Novel Prodrugs of SN38 Using Multiarm Poly ... - ACS Publications

Bioconjugate Chem. 2008, 19, 849–859

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Novel Prodrugs of SN38 Using Multiarm Poly(ethylene glycol) Linkers Hong Zhao,* Belen Rubio, Puja Sapra, Dechun Wu, Prasanna Reddy, Prakash Sai, Anthony Martinez, Ying Gao, Yoany Lozanguiez, Clifford Longley, Lee M. Greenberger, and Ivan D. Horak Enzon Pharmaceuticals, Inc., 20 Kingsbridge Road, Piscataway, New Jersey 08854. Received August 31, 2007; Revised Manuscript Received January 30, 2008

CPT-11, also known as irinotecan, is a prodrug that is approved for the treatment of advanced colorectal cancer. The active metabolite of CPT-11, SN38 (7-ethyl-10-hydroxy-camptothecin), has 100- to 1000-fold more potent cytotoxic activity in tissue cell culture compared with CPT-11. However, parental administration of SN38 is not possible because of its inherently poor water solubility. It is reported here that a multiarm poly(ethylene glycol) (PEG) backbone linked to four SN38 molecules (PEG-SN38) has been successfully prepared with high drug loading and significantly improved water solubility (400- to 1000-fold increase). Three different protecting strategies have been developed in order to selectively acylate the 20-OH of SN38 to preserve its E-ring in the lactone form (the active form of SN38 with cytotoxic activities) while PEG is still attached. One chemical process has been optimized to make a large quantity of the PEG-SN38 conjugate with a high yield that can be readily adapted for scale-up production. The PEG-SN38 conjugates have shown excellent in vitro anticancer activity, with potency similar to that of native SN38, in a panel of cancer cell lines. The PEG-SN38 conjugates also have demonstrated superior anticancer activity in the MX-1 xenograft mice model compared with CPT-11. Among the four conjugates, PEG-Gly-(20)-SN38 (23) has been selected as the lead candidate for further preclinical development.

INTRODUCTION Camptothecin and its analogues have potent cytotoxic activity in tumor cells grown in tissue culture and moderate anticancer efficacy in animal models. The compounds selectively inhibit topoisomerase I (TOP1) by trapping the enzyme during the cleavage of DNA (1), thereby prohibiting faithful cell division and protein production. This ultimately leads to cell death. Early clinical development of camptothecin carboxylate in the 1970s demonstrated that the compound had antitumor activity but also substantial toxicity (2). It was not until the discovery of more water-soluble derivatives of camptothecin that two TOP1 inhibitors (Hycamtin [topotecan] and Camptosar [CPT-11, irinotecan]) were approved as anticancer agents by the U.S. Food and Drug Administration (FDA) in 1997. While CPT-11 has clinical utility, there are many limitations that suggest further improvement of the compound may be possible. One major limitation is that the active ingredient, SN38 (7-ethyl-10-camptothecin), which is produced by esterasemediated hydrolysis of the solubilizing group on CPT-11 (the bispiperidine moiety), requires carboxylesterase-2. Although carboxylesterase-2 is present in abundance within the liver, its expression is far lower in the blood (3, 4). As a result, only 1-9% of an injected dose of CPT-11 is converted to SN38 (3, 5, 6) in humans (6). In addition, the lactone E-ring of CPT-11 (or SN38) can be readily converted to an open carboxylate form that is inactive against TOP1 (6–8) and binds tightly to human serum albumin. In particular, it is known that, 24 h after CPT-11 infusion in humans, approximately 25% and 55% of CPT-11 and SN38, respectively, are in the closed lactone form (3) compared with the total amount of CPT-11 and SN38. Hence, inhibition of the conversion to the inactive open carboxylate form of the molecule may have therapeutic benefit. Finally, while SN38 has 100- to 1000-fold more potent in vitro cytotoxic activity compared with CPT-11 (3), it has poor * Contact information: Telephone 732-980-4902, fax 732-885-2950, E-mail [email protected].

solubility in any pharmaceutically acceptable excipient and cannot be used for systemic applications (9). One solution to alleviate the above-mentioned limitations is to PEGylate SN38. PEGylation is well-known for its capability to solubilize very insoluble small molecule compounds, prolong circulation time, and alter the biodistribution of parent drugs (10). In fact, we previously have shown that dramatic enhancement of circulating half-life, solubility, and in vivo efficacy of the native anticancer drug camptothecin was achieved utilizing a prodrug strategy in conjunction with the nonimmunogenic 40 kDa polymeric poly(ethylene glycol) (PEG) (11). The active lactone form of camptothecin was largely preserved during circulation due to the acylation through the 20-hydroxyl functional group where PEG was attached (12). The lactone stabilization was further proven in a separate study using different acylation agents with this 20-hydroxyl group (13). However, the PEG used in the camptothecin delivery was the bifunctional linear PEG that had only two active sites for drug conjugation. As a result, there was about 1.7% of camptothecin (CPT) by weight per PEG-CPT conjugate. Subsequently, a large amount of the compound was given to patients during clinical trials; most of the compound was inactive PEG polymer, which could be as high as 14 g of PEG per dose. Due to the high potency of SN38 and our enhanced ability to increase the loading of the drug onto PEG, it was reasoned that PEG-SN38 would overcome the limitations of CPT-11 or PEG-CPT. We report here the description of PEGylated SN38 derivatives that have remarkable efficacy in animal tumor models and have recently entered into phase I clinical trials.

CHEMISTRY The most commonly used PEG is a linear polymer with either one or two functional groups at the two distal ends. Simple conjugation with such linear PEG will attach up to two drug molecules per PEG conjugate. To increase the payload, we previously developed methods by branching at the ends of PEG with trifunctional moieties such as aspartic acid and have successfully increased the loading of the water-soluble antican-

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850 Bioconjugate Chem., Vol. 19, No. 4, 2008

Zhao et al.

Scheme 1

cer drug cytarabine (14). However, this approach involved multiple steps of preparation of both the branching small molecule part as well as the PEG linkers. In addition, the sites for drug molecule attachment were too close to each other in some cases, which caused steric hindrance among the functional groups and resulted in incomplete conjugation of drug molecules. When the two aromatic drug molecules were close to each other, the stacking phenomenon became another issue that could impact the solubility of the drug molecules. This could be a bigger concern in the case of the delivery of very insoluble drug molecules. Previously, Gopin and co-workers developed dendritic prodrugs for delivery of camptothecin (15). To increase the loading of water-insoluble cytotoxic compounds, we sought here to use the multiarm PEG as the carrier. The active sites at the end of each “arm” of multiarm PEG are far away from each other and behave independently. This property will help to eliminate the steric hindrance and stacking phenomenon when the drug molecules are too close to each other. It also will help the completion of the loading and improve the solubility of the loaded drug molecules. It has been demonstrated conclusively that substantial increments to the area under the drug concentration-time curve or circulation time can be achieved by PEG conjugation to small molecule drugs if the molecular weight of the PEG exceeds 20 000 Da (10). In this study, we chose to use 40 kDa four-arm-PEG-OH (1), which has an active group at the distal end of each arm. Upon full loading, this PEG can conjugate up to four SN38 molecules. To do so, we first converted compound 1 to four-arm-PEG-acid (3) using a previously reported method (16). Hence, 1 was first reacted with t-butyl bromoacetate in the presence of potassium t-butoxide to form the ester 2, which was deprotected by trifluoroacetic acetic acid (TFA) to give the desired PEG acid 3 (Scheme 1). SN38 (4) has two hydroxyl groups, i.e., 10-OH and 20-OH. Since the 10-OH group is more active, in order to selectively acylate the 20-OH of SN38, the 10-OH has to be protected first. We sought to use different amino acids as the linking moiety between the PEG and SN38. During the study, alanine (Ala), methionine (Met), sarcosine (Sar), and glycine (Gly) had been chosen as the linkers based on our previous research (17). To find the best protecting strategy, three different approaches were tried. The first approach was the combination of acid-labile t-butylcarbonyl (Boc) group and base-labile 1,1-dioxobenzo[b]thiophen-2-ylmethyloxycarbonyl (Bsmoc) group. The Bsmoc group developed by Carpino et al. (18) can be removed by treatment with a secondary amine via Michael addition that is conducted under extremely mild conditions. We used this protecting group in the past to synthesize and isolate moistureand base-sensitive compounds such as 2′-paclitaxel glycinate (19). Thus, SN38 was first reacted with di-t-butyl dicarbonate to selectively protect the 10-OH with a Boc group to give compound 5 (Scheme 2), followed by reaction with BsmocAla to acylate the 20-OH to give compound 6. One equivalent

of 4-piperidinopiperidine was used to remove the Bsmoc group, and the resulting free amine was converted to HCl salt through an acid wash (7). It was found that the intermediate with a free amine group was not very stable and slowly converted into byproduct. Therefore, we had to make the HCl salt of this intermediate to avoid the free amine. A similar phenomenon has been disclosed by Liu and co-workers (20). Compound 7 was then coupled with four-arm-PEG acid (3) using N-ethylN′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) to give Boc-protected compound 8. One final treatment with TFA yielded the final product PEG-Ala-(20)-SN38 (9). Another conjugate, PEG-Met-(20)-SN38 (13), was prepared in a similar fashion (Scheme 3). In the second approach, Boc-protected compound 5 was reacted with Boc-Sar to give compound 14 (Scheme 4). TFA treatment removed both Boc groups on 10-OH and sarcosine to give 15 as the intermediate. At this time, the 10-OH was protected again through reaction with tert-butyldimethylsilyl chloride (TBDMS-Cl) to prevent side reactions in the following steps. The resulting molecule 16 was directly conjugated with 3 to give 17, and the TBDMS protecting group was removed by TFA to give the final product PEG-Sar-(20)-SN38 (18). The problem for this approach was that intermediate 16 was not very stable either and slowly lost the silyl protecting group TBDMS during storage in refrigerator. To resolve this stability issue, the more hindered t-butyldiphenylsilyl group (TBDPS) was used to protect the 10-OH directly in the third approach (Scheme 5). Thus, t-butyldiphenylsilyl chloride (TBDPS-Cl) reacted with SN38 selectively to give compound 19 in the first step. Subsequent acylation of 20OH with Boc-Gly gave compound 20. The Boc group can be removed by HCl in dioxane without affecting the TBDPS group. The resulting intermediate 21 was a very stable molecule even under elevated temperatures. This property ensured the possibility that key intermediate 21 could be scaled up to large quantities with high purity and stored either at room temperature or in the refrigerator for a prolonged period of time. The next conjugation with PEG acid (3) was done by using propane phosphonic acid anhydride (PPAC) as the coupling agent. The TBDPS group was finally removed with tetra-n-butylammonium fluoride (TBAF) to give pure conjugate 23. This last approach has been successfully optimized and scaled up to hundreds of grams per batch on a repetitive base.

EXPERIMENTAL PROCEDURES General Procedures. All reactions were run under an atmosphere of dry nitrogen or argon. Commercial reagents were used without further purification. All PEG compounds were dried under vacuum or by azeotropic distillation from toluene before use. 1H and 13C NMR spectra were obtained using a Varian Mercury 300 NMR spectrometer and deuterated chloroform and methanol as the solvents unless otherwise specified.

Novel Prodrugs of SN38 Using Multiarm PEG Linkers

Bioconjugate Chem., Vol. 19, No. 4, 2008 851

Scheme 2

Chemical shifts (δ) are reported in ppm downfield from tetramethylsilane (TMS). Mass spectra (MS) were obtained by electrospray ionization (ESI). HPLC Method. The reaction mixtures and the purity of intermediates and final products were monitored by a Beckman Coulter System Gold HPLC instrument. It employs a ZOBAX 300SB C8 reversed-phase column (150 × 4.6 mm) or a Phenomenex Jupiter 300A C18 reversed-phase column (150 × 4.6 mm) with a multiwavelength UV detector, using a gradient of 15–100% of acetonitrile in 0.05% TFA at a flow rate of 1 mL/min. Compound 2. 40 kDa four-arm-PEG-OH (compound 1, 12.5 g, 0.31 mmol) was azeotroped with 220 mL of toluene to remove 35 mL of toluene/water. The solution was cooled to 30 °C, and 1.0 M potassium t-butoxide in t-butanol (3.75 mL, 3.75 mmol) was added. The mixture was stirred at 30 °C for 30 min, and then t-butyl bromoacetate (0.975 g, 4.96 mmol) was added. The reaction was kept at 30 °C for 1 h and then was cooled to 25 °C. Ethyl ether (150 mL) was slowly added to precipitate the product. The crude product was filtered and washed with ethyl ether twice (2 × 125 mL). The resulting solids were dissolved in 50 mL of dichloromethane (DCM), precipitated with 350 mL of ethyl ether, filtered, and washed with ethyl ether (2 × 125 mL). The product was dried under vacuum at 40 °C to give compound 2 (12.25 g, 98% yield). 13C NMR (75.4 MHz, CDCl3): δ 27.71, 68.48–70.71 (PEG), 80.94, 168.97. Compound 3. Compound 2 (12 g, 0.30 mmol) was dissolved in 120 mL of DCM, and then, 60 mL of TFA was added. The mixture was stirred at room temperature for 3 h, and then the solvent was removed under vacuum at 35 °C. The resulting oil was dissolved in 37.5 mL of DCM, and the crude product was precipitated with 375 mL of ethyl ether. After filtration, the resulting solids were dissolved in 30 mL of 0.5% NaHCO3.

The product was extracted with DCM (2 × 150 mL), and the combined organic layers were dried over MgSO4. After filtration, the solvent was removed under vacuum at room temperature. The resulting residue was dissolved in 37.5 mL of DCM, and the product was precipitated with 300 mL of ethyl ether. The resulting solids were filtered, washed with ethyl ether (2 × 125 mL), and dried under vacuum at 40 °C to give compound 3 (10.75 g, 90% yield). 13C NMR (75.4 MHz, CDCl3): δ 67.93–71.6 (PEG), 170.83. Compound 5. To a suspension of SN38 (compound 4, 2.45 g, 6.25 mmol) in 250 mL of anhydrous DCM were added di-tertbutyl dicarbonate (1.764 g, 8.12 mmol) and anhydrous pyridine (15.2 mL, 185.5 mmol). The suspension was stirred overnight at room temperature. The hazy solution was filtered through celite, and the filtrate was washed with 0.5 N HCl (3 × 150 mL) and saturated NaHCO3 (1 × 150 mL). The organic phase was dried over MgSO4, filtered, and evaporated under vacuum at 30 °C. The resulting solids were dried under vacuum at 40 °C to give compound 5 (2.52 g, 82% yield). 1H NMR (300 MHz, CDCl3): δ 1.00 (3H, t, J ) 7.3 Hz), 1.40 (3H, t, J ) 7.7 Hz), 1.62 (9H, s), 1.81–1.97 (2H, m), 3.13 (2H, q, J ) 7.7 Hz), 4.39 (1H, s, OH), 5.23 (2H, s), 5.29 (1H, d, J ) 16.5 Hz), 5.73 (1H, d, J ) 16.5 Hz), 7.63 (1H, dd, J ) 2.6, 9.1 Hz), 7.69 (1H, s), 7.84 (1H, d, J ) 2.2 Hz), 8.23 (1H, d, J ) 9.1 Hz). 13C NMR (75.4 MHz, CDCl3): δ 7.90, 13.98, 23.17, 27.73, 31.62, 49.33, 66.18, 72.80, 84.26, 98.06, 113.88, 118.53, 124.98, 127.13, 127.19, 131.83, 145.15, 146.50, 147.00, 149.70, 150.02, 151.28, 151.60, 157.38, 173.53. HRMS (ESI) calcd for C27H28N2O7 [M + H]+: 493.1975, found: 493.1969. Compound 6. To a solution of compound 5 (0.85 g, 1.71 mmol) and Bsmoc-Ala-OH (0.68 g, 2.30 mmol) in 20 mL of anhydrous DCM at 0 °C were added EDC (0.51 g, 2.67 mmol) and 4-(dimethylamino)pyridine (DMAP, 65 mg, 0.53 mmol).


Zhao et al.

Scheme 3

The mixture was stirred at 0 °C for 45 min and then warmed up to room temperature. When completion of the reaction was confirmed by HPLC, the reaction mixture was washed with 1% NaHCO3 (2 × 50 mL), water (50 mL), and 0.1 N HCl (2 × 50 mL). The organic phase was dried over anhydrous MgSO4, filtered, and evaporated under vacuum. The resulting solid was dried under vacuum at 40 °C to give compound 6 (1.28 g, 95% yield). 1H NMR (300 MHz, CDCl3): δ 0.90 (3H, t, J ) 7.5 Hz), 1.37 (3H, t, J ) 7.3 Hz), 1.56 (3H, d, J ) 7.3 Hz), 1.61 (9H, s), 2.04–2.28 (2H, m), 3.13 (2H, q, J ) 7.5 Hz), 4.51 (1H, quint, J ) 7.2 Hz), 5.12–5.20 (2H, m), 5.23 (2H, s), 5.37 (1H, d, J ) 17.0 Hz), 5.66 (1H, d, J ) 17.0 Hz), 5.70 (1H, d, J ) 7.0 Hz), 7.18 (1H, s), 7.21–7.78 (5H, m), 7.65 (1H, dd, J ) 2.3, 9.1 Hz), 7.87 (1H, d, J ) 2.3 Hz), 8.20 (1H, d, J ) 9.4 Hz). 13C NMR (75.4 MHz, CDCl3): δ 7.53, 14.02, 17.94, 27.76, 31.7, 49.34, 49.96, 53.45, 56.66, 67.10, 76.64, 84.38, 95.90, 114.17, 120.01, 121.38, 125.14, 125.32, 127.08, 127.40, 130.33, 130.40, 130.45, 131.83, 133.60, 136.88, 139.08, 145.15, 145.35, 146.68, 147.17, 149.82, 151.33, 151.59, 154.78, 157.16, 166.83, 171.16. MS (ESI): 786.20 [M + H]+. HRMS (ESI) calcd for C40H39N3O12S [M + H]+: 786.2333, found: 786.2321. Compound 7. To a solution of compound 6 (4.2 g, 5.35 mmol) in 200 mL of anhydrous DCM was added 4-piperidinopiperidine (1.17 g, 6.96 mmol). The mixture was stirred at room temperature for 5 h and then washed with 0.1 N HCl (2 × 40 mL). The organic phase was dried over MgSO4, filtered, and evaporated under vacuum to give compound 7 (2.8 g, 87% yield). 1H NMR (300 MHz, CD3OD): δ 1.08 (3H, t, J ) 7.3 Hz), 1.37 (3H, t, J ) 7.6 Hz), 1.58 (9H, s), 1.80 (3H, d, J )

7.0 Hz), 2.31 (2H, sept, J ) 7.2 Hz), 3.18 (2H, q, J ) 7.3 Hz), 4.41 (1H, q, J ) 7.2 Hz), 5.25 (2H, s), 5.51 (1H, d, J ) 17.0 Hz), 5.65 (1H, d, J ) 17.0 Hz), 7.32 (1H, s), 7.65 (1H, dd, J ) 2.3, 9.4 Hz), 7.95 (1H, d, J ) 2.3 Hz), 8.14 (1H, d, J ) 9.4 Hz). 13C NMR (75.4 MHz, CD3OD): δ 8.11, 14.27, 16.26, 23.90, 27.94, 32.12, 50.78, 67.86, 79.54, 85.11, 97.14, 115.80, 120.68, 126.66, 128.76, 129.26, 131.95, 146.74, 147.68, 147.82, 148.09, 151.37, 152.59, 152.90, 158.63, 168.30, 169.99. HRMS (ESI) calcd for C30H33N3O8 [M + H]+: 564.2346, found: 564.2357. Compound 8. To a solution of compound 7 (1.50 g, 2.5 mmol) and compound 3 (10.01 g, 0.25 mmol) in 100 mL of anhydrous DCM at 0 °C were added EDC (0.29 g, 1.5 mmol) and DMAP (0.30 g, 2.5 mmol). The mixture was stirred at 0 °C for 1 h and at room temperature overnight. The solvent was evaporated under vacuum. The residue was dissolved in 40 mL of DCM, and the crude product was precipitated with ethyl ether (300 mL). After filtration, the resulting solids were recrystallized with a mixture of dimethyl formamide/isopropyl alcohol (DMF/ isopropyl alcohol [IPA], 60 mL /240 mL). Then, the solids were filtered, washed with ethyl ether (2 × 200 mL), and dried under vacuum at 40 °C to give compound 8 (8.5 g, 80% yield). 13C NMR (75.4 MHz, CDCl3) δ: 7.46, 13.87, 17.68, 23.04, 27.59, 31.53, 47.17, 49.13, 66.85, 69.67–70.88 (PEG), 84.11, 95.52, 113.83, 119.72, 124.85, 126.84, 127.14, 131.72, 145.05, 146.59, 146.97, 149.60, 151.09, 151.32, 156.92, 166.54, 169.29, 170.83. Compound 9. Compound 8 (7.98 g, 0.19 mmol) was dissolved in 130 mL of 30% TFA in anhydrous DCM. The mixture was stirred for 3 h, and then the solvent was removed



Scheme 4

under vacuum at 35 °C. The residue was dissolved in 50 mL of DCM, and the crude product was precipitated with 350 mL of ethyl ether. After filtration, the resulting solids were recrystallized from a mixture of DMF/IPA (50 mL/200 mL). The solid was filtered, washed with ethyl ether (2 × 200 mL), and dried under vacuum to give compound 9 (6.7 g, 84% yield). 13C NMR (75.4 MHz, CDCl3): δ 7.46, 13.52, 17.78, 22.95, 31.53, 47.14, 49.14, 66.90, 69.57–71.15 (PEG), 78.08, 94.57, 105.10, 118.79, 122.47, 126.42, 128.26, 131.49, 143.00, 144.29, 145.03, 147.19, 148.36, 156.31, 157.00, 166.65, 169.30, 170.75. Compound 10. To a solution of compound 5 (2.73 g, 5.53 mmol) and Bsmoc-Met-OH (3.19 g, 8.59 mmol) in 50 mL of anhydrous DCM at 0 °C were added EDC (1.64 g, 8.59 mmol) and DMAP (0.21 g, 1.72 mmol). The mixture was stirred at 0 °C for 45 min and at room temperature for 1 h. The reaction mixture was washed with 1% NaHCO3 (2 × 100 mL), water (100 mL), and 0.1 N HCl (2 × 100 mL). The organic phase was dried over MgSO4, filtered, and evaporated under vacuum. The resulting solids were dried under vacuum to give compound 10 (4.2 g, 88% yield). 1H NMR (300 MHz, CDCl3): δ 0.89 (3H, t, J ) 7.3 Hz), 1.39 (3H, t, J ) 7.7 Hz), 1.61 (9H, s), 2.03–2.15 (2H, m), 2.10 (3H, s), 2.19–2.31 (2H, m), 2.61 (2H, t, J ) 7.0 Hz), 3.15 (2H, q, J ) 7.5 Hz), 4.61 (1H, dt, Jd ) 5.3 Hz, Jt ) 7.9 Hz), 5.11–5.28 (4H, m), 5.39 (1 H, d, J ) 17.1 Hz), 5.67 (1H, d, J ) 17.1 Hz), 5.70 (1H, d, J ) 7.0 Hz), 7.18 (1H, s), 7.23–7.68 (5H, m), 7.65 (1H, dd, J ) 2.2, 9.2 Hz), 7.89 (1H, s), 8.21 (1H, d, J ) 9.2 Hz). 13C NMR (75.4 MHz, CDCl3): δ 7.45, 13.92, 15.38, 23.12, 27.68, 29.65, 30.97, 31.57, 49.32, 53.54, 56.68, 66.99, 76.58, 84.28, 96.05, 114.09, 119.75, 121.17, 125.08, 125.27, 127.05, 130.15,130.29, 130.54, 131.65,

133.52, 136.69, 138.86, 145.10, 145.29, 146.55, 146.97, 149.69, 151.25, 151.39, 155.16, 157.08, 166.78, 170.33. HRMS (ESI) calcd for C42H43N3O12S2 [M + H]+: 846.2366, found: 846.2364. Compound 11. To a solution of compound 10 (4.1 g, 4.85 mmol) in 200 mL anhydrous DCM was added 4-piperidinopiperidine (1.06 g, 6.31 mmol). The reaction mixture was stirred at room temperature for 5 h and then washed with 0.1 N HCl (2 × 40 mL). The organic phase was dried over MgSO4, filtered, and evaporated under vacuum to give compound 11 (2.8 g, 88% yield). 1H NMR (300 MHz, CD3OD): δ 1.08 (3H, t, J ) 7.3 Hz), 1.37 (3H, t, J ) 7.6 Hz), 1.58 (9H, s), 2.19 (3H, s), 2.26–2.37 (3H, m), 2.51–2.58 (1H, m), 2.77 (2H, t, J ) 7.3 Hz), 3.17 (2H, t, J ) 7.5 Hz), 4.51 (1H, t, J ) 6.4 Hz), 5.25 (2H, s), 5.52 (1H, d, J ) 17.0 Hz), 5.66 (1H, d, J ) 17.0 Hz), 7.35 (1H, s), 7.67 (1H, dd, J ) 2.3, 9.1 Hz), 7.95 (1H, d, J ) 2.3 Hz), 8.17 (1H, d, J ) 9.1 Hz). 13C NMR (75.4 MHz, CD3OD): δ 8.15, 14.28, 15.13, 23.90, 27.94, 30.12, 30.84, 32.07, 50.83, 52.80, 67.92, 79.95, 85.14, 97.27, 115.78, 120.68, 126.66, 128.76, 129.25, 132.04, 146.58, 147.62, 147.85, 148.12, 151.36, 152.56, 152.90, 158.65, 168.24, 169.14. MS (ESI): 624.29 [M + H]+. HRMS (ESI) calcd for C32H37N3O8S [M + H]+: 624.2380, found: 624.2381. Compound 12. To a solution of compound 11 (1.48 g, 2.25 mmol) and compound 3 (9.0 g, 0.22 mmol) in 80 mL of anhydrous DCM at 0 °C were added EDC (0.26 g, 1.35 mmol) and DMAP (0.27 g, 2.25 mmol). The mixture was stirred at 0 °C for 1 h and at room temperature overnight. The reaction mixture was diluted with 70 mL of DCM and then extracted with 30 mL of 0.1 N HCl. The organic phase was dried over MgSO4, filtered, and evaporated under vacuum. The residue was


Zhao et al.

Scheme 5

dissolved in 40 mL of DCM, and the crude product was precipitated with 300 mL of ethyl ether. After filtration, the resulting solids were recrystallized from a mixture of DMF/ IPA (60 mL /240 mL). Then, the solid was filtered, washed with ethyl ether (2 × 200 mL), and dried under vacuum at 40 °C to give compound 12 (7.0 g, 74% yield). 13C NMR (75.4 MHz, CDCl3): δ 7.47, 13.85, 15.32, 23.06, 27.59, 29.62, 31.19, 31.53, 49.16, 50.68, 66.88, 69.93–70.97 (PEG), 78.10, 84.10, 95.53, 113.85, 119.66, 124.87, 126.81, 127.14, 131.68, 144.99, 146.62, 146.96, 149.58, 151.09, 151.24, 156.89, 166.49, 169.64, 169.82. Compound 13. To a solution of 30% TFA in anhydrous DCM (100 mL) were added dimethyl sulfide (2.5 mL) and compound 12 (6.0 g, 0.14 mmol). The mixture was stirred for 3 h, and then the solvent was removed under vacuum at 35 °C. The residue was dissolved in 50 mL of DCM, and the crude product was precipitated with 350 mL of ether and filtered. The solids were recrystallized from a mixture of DMF/IPA (60/300 mL). Then, the solids were filtered, washed with ethyl ether (2 × 200 mL), and dried under vacuum to give compound 13 (5.1 g, 86% yield). 13C NMR (75.4 MHz, CDCl3): δ 7.49, 13.53, 15.35, 22.96, 29.65, 31.30, 31.56, 49.19, 50.71, 66.96, 69.58–71.02 (PEG), 78.11, 94.61, 105.12, 118.72, 122.46, 126.41, 128.27, 131.54, 142.94, 144.39, 144.96, 147.31, 148.37, 156.28, 157.01, 166.63, 169.74. Compound 14. To a solution of Boc-Sar-OH (432 mg, 2.287 mmol) and compound 5 (750 mg, 1.52 mmol) in 75 mL of DCM at 0 °C were added EDC (837 mg, 0.686 mmol) and DMAP (432 mg, 2.287 mmol). The reaction mixture was stirred at 0 °C for 30 min and at room temperature for 1 h. The mixture was then washed with 0.5% NaHCO3 (2 × 75 mL), water (2 × 75 mL), and 0.1 N HCl (75 mL). The organic layer was dried over MgSO4 and filtered; the solvent was evaporated under vacuum and dried to give compound 14 (0.9 g, 89% yield). 1H

NMR (300 MHz, CDCl3, rotamers, ratio 60:40): δ 1.05 (3H, t, J ) 7.5 Hz), 1.39 (3H, t, J ) 7.3 Hz), 1.43 (3.6H), 1.44 (5.4H, s), 1.62 (9H, s), 2.11–2.28 (2H, m), 2.95 (3H, s), 3.13–3.19 (2H, m), 4.05 (0.4H, d, J ) 17.6 Hz), 4.13 (1.2H, s), 4.28 (0.4H, d, J ) 17.6 Hz), 5.23 (0.8H, s), 5.24 (1.2H, s), 5.40 (1H, d, J ) 17.2 Hz), 5.70 (1H, d, J ) 17.2 Hz), 7.14 (0.6H, s), 7.32 (0.4H, s), 7.66 (1H, d, J ) 9.2 Hz), 7.88 (0.4H, s), 7.89 (0.6H, s), 8.17 (0.6H, d, J ) 9.2 Hz), 8.24 (0.4H, d, J ) 9.2 Hz). 13C NMR (75.4 MHz, CDCl3): δ 7.68, 14.04, 23.25, 27.75, 28.31, 31.84, 35.42, 49.31, 50.27, 50.67, 66.99–70.97, 80.55, 84.40, 95.50, 96.47, 114.17, 119.80, 125.12, 126.98, 127.35, 131.59, 145.26, 145.58, 146.69, 147.09, 149.75, 151.33, 155.04, 157.13, 166.82, 168.81. HRMS (ESI) calcd for C35H41N3O10 [M + H]+: 664.2870, found: 664.2877. Compound 15. To a solution of compound 14 (0.9 g, 1.36 mmol) in 16 mL of DCM was added 4 mL of TFA. The reaction mixture was stirred at room temperature for 1 h, and then the solvent was evaporated under vacuum. The residue was dissolved in 10 mL of CHCl3 and then precipitated by addition of ethyl ether. The product was filtered and dried to give compound 15 (0.7 g, 78% yield). 1H NMR (300 MHz, CD3OD): δ 1.08 (3H, t, J ) 7.3 Hz), 1.34 (3H, t, J ) 7.6 Hz), 2.16–2.34 (2H, m), 2.77 (3H, s), 3.08 (2H, q, J ) 7.6 Hz), 4.31 (1H, d, J ) 17.3 Hz), 4.40 (1H, d, J ) 17.3 Hz), 5.20 (2H, s), 5.48 (1H, d, J ) 16.7 Hz), 5.63 (1H, d, J ) 17.0 Hz), 7.30 (1H, s), 7.37–7.42 (2H, m), 7.95 (1H, d, J ) 9.1 Hz). 13C NMR (75.4 MHz, CD3OD): δ 8.07, 13.86, 23.81, 31.81, 33.55, 49.46, 50.79, 67.69, 79.86, 96.72, 105.93, 119.42, 124.01, 128.80, 130.02, 131.85, 145.00, 147.23, 148.59, 149.40, 158.61, 158.79, 167.04, 168.36. HRMS (ESI) calcd for C25H25N3O6 [M + H]+: 464.1822, found: 464.1832. Compound 16. To a solution of compound 15 (2.17 g, 3.75 mmol) in DMF/DCM (30 /200 mL) were added triethylamine (Et3N, 2.4 mL, 17.40 mmol) and t-butyldimethylchlorosilane


(TBDMSCl, 2.04 g, 13.53 mmol). The reaction mixture was stirred at room temperature for 1 h. The organic layer was washed with 0.5% NaHCO3, water, and 0.1 N HCl saturated with brine, and then dried over MgSO4. After filtration and evaporation of the solvent under vacuum, the resulting oil was dissolved in DCM and precipitated by the addition of ethyl ether. The resulting solids were filtered to give compound 15 (2.00 g, 87% yield). 1H NMR (300 MHz, CD3OD): δ 0.23 (6H, s), 0.96 (9H, s), 0.98 (3H, t, J ) 7.3 Hz), 1.30 (3H, t, J ) 7.6 Hz), 2.13–2.18 (2H, m), 2.67 (3H, s), 3.11 (2H, q, J ) 7.6 Hz), 4.10 (1H, d, J ) 17.6 Hz), 4.22 (1H, d, J ) 17.6 Hz), 5.23 (2H, s), 5.40 (1H, d, J ) 16.7 Hz), 5.55 (1H, d, J ) 16.7 Hz), 7.32 (1H, s), 7.38–7.43 (2H, m), 8.00 (1H, d, J ) 9.1 Hz). 13C NMR (75.4 MHz, CD3OD): δ –4.14, 8.01, 14.10, 19.30, 23.98, 26.16, 31.78, 33.52, 49.46, 50.95, 67.66, 79.80, 97.41, 111.96, 119.99, 127.75, 129.28, 129.67, 131.57, 145.24, 146.86, 147.16, 148.02, 150.34, 156.69, 158.72, 167.02, 168.27. MS (ESI): 578.35 [M + H]+. HRMS (ESI) calcd for C31H39N3O6Si [M + H]+: 578.2686, found: 578.2694. Compound 17. To a solution of compound 3 (10 g, 0.25 mmol) in 150 mL of anhydrous DCM was added a solution of compound 16 (1.53 g, 2.5 mmol) in 20 mL of anhydrous DMF. The mixture was cooled to 0 °C. To this solution were added EDC (767 mg, 4 mmol) and DMAP (367 mg, 3 mmol). The reaction mixture was allowed to warm to room temperature slowly and stirred at room temperature overnight. The reaction mixture was then evaporated under vacuum, and the residue was dissolved in a minimum amount of DCM. After addition of ethyl ether, solid was formed and filtered under vacuum. The residue was dissolved in 30 mL of anhydrous acetonitrile (CH3CN) and precipitated by addition of 600 mL IPA. The solids were filtered and washed with IPA and ethyl ether to give compound 17 (9.5 g, 89% yield). 13C NMR (75.4 MHz, CDCl3): δ –4.49, 7.29, 13.44, 18.02, 22.78, 25.38, 31.36, 34.90, 48.93, 66.68, 69.98–71.15 (PEG), 94.94, 109.97, 118.64, 125.45, 126.32, 127.66, 131.55, 143.08, 144.82, 146.83, 149.08, 154.53, 156.71, 166.72, 167.70, 169.24. Compound 18. Compound 17 (7.8 g, 0.18 mmol) was dissolved in a 50% mixture of TFA in H2O (200 mL). The reaction mixture was stirred at room temperature for 10 h. The mixture was then diluted with 100 mL of H2O and extracted with DCM (2 × 300 mL). The combined organic phases were washed with H2O (2 × 100 mL), dried over MgSO4, filtered, and evaporated under vacuum. The residue was recrystallized with a mixture of DMF/IPA (100/400 mL). Then, the solids were filtered, washed with ethyl ether, and dried under vacuum at 40 °C to give compound 18 (6.8 g, 89% yield). 13C NMR (75.4 MHz, CDCl3): δ 7.51, 13.49, 22.89, 31.55, 35.18, 48.80, 49.11, 66.90, 69.57–71.13 (PEG), 78.08, 94.88, 105.00, 118.45, 122.45, 126.36, 128.14, 131.47, 142.79, 144.25, 145.09, 147.25, 148.37, 156.34, 156.96, 167.00, 167.86, 169.63. Compound 19. To a suspension of compound 4 (2.0 g, 5.10 mmol) in 100 mL of anhydrous DCM were added Et3N (3.2 mL, 22.93 mmol, 4.5 equiv) and t-butyldiphenylchlorosilane (TBDPSCl, 5.2 mL, 20.39 mmol). The reaction mixture was heated to reflux overnight and then was washed with 0.2 N HCl (2 × 50 mL), saturated NaHCO3 (100 mL), and saturated NaCl (100 mL). The organic layer was dried over MgSO4, filtered, and evaporated under vacuum. The residue was dissolved in DCM and precipitated by addition of hexane. The solids were filtered and dried under vacuum to give compound 19 (2.09 g, 65% yield). 1H NMR (300 MHz, CDCl3): δ 0.90 (3H, t, J ) 7.6 Hz), 1.01 (3H, t, J ) 7.3 Hz), 1.17 (9H, s), 1.83–1.92 (2H, m), 2.64 (2H, q, J ) 6.9 Hz), 3.89 (1H, s), 5.11 (2H, s), 5.27 (1H, d, J ) 16.1 Hz), 5.72 (1H, d, J ) 16.4 Hz), 7.07 (2H, d, J ) 2.63 Hz), 7.36–7.49 (7H, m), 7.58 (1H, s), 7.75–7.79 (4H, m), 8.05 (1H, d, J ) 9.4 Hz). 13C NMR (75.4 MHz, CDCl3): δ


7.82, 13.28, 19.52, 22.86, 26.48, 31.52, 49.23, 66.25, 72.69, 97.25, 110.09, 117.57, 125.67, 126.57, 127.65, 127.81, 130.02, 131.69, 131.97, 135.26, 143.51, 145.05, 147.12, 149.55, 149.92, 154.73, 157.43, 173.72. HRMS (ESI) calcd for C38H38N2O5Si [M + H]+: 631.2628, found: 631.2622. Compound 20. To a solution of compound 19 (3.78 g, 5.99 mmol) and Boc-Gly-OH (1.57 g, 8.99 mmol) in 100 mL of anhydrous DCM at 0 °C were added EDC (1.72 g, 8.99 mmol) and DMAP (329 mg, 2.69 mmol). The reaction mixture was stirred at 0 °C until HPLC showed complete disappearance of the starting material (approximately 2 h). The organic layer was washed with 0.5% NaHCO3 (2 × 50 mL), water (1 × 50 mL), 0.1 N HCl (2 × 50 mL), and saturated NaCl (1 × 50 mL), and then dried over MgSO4. After filtration, the solvent was evaporated under vacuum to give compound 20 (4.94 g, 100% yield). The solids were used in the next reaction without further purification. 1H NMR (300 MHz, CDCl3): δ 0.89 (3H, t, J ) 7.6 Hz), 0.96 (3H, t, J ) 7.5 Hz), 1.18 (9H, s), 1.40 (9H, s), 2.07–2.29 (3H, m), 2.64 (2H, q, J ) 7.5 Hz), 4.01–4.22 (2H, m), 5.00 (1H, br s), 5.01 (2H, s), 5.37 (1H, d, J ) 17.0 Hz), 5.66 (1H, d, J ) 17.0 Hz), 7.08 (1H, d, J ) 2.34 Hz), 7.16 (1H, s), 7.37–7.50 (7H, m), 7.77 (4H, d, J ) 7.6 Hz), 8.05 (1H, d, J ) 9.4 Hz). 13C NMR (75.4 MHz, CDCl3): δ 7.52, 13.30, 19.50, 22.86, 26.45, 28.21, 31.64, 42.28, 49.14, 67.00, 76.65, 79.96, 95.31, 110.13, 118.98, 125.75, 126.45, 127.68, 127.81, 130.03, 131.54, 131.92, 135.25, 143.65, 144.91, 145.19, 147.08, 149.27, 154.75, 155.14, 157.10, 166.98, 169.17. HRMS (ESI) calcd for C45H49N3O8Si [M + H]+: 788.3367, found: 788.3345. Compound 21. To a solution of compound 20 (1 g, 1.27 mmol) in 5 mL anhydrous dioxane were added 5 mL of a 4 M solution of HCl in dioxane. The reaction mixture was stirred at room temperature until HPLC showed complete disappearance of the starting material (approximately 1 h). The reaction mixture was added to 50 mL of ethyl ether, and the resulting solid was filtered. After the solids were dissolved in 50 mL DCM and washed with brine, the pH was adjusted to 2.5 by addition of saturated NaHCO3. The organic layer was dried over MgSO4, filtered, and evaporated under vacuum. The residues were dissolved in 5 mL of DCM and precipitated by addition of 50 mL ethyl ether. Filtration afforded compound 21 (0.770 g, 84% yield). 1H NMR (300 MHz, CDCl3): δ 0.84 (3H, t, J ) 7.6 Hz), 1.05 (3H, t, J ) 7.3 Hz), 1.16 (9H, s), 2.15–2.30 (3H, m), 2.59 (2H, q, J ) 7.6 Hz), 4.16 (1H, d, J ) 17.9 Hz), 4.26 (1H, d, J ) 17.9 Hz), 5.13 (2H, s), 5.46 (1H, d, J ) 17.0 Hz), 5.60 (1H, d, J ) 17.0 Hz), 7.11 (1H, d, J ) 2.34 Hz), 7.30 (1H, s), 7.40–7.51 (6H, m), 7.56 (1H, dd, J ) 2.34, 9.4 Hz), 7.77 (4H, dd, J ) 7.6, 1.6 Hz), 7.98 (1H, d, J ) 9.1 Hz). 13C NMR (75.4 MHz, CDCl3): δ 8.09, 13.72, 20.26, 23.61, 26.94, 31.83, 41.01, 50.71, 67.62, 79.51, 97.03, 111.65, 119.69, 127.13, 128.97, 128.99, 129.11, 131.43, 131.96, 133.00, 133.03,136.51, 145.62, 145.81, 147.24, 148.29, 150.58, 156.27, 158.68, 167.81, 168.34. MS (ESI): 688.40 [M + H]+. HRMS (ESI) calcd for C40H41N3O6Si [M + H]+: 688.2843, found: 688.2844. Compound 22. To a solution of compound 3 (1.4 g, 0.036 mmol) in 14 mL of anhydrous DCM were added compound 21 (0.207 g, 0.29 mmol), DMAP (0.175 g, 1.44 mmol), and PPAC (0.85 mL of a 50% solution in ethyl acetate, 1.44 mmol). The reaction mixture was stirred at room temperature overnight and then evaporated under vacuum. The resulting residue was dissolved in DCM, and the product was precipitated with ethyl ether and filtered. The residue was recrystallized with DMF/ IPA to give compound 22 (1.25 g, 81% yield). 13C NMR (75.4 MHz, CDCl3): δ 7.45, 13.20, 19.39, 22.73, 26.42, 31.67, 40.21, 49.01, 66.83, 69.80–71.12 (PEG), 78.08, 95.16, 110.02, 118.83, 125.58, 126.40, 127.53, 127.73, 129.96, 131.49, 131.76, 131.82, 135.12, 143.51, 144.78, 145.13, 146.95, 149.21, 154.61, 156.92, 166.70, 168.46, 170.30.


Zhao et al. Table 1. Properties of PEG Conjugates compound

% of loading

solubility (mg/mL)

SN38 9 13 18 23

2.5 2.1 2.6 3.7

0.0072 121 (3.0a) 142 (2.9a) NA 180 (6.7a)

a

Figure 1. Stability of compound 23 in different pH conditions. Stability profile was determined using HPLC methods, by plotting the percentage of remaining compound 23 over a time course. The percentage was calculated on the basis of the ratio of the peak area of the analyte at 0, 1, 3, 5, 8, and 23 h vs the initial area peak. Each stability profile represents the average of two independent runs with the same sampling schedules. The standard deviation of each point is typically 2% or less.

Compound 23. Compound 22 (1.25 g, 0.029 mmol) was dissolved in a solution of TBAF (0.122 g, 0.46 mmol) in a 1:1 mixture of THF and 0.05 M HCl (12.5 mL). The reaction mixture was stirred at room temperature for 4 h and then extracted with DCM (2 × 30 mL). The combined organic phases were dried over MgSO4, filtered, and evaporated under vacuum. The residue was dissolved in 7 mL of DMF and precipitated with 37 mL IPA. The solids were filtered and washed with IPA. Finally, the residue was dissolved in 2.5 mL of DCM and precipitated by the addition of 25 mL of ethyl ether. The solids were filtered and dried at 40 °C in vacuum oven overnight to give compound 23 (0.860 g, 71% yield). 13C NMR (75.4 MHz, CDCl3): δ 7.48, 13.52, 22.91, 31.67, 40.22, 49.12, 66.95, 69.70–70.87 (PEG), 78.14, 94.82, 105.03, 118.68, 122.54, 126.37, 128.20, 131.36, 142.92, 144.20, 144.98, 147.25, 148.29, 156.44, 156.98, 166.82, 168.49, 170.39. Plasma Stability Study. PEG-SN38 conjugate (0.8 mg) was incubated with plasma (Biomeda, 1849 Bayshore Blvd., STE 200, Burlingame, CA 94010, 150 µL) at 37 °C for 0, 0.5, 2, 4, 6, 20, and 24 h. The mixtures were quenched with a 1:1 mixture of CH3CN/MeOH (800 µL), vortexed for 1 min, and then filtered through a 0.2 µm filter membrane; 30 µL of the filtrate was analyzed by HPLC, measuring disappearance of the conjugates. pH Stability Study. PEG-SN38 conjugate (10 mg/mL) was diluted into phosphate buffered saline adjusted to pH 6.1, 7.3, and 8.1 and incubated at 37 °C. Aliquots were removed at different time points and, after addition of an equal amount of DMSO, the samples were analyzed by HPLC, measuring disappearance of the conjugate. Stability profile graph was generated by plotting the percentage of remaining starting material over a time course (Figure 1). The percentage was calculated on the basis of the ratio of the peak area of the sample at 0, 1, 3, 5, 8, and 23 h vs the initial area peak. Each stability profile represents the average of two independent runs with the same sampling schedules. The standard deviation of each point is typically 2% or less. In Vitro Cytotoxicity. All the cell lines were obtained from American type Culture Collection, Manassas, VA, and were grown in the listed medium: COLO 205 and OVCAR 3 (RPMI 1640 with 4.5 g/L glucose, 10 mM HEPES, 1 mM sodium pyruvate, 10% FBS); HT-29 (McCoy’s 5a with 1.5 mM L-glutamine, 10% FBS) and A549 (Ham’s F12K with 10% FBS). All cell lines were maintained at 37 °C (humidified, 5% CO2). The medium was changed every 3 to 4 days and the cultures trypsinized and recultured when they reached 85% confluence. Media was obtained from Invitrogen corporation,

t½ (min)b human plasma

rat plasma

12.5 26.8 19.0 12.3

6.3 12.4 10.5 3.5

Equivalent to native SN38. b Based on the release of SN38.

CA. The in vitro cytotoxicity of PEG-SN38, SN38, and CPT11 was determined using a cell proliferation (MTS) tetrazolium dye reduction assay. Briefly, adherent cells (10 000–20 000/well) were plated in 96-well plates and incubated overnight at 37 °C. The next morning, the cells were treated with serial dilutions of PEG-SN38, CPT-11, or SN38 dissolved in dimethyl sulfoxide (DMSO) and further incubated for 3–4 days at 37 °C. At the end of the incubation period, MTS dye was added, and formation of a colored product, formazan, was measured at 490 nm using a Spectramax 340PC reader (Molecular Devices, CA). In Vivo Efficacy Studies. Human mammary carcinoma (MX1) tumor fragment were obtained from DTP, DCTD tumor repository, NCI, Bethesda, MD. Subcutaneous (s.c.) tumor xenograft models were established in the right axillary flank region of female nude mice (4–5 weeks) by injecting either human cancer cells or tumor fragments. MX-1 tumors were established by implanting a 4- to 5-mm3 tissue fragment of MX-1 tumor collected from donor mice into the axillary flank of recipient nude mice.

RESULTS AND DISCUSSION The objective of this study was to solubilize the insoluble anticancer molecule SN38, alter its pharmacokinetic profile, protect it in its active lactone form during transportation, and enhance its antitumor activity compared with CPT-11. To do so, we chose to use the four-arm-PEG to increase the loading of SN38 and developed highly efficient methods to make such PEG-SN38 conjugates. Three different protecting strategies had been tried, and four PEG-SN38 conjugates with different amino acid spacers were obtained successfully. The TBDPS-Boc protecting strategy (Scheme 5) had the advantage that every intermediate was stable; hence, they could be isolated, analyzed, and scaled up. The optimized method and process can be easily scaled up to multiple hundreds of grams with high quality and yield. Since the active ends of each PEG “arm” were far away from each other, they reacted independently with SN38 small molecule derivatives without steric hindrance compared with our previous branching approach. In addition, high water solubility of the PEG-SN38 conjugates was obtained by avoiding the stacking effect between lipophilic SN38 molecules. In the current approach, the loading by weight of SN38 could be as high as 3.7% compared with about 1.7% for PEG-CPT when linear bifunctional PEG was used. As predicted, PEGylation has substantially increased the solubility of SN38, which is an insoluble molecule by itself (Table 1). The solubility of SN38 lactone in aqueous buffers published by Zhang et al. (9) is approximately 18 µM or 0.0072 mg/mL. All the PEG-SN38 conjugates have 120 to 180 mg/ mL solubility in water, which is equivalent to as high as 6.7 mg/mL of SN38. This highly increased solubility makes it possible to systemically evaluate the therapeutic efficacy of SN38 in vivo. The PEG conjugates were stable under neutral conditions in solution. Stability studies have demonstrated that PEG compounds release a minimum amount of free SN38 over 24 h at room temperature in clinically relevant solutions (Table 2). Since the linkage between the amino acid spacer and SN38 is an ester


Bioconjugate Chem., Vol. 19, No. 4, 2008 857 Table 5. Maximum Tolerated Dose Results

Table 2. Stability at Room Temperature compound

solution

PEG-Ala-SN38 (9)

saline (0.9% NaCl)

time of detection (h)

released SN38 (%)

0 23 0 24 0 24 0 24

0.1 0.3 0.2 1.6 0.09 0.29 0 0.29

water for injection PEG-Gly SN38 (23)

saline (0.9% NaCl) water for injection

Table 3. pH Stability Studies for Compound 23 at 37 °C pH t½ (h)

6.1 256.7

7.4 14.3

conjugate 9

23

a

8.1 12.2

Table 4. In Vitro Cytotoxicity of PEG Conjugates (IC50, µM) compound

COLO 205 (colorectal)

HT29 (colorectal)

OVCAR-3 (ovarian)

A549 (lung)

SN38 (4) CPT-11 9 13 18 23

0.2 ( 0.2 11 ( 1.4 0.13 ( 0.022 0.10 ( 0.024 0.18 ( 0.013 0.14 ( 0.032

0.1 ( 0.04 27 ( 8.6 0.21 ( 0.068 0.21 ( 0.049 0.34 ( 0.081 0.52 ( 0.066

0.1 ( 0.02 20 ( 7.1 0.22 ( 0.01 0.21 ( 0.047 0.27 ( 0.11 0.16 ( 0.032

1.0 ( 0.1 67 ( 17 3.9 ( 0.97 2.1 ( 0.28 5.1 ( 0.95 3.1 ( 0.43

bond, it will hydrolyze and release the intact SN38 under basic conditions. As predicted, when treated with buffers, the releasing rate of SN38 increased as the pH was increased above 6.0 in phosphate buffer at 37 °C (Figure 1, Table 3). In the preclinical studies, the release of SN38 could be due to several mechanisms as previously proposed (17). The pH-dependent study clearly showed that SN38 could be release at pH 7.4 and 37 °C with a half-life of about 14 h (Table 3). However, the half-life for the same compound in rat plasma was around 12 min (Table 1), which indicates that enzymes play a role in the release. These enzymes could be either esterases or aminopeptidases (17, 21). Enzymes that catalyze the hydrolysis of tertiary esters, although rare, have been previously reported in the literature (21). All the PEG-SN38 conjugates were first tested for their in vitro anticancer activities in different cancer cell lines using a cell proliferation [MTS] tetrazolium dye reduction assay. Briefly, adherent cells (10 000–20 000 /well) were plated in 96-well plates and incubated overnight at 37 °C. The next morning, the cells were treated with serial dilutions of PEG-SN38, CPT-11, or SN38 dissolved in dimethyl sulfoxide (DMSO) and further incubated for 3–4 days at 37 °C. At the end of the incubation period, MTS dye was added and formation of a colored product, formazan, was measured at 490 nm using a Spectramax 340PC reader (Molecular Devices, CA.). All conjugates were found to be very potent against a panel of cell lines (Table 4). Sensitivity of cells to PEG-SN38 is in the order COLO 205 > HT29 ) OVCAR-3 > A549. PEG-SN38 conjugates were equipotent to native SN38 and about 6.5- to 666-fold more potent than CPT11. One unique property of camptothecin and its analogues is that the E-ring has to be in the closed lactone form in order to have any anticancer activities (3, 5, 6). Our previous studies (12, 13) had proven that acylation through the 20-OH of camptothecin and its analogues will lock the E-ring in the active lactone form. Since PEG is attached to SN38 through the acylation at 20-OH position, all the PEG-SN38 conjugates will ensure the SN38 existing in its active form during circulation without any loss of activity. In comparison, the water-solubilizing bispiperidine group is attached to the 10-OH group of SN38 in CPT-11. As a result, about 75% of CPT-11 has the potential to lose its anticancer activity due to the lactone opening even before SN38 is released. The demonstrated potent in vitro anticancer activity

dose level survival/ (mg/kg) total 5 10 20 40

4/4 4/4 4/4 0/4

5 10 20 40

4/4 4/4 4/4 3/4

commentsa

1 found dead, 3 sacrificed due to body weight loss

1 sacrificed due to body weight loss

Total of 4 mice per study group.

of all PEG-SN38 conjugates further suggests that intact SN38 in its active closed lactone form was regenerated during the incubation with tumor cells. In order to conduct in vivo efficacy studies, the maximum tolerable doses (MTD) were first obtained in mice. Female naive athymic nude mice were injected intravenously with a single dose of PEG-SN38 ranging from 5 to 40 mg/kg. Mice were monitored daily for visible signs of toxicity and weighed biweekly. The MTD was defined as the highest dose at which no death occurred, and body weight loss was less than 20% of pretreatment animal weight (approximately 20 g). Two PEG conjugates were selected for this study. It was found that the MTD of PEG-Ala-SN38 (9) or PEG-Gly-SN38 (23) was between 20 and 40 mg/kg when injected intravenously as a single dose in nude mice (Table 5). For in vivo efficacy studies, MX-1 tumors were established by implanting a 4–5 mm3 tissue fragment of MX-1 tumor collected from donor mice into the right axillary flank of recipient nude mice. When tumors reached an average volume of 100 mm3, mice were injected with either PEG-SN38 conjugates or CPT-11 as a single dose of 20 mg/kg or multiple 5 mg/kg doses (every 2 days [q2d] × 6). Mouse weight and tumor sizes were measured at the beginning of the study and twice weekly throughout the study. Mice were euthanized when individual tumor volumes reached 1670 mm3, and all surviving mice were euthanized at 12 weeks. Treatment with either of the four conjugates as a single dose of 20 mg/kg or multiple doses of 5 mg/kg (q2d × 6) PEG-SN38 led to >99% tumor growth inhibition (TGI) and complete cures (no evidence of tumor upon visual external examination and palpation of injection site) of the animals up to 12 weeks after which the animals were humanely sacrificed (Figure 2, Tables 6 and 7). There was only one unexplained death, which occurred in the PEG-Ala-SN38 20-mg/kg group early within the study. At equivalent dose levels, treatment with CPT-11 caused a 26% and 44% TGI when given as a single dose or multiple injections, respectively. There was no body weight loss in all the cured mice. The mice were followed up to 100 days, and no tumor was detected after animals were sacrificed. Therefore, all PEG-SN38 conjugates demonstrated excellent anticancer activities and warrant further preclinical development. They all were superior to CPT11 since treatment with CPT-11 resulted in either no TGI (single dose) or partial TGI sustained for a short period of time (multiple-dose regimen). The much enhanced antitumor efficacy of the high molecular weight PEG-SN38 conjugates could be partially attributed to the improved biodistribution and passive tumor targeting effect due to the enhanced permeation and retention (EPR) effect (22). In conclusion, treatment with PEGSN38 conjugates was significantly more effective than treatment with CPT-11 in the MX-1 preclinical xenograft model. Due to the excellent anticancer efficacy as well as straightforward chemistry with little or no inherent impurities generated during the transformations, compound 23 was selected as the lead


Zhao et al.

Figure 2. Therapeutic efficacy of PEG-SN38 conjugates in a xenograft model of breast cancer (MX-1). Female athymic nude mice (6 mice per group), were inoculated subcutaneously with 4- to 5-mm3 tumor fragments of MX-1 breast tumors. When tumors reached an average volume of 100 mm3, mice were injected with either PEG-SN38 conjugates or CPT-11 as a single dose of 20 mg/kg (panel A) or multiple 5-mg/kg doses (q2d × 6) (panel B). Data represent % change in tumor volume ( standard deviation. Table 6. MX-1 (Breast) Xenograft Model (20 mg/kg single dose): Efficacy Comparison

compound

mean TV ( SD (mm3)a

% change in TV ( SDa

TGI (%)a

Cures (%)b

T/C at 1000 mm3 (%)

control 9 13 18 23 CPT-11

1136 ( 686 13 ( 9 8 ( 12 7 ( 10 7(9 845 ( 50

1399 ( 658 -87 ( 11 -95 ( 8 -94 ( 10 -94 ( 10 1424 ( 432

0 99 99 99 99 26

0 83 100 100 100 0

— 1 0 0 0 122

a Mean tumor volume (TV), % change in TV, and % TGI data were taken at day 31. (By day 31, a significant percentage of control animals were euthanized due to excess tumor burden.) b % cures were taken at the termination of the study (12 weeks).

candidate for further preclinical development and has entered into phase I clinical trials.

CONCLUSION PEGylation is well-known for its ability to improve pharmaceutical properties of small molecules and to enhance the efficacy due to the EPR effect. Using multiarm PEG linkers, we successfully developed several novel PEG-SN38 conjugates that have high drug loading and high water solubility. The chemistry for the conjugation of SN38 ensures that the anticancer molecule is locked into its active closed lactone form in the body until intact SN38 is released from the PEG

Table 7. MX-1 (breast) Xenograft Model (5 mg/kg q2d × 6): Efficacy Comparison

compound

mean TV ( SD (mm3)a

% change in TV ( SDa

TGI (%)a

cures (%)b

T/C (%)

control 9 13 18 23 CPT-11

1136 ( 686 7 ( 10 4(4 9 ( 10 12 ( 18 632 ( 698

1399 ( 658 -95 ( 8 -96 ( 7 -90 ( 13 -91 ( 10 423 ( 243

0 99 99 99 99 44

0 100 100 100 100 0

— 0 0 0 0 52

a Mean tumor TV, % change in TV, and % TGI data were taken at day 31. (By day 31, a significant percentage of control animals were euthanized due to excess tumor burden.) b % cures were taken at the termination of the study (12 weeks).

conjugate. Of the three different protecting strategies used to make such PEG-SN38 conjugates, one process has been optimized and scaled up with high reproducibility and yields. The improved pharmacokinetic profile of SN38, especially the possible passive accumulation of PEG-SN38 conjugates at the solid tumor sites, resulted in much enhanced anticancer activity of SN38 in the MX-1 xenograft mice model compared with CPT-11. Compound 23 was selected as the lead candidate for further preclinical development. In vitro and in vivo activities of the lead compound have been reported in another communication (23).


ACKNOWLEDGMENT We wish to thank Ms. Mary Mehlig and Ms. Jennifer Malaby for their in vitro and in vivo work, Mr. Syed Ali and Mr. Nish Sanghvi for their contribution in the scale up of PEG conjugate, Mr. Honghue Hsu, Ms. Michelle Boro, and Ms. Chia Chang for their analysis of PEG compounds, and Dr. Zhihua (John) Zhang for his helpful discussion. Supporting Information Available: HPLC of compounds 9, 13, 18, and 23 and pH stability studies for compound 23. This material is available free of charge via the Internet at http:// pubs.acs.org/BC.

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