Peptides 30 (2009) 18741881

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Peptides
journal homepage: www.elsevier.com/locate/peptides

A novel DPP-IV-resistant analog of glucagon-like peptide-1 (GLP-1): KGLP-1 alone

or in combination with long-acting PLGA microspheres
Zhihui Gao a, Yu Tang a, Jiaqi Chen a, Ru Bai b, Qi Zhang b, Yuanyuan Hou b, Yaxin Lu b, Gang Bai a,b,c,*
a

Department of Microbiology, College of Life Sciences, Nankai University, Tianjin, 300071, China
College of Pharmacy, Nankai University, Tianjin 300071, China
c
Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Tianjin State Laboratory of Microbial Functional Genomics, Tianjin, China
b

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 19 June 2009
Received in revised form 23 July 2009
Accepted 23 July 2009
Available online 6 August 2009

Glucagon-like peptide-1 (GLP-1) is an important hormone peptide secreted from the gastrointestinal
tract in response to nutrient ingestion. Its multifaceted actions make GLP-1 attractive as a candidate for
the treatment of type 2 diabetes mellitus. However, its main limitation is an extremely short half-life,
which is due to rapid inactivation by a ubiquitous enzyme, dipeptidyl peptidase-IV (DPP-IV). Therefore,
here we describe the development of a novel GLP-1 analog, designated KGLP-1. Initial in vitro
experiments revealed that KGLP-1 bound to and activated GLP-1R with similar efcacy as native GLP-1.
Importantly, KGLP-1 showed marked resistance to inactivation by DPP-IV. Further in vivo studies
conrmed that KGLP-1 had antihyperglycemic and insulinotropic actions after intraperitoneal injection
to KM mice and alloxan-induced diabetic mice. Finally, we prepared KGLP-1-loaded poly (D,L-lactic-coglycolic acid) microspheres (PLGA MS) using the solid in oil in oil (s/o/o) solvent extraction method,
which achieved controlled release and biological efcacy over a period of 10 days after a single
subcutaneous injection in alloxan-induced diabetic rats.
2009 Elsevier Inc. All rights reserved.

Keywords:
GLP-1
Type 2 diabetes
DPP-IV-resistant analog
Antihyperglycemic
Insulinotropic
Poly (D,L-lactic-co-glycolic acid)
microspheres (PLGA MS)

1. Introduction
Glucagon-like peptide-1 (GLP-1) is an incretin hormone that is
released from intestinal L-cells into the circulation in response to
orally ingested nutrients [15,18]. The therapeutic potential of GLP1 is exemplied by its spectrum of physiological actions, including
stimulation of insulin gene expression, stimulation of insulin
secretion, trophic effects on pancreatic islet b-cells, inhibition of
glucagon secretion, promotion of satiety, inhibition of food intake
and protracting gastric emptying [17]. The primary advantage of
GLP-1 over other antihyperglycemic drugs is that its administration does not induce hypoglycemia [25,31]. In recent years, the
potential for GLP-1 as a novel treatment strategy for type 2
diabetes has received considerable interest.
However, the benets of GLP-1 as a therapeutic agent are
limited by its short half-life, which is only 23 min because of its
rapid degradation and inactivation under physiological conditions
by dipeptidyl peptidase-IV (DPP-IV) [9]. DPP-IV is ubiquitously
expressed in mammalian organs and tissues and cleaves peptides
that contain penultimate Pro, Ala or hydroxyl Pro residues [22].

* Corresponding author at: College of Pharmacy, Nankai University, Weijin Road

94#, Tianjin, China. Tel.: +86 22 23508371; fax: +86 22 23508371.
E-mail address: [email protected] (G. Bai).
0196-9781/$ see front matter 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.peptides.2009.07.020

With respect to GLP-1, DPP-IV rapidly cleaves the His7Ala8

dipeptide from the N-terminus generating GLP-1 (9-36) amide. The
truncated form of GLP-1 is inactive and may even behave as a
receptor antagonist [20]. Therefore, various attempts to modulate
the level of GLP-1 to therapeutic advantage have recently
concentrated on two broad strategies: (1) subtle modications
of the GLP-1 molecule to generate analogs that are resistant to
DPP-IV; and (2) the use of selective DPP-IV inhibitors that are
capable of preventing in vivo GLP-1 degradation to increase the
circulation levels of the intact, biologically active peptide [8].
We previously screened a phage displayed (PhD) dodecapeptide library using a functional receptor gene high-throughput
screening assay system [4]. Consequently, KS-12 was chemically
synthesized, designated and tested further. Functional testing
revealed that KS-12 activated GLP-1R in vitro and reduced the
blood glucose level after intraperitoneal administration to KM
mice in a dose-dependent manner. Compared with GLP-1, the
bioactivity of KS-12 was limited, but it showed signicant
resistance to DPP-IV both in vitro and in vivo. It was proposed
that the N-terminal Lys of KS-12 was crucial for its resistance to
DPP-IV. Therefore, in continuation of our ongoing research
program, in this current study, we report a novel GLP-1 analog
designated KGLP-1, which was designed with an extra Lys in front
of the N-terminal His of the native GLP-1. We determined its
plasma stability and biological activity in vitro and in vivo.

Z. Gao et al. / Peptides 30 (2009) 18741881

Furthermore, KGLP-1-loaded poly (D,L-lactic-co-glycolic acid)

microspheres (PLGA MS) were prepared by solvent extraction
using solid in oil in oil (S/O/O) dispersion. The sustained-release
KGLP-1-loaded PLGA MS formulation achieved controlled release
in vivo to exert biological efcacy over a period of 10 days after a
single subcutaneous injection.

1875

to the animal facility for a minimum of 1-week before studies. The

experimental protocol was approved by the Animal Ethics
Committee of Nankai University, in accordance with Principles
of Laboratory Animal Care and Use in Research (Ministry of Health,
Beijing, China).
2.6. Induction of diabetes

2. Materials and methods

2.1. Reagents
Standard GLP-1 was from Sigma (St. Louis, MO, USA) and the
novel analog was synthesized chemically by GL Biochem
(Shanghai, China), designated as KGLP-1 (KHAEGTFTASVSSYLEGQAAKEFIAWLVKGR*). The cell culture media and reagents were
obtained from GibcoBRL Life Technologies (Gaithersburg, MD,
USA). The glucose kit was from BIOSINO Biotechnology and Science
Inc. (Beijing, China). The rat/mouse insulin enzyme-linked
immunosorbent kit was from Linco research (Linco Research,
Charles, USA). The poly (D, L-lactic-co-glycolic acid) (PLGA, 50:50,
molecular weight 15 kDa) was from Sigma (St. Louis, MO, USA). The
other reagents used in this study were of analytical grade and
commercially available.
2.2. Cell culture
Chinese hamster ovary (CHO) cells stably transfected with the
rat GLP-1 receptor and enhanced green uorescent protein (EGFP)
(carrying a cAMP-responsive EGFP reporter gene)(CHO/EGFP/GLP1R) [4] were cultured using RPMI-1640 tissue culture medium
containing 10% (v/v) fetal bovine serum, 105 U/L of penicillin and
0.1 g/L of streptomycin. All cells were maintained in sterile tissue
culture asks (Corning Inc, NY, USA) at 37 8C in an atmosphere of
5% CO2 and 95% air using a Heraeus BB16 incubator (Heraeus
Instruments, Hanau, Germany).
2.3. Receptor binding and activating studies
CHO/EGFP/GLP-1R cells were seeded in 96-well plates (Corning
Inc) at a density of 2  104 cells/well. After overnight culture
(37 8C; 5% CO2), various concentrations of GLP-1 or KGLP-1 diluted
with serum-free culture medium were added to the wells (100 mL/
well). After incubation at 37 8C in 5% CO2 for 8 h, the cells were
observed by inverted uorescence microscope, and the uorescence intensity was analyzed using Image Pro Plus 5.1 and
quantied using GraphPad Prism 5.0 software.
2.4. Stability test for DPP-IV in vitro
GLP-1 (1 nmol) or an equivalent amount of KGLP-1 was
incubated at 37 8C with 1 mL pig kidney microsomal membrane
extract (corresponding to 5 mU DPP-IV) for different times in
100 mL 0.1 M triethylamine/HCl buffer (pH 7.4). The reactions
were terminated by adding three volumes of acetonitrile. After
incubation for 2 h, the mixtures were centrifuged at 12,000 rpm for
10 min. Acetonitrile was removed from the supernatant by
vacuum evaporation, and the activities of the treated samples
were evaluated in CHO/EGFP/GLP-1R cells, as described above.

Diabetic mice and rats were established by a single intraperitoneal injection of 200 mg/kg alloxan monohydrate (dissolved in
saline just before use) (Sigma, USA). Animals had free access to
food and water after the alloxan injection. The animals were
considered diabetic if hyperglycemia (fasting blood glucose
levels > 11.0 mmol/L) was conrmed 72 h after the alloxan
injection.
2.7. Oral glucose tolerance test (OGTT)
Oral glucose tolerance tests were performed in KM mice and
alloxan-diabetic mice [29]. After an overnight fast (1618 h), the
animals were given peptides or saline by intraperitoneal injection.
At the appropriate time after dosing, the animals were given 1.5 g/
kg glucose orally through a gavage tube. A blood sample was drawn
from the tail vein at different times after glucose administration
and blood glucose levels were measured using a glucose kit from
BIOSINO Biotechnology and Science INC (Beijing, China). Blood
insulin levels were determined using a rat/mouse insulin enzymelinked immunosorbent kit (Linco Research).
2.8. Preparation of KGLP-1-loaded PLGA MS
Zinc-KGLP-1 compound microparticles were prepared rst, as
previously described [23,24]. Briey, a total of 10 mg KGLP-1 was
dissolved in distilled water and 2 mM zinc acetate solution was
added with KGLP-1 to zinc acetate at a molar ratio of 2:1. The pH of
the zinc-KGLP-1 solution was adjusted to pH 7.0. Then, 50 mg
PEG8000 was added to the solution and completely dissolved. The
solution was then lyophilized. The lyophilized powers were
reconstituted in 1 mL acetonitrile by vortexing for 5 min, and
centrifuged at 15,000 rpm for 5 min to remove the supernatant
which comprised PEG8000. The procedure was repeated three
times. The residue was placed under vacuum drier at 20 8C for 12 h
under a pressure of about 0.1 mPa.
Then, KGLP-1-loaded PLGA MS were prepared using the S/O/O
solvent extraction technique [28,30,33]. Briey, 1.5 mg of the zincKGLP-1 complex microparticles and 3 mg zinc carbonate microparticles (diameter < 5 mm) were added to a solution of PLGA
(100 mg) in 1 mL acetonitrile and suspended by sonication in
water bath. The suspension was added to 30 mL of peanut-blended
oil containing span80 (1 mL) and mechanically stirred (450 rpm for
2 h) (RCT B S25, IKA, China). Finally, 15 mL of petroleum ether was
added to harden the MS by continued stirring for 30 min. The MS
were collected by centrifugation and washed with petroleum ether
to remove the oil. The MS were dried under a vacuum (20 8C,
0.1 mPa) for 12 h to remove the residual solvent before further
assay.
2.9. Surface morphology, encapsulation efciency and in vitro release
of KGLP-1-loaded PLGA MS

2.5. Animals
Female Chinese Kunming (KM) mice and female Wister rats
(The Academy of Military Medical Science, Beijing, China) were
maintained on a 12 h light:12 h dark cycle and allowed free access
to standard rodent food and water, except where noted. KM mice
(78 weeks old) and Wister rats (67 weeks old) were acclimatized

The surface morphology of the MS was monitored by scanning

electron microscopy (SEM) (Quata200, USA), after they were
coated with gold palladium.
To determine the encapsulation efciency, the KGLP-1-loaded
PLGA MS were dissolved in dichloromethane (DCM) and KGLP-1
was extracted with isotonic phosphate-buffered saline (PBS,

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Z. Gao et al. / Peptides 30 (2009) 18741881

10 mM, pH 7.4) followed by reverse-phase high-performance

liquid chromatography (RP-HPLC) analysis. Briey, 10 mg of KGLP1-loaded PLGA MS were dissolved in DCM. Then, acetone was
added, and the vial vortexed and centrifuged at 12,000 rpm for
5 min. After the supernatant was removed, a 3:1 acetone: DCM
mixture was added. This washing procedure was repeated three
times. Finally, the residue was dissolved in PBS. KGLP-1 was
measured by RP-HPLC. A C18 (5 mm particles) 4.6 mm  250 mm
column was used and the elution condition was at a ow rate of
1 mL/min with an acetonitrile gradient in 0.1% triuoroacetic acid
(TFA) (3055% acetonitrile from 0 to 25 min, followed 100%
acetonitrile for 5 min). The detection wavelength was set at
220 nm. The encapsulation efciency of KGLP-1 was determined
based on the ratio of the actual amount of encapsulated peptides to
the amount of peptides used to prepare the MS (actual loading/
nominal loading).
KGLP-1 release from MS was studied in isotonic PBS (0.1 M,
pH 7.4) [10] containing 0.02% (v/v) Tween-80 and 0.02% (w/v)
sodium azide in a eppendorf tube at 37 8C using a water bath/
shaker (100 rpm), supernatants were collected after centrifugation at predetermined intervals. The removed medium was
replaced with fresh buffer to maintain the same pH and sink
condition. The withdrawn supernatants were analyzed by RPHPLC to measure the concentration of KGLP-1, as described
above. Meanwhile, the biological activities of the released KGLP1 samples were determined in CHO/EGFP/GLP-1R cells, as
described above, and compared with the KGLP-1 standard
solution.
2.10. Evaluation of the bioactivity of the KGLP-1 MS in diabetic rats
The in vivo biological activity of the KGLP-1-loaded PLGA MS
was determined using Wistar rats with alloxan-induced diabetes.
Briey, the alloxan-diabetic rats were divided into two groups of
10 animals each. Drinking water and a standard rodent maintenance diet were freely available. After an overnight fast, the
experimental animals were given a single subcutaneous injection
of KGLP-1-loaded or empty PLGA MS suspension. The KGLP-1 dose
was 5 mg/kg. Blood samples were obtained from the tail vein at
8:00 am everyday until the 14th day. Food was withdrawn at 18 h
prior to blood samples collection. At the designated times, 100 mL
of blood samples were obtained from the tail vein. The blood
glucose levels were monitored using a glucose kit from BIOSINO
Biotechnology and Science Inc. (Beijing, China). The blood insulin
levels were determined using a rat/mouse insulin enzyme-linked
immunosorbent kit (Linco Research).

2.11. Data analysis

All data are expressed as means  standard deviation (SD). The
areas under the curves (AUC) were calculated using GraphPad PRISM
version 5.0 (GraphPad Software, San Diego, USA), which used the
trapezoidal rule [2]. Statistical signicance was calculated using
Students t-test, one-way ANOVA followed by Tukeys post hoc test, or
two-way ANOVA followed by the Bonferoni post hoc test. Signicance
was set at P < 0.05.
3. Results
3.1. In vitro bioactivity analysis of KGLP-1
To determine whether KGLP-1 activates GLP-1R in CHO/EGFP/
GLP-1R cells in vitro, the intensity of EGFP expression was
evaluated. As shown in Fig. 1(A), KGLP-1 activated GLP-1R in a
dose-dependent manner. The EC50 for KGLP-1 was 2.44 nmol/L,
while the positive control, native GLP-1, exhibited a slightly
smaller EC50 of 0.83 nmol/L.
To evaluate the stability of KGLP-1 against degradation via DPPIV in vitro, a DPP-IV extract was prepared as described previously
[7] and the CHO/EGFP/GLP-1R cell line was used to evaluate the
activity of KGLP-1. GLP-1 or KGLP-1 was incubated with the DPP-IV
extract at 37 8C for times ranging from 0.5 to 12 h and the
percentage of active peptide remaining was assessed. After
incubation for 1 h, only 18.2% of active GLP-1 remained whereas
88.5% of active KGLP-1 remained. Furthermore, after 12 h
incubation, the EGFP expression stimulated by GLP-1 decreased
to 4.8%. In contrast, KGLP-1 revealed a marked enzymatic
resistance; and the activity was 79.8% after incubation for 12 h
(Fig. 1(B)).
3.2. Acute dose-dependent glucose-lowering effects of KGLP-1 in
normal KM mice
To investigate the dose-response relationship between KGLP-1
and glucose tolerance, an OGTT was performed after an
intraperitoneal injection of KGLP-1 (1, 10 or 100 nmol/kg) or
native GLP-1 (10 nmol/kg). As shown in Fig. 2(A), the blood
glucose level was signicantly lower in the peptide-treated
groups compared with saline-treated controls. Moreover, intraperitoneal KGLP-1 administration dose-dependently decreased
the glycemic excursion after glucose loading. In addition, the
analysis of blood glucose levels by DAUC0120 (Fig. 2(B))
conrmed these ndings and revealed that there was no

Fig. 1. Effects of KGLP-1 on EGFP expression and stability. (A) In vitro activity of GLP-1 (&) or KGLP-1 (&) in CHO/EGFP/GLP-1R cells. (B) Stability of KGLP-1 (&) against
enzymatic degradation by DPP-IV in comparison with native GLP-1 (&). All values were determined by assays in the CHO/EGFP/GLP-1R cell line, and are presented as
means  SD of triplicate measurements. The percent of maximal EGFP expression (% Maximal response) is shown.

Z. Gao et al. / Peptides 30 (2009) 18741881

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Fig. 2. Acute dose-dependent glucose-lowering activity of KGLP-1 in overnight fasted KM mice. (A) The OGTTs were performed after intraperitoneal administration of saline
(*) 10 nmol/kg of GLP-1 (^) or one of three doses of KGLP-1 (&) 1 nmol/kg; (~) 10 nmol/kg; (^) 100 nmol/kg). (B) Glucose DAUC0120 values for 0 to 120 min post glucose
loading. Values are means  SD for groups of six mice. *P < 0.05;**P < 0.01; and ***P < 0.001 compared with the saline group. (~) P < 0.05; (~~) P < 0.01; and (~~~) P < 0.001
compared with the GLP-1 group.

statistically signicant difference in the lowering of blood glucose

levels between the KGLP-1- and the GLP-1-treated groups at the
same dose (10 nmol/kg).
3.3. Sustained glucose-lowering and insulin-releasing effects of KGLP1 in normal KM mice
To evaluate the sustained metabolic actions of a single dose of
KGLP-1, an OGTT was performed at 0, 1, 2 and 4 h after
intraperitoneal administration of a single 10 nmol/kg dose of
KGLP-1 or GLP-1 in KM mice. As shown in Fig. 3(A), the glucoselowering effect of KGLP-1 was still evident at up to 2 h after
administration. There was a trend towards a decrease at 4 h after
dosing, although the difference did not reach statistical signicance. In contrast, the effect of native GLP-1 on glucose-lowering
lasted for less than 1 h.
The corresponding blood insulin responses are shown in
Fig. 3(B) and the results were consistent with those of the blood
glucose responses. KGLP-1 and GLP-1 both enhanced glucoseinduced insulin secretion when compared with the saline-treated
controls, but this effect of native GLP-1 was lost within 1 h.
However, in animals treated with 10 nmol/kg KGLP-1 at 2 h before
the OGTT, the increase in blood insulin levels was nearly twice as
high as the responses observed in saline or GLP-1-treated animals
(P < 0.001, respectively).

3.4. Acute glucose-lowering effects of KGLP-1 in alloxan-induced

diabetic mice
To ascertain the antihyperglycemic effect of KGLP-1 in the
diabetic model, an OGTT was performed at either 0 or 1 h after
peptide administration. The basal glucose levels in alloxaninduced diabetic mice treated with saline or with one of the
two peptides ranged from 11.71 to 12.69 mmol/L and did not differ
signicantly between the three groups. At 0 h (the time between
peptide injection and glucose loading was 0 h) after glucose
loading, the mean blood glucose level in the saline-control animals
increased to 20.21  1.41 mmol/L within 20 min, and was maintained during the entire study period. By contrast, in the KGLP-1- and
GLP-1-treated groups, the blood glucose levels increased to a peak
(16.41  1.22 mmol/L and 15.55  2.94 mmol/L, respectively) within
20 min and then decreased, nearly to the basal level, 120 min later.
Blood glucose levels analysis by DAUC0120 conrmed this and
revealed that the glucose-lowering effects of KGLP-1 and GLP-1 in this
0 h-study were equipotent (Fig. 4(A and C)).
However, when the time interval between peptide injection and
glucose loading was 1 h, a signicant difference was observed
between KGLP-1 and GLP-1. The mean blood glucose level in the
GLP-1-treated group increased to 18.70  2.76 mmol/L, but did not
decline signicantly during the 120 min period. Furthermore, there
were no signicant decreases in the glucose level or the corresponding

Fig. 3. Blood glucose levels (A) and blood insulin levels (B) in KM mice during an OGTT performed at different times after intraperitoneal administration of GLP-1 and KGLP-1.
Blood glucose levels and blood insulin levels 30 min after an OGTT performed at 0, 1, 2 and 4 h following intraperitoneal administration of GLP-2 or KGLP-1 (blank bars, saline;
striped bars, 10 nmol/kg GLP-1; lled bars, 10 nmol/kg KGLP-1). Values are means  SD. for groups of six mice. *P < 0.05; **P < 0.01; and ***P < 0.001 compared with the saline
group. (~) P < 0.05; (~~) P < 0.01; and (~~~) P < 0.001 compared with the GLP-1 group.

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Z. Gao et al. / Peptides 30 (2009) 18741881

Fig. 4. Glucose-lowering activity during an OGTT in alloxan-induced diabetic mice. Overnight fasted alloxan-induced diabetic mice were given saline (~) or 10 nmol/kg of
GLP-1 (&) or KGLP-1 (&) by intraperitoneal injection, and 0 h (A) or 1 h (B) later an OGTT was performed. The glucose DAUC0120 was determined and the percentage
reduction due to each treatment was calculated (C) (blank bars, saline; striped bars, 10 nmol/kg GLP-1; lled bars, 10 nmol/kg KGLP-1). Values are means  SD for groups of six
mice. *P < 0.05; **P < 0.01; and ***P < 0.001 compared with the saline group. (~) P < 0.05; (~~) P < 0.01; and (~~~) P < 0.001 compared with the GLP-1 group.

glucose DAUC0120 (2103.62  149.26 mmol/L  min for GLP-1 versus

2170.03  134.78 mmol/L  min for saline). By contrast, KGLP-1
markedly improved glucose tolerance, with signicantly lower blood
glucose levels throughout the study period compared with both saline
and native GLP-1 (Fig. 4(B and C)).

1stimulation. The amount of KGLP-1 released as measured by the

cell line or by HPLC was generally comparable. Considering the
KGLP-1 concentration measured by HPLC, the bioactivity evaluation test conrmed that the KGLP-1 released from MS maintained
its activity for activating GLP-1R (Fig. 6(B)).

3.5. Surface morphology and in vitro release of KGLP-1-loaded

PLGA MS

3.6. Evaluation of the bioactivity of KGLP-1-loaded PLGA MS in

diabetic rats

Surface morphology of the KGLP-1 MS was examined by SEM

and micrographs are shown in Fig. 5. The MS had a regular
spherical shape with a relatively smooth surface and a homogeneous size. The particle diameter was 33.5  6.1 mm.
The encapsulation efciency of the KGLP-1-loaded PLGA MS
was 85.5%. We determined the in vitro release of KGLP-1 from the
PLGA polymer, and the data are shown in Fig. 6(A). The graph
shows the percentage cumulative amount of KGLP-1 released over
2 weeks. The KGLP-1-loaded PLGA MS released the entrapped
peptide with a release rate of approximately 15.3% peptide during
the rst 6 h, followed by the release of 54.2% of the peptide over 2
weeks.
In addition, the biological activity of KGLP-1 released from MS
was conrmed using CHO/EGFP/GLP-1R cells. The assay measured
the intensity of EGFP expression in the cell line owing to KGLP-

We investigated the bioactivity of KGLP-1-loaded PLGA MS in

vivo using alloxan-induced diabetic rats. Fig. 7(A) shows the blood
glucose levels over 14 days. Injection of the empty polymer had no
effect on the blood glucose level, which was maintained at 15.49
19.15 mg/dL during the 14-day period. In contrast, a rapid decrease
in blood glucose level decrease was observed within 6 h after the
injection of KGLP-1-loaded PLGA MS. Furthermore, signicant
glucose-lowering activity was found up to day 10 after administration. This indicates that the KGLP-1-loaded PLGA MS provided
adequate initial release of KGLP-1, and controlled the KGLP-1
release in vivo to maintain the blood glucose levels.
The blood insulin prole in response to KGLP-1 release from the
PLGA MS is shown in Fig. 7(B). Injection of the empty MS had no
effect on insulin levels, which were maintained at a constant level
of 0.370.45 ng/mL. Injection of the KGLP-1 MS increased the

Fig. 5. Scanning electron micrographs showing the surface morphology of KGLP-1-loaded PLGA MS. (A) An individual KGLP-1-loaded PLGA MS; (B) A group of KGLP-1-loaded
PLGA MS.

Z. Gao et al. / Peptides 30 (2009) 18741881

1879

Fig. 6. In vitro release of KGLP-1-loaded PLGA MS. (A) Cumulative release of KGLP-1 from MS. (B) Actual KGLP-1 levels at selected time points. The amount of KGLP-1 released
from KGLP-1-loaded MS was measured at the indicated times using HPLC (&) or CHO/EGFP/GLP-1R cells (&). Values are means  SD; n = 3.

Fig. 7. Blood glucose level and blood insulin level in alloxan-induced diabetic rats after injection of KGLP-1-loaded PLGA MS. Overnight fasted alloxan-induced diabetic rats
were given empty (&) or KGLP-1-loaded PLGA MS (*) by intraperitoneal injection. The blood glucose levels (A) and blood insulin levels (B) were assayed at the indicated
times. Values are means  SD for groups of ve rats.

insulin levels to 0.480.54 ng/mL initially, a level that was

maintained for several days with a subsequent gradual decrease
over 10 days. The change in blood insulin level over time was
consistent with the blood glucose prole. Subcutaneous injection
of KGLP-1 MS was well tolerated, and there were no signs of
chronic inammation around the injection sites during the
experiment.
4. Discussion
The gastrointestinal hormone GLP-1 possesses a number of
antidiabetic actions, across a range of physiological systems to
enhance blood glucose tolerance. Of note, the potent insulinotropic
activity of GLP-1 is in a glucose-dependent mode, and is, therefore,
a key advantage in reducing the risk of hypoglycemia [1,14].
Clinical studies using GLP-1 in people with type 2 diabetic have
demonstrated that there is a considerable therapeutic potential to
be gained by using this hormone [16,34]. However, the rapid
degradation of GLP-1 by the proteolytic enzyme DPP-IV is the main
obstacle to its potential use as a new treatment modality. An
alternative approach is to develop DPP-IV-resistant and potentially
more potent GLP-1 analogs by modifying the primary structure of
GLP-1 [32].
The rst amino acid present in GLP-1 is His and numerous
modications of this amino acid have been evaluated [6,12,13,27].
Most modications convey DPP-IV resistance, but receptor
activation and biological activity can vary signicantly. For
example, N-acetyl-GLP-1 and N-ptyoglutamyl-GLP-1 are His7modied analogs of GLP-1 that are completely resistant to
degradation by either DPP-IV or human plasma, and bound to
the GLP-1 receptor, but with less afnity compared with native

GLP-1. Both analogs decreased the plasma glucose concentrations

and increased the insulin levels when administered in conjunction
with glucose in adult obese diabetic mice [7]. In addition to these
analogs in study, some of the most advanced development
candicates are in clinical stage, such as exenatide (the best studied
GLP-1 receptor activator) and liraglutide (a human GLP-1 analog).
Exenatide has been approved for combination administration with
metformin and sulfonylureas by FDA, and mild to moderate nausea
is the most common adverse event [26]. Liraglutide is in the clinical
stage of phase III. It once a day provided signicantly greater
improvements in glycaemic control than did exenatide twice a day,
and was generally better tolerated. Liraglutide might be a
treatment option for type 2 diabetes, especially when weight loss
and risk of hypoglycemia are major considerations [3]. Additionally, long-acting release formulations of GLP-1 and exenatide have
been studied [5,19,33]. Outstandingly, exenatide-LAR developed
by Amylin once weekly resulted in signicantly greater improvements in glycaemic control than exenatide given twice a day, with
no increased risk of hypoglycemia and similar reductions in
bodyweight [11].
In this present study, we developed a novel GLP-1 analog,
designated KGLP-1, which had an extra Lys in front of the Nterminal His. First, the in vitro studies demonstrated that KGLP-1
was resistant to DPP-IV degradation. XaaPro and XaaAla
dipeptides at the N-terminus are the recognizing sequence for
DPP-IV action. Accordingly, GLP-1 undergoes degradation by DPPIV because the N-terminal dipeptide is HisAla. In the case of
KGLP-1, the N-terminal of GLP-1 was extended by attaching an
extra Lys to His7. Thus, its N-terminus dipeptide LysHis, instead of
HisAla, conferred signicant resistance to DPP-IV. Second,
functional testing using CHO/EGFP/GLP-1R cells revealed that

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Z. Gao et al. / Peptides 30 (2009) 18741881

KGLP-1 showed receptor afnity and in vitro biological activity that

were comparable with native GLP-1 (The EC50 values for KGLP-1
and GLP-1 were 2.44 nmol and 0.83 nmol, respectively). This
indicates that this modication of the N-terminus of GLP-1 did not
lead to a serious loss of afnity of the synthetic ligand towards the
GLP-1 receptor. The slight decrease in receptor binding and
activation efciency may be attributed, in part, to the steric
hindrance between the imidazole ring of His7 and Lys. Meanwhile,
further animal experiments in normal KM mice or alloxan-induced
diabetic mice conrmed that KGLP-1 retained pharmacological
properties that were similar to native GLP-1, and signicantly
extended the timeaction proles (e.g. glucose-lowering and
insulin-releasing). KGLP-1 reduced the blood glucose levels after
intraperitoneal administration to KM mice in a dose-dependent
manner. At the same moderate dose, the glucose-lowering ability
of KGLP-1 was comparable with native GLP-1. Although native
GLP-1 immediately showed signicant glucose-lowering and
insulin-secreting effects after administration in normal or diabetic
model mice, the effect disappeared 1 h after administration.
Compared with native GLP-1, the signicant blood glucoselowering and insulinotropic activity of KGLP-1 lasted for at least
2 h after administration, demonstrating that KGLP-1 has a longer
biological action. The improved glycemic control of KGLP-1
compared with native GLP-1 probably resulted from an increasing
circulating half-life due to its resistance to DPP-IV. The inhibition of
KGLP-1 degradation would increase the availability of the intact
peptide, while also reducing the possible effect of feedback
antagonism at the receptor level through a reduction in the
formation of the receptor antagonist GLP-1 (9-36) amide.
It should be noted, however, that any GLP-1 derivative that is
only modied so that it is not degraded by DPP-IV would probably
still be characterized by rapid renal clearance [21]. Based on the
prolonged in vivo bioactivity of KGLP-1, in the range of 24 h, twice
or three times daily administration is still necessary to maintain
the plasma concentrations at appropriate levels.
Therefore, a sustained-release formulation may be more
convenient, reducing the need for multiple daily injections, and
improving compliance and the control of glycemia. For this
purpose, we prepared KGLP-1 entrapped in long-acting injectable
MS using the s/o/o solvent extraction method. In vitro, the
bioactivity of the controlled release KGLP-1 was sustained for
14 days. Animal studies using alloxan-induced diabetic rats have
been performed to evaluate the bioactivity of the KGLP-1 released
from the MS. After a single subcutaneous injection of KGLP-1loaded PLGA MS, the blood glucose level decreased to a level that
was signicantly lower than that with the empty MS, an effect that
lasted for 10 days. Similarly, the insulin level reached and stayed at
higher levels for nearly 10 days. This means that the continuous
release of KGLP-1 from the subcutaneously injected KGLP-1loaded PLGA MS had sustained glucose-lowering and insulinotropic effects. The KGLP-1 MS formulation achieved controlled
release in vivo for 10 days and exhibited sustained long-term
pharmacological efcacy to maintain stable blood glucose level in a
diabetic rat model.
5. Conclusions
In this study, we engineered a novel GLP-1 analog KGLP-1. By
assessing the stability, receptor binding and activating, insulinsecreting and antihyperglycemic effects of KGLP-1, we found that
KGLP-1 is an effective GLP-1 analog with marked resistance to DPPIV degradation and retained bioactivity that is normally associated
with native GLP-1 both in vitro and in vivo. Furthermore, KGLP-1loaded PLGA MS were prepared and, after a single injection in
diabetic rats, the effects of KGLP-1 on glucose control were
sustained for 10 days. The preliminary studies predict that this

sustained-release formulation may be more convenient, reducing

the need for multiple daily injections, and improving compliance
and the control of glycemia, and is a considerable therapeutic
potential for type 2 diabetes.
Acknowledgments
This work was supported in part by the National Key Basic
Research and Development Program of China (973 Program)
(2007CB914803) and Grant No.08QTPTJC28600 from the Natural
Science Foundation of Tianjin, China.
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