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The hypolipidemic activity of N(4-methyl-phenyl)diphenimide in rodents
Pharmacological
Research
Communications,
TEE WPOLIPIDEMIC
Iris
8.
Rail,
ACTIVITY
Vol. 19. No.
OF N(4-~~YL-PtFEMCL)DIPKIZNIMUlE IN RODENTS
Oi T. Wang, Akula and John
Division
of Medicinal
of North
R.K. Murthy, Calvin
Chemistry
School University
Patricia
and Natural
A. Day
Products
of Pharmacy
Carolina
Receivedin
839
12, 1987
at Chapel
Hill,
29 October
1987
final form
N.C.
27514
SUMMARY
N(&Methyl-phenyl)diphenimide hypolipidemic
activity
were
reduced
rats
by the
lipids
drug
after
Rate
limiting
in
Cholesterol
the
bile
compartment
were
VLDL and WL
suggest
that
class
0031-6989/87/l
triglyceride
were removed
not
after
two
the
drug
in
the
Lipids
content drug
weeks
of chemicals 20839-20/$03.00/O
the --de novo by the body
with
of administration.
thus,
from of
the the
drug blood
body. reduced
increase
in HDL These
in hyperlipidemic further
20
drug.
due to the
organs
marked
at
synthesis
was significantly
may be useful in man,
66% in
orally
removed
in the
levels
lowered
administration
from
deposited
the
were
suppressed
excretion.
with
and atherosclerosis this
liver
potent
Serum cholesterol
enzymes
cholesterol
treated
cholesterol
rodents.
two weeks
was also
accelerating
in rats
in
67% and serum
mg/kg/day. of
afforded
investigation
data states of
is warranted. 0 1987
The Italian
Pharmacological
Society
840 KEY
Pharmacological
Hypolipidemic
WORDS:
Agents,
Research
Cyclic
Communications,
iaides,
Atherosclerosis INTRODUCTION An investigation structure
[Chapman
et al.,
19841
[(Murthy
studies
showed
maximum
tested
mode of
hypolipidemic novo
lipoproteins
Fig
at
1
action
of
MATERIAL Source
of
1984,
cyclic
imides,
hypolipidemic
1985].
effects
20 mg/kg/day is
i.e. in
activity possessed
of all
of
in mice
a more
Wyrick
activity
The structure
rodents
the
[Murthy
in depth
lipids
to evaluate
and its
--in vivo
effects
et al.,
investigation
its
as a effects
on e
on serum
administration.
of N(bHethyl-Phenyl)Dipheninide
AND METHODS Compound
N(4-Methyl-phenyl)diphenimide by a previous chemical
ring
of N(4-methyl-phenyl)diphenimide in
after
Structure
member
potent
study
agent
synthesis
varying
N(4-Methyl-phenyl)diphenimides
The following
the
of
1983 a.b.c.d.,
seven
pharmacological
derivatives
of
that
--et al., that
imides
1979,
demonstrated
rodents
19851.
cyclic
et al.,
showed
diphenimides,
the
of
published
and physical
method
(Fig [Murthy
characteristics
1) was synthesized et al., were
19851.
identical
The to
Vol. 19, No.
I.?, 1987
Pharmacological
Research
those reported
Communications,
[Murthy --et al.,
Eypolipidemic
Screen
The test
in Normal
19851. Rodents
and administered
to male CF1 mice (Q
for 16 days or to Sprague Dawley male rats
g) orally
by intubation
needle for 14 days.
or 16, blood was obtained was separated cholesterol
by tail
by centrifugation levels
Burchard reaction levels
841
12, 1987
compound was suspended in 1% aqueous
carboxymethylcellulose 25 g) I.P.
Vol. 19. No.
and serum
The serum
for 3 min.
[Ness --et al.
were determined
On day 9, 14,
vein bleeding
were determined
(% 300
by the Liebermann-
19641.
Serum triglyceride
with a commercial kit
(Bio-
Dynamicsfbmc). Hyperlipidemic
Induced
Mice
Male CF1 mice ( a25 g) were placed on a commercial which contained
butterfat
(400 g), celufil
cholesterol
(52 g), choline
dihydrogen
mixture
(40 g), sodium cholate
oil
vitamin-free
casein
for 14 days. were
After
(60 g),
citrate
vitamin
additional
the test
triglyceride
levels
ip for an
Serum cholesterol
were measured after
levels
the mice were
drug at 20 mg/kg/day
14 days period.
supplement
and triglyceride
assayed and observed to be elevated,
administered
(4 g), salt-
(20 g), sucrose (223 g),
(ZOO g), and total the cholesterol
diet
and
14 days of drug
administration. Toricity
Studies
Animal weights experiments
periodically
and expressed as a percentage
the animal on day 0. drug,
were obtained
selected
After
during
the
of the weight
of
dosing for 14 days with the test
organs were excised,
trimmed of fat,
and
842
Pharmacological
weighed. of
The organ
the
total
body
was assessed
daily.
[Litchfield
et
administering mg/kg
weight
al.,
the
Enzymatic
test
drug
animal.
test
--In vivo
drug.
10% liver
from
mouse
liver
enzymatic
in male
CFl mice
from
each
by
5 to 1000
recorded
over
dosage.
adenosine
of
UM of
the
per
were
coenzyme
agent
mitochondrial
[Robinson
et al.,
19701;
cholesterol
[Haven
--et al.
coenzyme
A carboxylase synthetase
glycerol-3-phosphate phosphatidylate
1973, activity
activity acyl
16 days
at a
The enzyme
[Goodridge citrate
et al., lyase
citrate
exchange [Shefer
coenzyme
Wada ot&., [Greenspan [Brady
transferase
phosphohydrolase
19781;
cholesterol
acyl
transferase
al.,
19781;
cholesterol
ester
hydrolase
activated
M
7o-hydroxylase
al.,
and heparin
+ 0.001
literature
3-hydroxy-3-methylglutaryl
reductase,
for
day.
dependent
19781;
using
after
A synthetase
triphosphate
performed
M sucrose
by following
et al.,
acid
and 25-100
the
[Hoffmann
19781;
10%
obtained
10 to 60 mg/kg
acetyl
using
in 0.25
mice
determined
performed
studies
prepared
CF1 male
from were
procedures:
press]
value)
of deaths
were
administration
ranging
activities
19771;
(LD50
for
studies
CFl male
intraperitoneal
fatty
consumption
intraperitoneally
was determined
homogenates
pH 7.2
--et al.,
as a percentage Food
toxicity
The number
enzymatic of
1973);
the
Communications,
Studies
homogenates
dose
expressed
was determined
dose.
group
--In vitro
EDTA,
of
19461
the
in
were
The acute
as a single
7 days
weights
Research
hepatic
et
A
19691;
acetyl
al.,
19681;
et al.,
activity activity
19601;
E-
[Lamb
et al.,
[Mavis
[Balasubramaniam [Hall lipoprotein
--et al. lipase
et s
in
Vol. 19. No.
12, 7987
Pharmacological
Research
Communications,
[Chait
--et
al.
19821.
Protein
assays
by the
method
of Lowry
Liver,
Small
Intestine,
In male for (24-h
the
and Bligh
milligrams
of
extract
et
[Ness
lipid
lipid
content
content
[Bragdon
--et.
3 [ Hi-Cholesterol
for (40.7 were
( ~350
mCi/mmol)
administration,
and samples
of
Homogenates
(10%)
paper, 40°C, tissue Bile
blood,
dried,
digested
and counted.
Cannulation Male
20 mg/kg/day
rats
orally
test
in
rats,
Twenty-four
and feces hours
organs
were
and urine
were
obtained.
were
prepared,
were
plated
h in Hyamine are
expressed
agent
[3H]-cholesterol
major
samples
Results
the
10 pCi of
24 h.
for.24
and dpm/total
determined.
administered
tissues
Some tissue
The lipid
and phospholipid
were
al.
of
19511,
the
the
number
--et
in Rats
thyme,
of
of Folch
neutral
al
next
cholesterol
(2 mL) of
levels,
19351
the
in 0.25
triglyceride
On day 13,
for
and counted.
methods
and the
was administered
collected
materials
cholesterol
g) were
orally.
drug
and the
Distribution
rats
14 days
al.
test
An aliquot
was determined.
19641,
--et
the and fecal
up in methylchloride,
[Stewart
Male
[1959]
al.,
enzyme
and a 10% homogenate
by the
extract
all
Extraction
administered
removed,
and Dyer
was taken
levels
Lipid
intestine,
was extracted
for
[1951]
M EDTA was prepared.
homogenate
[1957]
were
small
were
M sucrose-O.001 the
that
liver,
collection)
--et al.
843
12, 1987
was determined
and Fecal
CFl mice
16 days,
Vol. 19, No.
after
excised,
cornbusted, on filter
hydroxide as dpm/mg
at of wet
organ. Study
($400 orally
g) were for
treated
14 days.
with
The rats
the were
test
drug
anesthetized
at
844
with
chlorpromazine
pentobarbital the pyloric
intestine,
of the
saline the
into
duct
around
it,
the the
was tied
in place,
milliliters).
analyzed
for
cholesterol
phospholipids
from
whole
mL) were
Each
of
triglyceride,
rats
the
ligature
duct.
tube
ligature,
the
the
next
counted
triglyceride,
was tube
duodenum
the
were
Once
was placed plastic
6 h and and
neutral
(c 400 g) were for
abdominal
of Hatch
chylomicrons,
fractions neutral
14 days. aorta. at
very
lipid,
On day
3500
14,
rpm.
Aliquots
ultracentrifugation
[1968]
and Have1 --et
low-density
and low-density was analyzed
administered
for
al.
lipoproteins, lipoproteins.
cholesterol,
phospholipid
and protein
levels.
RESULTS N(4-Methyl-phenyl)diphenimf.de
blood
Serum was separated
gradient
and Lees
lipoproteins, the
common bile
and PE-10
Aliquots
by density
high-density
The
over
separated
the
segment.
was collected
by centrifugation
into
duodenal
around
blood
[1955]
onesaline
ligatures
20 mg/kg/day
from
methods
approximately
the
Dawley
at
by the
the
Fractions
Sprague
was collected
around
isotonic
and the
content,
from
and proteins.
Plasma Lipoprotein
drug
placed
Once past
and the
The bile
was isolated
Sterile
a loose
duct.
(in
test
duodenum.
was made,
measured
the
were to a site
duodenum
the
removed.
Male
by
sectioned-off
an incision into
lipids,
30 min later
The duodenum
was identified,
introduced
were
followed
and distally
expanded bile
ip).
way down the
was injected
Communications,
and ligatures
sphincter
third
Research
(25 mg/kg)
(22 mg/kg
small
Pharmacological
proved
to be a potent
(3
Vol. 19, No.
12, 1987
Pharmacological
Research
hypolipidemic
Communications,
agent in mice and rats.
in mice showed that hypolipidemic
20 mg/kg/day
activity
lowering
and serum triglyceride Table 1:
Vol. 19, No.
levels
12,1987
A dose response curve
I.P.
afforded
the maximum
serum cholesterol 46% after
levels
16 days (Table
43% I).
The Effect of N(4-Methylphenyl)Diphenimide on the Serum Lipid Level of Mice After -In Vivo Administration Serum Serum Cholesterol (Mg%) Triglyceride (Mg%) Day 9 Day 16 Day 16
(N=6 )
Control 1% CMC 118 Treated I.P. 10 mg/kg/day 74 20 mg/kg/day 87 40 mg/kg/day 84 60 mgfkglday 90 * P( 0.001 Student's -------------------
Lt 7 + -i f +
122 +- 7
5* 6* 5* 6*
74 69 83 76
"t"
test
137 + 7
+ 7* t 5* f 6* +- 6*
96k 9" 74 XLa* 982 5* 97 + 8*
In rats when the drug was administered cholesterol levels
levels
orally,
serum
was reduced by 67% and serum triglyceride
were lowered by 66% after
14 days administration
(Table 2). Table 2:
The Effect of N(4-Methyl-phenyl)Diphenimide on the Serum Lipid Level of Sprague Dawley Rats After Oral Administration at 20 Mg/Kg/Day Serum
Serum Triglyceride
Ci (N=6 1
Day 7
Control.l% CMC 73 C qd Treated Orally 20 mgfkgfday 45 + 4*
In hyperlipidemic serum cholesterol 62% after
Day 14
Day 7
78 k 5
110 + 3
26 + 3*
diet
53 t 4*
Day 14 112 + 7 38 i 6*
induced mice the drug lowered
by 47% and serum triglyceride
14 days (Table 3).
(Mg%)
levels
by
846
Pharmacological
Table
3:
Research
Communications,
The Effects of N(4-Methyl-phenyl)Diphenimide Serum Lipids of Hyperlipidemic Diet Induced CFl Mice Administration at 20 ma/kg/day I.P.
(~=6 > Control - Normal Hyperlipidemic - Diet Hyperlipidemic-Diet Drug
Serum Cholesterol
Serum Triglyceride
128 f 7 mg% 409 -+ 9* mg%
137 * 8 mg% 517 2 7* mg%
215 + 6* mg%
198 f 6* mg%
* p (0.001 The body weights
of
control
over
increase
in weight.
The food
controls
and 23.26
gm/day
including
the
for
of
organ
the
the
control
weights
of
enzyme showed
exchange
was reduced
significantly
that
18% at
CoA synthetase
23-24%
at
transferase
acetyl
liver
were
a 6%
was 27.36 rats.
All
unchanged
from
on rate lipid
25
pM and lyase
by 75% at activity activity
citrate
20% at
100
activity
activity
50 and 100 nM (Table 18% at
50pM.
was marginally
inhibited
by
phosphohydrolase activities
100 nM (Table
had no effect acyl
5).
were
73% at
acyl 100 pM,
and heparin
induced
inhibited
by the
agents
N(Methyl-phenyl)diphentmide
on the activities transferase,
CoA carboxylase
4).
was inhibited
was inhibited
lipase
pM of
was
50 and 100 nM, E-glycerol-3-phosphate
phosphatidylate
cholesterol
treated
mitochondrial
citrate
inhibited
Acetyl
vitro
for
a 13% only
in rats
in hepatic
--in vitro
HMG CoA reductase
by 16% at
showed
consumption
involved
and ATP-dependent
lipoprotein
rats
N(4-methyl-phenyl)diphenimide
activity
metabolism
drug
treated
showed
values.
The effects limiting
whereas
animals
increase
gm/day
14 days
the
and fatty
of acyl
cholesterol acid
-in
CoA
7u-hydroxylase, synthetase
from
25 to
Vol. 19, No.
12, 1987
Pharmacological
Research
Communications.
LOOnM concentration
of drug.
Table 4:
Control
The Effects on -In Vitro Metabolites
Vol. 79, No.
847
12, 1987
of N(4-Methyl-phenyl)Diphenjimide Enzyme Activities of Cholesterol in CPl Mouse Liver
ATP Dependent citrate lyase Mg citrate hydrolyzed/ gm wet tissue
Acetyl CoA synthetase Mg acetyl CoA formedJgm wet tissue
HMGCoA reductase
Cholesterol side chain oxidation
dpm/gm
dpmJgm
wet tissue
wet tissue
30.5 +2.7
28.5 t1.9
38400 23456
31656 21881
26.5 21.7 21.9 k1.8* 21.7 +_1.4*
35328 k2304 31408 +2688* 37632 22316
44056 k2630 51689 ir3012* 42194 k2691
Drug 25 uM 25.6 +2.1* 50 nM 7.3 +0.9* 100 nM 7.0 +0.9* *p 5 0.001 Table 5:
The Effects of N(4-Methyl-phenyl)Diphenimide on -In Vitro Enzyme Activities of Triglyceride Metabolism in CFl Mouse Liver sn-Glycerol-3phosphate acyl transferase
Control
Heparin induced lipoprotein lipase
dpmJgm wet tissue
Phosphatidylate phosphohydrolase hydrolase g Pi released/ gm wet tissue
537,000 + 42960
16.7 + 1.2
278,583 f 16715
15.8 k 0.9 14.1 + 1.1" 13.8 * 0.9*
264,603 + 19443 236,795 k16557 233,995 +13876*
Drug 25 uM 375,900 _+26858* 50 uM 408,120 +16127X 100 nM 144,990 -r 21502X *p 5 0.001
When these same parameters administration dependent citrate
dpmJgm wet tissue
were examined after
of N(4-methyl-phenyl)diphenimide, lyase activity
was inhibited
--in vivo
ATP at all
doses
848
PharmacologicalResearch
with the maximum reduction Acetyl
CoA synthetase
of 77% at 20 mg/kg (Table 6).
activity
at 20, 40 and 60 mg/kg/day.
was reduced greater HMGCoh reductase
decreased 37% at 20 mg/kg/day. activity
and acetyl
unaffected
86% at
40
Table 6:
Cholesterol
Coh carboxylase
by drug treatment.
transferase
activity
Communications,
hcyl
than 60%
activity
was
7c-hydroxylase
were essentially CoA cholesterol
acyl
was suppressed 68% at 20 mg/kg/day
mg/kg/day.
sn-Glycerol-3-phosphate
and
acyl
The --In Vivo Effects of N(L-Methylphenyl)Diphenimide on Enzymes Involved in Lipid Metabolism of CF Mice After 16 Days Administration I!P.
(N=6 >
Control 10% CMC Treated 10 mg/kg/day 20 &kg/day 40 mg/kg/day 60 n&kg/day
ATP dependent citrate lyase Mg citrate hydrolase/ gm wet tissue
Acetyl CoA synthetase Mg acetyl CoA formed/ gm wet tissue
dpm/gm wet tissue
30.5 Ii 2.1
28.5 + 1.9
38400 t 3456
20.4 7.0 17.7 26.5
24.2 108. 5.9 9.7
28032 24895 26874 32241
f 1.5 +- 0.9 ?: 1.2 + 1.2
Acyl CoA cholesterol acyl transferase Control 10% CMC 224,167 Treated 10 mg/kg/day 138,983 20 mg/kg/day 71,733 40 mg/kg/day 31,382 60 mgfkglday 199,498
5.0 5.2 1.8 3.6
f + f +
-sn-Glycerol-3-Phosphate acyl transferase dpm/gm wet tissue
+ 15656
537,000 1 42960
+ 6737 f 11208 +- 6721 + 15643
542,370 483,301 433,395 327,565
Phosphatidylate Phosphohydrolase g Pi released/ gm wet tissue 1% CMC 16.7 * 1.2
Control Treated 10 u&kg/day 20 mg/kg/day 40 mg/kg/day 60 mg/kg/day "p < 0.001
?r 1.7 ?r 0.8 f 1.4* f 1.1*
HMGCoA reductase
0.8X 0.7* 0.3* 0.7*
2 32238 + 42843 -c 32206 + 42841
Heparin induced lipoprotein lypase 278503 _+13921 300831 317592 356581 384334
_+16715 k 16667 f 19500 2 15054
-+ 2286 f 2299 2 1916 + 2197
Vol. 19, MO. 72. 1987
Pharmacological
Research
transferase
activity
Communications,
was inhibited
dependent manner with Phosphatidylate greater
mg/kg/day
affording
activity
doses employed.
activity
39% reduction.
was inhibited Heparin induced
was increased
28% at 40
and 38% at 60 mglkgfday.
Lipid
content
days with levels
phosphohydrolase
lipase
849
12, 1987
by the drug in a dose
60 mg/kg/day
than 68% at all
lipoprotein
Vol. 19, No.
of CF1 mouse liver
after
being treated
the drug at 20 mg/kg day showed that
were reduced greater
than 15% for all
16
cholesterol doses employed
(Table 7). Table 7:
The Effects of N(4-Methyl-phenyl)Diphenimide on the Lipid Content of CF1 Mice Liver After ' 16 Days Administration I.P. Mg/gm wet tissue
(~~61 Control 10 mg/kg/day 20 mg/kg/day 40 mg/kg/day 60 mg/kg/day
mg Lipid Extracted
Total Cholesterol
Triglyceride
35.6 38.1 23.1 28.5 27.4
5.69 4.78 4.61 4.72 4.56
4.77 4.14 3.02 4.42 3.75
t + 2 2 t
Neutral Control 10 mg/kg/day 20 mg/kgiday 40 mg/kg/day 60 mg/kg/day
a.85 a.42 7.32 8.65 8.14
1.8 1.4 1.3* l.O* 1.7* lipids
+ 0.43 f 0.41 -+0.34* It 0.55 -+0.31
p ~0.001 **neutral lipids = fatty cholesterol esters Triglyceride neutral
content
lipids
phospholipids Dawley rats
+ t k t *
0.34 0.27* 0.33* 0.39* 0.21*
+ + c * *
0.19 0.23 0.13 0.33 0.28*
Phospholipids
Protein
18.43 18.06 21.17 23.04 13.45
4.51 4.213 4.06 4.39 4.03
acids,
t + + f f
1.29 1.10 1.09 1.15 0.56*
+ 0.27 ?r 0.24 _+0.22 + 0.32 _+0.26
mono and diglyceride,
was reduced 37% at 20 mglkgfday,
were reduced 17% at 20 mg/kg and were reduced 27% at 60 mg/kg/day.
the liver
decrease in cholesterol,
lipids
after
treatment
28% in triglyceride,
In Sprague
showed a 23% 13% in neutral
850
Table
PharmacologicalResearch
8:
Communications,
The Effects of N(4-Methyl-phenyl)Diphenimide on Sprague Dawley Rats Tissue Lipids Days at 20 Mg/Kg/Day Orally
Organ (~=6) Liver Control Treated
Mg/gm wet Total Cholesterol
Mg lipid/ gm wet tissue
After
14
tissue Triglyceride
58.52 33.35
-+ 4.08, ?I 3.50
9.18 7.05
+ 0.63, + 0.44
6.37 4.52
+ 0.38, +- 0.36
Small Intestines Control 68.20 Treated 92.67
-+ 2.27, + 2.21
12.07 11.12
i- 0.84 + 0.61
11.20 9.85
+ 0.55 + 0.34
Fecal Control Treated
-+ 0.81 _+ 0.56
2.84 3.23
t 0.17 k 0.23
1.86 1.53
f 0.11 2 0.07
11.58 11.00
Bile (Mg%) Control -Treated --
118 + 5.9, 216 -c 7.1 Phospholipids
350 + 3.0, 177 + 2.9
Protein
Liver Control Treated
27.19 21.75
_+ 2.14, rt 1.09
12.02 it 0.73 12.21+ 0.82
Small Intestines Control Treated
20.06 19.83
+ 1.40 + 1.24
42.03 35.31
5 2.91 + 2.51
Fecal Control Treated
5.70 c 0.40 3.59 i 0.29*
6.99 7.12
f 0.33 + 0.42
Bile (mg%) Control Treated
1.75 t 0.63+
"p _jO.OOl ---------------------
lipids Small
0.337 _+ 0.020 0.367+ 0.019
(0.05
and 20% in phospholipids intestine
decrease content. increase triglyceride studies
**p
0.14 0.05*
in
lipids cholesterol
Fecal in
of
(Table
drug
treated
after
drug
cholesterol
(pi
0.05)
and phospholipids after
rats
and a 12% decrease
lipids
in rats
8).
two
in
treatment with
showed
administration
Bile
an 8%
triglyceride
a reduction
content. weeks
showed
a 14% in the
cannulation of drug
Vol. 19, No.
12. 1987
Pharmacological
Table
Research
9
Communications,
Vol. 19, No.
The Effects N(4-Methyl-Phenyl)Diphenimide Sprague Dawley Rat Serum Lipoprotein Days at 20 MGIG/Kg/Day Orally
on 14
After
g/ml of Sucrose Gradient Triglyceride Neutral Lipids
Cholesterol Chylomicron Control Treated
(4 ml) 337 * 23, 142 + 10
VLDL (2 ml) Control Treated LDL (2 ml) Control Treated HDL (6.5 Control Treated
851
12. 1987
420 273
f +
29, 17
67 31
+ 5, * 3
190 i 11 161 + 10
22 2 7_+1
1,
98 138
+ 7 -+ 6
210 + 7 67 + 13*
45 22
+ i
3 2*
10 f 1
544 + 38, 1121 * 27
27 16
i +
2, 2
62O.k 43, 384 2 25
2 -+ 1"
ml)
Chylomicron Control Treated
Phospholipid
Protein
149 +- 10
184 k 11 103 + 9*
(4 ml)
VLDL (2 ml) Control Treated
+ 6*
26
*
2
50 47
12 _+ 1"
LDL (2 ml) Control Treated HDL (6.5 Control Treated
63
41 + 3 7 f 1"
+ 3 + 3
122 + 13 101 + 9
ml) 1.53
+ 9
657 539
119 + 5*
f f
38 45"
p 5 0.001 showed
an 83% increase
lipid
class
was elevated
The drug content
(Table
triglyceride the
did
in
the
modulate
9). (35%)
chylomicron
reduction
in cholesterol
the
rat
and phospholipids
in cholesterol,
but
no other
bile.
Cholesterol
fraction.
content,
serum
(58%),
lipoprotein
neutral (58%)
were
The VLDL demonstrated a 57% reduction
lipid
lipids reduced a 14%
in triglycerides,
(53%) in
852
Pharmacologidd
a 55% reduction neutral
in phospholipid,
lipid
contents.
in cholesterol (80%)
the
triglycerides (22%)
chylomicron,
The effects cholesterol showed
the
radioactivity, contained
Table
Tissue
The drug
increased
14 days
drug
of
whereas
the
thyme,
feces
of
3
content
the
the
body
treatment.
steroidogenesis
3
H-
administration
contained
less
and large
intestine
H-cholesterol
Organ Treated
and its
24483
1.673 2.633 0.771 1.340 3.20 10.083 2.267 6.800 3.767 6.133 9.10 0.0807 368
25602 23699 70892
469188 42723 400597
170912 292985 879986 --
effect the in
on the
adrenal the
which,
After
Organ Weight (g) -Control -Treated
45729
had no major
hypertrophy
the
10).
34106 64746 35636 33198 103715 1228432 87971 466375 139044 250727 751203 ---
in particularly
of
on
drug
while
and
content
after
organs
DPM/Total --Control
Brain Kidney Spleen Heart Lung Liver Stomach Small intestine Large intestine Chyme Feces Adrenals Final body Wt.
compensatory
106X,
The Effects of N(4-Methyl-phenyl)Diphenimide on the Distribution of 3H-Cholesterol 14 Days Administration at 20 Mg/Kg/Day Orally
(~=6)
body
Protein
reduced
after
(p 2 O.O5)(Table
10:
of
(38%)
major
a larger
metabolites
lipids
of N(4-methyl-phenyl)diphenimide
distribution that
lipids
reduced.
LDL and HDL were
neutral
content
neutral
were
reductions
The HDL fraction
cholesterol.
(40%),
Vol. 19, No.
in
showed
(52X),
(85%).
an elevated
phospholipids
the
triglyceride
and phospholipids
Communications,
a 41% increase
The LDL fraction
(68X),
demonstrated
but
Research
weights
adrenal if
organ
present,
cortex
1.701 1.503 0.533 1.400 2.033 9.40 1.90 7.433 3.30 5.900 6.16 0.0746 359 weights demonstrated
of no
due to would
have
been
I.?, 1987
Pharmacological
Research
Communications,
reflected
in the
mice
was 500 &kg
I.P.
adrenal
Vol. 19, No.
weight.
The LD50 of
as a single
853
12. 1987
the
drug
in
dose.
DISCUSSION
N(4-Methyl-phenyl)diphenimi.de hypolipidemic
activity
hyperlipidemic
mice.
hypolipidemic
activity
intraperitoneally of
150-200
Rather
The drug
lowered
the --de nova
was the
for
inhibited
the
maximum effect
serum
carboxylase
acid
acid
--in vitro
regulatory
acid
enzymes
of --de novo
glycerol-3-phosphate phosphohydrolase
the
acyl were
The former
enzymes
positively
enzyme
However,
the
was at with
the
acids in
dose
acetyl
drug
but
the
CoA were for
both
limiting
inhibited of
by
the
synthesis,
E-
and phosphatidylate
inhibited
--in vivo
followed by the
required
Not
cholesterol
not
triglyceride
dose,
and
CoA
were
inhibition
lower
Firstly
liver.
The rate
The activities
inhibition
the
the
required
transferase
doses.
of means.
acetyl
i.e.
moderately
lower
by the
synthetase
or --in viva.
response
--de novo
synthesis.
dose
The --in vivo
the
drug
CoA is
synthesis,
and fatty
optimum
of cytoplasmic
The acetyl
fatty
to the
a dose
inhibited
generation
or
fatty
for
potent
orally
by a number
enzyme
as
to afford
was at
by the
limiting
and fatty for
follow
of cholesterol,
as well.
enzymes
effect.
not
lipids
as well
in rats.
did
the
cholesterol
drug.
clofibrate
HMG CoA reductase,
enzymes
drug
for
activities
rate
dose
low compared
was suppressed
synthesis,
the
is
potent
and mice
was 20 mgfkgfday
synthesis
triglycerides
rat
The therapeutic
mg/kg/day
effect.
only
in normal
which
hypolipidemic
demonstrated
for
a dose
drug
which the
by the
for
correlated optimum
response
a number
of
854
Pharmacological
hypolipidemic inhibition
activity,
i.e
and phosphatidylate
be due to a metabolite
e.g.
tissue,
lipase
liver
activity
was moderately
phenyl)diphenimide demonstrated
that
stimulated
by the drug &
et al.,
effect
of liver,
studies
alkaline
CP Kinase,
phosphotase, hydrolytic
LDH, glucose,
enzyme
the drug appeared
to cause an
increase
in the clearance
of cholesterol
the bile
into
Additional
the feces. et al.
secretion
Enterohepatic
or liver
enzyme and peroxidase
Secondly,
salt
have
in serum BUN, SPGT,
activities.
bile
with N(&methyl-
kidney or spleen morphology.
of elevations
[Hall
may be due to a
submitted]
There was no evidence
studies
in
the agent in mice at 20, 50 or 100 mg/kg
caused no alteration
lysosomal
esters
induced lipoprotein
Additional
[Hall
by the
Heparin
Again this
of the drug.
of
Inhibition
activity
of cholesterol
and plaque.
vivo but not I-in vitro. metabolite
--in vitro.
acyl transferase
drug would lead to a reduction
--in vivo may
since the inhibition
was not evident
acyl CoA cholesterol
The
acyl
phosphohydrolase
of the drug,
the enzyme activities
Communications,
20 rag/kg/day --in vivo.
by the drug of acyl CoA cholesterol
transferase
of
Research
in press]
rat bile
acids via
cannulation
have shown an increase
with an increase
reabsorption
and bile
in bile
flow.
and cholic
acid
were reduced by the drug [Reynold et al., submitted]. 3 Higher content of U-cholesterol and its metabolites
was
observed in the fecal as in the bile,
material
thus reducing
plasma compartment. not deposited
of cholesterol
in
in other
The lipids tissues.
after
drug treatment
the cholesterol
as well
levels
in the
removed from the blood were This was evident
from the
Vol. 19, No.
12. 1987
Pharmacological
lipid 3
Research
analysis
of
H-cholesterol
liver
or its
increased
gut
or decreased
by the
of
the
deposition
of
plaques
cells.
HDL is
excretion. favors
tissues
lipid
therapy cholesterol
high
reported
clearance
of cholesterol
hypolipidemic
ratio
so that
man,
e.g.
the
to protect
[Miettinen
et
from
HDL cholesterol
that
lipoprotein the
elevation
drug
This is
may have
highly
liver
This
should
favor
Unfortunately,
do not
modulate
significatly
increase.
of
N(4-Methyl-
the
by
after
lipid
and would clinical
this in
LDL cholesterol
significant
in rats
ratio
myocardial
elevated
the
for
HDL
by 106% in rats
a use in
of HDL cholesterol
cholesterol
of drug
cells.
modulation
tissue, to these
low.
and it
lowers
This
patients
is
market
shows a 4-16%
68% and elevated
the
of
so that
plaque
is
significantly
of
lipid
objectives
19811
on the
HDL cholesterol
administration.
low.
to the
man against
phenyl)diphenimide
weeks
it
the
to LDL-
peripheral
removal
ratio
al.,
agents
clofibrate
is
in hyperlipidemic one of
serum
approach
the
and LDL cholesterol
infaraction
most
the
and returning ratio
the
in man the
into
LDL conveys
be to reverse is
has been
the
of
states
for
deposition,
would
H-
of cholesterol
a chemotherapy
cholesterol
Since
Since
3
of
may be due to
HDL cholesterol
responsible
peripheral
feces
content
from
and the
tissues, from
lipids
is high
allows
levels
absorption
In hyperlipidemic
cholesterol
e.g.
the
was desirable
atherosclerosis.
as the
drug.
The modulation lipoproteins
as well
Increased
in the
855
12, 7987
intestine
study.
metabolites
excretion
the
Vol. 79, No.
and small
distribution
cholesterol
from
Communications,
two content
suggest
situation.
was much quicker
The with
856
Pharmacological
the
seven
member
derivatives,
e.g.
five
member
of
After
drug
would
potential
to other
two weeks
were et al.,
suggest
imide
19871. the
to investigate use
Acknowledgement: National Institute
in
drug
the
has a
administration rather
required
that
Communications,
which
of HDL cholesterol
[Hall
may be desirable for
compared
administration
in HDL
this
ring.
no elevation
of drug
increase
ring
o(N-phthalimido)acetophenone
imide
demonstrated weeks
imide
Research
eight
to obtain
a 41%
The therapeutic drug
drugs
is
of
safe.
this
index Thus
chemical
it class
clinic.
This work of Heart,
was supported by a grant from Lung and Blood # HL25680.
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Hatch,
F.T.
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Adv. Lipid
Havel,
R.J.,
and Bragdon,
J.H.
Invest.
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S.A.
H.A.
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G.T.,
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Coumm. Chem. Pathol. Hoffman,
M.,
Weiss,
and Nguyen,
T.T.
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Pharmacol. a, 253. L. and Wieland,
O.H.
(1978)
Anal.
Biochem. 84, 441. Lamb,
R.G.,
Wyrick,
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T.A., V.,
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N.J., Farr, 265.
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I.H., (1970)
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Shefer, Res. 2, Stewart,
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J. Biochem., and Hall,
I.H.
Vol. 19. No.
72, 1987
Research
Communications,
TEE WPOLIPIDEMIC
Iris
8.
Rail,
ACTIVITY
Vol. 19. No.
OF N(4-~~YL-PtFEMCL)DIPKIZNIMUlE IN RODENTS
Oi T. Wang, Akula and John
Division
of Medicinal
of North
R.K. Murthy, Calvin
Chemistry
School University
Patricia
and Natural
A. Day
Products
of Pharmacy
Carolina
Receivedin
839
12, 1987
at Chapel
Hill,
29 October
1987
final form
N.C.
27514
SUMMARY
N(&Methyl-phenyl)diphenimide hypolipidemic
activity
were
reduced
rats
by the
lipids
drug
after
Rate
limiting
in
Cholesterol
the
bile
compartment
were
VLDL and WL
suggest
that
class
0031-6989/87/l
triglyceride
were removed
not
after
two
the
drug
in
the
Lipids
content drug
weeks
of chemicals 20839-20/$03.00/O
the --de novo by the body
with
of administration.
thus,
from of
the the
drug blood
body. reduced
increase
in HDL These
in hyperlipidemic further
20
drug.
due to the
organs
marked
at
synthesis
was significantly
may be useful in man,
66% in
orally
removed
in the
levels
lowered
administration
from
deposited
the
were
suppressed
excretion.
with
and atherosclerosis this
liver
potent
Serum cholesterol
enzymes
cholesterol
treated
cholesterol
rodents.
two weeks
was also
accelerating
in rats
in
67% and serum
mg/kg/day. of
afforded
investigation
data states of
is warranted. 0 1987
The Italian
Pharmacological
Society
840 KEY
Pharmacological
Hypolipidemic
WORDS:
Agents,
Research
Cyclic
Communications,
iaides,
Atherosclerosis INTRODUCTION An investigation structure
[Chapman
et al.,
19841
[(Murthy
studies
showed
maximum
tested
mode of
hypolipidemic novo
lipoproteins
Fig
at
1
action
of
MATERIAL Source
of
1984,
cyclic
imides,
hypolipidemic
1985].
effects
20 mg/kg/day is
i.e. in
activity possessed
of all
of
in mice
a more
Wyrick
activity
The structure
rodents
the
[Murthy
in depth
lipids
to evaluate
and its
--in vivo
effects
et al.,
investigation
its
as a effects
on e
on serum
administration.
of N(bHethyl-Phenyl)Dipheninide
AND METHODS Compound
N(4-Methyl-phenyl)diphenimide by a previous chemical
ring
of N(4-methyl-phenyl)diphenimide in
after
Structure
member
potent
study
agent
synthesis
varying
N(4-Methyl-phenyl)diphenimides
The following
the
of
1983 a.b.c.d.,
seven
pharmacological
derivatives
of
that
--et al., that
imides
1979,
demonstrated
rodents
19851.
cyclic
et al.,
showed
diphenimides,
the
of
published
and physical
method
(Fig [Murthy
characteristics
1) was synthesized et al., were
19851.
identical
The to
Vol. 19, No.
I.?, 1987
Pharmacological
Research
those reported
Communications,
[Murthy --et al.,
Eypolipidemic
Screen
The test
in Normal
19851. Rodents
and administered
to male CF1 mice (Q
for 16 days or to Sprague Dawley male rats
g) orally
by intubation
needle for 14 days.
or 16, blood was obtained was separated cholesterol
by tail
by centrifugation levels
Burchard reaction levels
841
12, 1987
compound was suspended in 1% aqueous
carboxymethylcellulose 25 g) I.P.
Vol. 19. No.
and serum
The serum
for 3 min.
[Ness --et al.
were determined
On day 9, 14,
vein bleeding
were determined
(% 300
by the Liebermann-
19641.
Serum triglyceride
with a commercial kit
(Bio-
Dynamicsfbmc). Hyperlipidemic
Induced
Mice
Male CF1 mice ( a25 g) were placed on a commercial which contained
butterfat
(400 g), celufil
cholesterol
(52 g), choline
dihydrogen
mixture
(40 g), sodium cholate
oil
vitamin-free
casein
for 14 days. were
After
(60 g),
citrate
vitamin
additional
the test
triglyceride
levels
ip for an
Serum cholesterol
were measured after
levels
the mice were
drug at 20 mg/kg/day
14 days period.
supplement
and triglyceride
assayed and observed to be elevated,
administered
(4 g), salt-
(20 g), sucrose (223 g),
(ZOO g), and total the cholesterol
diet
and
14 days of drug
administration. Toricity
Studies
Animal weights experiments
periodically
and expressed as a percentage
the animal on day 0. drug,
were obtained
selected
After
during
the
of the weight
of
dosing for 14 days with the test
organs were excised,
trimmed of fat,
and
842
Pharmacological
weighed. of
The organ
the
total
body
was assessed
daily.
[Litchfield
et
administering mg/kg
weight
al.,
the
Enzymatic
test
drug
animal.
test
--In vivo
drug.
10% liver
from
mouse
liver
enzymatic
in male
CFl mice
from
each
by
5 to 1000
recorded
over
dosage.
adenosine
of
UM of
the
per
were
coenzyme
agent
mitochondrial
[Robinson
et al.,
19701;
cholesterol
[Haven
--et al.
coenzyme
A carboxylase synthetase
glycerol-3-phosphate phosphatidylate
1973, activity
activity acyl
16 days
at a
The enzyme
[Goodridge citrate
et al., lyase
citrate
exchange [Shefer
coenzyme
Wada ot&., [Greenspan [Brady
transferase
phosphohydrolase
19781;
cholesterol
acyl
transferase
al.,
19781;
cholesterol
ester
hydrolase
activated
M
7o-hydroxylase
al.,
and heparin
+ 0.001
literature
3-hydroxy-3-methylglutaryl
reductase,
for
day.
dependent
19781;
using
after
A synthetase
triphosphate
performed
M sucrose
by following
et al.,
acid
and 25-100
the
[Hoffmann
19781;
10%
obtained
10 to 60 mg/kg
acetyl
using
in 0.25
mice
determined
performed
studies
prepared
CF1 male
from were
procedures:
press]
value)
of deaths
were
administration
ranging
activities
19771;
(LD50
for
studies
CFl male
intraperitoneal
fatty
consumption
intraperitoneally
was determined
homogenates
pH 7.2
--et al.,
as a percentage Food
toxicity
The number
enzymatic of
1973);
the
Communications,
Studies
homogenates
dose
expressed
was determined
dose.
group
--In vitro
EDTA,
of
19461
the
in
were
The acute
as a single
7 days
weights
Research
hepatic
et
A
19691;
acetyl
al.,
19681;
et al.,
activity activity
19601;
E-
[Lamb
et al.,
[Mavis
[Balasubramaniam [Hall lipoprotein
--et al. lipase
et s
in
Vol. 19. No.
12, 7987
Pharmacological
Research
Communications,
[Chait
--et
al.
19821.
Protein
assays
by the
method
of Lowry
Liver,
Small
Intestine,
In male for (24-h
the
and Bligh
milligrams
of
extract
et
[Ness
lipid
lipid
content
content
[Bragdon
--et.
3 [ Hi-Cholesterol
for (40.7 were
( ~350
mCi/mmol)
administration,
and samples
of
Homogenates
(10%)
paper, 40°C, tissue Bile
blood,
dried,
digested
and counted.
Cannulation Male
20 mg/kg/day
rats
orally
test
in
rats,
Twenty-four
and feces hours
organs
were
and urine
were
obtained.
were
prepared,
were
plated
h in Hyamine are
expressed
agent
[3H]-cholesterol
major
samples
Results
the
10 pCi of
24 h.
for.24
and dpm/total
determined.
administered
tissues
Some tissue
The lipid
and phospholipid
were
al.
of
19511,
the
the
number
--et
in Rats
thyme,
of
of Folch
neutral
al
next
cholesterol
(2 mL) of
levels,
19351
the
in 0.25
triglyceride
On day 13,
for
and counted.
methods
and the
was administered
collected
materials
cholesterol
g) were
orally.
drug
and the
Distribution
rats
14 days
al.
test
An aliquot
was determined.
19641,
--et
the and fecal
up in methylchloride,
[Stewart
Male
[1959]
al.,
enzyme
and a 10% homogenate
by the
extract
all
Extraction
administered
removed,
and Dyer
was taken
levels
Lipid
intestine,
was extracted
for
[1951]
M EDTA was prepared.
homogenate
[1957]
were
small
were
M sucrose-O.001 the
that
liver,
collection)
--et al.
843
12, 1987
was determined
and Fecal
CFl mice
16 days,
Vol. 19, No.
after
excised,
cornbusted, on filter
hydroxide as dpm/mg
at of wet
organ. Study
($400 orally
g) were for
treated
14 days.
with
The rats
the were
test
drug
anesthetized
at
844
with
chlorpromazine
pentobarbital the pyloric
intestine,
of the
saline the
into
duct
around
it,
the the
was tied
in place,
milliliters).
analyzed
for
cholesterol
phospholipids
from
whole
mL) were
Each
of
triglyceride,
rats
the
ligature
duct.
tube
ligature,
the
the
next
counted
triglyceride,
was tube
duodenum
the
were
Once
was placed plastic
6 h and and
neutral
(c 400 g) were for
abdominal
of Hatch
chylomicrons,
fractions neutral
14 days. aorta. at
very
lipid,
On day
3500
14,
rpm.
Aliquots
ultracentrifugation
[1968]
and Have1 --et
low-density
and low-density was analyzed
administered
for
al.
lipoproteins, lipoproteins.
cholesterol,
phospholipid
and protein
levels.
RESULTS N(4-Methyl-phenyl)diphenimf.de
blood
Serum was separated
gradient
and Lees
lipoproteins, the
common bile
and PE-10
Aliquots
by density
high-density
The
over
separated
the
segment.
was collected
by centrifugation
into
duodenal
around
blood
[1955]
onesaline
ligatures
20 mg/kg/day
from
methods
approximately
the
Dawley
at
by the
the
Fractions
Sprague
was collected
around
isotonic
and the
content,
from
and proteins.
Plasma Lipoprotein
drug
placed
Once past
and the
The bile
was isolated
Sterile
a loose
duct.
(in
test
duodenum.
was made,
measured
the
were to a site
duodenum
the
removed.
Male
by
sectioned-off
an incision into
lipids,
30 min later
The duodenum
was identified,
introduced
were
followed
and distally
expanded bile
ip).
way down the
was injected
Communications,
and ligatures
sphincter
third
Research
(25 mg/kg)
(22 mg/kg
small
Pharmacological
proved
to be a potent
(3
Vol. 19, No.
12, 1987
Pharmacological
Research
hypolipidemic
Communications,
agent in mice and rats.
in mice showed that hypolipidemic
20 mg/kg/day
activity
lowering
and serum triglyceride Table 1:
Vol. 19, No.
levels
12,1987
A dose response curve
I.P.
afforded
the maximum
serum cholesterol 46% after
levels
16 days (Table
43% I).
The Effect of N(4-Methylphenyl)Diphenimide on the Serum Lipid Level of Mice After -In Vivo Administration Serum Serum Cholesterol (Mg%) Triglyceride (Mg%) Day 9 Day 16 Day 16
(N=6 )
Control 1% CMC 118 Treated I.P. 10 mg/kg/day 74 20 mg/kg/day 87 40 mg/kg/day 84 60 mgfkglday 90 * P( 0.001 Student's -------------------
Lt 7 + -i f +
122 +- 7
5* 6* 5* 6*
74 69 83 76
"t"
test
137 + 7
+ 7* t 5* f 6* +- 6*
96k 9" 74 XLa* 982 5* 97 + 8*
In rats when the drug was administered cholesterol levels
levels
orally,
serum
was reduced by 67% and serum triglyceride
were lowered by 66% after
14 days administration
(Table 2). Table 2:
The Effect of N(4-Methyl-phenyl)Diphenimide on the Serum Lipid Level of Sprague Dawley Rats After Oral Administration at 20 Mg/Kg/Day Serum
Serum Triglyceride
Ci (N=6 1
Day 7
Control.l% CMC 73 C qd Treated Orally 20 mgfkgfday 45 + 4*
In hyperlipidemic serum cholesterol 62% after
Day 14
Day 7
78 k 5
110 + 3
26 + 3*
diet
53 t 4*
Day 14 112 + 7 38 i 6*
induced mice the drug lowered
by 47% and serum triglyceride
14 days (Table 3).
(Mg%)
levels
by
846
Pharmacological
Table
3:
Research
Communications,
The Effects of N(4-Methyl-phenyl)Diphenimide Serum Lipids of Hyperlipidemic Diet Induced CFl Mice Administration at 20 ma/kg/day I.P.
(~=6 > Control - Normal Hyperlipidemic - Diet Hyperlipidemic-Diet Drug
Serum Cholesterol
Serum Triglyceride
128 f 7 mg% 409 -+ 9* mg%
137 * 8 mg% 517 2 7* mg%
215 + 6* mg%
198 f 6* mg%
* p (0.001 The body weights
of
control
over
increase
in weight.
The food
controls
and 23.26
gm/day
including
the
for
of
organ
the
the
control
weights
of
enzyme showed
exchange
was reduced
significantly
that
18% at
CoA synthetase
23-24%
at
transferase
acetyl
liver
were
a 6%
was 27.36 rats.
All
unchanged
from
on rate lipid
25
pM and lyase
by 75% at activity activity
citrate
20% at
100
activity
activity
50 and 100 nM (Table 18% at
50pM.
was marginally
inhibited
by
phosphohydrolase activities
100 nM (Table
had no effect acyl
5).
were
73% at
acyl 100 pM,
and heparin
induced
inhibited
by the
agents
N(Methyl-phenyl)diphentmide
on the activities transferase,
CoA carboxylase
4).
was inhibited
was inhibited
lipase
pM of
was
50 and 100 nM, E-glycerol-3-phosphate
phosphatidylate
cholesterol
treated
mitochondrial
citrate
inhibited
Acetyl
vitro
for
a 13% only
in rats
in hepatic
--in vitro
HMG CoA reductase
by 16% at
showed
consumption
involved
and ATP-dependent
lipoprotein
rats
N(4-methyl-phenyl)diphenimide
activity
metabolism
drug
treated
showed
values.
The effects limiting
whereas
animals
increase
gm/day
14 days
the
and fatty
of acyl
cholesterol acid
-in
CoA
7u-hydroxylase, synthetase
from
25 to
Vol. 19, No.
12, 1987
Pharmacological
Research
Communications.
LOOnM concentration
of drug.
Table 4:
Control
The Effects on -In Vitro Metabolites
Vol. 79, No.
847
12, 1987
of N(4-Methyl-phenyl)Diphenjimide Enzyme Activities of Cholesterol in CPl Mouse Liver
ATP Dependent citrate lyase Mg citrate hydrolyzed/ gm wet tissue
Acetyl CoA synthetase Mg acetyl CoA formedJgm wet tissue
HMGCoA reductase
Cholesterol side chain oxidation
dpm/gm
dpmJgm
wet tissue
wet tissue
30.5 +2.7
28.5 t1.9
38400 23456
31656 21881
26.5 21.7 21.9 k1.8* 21.7 +_1.4*
35328 k2304 31408 +2688* 37632 22316
44056 k2630 51689 ir3012* 42194 k2691
Drug 25 uM 25.6 +2.1* 50 nM 7.3 +0.9* 100 nM 7.0 +0.9* *p 5 0.001 Table 5:
The Effects of N(4-Methyl-phenyl)Diphenimide on -In Vitro Enzyme Activities of Triglyceride Metabolism in CFl Mouse Liver sn-Glycerol-3phosphate acyl transferase
Control
Heparin induced lipoprotein lipase
dpmJgm wet tissue
Phosphatidylate phosphohydrolase hydrolase g Pi released/ gm wet tissue
537,000 + 42960
16.7 + 1.2
278,583 f 16715
15.8 k 0.9 14.1 + 1.1" 13.8 * 0.9*
264,603 + 19443 236,795 k16557 233,995 +13876*
Drug 25 uM 375,900 _+26858* 50 uM 408,120 +16127X 100 nM 144,990 -r 21502X *p 5 0.001
When these same parameters administration dependent citrate
dpmJgm wet tissue
were examined after
of N(4-methyl-phenyl)diphenimide, lyase activity
was inhibited
--in vivo
ATP at all
doses
848
PharmacologicalResearch
with the maximum reduction Acetyl
CoA synthetase
of 77% at 20 mg/kg (Table 6).
activity
at 20, 40 and 60 mg/kg/day.
was reduced greater HMGCoh reductase
decreased 37% at 20 mg/kg/day. activity
and acetyl
unaffected
86% at
40
Table 6:
Cholesterol
Coh carboxylase
by drug treatment.
transferase
activity
Communications,
hcyl
than 60%
activity
was
7c-hydroxylase
were essentially CoA cholesterol
acyl
was suppressed 68% at 20 mg/kg/day
mg/kg/day.
sn-Glycerol-3-phosphate
and
acyl
The --In Vivo Effects of N(L-Methylphenyl)Diphenimide on Enzymes Involved in Lipid Metabolism of CF Mice After 16 Days Administration I!P.
(N=6 >
Control 10% CMC Treated 10 mg/kg/day 20 &kg/day 40 mg/kg/day 60 n&kg/day
ATP dependent citrate lyase Mg citrate hydrolase/ gm wet tissue
Acetyl CoA synthetase Mg acetyl CoA formed/ gm wet tissue
dpm/gm wet tissue
30.5 Ii 2.1
28.5 + 1.9
38400 t 3456
20.4 7.0 17.7 26.5
24.2 108. 5.9 9.7
28032 24895 26874 32241
f 1.5 +- 0.9 ?: 1.2 + 1.2
Acyl CoA cholesterol acyl transferase Control 10% CMC 224,167 Treated 10 mg/kg/day 138,983 20 mg/kg/day 71,733 40 mg/kg/day 31,382 60 mgfkglday 199,498
5.0 5.2 1.8 3.6
f + f +
-sn-Glycerol-3-Phosphate acyl transferase dpm/gm wet tissue
+ 15656
537,000 1 42960
+ 6737 f 11208 +- 6721 + 15643
542,370 483,301 433,395 327,565
Phosphatidylate Phosphohydrolase g Pi released/ gm wet tissue 1% CMC 16.7 * 1.2
Control Treated 10 u&kg/day 20 mg/kg/day 40 mg/kg/day 60 mg/kg/day "p < 0.001
?r 1.7 ?r 0.8 f 1.4* f 1.1*
HMGCoA reductase
0.8X 0.7* 0.3* 0.7*
2 32238 + 42843 -c 32206 + 42841
Heparin induced lipoprotein lypase 278503 _+13921 300831 317592 356581 384334
_+16715 k 16667 f 19500 2 15054
-+ 2286 f 2299 2 1916 + 2197
Vol. 19, MO. 72. 1987
Pharmacological
Research
transferase
activity
Communications,
was inhibited
dependent manner with Phosphatidylate greater
mg/kg/day
affording
activity
doses employed.
activity
39% reduction.
was inhibited Heparin induced
was increased
28% at 40
and 38% at 60 mglkgfday.
Lipid
content
days with levels
phosphohydrolase
lipase
849
12, 1987
by the drug in a dose
60 mg/kg/day
than 68% at all
lipoprotein
Vol. 19, No.
of CF1 mouse liver
after
being treated
the drug at 20 mg/kg day showed that
were reduced greater
than 15% for all
16
cholesterol doses employed
(Table 7). Table 7:
The Effects of N(4-Methyl-phenyl)Diphenimide on the Lipid Content of CF1 Mice Liver After ' 16 Days Administration I.P. Mg/gm wet tissue
(~~61 Control 10 mg/kg/day 20 mg/kg/day 40 mg/kg/day 60 mg/kg/day
mg Lipid Extracted
Total Cholesterol
Triglyceride
35.6 38.1 23.1 28.5 27.4
5.69 4.78 4.61 4.72 4.56
4.77 4.14 3.02 4.42 3.75
t + 2 2 t
Neutral Control 10 mg/kg/day 20 mg/kgiday 40 mg/kg/day 60 mg/kg/day
a.85 a.42 7.32 8.65 8.14
1.8 1.4 1.3* l.O* 1.7* lipids
+ 0.43 f 0.41 -+0.34* It 0.55 -+0.31
p ~0.001 **neutral lipids = fatty cholesterol esters Triglyceride neutral
content
lipids
phospholipids Dawley rats
+ t k t *
0.34 0.27* 0.33* 0.39* 0.21*
+ + c * *
0.19 0.23 0.13 0.33 0.28*
Phospholipids
Protein
18.43 18.06 21.17 23.04 13.45
4.51 4.213 4.06 4.39 4.03
acids,
t + + f f
1.29 1.10 1.09 1.15 0.56*
+ 0.27 ?r 0.24 _+0.22 + 0.32 _+0.26
mono and diglyceride,
was reduced 37% at 20 mglkgfday,
were reduced 17% at 20 mg/kg and were reduced 27% at 60 mg/kg/day.
the liver
decrease in cholesterol,
lipids
after
treatment
28% in triglyceride,
In Sprague
showed a 23% 13% in neutral
850
Table
PharmacologicalResearch
8:
Communications,
The Effects of N(4-Methyl-phenyl)Diphenimide on Sprague Dawley Rats Tissue Lipids Days at 20 Mg/Kg/Day Orally
Organ (~=6) Liver Control Treated
Mg/gm wet Total Cholesterol
Mg lipid/ gm wet tissue
After
14
tissue Triglyceride
58.52 33.35
-+ 4.08, ?I 3.50
9.18 7.05
+ 0.63, + 0.44
6.37 4.52
+ 0.38, +- 0.36
Small Intestines Control 68.20 Treated 92.67
-+ 2.27, + 2.21
12.07 11.12
i- 0.84 + 0.61
11.20 9.85
+ 0.55 + 0.34
Fecal Control Treated
-+ 0.81 _+ 0.56
2.84 3.23
t 0.17 k 0.23
1.86 1.53
f 0.11 2 0.07
11.58 11.00
Bile (Mg%) Control -Treated --
118 + 5.9, 216 -c 7.1 Phospholipids
350 + 3.0, 177 + 2.9
Protein
Liver Control Treated
27.19 21.75
_+ 2.14, rt 1.09
12.02 it 0.73 12.21+ 0.82
Small Intestines Control Treated
20.06 19.83
+ 1.40 + 1.24
42.03 35.31
5 2.91 + 2.51
Fecal Control Treated
5.70 c 0.40 3.59 i 0.29*
6.99 7.12
f 0.33 + 0.42
Bile (mg%) Control Treated
1.75 t 0.63+
"p _jO.OOl ---------------------
lipids Small
0.337 _+ 0.020 0.367+ 0.019
(0.05
and 20% in phospholipids intestine
decrease content. increase triglyceride studies
**p
0.14 0.05*
in
lipids cholesterol
Fecal in
of
(Table
drug
treated
after
drug
cholesterol
(pi
0.05)
and phospholipids after
rats
and a 12% decrease
lipids
in rats
8).
two
in
treatment with
showed
administration
Bile
an 8%
triglyceride
a reduction
content. weeks
showed
a 14% in the
cannulation of drug
Vol. 19, No.
12. 1987
Pharmacological
Table
Research
9
Communications,
Vol. 19, No.
The Effects N(4-Methyl-Phenyl)Diphenimide Sprague Dawley Rat Serum Lipoprotein Days at 20 MGIG/Kg/Day Orally
on 14
After
g/ml of Sucrose Gradient Triglyceride Neutral Lipids
Cholesterol Chylomicron Control Treated
(4 ml) 337 * 23, 142 + 10
VLDL (2 ml) Control Treated LDL (2 ml) Control Treated HDL (6.5 Control Treated
851
12. 1987
420 273
f +
29, 17
67 31
+ 5, * 3
190 i 11 161 + 10
22 2 7_+1
1,
98 138
+ 7 -+ 6
210 + 7 67 + 13*
45 22
+ i
3 2*
10 f 1
544 + 38, 1121 * 27
27 16
i +
2, 2
62O.k 43, 384 2 25
2 -+ 1"
ml)
Chylomicron Control Treated
Phospholipid
Protein
149 +- 10
184 k 11 103 + 9*
(4 ml)
VLDL (2 ml) Control Treated
+ 6*
26
*
2
50 47
12 _+ 1"
LDL (2 ml) Control Treated HDL (6.5 Control Treated
63
41 + 3 7 f 1"
+ 3 + 3
122 + 13 101 + 9
ml) 1.53
+ 9
657 539
119 + 5*
f f
38 45"
p 5 0.001 showed
an 83% increase
lipid
class
was elevated
The drug content
(Table
triglyceride the
did
in
the
modulate
9). (35%)
chylomicron
reduction
in cholesterol
the
rat
and phospholipids
in cholesterol,
but
no other
bile.
Cholesterol
fraction.
content,
serum
(58%),
lipoprotein
neutral (58%)
were
The VLDL demonstrated a 57% reduction
lipid
lipids reduced a 14%
in triglycerides,
(53%) in
852
Pharmacologidd
a 55% reduction neutral
in phospholipid,
lipid
contents.
in cholesterol (80%)
the
triglycerides (22%)
chylomicron,
The effects cholesterol showed
the
radioactivity, contained
Table
Tissue
The drug
increased
14 days
drug
of
whereas
the
thyme,
feces
of
3
content
the
the
body
treatment.
steroidogenesis
3
H-
administration
contained
less
and large
intestine
H-cholesterol
Organ Treated
and its
24483
1.673 2.633 0.771 1.340 3.20 10.083 2.267 6.800 3.767 6.133 9.10 0.0807 368
25602 23699 70892
469188 42723 400597
170912 292985 879986 --
effect the in
on the
adrenal the
which,
After
Organ Weight (g) -Control -Treated
45729
had no major
hypertrophy
the
10).
34106 64746 35636 33198 103715 1228432 87971 466375 139044 250727 751203 ---
in particularly
of
on
drug
while
and
content
after
organs
DPM/Total --Control
Brain Kidney Spleen Heart Lung Liver Stomach Small intestine Large intestine Chyme Feces Adrenals Final body Wt.
compensatory
106X,
The Effects of N(4-Methyl-phenyl)Diphenimide on the Distribution of 3H-Cholesterol 14 Days Administration at 20 Mg/Kg/Day Orally
(~=6)
body
Protein
reduced
after
(p 2 O.O5)(Table
10:
of
(38%)
major
a larger
metabolites
lipids
of N(4-methyl-phenyl)diphenimide
distribution that
lipids
reduced.
LDL and HDL were
neutral
content
neutral
were
reductions
The HDL fraction
cholesterol.
(40%),
Vol. 19, No.
in
showed
(52X),
(85%).
an elevated
phospholipids
the
triglyceride
and phospholipids
Communications,
a 41% increase
The LDL fraction
(68X),
demonstrated
but
Research
weights
adrenal if
organ
present,
cortex
1.701 1.503 0.533 1.400 2.033 9.40 1.90 7.433 3.30 5.900 6.16 0.0746 359 weights demonstrated
of no
due to would
have
been
I.?, 1987
Pharmacological
Research
Communications,
reflected
in the
mice
was 500 &kg
I.P.
adrenal
Vol. 19, No.
weight.
The LD50 of
as a single
853
12. 1987
the
drug
in
dose.
DISCUSSION
N(4-Methyl-phenyl)diphenimi.de hypolipidemic
activity
hyperlipidemic
mice.
hypolipidemic
activity
intraperitoneally of
150-200
Rather
The drug
lowered
the --de nova
was the
for
inhibited
the
maximum effect
serum
carboxylase
acid
acid
--in vitro
regulatory
acid
enzymes
of --de novo
glycerol-3-phosphate phosphohydrolase
the
acyl were
The former
enzymes
positively
enzyme
However,
the
was at with
the
acids in
dose
acetyl
drug
but
the
CoA were for
both
limiting
inhibited of
by
the
synthesis,
E-
and phosphatidylate
inhibited
--in vivo
followed by the
required
Not
cholesterol
not
triglyceride
dose,
and
CoA
were
inhibition
lower
Firstly
liver.
The rate
The activities
inhibition
the
the
required
transferase
doses.
of means.
acetyl
i.e.
moderately
lower
by the
synthetase
or --in viva.
response
--de novo
synthesis.
dose
The --in vivo
the
drug
CoA is
synthesis,
and fatty
optimum
of cytoplasmic
The acetyl
fatty
to the
a dose
inhibited
generation
or
fatty
for
potent
orally
by a number
enzyme
as
to afford
was at
by the
limiting
and fatty for
follow
of cholesterol,
as well.
enzymes
effect.
not
lipids
as well
in rats.
did
the
cholesterol
drug.
clofibrate
HMG CoA reductase,
enzymes
drug
for
activities
rate
dose
low compared
was suppressed
synthesis,
the
is
potent
and mice
was 20 mgfkgfday
synthesis
triglycerides
rat
The therapeutic
mg/kg/day
effect.
only
in normal
which
hypolipidemic
demonstrated
for
a dose
drug
which the
by the
for
correlated optimum
response
a number
of
854
Pharmacological
hypolipidemic inhibition
activity,
i.e
and phosphatidylate
be due to a metabolite
e.g.
tissue,
lipase
liver
activity
was moderately
phenyl)diphenimide demonstrated
that
stimulated
by the drug &
et al.,
effect
of liver,
studies
alkaline
CP Kinase,
phosphotase, hydrolytic
LDH, glucose,
enzyme
the drug appeared
to cause an
increase
in the clearance
of cholesterol
the bile
into
Additional
the feces. et al.
secretion
Enterohepatic
or liver
enzyme and peroxidase
Secondly,
salt
have
in serum BUN, SPGT,
activities.
bile
with N(&methyl-
kidney or spleen morphology.
of elevations
[Hall
may be due to a
submitted]
There was no evidence
studies
in
the agent in mice at 20, 50 or 100 mg/kg
caused no alteration
lysosomal
esters
induced lipoprotein
Additional
[Hall
by the
Heparin
Again this
of the drug.
of
Inhibition
activity
of cholesterol
and plaque.
vivo but not I-in vitro. metabolite
--in vitro.
acyl transferase
drug would lead to a reduction
--in vivo may
since the inhibition
was not evident
acyl CoA cholesterol
The
acyl
phosphohydrolase
of the drug,
the enzyme activities
Communications,
20 rag/kg/day --in vivo.
by the drug of acyl CoA cholesterol
transferase
of
Research
in press]
rat bile
acids via
cannulation
have shown an increase
with an increase
reabsorption
and bile
in bile
flow.
and cholic
acid
were reduced by the drug [Reynold et al., submitted]. 3 Higher content of U-cholesterol and its metabolites
was
observed in the fecal as in the bile,
material
thus reducing
plasma compartment. not deposited
of cholesterol
in
in other
The lipids tissues.
after
drug treatment
the cholesterol
as well
levels
in the
removed from the blood were This was evident
from the
Vol. 19, No.
12. 1987
Pharmacological
lipid 3
Research
analysis
of
H-cholesterol
liver
or its
increased
gut
or decreased
by the
of
the
deposition
of
plaques
cells.
HDL is
excretion. favors
tissues
lipid
therapy cholesterol
high
reported
clearance
of cholesterol
hypolipidemic
ratio
so that
man,
e.g.
the
to protect
[Miettinen
et
from
HDL cholesterol
that
lipoprotein the
elevation
drug
This is
may have
highly
liver
This
should
favor
Unfortunately,
do not
modulate
significatly
increase.
of
N(4-Methyl-
the
by
after
lipid
and would clinical
this in
LDL cholesterol
significant
in rats
ratio
myocardial
elevated
the
for
HDL
by 106% in rats
a use in
of HDL cholesterol
cholesterol
of drug
cells.
modulation
tissue, to these
low.
and it
lowers
This
patients
is
market
shows a 4-16%
68% and elevated
the
of
so that
plaque
is
significantly
of
lipid
objectives
19811
on the
HDL cholesterol
administration.
low.
to the
man against
phenyl)diphenimide
weeks
it
the
to LDL-
peripheral
removal
ratio
al.,
agents
clofibrate
is
in hyperlipidemic one of
serum
approach
the
and LDL cholesterol
infaraction
most
the
and returning ratio
the
in man the
into
LDL conveys
be to reverse is
has been
the
of
states
for
deposition,
would
H-
of cholesterol
a chemotherapy
cholesterol
Since
Since
3
of
may be due to
HDL cholesterol
responsible
peripheral
feces
content
from
and the
tissues, from
lipids
is high
allows
levels
absorption
In hyperlipidemic
cholesterol
e.g.
the
was desirable
atherosclerosis.
as the
drug.
The modulation lipoproteins
as well
Increased
in the
855
12, 7987
intestine
study.
metabolites
excretion
the
Vol. 79, No.
and small
distribution
cholesterol
from
Communications,
two content
suggest
situation.
was much quicker
The with
856
Pharmacological
the
seven
member
derivatives,
e.g.
five
member
of
After
drug
would
potential
to other
two weeks
were et al.,
suggest
imide
19871. the
to investigate use
Acknowledgement: National Institute
in
drug
the
has a
administration rather
required
that
Communications,
which
of HDL cholesterol
[Hall
may be desirable for
compared
administration
in HDL
this
ring.
no elevation
of drug
increase
ring
o(N-phthalimido)acetophenone
imide
demonstrated weeks
imide
Research
eight
to obtain
a 41%
The therapeutic drug
drugs
is
of
safe.
this
index Thus
chemical
it class
clinic.
This work of Heart,
was supported by a grant from Lung and Blood # HL25680.
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