Synthesis, Characterization, and Pharmacokinetic Studies of

Figure 6 Mass spectra of DPP-IV-catalyzed (A) intact GLP-1 and (B) ... PEG2k-Lys-GLP-1, 16.67 ± 5.77, 0.22 ± 0.04, 21.67 ± 9.36, 12.63 ± 1.75, 43...
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Bioconjugate Chem. 2005, 16, 377−382

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Synthesis, Characterization, and Pharmacokinetic Studies of PEGylated Glucagon-like Peptide-1 Sang-Heon Lee,† Seulki Lee,‡ Yu Seok Youn,† Dong Hee Na,† Su Young Chae,‡ Youngro Byun,‡ and Kang Choon Lee*,† Drug Targeting Laboratory, College of Pharmacy, SungKyunKwan University, 300 Chonchon-dong, Jangan-ku, Suwon City 440-746, Korea, and Center for Cell and Macromolecular Therapy, Gwangju Institute of Science and Technology, 1 Oryung-dong, Buk-gu, Gwangju 500-712, Korea. Received November 5, 2004; Revised Manuscript Received December 28, 2004

Glucagon-like peptide-1-(7-36) (GLP-1) is a hormone derived from the proglucagon molecule, which is considered a highly desirable antidiabetic agent mainly due to its unique glucose-dependent stimulation of insulin secretion profiles. However, the development of a GLP-1-based pharmaceutical agent has a severe limitation due to its very short half-life in plasma, being primarily degraded by dipeptidyl peptidase IV (DPP-IV) enzyme. To overcome this limitation, in this article we propose a novel and potent DPP-IV-resistant form of a poly(ethylene glycol)-conjugated GLP-1 preparation and its pharmacokinetic evaluation in rats. Two series of mono-PEGylated GLP-1, (i) N-terminally modified PEG2k-Nter-GLP-1 and (ii) isomers of Lys26, Lys34 modified PEG2k-Lys-GLP-1, were prepared by using mPEG-aldehyde and mPEG-succinimidyl propionate, respectively. To determine the optimized condition for PEGylation, the reactions were monitored at different pH buffer and time intervals by RP-HPLC and MALDI-TOF-MS. The in vitro insulinotropic effect of PEG2k-Lys-GLP-1 showed comparable biological activity with native GLP-1 (P ) 0.11) in stimulating insulin secretion in isolated rat pancreatic islet and was significantly more potent than the PEG2k-Nter-GLP-1 (P < 0.05) that showed a marked reduced potency. Furthermore, PEG2k-Lys-GLP-1 was clearly resistant to purified DPP-IV in buffer with 50-fold increased half-life compared to unmodified GLP-1. When PEG2k-LysGLP-1 was administered intravenously and subcutaneously into rats, PEGylation improved the halflife, which resulted in substantial improvement of the mean plasma residence time as a 16-fold increase for iv and a 3.2-fold increase for sc. These preliminary results suggest a site specifically monoPEGylated GLP-1 greatly improved the pharmacological profiles; thus, we anticipated that it could serve as potential candidate as an antidiabetic agent for the treatment of non-insulin-dependent diabetes patients.

INTRODUCTION

Glucagon-like peptide-1-(7-36) (GLP-1) is a proglucagon-derived peptide secreted from the L-cells in the gastrointestinal tract in response to orally ingested nutrients (1). Initial clinical interest in GLP-1 was based on its glucose-dependent insulinotropic effect, which was thought to be a potential advantage over conventional antidiabetic agents that secrete insulin via glucoseindependent mechanism (2, 3). GLP-1 also exerts actions suit for non-insulin-dependent diabetes mellitus (NIDDM) patients such as inhibition of glucagons secretion, inhibition of gastric emptying, and a decrease in appetite (4). Moreover, numerous recent studies indicated direct effects of GLP-1 on pancreatic β-cells. For example, GLP-1 is involved in β-cell growth and survival by stimulating cell proliferation and differentiation into new β-cells which could lead to increased β-cell mass (5, 6). On the basis of the advantages as described above, GLP-1 has a great therapeutic potential to treat NIDDM diabetes. However, GLP-1 is quite unstable in vivo (halflife; < 2 min for iv injection) and clinical application requires frequent administration at high dosages. These * To whom correspondence should be addressed. Phone: 8231-290-7704, Fax: 82-31-290-7724, E-mail: [email protected]. † SungKyunKwan University ‡ Gwangju Institute of Science and Technology

limitations pose great challenges in the development of GLP-1 as an antidiabetic pharmaceutical agent. The rapid clearance and degradation of GLP-1 in plasma is mainly due to N-terminal cleavage of the first two amino acids, His8-Ala9, by the enzyme dipeptidyl peptidase IV (DPP-IV) (7). Until now, the main approach that has been used to improve the therapeutic level of GLP-1 is development of enzyme-resistant GLP-1 analogues by substituting one or two amino acids of the peptide (8). Design of GLP-1 analogues showed a number of promising results; however, it still remains difficult to optimize conditions such as control of enzyme resistance and biological potency in vivo. Chemical conjugation of several substances to GLP-1 has been also proposed to improve clinical efficiency. For instance, fatty acid acylation, NN2211 (9), was found to extend action via binding to an existing serum protein derivative while albumin conjugation, CJC-1131 (10), showed prolonged half-life compared to the circulating half-life of albumin itself. Protein modification is one of the convenient alternative ways to improve therapeutic profiles of native proteins; however, little work was done with GLP-1. We previously reported site specifically poly(ethylene glycol) (PEG)-modified salmon calcitonin (sCT) exhibited improved pharmacokinetic and pharmacodynamic profiles with pronounced stability compared to intact sCT in vitro and in vivo (11-15). PEG-modified proteins often have extended plasma half-lives, decreased proteolytic

10.1021/bc049735+ CCC: $30.25 © 2005 American Chemical Society Published on Web 02/23/2005

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degradation, and reduced immunogenecity (16, 17). However, the nonspecific PEGylation process frequently results in substantial loss of biological activity when PEG binds around the protein’s active sites (18). This may show inconsistent pharmacodynamic profiles. Therefore, it is important to strike a balance between biological stability and therapeutic activity of PEGylated proteins under physiological conditions. In this respect, we hypothesized that covalent coupling of PEG to a specific site of GLP-1 may improve the overall therapeutic profile of GLP-1 while maintaining biological activity. In this work, we have prepared and characterized novel PEGylated GLP-1 conjugates in vitro and investigated pharmacokinetic profiles in vivo. MATERIALS AND METHODS

Materials. GLP-1 was purchased from Bachem (Torrance, CA). Succinimidyl propionate monomethoxypoly(ethylene glycol) (mPEG-SPA) and aldehyde monomethoxypoly(ethylene glycol) (mPEG-ALD) were purchased from Shearwater Polymers (Huntsville, AL). Trifluoroacetic acid (TFA) and acetonitrile (ACN) was purchased from Sigma (St. Louis, MO). All reagents and organic solvents used were at least ACS grade. Preparation of Mono-PEGylated GLP-1 Conjugates. The positional isomers of mono-PEGylated GLP-1 conjugates were prepared and purified according to the procedures described previously (11). In brief, GLP-1 (100 µL, 1 mg/mL) in various 50 mmol/L buffer solutions was reacted with 2 mole excess of mPEG2k-SPA (pH adjusted from 7.5 to 9.0 with 50 mmol/L Tris-HCl buffer) at room temperature for 1 h and mPEG2k-ALD (pH adjusted from 4.5 to 5.5 with 50 mmol/L sodium acetate buffer) at 4 °C for 2 h, respectively. The mono-PEGylated GLP-1 conjugates were monitored and purified by reversed-phase high-pressure liquid chromatography (RP-HPLC) on X-tera C18 (4.6 × 250 mm, 5 µm, Waters, Milford, MA) at room temperature. The mobile phase consisted of 0.1% TFA in distilled water (eluent A) and ACN containing 0.1% TFA (eluent B). The mobile phase was run with a linear gradient from 30 to 60% eluent B for 20 min at a flow rate of 1 mL/min and the UV absorbance of the eluent was monitored at 215 nm. The HPLC fractions corresponding to respective peaks were collected separately, purged with nitrogen, and lyophilized. Lys-C Digestion of Mono-PEGylated GLP-1. To determine the positions where the PEG molecules were attached, a lysyl endoproteinase, Lys-C, digestion was employed as described previously (19). Each purified mono-PEGylated conjugate was dissolved in 50 µL of triethylamine-HCl buffer (10 mmol/L; pH 7.4) to a concentration of 1 mg/mL. Following the addition of 50 µL of enzyme (1 mg/mL), the reaction was allowed to incubate for 1 h at 37 °C. To quench the digestion, 5 µL of 10% (v/v) TFA was added and reaction mixture was analyzed by MALDI-TOF MS. MALDI-TOF MS. Molecular weight was obtained from MALDI-TOF mass spectrometry using a Voyager Biospectrometry Workstation (PerSeptive Biosystem, Framingham, MA). Samples were prepared by mixing 1 µL of aliquot with 2 µL of the matrix solution, a saturated solution of R-cyanohydroxycinnamic acid in 50% of water/ ACN with 0.3% TFA. One microliter of the sample mixture was spotted into a well of the sample plate and dried by vacuum prior to mass spectrometry. Data for 2 ns pulses of the 337 nm nitrogen laser were averaged for each spectrum in a linear mode, and positive ion TOF detection was performed using an accelerating voltage of 25 kV.

Lee et al.

Insulinotropic Actions from Isolated Rat Pancreatic Islets. Pancreatic islets were isolated from male Sprague-Dawley rats weighting 250-270 g by the modified method of Lacy and Kostianovsky (20). Briefly, anesthesia was induced by intraperitoneal injection of pentobarbital sodium (50 mg/kg). The donor pancreas was inflated by injection of cold Hank’s balanced buffered salt solution (HBSS, pH 7.4, Sigma) containing 1.5 mg/mL type V collagenase (Sigma). Isolated islets were purified by centrifugation with a discontinuous Ficoll (Amersham Biosciences AB, Uppsala, Sweden) gradient at 2400 rpm for 25 min. Purified islets were cultured with RPMI-1640 culture medium (Sigma) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA) and 1% penicillin-streptomycin (Gibco) at 37 °C with an atmosphere of 95% air and 5% CO2. After the 2 day maintenance period, the islets were washed and incubated in a Krebs-Ringer bicarbonate buffer. Twenty islets were incubated for 2 h in 2 mL of KRB-HEPES at 37 °C with an atmosphere of 95% air and 5% CO2, containing the respective stimulus. The insulin release was measured by a radioimmunoassay kit (Insulin Kit, ICN Pharmaceuticals, Orangeburg, NY). Stability to DPP-IV in Vitro. GLP-1 (100 µL, 5 nmol/L) and an equivalent amount of purified monoPEGylated GLP-1 were prepared in triethylamine‚HCl buffer (10 mmol/L; pH 7.4). DPP-IV (5 mU, 900 µL) was added, and the solutions were incubated at 37 °C. At the indicated time points, 100 µL was removed from the reaction mixture, and reactions were terminated by the addition of 5 µL of 10% (v/v) TFA. Each sample was analyzed by MALDI-TOF MS and RP-HPLC as described. Pharmacokinetics in Vivo. Male Sprague-Dawley (SD) rats were cannulated in jugular vein day before the experiment. The animals were randomly divided into four groups, and the GLP-1 and molar equivalent amount of mono-PEGylated GLP-1 dissolved in saline was intravenously (1 µg/kg) and subcutaneously (10 µg/kg) administered. Blood samples were drawn at predetermined time points in an ice-chilled polyethylene tube containing DPP-IV inhibitor (10 µL per mL of blood; Linco Research Inc., St. Charles, MO). The plasma samples were obtained by centrifugation and stored at -70 °C until assay. The concentration of GLP-1 in rat plasma was analyzed by a commercial sandwich enzyme-linked immunosorbent assay (ELISA) kit (Linco Research Inc.) with GLP-1 and PEGylated conjugates as standards. The peptide concentration beyond the calibration range was diluted by addition of blank rat plasma. Data Analysis. Data are expressed as mean ( SD. The pharmacokinetic parameters were calculated from the plasma concentrations after administration. The area under the time curves was calculated using the linear trapezoidal method. Differences between treatments were evaluated using paired Student’s t tests. P < 0.05 was considered statistically significant. RESULTS AND DISCUSSION

Monitoring of PEGylation. The GLP-1 molecule has three possible sites for the PEGylation; they are the primary amino group of N-terminus (His7) and two lysine residues (Lys26 and Lys34) (Figure 1). Two different chemical approaches were used to PEGylate free amino functional group of the peptide. In the first approach, mPEG-SPA was used to form an amide bond with Lys26 and Lys34. In the second approach, reductive alkylation was carried out by reaction mPEG-ALD with His7, followed by NaCNBH3 reduction. To determine the

PEGylated Glucagon-like Peptide-1

Figure 1. Primary structure of GLP-1. Possible PEGylated sites are His7, Lys26, and Lys34.

optimized condition for PEGylation that would selectively label the target site, the feed molar ratio between GLP-1 and PEG was fixed as 1 to 2 and reactions were carried out at different pH and were monitored at different time intervals by HPLC over an 8 h period. For the lysine, pH was varied from 7.5 to 9.0 and for the histidine, pH was varied from 4.5 to 5.5. Figure 2 illustrates the RPHPLC analysis of the reaction mixture obtained after the termination of the each reaction. Increasing the pH from 7.5 to 9.0 decreased the yield of PEG2k-Lys-GLP-1, while decreasing the pH from 5.5 to 4.5 led to an increased yield of PEG2k-Nter-GLP-1. Selective pH affected the final yield of PEGylated product; however, the reaction time did not influence the production yield much. Each of the purified mono-PEGylated GLP-1 derivatives was then subjected to the lysyl endoproteinase, Lys-C, proteolysis to determine the positional substitution of PEG molecules. After incubation with Lys-C, reaction mixtures were directly characterized by MALDI-TOF MS (Figure 3). By peptide mapping (Table 1), it was confirmed that (i) purified PEGylated GLP-1 conjugates were purely monosubstituted and (ii) PEG2k-Lys-GLP-1 consisted of two different isomers, PEG2k-Lys26-GLP-1 and PEG2k-Lys34-GLP1. In addition, the amount of proteolytic fragment of Glu27-Arg36 is more predominant than His7-Lys34 (Figure 3C). MALDI-TOF MS is one of the direct determination techniques for the peptide content (21), and the result indicates that most of the PEG2k-Lys-GLP-1 consisted of PEG2k-Lys34-GLP-1. This result well matches what we expected based on the natural characteristics of PEG molecule. Once PEG binds to Lys34-GLP-1, the Lys26 position has a reduced chance to form a chemical bond with PEG due to the steric hindrances induced by the closely attached PEG molecule at the Lys34 position. However, we could not completely purify two different

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PEG2k-Lys-GLP-1 isomers by using HPLC, mainly due to the small physicochemical differences between PEG2kLys26-GLP-1 and PEG2k-Lys34-GLP-1. Similarly, the chemical structure of N-terminally PEGylated PEG2k-NterGLP-1 was also confirmed, with good yield (Figure 3D,E, Table 1). After characterization of mono-PEGylated GLP-1 conjugates, the fractions were collected, pooled, and lyophilized. The BCA protein analysis was then carried out to determine the concentration of peptide. Biological Activities. The influence of GLP-1 and mono-PEGylated GLP-1 conjugates on insulin secretion was investigated in isolated rat islets to determine biological activities indirectly in vitro. The primary physiological response to GLP-1 observed is glucosedependent insulin secretion. Since the effect of GLP-1 under low glucose concentration (2.8 mmol/L) is not that significant compared to high glucose concentration (16.8 mmol/L), the biological activity of GLP-1 conjugates was evaluated under high glucose conditions. Culturing only islets in 16.8 mmol/L glucose resulted in increases in the rate of insulin secretion of 7.8 ( 1.2-fold per islet over those observed in the presence of 2.8 mmol/L glucose (Figure 4). Insulin release was clearly stimulated by the addition of 100 nmol/L GLP-1, a significant enhancement to 3.4 ( 0.7-fold (p < 0.001) per islet compared to without stimulant in 16.8 mmol/L glucose. The high concentration of peptide was chosen for in vitro study because the reported receptor binding (EC50) value of GLP-1 is around 1 nmol/L and was also shows no further increase of insulin release in islets at concentrations above 100 nmol/L of GLP-1 (22). At equivalent molar concentrations of mono-PEGylated conjugates, PEG2k-Lys-GLP-1 was at least as potent as native peptide in stimulating insulin secretion (306.6 ( 72.8 vs 368.9 ( 82.9 pmol/L, P ) 0.117) and was significantly more potent than PEG2k-Nter-GLP-1 which showed 1.5 ( 0.5-fold increase of insulin secretion per islet compared to that without stimulant. It is wellknown that certain residues or regions of residues are important for biological activity of therapeutic proteins, and for GLP-1, the N-terminus plays an important role in receptor binding which could directly affect the therapeutic action in vivo. It is postulated that conjugation of PEG moieties to the N-terminus of GLP-1 pro-

Figure 2. RP-HPLC analysis of the reaction mixture obtained by reaction of (A) mPEG-SPA and (B) mPEG-ALD with GLP-1 at different pHs as indicated. Analysis was performed on a X-tera C18 column with water/acetonitrile/TFA as eluent and a flow rate of 1 mL/min, and peaks were monitored at 215 nm.

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Figure 4. Stimulation of insulin secretion from rat pancreatic islets by 100 nM of GLP-1 and an equivalent amount of PEGylated GLP-1 conjugates. Rat islets were isolated by collagenase digestion, and static incubation of 20 islets per sample was performed for 2 h at 37 °C in the presence of 16.8 mM glucose (2.8 mM indicates basal). Insulin release was measured by RIA as described in Materials and Methods. Values are mean ( SD (n ) 6-8).

Figure 3. Mass analysis of (A) intact GLP-1, (B) purified PEG2k-Lys-GLP-1, (C) proteolytic fragments of PEG2k-Lys-GLP1, (D) purified PEG2k-Nter-GLP-1, and (E) proteolytic fragments of PEG2k-Nter-GLP-1. Proteolysis was performed by incubating each purified peptide with lysyl endoproteinase, Lys-C, and the mass of each proteolytic fragment was directly evaluated by MALDI-TOF MS.

duces conformational disturbances mainly by steric hindrance, which may perturb the receptor-ligand interaction and lead to lower biological activity. Because N-terminally modified GLP-1 conjugates showed a markedly reduced potency in stimulating insulin secretion, PEG2k-Lys-GLP-1 was selected for further experiments. Stability of Mono-PEGylated GLP-1. Enzymatic resistance to DPP-IV of intact GLP-1 and PEG2k-LysGLP-1 was evaluated. GLP-1 was degraded by the purified DPP-IV in vitro at 37 °C, with a half-life value of 15.5 min. In contrast, PEGylated GLP-1 revealed a significantly prolonged half-life compared to intact GLP-1 itself with a value of 770 min (Figure 5). Degradation profiles was also monitored and indirectly quantified by MALDI-TOF MS as GLP-1 (3297.1 Da) and N-terminally truncated GLP-1 metabolite, GLP-1 (9-36) amide (3087.1 Da) (Figure 6). The mass difference between two peaks (210.0 Da) corresponds to mass of cleaved two amino acids (His7, Ala8) at the N-terminals. The relative height of the metabolite peak was increased as time passes. At 10 min postincubation with DPP-IV, nearly one-third was recovered as GLP-1 (9-36), and at 60 min, most of the intact GLP-1 was degraded. In contrast, mono-PEGylated GLP-1 was significantly resistant to proteolytic enzyme. Most of the PEG2k-Lys-GLP-1 remained in original form after 60 min incubation and could still be detected even

Figure 5. Stability of PEG2k-Lys-GLP-1 toward enzymatic degradation by DPP-IV in comparison with intact GLP-1. Unmodified GLP-1 (b) and PEGylated GLP-1 (O) were incubated with purified DPP-IV at 37 °C. At the indicated time points, aliquots were subjected to analytical HPLC. The quantity of the peptide (peak area) at t ) 0 was assigned to 100%. Values are mean ( SD (n ) 3).

at 2 h postincubation. From the literature, some of the N-terminally substituted GLP-1 analogues by addition of various different kinds of amino acids showed potent biological activity with decreased degradation profiles (23). However, this process generally needs a large screening process to search for analogues with balanced stability and activity. In the case of our system, PEG2kLys-GLP-1, even simple selective attachments of PEG not far from the site of cleavage showed significantly improved proteolytic stability in vitro while maintaining

Table 1. Molecular Mass of PEGylated GLP-1 after Lys-C Protease Digestion As Determined by MALDI-TOF (calcd/found) proteolytic fragment

calcd mass (GLP-1 std)

His7-Lys26

2098.2 1005.2 231.2 3085.4 1218.5

Glu27-Lys34 Gly35-Arg36 His7-Lys34 Gly27-Arg36 a

PEGylated fragments.

b

Not determined.

PEG2k-Nter-GLP-1 4217.3/4219.0a 1005.2/1008.7 N.D.b -

PEG2k-Lys26-GLP-1 N.D.b 5391.3/5393.0a -

PEG2k-Lys34-GLP-1 2098.2/2099.9 3524.4/3526.4a

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PEGylated Glucagon-like Peptide-1

Figure 6. Mass spectra of DPP-IV-catalyzed (A) intact GLP-1 and (B) PEG2k-Lys-GLP-1 upon degradation. Each peptide was incubated with purified DPP-IV, and reaction mixtures were monitored at different time intervals by MALDI-TOF MS over 2 h periods. GLP-1 (9-36) indicates the N-terminally truncated GLP-1 metabolite.

Figure 7. Plasma concentration of GLP-1 and PEG2k-LysGLP-1 versus time after intravenous (1 µg/kg) and subcutaneous (10 µg/kg) injection in rats. Values are mean ( SD (n ) 3).

biological activity, which is a decisive and potential advantage of our system in terms of efficiency. Pharmacokinetics. The pharmacokinetic properties of GLP-1 and PEG2k-Lys-GLP-1 were evaluated by non-

compartmental and two-compartmental analysis following sc and iv administration in male SD rats, respectively. Figure 7 illustrates the plasma concentration profiles during 2 h experimental time periods, and pharmacokinetic parameters calculated from the data are summarized in Table 2. Plasma concentration of both intravenously administered GLP-1 and conjugate showed a biexponential disposition. Plasma activity of GLP-1 declined rapidly, with a half-life of 2.6 min, whereas the mono-PEGylated GLP-1 showed increased half-life by 10fold, reduced clearance by 1.5-fold, and a 16-fold increase in mean residence time. Again, PEGylation had a profound effect on the pharmacokinetic parameters after sc administration as evident from the increase in the area under the curve (AUC) of the mono-PEGylated GLP-1. The plasma concentration of GLP-1 peaked 5 min after the sc injection; however, Tmax of PEG2k-Lys-GLP-1 observed 16 min after the injection. The pharmacokinetic profile of intact sc GLP-1 was unsuitable, showing a rapid peak followed by a fast decay in plasma, which is in accordance with human trials (24). In contrast, PEG2kLys-GLP-1 showed a 2-fold increase in Cmax, a 7.5-fold increase in AUC, and a 2.5-fold increase in half-life, noting a low variability among individuals. Consequently, PEG2k-Lys-GLP-1 has a significantly improved plasma exposure and stability compared to intact GLP-1. We have designed and characterized two series of mono-PEGylated GLP-1, PEG2k-Nter-GLP-1, and PEG2k-

Table 2. Pharmacokinetic Parameters after GLP-1 and PEGylated GLP-1 Administration to SD Ratsa. sc (10 µg/kg)

Tmax (min)

Cmax (ng/mL)

T1/2 (min)

AUC (ng × min × mL-1)

MRT (min)

intact GLP-1 PEG2k-Lys-GLP-1

5.00 ( 0.00 16.67 ( 5.77

0.14 ( 0.01 0.22 ( 0.04

9.09 ( 3.49 21.67 ( 9.36

1.73 ( 0.13 12.63 ( 1.75

14.35 ( 3.66 43.81 ( 5.90

iv (1 µg/kg)

T1/2 (min)

MRT (min)

Cl (mL/min)

AUC (ng × min × mL-1)

AUMC (ng × min2 × mL-1)

intact GLP-1 PEG2k-Lys-GLP-1

2.61 ( 0.82 33.36 ( 20.84

3.34 ( 1.45 54.21 ( 21.10

581.4 ( 339.8 307.2 ( 64.06

2.11 ( 1.04 3.35 ( 0.67

7.86 ( 6.85 181.1 ( 76.69

a Data are means ( SD (n ) 3). T max, time to reach maximum plasma concentration; Cmax, maximal plasma concentration;, T1/2. halflife; AUC, area under the curve; MRT, mean residence time; Cl, clearance; AUMC; area under the moment curve. Individual data from sc and iv doses were analyzed by noncompartment and two-compartment models, respectively.

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Lys-GLP-1. Biological activity was altered by site modification, in which PEG2k-Nter-GLP-1 showed a marked reduction in activity, whereas the PEG2k-Lys-GLP-1 retained biological activity. The enzyme proteolytic stability of mono-PEGylated GLP-1 was significantly enhanced after PEGylation mainly by steric hindrance induced by the PEG molecule. When lead compound PEG2k-Lys-GLP-1 was administered to rats, PEGylation improved the pharmacokinetic properties of the GLP-1. Increase in half-life and decrease in clearance resulted in substantial improvement in the plasma exposure of the mono-PEGylated GLP-1. GLP-1 has been suggested to be a useful novel therapy in the treatment of NIDDM patients. However, the instability of GLP-1 against the proteolytic enzyme, DPP IV, is the most daunting factor for its therapeutic use as a new treatment modality. Although, the short metabolic stability of the native peptide can be offset by constant iv infusion, this does not fundamentally overcome the effect of proteolytic enzymes. Moreover, an iv infusion may constrain the potential use of GLP-1 in the clinical setting. Thus, the development of a prolonged-acting or slow release form of GLP-1 in vivo has been the focus of much recent research. In this respect, the enzymeresistant form of site-specifically PEGylated PEG2k-LysGLP-1 could serve as a potential GLP-1-based antidiabetic agent for clinical application. While our prototype of PEGylated GLP-1 showed prolonged action in vivo compared with the native GLP1, the total plasma residence time is still in the range of 1 to 2 h. Therefore, our further investigation will examine the effect of the molecular weight of PEG on extended metabolic stability of GLP-1 in vivo. In addition, pharmacodynamic responses using a NIDDM disease animal model are required to evaluate whether the improved half-life of PEGylated GLP-1 is sufficient for the treatment of diabetes. ACKNOWLEDGMENT

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