Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6895
were performed using rat insulinoma of RIN-m5F cells (ATCC,
Manassas, VA) using a slight modification of a previously
described method.24 Briefly, RIN-m5F cells were maintained
in RPMI 1640 medium containing 10% fetal bovine serum and
1% penicillin/streptomycin at 37 °C in a humidified 5% CO2
incubator. They were seeded in 12 well plates at 3 ꢀ 105 cells per
well and grown for 48 h. They were washed twice with binding
buffer (120 mM NaCl, 1.2 mM MgSO4, 13 mM sodium acetate,
5 mM KCl, 1.2 g/L TRIS, 2 g/L bovine serum albumin, and
1.8 g/L glucose, pH 7.6) and cotreated with unlabeled BA-Ex4
samples (final concentration range 10-4-103 nM) and 30 pM of
125I-Exendin-4 (exendin-4 (9-39), PerkinElmer, Boston, MA)
for 2 h at room temperature. The cells were washed three times
with chilled PBS containing 1 mg/mL of bovine serum albumin,
lysed with cell lysis buffer (0.5 N NaOH with 1% SDS) for
15 min, and 125I contents in lysates were measured using a
γ-counter (GMI, Inc., Ransey MN).
Protein assay kit (PIERCE, Rockford, IL). In a similar manner,
the nonspecific absorptions of the peptides on albumin-free
resin were determined using NHS-inactivated resin.
The pharmacokinetic profiles of s.c. exendin-4 and BAM1-
Ex4s were assessed as previously described.24,29 Briefly, the
jugular veins of male Sprague-Dawley rats (body weight
200 g, 4 rats per group) were cannulated one day before the
experiment. Exendin-4 or its derivatives (10 nmol/rat, 200 μL,
s.c.) were then administered. Blood samples, drawn at prede-
termined times, were placed in chilled polyethylene tubes con-
taining anticoagulant (heparin solution, 5000 U/mL, 1/100
volume of blood), and plasma samples were obtained by cen-
trifugation and stored at -70 °C until required. The plasma
concentrations of exendin-4 and its derivatives were determined
using exendin-4 EIA kits (Phoenix Pharmaceuticals, Inc.,
Burlingame, CA). Data were analyzed for individual rats using
noncompartment models.
The insulinotropic activities of exendin-4 and BAM1-Ex4s on
rat pancreatic islets were determined as previously described.9,24
Briefly, male Sprague-Dawley rats (SD rats, 250 g body weight)
were anesthetized with ketamine and xylazine (90/10 mg/kg,
i.p.). After opening the abdominal cavity, the pancreas was
expanded by injecting cold Hank’s balanced salt solution
(HBSS) containing 1.5 mg/mL of type V collagenase, disinte-
grated by enzyme activation (37 °C, 15 min), and islets were
isolated and purified by discontinuous Ficoll (Amersham Bios-
ciences AB, Uppsala, Sweden) gradient centrifugation at 1900g
for 25 min. Purified islets were cultured in RPMI-1640 culture
medium supplemented with 10% fetal bovine serum (FBS,
Gibco, Carlsbad, CA) and 1% penicillin-streptomycin
(Gibco) at 37 °C in a 95% air/5% CO2 atmosphere. After a 2-
day maintenance period, islets were washed and incubated in
Krebs-Ringer HEPES (KRH) buffer (119 mM NaCl, 4.74 mM
KCl, 2.54 mM CaCl2, 1.19 mM MgCl2, 1.19 mM KH2PO4,
25 mM NaHCO3, 10 mM HEPES, and 1 g/L BSA), seeded at
20 islet/well on 24-well plates in 1 mL of KRH buffer containing
16.7 mM glucose or 2.2 mM glucose, and then were incubated
with 10 nM of exendin-4, CAM1-Ex4, DAM1-Ex4, or LAM1-
Ex4 for 2 h. Insulinotropic activities were evaluated by measur-
ing the amount of insulin released to media using an insulin
EIA kit.
In Vivo Antidiabetic Activities of BA-Ex4s. Six-week-old male
C57BL/6 db/db mice were used for the acute antidiabetic
activity tests, after being acclimatized for 1-week in our animal
facility. Under nonfasting conditions with free access to water
and food, mice were administered a single subcutaneous injec-
tion of exendin-4 or BAM1-Ex4s (15 nmol/kg, 200 μL, s.c.,
6 mice per group), and blood glucose levels were then monitored
using a glucometer and tail-tip blood samples (0, 0.5, 1, 2, 4, 6, 8,
12, 20, and 24 h after administration).
Statistical Analysis. Data are expressed as the means ( SDs.
The student’s t-test was used throughout, and values of p < 0.05
were considered statistically significant.
Acknowledgment. This work was supported by SEOK
CHUN Research Fund, Sungkyunkwan University, 2008.
Supporting Information Available: Purity table, HPLC chro-
matography and reaction optimization, HPLC analysis of DA-
Ex4s and LA-Ex4s, MALDI-TOF MS spectra of BA-Ex4s and
their Lys-C digested fragments, and the receptor binding char-
acteristics of BAM1-Ex4s and BADi-Ex4s. This material is
References
The in vivo bioactivities of exendin-4 and BAM1-Ex4s were
evaluated by intraperitoneal glucose tolerance testing (IPGTT)
in type 2 diabetic db/db mice after subcutaneous drug admin-
istration.24 In brief, 18 h-fasted mice were randomly allocated to
five groups (4 mice per group) and then administered normal
saline, exendin-4, or one of the three BAM1-Ex4s (10 nmol/kg,
s.c., 30 min prior to glucose administration). A 1.0 g/kg dose of
glucose was then administered intraperitoneally to each mouse.
At predetermined times, blood glucose levels were monitored
using a glucometer (Accu-Chek Sensor, Roche Diagnostics
Corp., Mannheim, Germany).
Physicochemical and Pharmacokinetic Characterizations of
the BAM1-Ex4s. The albumin binding characteristics of the
BAM1-Ex4s were investigated using a modified albumin-bind-
ing assay using albumin-conjugated Sepharose resin, according
to the manufacturer’s instructions. Briefly, human serum albu-
min (HSA; 30 mg in 10 mM PBS, pH 7.4) and NHS-activated
Sepharose 4 fast flow (4 mL, wet resin volume, Amersham
Bioscience, Sweden) were mixed and allowed to react by gentle
shaking at room temperature for 4 h. The albumin-conjugated
resin was then recovered by centrifugation, followed by triple
PBS washing. The HSA content in the resin used was 6-7 mg/
mL of wet resin. An albumin-free control resin was prepared by
inactivating the resin with hydrolysis of the NHS-active ester.
For the albumin binding study, exendin-4 or BAM1-Ex4s
(100 μg/mL in PBS, 50 μL) were mixed with HSA resin and
incubated for 2 h at room temperature. The resin and super-
natant were separated by centrifugation (1000 g, 10 min), and
unbound peptide contents were determined using a Micro BCA
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Son, Sohee
