ARTICLE pubs.acs.org/bc
PEGylated TNF-Related Apoptosis-Inducing Ligand (TRAIL) Analogues: Pharmacokinetics and Antitumor Effects Tae Hyung Kim,† Yu Seok Youn,† Hai Hua Jiang,† Seulki Lee,§ Xiaoyuan Chen,§ and Kang Choon Lee*,† †
College of Pharmacy, SungKyunKwan University, 300 Chonchon-dong, Jangan-ku, Suwon City 440-746, Korea Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH), Bethesda, Maryland 20892, United States
§
ABSTRACT: The low stability and fast clearance of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) are the main obstacles to its implementation as an antitumor agent. Here, we attempted to improve its pharmacokinetic and pharmacodynamic profiles by using PEGylation. N-terminal PEGylated TRAIL (PEG TRAIL) was synthesized using 2, 5, 10, 20, and 30 kDa PEG. Antitumor effect assessments in HCT116 tumor bearing nude mice showed that all PEG TRAIL analogues efficiently suppressed mean tumor growth, with mean tumor growth inhibition (TGI) values (5K-, 20K-, 30K-PEG TRAIL) of 43.5, 61.7, and 72.3%, respectively. In particular, 30K-PEG TRAIL was found to have antitumor efficacy for five days after a single administration (1 mg/mouse, ip). The different antitumor effects of these PEG TRAIL analogues were attributed to augmented pharmacokinetics and metabolic resistance. All analogues were found to have higher metabolic stabilities in rat plasma, extended pharmacokinetic profiles, and greater circulating half-lives (3.9, 5.3, 6.2, 12.3, and 17.7 h for 2, 5, 10, 20, and 30K-PEG TRAIL, respectively, versus 1.1 h for TRAIL, ip) in ICR mice. Our findings suggest that TRAIL derivatized with PEG of an appropriate Mw might be useful antitumor agent with protracted activity.
’ INTRODUCTION Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily, members of which selectively induce apoptosis in tumor cells but not in most normal cells. This novel selectivity and cytotoxicity are due to the ability of trimeric TRAIL to bind to the death receptors DR4 and DR5, which are overexpressed on tumor cells. Furthermore, only the complex formed between DR4, DR5, and trimeric TRAIL has apoptotic activity. During apoptosis, the activation of caspases leads to the cleavage of vital cellular substrates and subsequent cell death.1 5 However, although TRAIL has a potent apoptotic effect on tumor cells, its rapid inactivation, poor stability and solubility, rapid renal clearance, and severe hepatotoxicity6 8 after systemic delivery represent critical obstacles to its clinical application. PEGylation, a type of covalent modification with polyethylene glycol (PEG), is viewed as a practical way of improving the therapeutic values of peptide and protein drugs,3,9,10 and it has been well documented that this technique increases metabolic resistance and reduces renal clearance and tissue affinity.11,12 Furthermore, the pharmacokinetic profiles of PEGylated peptides and proteins can be controlled by simply changing the Mw of the PEG and thus increasing PEG Mw enhances the above properties.11,12 Nevertheless, increases in PEG Mw can reduce the biological activities of PEGylated proteins due to the steric effect of the pendant PEG. r 2011 American Chemical Society
PEGASYS (a 40 kDa branched PEG-conjugated interferon (IFN) R-2a, Roche), has only 5 10% of the in vitro bioactivity of wild type IFN-R-2a, and yet it was significantly more effective in vivo activity in human than the standard interferon so its pharmacokinetic potency is superior.13,14 Accordingly, it is important that PEG size be optimized to achieve an appropriate balance between in vitro biological activity and in vivo pharmacokinetics. We previously reported on an N-terminal specific PEGylated (PEG Mw: 5 kDa) TRAIL.3 This 5K-PEG TRAIL displayed preserved biological activity and profoundly improved physicochemical stability. Moreover, 5K-PEG TRAIL showed a much longer pharmacokinetic profile and enhanced antitumor activity than native TRAIL, with fewer side effects (hepatotoxicity). However, despite these encouraging results, the antitumor activity of 5K-PEG TRAIL was achieved by daily administration. To reduce the administration frequency and thus increase convenience to patients, we decided to develop longer lasting PEG TRAIL analogues. Accordingly, in the present study, we investigated the antitumor activities of PEG TRAIL analogues substituted with different Mw PEG (2, 5, 10, 20, 30 kDa). In addition, we performed experiments to determine the
Received: April 13, 2011 Revised: July 6, 2011 Published: July 14, 2011 1631
dx.doi.org/10.1021/bc200187k | Bioconjugate Chem. 2011, 22, 1631–1637
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physicochemical stabilities, biological activities, and pharmacokinetic behaviors of these analogues.
’ EXPERIMENTAL PROCEDURES Materials. PEG TRAIL analogues were prepared as previously described.3 Briefly, trimeric TRAIL was constructed by incorporating trimer-forming zipper sequences and then PEGylated in an N-terminal specific manner. Monomethoxy PEG aldehyde (mPEG ALD, Mw 2, 5, 10, 20, 30 kDa) was purchased from Nippon Oil and Fats (NOF, Tokyo). All other chemicals were of analytical grade and were used as obtained. Cell lines were obtained from the Korean Cell Line Bank (Seoul). Experimental Animals. BALB/c athymic nude mice (male, 4 5 weeks old) and ICR mice (male, 4 5 weeks old, weighing 16 18 g) were purchased from the Hanlim Experimental Animal Laboratory (Seoul). Animals were cared for in accordance with the guidelines issued by the National Institutes of Health (NIH) for the care and use of laboratory animals (NIH publication 80 23, revised in 1996). Animals were housed in groups of 6 8 under a 12 h light/dark cycle (lights on 6 a.m.), allowed food and water ad libitum, and acclimatized for 2 weeks. This study was approved by the Ethical Committee on Animal Experimentation of SungKyunKwan University. Preparation and Characterization of PEG TRAIL Analogues. PEG TRAIL analogues were prepared using 2, 5, 10, 20, and 30 kDa mPEG ALD in the presence of 20 mM sodium cyanoborohydride (NaCNBH3) in 50 mM sodium acetate buffer at pH 5, as described previously.3 In brief, PEG/TRAIL molar ratios and reaction times were optimized by size exclusion chromatography monitoring. PEG TRAIL analogues were recovered by gel-filtration chromatography, concentrated by ultrafiltration, and stored at 20 °C until required. Analogues were identified by SDS-PAGE. Coomassie blue was used to visualize the PEG TRAIL analogues. In Vitro Biological Activities of PEG TRAIL Analogues. The in vitro cytotoxicities of PEG TRAIL analogues were determined using MTT assays, as previously described.3 In brief, in vitro cytotoxicities were investigated using human colon tumor cells (HCT116). The cells were maintained in DMEM supplemented with 10% FBS containing 1% penicillin/streptomycin. For dose-dependent cytotoxicity assays, cells were seeded in 96-well plates at 1 104 cells/well and preincubated for 24 h. Media were then replaced with fresh serum-free DMEM, and predetermined amounts of TRAIL or PEG TRAIL analogues were added to final concentrations of 0 1000 ng/mL. The in vitro cytotoxicities of TRAIL and of the PEG TRAIL analogues were determined by performing MTT assays after incubation for 24 h. To determine apoptotic activities, apoptosis and necrosis were determined using Annexin-V-FLUOS staining kits (Roche, Applied Science, Mannheim, Germany). The apoptotic activities of free TRAIL and PEG TRAIL analogues (each IC50 concentration treated) were compared using a FACS Calibur unit (BD Bioscience Mountain View, CA). In Vitro Stabilities of PEG TRAIL Analogues. In vitro stabilities in PBS were investigated by measuring solubility changes in isotonic buffer solution, as previously described.3 In brief, solutions of TRAIL or of the PEG TRAIL analogues (400 μg/mL in PBS pH 7.4) were incubated at 37 °C for 0, 10, or 30 min and 1, 2, 3, 6, 12, 24, 48, or 72 h. Aliquots (100 μL) were
Figure 1. Characterization of PEG TRAIL analogues. SDS-PAGE patterns of PEG TRAIL analogues obtained by N-terminal specific PEGylation.
then centrifuged at 10000 rpm for 20 min to remove denatured protein aggregates. Protein concentrations in supernatants were determined using BCA protein assays using BSA as a standard. The biological stabilities of TRAIL and of the analogues were also investigated by measuring their cytotoxic effects on HCT116 colon tumor cells. Briefly, after preparing TRAIL or PEG TRAIL analogue solutions (10 μg/mL) in 50% rat plasma, samples were incubated at 37 °C for 0, 15, and 30 min, and for 1, 3, 6, 12, and 24 h. Aliquots (100 μL) were the centrifuged at 10000 rpm for 20 min to remove debris, and HCT116 cells in 96well cell culture plates were treated with supernatants for 24 h. Cell viabilities were determined using MTT assays. Radioiodine (125I) Labeling of PEG TRAIL Analogues. Radioiodinated TRAIL and PEG TRAIL analogues were prepared using a modification of the IODO-GEN method previously described.4,15,16 Briefly, 100 μL of an IODO-GEN (Pierce, Rockford, IL) solution in methylene chloride (1.0 mg/mL) was dispensed into a fresh tube, and evaporated under a nitrogen stream. Aliquots (100 μL) of TRAIL or PEG TRAIL analogues (0.5 mg/mL) and 50 μCi of Na125I (Perkin-Elmer, Boston, MA) diluted to 100 mM with PBS (pH 7.4) were then added. The reaction was allowed to proceed for 2 min, and supernatants were then loaded into a Superose 12 HR 10/30 column connected to a flow-through radioisotope detector (Ramona 200, Raytest, Straubenhardt, Germany). 125I-labeled protein fractions were collected and stored at 4 °C until required. In Vivo Pharmacokinetic Disposition Study. Pharmacokinetic dispositions were examined using a modification of a previously described method.4,15,16 Radioiodinated TRAIL or PEG TRAIL analogues were administered to ICR mice (n = 4) by ip injection (approximately 10 μg, 13000 cpm), and mice were then sacrificed at predetermined times. Blood samples were obtained by heart puncture, and livers and kidneys were quickly excised and rinsed three times with saline solution. Radioactivities of blood (100 μL) and whole tissue organs were measured by γ-counting (Cobra, Packard Instruments Co., Groningen, The Netherlands), and the results obtained were used to calculate percentage radioactivities in blood and organs versus total dose administered. In the case of blood, total blood volume 1632
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Figure 3. In vitro physicochemical characterizations of PEG TRAIL analogues. (A) Time-dependent solubility changes of TRAIL and PEG TRAIL analogues (n = 3) in PBS (pH 7.4) at 37 °C. (B) timedependent activity changes of TRAIL and PEG TRAIL analogues (n = 3) in 50% rat plasma (1:1 mixture of plasma and PBS, 37 °C for 24 h, activity test was conducted using HCT116 cells using MTT). Figure 2. In vitro biological activity of PEG TRAIL analogues. (A) Cytotoxicity of TRAIL and PEG TRAIL analogues on HCT116. (B) Apoptotic activities of TRAIL and PEG TRAIL analogues (each IC50 concentration treated) were compared using a FACS Calibur unit.
to body weight for ICR mice was presumed to be 70 mL/kg, and this figure was used to calculate percentages of radioiodinated TRAIL and radioiodinated PEG TRAIL analogues in whole blood. All pharmacokinetic parameters were calculated by twocompartmental analysis using WinNolin version 1.1 (Scientific Consulting, Inc., Cary, NC). In particular, the AUCinf value was obtained by the computational calculation of the area under the curve from zero to infinite time based on the trapezoidal rule, and the circulating half-life (t1/2) was obtained by the same method about the elimination phase after ip injections.
In Vivo Antitumor Activity Study. The antitumor effects of PEG TRAIL analogues were investigated in HCT116 tumor bearing mice (n = 6). Briefly, freshly harvested HCT116 cells (3 106 cells/mouse) were inoculated sc into BALB/c athymic mice, and 5 days later, mice were treated with TRAIL (1 mg/ mouse, ip) or the PEG TRAIL analogues (1 mg/mouse, ip) every 5 days. Tumor volumes were monitored for 24 days after tumor cell administration. Tumor volumes were calculated using longitudinal (L) and transverse (W) diameters using V = (L 3 W2)/2, and tumor growth inhibition (TGI) percent values were calculated using the formula TGI % = (1 TVsample/ TVcontrol) 100, where TV is tumor volume. In vivo tumor cell apoptosis were also investigated in HCT116 tumor-bearing BALB/c athymic mice. Briefly, at 24 days after HCT116 administration, tumor tissues were recovered from 1633
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Figure 4. Pharmacokinetic profiles of TRAIL (A) and PEG TRAIL analogues ((B) PEG 2 kDa, (C) 5 kDa, (D) 10 kDa, (E) 20 kDa, and (F) 30 kDa) in blood and tissue organs (liver and kidney) of ICR mice (n = 4). Results were then used to calculate percentages versus injected doses in blood and tissue organs (liver and kidney). 1634
dx.doi.org/10.1021/bc200187k |Bioconjugate Chem. 2011, 22, 1631–1637
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Table 1. Pharmacokinetic Parameters of TRAIL and PEG TRAIL Analogues in ICR Micea PK parameters
TRAIL
2K-PEG TRAIL
5K-PEG TRAIL
10K-PEG TRAIL
20K-PEG TRAIL
30K-PEG TRAIL
AUCinf (% 3 h)
28.4 ( 2.8
66.3 ( 1.7
273.7 ( 5.4
338.5 ( 23.0
612.7 ( 49.2
957.2 ( 67.8
Cmax (%)
5.8 ( 0.1
9.2 ( 1.2
14.7 ( 0.2
17.3 ( 1.4
18.8 ( 0.8
22.7 ( 1.2
t1/2 (h)
1.1 ( 0.0
3.9 ( 0.7
5.3 ( 0.3
6.2 ( 0.2
12.3 ( 2.2
17.7 ( 2.6
Data are means ( SDs. AUCinf = area under the curve from zero to infinity; Cmax = maximum concentration expressed as a percentage of injected dose (%); t1/2: half-life during the elimination phase. a
euthanized animals. Sections (5 μm) were then cut from 10% neutral buffered, formalin-fixed, paraffin-embedded tissue blocks. Apoptotic cell death in tumor tissues was visualized by performing TdT-mediated dUTP nick end labeling (TUNEL) assays using a commercial kit (In Situ Cell Death Detection Kit, Roche, Applied Science, Mannheim, Germany). Data Analysis. Data are presented as mean ( SDs. Statistical significances were determined using One-Way ANOVA, and P values of
