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Hypolipidemic Effects of 2-Furoic Acid in Sprague-Dawley Rats.

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15
Hypolipidemic Effects of 2-Furoic Acid
The Hypolipidemic Effects of 2-Furoic Acid in Sprague-Dawley Rats
Iris H. Halla)*,Oi T. Wongal, David J. Reynoldsa),and J. J. Changb)
a) Division of Medicinal Chemistry and Natural Products School of Pharmacy, University of North Carolina, Chapel Hill, N.C. 27759-7360, USA
b) Department of Pathology, School of Medicine, University of North Carolina, Chapel Hill, NC 27559, USA
Received May 29, 1992
2-Furoic acid was shown to be effective in lowering both serum cholesterol and serum triglyceride, levels significantly in rats with an elevation of
HDL cholesterol level at 20 mg/kg/day orally. LDL receptor activity was
reduced in hepatocytes, aorta foam cells, small intestinal epithelium cells
and fibroblasts. HDL receptor activity was elevated in the rat hepatocytes
and small intestinal cells. These activities were correlated with inhibition
of acyl CoA cholesterol acyl transferase activity. Neutral cholesterol ester
hydrolase activity was elevated in rat hepatocytes and human fibroblasts.
Thus, 2-furoic acid appears to interfere directly with activity of intracellular enzymes rather than affecting high affinity-mediated lipoprotein membrane receptors. In vivo treatment with 2-furoic.acid led to reduction in the
liver and small intestine ATP dependent citrate lyase, acetyl CoA synthetase, acyl CoA cholesterol acyl transferase, sn-glycerol 3-phosphate acyl
transferase, phosphatidylate phosphohydrolase and heparin induced lipoprotein lipase activities.
2-Furoic acid reduced biliary cholesterol levels but the agent increased bile
salts which are lithogenic. Acute toxicity studies in mice suggest that the
agent has some hepatic toxicity effects. The LD,, was relatively low at 250
m a g IP in mice.
Senkung des Lipidblutspiegels durch Furan-2-carbonsaure bei SpragueDawley-Ratten
Previously a series of furoic acids has been shown to possess potent
hypolipidemic activity in rodents lowering both serum cholesterol and
triglyceride levels’). 2-Furoic acid was the most potent of the series. In
mouse liver, a number of enzymes involved in lipid metabolism were inhibited by 2-furoic acid. Tissue lipid levels and cholesterol distribution in
peripheral tissues were reduced over a 14-day period by the agent.’)
mals were maintained in an environment of 12 h light followed by 12 h
dark cycles at 22°C in plastic cages.
On day 7 and 14, the animals were bled between 7:30-8:30 a.m. Serum
cholesterol*) and triglycerides (Dow Kit) were determined. The animals
were sacrificed on day 14.
The present study involves the investigation of this agent
in Sprague-Dawley rats and tissue culture cells for its effect
on modulation of lipid synthetic enzymes and their relationship to LDL and HDL receptor activities as well as its
acute toxicity in rodents.
Animal Weights and Food Intake
Periodic animal weights were obtained during the experiments and
expressed as % of the animal’s weight on day 0. After dosing for 14 days
with test drugs, selected organs were excised, trimmed of fat, and
weighed3).Food consumption was determined daily.
Enzymatic Studies
Materials and Methods
Source of Compounds
2-Furoic acid: Aldrich Chemical Company, 1nc.- Isotopes: New England
Nuclear (DuPont). Substrate and cofactor: Sigma Chemical Co.
Hypolipidemic Activity in Normalogenic Rats
Sprague Dawley male rats (- 300 g) were administered 2-furoic acid
prepared as a homogenate in I % CMC at 20 mg/kg/day orally. The ani-
Arch. Pharm. (Weinheim) 326,15-23 (1993)
In Ratten erniedrigt Furan-2-carbonsaure signifikant sowohl das
Serumcholesterol als auch die Triglyceride, verbunden mit einer Erhohung
des HDL-Cholesterols bei oraler Gabe von 20 mg/kg/Tag. Die Aktivitat
des LDL-Rezeptors wurde in Hepatocyten, Schaumzellen der Aorta, in den
Epithelzellen des Dunndarms und Fibroblasten erniedrigt, wahrend die
HDL-Rezeptor-Aktivitat in Rattenhepatocyten und in den Zellen des
Dunndarms erhoht wurde. Diese Aktivitaten korrelieren mit der Hemmung
des Acyl-COA-Cholesterol Acyltransferase-Aktivitat. Die Aktivitat der
neutralen Cholesterolester-Hydrolase war in Rattenhepatocyten und in
menschlichen Fibroblasten erhoht. In vivo Behandlung mit Furan-2-carbonsaure reduzierte in der Leber und im Diinndarm die Aktivitaten der
ATP-abhhgigen Citratlyase, Acyl-COA-Synthetase, Acyl-COA-Cholesterol Acyltransferase, sn-Glycerol-3-phosphat-Acyltransferase,
Phosphatidylat-Phosphorylase und der heparininduzierte Lipoprotein-Lipase.- Furan-2carbonsaure reduziert zwar den Spiegel des biliaen Cholesterols, erhoht
aber den der lithogenen Gallensluren.- Studien zur akuten Toxizitat an
Mausen konnten nahelegen, dai3 die Verbindung hepatotoxische Effekte
hat. Die LD,, war mit 250 mg/kg nach i.p.-Gabe bei Mausen ziemlich
niedrig.- Die Studien lassen den Schlua zu, dal3 Furan-2-carbonsauren den
Lipidstoffwechsef nicht iiber intraze1luEre Enzyme sondern uber mernbranstandige Rezeptoren beeinflub.
A 10%homogenate was prepared of the liver and small intestinal mucosa in 0.25 M sucrose + 0.001 M EDTA, pH 7.2. The enzyme activities
were determined by following procedures: acetyl coenzyme A
synthetase4), adenosine triphosphate-dependent citrate lyase5), cholesterol7-a-hydroxylase6), acyl CoA cholesterol acyl transferase’), 3-hydroxy-3methylglutaryl coenzyme A8*9),acetyl coenzyme A carboxylase activity’O),
sn-glycerol-3-phosphate acyl transferase activity”), phosphatidate phosphohydrolase activity”), and heparin-activated hepatic lipoprotein
lipase13).Protein was determined for all enzyme assays by the technique of
Lowry et ai.I4’.
0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1993
0365-6233/93/0101-0015 $3.50 + .25/0
16
Bile Cannulation Study
Sprague Dawley male rats (- 300 gj were administered 2-furoic acid at
20 mg/kg/day. orally for 14 days. The rats were anesthetized with chlorpromazine (25 m a g ) followed 30 min later by pentobarbital (22 mg/kg
i.p.)'". Once the bile duct was identified, a loose ligature was placed around it and the duct was nicked. PE-I0 plastic tubing was placed in the duct
and tied in place. The ligatures were removed from the duodenum. The
bile was collected over the next 6 h.
Hall and coworkers
350 mCi/ml) for 10 min at 4°C in 0.5 M glycine-NaOH buffer, pH 10 with
4 equivalents of IC1 reagent per mole of lipoprotein and dialyzed against
0.15 M NaCI-0.1 % EDTA, pH 7.4 for 48 h changing the dialysis medium
six times. The lipoprotein was sterilized by using a 0.45 p filter.
Tissue Culture Cczlls
Normal human fetal foreskin (BG9) fibroblasts were obtained from the
University of North Carolina Cancer Research Center. The cells were
maintained as I monolayer culture in Eagles minimum essential medium
(MEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics
Bile Lipid Lrvels
in a humidified incubator (5% C02) at 37OC.
The flow rate of the bile was calculated for each group as ml/inin. .41iIsolated rat hepatocytes were obtained from Sprague Dawley male rats
quots were extracted for lipids by the Folch et al.") and Bligh and Dyer")
(- 350 g) by perfusing the livers with calcium free HEPES buffer, pH 7.4
methods. Cholesterol'), triglyceride, neutral lipids"', and pho~pholipid'~' for 5 min and then collagenase buffer (SO nil/min) for 10 min which afforcontent was determined. Protein was determined in the original bile'4).
ded a single cell suspension. The parenchymal cells were purified by centrifugation and plated in organ tissue plates at 1.5 x lo6 cells in MEM supplemented with antibiotics and 10% fetal calf serum for 24 h in the CO,
HPLC Airalvsis ofBile Acids Conrenr
incubator2'?
Samples were frozen overnight and thawed. The bile lipids were extracSmall intestinal rat epithelium cells were obtained from American Tisted with EtOH 1:20 and filtered20'. Aliquots (250 pl) of the bile were
sue Culture and were maintained in D-MEM + 10% FBS and P/S + glucoadded to 100 pI of the internal standard (200 mg% testosterone). Then
se, 5% insulin. Mouse macrophages were obtained from American Tissue
4.75 ml of hot analytical grade ethanol was added, vortexed, and allowed
Culture and were maintained in D-MEM + 10%FBS and P/S.
to evaporate in a boiling water bath. The residues were dissolved in 250 pl
Sprague Dawley male rats (- 450 g) were anesthetized with ether, the
of the mobile phase A and 100 pl were injected onto the HPLC column (p
thoracic cavity was opened and the aorta exposed. Excessive connective
tissue was removed and the aorta (- 2 cm) was excised and immediately
Bondapak C18 column) (Waters) with a guard column (Bondapak
C 1 H/Corasi) (Waters) eluted with acetonitrile:methanol:O.03 M phosphate
placed in cold MEM. 10% newborn calf serum, non-essential amino acids
buffer, pH 3.4 (l:6:3 v/v/v). The flow rate was 0.5 ml/min (600 psi isobaand antibiotics. The aorta was cut into small circular segments using a
ric flow) with detection at 210 nm. Standard bile acids (Sigma Chemical
dissecting microscope. The rings of aorta were cut longitudinally to expose
C0.j were purchased and were treated identically as the bile biological
the intimal surface. This surface was carefully stripped from the adventitia,
placed in 6 ml fresh medium, and placed in a 5% CO, incubator at 37°C.
samples.
After 3 days, new MEM medium was added to a total volume of 10 ml.
From this point, medium was changed twice a week. Usually the cells had
Absorptionfiom In Siru Duodenum Loops
become confluent within 2 weeks, at which time they were treated with
0.25% trypsin and 1 x 10' cells were transferred to Petri dishes (35 mm)
Sprague Dawley male rats were treated and anesthetized as indicated
and allowed to grow until they were confluent after three days25).This preabove and the duodenum was isolated and nicked. Glass L-shaped cannuparation contains more than one cell type as derived from the plaques.
laes were placed at the proximal end of the duodenum and 20 cm distally
down the intestine2". The segment was perfused with isotonic PBS buffer,
Tissue CulfureReceptor Acriviry for I D L or HDf.
pH 7.2 until the lumen was clear and all material expelled. Drug solution
(0.2 ml) (20 mgkg) was placed in the loop and either 1,2-'H-cholesterol
Receptor activity binding and internalization as well as lipoprotein deg(54.8 Ci/mol) or 2,4-3H-cholic acid (25 mCi/mol) (2 pCi) were also placed
radation were determined by the method of Brown and G o l d ~ t e i n * ~ ~ ~ ~ J .
in the loop. Aliquots (50 pl) were removed over the next 210 min and the
Cells [ I S x lo6 hepatocytes, 2 x 10' fibroblasts, macrophages or small
radioactivity determined using a Packard scintillation counter after conecintestinal cells, or I x lo4 aorta cells] were added to 2 ml of MEM + 10%
ting for quenching. The disappearance of the isotope from the loop over
LPDS and 10 pCi of "'I-LDL or %HDL (I00 mg). Sterile drugs (0.2
time was plotted for the control and treated animals.
ml) in phosphate saline buffer (PBS) were added to obtain final concentrations of 5 , 10, 25 or 50 pM. Incubation was for 5 h in the COl incubator at
37°C after which the medium was removed and processed for the lipoproSeparation of Lipoproteins
tein degradation assays by treating with trichloroacetic acid, K1, and H20,
Blood was collected from Sprague Dawley rats (- 400 g) from the abdoand the lipids removed by CHCI? extraction. The whole cells were washed
minal vein (- 10 ml) under light ether anesthesia and the serum lipoproand then taken up in 0.1 M NaOH and counted in a ycoumer for Iz5I-LDL
teins were separated by ultracentrifugation. Human serum from healthy
or "SI-HDL receptor binding and internalization. Aliquots were analyzed
male suhjects was obtained from the North Carolina Memorial Hospital
for protein content by Lowry techniquesi4).
Blood Bank. Human low density lipoprotein (LDL: density 1.019-1.063
g/ml) and high density lipoprotein (HDL: density 1.063-1.210 g/ml) as
Enzyme Activity
well as lipoprotein-deficient serum (LPDS: density 1.215 d m l ) were determined. Rat lipoproteins were separated by differential ultracentrifugation
Cholesterol synthesis (HMG-Co4 reductase activity) was determined
which have slightly different densities than humans2".
using 2 pCi (l-'4C)-acetyl CoA (56 mCi/mmol)*'. After 5 h incubation in
medium and 10% LPDS, pH 7.4, the cells were taken up in lysis buffer.
The cholesterol was precipitated as the digitonide and isolated by the proLabeling of Lipoproteins
cedure of Wadu'!.
Acyl CoA cholesterol acyl transferase activity was determined by the
Radiolabeling of LDL and HDL fractions was carried out using a modiBalasuhramanium method" using 20 pCi of I-14C-oleic acid (57.3
fication of the iodine monochloride method*". Purified LDL or HDL was
mCi/mmol) as an albumin complex which was incubated with cells in 2 ml
iodinated with 250 pCi or carrier free '251-(New England Nuclear,
Arch. Pharm. (Weinheim) 326, 15-23 (1993)
17
Hypolipidemic Effects of 2-Furoic Acid
medium containing LDL (100 mg protein/ml) and drug for 5 h. The cells
were taken up in PBS extracted with CHCI,:MeOH (2:1), and the org. solvent evaporated, The org. layer residue was taken up in 250 p1 of ethyl
acetate and 100 pl were plated on tlc silica gel plates. After eluting heptane:ethyl ether:glacial acetic acid (85: 15:2), the area where the cholesterol
ester eluted (Rf value = 0.9) was scraped and counted in a Packard p-scintillation counter.
Cholesterol ester hydrolase activity was determined by using 2 pCI of
cholesterol oleate-l-I4C (56.6 mCi/mol) for a 5 h incubation period2”. The
cells were taken up in PBS and extracted with CHC1,:MeOH (2: 1j and the
org. layer was evaporated. The residue of the org. layer was taken up in
250 pl of ethyl acetate and 100 pl were plated on tlc silica gel plates. After
eluting the plates with heptane:ethyl ether:glacial acetic acid (85: 15:2)16’,
the free I4C-oleate (Rf = 0.35) area was scraped and counted.
Cholesterol-7-a hydroxylase activity was determined in hepatocytes by
the method of Shefer” with 1,2-3H-cholesterol (54.8 Ci/mmol) by incubating for 5 h in MEM, antibiotic, 10% LPDS. 7-a-Hydroxy-cholesterol was
separated on silica gel plates by tlc and an area corresponding to a Rf value
of 0.42 was scanned and counted.
az-Glycerol-3-phosphate acyl transferase activity was determined essentially by the method of Lamb1”. sn[ 1 ,3-1JC]Glycerol-3-phosphate
(144
mi/mmol) (5 pCi) was added to medium and 10% LPDS and allowed to
incubate for 5 h. The cells were washed, lysed and taken up in phosphate
saline buffer. The cells were extracted with ether containing 1% glacial
acetic acid. The org. phase was transferred to a scintillation vial, allowed
to evaporate and then counted.
Lipoprotein lipase activity was measured according to ChairL3’using
gly~erol-tri-(‘~C)
palmitate (64 mCi/mmol) emulsified with lecithin. The
cells were treated with 5 U/ml heparin, 4% albumin, 10%glycerol, 30 min
prior to the assays to release the lipase from the cell surface. The 5 pCi of
isotope and drugs were added and incubated for 5 h. The cells were washed, taken up in phosphate saline buffer and extracted with chloroformmethanol-heptane (l.41:1.25:1), After 30 min, 0.14 M boric acid was
added and the tubes were centrifuged. The aqueous layer was transferred
to a scintillation vial and counted.
Protein synthesis was determined by incubating cells with 2 pCi L - ~ H 4,5 leucine (59.8 Ci/nimol) for 5 h. The trichloroacetic acid precipitated
3H-protein was collected on Whatman no. 1 filters by vacuum suction, and
after washing the filters with acid they were countedlX?
Clinical Chemistry
At the time of the sacrifice, the major organs were excised, trimmed of
fat and weighed. Blood was obtained from the carotid and centrifuged at
3500 g x 10 min to obtain the serum. Chemical or enzymatic assays were
performed with Sigma Chemical kits: urea nitrogen (BUN, No. 640),
alanine amino transferase (SGPT. No. 505), alkaline phosphatase (AP. No.
104), glucose (No. SlO), lactic dehydrogenase (LDH, No. SOO), creatine
phosphokinase (CP-kinase, No. 661), and total and direct bilirubin (No.
605). Serum triglycerides were determined with a diagnostic kit from
Boehringer Mannheim; serum cholesterol was determined by the method
of Ness”. Albumin and total protein were determined by the method of
Lowry’“’. Cholic acid and uric acid were determined as outlined by
Tietz”).
Blond Collection and Parameters
Blood was obtained from the carotid, a drop was placed on glass slides
and fixed in Wright’s stain. Differential white blood cell counts, platelet
counts and hematocrits were obtained for each mouse group sacrificed at
the specified times.
Histological Section
The animals were killed by C 0 2 asphyxiation. After all vital signs had
ceased, a midline incision was made from the lower jaw to the inguinal
area. Thymus, spleen, liver, and kidney were excised and weighed, and
representative tissue samples were fixed in 10% buffered formalin, trimmed and sectioned at 6 p in thickness and stained with hematoxylin and
eosin.
Female Fertility
CF, female mice (- 30 g) were administered 20, SO or 100 mg/kg/day
for three weeks. While continuing dosing the females were exposed to
males (2:l j for another three weeks. The males were rotated every 7 days
to eliminate infertility. After three weeks the males were removed. The %
pregnancy, number of live births, deaths and birth weights were noted.
Four weeks after birth the pups weight, % survival and sex were noted for
each group.
Results
HDL Uptake of Cholesterol from Fibroblusts Cells
Human fibroblasts (BG-9) were grown to confluence. ‘H-Cholesterol ( I
pCi) was added to the Petri dishes in fresh medium for 24 h at 37°C in a
C 0 2 incubator, after which the medium was decanted and the cells washed
four times in PBS, pH 7.4. Fresh MEM + 100 ml of HDL (human)29)+
antibiotics was added to the Petri dishes which were incubated for 24 h at
37°C in a CO, incubator”? The extracellular medium was collected, and
the protein precipitated with 10% trichlorodcetic acid (TCA). The denatured protein was collected on a Whatman no. 1 filter disc, washed and corrected. Preliminary studies demonstrated that with BG-9 fibroblasts, a concentration-dependent binding of 3H-cholesterol derived from the cells to
the extracellular HDL occurred from I to 100 pI of human HDL.
Mouse Toxicirq
LD5, acute toxicity was determined in CF, male mice (28 g) i.p. using
doses of 5 mgkg to 1 g/kg as a single dose. The number of deaths in each
group was noted over the next 7 days.
For the mouse toxicity study, CF, male mice (- 28 g) were dosed at 20,
40 and 100 mg/kg/day i.p. for 7 days with 2-furoic acid. The food consumption was determined daily and water was ad libitum. The animals
were maintained in 12 h light and dark cycles at 22°C.
Arch. Pharm. (Weinheim)326,15-23 (1993)
2-Furoic acid at 20 mg/kg/day orally suppressed the
serum cholesterol levels 50% and serum triglyceride levels
42% after 14 days in rats. The HDL cholesterol content was
elevated 34% on day 14 (Table 1). The VLDL and LDL
cholesterol levels were not reduced at this dose. When the
de now lipid enzyme activities were examined after in vivo
treatment to rats (Table 2), ATP dependent citrate lyase and
acetyl CoA synthetase activities were reduced both in liver
and small intestine mucosa. HMG CoA reductase activity
was significantly elevated in rat liver but reduced in the
small intenstine mucosa. Acyl CoA cholesterol acyl transferase, sn-glycerol-3-phosphate acyl transferase, phosphatidylate phosphohydrolase, and lipoprotein lipase activities
were reduced in both tissues. Neutral cholesterol ester
hydrolase activity was elevated in both tissues. Cholesterol7-a-hydroxylase activity was only reduced in the small
intestine mucosa. Acetyl CoA carboxylase activity was
marginally inhibited in the small intestine mucosa. When
tissue culture cells were examined (Table 3), 2-furoic acid
suppressed LDL receptor activity, i.e. binding and interna-
18
Hall and coworkers
Table I: Effects of 2-Furoi~Acid on Serum Lipid and Lipoprotein Cholesterol Levela of Sprague
Ddwkey Rats after 14 Days Adminimdtion at 20 mg/kg/Day Orally
-
-_
__.
-~
Percent of Control (XfSD)
(N-6)
Control
Serum Lipids
Cholesterol Trielvcerides
100+7'
Treated
50+5*
100+sb
100+6c
10026d
100+6e
58+6*
112+7
100+7
134+7*
a7a mg/dl
b112 mg/dl
*P<O.OOl
Lipoprotein
Cholesterol Content
U L
c190 pg/ml
d 2 1 ~pg/ml
Student's "t" test
Table 2: Effects of 2-Furoic Acid on Sprague Dawley Rat Enzyme Activities of Liver and Small
Intestine after Treatment at 20 ing/kg/Day Orally
Enzyme Activity
(N-6)
Percent of Control (X+SD)
Liver
Small Intestine
Control Treated
Control Treated
-
ATP-dependent
100+4a
27+2*
citrate lyase
10o+_sb 43+3*
Acetyl CoAsynthetase
100+6c 17358"
HMG - COA
reductase
ioo+sd
7a+5*
Acyl-CoA-cholesterolacyl transferase
i00+6~ i5a+7*
Cholesterol ester
hydrolase
Cholesterol-7a
100+_3f 9454
hydroxylase
100+4g
99+5
Acetyl CoA
carboxylase
54+4*
~-Glycerol-3-phosphate-100+5h
acyl-transferase
100+6'
52+4*
Phosphotidylate
Phosphohydrolase
100+5j
60+5*
Lipoprotein lipase
37**
46+6*
6925*
65+4*
145+4*
65+5*
8256
22+3*
66+7*
52+5*
* p < 0.001; a) 9.2 mg citrate hydrolyzed/g wet tissue; h,
10.0 mg acetyl CoA formed/g wet tissue; cy
103020 dpin/g wet tissue; dl 86640 dpm/g wet tissue; "22443 dpm/g wet tissue; 289450 dpm/g wet
tissue: f ) 43000 dpm/g wet tissue; h, 87620 dpm/g wet tissue; I ) 11 mg Pi released/gm wet tissue; J).
3 1 12 dpm/g wet tissue; k, 9.17 mg citrate hydrolyzed/g wet tissue; ') 5.27 mg Acetyl CoA formed/g
wet tissue: 113322 dpm/gm wet tissue; ") 64819 dpm/gm wet tissue: ('1 259099 dpm/gm wet tissue;
P ) 230.99 dpm/gm wet tissue; 4) 54892 dpm/gm wet tissue; ') 73219 dpm/gm wet tissue: ') 111 ug Pi
released/gm; ') 43128 dpm/g wet tissue.
lization, in a dose dependent manner except for the macrophage system which was elevated. LDL degradation was
reduced except for the liver and macrophages. HDL receptor activity was elevated by 2-furoic in the liver and small
intestinal epithelium but suppressed in the other cells. HDL
degradation was accelerated by the drug in liver, aorta, and
small intestine but not in fibroblasts or macrophages. HMG
CoA reductase activity was suppressed in the liver, aorta
foam cells, and macrophages but elevated 30-38% in the
small intestinal epithelium and fibroblasts. Acyl CoA cholesterol acyl transferase activity was inhibited in a concentration dependent manner in all cells except the macropha-
ges. Cholesterol-7-a-hydroxylaseactivity was elevated at
50 yM in the liver and small intestine mucosa. Neutral cholesterol ester hydrolase was accelerated in concentration
dependent manner in hepatocytes and human fibroblasts but
inhibited in rat foam cells, small intestinal epithelium and
macrophages. sn-Glycerol-3-phosphate acyl transferase
activity was inhibited in all cell lines except the rat foam
cells. Heparin induced lipoprotein lipase activity was suppressed in hepatocytes and fibroblasts in a concentration
dependent manner. 'H-Leucine incorporation into protein
was only significantly elevated 37% at 50 yM in the rat
foam cells. Human fibroblasts demonstrated reductions of
Arch. Phurm. (Weinheim) 326, I S - 2 3 (1993)
19
Hypolipidemic Effects of 2-Furoic Acid
Table 3: Effects of 2-Furoic Acid on Tissue Culture Receptor and Enzyme Activities
Control
(N-5 1
Rat Hepatocyte
R a t Foam Cell
R a t SI Epithelium
Human Fibroblast
Mouse Macrophage
5uM
lOuH
Percent of Control &SD)
25UM
50124 Control 5uM
'954
b350
'993
d577
e107
74&5*
9426
12127
41&5
11626
cpm/mg
cpm/mg
cpm/mg
cpm/mg
cpm/mg
of
of
of
of
of
74+4* 65+6*
75+5* 75+4*
7725* 75&4*
2023
18+5
11957 136+8*
57+5*
78+6
53&5*
1424
139+7*
10027f 9226 133+7*
100258 1 0 4 ~ 7 10926
100+6h 11328 82&6*
10025' 54+_66* 23&*
100+6j 102+5 11025
Human Fibroblast
Mouse Macrophage
100+5a
100+4b
100+8c
100+7d
100+8e
159+6*
76+7*
68+4*
21&3*
11726
171+6*
9329
60+5*
19+4*
12326
HDL Degradation
10426 10427 156&6* 157+7* 100+6f 10727 11628 169+7* 178+6*
8127
79+6* 100+6g 49+_6* 9 7 ~ 6 12426 137+6*
90+,5 8326
11826 13028 18127* 261+6* 100+ah 11025 11427 324~7*455+6*
109+7 73+6* 55+4* 40+4* 100+4' 112+6
53+6* 4826* 40+5*
8626
75+5* 10026j 71+5* 5 9 ~ 5 * 57+5* 46+5*
11026 9825
a322 cpm/mg of p r o t e i n
b277 cpm/mg of p r o t e i n
'584 cpm/mg of p r o t e i n
%93 cpm/mg of p r o t e i n
e17 cpm/mg of p r o t e i n
rol excretion was accelerated 144%; triglycerides were
reduced 29% and phospholipids 14% after 14 days treatment with 2-furoic acid. The bile flow was reduced 15%
after drug treatment. There did appear to be an increase
concentration in bile acids (Table 4).For example, taurochoiic acid was elevated five fold above normal; lithocholic
acid was elevated 57% and tauroursodeoxycholic acid 65%.
Glycocholic acid was not present in the treated animals.
Nevertheless, the in situ intestinal loop studies showed that
2-furoic acid interfered with absorption or reabsorption of
cholic acid with 70% remaining in the lumen after 181 min
(Fig. 1). Cholesterol reabsorption was not affected significantly by the presence of drug (Fig. 2).
When the acute toxicity of 2-furoic acid was examined,
the LD,o in CF1 male mice was 250 m a g i.p. Multiple
dosing in mice of 20, 40 and 80 mgkg for seven days was
well tolerated and all animals survived. The total weight
increase over seven days was reduced at 40 and 80
mg/kg/day . The hematocrit percentage and platelet estimates were decreased slightly in the treated group but were not
of a serious concern. The differential white cell counts were
Arch. Pharm. (Weinheim)326,15-23 (1993)
50uM
f1094 cpm/mg of protein
8712 cpm/mg o f protein
h502 cpm/mg of protein
'2744 cpm/mg of protein
j37 cpm/mg of protein
protein
protein
protein
protein
protein
HDL Receptor binding
and I n t e r n a l i z a t i o n
R a t Hepatocyte
R a t Foam Cell
R a t SI Epithelium
25124
LDL Degradation
LDL Receptor Binding
and I n t e r n a l i z a t i o n
100+6a
10024'
10025'
100+3d
100+6e
lOuM
f1327 cpna/mg of protein
g1722 cpm/mg of protein
h507 cpm/mg of protein
'654 cpm/mg of protein
j34 cpm/mg of protein
The Effects of 2-Furoic Acid on
3H-Cholic Acid Reabsorption from Gut
110
+Contiol
1-Treatad
2
m
L
0)
E
40 -
2
(D
30 -
c
0
20
I
0
Figure
100
200
20
Hall and coworkers
Table 3: Continued
a-5 1
Control
5uM
lOuM
Percent o f Control &SD)
25uM
50uM Control 5uM
10025'
96+7
100+6b 14527
100+5' 10627
100+7d 10827
100+5e 8856
92+6
71+5* 63+4*
95+6
69+5* 6055*
108+6 11527 13028*
12527 133+6* 138+6*
67+5* 64+6* 48+5*
a6556 dpm/mg o f p r o t e i n
b607 dpm/mg o f p r o t e i n
'642 dpm/mg o f p r o t e i n
'2589 dpm/mg of p r o t e i n
e552 dpm/mg of p r o t e i n
50uM
100+4f
100248
100+6h
100+5'
100+6j
8124
84+5
9325
9726
9526
75+5* 74+6* 6627*
8225
50+5* 39+4*
79+6* 65+6* 53+5*
9224
8426
68+5*
98+7 1 2 h 5 135+7*
€684 dpm/mg of p r o t e i n
g1.22 dpm/mg o f p r o t e i n
h134 dpm/mg of p r o t e i n
'2326 dpm/mg of p r o t e i n
j 2 4 dpm/mg of p r o t e i n
N e u t r a l C h o l e s t e r o l Ester
Hydrolase
R a t Hepatocyte
Rat Foam Cell
Rat SI Epithelium
Human F i b r o b l a s t
Mouse Macrophage
25uM
Acyl CoA C h o l e s t e r o l Acyl
Transferase
HMG CoA Reductase
R a t Hepatocyte
R a t Foam C e l l
Rat SI Epithelium
Human F i b r o b l a s t
Mouse Macrophage
lOuM
~-Glycerol-3-Phosphate
Acyl T r a n s f e r a s e
78+5* 63+6* 58+5* 53+5*
97+6
95+5 104&6 11656
131+7* 140+5* 97+6
77+5*
9526
8226
47&* 41+6*
73+5* 70+5* 60+5* 54+6*
100+6a 149+6* 157+6* 208+8* 244+9*
100+5b 61+7* 59+5* 51+6* 4425*
100+9c 65+6* 59+6* 5924* 53+4*
100+5d 149+7* 157+5* 208+6* 244+8*
100+7e 11628
8325
68+7* 47+5*
100+6f
100268
100+6h
100+6'
100+7j
a670 dpm/mg of p r o t e i n
b2876 dpm/mg of p r o t e i n
63 dpm/mg of p r o t e i n
68 dpm/mg of p r o t e i n
e l l o dpm/mg of p r o t e i n
f1804 dpm/mg of p r o t e i n
g242 dpm/mg of p r o t e i n
h266 dpm/mg of p r o t e i n
'1473 dpm/mg of p r o t e i n
j 3 8 0 dpm/mg of p r o t e i n
3H Leucine I n c o r p o r a t i o n
3H C h o l e s t e r o l uptake by HDL
into Protein
R a t Hepatocyte
R a t Foam C e l l
R a t SI Epithelium
Human F i b r o b l a s t
Mouse Macrophage
1 0 0 ~
10027b
100+7c
100+7d
100+6e
11525
6 ~
8024
8756
170+7*
132+6*
11257
8355
105+6
10656
11626
115+6 10925
8556 137+6*
10857 112+7
75+6* 73+6*
9725
9727
C h o l e s t e r o l 7 - a l p h a Hydroxylase
100+5a 11125 117+6 309+7*
1 0 0 ~ 176+6*
7 ~
182+8* 174+8*
Human F i b r o b l a s t
a288 dpm/mg of p r o t e i n
b685 dpm/mg of p r o t e i n
40 and 80 mg/kg treated groups. The % total body weight
values for most organs were within normal limits. The kidney weight demonstrated an increase in all of the treated
groups and the spleen showed a reduction in weight of the
treated groups. Stomach showed an increase at 20 m g k g
160+7*
d1380 dpm/mg of p r o t e i n
e197 dpm/mg of p r o t e i n
f576 dpm/mg of p r o t e i n
a980 dpm/mg of p r o t e i n
b760 dpm/mg o f p r o t e i n
'116 dpm/mg of p r o t e i n
R a t Hepatocyte
R a t SI Epithelium
100+5f
9426
8226
Heparin Induced Lipoprotein Lipase
100&4c 7755* 61+6*
44+6*
33+5*
100+4d 12027
61+6*
57+5*
73+5*
'25 dpm/mg of p r o t e i n
d269 dpm/mg of p r o t e i n
followed by a decrease in weight at 40 and 80 mg/kg. The
reproductive organs demonstrated an increase at all doses.
The liver showed reduced cellularity and increased cytoplasmic vacuolation in the 80 rng/kg/day treated animals.
There were many multifocal granulomas consisting with
Arch. Pharm. (Weinheim)3 2 6 , 1 5 2 3 (1993)
21
Hypolipidemic Effects of 2-Furoic Acid
Table 4: Effects of 2-Furoic Acid on Bile Lipids and Bile Acids of Sprague Dawley RatS after 14
Days Treatment at 20 mg/kg/Day Orally
Control
Treated
Bile Flow Rate (ml/h)
0.62
0.53
Lipid Content
Percent of Control
(N-6)
~~~
(X+SD)
~
244+7*
71+5*
103+5
8625
119+9
10726
Cholesterol
Triglyceride
Neutral Lipids
Phospholipids
Weight of Lipids
Proteins
21.42+2.7*
13.289.1
Bile Acid Content (pg/ml)
Tauroursodeoxycholic Acid
Glycoursodeoxycholic Acid
Taurocholic Acid
Glycocholic Acid
Taurodeoxycholic Acid
Lithocholic Acid
Taurochenoxycholic Acid
Glycochenoxycholic Acid
100258
100+4h
10026i
10028j
100+4k
100~7~
100+6m
10025"
165+6*
79+5*
517+12*
Total Bile Acids
100+6O
174;t;4*
a 1.18 mg/dl
0.5 mg/dl
1.70 mg/dl
1.75 mg/dl
2.60 mg/dl
p 5 0.001
f0.033 mg/dl
g2.50 pg/ml
h5.61 pg/ml
'1.53 pg/ml
j0.62 pg/ml
10323
157+6*
116+5
108+6
k0.50
'2.82
%.24
"0.09
'14.03
pg/ml
pg/ml
pg/ml
pg/ml
pg/ml
-
collections of chronic inflammatory cells. The spleen and
kidney were normal and demonstrated no lesions at any
doses employed as were the livers collected from animals
treated at 20 and 40 mg/kg/day.
The Effects of 2-Furoic Acid on
3H-Cholesterol Reabsorption From the Gut
GuiE2
The clinical chemistry values also reflect changes in the
liver induced by the drug. For example SGPT values are
elevated at all doses used, and alkaline phosphatase is elevated at 20 mg/kg/day (Table 5). Serum glucose and uric
acid levels were reduced over time which may reflect changes in liver function. There were effects on the fertility of
female mice (Table 6). After administered 2-furoic acid at
100 mg/kg/day, all of the female mothers died. The number
of fetusesflitter was reduced at 20 mg/kg from 9.33 to 7.75.
The average weight of the fetuses at birth was reduced from
1.6 g to 1.37 g at 50 mg/kg/day. The survival of the fetuses
was not affected at 20 mg/kg but was reduced from 10.6 at
birth to 7.75 after four weeks at 50 mg/kg/day treatment of
the mother. The percentage of maleshitter was lower in the
treated groups and the average weight of the fetuses after
four weeks was elevated in the treated groups.
Discussion
10
I
I
Figure 2
Arch. Pharm. (Weinheim) 326,15-23 (1993)
,
2-Furoic acid demonstrates potent hypolipidemic acitivity
lowering both serum cholesterol and triglyceride levels.
One of the disappointing features of this agent is that
VLDL and LDL-cholesterol levels are not reduced. After
14 days HDL-cholesterol levels are elevated after treatment
with 2-furoic acid. Increased HDL-cholesterol levels supposedly protect man against myocardial infarction3*,"). The
22
Hall and coworkers
Table 5: Effects of 2-Furoic Acid
80 mg/kg/Day i.p.
(N-5)
011
Control
Weieht Increase
Survival
Hematocrit
Platele Estimate
x 10
%
CF, Mice when Administered at 20, 40 and
Percent of Control (X+SD)
20 mg/kg
40 mg/kg
80 mg/kg
100+3
5/5
4722
19.9
12525
5/5
45+3
17.3
102+4
102+5
5/5
5/5
4326
16.3
4224
15.3
Differential White Cell Count (%l
Lymphocyte
53.8
52.0
PMNS
42.5
44.7
Bands
1.5
1.3
1.0
0.7
Monocytes
1.0
1.3
Eosinophils
0
Basophils
0.2
55.6
42.3
1.3
0.3
0.3
0.2
52.0
45.7
1.3
0.7
0.3
0
Weieht ( % Tota1 Body Weiuhtl
Brain
0.417
0.417
Lung
0.200
0.200
Heart
0.167
0.150
Liver
1.750
1.883
Kidney
0.388
0.466
Spleen
0.183
0.117
Stomach
0.966
1.250
Small intestine
2.042
2.133
Large intestine
1.500
1.500
Reproductive organs 0.450
0.566
0.383
0.200
0.200
1.675
0.400
0.133
0.550
1.983
1.333
0.500
0.417
0.233
0.200
1.733
0.450
0.133
0.540
2.317
1.483
0.817
Flinical Chemistry Values
Total protein
100+3
Glucose
loo&
Cholesterol
10026
Triglyceride
100+5
SGPT
10024
BUN
10026
CP-kinase
10027
Alkaline phosphatase 10024
Uric acid
10026
107k3
9555
8024
8655
250+5*
10357
25+5*
10355
8456
9852
91+2
84+5
7657
27757*
96+3
53+3
8256
6125
z
102+3
10257
83+3
94+7
276+6*
10055
31+6*
17056
91+5
Table 6: Effects of ZFuroic Acid on the Fertility of CF, Female Mice
(N-6 )
Control
20 mg/kg
50 mg/kg
%
pregnancy
100
80
#
alive fetuses/litter
9.33
7.75
Wt. of fetuses/litter
1.60
1.65
1.37
Survival of fetuses, 4 wks
9.20
8.0
7.66
60
0
10.6
Wt. of fetuses, 4 wks
12.33
15.03
13.11
Percent male/litter
54.5
48.7
43.96
mode of action in lowering lipid de novo synthesizing enzymes was at feasible sites where other small ring structur e ~ ~have
~ ,their
~ ~effects,
)
e.g. cyclic i m i d e ~ ~benzenetri~),
carboxylic acid37), p y r r ~ l i d i n o n e ~ ~benzohydroxamic
),
acids’9), d i l a t ~ t i n ~4-pyrimidine
~),
carboxylic acid3) and N benzyl~ulfamates~~).
2-Furoic acid inhibited LDL receptor activity which suggests that the agent blocked lipid uptake in peripheral tissue
100 mg/kg
that is transported by apo B lipoproteins. The macrophage
system is supposedly a scavenger pathway which clears
chemically altered LDLs from the serum. Treatment with 2furoic acid accelerated this process. HDL binding activity
in the liver was accelerated which may be indicative of the
agent’s ability to accelerate the cholesterol reverse transport
of lipids from the peripheral tissue to the liver for excretion
in the bile. One of the disturbing aspects of the effects of 2-
Arch. Pharm. (Weinheim)326,15-23 (1993)
23
Hypolipidemic Effects of 2-Furoic Acid
furoic acid on the enzyme activities was the effects afforded
by the agent were not consistent from cell line to cell line.
Inhibition of acyl CoA cholesterol acyl transferase activity
and acceleration of neutral cholesterol ester hydrolyase
would lead to the reduction of the synthesis and storage of
tissue cholesterol esters. The inhibition of cholesterol synthesis did not correlate well with the LDL receptor activity.
According to the theory of Brown and Goldstein26%27’,
LDL
receptor activity should increase as HMG CoA reductase
activity is inhibited. The present evidence would suggest
that 2-furoic acid does not function on membrane receptors
in a consistent manner to regulate lipid regulatory enzymes
but may act on enzymes directly in the cell. The inhibition
of sn-glycerol-3-phosphate acyl transferase and heparin
induced lipoprotein lipase by 2-furoic acid would be of sufficient magnitude to explain the reduction of serum triglyceride levels. It is not known whether these two enzyme
activities are regulated by lipoprotein receptor mechanisms.
The increase in biliary excretion of cholesterol afforded
by 2-furoic acid would cause a net reduction in serum total
cholesterol levels. Similar types of increases in biliary
cholesterol have been observed with clofibrate and
2-Furoic acid accelerated cholesterol-7-ahydroxylase activity indicative of an increase in the de novo
synthesis of bile acids. This enzyme presence in GI mucosa
cells has been reported in the rat43) and is inhibited by
hypolipidemic agents. In vivo rat studies showed there was
an observable 74% increase in bila acids. Unfortunately, a
number of these bile acids have lithogenic effects, e.g.
lithocholic acid and taurocholic acid. The reduction in the
bile flow may also reflect biliary tract problems induced by
the agent. The morphology of the liver and the clinical chemistry values, e.g. elevated SGPT, alkaline phosphatase and
decreased glucose, again suggest that 2-furoic acid has
some hepatotoxicity effects. The hematopoietic parameters
and other organ histologies do not appear to be markedly
affected by 2-furoic acid. 2-Furoic acid appears to have
some toxic effects in the fetus with reduced pregnancies
and number of fetuses/litter, particularly at higher doses.
Even though 2-furoic acid has a number of ideal characteristics regarding lipid metabolism, the liver and biliary deleterious effects of 2-furoic acid may render it unattractive for
development as a hypolipidemic agent in man. The low
LD50 value suggests that the safety margin of the agent is
not as favorable as other simple ring structures, e.g. cyclic
imides.
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
3I
32
33
34
35
36
37
38
39
40
References
1
2
3
4
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