Design, synthesis, and evaluation of novel and selective G-protein coupled receptor 120 (GPR120) spirocyclic agonists

Article abstract of DOI:10.1021/acsmedchemlett.6b00360

Type 2 diabetes mellitus (T2DM) is an ever increasing worldwide epidemic, and the identification of safe and effective insulin sensitizers, absent of weight gain, has been a long-standing goal of diabetes research. G-protein coupled receptor 120 (GPR120) has recently emerged as a potential therapeutic target for treating T2DM. Natural occurring, and more recently, synthetic agonists have been associated with insulin sensitizing, anti-inflammatory, and fat metabolism effects. Herein we describe the design, synthesis, and evaluation of a novel spirocyclic GPR120 agonist series, which culminated in the discovery of potent and selective agonist 14. Furthermore, compound 14 was evaluated in vivo and demonstrated acute glucose lowering in an oral glucose tolerance test (oGTT), as well as improvements in homeostatic measurement assessment of insulin resistance (HOMA-IR; a surrogate marker for insulin sensitization) and an increase in glucose infusion rate (GIR) during a hyperinsulinemic euglycemic clamp in diet-induced obese (DIO) mice.

Full text of DOI:10.1021/acsmedchemlett.6b00360

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Letter
Design, Synthesis, and Evaluation of Novel and Selective G-
protein Coupled Receptor 120 (GPR120) Spirocyclic Agonists
Jason M. Cox, Hong D. Chu, Mariappan Vasu Chelliah, John S Debenham, Keith Eagen, Ping Lan,
Matthew Lombardo, Clare London, Michael A. Plotkin, Unmesh Shah, Zhongxiang Sun, Henry M.
Vaccaro, Srikanth Venkatraman, Takao Suzuki, Nengxue Wang, Eric R Ashley, Alejandro Crespo,
Maria Madeira, Dennis H. Leung, Candice Alleyne, Aimie M. Ogawa, Sarah Souza, Brande Thomas-
Fowlkes, Jerry Di Salvo, Adam B. Weinglass, Melissa Kirkland, Michele Pachanski, Mary Ann
Powles, Effie Tozzo, Taro E. Akiyama, Feroze Ujjainwalla, James R. Tata, and Christopher J Sinz
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00360 • Publication Date (Web): 17 Nov 2016
Downloaded from http://pubs.acs.org on November 18, 2016
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Design, Synthesis, and Evaluation of Novel and Selective G-protein
Coupled Receptor 120 (GPR120) Spirocyclic Agonists
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Jason M. Cox,* Hong D. Chu, Mariappan V. Chelliah,† John S. Debenham, Keith Eagen,‡ Ping Lan,
Matthew Lombardo, Clare London,≠ Michael A. Plotkin,ǁ Unmesh Shah,# Zhongxiang Sun, Henry M.
Vaccaro,┴ Srikanth Venkatraman, Takao Suzuki,¢ Nengxue Wang,¢ Eric R. Ashley, Alejandro Crespo,
Maria Madeira, Dennis H. Leung,£ Candice Alleyne, Aimie M. Ogawa, Sarah Souza, Brande Thomasꢀ
Fowlkes, Jerry Di Salvo, Adam Weinglass, Melissa Kirkland,^ Michele Pachanski, Mary Ann Powles,
Effie Tozzo,§ Taro E. Akiyama, Feroze Ujjainwalla, James R. Tata and Christopher J. Sinz
Merck & Co., Inc., Kenilworth, NJ 07033, USA and Merck & Co., Inc., Rahway, NJ 07065, USA
¢WuXi AppTec, Shanghai, 200131, China
KEYWORDS: GPR120, FFAR4, insulin sensitization, type 2 diabetes, spirocyclic
ABSTRACT: Type 2 diabetes mellitus (T2DM) is an ever increasing worldwide epidemic, and the identification of safe and effecꢀ
tive insulin sensitizers, absent of weight gain, has been a long standing goal of diabetes research. Gꢀprotein coupled receptor 120
(GPR120) has recently emerged as a potential therapeutic target for treating T2DM. Natural occurring, and more recently, synthetic
agonists have been associated with insulin sensitizing, antiꢀinflammatory and fat metabolism effects. Herein we describe the
design, synthesis, and evaluation of a novel spirocyclic GPR120 agonist series, which culminated in the discovery of potent
and selective agonist 14. Furthermore, compound 14 was evaluated in vivo and demonstrated acute glucose lowering in an
oral glucose tolerance test (oGTT), as well as improvements in homeostatic measurement assessment of insulin resistance
(HOMAꢀIR; a surrogate marker for insulin sensitization) and an increase in glucose infusion rate (GIR) during a hyperinsuꢀ
linemic euglycemic clamp in dietꢀinduced obese (DIO) mice
.
Type 2 diabetes mellitus (T2DM) is an ever increasing epiꢀ
demic that affects over 415 million people worldwide.1 While
current therapies such as metformin, sulfonylureas, glitazones,
DPPꢀ4 inhibitors, GLPꢀ1 analogs, and SGLT2 inhibitors proꢀ
regarding the insulin sensitizing and chronic antiꢀ
inflammatory effects of selective GPR120 spirocyclic agonist
compound A (14).16 Herein we describe the design, synthesis,
structure activity relationship (SAR), in vitro selectivity, and
expanded in vivo data for the spirocyclic series.
,
vide physicians many options for treating T2DM patients,2 3
eventually patients may become insulin resistant and must be
treated with exogenous prandial and/or basal insulin.4 It has
been a long standing goal of diabetes research to identify safe
,
and effective insulin sensitizers5 6 which do not have the
weight gain side effect associated with the glitazones7 (peroxiꢀ
some proliferatorꢀactivated receptor (PPAR) agonists).8
Gꢀprotein coupled receptor 120 (GPR120), encoded by the
free fatty acid receptor 4 (FFAR4) gene, has gained attention
,
in recent years as a potential target to treat T2DM.9 10 GPR120
is a member of the rhodopsin family of 7ꢀtransmembrane doꢀ
main Gꢀprotein coupled receptors (GPCRs) and has been assoꢀ
ciated with insulin sensitizing, antiꢀinflammatory and fat meꢀ
tabolism effects.11 GPR120 is expressed primarily in the intesꢀ
tine, adipocytes and proꢀinflammatory macrophages and can
be activated by long chain free fatty acids.12 In mice, GPR120
is preferentially expressed in pancreatic delta cells and reguꢀ
lates somatostatin secretion from islets.13
Figure 1. Alternate cyclization modes of early lead 1.
The inspiration for the spirocyclic series originated from
early lead 1 (Figure 1). As we recently disclosed, 1 was deꢀ
rived from an ultra highꢀthroughput screening (uHTS) hit, and
we utilized the common medicinal chemistry strategy of rigidꢀ
ifying through cyclization of compound 1, via pathway “a” to
form a novel benzofuran series represented by compound 2.17
Both acyclic analog 1 and cyclic analog 2 had similar human
(h) and mouse (m) IP1 and βꢀArrestin2 (βꢀArr2) GPR120 poꢀ
tency,18 however cyclic analog 2 lost selectivity versus GPR40
(Table 1). We envisioned using a similar rigidifying strategy,
but in this case cyclizing via route “b” and changing the ether
linkage to an amine, to provide piperidine 3 (Figure 1). While
Agonism of the related GPCR, GPR40 (FFAR1), has also
gained attention in recent years as a potential treatment target
for T2DM.14,15 Both GPR120 and GPR40 are activated by long
chain fatty acids (LCFA), and in order to better understand the
antiꢀdiabetic role of GPR120, identification of selective
GPR120 agonists versus GPR40 agonists is essential. Recentꢀ
ly, in collaboration with the Olefsky group, we published data
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the carboxylic acid to adopt the appropriate conformation for
this change resulted in loss of potency on h/m IP1 and h/m βꢀ
1
2
3
potency and inspired by structural minimization overlays with
previous potent analogs (see supporting information), we enviꢀ
sioned adding a methylene spacer between the cyclohexyl Aꢀ
ring and the carboxylic acid, as well as Aꢀ/Bꢀring spirocycle
formation to generally maintain similar spacing of the acid
moiety and the aromatic Cꢀring. The aforementioned transꢀ
formations resulted in the discovery of the novel GPR120
agonist spirocyclic series 6 (Figure 2, Table 2 and Table 3).19
Arrestin2 GPR120 assays for 3 (Table 1), it provided an initial
baseline point suggesting that the alternate cyclization strategy
was a reasonable approach to new analog design.
4
5
6
7
8
9
Table 2. C-Ring SARa
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12
13
14
15
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17
18
19
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GPR120 EC50 (nM)
hGPR40 IP1
hβꢀ
Arr2
mβꢀ
Arr2
EC50 (nM)
Entry
R
hIP1
2100
mIP1
Figure 2. Conceptual formation of the spirocyclic series.
12
570
2500
5300
>10000
The team was keenly interested in developing a viable novel
series with which to build upon, and we felt it was important
to diversify our structure class and explore broader chemical
space. To this end, we made an initial ring switch of the Aꢀ
and Bꢀrings on piperidine 3, to provide the transꢀlocated piperꢀ
idine 4 (Figure 2), which would also facilitate more rapid
investigation of the Cꢀring SAR. Unfortunately, this change
resulted in complete loss of GPR120 potency for 4 (Table 1).
13
14
130
98
49
33
700
66
470
54
>10000
>10000
Table 1. In Vitro Potency of Select GPR120 Agonists a
F
15
16
17
18
1200
590
2200
3000
>10000
>10000
2500
GPR120 EC50 (nM)
hGPR40 IP1
F3C
hβꢀ
Arr2
mβꢀ
Arr2
EC50 (nM)
Entry
hIP1
mIP1
>10000 >10000 >10000 >10000
1
2
3
4
5
94
63
300
>10000
700
16
43
89
>10000
1490
100
16
1600
>10000
NA
35
21
320
>10000
990
>10000
1829
>10000
>10000
>10000
200
420
66
370
290
aValues are the average of two experiments, each in 10ꢀpoint titraꢀ
tions. All analogs tested were generally deemed to be full
GPR120 agonists under assay conditions.
Scheme 1. Synthesis of Spirocyclic Analogsa
190
1400
1100
8100
19
20
3300
220
670
51
3100
190
4000
120
>10000
6300
aReagents and conditions: (a) MVK, KOH, EtOH, water, 100 °C,
39% (b) 9, NaH, THF, 0 °C to 50 °C, 83% (c) Pd/C, H2 50 psi,
Boc2O, MeOH, 40 °C, 91% (d) TFA, DCM, rt, 91% (e) RꢀBr,
BINAP, Pd2(dba)3, Cs2CO3, dioxane, 100 °C, (f) LiOH, MeOH,
THF, water, rt, 15ꢀ75% over 2 steps.
21
22
360
44
480
140
>10000
>10000
3100
1300
9200
4200
aValues are the average of two experiments, each in 10ꢀpoint titraꢀ
tions. All analogs tested were generally deemed to be full
GPR120 agonists under assay conditions.
We hypothesized that the piperidine Bꢀring was not the isꢀ
sue since the preceding molecule (3) also contained a piperiꢀ
dineꢀphenyl moiety, but that the phenyl Aꢀring was not allowꢀ
ing the carboxylic acid to adopt the appropriate conformation
for potency. To test this hypothesis, we prepared the cycloꢀ
hexyl carboxylic acid derivative 5 (Figure 2) as a mixture of
cisꢀ and transꢀisomers. Gratifyingly, some of the potency lost
during the Aꢀ/Bꢀring switch was recaptured by analog 5 (Ta-
ble 1), even as a mixture of isomers. To enhance the ability of
The synthesis of the spirocyclic series commenced by ring
annulation of aldehyde 7 with methyl vinyl ketone (MVK), in
the presence of potassium hydroxide, to afford spirocycle 8
(Scheme 1). Key intermediate piperidine spirocycle 10 was
prepared by HornerꢀWadsworthꢀEmmons (HWE) reaction of
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ketone 8 with phosphonate 9, followed by concomitant Cbz moderate loss of GPR120 potency and reduced the GPR40
1
2
3
4
5
6
7
8
removal and olefin reduction with hydrogen, in the presence of
Boc anhydride (to facilitate purification), and finally Boc reꢀ
moval with TFA. With piperidine spirocycle 10 in hand, we
were poised to rapidly evaluate the Cꢀring SAR of the class by
utilizing palladium Nꢀarylation chemistry to provide final anaꢀ
logs 11 after saponification of the methyl ester with lithium
hydroxide.
selectivity relative to previous analogs. Triꢀsubstituted Cꢀring
variants represented by analogs 18 and 19 led to variable
GPR120 potencies, however in all cases the potency was reꢀ
duced relative to compound 14. Analogs 20 and 21 are repreꢀ
sentative analogs with bisꢀmetaꢀsubstitution and in general
demonstrated worse potency when compared to 14. In an efꢀ
fort to have greater effect on the physicochemical properties of
the molecules, we replaced the orthoꢀhalide as a pyridine,
resulting in only a moderate loss of potency (analog 22 versus
analog 15). Unfortunately, other pyridine analogs resulted in
significant loss of GPR120 potency for the series (data not
shown). In general, the SAR around the Cꢀring was challengꢀ
ing and only limited modifications were tolerated, with comꢀ
pound 14 proving to have the optimal Cꢀring substitutions and
substitution pattern to maintain reasonable GPR120 potency
and GPR40 selectivity.
Table 3. Spirocycle Core and Acid Linker SARa
9
GPR120 EC50 (nM)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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Entry
Structure
hIP1
hβꢀArr2
23
3500
4400
With the identification of the orthoꢀCl, metaꢀOCF3 substituꢀ
tion pattern for the Cꢀring, we next turned our attention toward
modifying the spirocyclic core (Table 3). Substitution on the
methylene spacer between the acid and the cyclohexyl ring
resulted in significant loss of GPR120 potency, either as a
methyl (23) or 2,3ꢀcyclopropyl substitution (24). Introduction
of a methyl group on the cyclohexyl ring also resulted in loss
of GPR120 potency (compounds 25 and 26) relative to 14.
Extending the carboxylic acid by one methylene (27) or reducꢀ
ing the acid of 14 to an alcohol (28), resulted in significant
potency loss. In a similar vein as with the Cꢀring SAR, introꢀ
duction of heteroatom replacements for the cyclohexyl (pyran
29 or piperidine 30) also resulted in substantial loss of
GPR120 potency. The SAR observations within the spirocyꢀ
clic core modifications reaffirmed the challenges we encounꢀ
tered with overall optimization of the series, resulting in no
analog having superior GPR120 potency and GPR40 selectiviꢀ
ty when compared to spirocyclic lead 14.
24
25
26
27
28
29
30
2200
4100
440
3900
2700
580
2700
4300
5700
1000
330
Table 4. PK profile of compound 14a
2100
Property
Rat
17.5
2.3
Mouse
14.6
8.4
Dog
4.6
9.8
6.7
11
Rhesus
8.7
>10000 >10000
AUC iv (ꢀM•h)
Clp (mL/min/kg)
Vdss (L/kg)
t1/2 (h)
2.7
aValues are the average of two experiments, each in 10ꢀpoint titraꢀ
tions. All analogs tested were generally deemed to be full
GPR120 agonists under assay conditions. In cases of possible
stereoisomers, the isomers were separated and the data of the most
potent is shown but the stereochemistry was not determined.
0.4
8.9
1.7
1.4
18.1
9.1
MRT (h)
3.0
95
26.5
100
10.5
100
10.1
77
Foral (%)
Due to the nature of our synthesis, we were able to rapidly
explore the Cꢀring SAR, as highlighted by select analogs in
Table 2. The initial Cꢀring analogs prepared were based on
our previous SAR knowledge from both the acyclic and cyclic
analogs 1 and 2 respectively. The first three analogs prepared
in the spirocyclic series are listed in order of preparation, with
the first (12) demonstrating modest GPR120 potency, the seꢀ
cond (13) demonstrating improved GPR120 potency and the
third (14) demonstrating subꢀ100 nM GPR120 potency while
maintaining hGPR40 selectivity. The subꢀ100 nM GPR120
potency for compound 14 was similar to benzofuran lead 2 at
h/m IP1 and h/m βꢀArrestin2, but proved to have superior
GPR40 selectivity.
PPB (% bound)
Unbound Cl (Clp/funb
99.87
1769
99.79
4000
99.85
6533
99.72
964
b
)
aClp: plasma clearance; t1/2: terminal halfꢀlife; MRT: mean resiꢀ
dence time; Foral: oral bioavailability; AUC: area under the plasma
concentration vs time curve following iv dosing; Vdss: volume of
distribution at steady state; PPB: plasma protein binding. funb
(100ꢀ(%PPB))/100.
b
=
Often early leads have off target and pharmacokinetic (PK)
issues, limiting their utility as tools to better understand bioꢀ
logical processes; therefore, while investigating the SAR of
the spirocyclic series, we began to profile compound 14 versus
common counter screen assays. Ion channel activity for calciꢀ
um (Cav1.2 = 21 ꢀM),20 potassium (IKr > 60 ꢀM),21 sodium
(Nav1.5 = 13 ꢀM)22 and cytochrome Pꢀ450 (CYP3A4,
CYP2D6, CYP2C9 > 48 ꢀM)23 all offered acceptable values.
Furthermore, 14 demonstrated minimal binding activity on
Changing the metaꢀsubstitution from ꢀOCF3 (13) to ꢀCF3
(15) resulted in approximately an order of magnitude loss in
GPR120 potency and removing the metaꢀsubstitution as in
entry 16 led to complete loss of GPR120 potency. Changing of
the ortho position from a halogen to ꢀCN (17) resulted in a
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PPAR alpha, delta and gamma (>10 ꢀM; see supporting inꢀ
(A)
(B)
(C)
1
2
3
4
5
6
7
8
formation).
In light of the minimal offꢀtarget activity, we continued to
profile spirocycle 14 by conducting rat, mouse, dog, and rheꢀ
sus monkey PK studies (Table 4). The clearance and volume
of distribution at steady state resulted in a 1.4 h halfꢀlife and
3.0 h mean residence time for the rat, while resulting in longer
halfꢀlife and MRT in the mouse, dog and rhesus. The oral bioꢀ
availability across species was high (> 77%), but so too was
the unbound clearance (> 964). Unfortunately, all attempts to
reduce clearance by introduction of more polar groups or addiꢀ
tional substitutions, resulted in significant loss of potency on
the GPR120 receptor (vide supra). At this time it became clear
that 14 was the lead compound in the spirocyclic series, since
the overall PK profile allowed for evaluation in more adꢀ
vanced preclinical in vivo studies.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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(A)
(B)
(D)
Figure 3. Mouse oral glucose tolerance test (oGTT) dose titration
of compound 14 in comparison to GPR120 positive control comꢀ
pound 2. (A) Blood glucose (mg/dl) over time (minutes). (B) Net
blood glucose area under the curve (AUC). Vehicle (veh); dexꢀ
trose (dex) challenge; Tx = vehicle and compound administration.
* P < 0.05 compared to vehicle, ** P < 0.001 compared to vehiꢀ
cle; twoꢀway ANOVA. Data are represented as mean ± SEM.
Figure 4. Five week diet induced obese (DIO) mouse study with
compound 14 and pioglitazone. (A) Glucose changes over time.
(B) Insulin changes over time. (C) Homeostatic model assessment
of insulin resistance (HOMAꢀIR) changes over time; HOMAꢀIR =
glucose (mM) x insulin (ꢀU/mL)/22.5. (D) Glucose infusion rate
(GIR) change during hyperinsulinemic euglycemic clamp. For (Bꢀ
D), * P < 0.05 compared to vehicle; oneꢀway ANOVA for (B) and
(C); twoꢀway ANOVA for (D). Data are represented as mean ±
SEM.
Spirocyclic lead 14 was assessed for its ability to improve
glucose tolerance in lean mice (Figure 3). A dose titration, in
an oral glucose tolerance test (oGTT),24 was conducted with
three doses of 14 (10, 30, 100 mpk), utilizing compound 2 (30
mpk) as a positive control and vehicle controls with and withꢀ
out a dextrose challenge. 2 and 14 were orally administered 1
h prior to dextrose challenge and 14 dose dependently reduced
blood glucose, with the 100 mpk dose demonstrating compaꢀ
rable reduction to 2 at 30 mpk. As previously disclosed, both
1416 and 217 demonstrated onꢀtarget effects in wildꢀ
type/GPR120 knock out acute mouse oGTT studies.
With a superior in vitro GPR40 selectivity profile of spiroꢀ
cyclic lead 14 versus 2, we further evaluated 14 for its ability
to improve insulin sensitivity following five week dosing in
insulin resistant diet induced obese (DIO) mice using pioglitaꢀ
zone (PPAR alpha and gamma active) as a positive control
(Figure 4). While only modest, nonꢀstatistically significant
reductions in glucose were observed for 14 and pioglitazone
(Figure 4A), both showed statistically significant reductions
in insulin levels on days 14 and 28 of the study (Figure 4B).
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Furthermore, compound 14 demonstrated statistically signifiꢀ
§ (E.T.) Mitobridge Inc., 1030 Massachusetts Ave., Suite 200,
Cambridge, MA 02138, USA
1
2
3
cant reduction in homeostatic model assessment of insulin
resistance (HOMAꢀIR),25 a surrogate marker for insulin reꢀ
sistance, at days 14 and 28 (Figure 4C).
.
Notes
The authors declare no competing financial interest.
4
5
6
7
8
9
As shown in Figure 4D, selective GPR120 agonist 14
demonstrated a statistically significant increase in the glucose
infusion rate (GIR), which was similar to the known insulin
sensitizer pioglitazone, during the hyperinsulinemic euglyceꢀ
mic clamp. These results are indicative of an insulin sensitizꢀ
ing effect of 14, consistent with previous reports.16 Notably,
after five weeks of treatment, no body weight changes were
observed for 14, which is in contrast to the body weight inꢀ
crease for pioglitazone (see supporting information) and body
weight decrease observed with a recently described benꢀ
zosultam GPR120 agonist.26
ACKNOWLEDGMENT
The authors would like to acknowledge Junling Gao and
Mengfen (Melissa) Lin for analytical and WuXi AppTec for
synthetic chemistry support.
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ABBREVIATIONS
T2DM, type 2 diabetes mellitus; DPPꢀ4, dipeptidyl peptidase
IV; GLPꢀ1, glucagon like peptide 1; SGLT2, sodium glucose
coꢀtransporter 2; GPR120, Gꢀprotein coupled receptor 120;
FFA4, free fatty acid receptor 4; GPCR, Gꢀprotein coupled
receptor; GPR40, Gꢀprotein coupled receptor 40; FFAR1, free
fatty acid receptor 1; LCFA, longꢀchain fatty acid; uHTS, ultra
highꢀthroughput screen; IP1, Dꢀmyoꢀinositolꢀ1ꢀphosphate;
oGTT, oral glucose tolerance test; DIO, diet induced obese;
GIR, glucose infusion rate; HOMAꢀIR, homeostatic model
assessment for insulin resistance.
CONCLUSION
Interest in GPR120 has increased in recent years as a potential
type 2 diabetes mellitus target, in particular for its potential
role in insulin sensitization. Through systematic modification
of an internal hit, we identified a spirocyclic lead series that
resulted in the discovery of lead compound 14. Analog 14 had
suitable offꢀtarget (including high selectivity versus GPR40
and PPARs) and PK profiles to further evaluate the physiologꢀ
ical effects of GPR120 agonism. Lead 14 doseꢀdependently
increased glucose excursion (oGTT) and upon five week adꢀ
ministration, reduced insulin levels, decreased HOMAꢀIR and
increased GIR during a hyperinsulinemic euglycemic DIO
mouse clamp. The disclosure of further efforts on GPR120
agonism will be forthcoming.
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ASSOCIATED CONTENT
Supporting Information
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AUTHOR INFORMATION
Corresponding Author
* Phone: 908ꢀ740ꢀ0425. Eꢀmail: jason_cox@merck.com
Present Addresses
†(M.C.) FDA, 10903 New Hampshire Ave., Silver Spring, MD
20993, USA
.
‡ (K.E.) Leidos, 356 Ninth Ave., Suite 106, Picatinny Arseꢀ
nal, NJ 07806, USA.
≠ (C.L.) Dotmatics, 800 W. Cummings Park, Suite 2950, Woꢀ
burn, MA 01801, USA
.
ǁ (M.A.P.) Merck and Co., Inc., West Point, PA 19486, USA
.
# (U.S.) Jones Day, 250 Vesey Street, New York, NY
10281, USA.
┴ (H.M.V.) Retired.
£(D.L.) Genentech, 1 DNA Way, South San Francisco, CA
94080, USA.
^ (M.K.) Charles River Laboratories, 334 South St.,
(14) Sekiguchi, H.; Kasubuchi, M.; Hasegawa, S.; Pelisch, N.; Kiꢀ
mura, I.; Ichimura, A. A Novel Antidiabetic Therapy: Free Fatty Acid
Shrewsbury, MA 01545, USA
.
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