Copyright © 2005 by the American Society for Biochemistry and Molecular Biology, Inc.
This article is available online at http://www.jlr.org
Journal of Lipid Research
Volume 46, 2005
Dual-action hypoglycemic and hypocholesterolemic
agents that inhibit glycogen phosphorylase and
H. James Harwood, Jr.,
Victor F. Soliman,* Eliot D. Sugarman,* Bernard Hulin,* Younggil Kwon,* E. Michael Gibbs,*
James T. Mayne, and Judith L. Treadway*
Departments of Cardiovascular and Metabolic Diseases,* and Exploratory Toxicology,
and Development, Groton Laboratories, Pfizer Inc., Eastern Point Road, Groton, CT 06340
* Stephen F. Petras,* Dennis J. Hoover,* Dayna C. Mankowski,*
Pfizer Global Research
ment with hypoglycemic agents and lipid-modulating drugs.
We recently described glycogen phosphorylase inhibitors that
reduce glycogenolysis in cells and lower plasma glucose in
ob/ob mice (
J. Med. Chem.
, 41: 2934, 1998). In evaluating the
series prototype, CP-320626, in dogs, up to 90% reduction in
plasma cholesterol was noted after 2 week treatment. Choles-
terol reductions were also noted in ob/ob mice and in rats.
In HepG2 cells, CP-320626 acutely and dose-dependently
inhibited cholesterolgenesis without affecting fatty acid syn-
thesis. Inhibition occurred together with a dose-dependent
increase in the cholesterol precursor, lanosterol, suggesting
that cholesterolgenesis inhibition was due to lanosterol 14
demethylase (CYP51) inhibition. In ob/ob mice, acute treat-
ment with CP-320626 resulted in a decrease in hepatic cho-
lesterolgenesis with concomitant lanosterol accumulation,
further implicating CYP51 inhibition as the mechanism of
cholesterol lowering in these animals. CP-320626 and ana-
logs directly inhibited rhCYP51, and this inhibition was highly
correlated with HepG2 cell cholesterolgenesis inhibition
0.77). These observations indicate that CP-320626
inhibits cholesterolgenesis via direct inhibition of CYP51,
and that this is the mechanism whereby CP-320626 lowers
plasma cholesterol in experimental animals. Dual-action gly-
cogenolysis and cholesterolgenesis inhibitors therefore have
the potential to favorably affect both the hyperglycemia and
the dyslipidemia of type 2 diabetes.
S. F. Petras, D. J. Hoover, D. C. Mankowski, V. F. Soliman, E. D.
Sugarman, B. Hulin, Y. Kwon, E. M. Gibbs, J. T. Mayne, and
J. L. Treadway.
Dual-action hypoglycemic and hypocholes-
terolemic agents that inhibit glycogen phosphorylase and
J. Lipid Res.
Diabetic dyslipidemia requires simultaneous treat-
—Harwood, Jr., H. James,
Supplementary key words
type 2 diabetes
Type 2 diabetes is a severe and prevalent disease in the
Western world and affects roughly 16 million persons in
the US, and another 14 million people have impaired glu-
cose tolerance (1). Projections indicate that the incidence
of type 2 diabetes will increase to over 25 million by 2010
in the US, and to over 300 million worldwide by 2025 (1–3).
The annual direct medical costs associated with type 2 dia-
betes, which in the United States was in excess of 44 bil-
lion dollars in 1997 (4), result primarily from secondary
hyperglycemia-related complications, such as retinopathy,
nephropathy, peripheral neuropathy, and cardiovascular,
peripheral vascular, and cerebrovascular disease.
A high correlation exists between tighter glycemic control
and reduction of these long-term complications in type 2
diabetes (5). Type 2 diabetics are currently treated with in-
terventions to improve glycemia through a progressive regi-
men of diet, exercise, oral antidiabetic drugs (as monother-
apy or in combination), and insulin (6). However, there is
an ongoing need for additional oral antidiabetic agents
that will achieve better glycemic control as monotherapy
and/or work more safely or effectively in combination.
In addition to their hyperglycemia, patients with type 2
diabetes often present with a concomitant atherogenic
dyslipidemia (elevated triglycerides, low HDL cholesterol,
and small, dense LDL) that increases their risk of cardio-
vascular disease (7, 8). Because there is a high incidence
of mortality for type 2 diabetics with their first myocardial
infarction (7), aggressive therapy for treating diabetic dys-
lipidemia is recommended (7). It is suggested that initial
lipid-modulating therapy be directed toward reducing LDL
cholesterol levels to below 100 mg/dl through administra-
tion of a cholesterol synthesis inhibitor, such as a statin
(HMG-CoA reductase inhibitor), and that this treatment
Abbreviations: AUC, area under the curve; CYP, cytochrome-P450;
DMEM, Dulbecco’s modified Eagle’s medium.
To whom correspondence should be addressed.
Manuscript received 2 November 2004 and in revised form 3 December 2004.
Published, JLR Papers in Press, December 16, 2004.
by guest, on June 1, 2013
548 Journal of Lipid Research
Volume 46, 2005
be combined with a fibric acid derivative, such as fenofi-
brate, for patients with HDL cholesterol levels below 40
mg/dl and for patients with triglycerides that remain ele-
150 mg/dl) after both improvement of glycemic
control and initiation of statin therapy (7).
In pursuing new treatments for type 2 diabetes, we have
targeted inhibition of glycogen phosphorylase (E.C. 184.108.40.206),
the enzyme that catalyzes the hydrolytic release of glucose-
1-phosphate from glycogen, as an approach to reducing he-
patic glycogenolysis and thereby controlling plasma glucose
levels (2). Through these efforts, we have identified a series
of indole-2-carboxamide glycogen phosphorylase inhibitors
(9, 10) that inhibit the human liver isoform of glycogen phos-
phorylase by binding at a unique allosteric regulatory site on
the enzyme (11), reduce forskolin-induced glycogenolysis
in SK-HEP-1 cells (10, 12), and exhibit glucose-lowering
activity when given orally to diabetic ob/ob mice (10, 12).
Because of the potential pharmacological utility of this
series of glycogen phosphorylase inhibitors, we have eval-
uated a representative analog, CP-320626 (
human liver glycogen phosphorylase, 205 nM (10)], for its
subchronic effects in diabetic ob/ob mice (10, 12), in rats,
and in dogs. During the course of these studies, we discov-
ered that CP-320626 reduced plasma cholesterol levels in a
variety of normoglycemic, nondiabetic animals in a manner
and magnitude inconsistent with its expected action as a
glycogen phosphorylase inhibitor. Herein, we report iden-
tification of the mechanism responsible for the cholesterol-
lowering action of CP-320626 as inhibition of the choles-
terolgenic enzyme lanosterol 14
characterize structure–activity relationships for this activ-
ity within the series.
-demethylase (CYP51), and
isocitrate dehydrogenase, dioleoyl
terol, tyloxapol, ketoconazole, quinidine, sulfaphenazole, cyto-
chrome-P450 (CYP) reductase, PEG400, and diagnostic kits for
measuring plasma lactate and
-hydroxybutyrate were from Sigma
Chemical Co. (St. Louis, MO). TMSI
1 ml aliquots were from Supelco (Bellefonte, PA). Sodium [2-
acetate (56 mCi/mmol), R,S-[2-
mmol), and Aquasol-2 were from New England Nuclear (Boston,
MA). Ready-Safe was from Beckman Instruments (Fullerton, CA).
H]lanosterol was from American Radiolabeled
Chemicals, Inc. (St. Louis, MO). Dulbecco’s modified Eagle’s me-
-glutamine, and gentamicin were from GIBCO
, glucose-1-phosphate, glycogen, isocitrate,
Pyridine, 1:4 (Sylon TP) in
C]mevalonolactone (58 mCi/
Laboratories (Grand Island, NY). Heat-inactivated fetal bovine
serum was from HyClone Laboratories (Logan, UT). Silica gel 60C
TLC plates were from Eastman Kodak (Rochester, NY). BCA pro-
tein assay reagent was from Pierce (Rockford, IL). A-Gent™ Glu-
cose-UV, A-Gent™ Triglyceride and A-Gent™ Cholesterol Test re-
agent systems were from Abbott Laboratories (Irving, TX). Pluronic
P105 Block Copolymer Surfactant was from BASF (Parsippany, NY).
Sprague Dawley rats and Beagle dogs were from Charles Rivers
(Boston, MA). C57BL/6J-ob/ob mice were from Jackson Labora-
tory (Bar Harbor, ME). RMH 3200 laboratory meal and Agway
Respond 2000 laboratory dog chow were from Agway, Inc. (Syra-
cuse, NY). HepG2 cells were from the American Type Culture
Collection (Rockville, MD). All other chemicals and reagents
were from previously listed sources (13–16).
Studies using experimental animals
All procedures using experimental animals were approved by
the Institutional Animal Care and Use Procedures Review Board.
Sprague Dawley rats, C57BL/6J-ob/ob mice, and Beagle dogs were
given food and water ad libitum and treated orally at a volume of
1.0 ml/200 g body weight (rats), 0.25 ml/25 g body weight (mice),
or 1.0 ml/kg body weight (dogs) with either an aqueous solution
of 0.1% pluronic P-105 in 10% DMSO (vehicle) or an aqueous
solution of 0.1% pluronic P-105 in 10% DMSO plus CP-320626.
Measurement of plasma, cholesterol, triglyceride,
glucose, and related metabolite levels
Serum glucose triglyceride and total cholesterol concentrations
were determined by the Abbott VP™ and VP Super System
analyzer using the A-Gent™ Glucose-UV, A-Gent™ Triglyceride
and A-Gent™ Cholesterol Test reagent systems. Serum insulin and
glucagon concentrations were determined by radioimmunoassay
(RIA) using kits from Binax (Portland, ME) and Amersham Corp.
(Arlington Heights, IL), respectively.
tration was determined spectrophotometrically using kits from
Sigma. Free fatty acid concentration was determined using a kit
from Wako (Richmond, VA). The serum glucose, insulin, glucagon,
triglyceride, total cholesterol,
-hydroxybutyrate, and free fatty acid–
lowering activity of test compounds were determined by statisti-
cal analysis (unpaired
t -test) with the vehicle-treated control group.
Measurement of plasma CP-320626 levels
l aliquots of plasma were added 50
g/ml CP-89816 in methanol), 5 ml methyl tert-butyl
ether, and 1 ml of 0.5 M sodium carbonate (pH 9). After vigor-
ous mixing and centrifugation, the ether layers were removed
and evaporated to dryness, and the resulting solid was reconsti-
tuted with 75
l mobile phase [45% acetonitrile, 55% 50 mM so-
dium phosphate monobasic, and 30 mM triethylamine (pH 3)].
l) of reconstituted samples were injected onto a
m Waters Nova-Pak C-18 (Waters Corp, Bedford, MA) reverse
phase column (3.9
150 mm), with a mobile phase flow rate of
1 ml/min. CP-320626 and internal standard were detected by
fluorescence (excitation at 290 nm and emission at 348 nm). The
linear dynamic range was between 0.1
tification) and 1
g/ml (upper limit of quantification). C
the concentration in the blood sample in which the highest
plasma concentration was measured. The area under the plasma
concentration time curve (AUC) from 0 to tlast (AUC
calculated using a linear trapezoidal approximation, where tlast
is the time point of the last quantifiable plasma concentration.
l of an internal
g/ml (lower limit of quan-
Measurement of liver and plasma precursor sterols by gas
Nonsaponifiable lipids were isolated from liver and plasma
and quantitated by gas chromatography-mass spectrometry (GC/
The structure of CP-320626.
by guest, on June 1, 2013
Harwood et al.
Dual-action glycogenolysis and cholesterolgenesis inhibitors549
MS). Samples of plasma (0.75 ml) and liver (0.75 g) were saponi-
fied at 70
C for 120 min in 2.5 ml of 2.5 M NaOH, then 5 ml of
absolute EtOH was added to each sample and the solutions were
mixed. Ten milliliters of petroleum ether was then added to each
sample, and the mixtures were shaken vigorously for 2 min then
centrifuged at 2,000
g in a bench-top Sorvall for 10 min. After a
second petroleum ether extraction, the resultant petroleum ether
layers were removed, dried under nitrogen, and dissolved in 500
l dry pyridine solution. A 500
(Sylon TP) was then added to each sample, and derivatization was
allowed to continue at room temperature (RT) for 1 h. Derivatized
samples were analyzed using an HP-6890 Series gas chromato-
graph equipped with a 6890 Series GC Injector and interfaced
with an HP-5973N mass selective detector. Separation was achieved
using a Supelco SAC-5 (15 m
The oven temperature was held at 250
C at a rate of 2
C/min. The ions monitored using full scan
electron ionization (70 eV) corresponded to the molecular ions
of the trimethylsilyl derivatives. Retention times of detectable pre-
cursor sterols relative to that of cholesterol were cholestanol (di-
hydrocholesterol), 1.03; 8-dehydrocholesterol, 1.06; desmosterol,
1.07; 7-dehydrocholesterol, 1.07; lathosterol, 1.10; 4-methylsterol,
1.20; 4,4-dimethylsterol, 1.20; lanosterol, 1.42; and dihydrolanos-
terol, 1.42, similar to those previously reported for C-3 hydroxyl-
derivatized sterols (17, 18).
Based on GC/MS analysis, the sterol composition of con-
trol rat liver was
98% cholesterol, with 0.04% cholestanol and
8-dehydrocholesterol, 0.17% desmosterol and 7-dehydrocholesterol,
0.13% lathosterol, 1.11% monomethyl and dimethyl sterols, and
0.49% trimethylsterols (lanosterol and dihydrolanosterol). The
sterol composition of control rat plasma was
0.57% monomethyl and dimethyl sterols and 0.38% trimethyl-
sterols (lanosterol and dihydrolanosterol). Levels of cholestanol,
8-dehydrocholesterol, desmosterol, 7-dehydrocholesterol, and la-
thosterol in control rat plasma were all below the limits of detection.
l aliquot of TMSI
m) GC column.
C for 0 min, then heated
99% cholesterol, with
Measurement of CYP51 activity
The activity of recombinant human CYP51, expressed in TOPP3
cells and partially purified by the method of Stromstedt, Rozman,
and Waterman (19), was determined by measuring the conversion
of lanosterol to 4,4-dimethylcholesta-8,14,24-trien-3
ously described (19), with the following modifications: Briefly, 25
l of a 1 mM suspension of lanosterol in a mixture of tyloxapol-
acetone (1:1; v/v), dioleylphosphatidylcholine micelles (5 mg/ml),
methanol, and 100 mM potassium phosphate buffer (pH 7.4)
was added to 5 ml glass tubes to provide a final lanosterol con-
centration of 50
methanol for final concentrations ranging between 0.01 and 200
M, were then added, and the lanosterol/inhibitor mixtures
were allowed to dry under nitrogen for 10 min. To the residues were
added 20 pmol partially purified recombinant human CYP51,
125 pmol human CYP reductase, and 50
bation at RT for 10–15 min for enzyme reconstitution, 540
100 mM potassium phosphate buffer (pH 7.4) containing 20%
glycerol, 0.1 mM DTT, 0.1 mM EDTA, and 0.5 mM KCN was
added to each tube. Reaction mixtures were preincubated for 2
min at 37
C, then reactions were initiated by the addition of 50
l of an NADPH regenerating system (final incubation concen-
tration, 10 mM MgCl
, 0.54 mM NADPH, 6.2 mM DL-isocitric
acid, 0.5 U/ml isocitrate dehydrogenase). After 60 min incuba-
tion at 37
C, reactions were terminated by addition of a 25
ume of ethyl acetate that also contained the internal standard,
ergosterol (1 mg/ml), followed by extraction with 5 ml ethyl ace-
tate. After vigorous mixing and centrifugation to facilitate phase
separation, 3–4 ml of the ethyl acetate phase was transferred to
fresh tubes and evaporated to dryness under nitrogen at 50
-ol as previ-
). Inhibitors, dissolved in
l rat lipid. After incu-
Samples were reconstituted in 150
l was applied to an HPLC system. Ergosterol (inter-
nal standard) and 4,4-dimethylcholesta-8,14,24-trien-3
tion product) were separated on a Waters Novapak C18 column
M, 150 mm
3.9 mm), with a mobile phase consisting of
methanol-acetonitrile-HPLC-grade water (45:45:10 v/v/v), at a
flow rate of 1.5 ml/min. Ergosterol and 4,4-dimethylcholesta-
-ol were monitored by UV detection at 248 nm
using a SpectroMonitor variable wavelength detector (LDC Ana-
lytical; Riviera Beach, FL), and the Multichrom™ data acquisiton
system (Version. 2.11; Fisons Instruments, Beverly, MA) was used
for data collection and analysis. Approximate retention times for
and 30 min, respectively.
l mobile phase (see below),
-ol and ergosterol were 25
Measurement of human liver glycogen phosphorylase
The activity of recombinant human liver glycogen phosphory-
lase, expressed in baculovirus, purified to
and fully activated by phosphorylase kinase as previously de-
scribed (20–22), was determined by measuring glycogen synthe-
sis from glucose-1-phosphate by assessing the release of inorganic
phosphate (reverse reaction) at 22
buffer (pH 7.2) containing 100 mM KCl, 2.5 mM EGTA, 2.5 mM
, 0.5 mM dithiothreitol, 0.63 mM glucose-1-phosphate, 1.25
mg/ml glycogen, 9.4 mM glucose, 0.7% DMSO, and up to 2
of partially purified, activated human liver glycogen phosphory-
lase, based on the method of Engers, Shechosky, and Madsen (22),
as previously described (9). The inorganic phosphate released
during a 60 min incubation was measured at 620 nm, 20 min af-
ter the addition of 150
l of 1 M HCl containing 10 mg/ml am-
monium molybdate and 0.38 mg/ml malachite green (23).
C in 100
l of 50 mM HEPES
Measurement of sterol and fatty acid synthesis in
Sterol and fatty acid synthesis were evaluated in HepG2 cells
by measuring incorporation of [2-
as previously described (13, 14), with modifications (15, 16) to
allow simultaneous assessment of both sterol and fatty acid syn-
thesis. HepG2 cells grown in T-75 flasks as previously described
(13, 14) were seeded into 24-well plates at a density of 1.2
cells/well and maintained in 1.0 ml of supplemented DMEM
(DMEM containing 10% heat-inactivated fetal bovine serum, 2 mM
g/ml gentamicin) for 7 days in a 37
incubator with medium changes on days 3 and 5. On day 8, the
medium was removed and replaced with fresh medium contain-
ing 1% DMSO
effector compounds. Immediately after com-
pound addition, 25
l of media containing 4
tate (56 mCi/mmol) was added to each incubation well. Plates
were then sealed with parafilm to prevent evaporation, and cells
were incubated at 37
C for 6 h with gentle shaking. After incuba-
tion, the samples were saponified by addition to each well of 1 ml
of 5 N KOH in MeOH, followed first by incubation for 2 h at
C and then by overnight incubation at RT. Mixtures were
transferred to glass conical tubes and extracted three times with
4.5 ml hexane. The pooled organic fractions (containing choles-
terol, post-squalene cholesterol precursors, and other nonsapon-
ifiable lipids) were dried under nitrogen, resuspended in 25
chloroform, and applied to 1
20 cm channels of Silica Gel 60C
TLC plates. Channels containing nonradioactive cholesterol, lanos-
terol, and squalene were included on selected TLC plates as sep-
aration markers. TLC plates were developed in hexane-diethyl
ether-acetic acid (70:30:2 v/v/v), air dried, and assessed for ra-
dioactivity using a Berthold Linear Radioactivity Analyzer (Oak
Ridge, TN) that reports radioactive peak location and integrated
peak area. The desmethylsterol peak (R
C]acetate into cellular lipids
C, 5% CO
Ci of [2-
0.27) was predomi-
by guest, on June 1, 2013
562 Journal of Lipid Research
Volume 46, 2005
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