Phenylbutazone, a New Long-Acting Agent that can Improve the Peptide Pharmacokinetic Based on Serum Albumin as a Drug Carrier

Article abstract of DOI:10.1111/cbdd.12726

As a NPY-2 receptor agonist, PYY_24-36-Leu₃₁ is reported to suppress appetite and has a potential in obesity treatment, but its short half-life limits the clinical application. The use of chemical modification to improve interactions

Full text of DOI:10.1111/cbdd.12726

Chem Biol Drug Des 2016; 87: 936–945
Research Article
Phenylbutazone, a New Long-Acting Agent that can
Improve the Peptide Pharmacokinetic Based on Serum
Albumin as a Drug Carrier
regulation of food intake is the NPY family (3–5). The
observation that acetylation of PYY24–36-Leu31(PYY24–36
Jie Zhou, Xue Li, Xiaoyun Zhu, Jian Sun,
Qianqian Qiu, Wenlong Huang* and Hai Qian*
)
(6–8) increases NPY-2 receptor affinity while maintaining
specificity against the NPY-1 and NPY-5 receptors sug-
gests that further improvements may be obtained with
more complex modifications of the N-terminal amino group
of PYY24–36 (9,10). But short half-life limits its clinical appli-
cation (11). Albumin is emerging as a versatile protein car-
rier for drug targeting and for improving the
pharmacokinetic profile of peptide or protein-based drugs
(12). The use of chemical modification to improve covalent
or non-covalent interactions with serum albumin is an
effective strategy for prolonging the half-lives of peptide
analogues (13,14). At present, the most common used
long-term peptide modified agents are fatty chain, PEG,
and bile acid. They are many successful case; acylation
(attachment of a fatty acid side chain) facilitated non-cova-
lent binding to serum albumin in the GLP-1 analogue
liraglutide (15). This delayed both the absorption and clear-
ance rates of the compound. D-Ala8,Lys37[2-[2-[2-maleimi-
Center of Drug Discovery, State Key Laboratory of Natural
Medicines, China Pharmaceutical University, Nanjing
210009, China
*Corresponding authors: Wenlong Huang,
ydhuangwenlong@126.com; Hai Qian,
qianhai24@163.com
As
a NPY-2 receptor agonist, PYY24–36-Leu31 is
reported to suppress appetite and has a potential in
obesity treatment, but its short half-life limits the clini-
cal application. The use of chemical modification to
improve interactions with human serum albumin (HSA)
is an effective strategy for prolonging the half-lives of
peptide analogues. So based on the characteristics
that phenylbutazone has a good combination with
HSA, we selected a proper linker to link with PYY24–36
-
Leu31 to create long-acting and highly biologically
active PYY24–36-Leu31 conjugates, and successfully find
a novel, long-acting PYY24–36-Leu31 conjugate 8 that,
when dosed every other day in diet induce obese (DIO)
mice for 2 weeks, results in a significant reduction in
food intake and body weight and improvement in
blood parameter and hepatic steatosis.
dopropionamido(ethoxy)-ethoxy]acetamide
GLP-1(7–37)
(CJC1131), a GLP-1 analogue with a reactive maleimide
group at the carboxyl terminus, binds covalently to serum
albumin following injection (14).
This subject is to find a new long-acting method, which
can be widely used to improve peptides pharmacokinetic
profile. Previous studies have found that an anti-inflamma-
tory drug, phenylbutazone (16,17), has a strong combining
ability with human serum albumin (HSA). So based on this
characteristic, we carefully modified its proper group to link
with PYY24–36 (Figure 1) to create long-acting and highly
biologically active PYY24–36 conjugates.
Key words: anorexia, HSA, obesity, PYY24–36 conjugate,
weight loss
Abbreviations: DIO, diet induce obese; DMAP, 4-dimethyla-
minopyridine; HSA, human serum albumin; OGTT, oral glu-
cose tolerance test; PYY24–36
trifluoroacetic acid.
,
PYY24–36-Leu31
;
TFA,
Received 10 November 2015, revised 30 December 2015
and accepted for publication 7 January 2016
So here we synthesized three modifying groups (the two
known molecules—bile acid and fatty acid chain, and a
new molecule–phenylbutazone) that allow for amidation
with PYY24–36 N-terminus to improve peptide duration of
action in vivo. The structural properties (Table. 1), in vitro
biological activity, and physicochemical characteristics of
1–9 were explored. The studies result in a novel, long-act-
ing NPY2 receptor agonist 8 that, when dosed every other
day in DIO mice, results in a significant reduction in food
intake and body weight and improvement in blood param-
eter and hepatic steatosis.
Obesity presents an increasing public health burden that is
associated with several hyperlipemia, hypertension,
osteoarthritis, type 2 diabetes, and other illnesses, and
with reduced life expectancy. Current therapeutic options
are limited, and the unmet medical need for new, effective
treatments is high (1). Several neurological pathways con-
tribute to food intake and energy homeostasis that moder-
ate appetite, weight maintenance, and weight gain (2).
One family of G protein-coupled receptors involved in the
936
ª 2016 John Wiley & Sons A/S. doi: 10.1111/cbdd.12726

A New PYY24–36 Conjugate Greatly Reduces Weight
Figure 1: Phenylbutazone visually dock with HSA.
Table 1: Sequence and experimental data for PYY24–36-Leu31 conjugates
Molecular mass
Name
1
R
Calculated
Found
1081.8[M + 2H]2+
721.5[M + 3H]3+
1081.4[M + 2H]2+
721.5[M + 3H]3+
O
OAc
H
H
H
AcO
H
O
2
3
555.9 [M + 4H]4+
555.1[M + 4H]4+
N
H
2
OAc
O
H
H
H
AcO
H
O
1159.1[M + 2H]2+
773.3[M + 3H]3+
1159.4[M + 2H]2+
773.4[M + 3H]3+
N
H
5
O
OAc
H
H
H
AcO
H
O
4
5
1201.3[M + 2H]2+
801.4[M + 3H]3+
1201.3[M + 2H]2+
801.2[M + 3H]3+
N
H
11
O
OAc
H
H
H
AcO
H
1102.9[M + 2H]2+
1103.7[M + 2H]2+
Chem Biol Drug Des 2016; 87: 936–945
937

Zhou et al.
Table 1: continued
Molecular mass
Calculated
Name
R
Found
735.6[M + 3H]3+
735.6[M + 3H]3+
O
H
AcO
OAc
H
6
7
8
9
964.7[M + 3H]3+
643.5[M + 4H]4+
963.9[M + 3H]3+
643.2[M + 4H]4+
O
n
n = 5
992.7[M + 2H]2+
662.2[M + 3H]3+
992.4[M + 2H]2+
662[M + 3H]3+
O
n
n = 7
717.5[M + 3H]3+
538.4[M + 4H]4+
717.4[M + 3H]3+
538.3[M + 4H]4+
O
N
N
n
O
O
n = 4
712.9[M + 3H]3+
534.9[M + 4H]4+
712.7[M + 3H]3+
534.7[M + 4H]4+
O
N
N
n
O
O
n = 3
Institutes of Health (NIH publication 85–23, revised
1986). The structure of HSA crystalline complex (ID:
2BXP) was searched in PDB database and resolved by
LIGANDSCOUT V2.02 program (Inteligand GmbH, Vienna,
Austria). 1H NMR spectra were recorded on a 300 MHz;
chemical shifts are given in ppm (d) relative to TMS as
internal standard; coupling constants (J) are in hertz (Hz);
and signals are designated as follows: s, singlet; d, dou-
blet; t, triplet; m, multiplet; br s, broad singlet, etc.).
Methods and Materials
Materials
Peptide PYY24–36-Leu31 and analogues were synthesized
using a CEM microwave peptide synthesizer. Fmoc Rink
Amide MBHA resin and Fmoc-protected amino acids
were obtained from GL Biochem (Shanghai, China).
HPLC grade acetonitrile and methanol were purchased
from Merck (Darmstadt, Germany). All other reagents,
unless indicated, were purchased from Sigma-Aldrich
(Saint Louis, MO, USA). The HPLC analysis was per-
formed on a Shimadzu 2010C HPLC system (Nakagyo-
ku, Kyoto, Japan). For purification, Shimadzu LC-10
preparative RP-HPLC system was used. The ESI-MS
spectra of the peptides were obtained with Waters
ACQUITY UPLC Systems with Mass (Waters, Milford,
MA, USA). C57BL/6J and ICR mice (male, 8 weeks old)
were purchased from the comparative medical center of
Yangzhou University (Jiangsu, China). All animal experi-
mental protocols adhered to the Guide for the Care and
Use of Laboratory Animals published by the National
Peptide PYY24–36-leu31 and derivatives synthesis
and characterization
PYY24–36-Leu31 H-LRHWLNLLTRQRW-NH2 were synthe-
sized using standard solid-phase methodology on a Rink
amide MBHA resin with a microwave synthesizer (CEM,
Matthews, NC, USA). Microwave irradiation was applied in
coupling and deprotection steps at 2450 MHz continu-
ously. The final protected peptidyl-resin was deprotected
with 20% piperidine (v/v) in dimethylformamide to remove
Fmoc (18,19).
938
Chem Biol Drug Des 2016; 87: 936–945

A New PYY24–36 Conjugate Greatly Reduces Weight
The N-Terminal peptide modification
was extracted with DCM (3*30 mL). The combined
organic layer was washed with water (20 mL) and brine
(20 mL), dried over anhydrous Na2SO4, filtered, and con-
centrated to afford light yellow solid. The product was
directly put into use for next step. To the I-1 in dichloro-
methane was added dichlorosulfoxide (2equiv) and DMF
three drops. The solution was stirred for 3.5 h and con-
centrated under reduced pressure. The oil was dissolved
in 10 mL THF and waited for use.
To the above peptidyl-resin, five equivalents of each small
molecule were manually added into CEM vessel. And the
coupling procedures were carried on for 30 min with
microwave irradiation at 2450 MHz. Upon completion of
the synthesis, peptides derivatives were cleaved from the
solid support with removal of side chain protecting groups
by treatment with aqueous trifluoroacetic acid (TFA). Pep-
tides were purified using a Shimadzu LC-10 preparative
RP-HPLC and a C18 RP column (5, 340, 28 mm) with
gradient elution using (A) water with 0.1% TFA and (B)
methanol with 0.1% TFA as the mobile phase. The gradi-
ent was developed from 20% B to 80% B over 60 min at
a flow rate of 5.0 mL/min, with UV detection at 214 nm.
The purity of peptides administered to the mice was over
90%. HPLC analysis was performed on a Shimadzu
2010C HPLC system with gradient elution using (A) water
with 0.1% TFA and (B) acetonitrile with 0.1% TFA as the
mobile phase. The gradient was developed from 20% B to
80% B over 20 min at a flow rate of 1.0 mL/min, with UV
detection at 214 nm.
To a solution of b-alanine, 6-aminocaproic acid or 12-ami-
nolauric acid (1.5equiv) in 1N aqueous sodium hydroxide
respectively, added dropwise the above THF solution at
0 °C, and warmed to RT. After stirring for 4 h, the reaction
mixture was concentrated in vacuo and H2O was added.
The mixture was extracted with diethyl ether. The com-
bined organic extracts were washed with brine (15 mL),
dried over Na2SO4, filtered, and concentrated in vacuo.
The residue was purified by silica gel chromatography (PE:
EtOAc, 1:2?1:1) to furnish the desired product I-2, I-3,
I-4. They were directly used to amide with peptide.
Small molecule chemical synthesis
General procedure II for the preparation of II-3,
II-4
General procedure I for the preparation of I-1, I-2,
I-3, I-4
Methyl 6-bromohexanoic (2.6 g, 11.7 mmol) or methyl
5-bromopentanoic (2.45 g, 11.7 mmol) combined with
butazodine (3 g, 9.7 mmol) were dissolved in 20 mL THF.
A total of 60% sodium hydride (0.58 g, 14.6 mmol) was
added to the solution above and refluxed for 1.5 h and
add ice water to quench the reaction (Scheme 2). Then,
the mixture was extracted with DCM (3*30 mL). The
Deoxycholic acid (1 g), acetic anhydride (0.524 mL), and
DMAP in catalytic amount were dissolved in mixture sol-
vent of pyridine (10 mL) and DCM (10 mL) (Scheme 1). It
was stirred for 20 h at RT. The reaction was monitored by
TLC and added 1N HCl for termination. Then, the mixture
Scheme 1: Synthetic route for deoxycholic acid derivatives.
Scheme 2: Synthetic route for phenylbutazone derivatives.
Chem Biol Drug Des 2016; 87: 936–945
939

Zhou et al.
combined organic layer was dried over anhydrous
Na2SO4, filtered, and concentrated to be purified by silica
gel chromatography (PE:EtOAc, 1:2?1:1). The oil product
was dissolved in the mixtures of 10 mL methanol and
THF. Lithium hydroxide (0.32 g, 6.53 mmol) in 10 mL
water was added to the above solution, which was stirring
for 8 h at RT. The reaction was monitored by TLC and
added with 1N HCl to pH 5~6. Then, the mixture was
extracted with EA (3*30 mL). The combined organic layer
was dried over anhydrous Na2SO4, filtered, and concen-
trated. The residue was purified by flash column chro-
matography on silica gel using PE:EtOAc (1:4) as eluent to
obtain light yellow oil.
CH3), 1.12–1.60(m, 8H), 2.25(t, 2H, -CH2(CH2)3CH2COO),
2.41(t, 2H, -CH2(CH2)2CH3), 4.10 (t, 2H, -CH2(CH2)4COO),
5.13(s, 2H, PhCH2-), 7.04–7.5(m, 15H, 4 Ar-H).
Compound stability in rat plasma
The stability of these conjugates was assessed in rat
plasma collected from adult male SD rats, as previously
described (20). The plasma was stored at À80 °C until
needed. In vitro stability of 1–9 and PYY24–36 were mea-
sured using an initial concentration of 1000 ng/mL of each
peptide in rat plasma at 37 °C. An amount of 100 lL of
plasma was removed from the incubations at 0, 1, 2, 4, 6,
8, 12, and 24 h time-points and subjected to solid-phase
extraction on a Waters Oasis HLB 96-well plate (Milford).
An amount of 20 lL of extract was injected onto the
LC–MS/MS system. Peptides were detected by multiple
reactions monitoring (MRM) using electrospray ionization
mass spectrometry (ESI-MS) on an Applied Biosystems
Sciex API-4000 instrument (Foster City, CA, USA) fitted
with a TurboIonSpray source. RPLC separation was per-
formed on a Waters Acquity UPLC HSS T3 column
(1.8 lm, 2.1 9 100 mm) equilibrated in buffer A (water,
0.2% formic acid), and elution was with a linear gradient of
Synthesis of 6-(4-butyl-3,5-dioxo-1,2-diphenylpyrazolidin-4-
yl)hexanoic acid (II-3): Yield: 43.1%; MS (ESI) m/z: 423.6
(M + H)+;1H-NMR (CDCl3, 300 MHz): d(ppm) 0.94 (t, 3H, -
CH3), 1.14–1.60(m, 10H), 2.22(t, 2H, -CH2(CH2)4CH2COO),
2.39(t, 2H, -CH2(CH2)2CH3), 4.09 (t, 2H, -CH2(CH2)4COO),
5.1(S, 2H, PhCH2-), 7.03–7.43(m, 15H, 4 Ar-H).
Synthesis of 6-(4-butyl-3,5-dioxo-1,2-diphenylpyrazolidin-4-
yl) pentanoic acid (II-4) Yield: 46%; MS (ESI) m/z: 411.7
(M + H)+; 1H-NMR (CDCl3, 300 MHz): d(ppm) 0.95 (t, 3H, -
Figure 2: Degradation of PYY24–36 and its
conjugates (1–9). Data are expressed as the
mean Æ SD.
Figure 3: Correlation between the plasma
stability and albumin binding of PYY24–36
conjugates (1–9).
940
Chem Biol Drug Des 2016; 87: 936–945

A New PYY24–36 Conjugate Greatly Reduces Weight
Figure 4: Acute effect on cumulative food
intake for 8 h after an overnight fast in ICR
mice, i.p. administration of PYY24–36 or its
conjugates all at 1 lmol/kg.
5–95% buffer B (acetonitrile, 0.2% formic acid) over 4 min
containing a preweighted amount of food that was
at a flow rate of 0.3 mL/min.
reweighted at 0.5, 1, 2, 4, 8 h after injection.
Albumin binding
Two-week study in lean ICR mice
The albumin-binding characteristics of the PYY24–36 conju-
gates 1–9 were investigated using an albumin-binding
assay using albumin-conjugated Sepharose resin (15).
NHS-activated Sepharose 4 fast flow (5 mL, wet resin vol-
ume, Amersham Bioscience, Uppsala, Sweden) and
human serum albumin (HSA; 33 mg in 10 mM PBS,
pH7.4) were mixed and allowed to react by gentle shaking
at room temperature for 4.5 h. The albumin-conjugated
resin was then recovered by centrifugation (278 9 g,
5 min) and followed by 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. PYY24–36 or conju-
gates 1–9 (1 lmol/mL in PBS, 50 mL) were mixed with
HSA resin and incubated for 3 h at room temperature.
The resin and supernatant were separated by centrifuga-
tion (278 9 g, 10 min), and unbound peptide contents
were determined using a Micro BCA Protein assay kit
(PIERCE, Rockford, IL, USA). The non-specific absorptions
of the peptides on albumin-free resin were determined
using NHS-inactivated resin in a similar manner.
In a follow-up experiment, the mice were dosed above
peptides intraperitoneally every other day for 2 weeks.
Body weight gain, food, and water intake of the mice were
recorded every days during the experiment.
Effect on body weight of every other day dosing
for 2 weeks in DIO mice
Male C57BL/6 mice (Qinglong Farms, Inc., Nanjing, China)
were fed a high-fat diet containing 45% calories from fat
(Research Diets, D12451) for 20–22 weeks and had an
average body weight over five standard deviations greater
than mice fed a standard laboratory diet (10% calories
from fat). A treatment group comprised six mice per treat-
ment group with body weights of approximately 44 g.
Mice were kept in standard animal rooms under controlled
temperature and humidity and a 12/12 h light dark cycle.
Food and water were continuously available. Mice were
assigned to treatment groups based on their body weight
so that the initial mean and SEM of body weight were sim-
ilar. Animals were administered peptide in 0.9% saline
intraperitoneally every other day before the dark phase,
and body weight and food consumption were measured.
On the final day, and body weight was measured.
Acute feeding studies in lean ICR mice
Forty-two lean male ICR mice (20–25 g) were maintained
under controlled conditions of temperature (21–23 °C) and
12 h light/12 h dark cycle. Mice were housed in pairs in
cages with water and food (standard chow pellet diet) con-
tinuously available. The mice were fasted overnight with
water available during the dark phase, and on the morning
of study, mice (n = 6/group) were distributed by weight into
Oral glucose tolerance test of 8 in DIO mice
Mice were fasted for 16 h with free access to water before
basal blood glucose was measured (t = À30 min). Com-
pound 8 (1 lmol/kg) or PYY24–36-Leu31 (1 lmol/kg) and
0.9% saline were then intraperitoneally injected, and an
oral glucose load of 2 g/kg was administered to all ani-
mals. Blood glucose levels were measured at 0, 30, 60,
90, and 120 min postglucose load using blood glucose
monitor (SanNuo; Changsha Company, Changsha, China).
treatment groups and dosed intraperitoneally with PYY24–36
,
2, 5, 7, 8, 9 in USP saline (1 lmol/kg) in the early light phase
(9–10 h). Control groups were dosed with 0.9% saline. After
injection, animals were returned to their home cages
Chem Biol Drug Des 2016; 87: 936–945
941

Zhou et al.
Bioengineering Institute, Nanjing, China). All assays were
performed according to the manufacturers’ instructions.
Statistical analysis
The statistical analyses were performed using GRAPHPAD
PRISM version 5.0 (GraphPad Software Inc., La Jolla, CA,
USA) and EXCEL 2010 (Microsoft Corp., Richmond, WA,
USA). The results are shown as mean Æ SEM and com-
pared using the unpaired Student’s t-test. Groups of data
were considered to be significantly different if p < 0.05.
Results and Discussion
Figure 5: Body weight increase of 2 weeks (bar graph) and total
food intake at day 0 (line chart) in ICR lean mice.
Stability of the PYY24–36 conjugates in rat plasma
Nine conjugates (1–9) were incubated with plasma taken
from SD rats over 24 h and compared with PYY24–36
.
Blood parameters
Solid-phase extraction was conduct at each time-point,
and the extract was analyzed using LC–MS/MS to exam-
ine plasma stability (Figure 2). PYY24–36 possessed a half-
life of less than 0.5 h at 37 °C. All PYY24–36 conjugates
showed better stability than PYY24–36. The half-lives of
Blood was collected after a 6-h fast from tail veins using
Microvette tubes and stored at room temperature for 2 h.
After 15 min of centrifugation at 3000 9 g and 4 °C,
plasma was stored at –80 °C. Plasma adiponectin and
leptin were measured by Elisa kits (Nanjing Jiancheng
Figure 6: Two-week body weight loss studies in C57BL/6 DIO mice with PYY24–36 and 8 (A–C): (A) Oral glucose tolerance at day 14. (B)
Dynamic body weight change for 2 weeks. (C) Epididymis cell number. **p < 0.01; ***p < 0.001 compared with control group;
#p < 0.001 compared with normal group.
A
B
C
D
Figure 7: Chronic effect on liver lipid droplets of DIO mice (A–D): (A) PYY24–36. (B) 8. (C) normal. (D) DIO vehicle.
942 Chem Biol Drug Des 2016; 87: 936–945

A New PYY24–36 Conjugate Greatly Reduces Weight
most of the conjugates were over 12 h, and especially, 8
exhibited the longest half-life of over 24 h (Figure 2). Nota-
bly, the plasma half-life values of 1–4 correlated with the
alkyl chain length of these Ac-deoxycholic acid, and com-
pound 2 was most stable (> 20 h). It might indicate that
the medium alkyl chain length was most proper. Fat acid
chain conjugates showed comparable results.
potency after first turn, which means PYY24–36 and its
analogues may have blood glucose regulation effect (21).
Histological changes in the liver and brown fat
tissue of DIO mice
As 8 treatment had pronounced weight loss effects in DIO
mice, liver, white fat, and brown fat tissue were histologi-
A
B
C
Binding to human serum albumin
We predicted that the increased albumin binding might
lead to the increased metabolic stability, and this was
investigated for 1–9. With PYY24–36 used as control, all the
conjugates exerted improved albumin affinity. Albumin
binding correlated well with plasma stability (Figure 3).
Effects of acute feeding studies and 2 weeks
treatment in lean ICR mice
Peptide conjugates 2, 5, 7, 8, 9 with their half-lives higher
than 20 h were chose for further pharmacology study. The
total amount of food intake in the mice for each time was
reduced (Figure 4), and the total consumption for 8 h was
significantly less than that of the control (Figure 5). The
results showed that all the tested compounds had appetite
control effect and had a longer duration of action than
PYY24–36. In particular, the anorexia effect of 8 was relatively
strong and stable, which is worthy of further development.
In addition, 2-week treatment was given to ICR mice to
illustrate body weight change. As shown in Figure 5, most
of the compounds exhibited good weight control com-
pared with control (saline) and PYY24–36. In particular,
treatment of
8
increased body weight of only
2.4 Æ 0.13 g, while control group increased body weight
of 4.25 Æ 0.36 g after 2 weeks.
Effect in DIO mice of every other day dosing on
body weight and oral glucose tolerance test
(OGTT)
Peptide
OGTT studies in DIO mice. Subcutaneous administration
of every other day resulted in dose-dependent
8 was further selected for weight loss and
8
a
reduction in body weight (Figure 6B). The injections
decreased body weight of DIO mice by 1.45 Æ 0.2 g,
respectively, compared to vehicle increased by
1.75 Æ 0.12 g (p < 0.01, n = 6 per group) (Figure 6B).
Food intake is reduced for the treated animals compared
with the untreated control groups. The initial reduction
and subsequent restoration of food intake are also
observed with PYY24–36 upon continuous dosing in DIO
mice, which is consistent with result in ICR mice (data
not shown). Oral glucose tolerance test was following
performed for evaluating hypoglycemic (Figure 6A). Com-
pared with control (0.9% saline), compound 8 exhibited
moderate hypoglycemic effect unexpectedly after two
round of injection glucose, while PYY24–36 lost its
Figure 8: Chronic effect on liver brown fat cell of DIO mice
(brown fat cells were not seen in DIO mice) (A–C): (A) PYY24–36
.
(B) 8. (C) normal.
Chem Biol Drug Des 2016; 87: 936–945
943

Zhou et al.
Table 2: Chronic treatment with
PYY24–36 in DIO mice: plasma determina-
tions at the end of the chronic study
8 and
Plasma parameters
Normal
8
PYY24–36
DIO
Insulin (ng/mL)
Leptin (ng/mL)
Adiponectin (lg/mL)
7.96 Æ 0.13
6.33 Æ 0.15
4.78 Æ 0.15
9.210 Æ 0.35
5.85 Æ 0.27
5.01 Æ 0.25
10.94 Æ 0.23
5.33 Æ 0.18
5.17 Æ 0.26
12.56 Æ 0.25
4.37 Æ 0.11
4.57 Æ 0.12
Data are means Æ SEM. n = 6, *p < 0.05 8 and PYY24–36 versus vehicle.
cally assessed in mice from each of the four experimental
groups. Liver cells of untreated DIO control mice appeared
to be full of lipid droplets (Figure 7), while the 8 treatment
group showed a little and small lipid droplets. The body
weight changes were associated with an increase in white
adipose (WAT) cell number in the same area
(18.24 Æ 1.58 for 8, 16.62 Æ 1.19 for PYY24–36, and
14.4 Æ 1.26 for controls, 29.4 Æ 5.83 for normal
p < 0.01) (Figure 6C). Brown adipose tissue can speed up
the metabolism and promote white fat consumption. It
helps to promote obesity treatment. In contrast to no
image of brown fat cell seen in control DIO mice, mice
treated with 8 showed a significant increase in brown fat
cell number. Images of representative liver and brown fat
for each group are shown (Figure 7 and 8).
Therefore, compound 8 may be considered as a promising
candidate for obesity treatment. It also has a certain hypo-
glycemic activity (Figure 6) (21), which may provide a new
way to find hypoglycemic peptide. In addition, even
phenylbutazone has long been known as anti-inflammatory
drug, but as a new type of long-acting chemical agent, it
can be easily modified with other peptides through amida-
tion to improve their pharmacokinetic profile, which may
provide a new strategy to prolong peptides lifetime.
Acknowledgments
This study was supported by grants from the National Natu-
ral Science Foundation of China (Grants 81273376),Funda-
mental Research Funds for the Central Universities of China
Pharmaceutical University (Grant Z13136), and Innovation
Project Program of Huahai Pharmaceutical (HH13B003).
Chronic treatment improves metabolism
Several other metabolic parameters in plasma were also
improved by chronic treatment with 8 (Table. 2). Leptin
and adiponectin are important factors of body mass index,
insulin resistance, and glucose metabolism. Increases in
adiponectin and leptin and decreases in insulin levels cor-
related with the decreased adiposity observed at the end
of the study in each treatment group.
Conflict of Interest
All authors report no conflict of interest.
References
Conclusion
1. Neovius M., Narbro K. (2008) Cost-effectiveness of
pharmacological anti-obesity treatments: a systematic
review. p. 1752–1763.
2. Neary M.T., Batterham R.L. (2009) Gut hormones:
implications for the treatment of obesity. Pharmacol
Ther;124:44–56.
3. Lumb K.J., DeCarr L.B., Milardo L.F., Mays M.R.,
Buckholz T.M., Fisk S.E., Pellegrino C.M. et al. (2007)
Novel selective neuropeptide Y2 receptor PEGylated
peptide agonists reduce food intake and body weight
in mice. J Med Chem;50:2264–2268.
4. Ortiz A.A., Milardo L.F., DeCarr L.B., Buckholz T.M.,
Mays M.R., Claus T.H., Livingston J.N. et al. (2007) A
novel long-acting selective neuropeptide Y2 receptor
polyethylene glycol-conjugated peptide agonist reduces
food intake and body weight and improves glucose
In this study, to develop long-acting and highly biologically
active PYY24–36 derivatives, we modified the carboxylic
groups of peptide by amidation with small molecules and
obtained a series of PYY24–36 conjugates. Besides two
known small molecules bile acid and fatty chain that com-
monly used in prolong peptide half-life, phenylbutazone
was firstly used based on its characteristic that it can
strongly bind to HSA (22). By visually resolved with HSA
(Figure 1), we know that the phenylbutazone structure is
very conservative. So we decided to add a linker to it and
connect it with PYY24–36 by amidation.
The results show that phenylbutazone-PYY24–36 conjugate
8 is most potent and has a significant weight reduction in
DIO mice. Other improvements are also accompanied by
weight loss. Other effects of compound 8 are better than
PYY24–36, but not that much. That may because the meta-
bolism improvement is a slow process and may need
longer time of treatment. In conclusion, anorexia effect,
energy expenditure, and its duration time may all possible
affect its potential of weight loss.
metabolism
in
rodents.
J
Pharmacol
Exp
Ther;323:692–700.
5. Shechter Y., Sasson K., Lev-Goldman V., Rubinraut S.,
Rubinstein M., Fridkin M. (2012) Newly designed modi-
fier prolongs the action of short-lived peptides and pro-
teins by allowing their binding to serum albumin.
Bioconjugate Chem;23:1577–1586.
944
Chem Biol Drug Des 2016; 87: 936–945

A New PYY24–36 Conjugate Greatly Reduces Weight
6. Challis B., Pinnock S., Coll A., Carter R., Dickson S.,
O’rahilly S. (2003) Acute effects of PYY 3–36 on food
intake and hypothalamic neuropeptide expression in the
mouse. Biochem Biophys Res Commun;311:915–919.
7. Potter E.K., Barden J.A., McCloskey M.J., Selbie L.A.,
Tseng A., Herzog H., Shine J. et al. (1994) A novel neu-
ropeptide Y analog, N-acetyl [Leu 28, Leu 31] neuropep-
tide Y-(24–36), with functional specificity for the
presynaptic (Y 2) receptor. Eur J Pharmacol Mol Pharma-
col;267:253–262.
8. DeCarr L.B., Buckholz T.M., Milardo L.F., Mays M.R.,
Ortiz A., Lumb K.J. (2007) A long-acting selective neu-
ropeptide Y2 receptor PEGylated peptide agonist
reduces food intake in mice. Bioorg Med Chem
Lett;17:1916–1919.
9. DeCarr L.B., Buckholz T.M., Coish P.D., Fathi Z., Fisk
S.E., Mays M.R., O’Connor S.J. et al. (2007) Identifica-
tion of selective neuropeptide Y2 peptide agonists.
Bioorg Med Chem Lett;17:538–541.
10. Cox J.E., Randich A. (2004) Enhancement of feeding
suppression by PYY 3-36 in rats with area postrema
ablations. Peptides;25:985–989.
11. Avraham Y., Katzhendler J., Vorobeiv L., Merchavia S.,
Listman C., Kunkes E., Harfoush F. et al. (2013) Novel
acylethanolamide derivatives that modulate body
weight through enhancement of hypothalamic pro-
opiomelanocortin (POMC) and/or decreased neuropep-
tide Y (NPY). J Med Chem;56:1811–1829.
12. Kratz F. (2008) Albumin as a drug carrier: design of
prodrugs, drug conjugates and nanoparticles. J Con-
trolled Release;132:171–183.
13. Chae S.Y., Choi Y.G., Son S., Jung S.Y., Lee D.S.,
Lee K.C. (2010) The fatty acid conjugated exendin-4
analogs for type 2 antidiabetic therapeutics. J Con-
trolled Release;144:10–16.
as a stable and bioactive GLP-1 (7–36) analog. Bioorg
Med Chem Lett;14:4395–4398.
15. Madsen K., Knudsen L.B., Agersoe H., Nielsen P.F.,
Thøgersen H., Wilken M., Johansen N.L. et al. (2007)
Structure-activity and protraction relationship of long-
acting glucagon-like peptide-1 derivatives: importance
of fatty acid length, polarity, and bulkiness. J Med
Chem;50:6126–6132.
16. Sharma J. (1977) Comparison of the anti-inflammatory
activity of Commiphora mukul (an indigenous drug)
with those of phenylbutazone and ibuprofen in experi-
mental arthritis induced by mycobacterial adjuvant.
Arzneimittelforschung;27:1455–1457.
€
17. Burns J., Yu T., Dayton P.G., Gutman A.B., Brodie
B.B. (1960) Biochemical pharmacological considera-
tions of phenylbutazone and its analogues. Ann NY
Acad Sci;86:253–262.
18. Yang B., Mei Y., Wang X., Deng X., Qian H., Huang
W. (2014) Design, synthesis and biological evaluation
of novel peptide MC62 analogues as potential antihy-
perglycemic agents. Eur J Med Chem;73:105–111.
19. Han J., Huang X., Sun L., Li Z., Qian H., Huang W.
(2013) Novel fatty chain-modified glucagon-like peptide-
1 conjugates with enhanced stability and prolonged
in vivo activity. Biochem Pharmacol;86:297–308.
20. Kim T.H., Jiang H.H., Lee S., Youn Y.S., Park C.W.,
Byun Y., Chen X. et al. (2011) Mono-PEGylated dimeric
exendin-4 as high receptor binding and long-acting con-
jugates for type 2 anti-diabetes therapeutics. Bioconju-
gate Chem;22:625–632.
21. Boey D., Sainsbury A., Herzog H. (2007) The role of
peptide YY in regulating glucose homeostasis. Pep-
tides;28:390–395.
22. MacAllister C.G., Morgan S.J., Borne A.T., Pollet R.A.
(1993) Comparison of adverse effects of phenylbuta-
zone, flunixin meglumine, and ketoprofen in horses.
J Am Vet Med Assoc;202:71–77.
ꢀ
14. Leger R., Thibaudeau K., Robitaille M., Quraishi O.,
van Wyk P., Bousquet-Gagnon N., Carette J. et al.
(2004) Identification of CJC-1131-albumin bioconjugate
Chem Biol Drug Des 2016; 87: 936–945
945