 37 °C incubation, cells were washed three times with 0.5 mL of PBS, pH 7.4, and then treated with 0.5 mL of ice-cold 5% trichloroacetic acid per well. After 15 min, the trichloroacetic acid was aspirated and the cells solubilized by the addition of 0.5 mL of 0.25 N sodium hydroxide for 15 min. Four hundred and fifty microliters of each solubilized sample were transferred to scintillation vials containing 3 mL of Ecolume scintillation cocktail and then counted in a liquid scintillation counter. Final tabulated results were expressed as the percentage of 3H-thymidine incorporation relative to untreated controls. Dose-Dependent Activity. Cells were heavily seeded in 12-well Falcon plates and allowed to form nearly confluent monolayers overnight. Following one rinse with 1 mL of fresh FFRPMI/HIFCS, each well received 1 mL of media containing increasing concentrations of EC72 (four wells per sample). Cells were pulsed for 10 min at 37 °C, rinsed four times with 0.5 mL of media, and then chased in 1 mL of fresh media up to 72 h. Cells were incubated with 3H-thymidine and processed as described above. Final tabulated results were expressed as the percentage of 3H-thymidine incorporation relative to untreated controls. Serum Stability. The stability of EC72 in human serum was determined by an in vitro assay which included a solid-phase extraction procedure for sample cleanup followed by HPLC analysis. EC72 was added to human serum at a 200 µM final concentration, and the sample was incubated in a 37 °C water bath for a total period of 24 h. Two hundred microliters of serum sample (n ) 3) was removed 1, 2, 4, 8, and 24 h after initiation of incubation, and EC72 was recovered from serum by solid-phase extraction with Oasis HLB cartridges (1 cm3/ 30 mg; Waters Corporation). First, 800 µL of 6 M guanidine (pH adjusted to 7.4 with 0.1 M NaOH) was added to each 200 µL serum sample to reduce nonspecific protein binding of the drug. Next, cartridges were conditioned with 1 mL of methanol and equilibrated with 1 mL of 6 M guanidine, pH 7.4. Samples (1 mL) were then loaded onto the cartridges followed by a wash with 5% methanol in water (1 mL). Finally, samples were eluted with 1 mL of methanol, evaporated to dryness, and reconstituted in 200 µL of phosphate-buffered saline, pH 7.4. Ten microliters of the reconstituted sample was injected on an HPLC equipped with a Sunfire C18 3.0 × 100 mm column, a UV detector at 280 nm, 10 mM

ammonium acetate, pH 7.0 (mobile phase A), and acetonitrile (mobile phase B). EC72 was eluted with a gradient from 95:5 (A:B) to 60:40 (A:B) in 15 min. Percent recoveries were calculated based on the integrated peak areas assuming that a t ) 0 serum extracted sample represented 100% recovery. The %RSD for each individual time point was 1000 >1000

a A panel of cell lines with varying levels of FR expression were treated for 10 min with increasing concentrations of EC72. Following a 70 h chase in fresh media, cells were incubated with 3H-thymidine for 2 h and then counted for radiolabel incorporation into newly synthesized DNA, as described in Experimental Procedures. IC50 values were calculated from nonlinear fitted dose curves.

moderate, 10-fold excess of folic acid. Although the therapeutic difference between the +/- excess folate cohorts was obvious, the data only support a p-value of 0.065. However, different from the highly controllable receptor competition studies that are often performed in vitro (15), competition in vivo can be particularly challenging due to (i) nonequilibrium conditions, (ii) dissimilar pharmacokinetics between folic acid and a given folate-drug conjugate, and (iii) the fact that repeated doses of high excess folic acid (e.g. 100-fold over the

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Figure 8. EC72 dose-response. 0.5 × 106 M109 cells were inoculated ip into each Balb/c mouse. Four days later, animals were treated intraperitoneally with (b) 100 nmol/kg, (O) 400 nmol/kg, or (1) 1800 nmol/kg EC72 following a qdx5, 4 wk schedule. A control cohort (9) was not treated. Animal survival was monitored daily (n ) 4 animals per cohort).

Figure 6. Serum stability and pharmacokinetics. Panel A, stability of EC72 in human serum at 37 °C (n ) 3 with 10 times within a 6 to 8 week cycle with “receptor-saturating” dose levels of EC72. Such a dose density, or metronomic, style of dosing may produce greater therapeutic outcomes. In regards to MMC, this targeted approach may also suddenly revitalize this old drug’s clinical utility. In conclusion, additional animal studies with EC72 are planned to better understand the effects of dose level and dose schedule. We also plan to study the effects of tumor growth rate and FR content on the overall observed antitumor response manifested by EC72 therapy. It is also important to study the effect of alternative dosing routes (ip versus iv or subcutaneous) as well as to vary the location of the tumor (i.e. ip versus subcutaneous). Since EC72 therapy appears to be rather safe, at least at the preclinical level, various combinations of EC72 plus other drugs should also be investigated for possible additive or synergistic responses. This is especially true for those drugs that cannot be dosed in combination with free MMC due to overlapping toxicities. ACKNOWLEDGMENT

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