Utility of Poly(ethylene glycol) Conjugation To Create Prodrugs of

Page 1. Utility of Poly(ethylene glycol) Conjugation To Create Prodrugs of. Amphotericin B. Charles D. Conover, Hong Zhao, Clifford B. Longley,* Kwok ...
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Bioconjugate Chem. 2003, 14, 661−666

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Utility of Poly(ethylene glycol) Conjugation To Create Prodrugs of Amphotericin B Charles D. Conover, Hong Zhao, Clifford B. Longley,* Kwok L. Shum, and Richard B. Greenwald Enzon Pharmaceutical, Inc., 20 Kingsbridge Road, Piscataway, New Jersey 08854-3998. Received December 23, 2002; Revised Manuscript Received February 6, 2003

This paper reports on the synthesis, safety, and efficacy of a series of water-soluble derivatives of poly(ethylene glycol) (PEG)-conjugated amphotericin B (AmB). PEG 40 000 attached to the sugar amino group of AmB via labile carbamate and carbonate linkages was examined. The synthetic program conducted for this investigation provided a series of disubstituted PEG-AmB derivatives which had in vitro PEG half-life of hydrolyses rates in rat plasma varying between 1 and 3 h. Importantly, all conjugates demonstrated less than 6% hydrolysis following 24 h incubation in pH 7.4 phosphate buffer at 25 °C and showed solubilities greater than 46 mg/mL in aqueous solutions. The solubility of AmB in the conjugates increased up to approximately 200 times compared to unmodified AmB in saline. As a major finding, this investigation demonstrated that conjugation of PEG to AmB could produce conjugates that were significantly (6×) less toxic than AmB-deoxycholate and maintained, or even had enhanced, in vivo antifungal activity.

INTRODUCTION

Available since 1960, amphotericin B (AmB) is an amphoteric polyene antibiotic prepared from the soil microorganism, Streptomyces nodosus. It binds selectively to ergosterol in the cell membrane of susceptible fungi, inducing changes in permeability that can produce lethal cell injury. Because this fungicide has such broadspectrum activity, it remains the gold standard agent for many life-threatening fungal infections. However, AmB is virtually insoluble in water and can only be formulated into a suspension. Intravenous administration of AmB formulated as a deoxycholate micellar suspension (Fungizone) is often complicated by both infusion-related adverse reactions (e.g., fever, chills, rigors, nausea, vomiting, and hypotension) and systemic toxicities (e.g., nephrotoxicity, acidosis, hypokalemia, hypomagnesemia, and anemia). To overcome the limitations of AmB, other modalities of AmB have been developed in which this agent is encapsulated in liposomes (20) or is bound to carriers (8). A number of the lipid carrier approaches have been successful in increasing the therapeutic index of AmB and a few have achieved clinical application (20). One methodology that has received only sparse consideration is the conjugation of AmB to poly(ethylene glycol) (PEG) (24). PEG is a condensation product of ethylene oxide having the general formula HO(CH2CH2O)nH. It is a linear amphiphilic polymer that is nonbiodegradable, nontoxic, and nonimmunogenic and has been widely studied for both protein (10) and small molecule modification (15). PEG has been extensively utilized due to its ability to increase the solubility, circulating life, safety, and permeable tissue accumulation level of proteins, while decreasing their immunogenicity and renal excretion (16, 18, 22, 26). This technology has also been extended into small molecule prodrug platforms (15). The use of prodrug design has been applied to numerous therapeutic areas with the goal to improving the pharmacologic properties of drugs. A * Corresponding author. Phone: 1-732-980-4832. Fax: 1-732885-2950. E-mail: [email protected].

prodrug is a biologically inactive derivative of a parent drug molecule that usually requires an enzymatic or chemical transformation within the body in order to release the active drug and has improved delivery properties over the parent molecule (25). Essential to drug delivery with a prodrug is the rate of release of the active drug. A rapid breakdown of the prodrug can result in spiking of the parent drug and possible toxicity, while too slow a release rate will compromise the drug’s effectiveness. The linkages between the active drug and the carrier molecule can theoretically be chosen so that either pH or enzymatic degradation mediate prolonged drug release. Therefore, the effectiveness of prodrug delivery is dependent on the stability of the drug conjugate linkage and its potential for controlled degradation. The amino group on the sugar moiety of AmB is essential for its antifungal activities (1, 21) and provides an ideal site for attachment of PEG. Recently we have developed two platform technologies for PEG conjugates through the controlled release of amino groups (13, 14). Both methods can be utilized for AmB; however, the 1,6benzyl elimination system is more easily applied. The chemistry of these conjugations continues our exploration of delivery of insoluble bioactive agents using PEG, strategies first initiated with paclitaxel (11) and camptothecin (12). For these two agents, PEG modifications were shown to extend circulatory exposure, reduce toxicity, and increase the therapeutic index of the parent compounds (5, 23). The objective of the current study is to assess the use of PEG conjugation to the antifungal agent AmB and evaluate its in vitro and in vivo activity. MATERIALS AND METHODS

Chemicals. Amphotericin B was purchased from Fluka Chemie AG, Switzerland, and all other reagents and solvents were purchased from Aldrich Chemical Co. (Milwaukee, WI) and Advanced ChemTech (Louisville, KY). Unless stated otherwise, they were used without further purification. All the PEGs used in this study had a molecular weight of 40 kDa and were dried under vacuum or by azeotropic distillation from toluene prior

10.1021/bc0256594 CCC: $25.00 © 2003 American Chemical Society Published on Web 03/29/2003

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Scheme 1. Syntheses of PEGylated AmB Conjugates Using Bifunctional PEG

to use. Organic solutions were dried over MgSO4. Solvents were removed by rotary evaporation. 13C NMR spectra were obtained on a JEOL JNM-GSX270 FT NMR System. Deuterated chloroform or pyridine was used as solvents unless otherwise specified. Analytical HPLC was conducted on a ZORBAX 300 SB C8 RP column (150 × 4.6 mm) with a gradient of 30 to 80% acetonitrile in 0.1 M triethylamine acetate buffer (pH ) 6.8) at a flow rate of 1 mL/min. Synthetic Approaches. The preparation of the various PEG 1,6 benzyl elimination linkers was carried out according to published procedures (13). Conjugation of PEG linkers with AmB was done in anhydrous dimethylformamide (DMF) in the presence of 4-(dimethylamino)pyridine (DMAP) (Scheme 1). We observed that once AmB was conjugated through the sugar amino group, the glycosyl bond between the sugar moiety and the macrocyclic heptaene in PEG-AmB conjugates were very sensitive to acid. Therefore, pyridine was added to recrystallization solvents in order to maintain basicity and prevent the breakdown of this bond. Compound 3. To a solution of PEG linker 2 (13) (4.0 g, 0.0983 mmol) in 40 mL anhydrous DMF were added AmB (0.272 g, 0.295 mmol) and DMAP (0.360 g, 2.95 mmol), and the mixture was stirred for 12 h at room temperature. Ethyl ether (100 mL) was added to the reaction solution with swirling and the PEG derivative precipitated. The mixture was stored at -20 °C for 12 h and filtered. The crude solid was recrystallized from a mixture of 100 mL of 2-propanol (IPA) and 1 mL of pyridine twice to give pure product 3 (3.80 g, 0.090 mmol, 91%). 13C NMR (67.8 MHz, C5D5N) δ 176.21, 171.73, 157.35, 153.98, 151.20, 137.67, 137.21, 134.85, 134.57, 134.46, 134.10, 133.80, 133.27, 133.02, 132.94, 132.78,

132.69, 132.55, 129.47, 127.97, 121.51, 98.25, 97.93, 78.34, 76.37, 75.75, 74.91, 74.33, 73.50-69.20 (PEG), 68.89, 68.23, 66.73, 66.29, 65.55, 63.46, 61.55, 59.11, 58.26, 47.29, 45.76, 45.00, 43.59, 42.79, 40.79, 40.52, 38.08, 36.41, 31.77, 18.91, 18.59, 17.20, 12.63. Compounds 5, 7, 9, 11, and 13 were prepared in a manner similar to that of 3. Compound 5. 13C NMR (67.8 MHz, C5D5N) δ 176.14, 171.66, 157.35, 155.37, 133.03, 131.05, 129.33, 127.74, 121.45, 98.35, 78.00, 74.50-69.00 (PEG), 66.83, 65.93, 63.33, 61.65, 59.29, 58.37, 47.38, 45.95, 45.18, 43.72, 42.98, 41.70, 41.16, 40.05, 36.59, 31.90, 26.15, 19.10, 18.81, 17.43, 12.84. Compound 7. 13C NMR (67.8 MHz, C5D5N) δ 176.06, 171.67, 169.41, 157.25, 134.47, 133.20, 132.96, 132.75, 129.86, 127.93, 121.93, 98.15, 97.80, 78.29, 76.70, 74.79, 74.59, 73.50-69.00 (PEG), 68.63, 68.35, 66.60, 65.70, 58.96, 58.61, 47.00, 44.87, 43.56, 42.64, 36.33, 31.76, 18.88, 18.52, 17.16, 12.59. Compound 9. 13C NMR (67.8 MHz, C5D5N) 176.14, 171.63, 168.76, 157.43, 147.69, 137.67, 137.10, 134.07, 133.76, 132.99, 130.40, 129.57, 128.61, 127.29, 98.30, 97.96, 78.41, 76.50-66.50 (PEG), 66.84, 66.35, 65.88, 61.65, 59.13, 58.35, 47.46, 45.88, 45.14, 43.72, 42.94, 40.66, 36.56, 31.92, 19.09, 18.76, 17.40, 16.36, 12.84. Compound 11. 13C NMR (67.8 MHz, C5D5N) δ 176.13, 171.63, 171.02, 170.33, 161.67, 132.98, 129.45, 127.18, 98.30, 78.47, 76.47, 75.01, 74.41, 73.50-69.00 (PEG), 66.84, 64.49, 63.32, 61.67, 59.23, 58.42, 47.41, 45.88, 45.15, 43.72, 42.91, 41.45, 40.79, 40.52, 36.57, 31.98, 19.08, 18.82, 17.42, 12.84. Compound 13. 13C NMR (67.8 MHz, C5D5N) δ 176.11, 171.63, 171.20, 157.34, 133.74, 133.01, 129.29, 127.74, 98.30, 98.01, 78.42, 76.50-67.00 (PEG), 66.86, 65.93,

Polyethylene Glycol Conjugation of Amphotericin B

63.76, 61.61, 59.27, 58.36, 47.45, 45.90, 45.15, 43.72, 42.97, 40.71, 39.84, 38.34, 36.51, 31.93, 19.07, 18.79, 17.40, 12.84. In Vitro Solubility, Stability, and Dissociation Profile. The solubility of the conjugates was determined by dissolution in saline. Briefly, a known amount of compound was added to 1 mL of 0.9% saline in a 4 mL glass vial. The mixture was vortexed for 5 min and left for 10 min. The mixture was filtered through a 0.45 µm hydrophilic filter membrane, and the filtrate was lyophilized to dryness and weighed. Rates of hydrolysis (dissociation of PEG conjugate) of the PEG-AmB derivatives were determined in phosphate-buffered saline (PBS, pH 7.4) and fresh rat plasma as previously described (12). In Vitro Efficacy. In vitro microbial proliferation was assessed by using an Alamar Blue based proliferation assay (11) to determine each conjugate’s IC50 (drug concentration inhibiting growth of cells by 50%). Briefly 2-fold dilutions of PEG conjugates were made in yeastpeptone-dextrose (YPD) broth. A 50 µL aliquot of Saccharomyces cerevisiae (ATCC 9763) was seeded into microwell plates at a final density of 2 × 103 cells per well. Plates were incubated at 30 °C in a humidified incubator with 5% CO2 for 3 days. Cell growth was measured by the addition of 10 µL/well of Alamar Blue (Alamar Biosciences, Inc., Sacramento, CA), and the plates were incubated further for 4 h at 30 °C. The IC50 values for each compound were determined from absorbance versus dilution factor plots. In addition, minimum inhibitory concentration (MIC) was determined by the broth dilution method (7). Constructs were dissolved and serially diluted in 100% DMSO. For each concentration tested, 0.01 mL of the aliquot was added to a 48-well plate containing 0.99 mL of potato dextrose broth (DIFCO, Detroit, MI) with 103 to 104 CFU/mL of Aspergillus niger (ATCC 8740). The final maximum concentration of DMSO was 1%, and the initial conjugate concentration was 100 µg/mL. The plates were incubated at 24 °C for 48 h and then visually examined and scored positive for inhibition of growth or turbidity or negative for no effect upon growth or turbidity. Vehicle-control and an AmB reference were used as blank and positive controls, respectively. Each concentration was evaluated in duplicate. Erythrocyte Permeability. Erythrocyte permeability was assessed by examining the effect of the conjugates on erythrocyte hemolysis (9). Rabbit erythrocytes were suspended in PBS and were washed twice in the same buffer by centrifugation (3000 × g for 10 min). The hemolysis reaction was conducted in glass tubes containing 0.1 mL of the serially diluted compounds and 0.9 mL of erythrocytes. Tubes were incubated for either 30 min or 3 h at 37 °C and then placed into a refrigerator for 5 min to halt the reaction. Samples were then centrifuged, and the supernatant was measured in duplicate for hemoglobin concentration (OSM3 Hemoximeter, Radiometer, Copenhagen). Circulatory Retention. Circulatory retention studies were performed in 260 g, non-tumor-bearing Sprague Dawley female rats (Charles River Laboratories, Stone Ridge, NY). Rats received an iv bolus of either 45.77 mg/ kg of 3 (2 mg/kg AmB equivalents) in saline or 2 mg/kg of a deoxycholate micellar suspension of amphotericin B (Fungizone, Sigma, St. Louis, MO) via the tail vein at an injection rate of 0.5 mL/min. Rats were bled over a 48 h period (three rats/time point). Bleeding was conducted in unconscious (70% CO2/30% O2) animals via retro-orbital plexus into a sterile EDTA containing tube. A minimum of 250 µL of whole blood was collected and

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centrifuged at 4 °C for 5 min at 5000 rpm. The plasma fraction was collected and frozen immediately at -80 °C on dry ice until analyzed. The plasma samples were thawed and analyzed by HPLC within 1 to 2 h of thawing using a modification of the method by Echevarria (6). Briefly, 10 µL of a 2.5 mg/mL solution piroxicam (Sigma, St. Louis, MO) was added to 60 µL of plasma as an internal standard. Compound 1 and 3 were extracted from the plasma by addition of 250 µL of acetonitrile/ methanol (1:1), incubating at room temperature for 5 min and then centrifuging the mixture at 14 000 rpm for 3 min in a microcentrifuge. The supernatant was transferred to a microcentrifuge tube. A 100 µL aliquot of the extract was applied to a Jupiter C18 (4.6 mm × 250 mm, Phenomenex) column previously equilibrated with solvent A (41% acetonitrile, 4.3% acetic acid, 54.7% water). The HPLC was eluted for 12 min with solvent A and stepped to 100% acetonitrile for 5 min, and the column was reequilibrated with solvent A for 5 min. The eluted peaks were detected by UV absorbance using 357 nm for the initial 7.5 min of the elution and 290 nm for the final 14.5 min. The amount of 1 and 3 in the plasma was calculated from the linear regression analysis of 1 and 3 standard curves performed in rat plasma identical to the rat plasma samples. The area under the plasma curve (AUC), elimination half-life (t1/2), and peak or maximum concentration (Cmax), were calculated using a one compartment, iv bolus, first-order elimination model (WinNonlin, Pharsight Corp., Moutain View, CA). In Vivo Safety. For in vivo administration, AmB solubilized in sodium deoxycholate (Fungizone, Gibco BRL, Life Technologies) was employed. AmB and PEGAmB dosages were dissolved in sterile physiological saline prior to in vivo dosing. All PEG-AmB dosages were given as their AmB equivalents (absolute amount of AmB given). The maximum tolerated single dose (MTD) of selected PEG-conjugated AmB derivatives was estimated by body weight loss in female ICR mice (7-8 weeks old, Harlan Labs). The MTD was determined by administering different groups of mice (N ) 4-5) ascending iv doses of between 4 and 12 mg (AmB content) at 1 to 2 mg/kg increments. Body weights were measured twice weekly for two weeks. The highest dose to cause a loss of less than 20% of initial weight within the time period was considered the MTD. All animals received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society of Medical Research and the “Guide for the Care and Use of Laboratory Animals” published by the National Institute of Health. This animal facilities’ Institutional Animal Care and Use Committee approved these experimental protocols. In Vivo Efficacy. Antifungal animal testing (17) was conducted at MDS Pharma Services (Bothhell, WA). Briefly, groups of 10 ICR-derived male mice (MDS Pharma Services- Taiwan Ltd.) weighing 22 ( 1 g were used. Mice were inoculated iv with an LD90-100 of Candida albicans (ATCC 10231, 1.1 × 107 CFU/mouse, American Type Culture Collection, Rockville MD) in 0.2 mL of PBS without mucin. Test substances and vehicle control, sterile saline, were administered iv (10 mL/kg) with doses ranging from 0.5 mg/kg to 6 mg/kg to test animals 1 h after the fungal inoculation. Mortality was recorded once daily for 10 days. An increased survival of 50% or more relative to the vehicle control group after the fungal inoculation was considered significant protective activity. All aspects of the study were performed according to the International Guiding Principles for

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Table 1. In Vitro Profilea of PEG-Conjugated Amphotericin B compd

solubility in saline (mg/mL)b

% buffer hydrolysis in 4 hc

% buffer hydrolysis in 24 hc

dissociation t1/2 (h) rat plasmac

IC50 S. cerevisiae (µM)d

MIC A. niger (µg/mL)d

1 3 5 7 9 11 13

100 NDe 30 ND ND 100

a All experiments were conducted at least in duplicate. Standard deviation of measurements ) (10%. b Solubility of AmB in conjugate (mg/mL) in brackets. c Rates of hydrolysis of the PEG-AmB derivatives were determined in phosphate-buffered saline (PBS, pH 7.4) at 25 °C and fresh rat plasma at 37 °C. d IC50 and MIC were measured by an Alamar-blue-based cytotoxicity assay and the broth dilution method, respectively. Values express as AmB equivalence. e ND not determined.

Biomedical Research Involving Animals (CIOMS Publication No. ISBN 92 90360194, 1985).

Table 2. Pharmacokinetic Parameters of Compound 1 and Compound 3 compound

Cmax (µg/mL)a,b

t1/2 (h)b

AUC (µg/mL‚h)a,b

RESULTS

AmB (1) PEG-AmB (3)

0.26 ( 0.02 6254 ( 990 [273 ( 43] 0.31 ( 0.04

1.88 ( 1.73 0.41 ( 0.03

0.68 ( 0.62 3715 ( 820 [162 ( 35.7] 0.83 ( 0.43

In Vitro Solubility, Stability, and Dissociation Profile. The in vitro profile of PEG-conjugated AmB derivatives are shown in Table 1. The solubility of the PEG-conjugated AmB compounds in saline ranged from 46.0 to 66.3 mg/mL, thus increasing AmB’s solubility from less than 0.01 mg/mL to as high as 2.5 mg/mL when presented in the conjugate form. The rates of hydrolysis of the PEG conjugates were determined in phosphatebuffered saline, pH 7.4 (PBS) and fresh rat plasma. The PEG-AmB conjugates appeared quite stable in PBS at room temperature with less than 7% hydrolysis occurring within 24 h. The hydrolysis half-life (t1/2) of PEG-AmB conjugates in fresh rat plasma varied between 1 and 3 h. In Vitro Efficacy. The in vitro antifungal activity of all the conjugates was tested using Saccharomyces cerevisiae. The inhibition of proliferation by AmB and the PEG-AmB conjugates are shown in Table 1. The IC50 of AmB was 35 nM, which is within its reported range (2). In contrast, it appears to take roughly 100 times greater quantity of AmB within the slower releasing PEG conjugates to show the same level of inhibition produced with free AmB. The MIC of conjugates 3 and 13 were also assessed using Aspergillus niger. Again, based on an AmB content, the PEG conjugates were approximated 100 times less effective than free AmB. Erythrocyte Membrane Permeability. The effect of PEG-AmB on perturbing plasma membranes was determined by examining the hemolysis of rabbit erythrocytes using varying concentrations of PEG-AmB. The two PEG-AmB conjugates representing the slowest in vitro dissociation rates 3 and 9 were assessed after 30 min and 3 h for these effects on erythrocyte plasma membranes. After 30 min incubation, neither PEG-AmB conjugate showed any hemolysis at concentrations up to 1 mg/mL, the highest concentration examined. In contrast, free AmB in a deoxycholate micellar suspension was hemolytic at concentrations greater than 10 µg/mL. The level of hemolysis for 1 mg/mL of AmB after a 30 min exposure was approximately 10% which increased to over 70% after the 3 h incubation. In contrast, the two PEG-AmB conjugates at 1 mg/mL (AmB content) showed less than 3% hemolysis after 3 h. Circulatory Retention. The modeled pharmacokinetic parameters for both 1 and 3 are shown in Table 2. A plot of the plasma concentration-time curve for 3 administered iv in rats is shown in Figure 1. The correlation between observed and predicted model time point values for individual rats gave coefficients of determination (r2) of greater than 0.95. Compound 1

AmB (1) released from 3

1.93 ( 1.22

a AmB in conjugate (µg/mL) in brackets. b Each value represents the mean ( standard deviation (n ) 3).

Figure 1. Mean plasma concentration-time profile for PEGAmB conjugate 3 administered intravenously at 45.77 mg/kg, equaling 2 mg/kg AmB equivalents, to Sprague-Dawley rats (n ) 3). Inset indicates plasma concentration-time curve of released compound 1 (AmB) from compound 3.

showed a Cmax of 0.26 ( 0.02 µg/mL with an elimination t1/2 of 1.88 ( 1.73 h and an AUC of 0.68 ( 0.62 µg/mL‚h. In contrast, 3 showed a Cmax of 273 ( 43 µg/mL AmB equivalents, a 1000-fold increase from that observed for 1 (AmB). The elimination t1/2 of 3 in the plasma was estimated to be 0.41 ( 0.03 h with an area under the curve (AUC) of 3.71 ( 0.82 mg/mL‚h or 162.3 ( 35.7 µg/ mL‚h AmB equivalents, an almost 200-fold increase compared to 1. The inset within Figure 1 shows the detected amount of 1 released in vivo from 3. The correlation between observed and predicted model time point values gave a r2 of 0.97. The estimated pharmacokinetic parameters for AmB released from 3 were similar to those observed for 1. The Cmax of released AmB was 0.31 ( 0.03 µg/mL with an estimated elimination t1/2 of 1.93 ( 1.2 h and an AUC of 0.83 ( 0.43 µg/mL‚h. In Vivo Safety. The maximum tolerated single dose (MTD) of all the PEG-AmB conjugates was estimated by body weight loss in female ICR mice (Figure 2). After a single dose of the PEG-AmBs, animals tend to show toxic effects within 24 h with severe morbidity occurring within 3 days. Free AmB in a deoxycholate micellar

Polyethylene Glycol Conjugation of Amphotericin B

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survival rate, was observed for doses at 1 mg/kg and greater for PEG-AmB. DISCUSSION

Figure 2. Acute toxicity of formulations. The maximum tolerated single dose (MTD) of selected PEG-conjugated AmB derivatives was estimated by body weight loss in female ICR mice. The MTD was determined following administration ascending iv doses of PEG-AmB to mice (n ) 4-5) at 1 to 2 mg/kg increments. Body weights were measured thrice weekly for two weeks. The highest dose to cause a loss of less than 20% of initial weight within the time period was considered the MTD.

Figure 3. In vivo efficacy of PEG-AmB conjugate 3. Survival of mice infected with C. albicans was determined for control mice and mice treated with either AmB (Fungizone) or doses of PEG-AmB compound 3 up to their MTD. Mortality was recorded once daily of 10 days. Untreated control (9); AmB 1 mg/kg (b); Compound 3: 6 mg/kg (0), 4 mg/kg (4), 2 mg/kg (]), 1 mg/kg (O), 0.5 mg/kg (3).

suspension had a MTD of 2 mg/kg, which is in line with the toxicity seen by others (4, 20). Within the PEG-AmB series, both 11 and 9 showed the lowest MTD at 4 mg/ kg followed closely by 7, 5, and 3 which were within the 5 to 6 mg/kg range (based on AmB content). Compound 13 appeared to be the safest conjugate with a MTD of 12 mg/kg. In Vivo Efficacy. The in vivo therapeutic efficacy of PEG-AmB conjugate 3, due to its comparatively elevated MTD and longer rat plasma half-life, and AmB [1] was studied with mice infected with Candida albicans. A dose/response with 3 is shown in Figure 3. Conjugate 3 when administered iv at its MTD (6 mg/kg) or at either 1/3 or 2/3 its MTD resulted in 100% and 90% survival, respectively. A dose of 1 mg/kg showed 80% survival, whereas, 0.5 mg/kg showed only 30% survival. Negative and positive controls showed 10% of the vehicle control mice survived as compared to 70% of mice treated with 1 mg/kg AmB. Noteworthy, was the observation that the deaths in the 1/3 to 2/3 MTD PEG-AmB treated mice occurred within the first 2 to 4 days, while AmB treated mice died more toward the middle of the 10-day monitoring period. Effective protection, as defined by a g50%

PEG conjugated to amino prodrugs that function via a 1,4-benzyl or 1,6-benzyl elimination has been demonstrated to be a feasible methodology to deliver anticancer drugs (15). Now this technology also has been proven to be useful for the delivery of antifungal agents as exemplified by AmB. In this PEG-linker-drug tripartate system, alteration of PEG conjugate pharmacokinetics can be easily achieved by changing the PEG-linker, thereby, altering the linker-drug cleavability and the introduction of steric hindrance (13). Modification of these parameters, therefore, can promote greater drug efficacy. The synthetic program conducted for this initial investigation of PEG-AmB prodrugs provided a series of disubstituted PEG-AmB, most of which demonstrated stability (>24 h) in pH 7.4 phosphate buffer at 25 °C, thus, physically the conjugates have potential clinical utility as injectable agents. These PEG conjugates, which carried two AmB molecules, were highly soluble (>46 mg/ mL in saline) and were engineered to vary in their rate of in vitro hydrolysis (dissociation of PEG conjugate in plasma) from one to 3 h. As a major finding, this investigation demonstrated that conjugation of PEG to AmB could produce prodrugs that were significantly (6×) less toxic than AmB-deoxycholate but maintained or even enhanced their in vivo antifungal effectiveness. Concomitantly, the prodrug characteristic of these PEG conjugates was likewise exemplified by the lack of antifungal activity with S. cerevisiae and A. niger and the minimal protuberance of erythrocyte membranes in vitro, suggesting that hydrolysis of AmB from the PEG may be required. In general, many small molecule drugs have poor solubility, including antifungal agents, like AmB, Nystatin, etc. By designing PEG conjugates of these insoluble molecules their water solubility can be greatly increased. In this study the water solubility of AmB increased from less than 0.01 mg/mL for the native AmB to 1.4 to 2.5 mg/mL as the PEG conjugate, an increase of approximately 150 to 250 times. The enhanced solubility of the PEG-AmB conjugate form makes it possible to use less of the drug to achieve the same level of efficacy, thereby reducing the side effects of the drug. As shown in this study, PEG-AmB conjugates were significantly less toxic than AmB-deoxycholate yet maintained their in vivo antifungal effectiveness. This study also demonstrates that the rate of hydrolysis can be decreased by not only changing the PEG hydrolytic linkage but also by introducing steric hindrance through the use of ortho substituents on the benzyl component of the prodrug. As expected, PEGylation of AmB increased in vivo plasma AmB concentration and the released AmB had an elimination t1/2 that was similar AmB. Future attempts to further increase plasma t1/2 of these conjugates by exploration of chemistries with different linker moieties are being considered. ACKNOWLEDGMENT

The authors wish to thank the following team members for their quality technical assistance on this project: Jenny Hsu, Ping Hu, Virna Browoski, and Mary Mehlig of the pharmacology and toxicology team, Prasanna Reddy, Anthony Martinez, and Shuiyun Guan of the medicinal and organic chemistry team, Michelle Boro and Dr. Chyi Lee of the analytical chemistry team.

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