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Bioconjugate Chem. 2008, 19, 334–341
Preparation, Characterization, and Application of Biotinylated and Biotin-PEGylated Glucagon-Like Peptide-1 Analogues for Enhanced Oral Delivery Su Young Chae,† Cheng-Hao Jin,† Han Jong Shin,† Yu Seok Youn,‡ Seulki Lee,# and Kang Choon Lee*,† Drug Targeting Laboratory, College of Pharmacy, SungKyunKwan University, 300 Chonchon-dong, Jangan-ku, Suwon City 440–746, Korea, College of Pharmacy, Pusan National University, Busan 609–735, Korea, and Biomedical Research Center, Korea Institute of Science and Technology, Seoul 136–791, Korea. Received August 2, 2007; Revised Manuscript Received October 28, 2007
Glucagon-like peptide-1 (GLP-1) (7–36) is a type of incretin hormone with unique antidiabetic potential. The introduction of orally active GLP-1 offers substantial benefits in the treatment of type 2 diabetes over conventional injection-based therapies. Because the intestinal absorption of GLP-1 is restricted by its natural characteristics, we developed a series of GLP-1 analogues via the site-specific conjugation of biotin-NHS and/or of biotin-poly(ethylene glycol)-NHS at Lys26 and Lys34 of GLP-1 (7–36), respectively, in order to improve oral delivery. The resultant GLP-1 analogues, Lys26,34-DiBiotin-GLP-1 (DB-GLP-1) and Lys26-Biotin-Lys34-(BiotinPEG)-GLP-1 (DBP-GLP-1), were prepared and studied in terms of their chemical, structural, and biological properties. DBP-GLP-1 demonstrated superior proteolytic stability against trypsin, intestinal fluid, and the major GLP-1 inactivation enzyme (dipeptidyl peptidase-IV (DPP-IV)) to native GLP-1 or DB-GLP-1 (p < 0.001). The in vitro insulinotropic effects of DB-GLP-1 and DBP-GLP-1 showed potent biological activity in a dose-dependent manner, which resembled that of native GLP-1 in terms of stimulating insulin secretion in isolated rat islets of Langerhans. Intraperitoneal glucose tolerance tests (IPGTT) after the oral administration of GLP-1 analogues in diabetic db/db mice demonstrated that DB-GLP-1 and DBP-GLP-1 significantly reduced the AUC0–180 min of glucose for 3 h by 14.9% and 24.5% compared to that of native GLP-1, respectively (p < 0.01). In particular, DBP-GLP-1 concentration in plasma rapidly increased 30 min after oral administration in rats, presumably due to improved intestinal absorption. These findings revealed that site-specific biotinylated and biotin-PEGylated GLP-1 is absorbed by intestine and that it has biological activity in vivo. Therefore, we propose that this orally active bioconjugated GLP-1 might be considered as a potential oral antidiabetic agent for type 2 diabetes mellitus.
INTRODUCTION Glucagon-like peptide-1 (GLP-1) is the product of the glucagon gene, and is secreted from enteroendocrine L-cells of the intestinal mucosa in response to orally ingested nutrients (1, 2) GLP-1 is viewed as an interesting antidiabetic agent for the treatment of type 2 diabetes because of its marked glucosedependent insulinotropic effect. GLP-1 has a number of beneficial effects in type 2 diabetes; for example, it inhibits glucagon secretion and gastric emptying and suppresses appetite (3–6) In addition, GLP-1 has strong disease-modifying possibilities in type 2 diabetes, as it inhibits β-cell apoptosis and stimulates β-cell proliferation and/or neogenesis (7, 8). Overall, GLP-1 is viewed as a potent therapeutic agent for type 2 diabetic patients, but its short half-life in physiological environments and limited oral bioavailability remain critical hurdles to its successful clinical application (3, 9). The short biological half-life (∼2 min) of GLP-1 is mainly due to its rapid inactivation by the ubiquitous protease (dipeptidyl peptidase-IV (DPP-IV)), which acts on its two N-terminal amino acids, histidine and alanin (10–12). To overcome this limitation, several GLP-1 receptor agonists have been developed and investigated to improve the therapeutic value of GLP-1 (13, 14). * Corresponding author. E-mail:
[email protected]. Tel: +82-31-2907704, Fax: +82-31-290-7724. † SungKyunKwan University. ‡ Pusan National University. # Korea Institute of Science and Technology.
However, although these injectable GLP-1 receptor agonists have produced promising results, there is a continuous demand for an orally active GLP-1, as such an agent offers significant advantages in terms of therapeutic and patient acceptability. Furthermore, since the absorption of orally active GLP-1 occurs in the intestine, a DPP-IV resistant GLP-1 efficiently absorbed in the intestine would have the potential to mimic the physiological effects of native GLP-1, which is secreted by intestinal L-cells. However, the development of such an orally active peptide drug presents an arduous challenge, due to the low intestinal absorption and proteolytic stability of GLP-1 in the gastrointestinal (GI) track (15, 16). Thus, the successful oral delivery of this peptide involves increasing its intestinal membrane permeability and overcoming the barrier of enzymatic degradation. Site-specific bioconjugation offers a convenient means of developing orally active peptide drugs. Many different types of peptide bioconjugation, with vitamins, fatty acids, bile acids, and PEG, have been used to develop orally active peptide agents (16–19). Here, we describe new strategies for producing orally active GLP-1 derivatives using site-specific biotinylation and biotinPEGylation. As a member of the vitamin B family, biotin can be recognized and absorbed through the Na+-dependent multivitamin transporter (SMVT) systems (20–22). Moreover, the natural physicochemical properties of biotins have been utilized to facilitate the intestinal absorption of peptide drugs through passive transport via enhanced hydrophobicity resulting from biotin conjugation and a predominant SMVT absorption
10.1021/bc700292v CCC: $40.75 2008 American Chemical Society Published on Web 12/14/2007
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system (22–24). We previously reported that site-specific PEGylated GLP-1 at the Lys34 position profoundly improves its enzymatic stability while retaining its biological activities, and thus enhances its therapeutic potential in type 2 diabetic db/db mice (25–27). The conjugation of water soluble polymers on the lysine residues GLP-1 also showed the preserved insulinotropic activity on pancreatic islets (28, 29). In view of these observations that site-specific PEGylated GLP-1 can increase enzymatic stability but retain biological activity, we hypothesized that site-specific biotinylation and biotin-PEGylation might enhance the intestinal absorption of GLP-1 due to its increased permeability and stability without prominent activity loss. In this study, we prepared and characterized site-specifically bioconjugated GLP-1s using sequential biotin and biotin-PEG conjugations on Lys26 and Lys34 of GLP-1, respectively. The therapeutic potentials of these bioconjugated GLP-1s were then investigated by examining their metabolic stabilities and insulinotropic activities in vitro and their hypoglycemic effects and intestinal absorptions in type 2 diabetic db/db mice.
Bioconjugate Chem., Vol. 19, No. 1, 2008 335 Scheme 1. Schematic Diagram of the Preparations of Site-Specific Biotinylated and Biotin-PEGylated GLP-1
MATERIALS AND METHODS Materials. GLP-1 was purchased from Bachem (Torrance, CA, USA), Fmoc-GLP-1 from the American Peptide Company (Sunnyvale, CA), and biotin-poly(ethylene glycol) N-hydroxysuccinimide ester (biotin-PEG, MW 3400) from Nektar Therapeutics (Huntsville, AL). Dipeptidyl peptidase inhibitor and GLP-1 radioimmunoassay (RIA) kits were purchased from Linco Research (St. Charles, MO), and insulin enzyme immunoassay (EIA) kits from Mercodia (Uppsala, Sweden). All other reagents, unless otherwise indicated, were purchased from Sigma-Aldrich Co. (Saint Louis, MO) and were used as received. Type 2 diabetic C57BL/6 db/db mice (male, 7–8 weeks old) were obtained from the Korea Research Institute of Bioscience and Biotechnology (Daejon, Korea), and male Sprague–Dawley (SD) rats (150–200 g) were purchased from the Hanlim Experimental Animal Laboratory (Seoul, Korea). Preparation of Lys26,34-DiBiotin-GLP-1 (DB-GLP-1). DBGLP-1 was synthesized by a coupling reaction between Nhydrosuccinimide-activated biotin (biotin-NHS) and the lysine residues (Lys26 and Lys34) of GLP-1 in organic solvent. Briefly, 20 µL of biotin-NHS (2 mg/mL in dimethylsulfoxide (DMSO) containing 0.3% triethylamine (TEA)) was reacted with 380 µL of GLP-1 (5 mg/mL in DMSO with 0.3% TEA) (GLP-1/ biotin-NHS molar ratio of 2) with gentle mixing at room temperature for 1 h. The reaction was then stopped by adding 100 µL of stop solution (deionized water containing 1% trifluoroacetic acid (TFA)). DB-GLP-1 was finally obtained by preparative high-performance liquid chromatography (HPLC). Preparation of Lys26-Biotin-Lys34-(Biotin-PEG)-GLP-1 (DBP-GLP-1). DBP-GLP-1 was prepared by the stepwise conjugation of biotin-PEG and biotin on Lys34 and Lys26 of GLP-1, respectively, using procedures similar to that described above for DB-GLP-1 synthesis. Detailed of the procedures used are illustrated in Scheme 1. First, Nter-FMOC-GLP-1 (FMOCGLP-1, 1 mg/mL in DMF with 0.3% TEA) was reacted with a 1.5 times molar excess of biotin-PEG-NHS (6 mg/mL in DMF with 0.3% TEA) for 1 h at room temperature. After stopping the reaction with stop solution, Lys34-(Biotin-PEG)-FMOCGLP-1 was separated by reverse-phase HPLC (RP-HPLC). The product was obtained from the collected fraction by lyophilization. Second, Lys34-(Biotin-PEG)-FMOC-GLP-1 (2 mg/mL in DMF containing 0.3% TEA) was further reacted with a 3-fold molar excess of biotin-NHS for 1 h at room temperature, as described above, following deprotection of the FMOC group of the final product by adding piperidine to a final concentration of 5% for 20 min, and then stopping the reaction with stop solution. DBP-GLP-1 was obtained by preparative HPLC.
Purification of DB-GLP-1, Lys34-(Biotin-PEG)-FMOCGLP-1, and DBP-GLP-1. The bioconjugated GLP-1 analogues mentioned above were isolated and purified from their reaction mixtures by preparative RP-HPLC using a Capcell-pak RP-18 column (250 × 4 mm, 5 µm, Shiseido, Japan) at ambient temperature at a constant flow rate of 1.0 mL/min under UV absorbance monitoring at 215 nm. The mobile phase consisted of 0.1% TFA in deionized water (eluent A) and acetonitrile (AN) containing 0.1% TFA (eluent B), and these were as linear gradients, as follows: DB-GLP-1, 35-42% B for 20 min; Lys34(Biotin-PEG)-FMOC-GLP-1, 40-43% B for 20 min; DBPGLP-1, 38-43% B for 15 min. HPLC fractions corresponding to respective peaks were collected separately, flushed off with nitrogen, and concentrated using an ultrafiltration unit (Ultracel YM3, MWCO 3000, Millipore, Billerica, MA). The products obtained were characterized by HPLC and MALDI-TOF mass spectroscopy, and stored at -70 °C until required for further experiments. Characterization of Bioconjugated GLP-1s. Bioconjugation and site-specific modification were confirmed by MALDI-TOF MS of GLP-1 analogues before and after lysyl endoproteinase Lys-C digestion, as reported previously (25). Bioconjugations were confirmed using MALDI-TOF-MS molecular weight measurements using a Voyager-RP Biospectrometry workstation (PerSeptive Biosystems, Cambridge, MA). Briefly, the samplematrix solution was prepared by mixing 1 µL of aliquot with 2 L of the matrix solution (a saturated solution of R-cyanohydroxycinnamic acid (CHCA) in 50% of water/ACN containing 0.1% (v/v) TFA). 1 µL of the sample-matrix solution was then deposited onto a well of a sample plate and dried by rapid vacuum evaporation. Data generated by 2 ns pulses of the 337 nm nitrogen laser were averaged for each spectrum in linear mode; positive-ion TOF detection was performed using on accelerating voltage of 25 kV. To determine the locations of the biotinylation and PEGylation sites, Lys-C digestion experiments were performed, as described previously (27). Briefly, 10 µL of Lys-C (10 µg/mL,
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Figure 2. MALDI-TOF mass spectra of native GLP-1, DB-GLP-1, and DBP-GLP-1.
Figure 1. RP-HPLC analysis of DB-GLP-1 and DBP-GLP-1. (A) Chromatogram of the biotinylation reaction mixture; and (B) Lys26(biotin-PEG)-FMOC-GLP-1 and DBP-GLP-1.
pH 7.4) was added to 20 µL of sample solution (50 mM Tris-HCl, buffer pH 8.5) containing each positional isomer of bioconjugated GLP-1s (10 µg/mL). Reaction mixes were incubated at 37 °C for 60 min, and after digestion, individual samples were analyzed by MALDI-TOF-MS. Proteolytic Stability Studies of the Bioconjugated GLP-1s. The proteolytic stabilities of the bioconjugated GLP1s against trypsin, DPP IV, and intestinal fluid were evaluated as previously described with slight modification (25). Intestinal fluid was obtained from 24 h prefasted SD rat intestine (jejunum) by flushing with PBS and centrifugation. A portion (20 µL) of either GLP-1 or bioconjugated GLP-1s (200 µg/mL each) were mixed with the same volume of enzyme solution (2 mM trypsin in PBS pH 6.5, 25 mU/mL DPP IV in Tris-HCl buffer pH 7.4, and intestinal fluid, preincubated at 37 °C for 30 min), and
further incubated at 37 °C. At predetermined times, the incubations were stopped by adding 100 µL of chilled stop solution (1% TFA in DW). The mixtures were then centrifuged at 12 000 rpm for 5 min, and supernatants were analyzed by RP-HPLC. Degradation half-lives were then calculated, assuming first-order kinetics. Biological Activities of the Bioconjugated GLP-1s. Insulinotropic activities of GLP-1 and bioconjugated GLP-1s on rat pancreatic islets were assessed as previously described (25–27). Briefly, male SD rats (250–270 g) were anesthetized with ketamine and xylazine (90/10 mg/kg, i.p.). The donor pancreas was swollen by injecting cold Hank’s balanced buffered salt solution (HBSS, pH 7.4, Sigma) containing 1.5 mg/mL of type V collagenase (Sigma). Isolated islets were purified by centrifugation in a discontinuous Ficoll (Amersham Biosciences AB, Uppsala, Sweden) gradient at 2400 rpm for 25 min. Purified islets were cultured in RPMI-1640 culture medium (Sigma) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA) and 1% penicillin-streptomycin (Gibco) at 37 °C in a 95% air/5% CO2 atmosphere for 2 days. The islets were then washed, incubated in Krebs-Ringer bicarbonate (KRB) buffer, seeded at 20 islet/well on a 24 well plate in 1 mL of KRB containing 16.8 mM glucose, and incubated with various concentrations of GLP-1 and bioconjugated GLP-1s (0.1, 1, 10, and 100 nM) for 2 h. Insulinotropic activities were evaluated by measuring the amount of insulin released to media using an enzyme immunoassay kit (Rat insulin ELISA kit, Mercodia AB, Uppsala, Sweden). The in vivo bioactivities of the bioconjugated GLP-1s were evaluated using oral glucose tolerance tests (OGTT) in type 2 diabetic db/db mouse (8-10 weeks old), after intraperitoneal drug administration (26, 27). In brief, 18 h fasted mice were administered bioconjugated GLP-1s (10 nmol/kg, i.p., at 30 min prior to oral glucose administration). A 1.0 g/kg dose of glucose was then administered orally to each mouse. At predetermined times, blood glucose levels were monitored using a glucometer (Accu-Chek Sensor, Roche Diagnostics Corp., Mannheim, Germany). Evaluation of the Hypoglycemic Efficacies of the Bioconjugated GLP-1s. The hypoglycemic efficacies of the bioconjugated GLP-1s administered orally were investigated using an intraperitoneal glucose tolerance test (IPGTT). Briefly, 18 h fasted mice were injected with a glucose solution (1.0 g/kg, 100 µL, at time 0 min, i.p.), and then orally administered gastric neutralizer (0.1 mL of 3% NaHCO3) and bioconjugated GLP1s (15 nmol/mouse, 200 mL, in a PBS/propylene glycol 50/50 mixture) at 30 min before glucose injection. Blood glucose levels
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Table 1. Molecular Masses of Site-Specific Biotinylated and Biotin-PEGylated GLP-1 after Lys-C Protease Digestion, as Determined by MALDI-TOF possible Lys-C fragments samples GLP-1 DB-GLP-1 Lys34-(Biotin-PEG)-FMOC-GLP-1 DBP-GLP-1 a
fragments
molecular mass
His7-Lys26 Glu27-Lys34 Gly35-Arg37 Lys26,34-Biotin-GLP-1 FMOC-His7-Lys26 Gln27-Lys34-(Biotin-PEG)-Arg36 Lys26-Biotin-Lys34-(Biotin- PEG)-GLP-1
2098.2 1005.2 231.2 3784.2 2322.8 4876.5 (Mn) 7192.3 (Mn)
observed mass 2099.6 1005.8 N.D.a 3784.6 2332.5 4926.7 (Mn) 7244.5 (Mn)
Not determined.
were then monitored using the above-mentioned glucometer. As controls, vehicle or GLP-1 were administered in an identical manner. Areas under the curve (AUC) were calculated in concentration per minute for the total experimental periods (0-180 min). Pharmacokinetics in Vivo. To investigate oral absorption, pharmacokinetic profiling of the bioconjugated GLP-1s was performed, as described previously (25). For this study, a jugular vein was cannulated in male SD rats at one day before the experiment. The animals were randomly divided into three groups and GLP-1 mol equiv amount of the bioconjugated GLP-1s (DBGLP-1 and DBP-GLP-1) was dissolved in PBS (20 nmol/rat) and administered i.v. or perorally (p.o.), respectively. Blood samples, drawn at predetermined times, were placed in ice-cold polyethylene tubes containing DPP IV inhibitor (10 µL per mL of blood; Linco Research Inc., St. Charles, MO). Plasma samples were obtained by centrifugation and stored at -70 °C until required for assay. Concentrations of GLP-1 in rat plasma were analyzed using a commercial GLP-1 radioimmunoassay kit (active GLP-1 RIA kit, Linco Research Inc., St. Charles, MO).
RESULTS AND DISCUSSION
Figure 3. The degradation profiles of GLP-1, DB-GLP-1 and DBPGLP-1 incubated with (A) trypsin, (B) intestinal fluid, and (C) isolated DPP IV enzyme. Residues were quantified by RP-HPLC. Data are the means ( SD of three determinations.
Preparation and Characterization of Bioconjugated GLP-1s. Unlike chemical conjugations to proteins where the degree of substitution plays a major role in their biological and pharmaceutical characteristics, peptide drug modifications, such as PEGylation, biotinylation, acylation, and other conjugations, show stronger relationships between conjugate position and therapeutic efficacy and potential (27, 30, 31). In particular, the chemical attachment of functional moieties to the primary amine groups of GLP-1 resulted in position-dependent characteristics. GLP-1 possesses three different sites for nucleophilic substitution via a primary amine group located at the N-terminal, i.e., His7, Lys26, and Lys34. In previous, we monitored the effect of the PEGylated GLP-1 on biological activity. Compared to the native GLP-1, the in vitro insulinotropic activity of Nter-PEGGLP-1 and Lys-PEG-GLP-1 were 44.1% and 83.1%, respectively (100 nM peptide, 16.8 mM glucose) (25, 26). More detailed investigations revealed that the PEGylation on Lys34 of GLP-1 resulted in 93% activity remaining (27). On the basis of these results, we concluded that conjugation of bulky biotin-PEG on the Lys34 and small m.w. of biotin on the Lys26 position could minimize the loss of GLP-1’s activity among the various combinations. To prepare the described bioconjugated GLP-1s, Lys-amine specific conjugation reactions were conducted in an organic medium, as described previously (25–27). The biotin modification of GLP-1 resulted in a mixture of biotinylated GLP-1 isomers, as illustrated in the RP-HPLC chromatogram shown in Figure 1A. Of the four distinct HPLC peaks observed in the biotinylated reaction mixture, the first three peaks were attributed to native GLP-1 (elution time 11.96 min) and monobiotin-GLP-1 (14.03 and 14.30 min), which had mass values of 3299.8 m/z and 3542.8 m/z, respectively. The fourth peak (16.53 min)
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Table 2. Summary of GLP-1, DB-GLP-1, and DBP-GLP-1 Stabilities in Various Digestive Systems trypsin
intestinal fluid
DPP IV
samples
half-life (s)
folds increase
half-life (s)
folds increase
half-life (s)
folds increase
GLP-1 DB-GLP-1 DBP-GLP-1
11.6 ( 0.8 85.6 ( 1.4 210.0 ( 5.3
7.4 18.1
0.57 ( 0.06 4.38 ( 0.79 13.94 ( 1.14
7.7 24.5
49.8 ( 3.6 117.4 ( 9.4 495.0 ( 9.3
2.4 9.9
represented Lys26,34-Dibiotinylated GLP-1 (DB-GLP-1), which had a mass of 3784.8 m/z, as illustrated in Figure 2. Further investigations of DB-GLP-1 using Lys-C digestion assays and following MALDI-TOF confirmed the biotinylation of GLP-1 at Lys26 and Lys34 (Table 1). To prepare heterogeneously bioconjugated GLP-1 with biotin and biotin-PEG at different lysine residues, N-terminal protected GLP-1 was utilized to improve reaction yield and selectivity (30, 31). Of the various N-terminal protected GLP1s examined, 9-fluorenylmethyl carbamate (FMOC) protected GLP-1 was found to have several advantages, e.g., superior stability in acidic HPLC conditions and ease of deprotection under mild basic conditions. By using FMOC-GLP-1, heterogeneous bioconjugations were conducted using sequential coupling reactions between lysine residues and biotin-PEG-NHS and biotin-NHS. As illustrated in Figure 1B, biotin-PEG
conjugation on FMOC-GLP-1 resulted in the formation of monoconjugated positional isomers (second and third peaks) and diconjugated GLP-1 on at Lys26 and Lys34. MALDI-TOF and Lys-C digestion assays revealed that the third peak was Lys34-(Biotin-PEG)-FMOC-GLP-1 (Table 1). The Lys34conjugated portion was separated, purified, and used as a substrate for further bioconjugation with biotin-NHS. Biotinylation at Lys26 and removal of the FMOC protecting group were conducted by sequentially reacting Lys34-(Biotin-PEG)FMOC-GLP-1 with biotin-NHS for 1 h and then treating with piperidine (final concentration 5%). HPLC of the reaction mixtures revealed that heterobioconjugated GLP-1 had been formed in high yield (Figure 1B, about 90% yield). MALDITOF and Lys-C digestion analysis confirmed the successful heterobioconjugations by biotin and biotin-PEG at Lys26 and Lys34, respectively. The observed mass of Lys26-Biotin-Lys34(Biotin-PEG)-GLP-1 (DBP-GLP-1) was 7244.5 (Mn, calcd. 7192.3). Moreover, DBP-GLP-1 did not produce fragments when treated with Lys-C (Figure 2 and Table 1).
Figure 4. In vitro (A) and in vivo (B) biological activities of GLP-1, DB-GLP-1, and DBP-GLP-1. (A) Dose-dependent insulinotropic activities of GLP-1, DB-GLP-1, and DBP-GLP-1 on isolated rat pancreatic islets at a glucose concentration of 16.8 mM. (B) Glucose stabilizing of the three agents in type 2 diabetic db/db mice (fasted for 18 h), after i.p. administration (-30 min, 10 nmol/kg) and oral glucose (0 min, 1.0 g/kg).
Figure 5. (A) Glucose clearance kinetics after the oral administrations of GLP-1, DB-GLP-1, or DBP-GLP-1 prior to intraperitoneal glucose injections. 18 h fasted db/db mice were administered the drugs orally (-30 min, 15 nmol/kg) followed by glucose (0 min, 1.0 g/kg), and plasma glucose levels were monitored for 180 min. (B) Calculated glucose AUCs of the experimental groups. * p < 0.05 vs DB-GLP-1 administered rats.
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Figure 6. Plasma concentrations of GLP-1 (20 nmol/kg, i.v.), DBGLP-1 (20 nmol/kg, p.o.), and DBP-GLP-1 (20 nmol/kg, p.o.). Values are means ( SD (n ) 4).
Proteolytic Stabilities of GLP-1 and Bioconjugated GLP-1s. The protection of peptide drugs from enzymatic digestion in the intestinal compartment must be accomplished to realize oral peptide drug delivery (1011). To investigate the effect of the described bioconjugations on the enzymatic stability of the GLP-1, a series of enzyme stability tests were conducted. Bioconjugations to the lysine residues of GLP-1 enhanced its gastrointestinal stability (Figure 3 and Table 2). In case of the trypsin, one of the most abundant pancreatic digestive enzymes, bioconjugation resulted in a dramatic improvement in proteolytic stability (Figure 3A), and this was further improved by introducing PEG in DBP-GLP-1. DB-GLP-1 and DBP-GLP-1 were found to have more than 7 and 18 times better proteolytic stabilities, respectively, than GLP-1 according to our trypsin degradation tests. Moreover, similar enhancements of proteolytic resistance (7- and 24-fold, respectively) were observed in the presence of intestinal fluid, which more closely represents intestinal enzymatic digestion. For GLP-1-based incretin mimetics, stability in the systemic circulation is also key for an efficient oral delivery system. The instability of GLP-1 in the circulation is mainly attributable to its degradation by DPP IV (1011). As shown in Figure 3, native GLP-1 was rapidly degraded by DPP IV with a half-life of 49.8 min. As previously reported, DPP IV mediated GlP-1 degradation resulted in the truncation of the two N-terminal amino acids and destroyed its insulinotropic activity (25). However, biotinylation and biotin-PEGylation enhanced the proteolytic resistances of DB-GLP-1 and DBP-GLP-1 against DPP IV by 2.4- and 9.9-fold, respectively, as compared with native GLP-1. Biological Activities of the Bioconjugated GLP-1s. Chemical modifications of protein or peptide biopharmaceuticals invariably alter their biological and pharmacological characteristics (17–19), and thus, such chemical modifications require meticulous investigation. One of the most prominent biological effects of GLP-1 is that it stimulates insulin secretion by pancreatic β-cells. Furthermore, this insulinotropic activity is strongly dependent on glucose concentration, and in fact, it has no effect on insulin secretion at glucose concentrations below
4.5 mM (32). Therefore, we performed experiments using isolated pancreatic islets in high-glucose media (16.8 mM glucose in KRH buffer). As shown in Figure 4A, GLP-1 and the bioconjugated GLP-1s showed concentration-dependent insulinotropic activity on pancreatic islets. As we reported previously for PEGylated GLP-1 derivatives (26, 27), GLP-1 and the two bioconjugated GLP-1s potentiated insulin secretion in a concentration-dependent manner and reached saturation at around 10 nM. Moreover, our results demonstrated that DBGLP-1 and DBP-GLP-1 robustly retained biological activity, and that they have insulinotropic effects on pancreatic islets. The in vivo bioactivities of GLP-1, DB-GLP-1, and DBPGLP-1 administered intraperitoneally into diabetic db/db mice were evaluated by performing oral glucose tolerance tests, as described previously (27). As was found by in vitro bioactivity testing, all three agents showed similar kinetic patterns of glucose clearance. As shown in Figure 4B, blood glucose levels in the placebo group (saline i.p. injection) rapidly increased to ca.450 mg/dL at 15 min after glucose administration and then decreased slowly. On the other hand, mice pretreated with GLP1, DB-GLP-1, or DBP-GLP-1 showed rapid glucose clearance kinetics and maximum plasma glucose levels of ca. 300 mg/ dL, mainly due to stimulated insulin secretion. Therefore, the in vitro insulinotropic activity tests and in vivo hypoglycemic activity tests performed during this study show that DB-GLP-1 and DBP-GLP-1 substantially maintained the biological activity of native GLP-1. Oral Hypoglycemic Effects of DB-GLP-1 and DBP-GLP-1. To evaluate the therapeutic efficacies of orally administered (p.o.) DB-GLP-1 and DBP-GLP-1, we used the intraperitoneal glucose tolerance test (IPGTT). Unlike the results described above after i.p. administration, p.o. administrations of GLP-1, DB-GLP-1, or DBP-GLP-1 had remarkably different glucose clearance kinetics. As illustrated in Figure 5A, the saline (placebo) and GLP-1 administered groups (30 min prior to glucose administration) showed a rapid increase in plasma glucose levels after the i.p. administration of glucose solution (0 min). 30 min after i.p. glucose administration, plasma glucose levels in saline and GLP-1 treated animals reached ca. 440 and 410 mg/dL, respectively, whereas in DB-GLP-1 and DBPGLP-1 treated animals, they reached only 315 and 296 mg/dL, respectively. Moreover, DB-GLP-1 and DBP-GLP-1 both enhanced glucose tolerance during the experimental periods, which we attribute to enhanced intestinal absorption, and although DBPGLP-1 treated animals showed initial blood glucose elevations that were similar to those observed for DB-GLP-1, DBP-GLP-1 treated animals achieved the normal glycemic state more quickly. Statistical analysis revealed that the mean glucose levels in DBP-GLP-1 treated animals at 90 and 120 min after glucose injection were significantly lower than in DB-GLP-1 treated animals (p < 0.05, t test). We attribute this enhanced stability of DBP-GLP-1 vs DB-GLP-1 to its enhanced proteolytic stability and biotin-facilitated intestinal absorption. Owing to the absence of a natural biotin synthesis system, humans and other mammals must obtain the vitamin exogenously through intestinal absorption (20, 21). Although the exact molecular mechanism is still unsolved, it is evident that the biotin intake is reliant on the carrier-mediated mechanism (SMVT system).
Table 3. Pharmacokinetic Parameters after Administering GLP-1, DB-GLP-1, or DBP-GLP-1 to SD Ratsa sample
AUC (ng × min × mL-1)
Cmax(ng/mL)
Tmax(min)
t1/2(min)
GLP-1 (i.v.) DB-GLP-1 DBP-GLP-1
9892 ( 913 507 ( 81 998 ( 104
N.A.b N.A. 7.72 ( 1.54
N.A. N.A. 28.0 ( 3.1
5.33 ( 0.76 N.A. 39.45 ( 10.15
Data are means ( SD (n ) 4). AUC, area under the curve; Cmax, maximal plasma concentration; Tmax, time to reach maximum plasma concentration; t1/2, half-life. b Not available. a
340 Bioconjugate Chem., Vol. 19, No. 1, 2008
The enhanced cellular and intestinal absorption of biotin-based peptides also have been reported (22–24). Sinco et al. reported that more than 3-fold enhanced absorption of R.I.-K-Tat9 peptide was achieved by biotin conjugation through passive and predominantly SMVT pathway (22). On the basis of these ideas, it can be expected that the effect of enhanced intestinal absorption and the following antidiabetic effect of DBP-GLP-1 is related to the SMVT system; however, the relationship between DBP-GLP-1 and the SMVT system should be elucidated. Pharmacokinetic Profiles of DB-GLP-1 and DBP-GLP-1 in the Rat. The pharmacokinetic characteristics of orally administered bioconjugated GLP-1s were also investigated, and the results are illustrated in Figure 6 and Table 3. As a control experiment, native GLP-1 was administered i.v., and its plasma concentrations were then monitored vs time. The plasma concentrations of GLP-1 rapidly reduced and reached the basal level at 3 h after injection, which we attribute to the rapid systemic clearance and DPP IV mediated inactivation. Orally administered DB-GLP-1 showed the maintenance of basal levels around 1-2 ng/mL. Compared to the blank plasma levels of GLP-1 (1.28 ( 0.32 ng/mL, time 0 point), the plasma GLP-1 concentrations observed by oral DB-GLP-1 administration revealed the negligible intestinal absorptions, except at the 15 min point (2.12 ( 0.35 ng/mL). Typical intestinal absorption patterns were observed for orally administered DBP-GLP-1. Active GLP-1 plasma concentrations in rats peaked at 7.72 ng/ mL (Cmax) at 28 min (Tmax) after DBP-GLP-1 oral administration. After 120 min, the plasma levels of DBP-GLP-1 groups also returned to the basal level. On the basis of their pharmacokinetic profiles, the calculated average AUCs for i.v. GLP-1, oral DB-GLP-1, and oral DBP-GLP-1 were 9892, 507, and 998 ng · min · mL-1, respectively. Although oral administration might provoke the intestinal secretion of natural GLP-1, the existence of apparent absorption peaks in plasma and the higher AUC value of DBP-GLP-1 strongly support its enhanced intestinal absorption. Moreover, the introduction of PEG molecules in DBP-GLP-1 also appeared to significantly increase plasma halflife, which concurs with our previous investigations (25–27). Moreover, as compared with GLP-1 administered i.v., orally administered DBP-GLP-1 had a plasma half-life that was more than 7-fold greater.
CONCLUSION In this report, we found that DB-GLP-1 and DBP-GLP-1 show enhanced GLP-1 intestinal absorption. Lys26,34-DiBiotinGLP-1 (DB-GLP-1) and Lys26-Biotin-Lys34-(Biotin-PEG)GLP-1 (DBP-GLP-1) were prepared using lysine-specific coupling reactions following HPLC separations. Both DB-GLP-1 and DBP-GLP-1, but especially DBP-GLP-1, had markedly better proteolytic stabilities than native GLP-1. Derivatization also preserved the biological and pharmacological activities of DB-GLP-1 and DBP-GLP-1. Furthermore, DBP-GLP-1 showed typical intestinal absorption patterns by pharmacokinetic profiling and a dramatically enhanced oral hypoglycemic effect versus native GLP-1. On the basis of these findings, we believe that DBP-GLP-1 is a good candidate antidiabetic peptide drug.
ACKNOWLEDGMENT This work was supported by a grant from the Ministry of Science and Technology of Korea (M10414030001-05N140300140).
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