In Vitro Cytotoxicity and in Vivo Distribution after Direct Delivery of

We implanted similar polymeric disks into rat brains and used a novel sectioning ... toward increasing drug distribution after direct, local delivery ...
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Bioconjugate Chem. 2004, 15, 1364−1375

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In Vitro Cytotoxicity and in Vivo Distribution after Direct Delivery of PEG-Camptothecin Conjugates to the Rat Brain Alison B. Fleming,† Kraig Haverstick,† and W. Mark Saltzman*,†,‡ School of Chemical and Biomolecular Engineering, Cornell University, 120 Olin Hall, Ithaca, New York 14853, and Department of Biomedical Engineering, Yale University, 227 Becton Center, New Haven, Connecticut 06511. Received September 30, 2003; Revised Manuscript Received September 2, 2004

Low water solubility and rapid elimination from the brain inhibits local delivery via implants and other delivery systems of most therapeutic drugs to the brain. We have conjugated the chemotherapy drug, camptothecin (CPT), to poly(ethylene glycol) (PEG) of molecular weight 3400 using previously established protocols. These new conjugates are very water-soluble and hydrolyze at a pH-dependent rate to release the active parent drug. We have studied the uptake of these conjugates by cells in vitro and quantified their cytotoxicity toward gliosarcoma cells. These conjugates were loaded into biodegradable polymeric controlled-release implants, and their release characteristics were studied in vitro. We implanted similar polymeric disks into rat brains and used a novel sectioning scheme to determine the concentration profile of CPT in comparison to conjugated CPT in the brain after 1, 7, 14, and 28 days. We have found that PEGylation greatly increases the maximum achievable drug concentration and greatly enhances the distribution properties of CPT, compared to corelease of CPT with PEG. Although only one percent of CPT in the conjugate system was found in the hydrolyzed, active form, drug concentrations were still significantly above cytotoxic levels over a greater distance for the conjugate system. On the basis of these results, we believe that PEGylation shows great promise toward increasing drug distribution after direct, local delivery in the brain for enhanced efficacy in drug treatment.

INTRODUCTION

Camptothecin, first isolated from the Chinese tree Camptotheca acuminata in the 1960s (1), is a potent anticancer agent that targets the DNA-unwinding enzyme topoisomerase I in the cell nucleus. Due to camptothecin’s extremely low aqueous solubility, there has been much interest in the synthesis of more water-soluble derivatives. This effort has resulted in FDA approval of two small molecule analogues, topotecan and irinotecan, and also in the creation of CPT1-polymer conjugates. Camptothecin has been conjugated to several polymers including poly(ethylene glycol) (2), dextran (3), poly(Lglutamic acid) (4), and N-(2-hydroxypropyl)methacrylamide (5). Camptothecin is a planar molecule composed of five rings (see Figure 1A). Of these rings, the R-hydroxy-δlactone moiety (ring E) is thought to be critical for * To whom correspondence should be addressed. Phone: (203) 432-4262. Fax: (203) 432-0030. Mail: Yale University, P.O. Box 208284, New Haven, CT 06520. E-mail: mark.saltzman@ yale.edu. † Cornell University. ‡ Yale University. 1 Abbreviations: AUC, area under the concentration-time curve; CM-PEG-CPT, carboxymethyl-poly(ethylene glycol) 3400camptothecin; CM-PEG-Gly-CPT, carboxymethyl-poly(ethylene glycol) 3400-glycyl-camptothecin; CM-PEG-Sar-CPT, carboxymethyl-poly(ethylene glycol) 3400-sarcosinyl-camptothecin; CPT, camptothecin; DIC, differential interference contrast; DMEM, Dulbecco’s Modified Eagle Medium; ELISA, enzymelinked immunosorbent assay; MTS [3-(4,5-dimethylthiazol-2-yl)5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PBS, phosphate-buffered saline; p(CPP:SA), poly(1,3-bis(pcarboxy phenoxy)-propane: sebacic acid; PEG, poly(ethylene glycol); PNPA, p-nitrophenyl acetate.

Figure 1. Camptothecin and camptothecin conjugates. (A) Chemical structure of camptothecin lactone and carboxylate forms. (B) Direct ester bond: CM-PEG-CPT. (C) Ester bond through a glycine residue: CM-PEG-Gly-CPT. (D) Ester bond through a sarcosine residue: CM-PEG-Sar-CPT.

cytotoxic activity. This lactone ring is subject to reversible hydrolysis to an open carboxylate form in aqueous solution with the dominant form at equilibrium depending on the pH of the solution. More specifically, it has been shown that the majority of camptothecin will be in the lactone form at pH 8 (6). This has important implications for the efficacy of delivered camptothecin, since the open-ring carboxylate (i.e. the inactive form) will be favored at physiological pH. Most attempts to increase the solubility of CPT have focused on stable substitutions at rings A and B (7). Although such substitutions may solve the problem of solubility, they have no effect on the rate of lactone hydrolysis (8). An alternative strategy is to modify the lactone moiety reversibly in order to create a watersoluble “prodrug” capable of releasing active-lactone CPT via hydrolysis or enzyme-mediated processes. The success of such PEG conjugates has been recently reviewed (9). This approach has been utilized in the creation of PEGCPT conjugates for systemic administration (1, 7, 10, 11). In these studies, attachment to PEG of molecular weight 40 000 (PEG40000) achieved water solubility, an increased residence time in the circulation, and stabilization of the essential lactone ring. CPT was linked to PEG40000 via a water-labile ester bond, and it was found that employing different spacer groups could alter the hydrolysis rate of the resulting conjugate (12). In previous work, we showed that local controlled delivery of anticancer compounds could provide cytotoxic levels of agents in the brain tissue for prolonged periods of time (13, 14); however, the volume of treatment is limited by the ability of drug molecules to penetrate into the local tissue before they are eliminated (13). We hypothesized that optimal local delivery requires agents that are both water-soluble, to prevent rapid clearance through capillary permeation, and stable in the brain extracellular space (15); conjugates of active drugs with inert polymers may provide these features (16). Therefore, we sought to synthesize PEG-CPT conjugates appropriate for local delivery to the brain using modifications of a previously published chemical approach (12). PEG-camptothecin conjugates theoretically meet basic requirements for a successful polymeric carrier system for localized, direct drug delivery to the brain. Previously reported promising PEGylated drug conjugates relied on high molecular weight PEG to increase the circulation time of the conjugate during systemic delivery (9). In the present case of local delivery to the brain by diffusion from a polymeric implant, the molecular weight of the PEG must be optimized. Although increasing the molecular weight of the PEG increases the solubility of the drug, it decreases the rate of diffusion of the conjugate, which is the primary means of distribution of the conjugate in the brain. We expected conjugation to PEG3400 to solubilize CPT and increase the effective molecular weight beyond the theoretical threshold for blood-brain barrier penetration (17); these PEG3400 conjugates should also diffuse readily through the extracellular space (18). In addition, these conjugates should be capable of releasing free, active CPT upon ester hydrolysis. Three different PEG3400-CPT conjugates were synthesized. All conjugates were linked via an ester bond, either directly or through a single amino acid (Figure 1B-D). The variation in the nature of the linkage chemistry resulted in conjugates with different stabilities in aqueous solutions. The primary objectives of this study were to measure solubility and stability of CPT conjugates in biological fluids, to determine if CPT released from our synthesized conjugates was capable of inducing cell death in culture, and to determine the drug distribution in vivo from a controlled release device implanted in the rat brain. Since it is known that large water-soluble polymers do not generally diffuse across cell membranes (19), these

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conjugates are likely taken up actively by endocytosis. It was thus of interest to determine if PEG-CPT conjugates release drug via hydrolysis outside of cells or if it is possible for the conjugate to enter the cell and release drug closer to its nuclear target. EXPERIMENTAL PROCEDURES

Materials. (S)-(+)-Camptothecin (CPT) and poly(ethylene glycol), average Mn 3400 (PEG3400) were purchased from Aldrich Chemical Co. (Milwaukee, WI). Stock solutions of CPT were created by dissolving drug in dimethyl sulfoxide (DMSO; Sigma Chemical Company, St. Louis, MO), and the solutions were stored at 4 °C. Carboxymethyl-PEG3400 was purchased from Shearwater Polymers (now Nektar Therapeutics, Huntsville, AL). The MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay reagent kit was purchased from Promega (CellTiter 96 Aqueous Non-Radioactive Cell Proliferation kit; Madison, WI). The LysoTracker Red probe for acidic organelles was purchased from Molecular Probes (Eugene, OR). Poly-L-lysine, type VI-S acetylcholinesterase from electric eel, crude esterase from porcine liver (liver esterase), type I protease from porcine pancreas, and p-nitrophenyl acetate were purchased from Sigma Chemical Company (St. Louis, MO). Poly(1,3-bis(p-carboxyphenoxy)propane: sebacic acid) 20:80 (p(CPP:SA)) was donated by Guilford Pharmaceuticals. The Coomassie Protein Reagent was purchased from Pierce Biotechnology (Rockford, IL). Methods. Analysis of Conjugates. MALD/I TOF mass spectrometry was done at the Biotechnology Resource Center at Cornell University on a Bruker Daltonics Biflex III with R-cyano-4-hydroxycinnamic acid (ACCA) as the matrix. The raw MALD/I data and the list of major MALD/I peaks are shown in the Supporting Information. 1 H and 13C nuclear magnetic resonance spectroscopy was done on an INOVA 500 MHz spectrometer at the NMR Facility at Cornell University. CDCl3 was the solved used, and CPT peaks were correlated to published results for CPT in DMSO (20). Synthesis of Camptothecin-Poly(ethylene glycol) Conjugates. These procedures were modified for PEG3400 from previously published protocols (12). The structures of the three conjugates appear in Figure 1. NMR peaks are labeled using the atom numbering shown for CPT in Figure 1B. CM-PEG-CPT. Carboxymethyl-PEG3400 (244 mg, 0.068 mmol) was dried by azeotroping in 75 mL of toluene for 2 h. Remaining toluene was removed under vacuum, and the PEG and CPT (50 mg, 0.144 mmol) were dissolved in 100 mL of methylene chloride under stirring. The solution was cooled to 0-5 °C in an ice bath. 2-Chloro-1-methylpyridinium iodide (65 mg, 0.253 mmol) and 4-(dimethylamino)pyridine (67 mg, 0.547 mmol) were added. The solution was allowed to warm to room temperature, and the reaction was continued for 48 h. The organic solution was then washed with 0.5 M HCl (2 × 25 mL) and dried over MgSO4. Solvent was removed under vacuum. The product was redissolved in 1-2 mL of methylene chloride and precipitated upon addition of 200 mL of 2-propanol. The precipitate was collected by filtration and dried under vacuum. The product was redissolved in 1-2 mL of methylene chloride and precipitated upon addition of 200 mL of ether. The precipitate was collected by filtration and dried under vacuum. The product was tested for purity by TLC with 5% dimethylformamide in chloroform. Yield ) 90% CMPEG-CPT. 1H NMR (CDCl3): δ(ppm) ) 0.97 (t, 6, C(18)-

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H3), 2.17, 2.28 (mm, 4, C(19)H2), 3.63 (m, >300, PEG), 4.34 (q, 4, OCH2CO), 5.29 (t, 4, C(5)H2), 5.41, 5.68 (dd, 4, C(17)H2), 7.21 (s, 2, dC(14)H), 7.67 (t, 2, dC(10)H), 7.83 (t, 2, dC(11)H), 7.94 (d, 2, dC(9)H), 8.20 (d, 2, dC(12)H), 8.40 (s, 2, dC(7)H). 13C NMR (CDCl3): δ(ppm) ) 7.83 (C18), 32.02 (C19), 50.23 (C5), 67.45 (C17), 68.36-71.27 (PEG), 76.60 (C20), 96.19 (C14), 120.59 (C16), 128.35 (C10), 128.45 (C8), 128.50 (C9), 128.68 (C12), 129.86 (C6), 130.99 (C11), 131.53 (C7), 145.64 (C3), 146.64 (C13), 149.10 (C15), 152.49 (C2), 157.57 (C16a), 167.50 (COOCPT), 169.95 (C21). MALD/I TOF: [CM-PEG3400-CPT + 2ACCA +H]+ predicted m/z: [CM-PEG3400 + 2ACCA +H]+ + 660.69, observed: + 662.94. CM-PEG-Gly-CPT. H-Gly-CPT‚TFA was synthesized using a previously established protocol (12). Carboxymethyl-PEG3400 (200 mg, 0.056 mmol) was dried by azeotroping in 100 mL of toluene for 2 h. The volume of toluene was reduced to 20 mL, and the solution was cooled to room temperature. H-Gly-CPT‚TFA (140 mg, 0.27 mmol), 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (62 mg, 0.25 mmol), and DMAP (110 mg, 0.90 mmol) were added. The solution was stirred at 55-60 °C for 16 h. Solvent was removed under vacuum, and the product was isolated by recrystallizing in 100 mL of 2-propanol, filtering, washing with ice-cold ether, and drying under vacuum. A second recrystallization was done when necessary to improve purity. Yield ) 85% CM-PEG-GlyCPT. 1H NMR (CDCl3): δ(ppm) ) 0.96 (t, 6, C(18)H3), 2.14, 2.25 (mm, 4, C(19)H2), 3.61 (m, >300, PEG), (q, 4, OCH2CO), 3.97 (m, 4, NHCH2CO), 4.15, 4.46 (dd, 4, COCH2O), 5.25 (t, 4, C(5)H2), 5.37, 5.65 (dd, 4, C(17)H2), 7.31 (s, 2, dC(14)H), 7.63 (d, 2, dC(10)H), 7.66 (q, 2, CONH), 7.80 (t, 2, dC(11)H), 7.90 (d, 2, dC(9)H), 8.22 (d, 2, dC(12)H), 8.38 (s, 2, dC(7)H). 13C NMR (CDCl3): δ(ppm) ) 7.82 (C18), 32.02 (C19), 40.66 (COCH2NH), 50.22 (C5), 67.32 (C17), 70.27-71.32 (PEG), 76.96 (C20), 96.52 (C14), 120.25 (C16), 128.32 (C10), 128.38 (C8), 128.40 (C9), 128.67 (C12), 129.92 (C6), 130.95 (C11), 131.52 (C7), 145.78 (C3), 146.62 (C13), 149.00 (C15), 152.44 (C2), 157.55 (C16a), 167.37 (COO-CPT), 169.16 (NHCO), 171.09 (C21). MALD/I TOF: [CM-PEG3400Gly-CPT + 2ACCA +H]+ predicted m/z: [CM-PEG3400 + 2ACCA +H]+ + 774.79, observed: + 774.15. CM-PEG-Sar-CPT. H-Sar-CPT‚TFA was synthesized using a previously established protocol (12). Carboxymethyl-PEG3400 (239 mg, 0.067 mmol) was dried by azeotroping in 100 mL of toluene for 2 h. Remaining toluene was removed under vacuum, and the PEG was dissolved in 20 mL of methylene chloride under stirring. The solution was cooled to 0-5 °C in an ice bath. H-SarCPT‚TFA (150 mg, 0.28 mmol), diisopropylcarbodiimide (42 µL, 0.27 mmol), and DMAP (33 mg, 0.27 mmol) were added. The solution was allowed to warm to room temperature, and the reaction was continued for 16 h. Solvent was removed under vacuum, and the product was isolated by recrystallizing in 100 mL of 2-propanol, filtering, washing with 2-propanol, and drying under vacuum. A second recrystallization was done when necessary to improve purity. Yield ) 70% CM-PEG-SarCPT. 1H NMR (CDCl3): δ(ppm) ) 0.97 (t, 6, C(18)H3), 2.13, 2.24 (mm, 4, C(19)H2), 3.04 (s, 6, N(CH3)), 3.61 (m, >300, PEG), 4.03, 4.64 (dd, 4, COCH2N(CH3)), 4.20 (q, 4, OCH2CO), 5.24 (t, 4, C(5)H2), 5.37, 5.66 (dd, 4, C(17)H2), 7.31 (s, 2, dC(14)H), 7.64 (t, 2, dC(10)H), 7.80 (t, 2, dC(11)H), 7.90 (d, 2, dC(9)H), 8.23 (d, 2, dC(12)H), 8.37 (s, 2, dC(7)H). 13C NMR (CDCl3): δ(ppm) ) 7.84 (C18), 31.96 (C19), 35.64 (N(CH3)), 49.27 (COCH2N(CH3)), 50.22 (C5), 67.36 (C17), 70.07-70.78 (PEG), 77.06 (C20), 96.57 (C14), 120.06 (C16), 128.30 (C10), 128.38 (C8), 128.38

Fleming et al.

(C9), 128.66 (C12), 129.98 (C6), 130.90 (C11), 131.47 (C7), 145.80 (C3), 146.65 (C13), 149.01 (C15), 152.45 (C2), 157.54 (C16a), 167.60 (COO-CPT), 168.58 (N(CH3)COCH2), 170.31 (C21). MALD/I TOF: [CM-PEG3400Sar-CPT + 2ACCA +H]+ predicted m/z: [CM-PEG3400 + 2ACCA +H]+ + 802.84, observed: + 804.96. Stability of Conjugates in Neutral and Acidic Conditions. The hydrolysis rate of conjugates was determined in phosphate-buffered saline (PBS) at pH 7.4 and in citrate-phosphate buffer solutions at pH 5.5 and pH 6.0. Conjugates were dissolved at a concentration of 10 µg/mL and incubated at 37 °C. For HPLC analysis (Waters 2690 HPLC with photodiode array detector, Milford, MA), at each time point 10-40 µL of sample was injected and eluted on a 40-min, 80:0:20 to 0:80:20 (water: acetonitrile:water/0.5%TFA) gradient. Free CPT eluted at 12.9 min on a Zorbax 300SB-CN column (Phenomenex, Torrance, CA). Areas of absorbance peaks at 254, 354, and 369 nm were compared to CPT standards to determine concentrations. For extraction analysis, at specified time points, 250 µL was removed from each of the conjugate solutions and subjected to a modified extraction procedure (21). Since ethyl acetate preferentially extracts free CPT out of solutions containing free and conjugated drug, this procedure provided a rapid method to isolate free CPT. Briefly, each 250-µL sample was acidified to pH 3.0 with 1 M HCl. Water-saturated ethyl acetate (500 µL) was added to extract out CPT, and the sample was vortexed for 10 s. The sample was then centrifuged at 8160g for 5 min. Half of the ethyl acetate layer (250 µL) was transferred to a clean tube, while the remaining ethyl acetate was transferred to waste. An additional 500 µL of ethyl acetate was added to the aqueous sample, and the extraction procedure was repeated, with 250 µL of the organic phase retained. The combined ethyl acetate extracts (500 µL) were evaporated to dryness under a gentle stream of nitrogen, reconstituted in 95% ethanol (1 mL) and the fluorescence intensity of CPT was determined at an emission wavelength of 434 nm after excitation at 370 nm. Stability of Conjugates in Simulated Biological Fluids. Conjugate stability studies were carried out at 37 °C and pH 7.4 in simulated biological fluids. Conjugates were present at a concentration of approximately 10 µg/mL in all cases. At specified time points, 250-µL samples were removed from the conjugate solutions and free CPT was quantified after preferentially extracting out the drug. The extraction procedure was the same as used for neutral and acidic stability experiments (see above), with the following changes. Ice cold citratephosphate buffer was used to reduce the pH to 3, and centrifugation was for 10 rather than 5 min. Six in vitro solutions were tested: PBS, acetylcholinesterase in PBS, liver esterase in PBS, pancreatic protease in PBS, 9L lysates in PBS, and brain homogenate in PBS. To create the test solutions, acetylcholinesterase, esterase, and protease proteins were dissolved in PBS at 15 U/mL of enzymatic activity, based on activity reported by the vendor. Cell lysate solutions were created from 9L gliosarcoma cells in culture. Six 75-cm2 flasks of cells were trypsinized at confluence, washed twice in PBS, and resuspended in 20 mL of PBS. Cells were lysed with sonication (TEKMAR Soni Disruptor, TM300) on ice at 40% power for 2 min. The lysate was centrifuged at 1070g for 10 min. The supernatant was diluted with approximately 30 mL of PBS to create a total volume of 50 mL, and the resulting lysate was diluted 1:1 with PBS to create the final solution used in the release study. Brain homogenate solutions were created by homogeniz-

Distribution of PEGylated CPT in the Rat Brain

ing (PowerGen Homogenizer, Fisher Scientific, Pittsburgh, PA) the brain of an adult female rat (Harlan Sprauge-Dawley) at a concentration of 50 mg/mL in PBS. Total protein content in the cell lysate solution and brain homogenate was determined using the Coomassie Protein Reagent Kit. For the total protein assay, 100 µL of serially diluted samples and standards prepared from liver esterase were placed into the wells of a 96-well plate, followed by 100 µL of the Coomassie Protein Reagent. Absorbance was measured at 595 nm on a microplate reader (Molecular Devices, Sunnyvale, CA). Total protein content was based on comparison to a BSA standard provided with the assay kit. The esterase activity of brain homogenate and cell lysate solutions was measured using p-nitrophenyl acetate (PNPA) as a model substrate. PNPA was dissolved in DMSO to create a 100 mg/mL stock solution which was stored at -20 °C. From this stock, an assay solution of 0.1 mg/mL PNPA in PBS was made. Serially diluted 100-µL brain homogenate samples and standards prepared from liver esterase were placed into the wells of a 96-well plate, followed by 100 µL of the PNPA assay solution. Absorbance was measured at 405 nm using a microplate reader after 10 min of incubation at room temperature. Esterase activity was based on comparison to the liver esterase standards in PBS. Cell Line. The 9L gliosarcoma cell line (9L) was kindly provided by Dr. Henry Brem of Johns Hopkins University (Baltimore, MD). The cell line was maintained in DMEM media (with L-glutamine) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C and 5% CO2 in a humidified environment. All tissue culture reagents were purchased from Gibco, BRL (Grand Island, NY). Cells were subcultured every 3-4 days at a relative cell density of 1:8. In Vitro Cytotoxicity. The cytotoxicity of CPT conjugates and free CPT was measured against 9L cells in monolayer culture. Cells were plated in 96-well plates (200 cells/well) and allowed to attach. Drug was serially diluted in cell culture media; CPT conjugates were dissolved directly into media whereas CPT was added from a stock solution in DMSO (1 mg/mL). Plated cells were incubated in media containing free or conjugated drug for 24, 72, or 120 h. In the case of the 24 and 72-h time points, the free drug- or conjugate-containing media was removed and replaced with fresh media, and the cells were returned to the incubator. After 120 h, cell viability was determined in all wells using an MTS assay and a microplate reader. The fraction of cells surviving was calculated by dividing mean optical density produced by treated cells by the mean optical density from untreated control cells. Cellular Uptake in Monolayer Culture. Glass slides were placed in six-well tissue culture plates and coated with a solution of poly-L-lysine in PBS (25 µg/mL) for 1 h at 37 °C. After the slides were rinsed with PBS, 9L cells were seeded at a density of 1.5 × 104 cells/cm2 and allowed to attach overnight. The cells were then incubated in medium containing conjugated drug or free drug (100 µg/mL) for 5 h. In some cases a fluorescent marker for acidic organelles (LysoTracker Red) was added during the final 30 min of incubation at a concentration of 50 nM. After incubation, cells were rinsed three times with cold PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. The resulting cell preparations were studied by fluorescence microscopy using an Olympus BX-50 fluorescence/DIC microscope with a mercury lamp excitation source. Camptothecin

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and LysoTracker fluorescence were visualized using UV and red filters, respectively. Fabrication of Controlled-Release p(CPP:SA) Implants. Controlled-release devices containing CPT conjugates at 20 wt % were prepared by first codissolving 50 mg of each conjugate and 200 mg of p(CPP:SA) in methylene chloride (2 mL). Each solution was thoroughly vortexed and, after complete dissolution, placed under vacuum at room temperature for 24 h. The resulting material was crushed into a homogeneous powder consisting of relatively uniform particles. To produce implantable disks approximately 1 mm in thickness, 10 mg of the powder was placed in a stainless steel die (3 mm diameter) and 0.25 tons of pressure was applied with a Carver press for 20 s. Disks to release unmodified CPT were prepared containing CPT plus unconjugated PEG3400 (20 wt % total loading). CPT and free PEG were combined in methylene chloride at a CPT:PEG ratio of 1:6.14 to match the drug content of conjugated material. Unlike the CPT conjugates, CPT did not completely dissolve in methylene chloride. Measurement of Drug Release into PhosphateBuffered Saline. The in vitro release characteristics of matrixes made into disks were determined in continuously stirred phosphate-buffered saline (pH 7.4) at 37 °C. Three identical disks from each group were incubated in individual aliquots of 1.5 mL of PBS in test tubes. At various time points the entire volume of each tube was removed and replaced with fresh buffer. The amount of CPT released was quantified in the resulting samples by measurement of the intrinsic fluorescence of the drug. Individual samples were diluted with PBS to a total volume of 700 µL and acidified with 7 µL of 1 M hydrochloric acid to convert any free CPT in the carboxylate form back to the lactone. A 10% SDS solution was then added (100 µL) to break up any aggregation of CPT conjugates, the mixture was thoroughly vortexed, and bubbles were allowed to dissipate. The fluorescence of the samples was measured at 428 nm (excitation at 370 nm). In some cases the samples were briefly centrifuged at 8160g for 1 min to remove undegraded p(CPP:SA) wafer remnants prior to analysis. No significant change in the fluorescence of CPT conjugate solutions with time was detected, and it was assumed that CPT conjugates and the CPT released from the conjugates had the same fluorescence yield. Implantation of p(CPP:SA) Matrixes into the Normal Rat Brain. Male Fischer 344 rats (200 g, Harlan, Indianapolis, IN) were anesthetized by intraperitoneal injection of 0.6 mL of a solution containing 25 mL of ketamine hydrochloride (100 mg/mL, Abbott Laboratories, N. Chicago, IL), 2.5 mL of xylazine (100 mg/mL, Bayer Corporation, Shawnee Mission, KS), 14.2 mL of ethanol (95%), and 58 mL of physiological saline (The Butler Company, Columbus, OH). A 3-cm incision was made above the skull, and the bregma was exposed. An oval-shaped burr hole was drilled 1 mm anterior to the bregma and perpendicular to the midline. A slit 4 to 5 mm deep was made in the dura and brain parenchyma with the tip of a number-11 scalpel blade, and the polymer matrix was gently inserted into the tissue, until it was no longer visible. Once the bleeding had ceased, the skin was closed with surgical staples. All procedures were approved by the Institutional Animal Care and Use Committee at Cornell University. Brain Tissue Analysis. Rats were sacrificed 1, 7, 14, or 28 days following implantation. At the time of sacrifice, the brains were frozen in hexanes over dry ice. On the day of analysis, brains were cut into three bulk sections,

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Figure 2. Implantation and sectioning scheme. The implant (grey rectangle) was placed 0.5-1 mm anterior to bregma. The frozen brain was first cut down the midline and then directly behind the polymer implant. The tissue posterior to the implant was sectioned into 1 mm slices. The tissue anterior to the implant was sectioned into 50 µm slices with a cryo-microtome.

first down the midline, and then directly behind the p(CPP:SA) implant (see Figure 2). Posterior Sectioning. The portion of the brain behind the implant was sectioned in the coronal plane. Brains were placed in a chamber maintained at -20 °C, and razor blades, fastened at 1-mm intervals with screws and washers, were forced through the tissue under gentle pressure. The tissue was allowed to refreeze, and the 1-mm (thick) sections were weighed in tared vials. These tissue slices and the entire contralateral hemisphere were homogenized in citrate-phosphate buffer containing 1% SDS (pH 3.0) at a concentration of 20 mg tissue per mL buffer. One milliliter of each tissue sample was centrifuged at 16 000g for 5 min, and the supernatant (700 µL) was analyzed for the presence of CPT and conjugated CPT by spectrofluorimetry. Anterior Sectioning. The frontal section of brain containing the polymer implant was placed on a cryostat chuck, embedded in OCT embedding medium, and cut into 50-µm (thin) sections using a cryomicrotome (Microm, Heidelberg, Germany) at -20 °C. Alternating sections were collected into separate tubes: one 50-µm section for total drug analysis, the next two 50-µm sections together for extraction of liberated CPT, one for total drug analysis, and so on. Thus total CPT levels (conjugated plus unconjugated) were measured in 50-µm sections of brain, whereas the fraction of drug in the free, liberated form was quantified in 100-µm sections (2, 50µm sections). Total Drug Levels in Thin Brain Slices. The total amount of CPT (in either conjugated or free form) was quantified in 50-µm sections of brain tissue. The sections were homogenized in citrate-phosphate buffer containing 1% SDS (pH 3.0) by sonication (Tekmar Soni Disrupter, microtip #4) on ice at 30% power for 10 s. The samples were centrifuged at 16 000g for 5 min, and the resulting supernatant (200 µL) was analyzed for the presence of CPT and conjugated CPT by spectrofluorimetry. Amount of Liberated, Free CPT in Thin Brain Slices. Free drug in 100-µm sections of brain was quantified by an extraction method similar to that described for the simulated biological fluids with two alterations. After the solution was acidified to pH 3, the sample was homogenized by sonication at 30% power for 20 s on ice, and as the final step, 200 mL of ethanol was used to reconstitute the CPT residue. We expected that in brains implanted with p(CPP:SA) matrixes releasing unmodified CPT, procedures to measure the amount of

Fleming et al.

Figure 3. Stability of PEG-camptothecin conjugates in water at 37 °C. Experiments were done at pH 7.4 (white), at pH 6.0 (grey), and at pH 5.5 (black) for CM-PEG-CPT (]), CM-PEGGly-CPT (4), and CM-PEG-Sar-CPT (0). The presence of free CPT was monitored as a function of time. Data at pH 7.4 was acquired by HPLC analysis and corroborated by extraction technique. Data at pH 6.0 and 5.5 were obtained by extraction. Table 1. Conjugate Hydrolysis Half-Lives per Hour at Different pHs CM-PEG-CPT CM-PEG-Gly-CPT CM-PEG-Sar-CPT

pH 7.4

pH 6.0

pH 5.5

38.5 57.8 81.5

603 630 924

1000 1050 1820

total drug (conjugated plus free) and the amount of free drug would yield equivalent values since all drug was released from p(CPP:SA) in the free form. Since this was, in fact, the case for the first several animals studied, subsequent brains were grouped into 100-µm slices and subjected only to the extraction procedure. RESULTS

In Vitro Experiments. Conjugates were first placed in aqueous solutions that simulated biological fluids, and the rates of hydrolysis were measured either by HPLC analysis or an extraction procedure with fluorescence detection. The stability of the CPT conjugates was determined in buffered solutions at pH 7.4, pH 6, and pH 5.5 to simulate the cytoplasmic/extracellular space, the endosome, and the lysosome, respectively (22). The resulting hydrolysis data appear in Figure 3. Independent measurements by HPLC and extraction at pH 7.4 gave identical results. The curves in Figure 3 represent best first-order fits to the data, and the half-lives for these curves are given in Table 1. All conjugates were much more stable at pH 6.0 and 5.5 than at pH 7.4, with the trend in hydrolysis rates for the three conjugates remaining the same (CM-PEG-Sar-CPT