Bioconjugate Chem. 2010, 21, 1513–1519
1513
Synthesis and Evaluation of Human Serum Albumin-Modified Exendin-4 Conjugate via Heterobifunctional Polyethylene Glycol Linkage with Protracted Hypoglycemic Efficacy Insoo Kim,| Tae Hyung Kim,‡ Kyungwan Ma,† Eun Seong Lee,§ Dongin Kim,| Kyung Taek Oh,⊥ Don Haeng Lee,# Kang Choon Lee,‡ and Yu Seok Youn*,† College of Pharmacy, Pusan National University, 30 Jangjun-dong, Geumjeong-gu, Busan 609-735, Korea, College of Pharmacy, Sungkyunkwan University, 300 Chonchon-dong, Jangan-gu, Suwon City 440-746, Korea, Division of Biotechnology, The Catholic University of Korea, 43-1 Yeokgok 2-dong, Wonmi-gu, Bucheon-si, Gyeonggi-do 420-743, Korea, The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, 315 Ferst Drive, Atlanta, Georgia 30322, Chung-Ang University, 221 Heukseok dong, Dongjak-gu, Seoul 155-756, Korea, and Department of Internal Medicine, Inha University, 253 Yonghyun-dong Nam-gu, Incheon, 402-751, Korea. Received March 20, 2010; Revised Manuscript Received July 10, 2010
Albumin conjugation is considered to be one of the most effective means of protracting the short in ViVo lifespans of peptides and proteins. Here, we present a new long-acting antidiabetic exendin-4 conjugate linked with human serum albumin (HSA) via polyethylene glycol (PEG). As a first step toward synthesizing this conjugate, three artificial sulfhydryl groups were introduced in HSA using 2-iminothiolane at pH 8.0. This thiolated HSA was further reacted with the monomer fraction of exendin-4 (6 equiv) conjugated with maleimide-PEG5k-Nhydroxysuccinimide (MAL-PEG5k-NHS) for 3 h. Because of the presence of PEG molecules, the resulting conjugate (HSA-PEG-Ex4) was found to have a greater apparent molecular weight and a larger particle size (ca. 195 kDa and 9.48 ( 0.74 nm) than those of HSA-exendin-4 without the PEG linker (HSA-Ex4, ca. 84.3 kDa and 7.77 ( 0.98 nm). Although the receptor binding affinity of HSA-PEG-Ex4 on RIN-m5F cells was significantly lower than that of Ex4, its antihyperglycemic efficacy was slightly higher than that of Ex-4 and HSA-Ex4 in type 2 diabetic db/db mice. Furthermore, HSA-PEG-Ex4 had greater circulating t1/2 and AUCinf values than HSA-Ex and native exendin-4 by 2.1- and 10.3-fold, respectively. Accordingly, its hypoglycemic duration was greatly increased to 31.0 h at a dose of 250 nmol/kg vs that of native Ex4 (7.0 h). Results show that the HSA-PEG-Ex4 conjugate produced has distinct advantages over HSA-Ex4 without PEG. We believe that this exendin-4 derivative, which has the merits of albumin conjugation and PEGylation, has considerable potential as a novel type 2 antidiabetic agent.
INTRODUCTION The therapeutic efficacies of peptides and proteins are restricted by their rapid clearance from the body (1), which means that they must be frequently administered to maintain therapeutic levels. Protein-based drugs have circulating halflives (t1/2) of at most a few hours, and especially, the t1/2 levels of most peptides range from a few minutes to less than two hours. These short lifespans of therapeutic peptides and proteins are mainly due to renal filtration or proteolysis (2, 3). Exendin4, a potent glucagon-like peptide-1 (GLP-1) agonist, is no exception in this respect, although it has a much longer t1/2 (∼2 h) than GLP-1 (∼2 min) (4, 5), its commercial product, Byetta, must be injected at least twice daily (6), which markedly reduces patient compliance. Thus, a strategy is required to extend the therapeutic duration of exendin-4 and maximize its antidiabetic effects, which include appetite suppression and the enhancements of glucose-dependent insulin release and β-cell proliferation (7-9). * Corresponding author. Tel: +82-51-510-2800. Fax: +82-51-5136754. E-mail:
[email protected]. † Pusan National University. ‡ Sungkyunkwan University. § The Catholic University of Korea. | Georgia Institute of Technology. ⊥ Chung-Ang University. # Inha University.
Albumin is an attractive macromolecular carrier due to its biodegradability, nontoxicity, and nonimmunogenicity. Recently, Human serum albumin (HSA) has been used to alter the pharmacokinetic profiles of a number of drugs (10) because HSA is long-lived in plasma with an average t1/2 of 19 days and seldom undergoes kidney filtration because it is around the same size (∼67 kDa) as glomerulus pores (11). Accordingly, the renal clearances of HSA-modified peptides or proteins are much lower, and their in ViVo lifespans are considerably longer. Thus far, three different approaches have been used for binding HSA. The first involves the covalent conjugation of HSA to drugs. This method uses bifunctional spacers to facilitate direct attachment (2, 12, 13). The second approach involves HSA binding in ViVo with drugs derivatized with long chain fatty acids (14-16). Fatty acid acylated drugs bind HSA molecules because HSA possesses five available fatty acid binding sites (11). The third approach involves the production of a HSA-fused peptide or protein. This method involves the generation of modified peptides or proteins by fusing the HSA gene to a gene that encodes the active peptide or protein (17, 18). In particular, HSA-based antidiabetic drugs have been reported to have remarkably extended therapeutic durations. Some of these, such as Levemir and Victoza (Novo Nordisk), are already commercially available, and some others, such as CJC-1131/ 1134 (ConjuChem) and Albiglutide (Human Genome Sciences), are undergoing clinical trials. In an attempt to produce a long-acting exendin-4 (Ex4) derivative, we devised a new way of conjugating HSA and Ex4.
10.1021/bc100143c 2010 American Chemical Society Published on Web 07/23/2010
1514 Bioconjugate Chem., Vol. 21, No. 8, 2010
Kim et al.
Scheme 1. Syntheses of HSA-Ex4 and HSA-PEG-Ex4
Unlike previously described HSA-peptide conjugates, which involve direct short linking spacers, we linked Ex4 to HSA using a 5 kDa bifunctional PEG with the aim of producing a conjugate with the pharmaceutical merits associated with PEGylation and albumin modification. After establishing the conjugation process, we investigated the receptor-binding and pharmacokinetic characteristics of the HSA-PEG-Ex4 conjugate produced. In addition, its hypoglycemic duration was evaluated in type 2 diabetic db/db mice and compared with that of HSA-conjugated exendin-4 without a PEG linker.
EXPERIMENTAL PROCEDURES Materials. Exendin-4 (Ex4) and maleimide-polyethylene glycol-N-hydroxysuccinimide (MAL-PEG-NHS: Mw 5,000) were purchased from the American Peptide Company (Sunnyvale, CA) and NOF Corporation (Tokyo, Japan), respectively. Sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate) and HSA were purchased from Pierce (Rockford, IL) and Sigma-Aldrich (St. Louis, MO), respectively. All other reagents, unless otherwise specified, were obtained from Sigma-Aldrich. Experimental Animals. Type 2 diabetic C57BL/6 db/db mice (male, 4-5 weeks old) were purchased from Korean Research Institute of Bioscience and Biotechnology (Daejon, Republic of Korea). ICR mice (males, 4-5 weeks old), weighing 16-18 g, were purchased from Hanlim Experimental Animal Laboratory (Seoul). Animals were cared for in accordance with the National Institutes of Health (NIH) Guidelines 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 at 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 at Pusan National University. Synthesis of HSA-Conjugated Ex4s. HSA-conjugated Ex4 conjugates were prepared by using a modification of a previously
described procedure (19). First, the primary amines of HSA were thiolated. Briefly, a 100 µL aliquot of 2-iminothiolane (Traut’s reagent: 0.06 mg, 0.45 µmol, approximately 3 equiv) was mixed with 2 mL of HSA (10 mg, 0.15 µmol) in 50 mM of sodium carbonate (pH 8.0) and allowed to react for 3 h. The reaction mixture was then dialyzed with 10 mM PBS (pH 7.4) using a dialysis kit (Mw cutoff ) 8,000, Gene Bio-Application Ltd., Israel) and stored at a concentration of 0.5 mg/mL at 4 °C until needed. Second, 5 mg of MAL-PEG-NHS (Mw: 5 kDa, 1 µmol) was mixed with 6.3 mg of Ex4 (1.5 µmol) in 0.5 mL of 0.1% dimethylaminopyridine (DMAP)/dimethylsulfoxide (DMSO) at room temperature for 1 h. The buffer media in this mixture was then changed to 10 mM PBS (pH 7.0) by desalting using Sephadex G-25 and concentrated. The final mixture was then applied into a Superdex 200 HR 10/30 column (300 × 10 mm, GE Healthcare Life Sciences, Piscataway, NJ) and eluted with PBS at a flow rate of 0.6 mL/min, and the MAL-PEG-Ex4 monomer fraction was collected. An aliquot (4 mL) of the MAL-PEG-Ex4 monomer (500 µg/mL, 0.48 µmol, exendin-4 equiv) was then quickly transferred to 2 mL of thiolated HSA (2.7 mg/mL, 0.08 µmol) in PBS, and the reaction was allowed to continue at room temperature for 3 h. After an additional chromatography step, the first symmetric fraction of the final HSA-PEG-Ex4 conjugate was collected and stored at 4 °C until needed. Separately, a HSA-Ex4 conjugate (HSA-Ex4) was prepared using the same procedure, but sulfo-SMCC was used instead of MAL-PEG-NHS (Scheme 1). The Ex4 amount in the final conjugates was calculated reversely by determining the unreacted intermediate Ex4 species (sulfo-SMCC-Ex4 or MAL-PEG-Ex4) in the chromatography process, and their concentrations in storage conditions were determined again by using commercial exendin-4 enzyme immunoassay kits (Phoenix Pharmaceuticals, Burlingame, CA). Characterization of HSA-Conjugated Ex4s. HSA-conjugated Ex4s were characterized by SDS-PAGE, as previously reported (20). Briefly, SDS-PAGE was performed in the
Human Serum Albumin-Modified Exendin-4 Conjugate
Bioconjugate Chem., Vol. 21, No. 8, 2010 1515
Table 1. Pharmacokinetic Parameters and Pharmacological Scores of Ex4, HSA-Ex4, and HSA-PEG-Ex4 in ICR and db/db Micea PK parameters
Ex4
HSA-Ex4
AUCinf (ng/mL · h) 123.5 ( 31.5 733.3 ( 33.8 Cmax (ng/mL) Tmax (h) Cl/F (mL/h) t1/2 (h, β)
42.5 ( 8.0 1.0 ( 0.0 8.8 ( 2.7 2.1 ( 0.5
40.2 ( 1.9 1.3 ( 0.5 1.4 ( 0.1 11.4 ( 2.4
HSA-PEG-Ex4 1418.7 ( 138.1 40.7 ( 1.7 1.3 ( 0.5 0.7 ( 0.1 24.2 ( 2.5
pharmacological scores
Ex4
HSA-Ex4
HDtotal (25 nmol/kg)/OGTT (mmol · h/L) 14.2 ( 6.2 19.9 ( 5.3 114.9 ( 33.7 187.9 ( 56.2 HDtotal (25 nmol/kg) (mmol · h/L) HDtotal (250 nmol/kg) (mmol · h/L) 235.0 ( 42.7 374.6 ( 103.4 HDtotal (500 nmol/kg) (mmol · h/L) hypoglycemic duration (h) (250 nmol/kg) 7.0 15.2 hypoglycemic duration (h) (500 nmol/kg)
HSA-PEG-Ex4 20.4 ( 0.9 419.2 ( 117.2 660.3 ( 145.7 1217.9 ( 191.2 31.0 61.4
a Data are means ( SDs. AUCinf, area under the curve from zero to infinity; Cmax, maximum concentration after intraperitoneal administration; Tmax, time required to reach Cmax; Cl/F, systemic clearance from zero time to last time; t1/2, half-life in the elimination phase (β).
presence of 0.1% SDS using a 12% slab gel, and electrophoresis was performed using a constant current of 25 mA for 1 h. The gel was soaked in a Coomassie brilliant blue R250 staining solution and then immersed in a destaining solution. Separately, HSA-conjugated Ex4s were subjected to size-exclusion chromatography (SEC) on a Superdex 200 HR 10/30 column and eluted with 10 mM PBS (pH 7.4) at a flow rate of 0.6 mL/min. Chromatograms were recorded at 215 nm. The apparent molecular weights of HSA-conjugated Ex4s were estimated using a calibration curve based on the retention times vs logarithmic molecular weights of globular protein standards. The particle sizes of HSA-conjugated Ex4s in wet conditions were measured by using a Zetasizer Nano-S90 based on dynamic light scattering (DLS) (Malvern Instruments, USA) with a He-Ne Laser beam at a wavelength of 633 nm and a fixed scattering angle of 90°. Receptor-Binding Affinities of HSA-Conjugated Ex4s to RIN-m5F Cells. The receptor-binding affinities of Ex4 and HSA-conjugated Ex4s were determined using a modification of a previously described method (21, 22). Briefly, RIN-m5F cells (a rat insulinoma cell line, ATCC, Manassas, VA), which express high levels of GLP-1 receptors (GLP-1R), were seeded in 12 well plates at 3 × 105 cells per well, grown for 48 h, washed twice with binding buffer (120 mM NaCl, 1.2 mM MgSO4, 13 mM sodium acetate, 5 mM KCl, 1.2 g/L Tris, 2 g/L bovine serum albumin, and 1.8 g/L glucose, pH 7.6), and treated with unlabeled Ex4 derivatives (final concentration range: 0.001-1000 nM) and 30 pM 125I-exendin-4 (9-39, PerkinElmer, Boston, MA) for 2 h at room temperature. The cells were then washed three times with chilled PBS containing 1 mg/mL of bovine serum albumin and lysed with cell lysis buffer (0.5 N NaOH with 1% SDS) for 15 min, and the radioactive levels in lysates were measured using a γ counter (GMI, Inc., Ramsey, MN). Radioiodination of HSA-Conjugated Ex4s. Radioiodinated samples were prepared using a modification of the IODO-GEN method, as previously described (20, 23). For native exendin4, a water-soluble Bolton-Hunter reagent (N-sulfosuccinimidyl3-4-hydroxyphenyl propionate) was utilized to introduce a phenolic moiety to exendin-4 because it does not have tyrosine that is highly reactive with the electrophilic substitution of oxidized iodine. Briefly, a 100 µL aliquot of Bolton-Hunter reagent (0.13 mg/mL, 36 nmol) in 10 mM PBS (pH 7.0) was mixed with 150 µL of exendin-4 (0.5 mg/mL, 18 nmol) at room temperature for 90 min and briefly dialyzed to remove the reagent. Separately, 100 µL of an IODO-GEN (Pierce, Rockford, IL) solution in methylene chloride (0.5 mg/mL) was dispensed into fresh tubes and evaporated under a nitrogen stream. To these tubes were added 50 µL aliquots of either water-soluble Bolton-Hunter reagent-labeled Ex4 or HSA-conjugated Ex4s (approximately 50 µg/mL) and 50 µCi of Na125I (Perkin-Elmer, Boston, MA) diluted with 10 mM PBS (pH 7.4). The reaction was allowed to proceed for 5 min, and then supernatants were loaded into a Sephadex G-25 desalting column (GE Healthcare Life Sciences, Piscataway, NJ) previously equilibrated with 10 mM PBS. 125I-labeled fractions were collected and stored in 10 mM PBS (pH 7.4). Radioactivities were measured by gamma
counting (CobraTM Series Auto-Gamma Counting System, Packard Instruments Co., Groningen, The Netherlands). The specific radioactivities of the radioiodinated samples fell in the range 4.8-5.9 × 105 cpm/µg. Pharmacokinetics of HSA-Conjugated Ex4s in ICR Mice. The pharmacokinetics of Ex4 and HSA-conjugated Ex4s were evaluated using a modification of a previously described method (24-27). Radioiodinated samples were administered to ICR mice (∼2 µg, ∼25 nmol/kg body wt, i.p.). Mice were sacrificed by heart puncture for blood collection at predetermined times, and radioactivities corresponding to 0.2 mL blood samples were determined by gamma counting. These results were then used to calculate the concentration in blood. Pharmacokinetic parameters were calculated by two-compartmental analysis using WinNolin, version 1.1 (Scientific Consulting, Inc., Cary, NC). In particular, AUCinf values were obtained by calculating areas under the curves from zero to infinity time using the trapezoidal rule, and circulating half-lives (t1/2) were obtained using the same method (Table 1). Antihyperglycemic Efficacies of HSA-Conjugated Ex4s in Fasted Type 2 Diabetic db/db Mice. Oral glucose tolerance testing (OGTT) was performed to evaluate the antihyperglycemic efficacies of HSA-conjugated Ex4s after an overnight fast (18 h), as previously described (24-26, 28). One hour prior to the first oral glucose challenge (0.2 mL, 1.0 g/kg body wt) (0 h), male db/db mice (n ) 4/group, 8-9 weeks old) were administered saline, Ex4, HSA-Ex4, or HSA-PEG-Ex4 (i.p., 25 nmol/kg body wt). A drop of blood was drawn from the tail vein of each animal at 0, 0.25, 0.5, 0.75, 1, 1.5, 2, and 3 h, and blood glucose levels (BGL) were determined using a one-touch blood glucose meter (ACCU-CHEK Sensor, Roche Diagnostics Corp., USA). Antihyperglycemic efficacies were evaluated by calculating total hypoglycemic degrees (HDtotal; calculated using AUCsaline,0-3 h - AUCtest,0-3 h). Hypoglycemic Efficacies of HSA-Conjugated Ex4s in Nonfasted db/db Mice. Hypoglycemic efficacies of Ex4 and of HSA-conjugated Ex4s were evaluated using a modification of a previously described method (21, 22). Male db/db mice (6-7 weeks old) were used for hypoglycemia testing. Under nonfasting conditions with free access to food and water, the animals received a single i.p. injection of saline, Ex4, or of HSAconjugated Ex4s (25, 250, 500 nmol/kg body wt). Blood glucose levels (BGL) were monitored using the procedure mentioned above. In addition, a drop of blood was drawn from the tail vein of each animal at different times (0, 0.5, 1, 2, 3, 4, 6, 8, 12, 24, 36, 48, 60, 72, 96, and 120 h), and blood glucose levels (BGL) were determined using a one-touch blood glucose meter (ACCU-CHEK Sensor, Roche Diagnostics, USA). Hypoglycemic efficacies are expressed as HDtotal, as mentioned above, but the last times measured were different in each group as seen in Figure 6. Furthermore, hypoglycemic durations to maintain a BGL of 93% yield. PEG possessing only a MAL or NHS group is very likely to generate impurities of their own dimers for HSA and Ex4. As initially expected, increasing the number of sulfhydryl groups on HSA increased the reaction rate and resulted in a high reaction yield. PEGylation provides a practical means of increasing molecular size, protects metabolic and immunogenic sites and thus prolongs plasma half-life and in ViVo stability, and diminishes immunogenicity. Because of the flexibility of the PEG linker, HSA-PEG-Ex4 was much larger than HSA-Ex4. Furthermore, HSA-PEG-Ex4 has a hydrodynamic molecular size estimated by SEC to be 2.9 and 2.3 times greater than those of native HSA and HSA-Ex4, respectively, and similarly, its actual particle size by DLS was found to be significantly larger than those of HSA-Ex4 and native HSA (>1.7-2.8 nm). Additionally, we designed HSA-PEG-Ex4 to contain three each of Ex4s and connector PEGs because incorporating more than 4-6 Ex4s per HSA resulted in unacceptably large hydrodynamic volume fractions (>approximately 280-340 kDa). The receptor-binding affinities of peptide drugs after albumin conjugation are often less than 10% of those of parent molecules (13, 19), which is largely attributable to the steric hindrance of HSA. Similarly, we found that the receptor-binding affinity of HSA-PEG-Ex4 was only ∼5% of intact Ex4.
Kim et al.
However, its receptor binding was comparable to that of HSA-Ex4, despite the attachment of three long PEG molecules, which is somewhat surprising because PEGylated drugs usually have markedly lower receptor-binding affinities than native drugs, for example, in a previous study, we found that mono-PEG5k-GLP-1 had only 1% of the binding affinity of GLP-1 (24). Bearing in mind the greater size of HSA-PEG-Ex4 as compared with HSA-Ex4, it appears that the flexibility of PEG in HSA-PEG-Ex4 allows Ex4 to better access receptors than Ex4 in HSA-Ex4, in which Ex-4 is directly and thus closely attached to HSA. In contrast to these in Vitro results, our OGTT results showed that the antihyperglycemic efficacy of HSA-PEG-Ex4 is similar to that of Ex4. Therefore, our findings indicate that HSA-PEG-Ex4 is active enough to activate GLP-1R in ViVo and that the reduced receptor binding observed in Vitro is probably due to an inability to maintain binding with GLP-1R rather than a failure to bind and activate GLP-1R. Because of its substantially larger molecular size, the pharmacokinetic profile of HSA-PEG-Ex4 after i.p. administration was markedly better than that of Ex4 (its AUC was 10-fold greater, and its circulating t1/2 was 12-fold greater than those of Ex4). Furthermore, the t1/2 of HSA-PEG-Ex4 in the circulation was twice that of HSA-Ex4, probably due to the molecular size increase caused by the attached PEG. It should also be mentioned that HSA and exendin-4 are obvious foreign materials to mice because they have human and reptile origins, respectively. However, PEGylation diminishes immunogenicity, and thus, we suppose the incorporation of PEG protects the HSA-PEG-Ex4 from mice antibodies. The hypoglycemic duration of HSA-PEG-Ex4 in db/db mice was also greatly prolonged vs HSA-Ex4 or native Ex4, regardless of the doses administered. In the present study, a blood glucose level below 8.35 mmol/L (150 mg/dL) was regarded as normal, and the euglycemic duration under that value is considered a practical indication of the potential for antidiabetic treatment. The administration of HSA-PEG-Ex4 sustained normal glycemia for 2 and 4 times longer than native Ex4 or HSA-Ex4, respectively. In addition, drug clearance in humans is known to be much slower than that in rodents, and thus, the antidiabetic duration of HSA-PEG-Ex4 may be substantially greater in humans than the ∼60 h in db/db mice in the present study. In summary, we describe a new albumin-conjugated exendin-4 derivative, in which these two moieties are connected by a heterobifunctional PEG. Both the albumin and PEG components of HSA-PEG-Ex4 were found to confer therapeutic benefits. The present study also shows that that HSA-PEG-Ex4 has greater molecular size, acceptable GLP-1 receptor-binding affinity, better pharmacokinetics, and a markedly greater hypoglycemic duration than Ex4, specifically due to the incorporation of PEG. We believe that HSA-PEG-Ex4 should be viewed as a potential long-acting treatment for type 2 diabetes.
ACKNOWLEDGMENT This work was supported by the National Research Foundation of Korea (NRF), by the Korean government (MEST) (no. 2009-0087518), and by a grant from the Ministry of Education, Science and Technology in Korea (2009K001605).
LITERATURE CITED (1) Caliceti, P., and Veronese, F. M. (2003) Pharmacokinetic and biodistribution properties of poly(ethylene glycol)-protein conjugates. AdV. Drug DeliVery ReV. 55, 1261–1277. (2) Yoram, S., Marina, M., Sara, R., Ayala, S., Haim, T., and Mati, F. (2005) Albumin-insulin conjugate releasing insulin slowly under physiological conditions: A new concept for long-acting insulin. Bioconjugate Chem. 16, 913–920.
Human Serum Albumin-Modified Exendin-4 Conjugate (3) Goodman, L. Gilman, A. G. (1995) The Pharmacological Basis of Therapeutics, McGraw-Hill, New York. (4) Deacon, C. F. (2004) Therapeutic strategies based on glucagonlike peptide 1. Diabetes. 53, 2181–2189. (5) David, P., Carolyn, J., Pam, S., Sonali, N., Liz, R., Ron, G., Kim, C., and Andrew, Y. (2001) Pharmacokinetic actions of exendin-4 in the rat: Comparison with glucagon-like peptide-1. Drug DeV. Res. 53, 260–267. (6) Davidson, M. B., Bate, G., and Kirkpatrick, P. (2005) Exenatide. Nat. ReV. Drug DiscoVery 4, 713–714. (7) Xu, G., Stoffers, D. A., Habener, J. F., and Bonner-Weir, S. (1999) Exendin-4 stimulates both beta-cell replication and neogenesis, resulting in increased beta-cell mass and improved glucose tolerance in diabetic rats. Diabetes 48, 2270–2276. (8) Szayna, M., Doyle, M. E., Betkey, J. A., Holloway, H. W., Spencer, R. G., Greig, N. H., and Egan, J. M. (2000) Exendin-4 decelerates food intake, weight gain, and fat deposition in Zucker rats. Endocrinology 141, 1936–1941. (9) Edwards, C. M., Stanley, S. A., Davis, R., Brynes, A. E., Frost, G. S., Seal, L. J., Ghatei, M. A., and Bloom, S. R. (2005) Exendin-4 reduces fasting and postprandial glucose and decreases energy intake in healthy volunteers. Am. J. Physiol. 281, 155– 161. (10) Chuang, V. T., Kragh-Hansen, U., and Otagiri, M. (2002) Pharmaceutical strategies utilizing recombinant human serum albumin. Pharm. Res. 19, 569–577. (11) Kratz, F. (2008) Albumin as a drug carrier: Design of prodrugs, drug conjugates and nanoparticles. J. Controlled Release 132, 171–183. (12) Kim, J.-G., Baggio, L. L., Bridon, D. P., Castaigne, J.-P., Robitaille, M. F., Jette, L., Benquet, C., and Druker, D. J. (2003) Development and characterization of a glucagon-like peptide 1-albumin conjugate: The ability to activate the glucagon-like peptide 1 receptor in vivo. Diabetes 52, 751–759. (13) Thibaudeau, K., Le´ger, R., Huang, X., Robitaille, M., Quraishi, O., Soucy, C., Bousquet-Gagnon, N., van Wyk, P., Paradis, V., Castaigne, J.-P., and Bridon, D. (2005) Synthesis and evaluation of insulin-human serum albumin conjugates. Bioconjugate Chem. 16, 1000–1008. (14) Juhl, C. B., Hollingdal, M., Sturis, J., Jakobsen, G., Agersø, H., Veldhuis, J., Pørksen, N., and Schmitz, O. (2002) Bedtime administration of NN2211, a long-acting GLP-1 derivative, substantially reduces fasting and postprandial glycemia in type 2 diabetes. Diabetes 51, 424–429. (15) Kurtzhals, P., Havelund, S., Jonassen, I., and Markussen, J. (1997) Effect of fatty acids and selected drugs on the albumin binding of a long-acting, acylated insulin analogue. J. Pharm. Sci. 86, 1365–1368. (16) Markussen, J., Havelund, S., Kurtzhals, P., Andersen, A. S., Halstrøm, J., Hasselager, E., Larsen, U. D., Ribel, U., Scha¨ffer, L., Vad, K., and Jonassen, I. (1996) Soluble, fatty acid acylated insulins bind to albumin and show protracted action in pigs. Diabetologia 39, 281–288. (17) Baggio, L. L., Huang, Q., Brown, T. J., and Drucker, D. J. (2004) A recombinant human glucagon-like peptide (GLP)-1albumin protein (albugon) mimics peptidergic activation of GLP-1 receptor-dependent pathways coupled with satiety, gastrointestinal motility, and glucose homeostasis. Diabetes 53, 2492–2500.
Bioconjugate Chem., Vol. 21, No. 8, 2010 1519 (18) Subramanian, G. M., Fiscella, M., Lamouse´-Smith, A., Zeuzem, S., and McHutchison, J. G. (2007) Albinterferon alpha2b: a genetic fusion protein for the treatment of chronic hepatitis C. Nat. Biotechnol. 25, 1411–1419. (19) Temming, K., Meyer, D. L., Zabinski, R., Dijkers, E. C., Poelstra, K., Molema, G., and Kok, R. J. (2006) Evaluation of RGD-targeted albumin carriers for specific delivery of auristatin E to tumor blood vessels. Bioconjugate Chem. 17, 1385–1394. (20) Shin, B. S., Kim, C. H., Lee, M. N., Hur, M. W., Lee, K. C., Youn, Y. S., and Yoo, S. D. (2006) Pharmacokinetics of 125IGST-TatdMt, a recombinant fusion protein possessing potent anti-obesity activity, after intravenous, nasal, oral, and subcutaneous administration. Regul. Pept. 140, 74–80. (21) Chae, S. Y., Jin, C. H., Shin, J. H., Son, S., Kim, T. H., Lee, S., Youn, Y. S., Byun, Y., Lee, M. S., and Lee, K. C. (2010) Biochemical, pharmaceutical and therapeutic properties of longacting lithocholic acid derivatized exendin-4 analogs. J. Controlled Release 142, 206–213. (22) Jin, C. H., Chae, S. Y., Son, S., Kim, T. H., Um, K. A., Youn, Y. S., Lee, S., and Lee, K. C. (2009) A new orally available glucagon-like peptide-1 receptor agonist, biotinylated exendin4, displays improved hypoglycemic effects in db/db mice. J. Controlled Release 133, 172–177. (23) Youn, Y. S., Na, D. H., Yoo, S. D., Song, S. C., and Lee, K. C. (2005) Carbohydrate-specifically polyethylene glycolmodified ricin A-chain with improved therapeutic potential. Int. J. Biochem. Cell Biol. 37, 1525–1533. (24) Chae, S. Y., Chun, Y. G., Lee, S., Jin, C. H., Lee, E. S., Lee, K. C., and Youn, Y. S. (2009) Pharmacokinetic and pharmacodynamic evaluation of site-specific PEGylated glucagon-like peptide-1 analogs as flexible postprandial-glucose controllers. J. Pharm. Sci. 98, 1556–1567. (25) Lee, S. H., Lee, S., Youn, Y. S., Na, D. H., Chae, S. Y., Byun, Y., and Lee, K. C. (2005) Synthesis, characterization, and pharmacokinetic studies of PEGylated glucagon-like peptide-1. Bioconjugate Chem. 6, 377–382. (26) Lee, K. C., Chae, S. Y., Kim, T. H., Lee, S., Lee, E. S., and Youn, Y. S. (2009) Intrapulmonary potential of polyethylene glycol-modified glucagon-like peptide-1s as a type 2 anti-diabetic agent. Regul. Pept. 152, 101–107. (27) Youn, Y. S., Jung, J. Y., Oh, S. H., Yoo, S. D., and Lee, K. C. (2006) Improved intestinal delivery of salmon calcitonin by Lys18-amine specific PEGylation: stability, permeability, pharmacokinetic behavior and in vivo hypocalcemic efficacy. J. Controlled Release 114, 334–342. (28) Youn, Y. S., Chae, S. Y., Lee, S., Jeon, J. E., Shin, H. G., and Lee, K. C. (2007) Evaluation of therapeutic potentials of site-specific PEGylated glucagon-like peptide-1 isomers as a type 2 anti-diabetic treatment: Insulinotropic activity, glucose-stabilizing capability, and proteolytic stability. Biochem. Pharmacol. 73, 84–93. (29) Baggio, L. L., Huang, Q., Cao, X., and Drucker, D. J. (2008) An albumin-exendin-4 conjugate engages central and peripheral circuits regulating murine energy and glucose homeostasis. Gastroenterology 134, 1137–1147. (30) Shechter, Y., Mironchik, M., Rubinraut, S., Saul, A., Tsubery, H., and Fridkin, M. (2005) Albumin-insulin conjugate releasing insulin slowly under physiological conditions: a new concept for long-acting insulin. Bioconjugate Chem. 16, 913–920. BC100143C
