Mono-PEGylated Dimeric Exendin-4 as High Receptor Binding and

Mar 14, 2011 - College of Pharmacy, SungKyunKwan University, 300 Chonchon-dong, Jangan-ku, Suwon City 440-746, Korea ...... Mammen , M., Choi , S. K.,...
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Mono-PEGylated Dimeric Exendin-4 as High Receptor Binding and Long-Acting Conjugates for Type 2 Anti-Diabetes Therapeutics Tae Hyung Kim,† Hai Hua Jiang,† Seulki Lee,‡ Yu Seok Youn,|| Chan Woong Park,† Youngro Byun,§ 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, NIBIB, NIH, Bethesda, Maryland 20892, United States College of Pharmacy, Pusan National University, Busan 609-735, Korea § College of Pharmacy, Seoul National University, Seoul 151-742, Korea

)

‡

ABSTRACT: Dimerization is viewed as the most effective means of increasing receptor binding affinity, and both dimerization and PEGylation effectively prolong the life spans of short-lived peptides and proteins in vivo by delaying excretion via the renal route. Here, we describe the high binding affinities of two long-acting exendin-4 (Ex4) conjugates, dimerized Ex4 (Di-Ex4) and PEGylated Di-Ex-4 (PEG-Di-Ex4). Di-Ex4 and PEG-Di-Ex4 were prepared using cysteine and amine residue specific coupling reactions using Ex4Cys, bisMal-NH2 , and activated PEG. The Ex4 conjugates produced were of high purity (>98.5%), as determined by sizeexclusion chromatography and MALDI-TOF mass spectrometry. The receptor binding affinity of Di-Ex4 on RIN-m5F cells was 3.5-fold higher than that of Ex4, and the in vivo antihyperglycemic efficacy of Di-Ex4 was also greater than that of native Ex4 in type 2 diabetic db/db mice. Furthermore, Di-Ex4 and PEG-Di-Ex4 were found to have greater blood circulating t1/2 and AUCinf values than native Ex4 by 2.7- and 13.7-fold, and by 4.0- and 17.3-fold, respectively. Accordingly, hypoglycemic durations were greatly increased to 15.0 and 40.1 h, respectively, at a dose of 25 nmol/kg (native Ex4 7.3 h). The results of this study show that combined dimerization and PEGylation are effective when applied to Ex4, and suggest that PEG-Di-Ex4 has considerable potential as a type 2 anti-diabetic agent.

’ INTRODUCTION Exendin-4 (Ex4), a naturally occurring dipeptidyl peptidaseIV (DPP IV) resistant GLP-1 analogue and GLP-1 receptor agonist, was first isolated from the saliva of the Gila monster and shares 53% homology with glucagon-like peptide-1 (GLP-1).1-3 Ex4 has several beneficial anti-diabetic effects in type 2 diabetes. It increases the glucose-dependent enhancement of insulin secretion, suppresses inappropriately high glucagon secretion, reduces gastric mobility and food intake, and increases β-cell mass.4-6 Overall, Ex4 is viewed as a potent therapeutic agent for type 2 diabetic patients, but its short circulating life (4-6 h) means that high doses must be administered frequently. A previous detailed examination revealed that the short biological half-life of Ex4 is mainly due to its rapid glomerular filtration, which can be attributed to its low molecular weight (∼4.2 kDa).7,8 To overcome this problem, numerous approaches, such as the use of long-acting incretin mimetics and/or sustained release mimetics, have been devised to improve the therapeutic level r 2011 American Chemical Society

of Ex4.9-12 In particular, our group has previously investigated the use of Ex4 bioconjugated with vitamins, fatty acids, and bile acids for oral or long-acting delivery.13-16 More specifically, the strategies used included modifying GLP-1 receptor agonists by conjugating them with poly(ethylene glycol). The resulting PEGylated GLP-1 receptor agonists showed typical long-acting characteristics and enhanced circulation half-lives.9,10 Dimeric interactions are frequently used in nature to increase the affinity of ligand-receptor interactions,17,18 and thus, this strategy has attracted the interest of researchers. As a result, several research groups have already produced compounds that enhance interactions between ligands and target cells.19 Goel et al. radiolabeled divalent and tetravalent scFv’s of mAb CC49 with 99mTc and analyzed the imaging potentials of these Received: September 9, 2010 Revised: January 19, 2011 Published: March 14, 2011 625

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radio-immunoconjugates.20 Similarly, dimerized interferon-R2b21 and dimerized adducts of therapeutic antibodies22 have also been examined. The covalent attachment of poly(ethylene glycol) (PEG) to peptides and proteins (PEGylation) provides a means of enhancing their therapeutic values. This technique increases molecular size, shields metabolic sites, and masks immunogenic sites and, thus, increases plasma half-lives and in vivo stabilities and diminishes immunogenicity. Moreover, PEGylated peptides/proteins are much more resistant to renal clearance because the linear hydrophilic nature of PEG molecule effectively increases Stokes’ radius,23-25 and furthermore, PEGylation has been shown to prolong the pharmacokinetic and pharmacodynamic properties of these entities.26-28 On the basis of the above, we undertook to synthesize PEGconjugated dimeric Ex4. We hypothesized that PEG-conjugated dimeric Ex4 would retain the advantages of PEGylation and have higher affinity for pancreatic β-cells. In the present study, we prepared dimerized Ex4 and PEG conjugated dimerized Ex4 constructs and then explored their physicochemical and biological characteristics. In addition, the pharmacokinetic and anti-diabetic characteristics were investigated using a diabetic mouse model to determine which has much better anti-diabetic properties.

Scheme 1. Syntheses of Di-Exendin-4 and PEG-Di-Exendin-4

’ EXPERIMENTAL PROCEDURES Materials. Exendin-4-Cys (Mw: 4290.7) with the sequence HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSC was purchased from AnyGen Co. (Gwangju, Republic of Korea (ROK)). Bis-Maleimide amine (bisMal-NH2, Mw: 660.64) and succinimidyl activated monomethoxy PEG (Mw: 20 kDa) were purchased from Quanta Biodesign (Powell, Ohio) and Nektar Therapeutics (Huntsville, AL), respectively. All other reagents, unless otherwise indicated, were purchased from Sigma-Aldrich (Saint Louis, MO) and were used as received. Experimental Animals. Type 2 diabetic C57BL/6 db/db mice (male, 4-5 weeks old) were purchased from the Korean Research Institute of Bioscience and Biotechnology (Daejon, ROK). 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 National Institute 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 were acclimatized for 2 weeks. This study was approved by the Ethical Committee on Animal Experimentation at SungKyunKwan University. Preparation of Dimerized Exendin-4 (Di-Ex4). As shown in Scheme 1A, Di-Ex4 was synthesized by utilizing a coupling reaction between bis-maleimide amine (bisMal-NH2, Mw: 660.64) and the cysteine residues of Ex4 in organic solvent using a modification of a previously described procedure.13,14 Briefly, 100 μL of bisMal-NH2 (1.5 mg/mL in dimethylsulfoxide (DMSO) containing 0.3% triethylamine (TEA)) was reacted with 100 μL of Ex4 (10 mg/mL in DMSO with 0.3% TEA) (an Ex4/bisMal-NH2 molar ratio of 2) at room temperature with gentle mixing for 1 h. The reaction was then stopped by adding 100 μL of stop solution (deionized water containing 1% trifluoroacetic acid (TFA)). Di-Ex4 was purified from reaction mixtures using semipreparative reversed-phase HPLC (RP-HPLC) and a Capcell-pak RP-18 column (250  10 mm, 5 μm, Shiseido, Japan) operated at

room temperature and a constant flow rate of 5.0 mL/min. Eluate was monitored using a 215 nm UV detector. The mobile phase consisted of 0.1% TFA in deionized water (eluent A) and acetonitrile containing 0.1% TFA (eluent B) using a linear gradient (36-42% B over 30 min). Eluting peaks were collected separately, acetonitrile was then removed with nitrogen, and the solutions so obtained were concentrated using a Centricon-10 (Mw cutoff 3000, Millipore Corp., Billerica, MA). Products obtained were characterized by analytical size-exclusion chromatography and MALDI-TOF mass spectroscopy, and stored at 4 °C until required. Preparation of PEGylated Di-Exendin-4 (PEG-Di-Ex4). As shown in Scheme 1B, bisMal-NH2 conjugated Ex4 and PEG (PEG-Ex4 and PEG-Di-Ex4) were prepared using a modification of a previously described procedure.14,28 Briefly, 10 mg of activated PEG (Mw: 20 kDa) was mixed with 0.3 mg of bisMal-NH2 in 5 mL of a mixture of dimethylsulfoxide and dimethylformamide (70:30) containing 0.3% dimethylaminopyridine at room temperature for 1 h. To this mixture was added 2 mg of Ex4, and the reaction was then allowed to continue for another 1 h. The reaction was then quenched by adding 5 mL of 1% TFA/DW and shaking thoroughly. The resulting mixture was quickly transferred to a Sephadex G-25 desalting column (GE Healthcare Life Sciences, Piscataway, NJ, USA) previously equilibrated with 10 mM PBS. The first fraction was collected and centrifuged using a Centricon-10 concentrator (Millipore Amicon, Beverly, MA, USA), and a portion (0.2 mL) of the supernatant (containing PEG-Ex4 and PEG-Di-Ex4) was purified by size-exclusion chromatography using a Biosep SEC-S2000 column (300  7.8 mm, 5 μm, Phenomenex, Torrance, CA) and eluted with 10 mM PBS (pH 7.4) at a flow rate of 0.75 mL/min. Eluates were monitored at 626

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Figure 1. Purification and characterization of Di-Exendin-4 and PEG-Di-Exendin-4. (A) Reverse-phase HPLC chromatograms of Ex4 dimerization reaction mixtures. (B) Size-exclusion chromatography of Ex4 PEGylation reaction mixtures. (C) Size-exclusion chromatography of purified Ex4 analogues. (D) Total mass spectra of Ex4 analogues, obtained by MALDI-TOF MS.

215 nm. The products so obtained were characterized by analytical size-exclusion chromatography and MALDI-TOF mass spectroscopy, and stored at 4 °C until required. Characterizations of Di-Ex4 and PEG-Di-Ex4. Ex4 analogues were characterized by size-exclusion chromatography using a Superdex 200 HR 10/30 column (300  10 mm, GE Healthcare Life Sciences, Piscataway, NJ, USA) and eluted with 10 mM PBS (pH 7.4) at a flow rate of 0.6 mL/min. Chromatograms were recorded at 215 nm. MALDI-TOF MS and a Voyager-RP Biospectrometry Workstation (PerSeptive Biosystems, Cambridge, MA) were used to confirm molecular weights. Briefly, sample-matrix solutions were prepared by mixing a 1 μL aliquot with 2 μL of matrix solution (a saturated solution of R-cyanohydroxycinnamic acid (R-CHCA) in 50% of water/ACN containing 0.1% (v/v) TFA). One microliter of this sample-matrix solution was then deposited onto a well of a sample plate and dried rapidly under vacuum. Data generated by 2 ns pulses of a 337 nm nitrogen laser were averaged for each spectrum in reflective mode using positive ion TOF detection and an accelerating voltage of 25 kV. Receptor-Binding Affinities of Di-Ex4 and PEG-Di-Ex4 to RIN-m5F Cells. The receptor-binding affinities of Ex4 analogues

were determined using a modification of a previously described method.13,14 Briefly, RIN-m5F cells (ATCC, Manassas, VA) 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), co-treated with the unlabeled Ex4 analogues (final concentrations range: 0.001-1000 nM) with 30 pM of 125I-exendin-4 (9-39, PerkinElmer, Boston, MA), incubated for 2 h at room temperature, and washed three times with chilled PBS containing 1 mg/mL of bovine serum albumin. Cells were then lysed with cell lysis buffer (0.5 N NaOH with 1% SDS) for 15 min, and 125 I radioactivity levels were measured using a γ counter (GMI, Inc., Ramsey, MN). Proteolytic Stability Tests. The proteolytic stabilities of the Ex4 analogues against rat kidney homogenate and plasma were evaluated as previously described,13 by incubating samples at 37 °C in rat kidney homogenate and plasma and measuring their degradation profiles by reverse-phase HPLC. Radioiodination. Radioiodinated samples were prepared using a modification of the IODO-GEN method.29,30 In brief, 627

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100 μL of water-soluble Bolton-Hunter reagent (N-sulfosuccinimidyl-3,4-hydroxyphenyl propionate; 0.13 mg/mL, 36 nmol) in 10 mM PBS (pH 7.0) was mixed with 150 μL of the Ex4 analogues (0.5 mg/mL, 18 nmol) at room temperature for 90 min and briefly dialyzed to remove the reagent. Separately, a 100 μL aliquot of an IODO-GEN (Pierce, Rockford, IL) solution in methylene chloride (0.5 mg/mL) was dispensed into a fresh tube and evaporated under a nitrogen stream. To these tubes were added 50 μL aliquots of each sample (approximately 50 μg/mL) and 50 μCi of Na125I (Perkin-Elmer, Boston, MA) diluted with 10 mM PBS (pH 7.4). Reactions were allowed to proceed for 5 min, and then supernatants were loaded onto a Sephadex G-25 desalting column (GE Healthcare Life Sciences, Piscataway, NJ) previously equilibrated with 10 mM of PBS. 125I-labeled fractions were collected and stored in 10 mM PBS (pH 7.4), and their radioactivities were measured by γ counting (CobraTM Series Auto-Gamma Counting System, Packard Instruments Co., Groningen, The Netherlands). The specific radioactivities of radioiodinated samples were in the range (4.8-5.9)  105 cpm/μg. Pharmacokinetics Tests. The pharmacokinetics of the Ex4 analogues were evaluated using a modification of a previously described method.26,28 Radioiodinated samples were administered to ICR mice (∼2 μg, ∼25 nmol/kg body wt., i.p.). Mice were sacrificed at predetermined times by heart puncture for blood collection, and the radioactivities of 0.2 mL blood samples were determined by γ counting and used to calculate blood concentrations. Pharmacokinetic parameters were calculated by two-compartmental analysis using WinNolin v 1.1 (Scientific Consulting, Inc., Cary, NC). In particular, AUCinf values were obtained by computing areas under the curve from zero to infinity using the trapezoidal rule. Circulating half-lives (t1/2) were calculated using the same method. Antihyperglycemic Efficacies in Fasted Type 2 Diabetic db/db Mice. Oral glucose tolerance testing (OGTT) was performed to evaluate the antihyperglycemic efficacies of the Ex4 analogues after an overnight fast (18 h), as previously described.26,28 One hour prior to 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, or Di-Ex4 (i.p., 25 nmol/kg body wt.). Injection dose was prepared based on the peptide portion of the conjugate. A drop of blood was drawn from a tail vein of each animal at 0, 0.25, 0.5, 0.75, 1, 1.5, 2, and 3 h, and blood glucose levels were determined using a one-touch blood glucose meter (ACCUCHEK Sensor, Roche Diagnostics Corp., USA). Antihyperglycemic efficacies were evaluated by calculating the total hypoglycemic degrees (HDtotal; using HDtotal = AUCsaline, 0-3 h - AUCtest, 0-3 h). Hypoglycemic Efficacies Tests in Nonfasted db/db Mice. Hypoglycemic efficacies of Ex4 analogues were evaluated using a modification of a previously described method13-15 using male db/db mice (6-7 weeks old). Under nonfasting conditions with free access to food and water, animals received a single i.p. injection of saline, Ex4, Di-Ex4, PEG-Ex4, or PEG-Di-Ex4 (5, 25, 250 nmol/kg body wt.). Blood glucose levels were monitored using the procedure mentioned above. In addition, a drop of blood was drawn from a tail vein of each animal at different times (0, 0.5, 1, 2, 3, 4, 6, 8, 12, 24, 36, 48, 60, 72, 96, 108 h) and blood glucose levels were determined using a one-touch blood glucose meter (ACCU-CHEK Sensor, Roche Diagnostics Corp., USA). Hypoglycemic efficacies are expressed as HDtotal, as mentioned above, but measurement times differed in the saline, Ex4, Di-Ex4, PEG-Ex4, and PEG-Di-Ex4 groups (Figures 4B and 5). In addition,

Figure 2. In vitro biological activity and proteolytic stability testing of Di-Exendin-4 and PEG-Di-Exendin-4. (A) Receptor-binding affinities of Exendin-4 analogues in RIN-m5F cells. (B) Stability testing of Ex4 analogues in rat kidney homogenate. (C) Stability testing of Ex4 analogues in rat plasma. Data represent the means ( SDs of five results.

hypoglycemic durations to a blood glucose level of 0.2) in Di-Ex4-treated mice was lower than in Ex4 treated mice (12.6 ( 0.4 mmol/L). At a dose of 25 nmol/kg, the HDtotal values of Di-Ex4 and PEG-Di-Ex4 (504.1 ( 108.9 and 1427.6 ( 187.4 mmol  h/L, P < 0.005, respectively) were 1.65 and 4.66 times greater than that of native Ex4 (306.3 ( 86.3 mmol  h/L) (Figure 5A). Furthermore, hypoglycemic durations in Di-Ex4 or PEG-Di-Ex4 mice were much greater than Ex4, and the times required to rebound to a glucose level of 8.35 mmol/L were ∼15.0 h and ∼40.1 h in Di-Ex4 and PEG-Di-Ex4 mice as compared with ∼7.3 h in Ex4 treated mice (Figure 5B). In particular, at the highest dose administered (250 nmol/kg), the t∼8.35 mmol/L value of PEG-Di-Ex4 was 76.3 h, which means that a single PEG-Di-Ex4 administration stabilized blood glucose levels for nearly 3 days in db/db mice.

Figure 5. Hypoglycemic efficacies of high dose (25/250 nmol/kg) Exendin-4 analogues in nonfasted db/db mice (A). The focused profile of A (0-80 h, 3-12 mmol/L). Times depict hypoglycemic duration rebound to 8.35 mmol/L (B). Data represent five mice and are presented as means ( SDs.

’ DISCUSSION Type 2 diabetes is a chronic, deteriorative metabolic disease, characterized by progressive hyperglycemia followed by dysfunctional insulin secretion and/or its impaired utilization. Progressive β-cell deficiencies associated with apoptosis and a malfunctional GLP-1 response after ingestion have encouraged researchers to consider incretin-based diabetic therapies.1,7 Intensive research and developmental efforts have resulted in the clinical application of a DPP IV resistant GLP-1 receptor Ex4 agonist, which was first isolated from lizard saliva.2,31 Although the coadministrations of Ex4 and traditional hypoglycemic agents achieve better glycemic control and lower levels of glycated hemoglobins (HbA1C) in serum and lower body weights,31 the demand for more efficient incretin-based anti-diabetics has not been satisfied by Ex4 because of its short biological halflife and its rapid renal clearance.8 In addition, recent research on long-acting incretin mimetics or sustained-release formula Ex4 systems has achieved more efficient glycemic control than twicedaily Ex4 injections.32 To develop efficient incretin-based anti-diabetics with protracted hypoglycemic efficacy in type 2 diabetes, we produced high binding affinity, long-acting Ex4 analogues, that is, Di-Ex4 (dimerized Ex4) and PEG-Di-Ex4 (PEGylated Di-Ex4), by PEGylating dimerized Ex-4. Di-Ex4 and PEG-Di-Ex4 were successfully prepared using cysteine and amine residue-specific coupling

reactions using Ex4-Cys, bisMal-NH2, and activated PEG (Scheme 1, Figure 1A,B). Interestingly, cysteine-specific (C-terminal domain) conjugations prevented the serious activity reduction caused by N-terminal conjugation (the N-terminal domain is known to play a crucial role during cellular response to GLP-1 signaling9,33,34). The Ex4 analogues were obtained in high purity (>98.5%), as determined by size-exclusion chromatography and MALDI-TOF (Figure 1C,D). After dimerization, peptide and protein drugs often have higher binding affinities and therapeutic potencies than native drugs.17-22 In the present study, we found that Di-Ex4 had 3.5fold higher in vitro receptor-binding affinity than Ex4 (Figure 2A). Furthermore, in vivo OGTT results showed that the antihypoglycemic efficacy of Di-Ex4 was greater than that of native Ex4 (Figure 4A). However, the receptor binding affinity of PEG-DiEx4 was reduced by the presence of PEG (Mw: 20 kDa), which inhibits PEG-Di-Ex4 binding to GLP-1 receptor. Conversely, PEG-Di-Ex4 was found to have much greater resistance to rat kidney homogenate than Di-Ex4 (Figure 2B). The improvements achieved to date provide reasons to extend the functional lifespan of Ex4 in vivo (Figure 3). In the present 630

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Table 2. Pharmacological Scores of Ex4, Di-Ex4, PEG-Ex4, and PEG-Di-Ex4 in db/db Micea PK parameters

Di-Ex4

HDtotal (5 nmol/kg) (mmol  h/L)

77.3 ( 18.2

161.2 ( 27.7

HDtotal (25 nmol/kg) (mmol  h/L)

306.3 ( 86.3

504.1 ( 108.9

HDtotal (250 nmol/kg) (mmol  h/L) hypoglycemic duration (h) (25 nmol/kg) hypoglycemic duration (h) (250 nmol/kg) a

Ex4

PEG-Ex4 640.2 ( 124.2

PEG-Di-Ex4 1427.6 ( 187.4 1894.9 ( 193.3

-

-

-

7.3

15.0

16.9

40.1

-

-

-

76.3

Data are means ( SDs.

study, dimerization and PEGylation greatly improved the pharmacokinetic profile of Ex4 (Table 2 and Figure 3), as the pharmacokinetic profiles of Di-Ex4 and PEG-Di-Ex4 were markedly better than that of Ex4 (their AUCs were 3.95- and 17.25fold higher and their circulating t1/2 values were 2.66- and 13.67fold greater, respectively, than that of Ex4). Furthermore, the t1/2 of Di-PEG-Ex4 in the circulation was 5.13-fold greater than that of Di-Ex4, probably due to the hydrodynamic size increase caused by the attached PEG. It should also be mentioned that Ex4 is a foreign material to mice, because of its reptile origins. So, it has the possibility for immunological side effects and responses. However, PEGylation diminishes immunogenicity, and thus, we suppose that the incorporation of PEG protects PEG-Di-Ex4 from mouse antibodies. The hypoglycemic durations of Di-Ex4 and of PEG-Di-Ex4 in db/db mice were also much greater than that of Ex4, regardless of the dose administered. In the present study, a blood glucose level of