Atherosclerosis, 88 (1991) 183-192 0 1991 Elsevier Scientific Publishers ADONIS
183 Ireland,
Ltd. 0021-9150/91/$03.50
002191509100122S
ATHERO
04641
Hypolipidemic effects of NB-598 in dogs Masahiro
Horie, Yoshio Sawasaki, Hitoshi Fukuzumi, Keiko Watanabe, Yoshimi Tsuchiya and Toshio Kamei
Central Research Laboratories,
Banyu Pharmaceutical
Co., Ltd., 2-9-3, Shimomeguro,
Meguro-ku,
Yuri Iizuka,
Tokyo 153 (Japan)
(Received 10 December, 1990) (Revised, received 12 February, 1991) (Accepted 15 February, 1991)
Summary
NB-598, a new inhibitor of mammalian squalene epoxidase, was found to be a potent inhibitor of microsomal squalene epoxidase from dog liver. Hypolipidemic effects of NB-598 were compared with those of simvastatin (MK-733, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor) in dogs. NB-598 was found to decrease serum total cholesterol levels and increase serum squalene levels in a dose-dependent manner. MK-733 decreased serum total cholesterol and squalene levels. Both NB-598 and MK-733 decreased all classes of lipoprotein cholesterol, and they decreased low density lipoprotein cholesterol most potently. Both drugs decreased phospholipid levels in parallel with cholesterol levels. NB-598 also decreased triacylglycerol levels. After termination of drug administration, these levels returned to the control levels. The potency of NB-598 is thought to be as great or greater than that of MK-733. Moreover, NB-598 increased squalene concentrations in the feces and gallbladder bile, but it did not affect neutral sterol and bile acid concentrations. NB-598 did not affect the lithogenic index.
Key words: NB-598; Hypolipidemic drug; Squalene epoxidase acids; Lipoprotein; Dog
Introduction
Squalene epoxidase (squalene monooxygenase, EC 1.14.99.7) from rat liver has been extensively studied by Bloch’s group [l-3] and purified to
Correspondence to: Masahiro Horie, Central Research Laboratories, Banyu Pharmaceutical Co., Ltd., 2-9-3 Shimomeguro, Meguro-ku, Tokyo 153, Japan.
inhibitor;
Squalene;
Cholesterol;
Bile
homogeneity by Ono et al. [4]. We have reported that squalene epoxidase is an important site of regulation in the cholesterol synthetic pathway [5,6]. Squalene epoxidase activity is increased by the administration of hypolipidemic drugs such as cholestyramine (a bile acid sequestrant) and lovastatin (MK-803, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor), and it is decreased by the feeding of cholesterol in rats [6,71.
184
NB-598 ((E)-N-ethyl-N-(6,6-dimethyl-2-hepten4-ynyl)-3-[(3, 3’-bithiophen-5yl)methoxy]benzenemethanamine hydrochloride) has been found to be a potent mammalian squalene epoxidase inhibitor [8]. In our previous study [8], we reported that NB-598 inhibited human squalene epoxidase (from Hep G2 cells) in a competitive manner. NB-598 inhibited cholesterol synthesis from [14C]acetate in Hep G2 cells, associated with an increase in intracellular levels of [14C]squalene. A single oral administration of NB-598 suppressed cholesterol synthesis from [14C]acetate in rats. Moreover, multiple oral administrations of NB-598 to dogs decreased serum total and low density lipoprotein (LDL) cholesterol levels. In this experiment, we examined the hypolipidemic effect of NB-598 in dogs. Simvastatin (MK733) [9] was used as a reference drug. Materials and Methods Materials
NB-598 was synthesized in our laboratories. MK-733 was prepared in Merck, Sharp & Dohme Research Laboratories (Rahway, NJ, U.S.A.). The structure of NB-598 is shown in Fig. 1. Chromatographic standards, squalene, cholesterol, campesterol, fi-sitosterol, coprostane, lithocholic acid, deoxycholic acid, and cholic acid were obtained from Sigma (St. Louis, MO, U.S.A.). 7-Ketolithocholic acid, 12-ketolithocholic acid, and Sfi-pregnan3a,17a,20a-triol were purchased from Steraloids (Wilton, NH, U.S.A.). Taurocholic acid sodium salt, taurodeoxycholic acid sodium salt, taurochenodeoxycholic acid sodium salt, and glycocholic acid were obtained from Techno Chemical (Tokyo, Japan). [4,8,12,13,17,21-3H]Squalene (24.6 Ci/ mmol) was purchased from Du Pont - New England Nuclear (Boston, MA, U.S.A.) and [14C]de-
Fig. 1. Structure of NB-598.
oxycholic acid (58 mCi/mol) was obtained from Amersham International (Buckinghamshire, U.K.). All other chemicals used were standard commercial high purity materials. Animals
Male beagle dogs weighing 9-13 kg were purchased from Marshall Farms (North Rose, NY, U.S.A.) and were maintained under the following environmental conditions: room temperature 23 _t 2°C relative humidity 55 f 15%, illumination 12 h from 6 a.m. to 6 p.m. The dogs were housed individually in metal cages and were given a commercial chow pellet (CD-5, Clea, Tokyo, Japan) and water ad libitum. Squalene epoxidase assay
Microsomal squalene epoxidase activity was assayed according to Yamamoto et al. [l] with some modifications. Microsomes were prepared from dog liver and solubilized with 2% Triton X-100 in 0.1 M Tris-HCl buffer, pH 7.5 [3]. The standard assay mixture consisted of a total volume of 0.3 ml, 0.25 ml solubilized microsomes (1.17 mg protein/ml containing 0.3% Triton X-100), FAD (100 PM), NADPH (1 mM), EDTA (1 mM) and squalene (8 PM, about 30 000 dpm) dispersed with the aid of Tween 80. NB-598 was dissolved in dimethyl sulfoxide (Me,SO, final concentration 1%). Incubation was performed at 37°C for 30 min. The reaction was terminated by adding 0.3 ml of 10% methanolic KOH. The mixture was saponified for 1 h at 75°C. Non-saponifiable materials were extracted with petroleum ether and separated on a silica gel TLC plate. The radio-labeled 2,3oxidosqualene was counted by a liquid scintillation counter (2000CA, Packard, IL, U.S.A.). Experimental procedures
Dogs were fed 300 g of the diet at 9 a.m. NB-598 was administered orally at dose levels of 10, 3 and 1 mg/kg/day 1 h after feeding, and MK-733 was administered orally at a dose level of 10 mg/kg/day 1 h after feeding. The drugs were suspended in 0.5 % methylcellulose and given at lo-11 a.m. using a Nelaton catheter (Terumo, Tokyo, Japan). Methylcellulose (0.5%) was given to dogs in the control group. Each group consisted of 4 dogs. Fasting blood samples (3 ml) were
185 collected from a cephalic vein. Feces were collected for 24 h on day 14. In a separate experiment, 2 dogs were administered NB-598 (10 mg/kg/day) and 2 dogs were administered methylcellulose for 30 days and the gallbladder bile was collected for determination of biliary lipids. Preparation of lipoproteins Lipoprotein fractions were separated by ultracentrifugation using a lipoprotein rotor (RPL42T, Hitachi, Tokyo, Japan) and fractionated by a micro-fractionator (DGF-2, Hitachi) as described previously [lo]. For the determination of lipoprotein lipid composition, serum lipoprotein fractions were separated according to Hatch and Lees [ll] using an ultracentrifuge (L8-80M, Beckman, Palo Alto, CA, U.S.A.) equipped with a 40.3 rotor (Beckman). Determination of serum lipids Serum levels of cholesterol, triacylglycerol and phospholipids were measured enzymatically by an autoanalyzer (Centrifichem, Encore, Baker Instruments, Allenton, PA, U.S.A.), using Determiner TC 555, Determiner TG-S 555 and Determiner PL (Kyowa Medex, Tokyo, Japan), respectively. Lipoprotein cholesterol was determined manually using the above enzymatic assay kit. Serum squalene was determined by a high performance liquid chromatograph (HPLC) (LC-800 series, JASCO, Tokyo, Japan). Serum was mixed with isopropanol and centrifuged. The resulting supematant was injected into a Capcell pak Cl8 column (150 x 4.6 mm i.d., Shiseido, Tokyo, Japan) equipped with a pre-column (LiChrospher 100 RP-18e, E. Merck, Darmstadt, Germany). Acetonitrile/isopropanol/ water (70 : 28 : 2, v/v) was used as a mobile phase at a flow rate of 1.0 ml/mm. Squalene was detected by UV absorption at 210 nm. Determination of fecal lipids Feces were homogenized with 5 ~01s. of 50% ethanol, and the homogenate was saponified with KOH for 3 h at 75°C (final concentration: 10% KOH and 70% ethanol). Non-saponified materials were extracted with petroleum ether and dried under a stream of nitrogen. Fecal neutral sterols were determined by a gas liquid chromatograph (GLC) (GC-9A, Shimadzu, Kyoto, Japan) on an
HP-l fused silica megabore column (10 m X 0.53 mm i.d., 2.65 pm film thickness, Hewlett Packard, CA, U.S.A.) equipped with a hydrogen flame ionization detector. Coprostane was used as an internal standard. Fecal squalene was determined by an HPLC method. After applying the sample to HPLC, the column was washed with acetonitrile/ isopropanol/water (72 : 20 : 8, v/v). Squalene was eluted with acetonitrile/isopropanol/ water (82 : 15 : 3, v/v). The other analytical conditions were similar to those described in the determination of serum squalene. Determination of fecal bile acids Fecal bile acids were extracted and hydrolyzed according to the method of Imai et al. [12]. Fecal extracts were separated by a reverse-phase HPLC column (Bilepak II, 125 x 4.6 mm i.d., JASCO). Acetonitrile/methano1/30 mM ammonium acetate (20: 20 : 60 (A) or 30 : 30 : 40 (B), v/v) were used as mobile phases at a flow rate of 1.0 ml/mm. The gradient elution was started from 100% of A, increased linearly to 100% of B for 32 min, and then eluted with B for 38 min. The eluate was mixed with NAD reagent consisting of 0.3 mM NAD, 10 mM KH,PO,, 1 mM EDTA and 0.045% 2-mercaptoethanol, pH 7.8, at a flow rate of 1.0 ml/mm, and passed through an immobilized 3ahydroxysteroid dehydrogenase column (Enzymepak-HSD, 35 X 4.6 mm i.d., JASCO) at 25“C. NADH formed in the column was subsequently detected fluorimetrically (excitation at 345 nm, emission at 475 nm). 5P-Pregnan-3a,17~,20a-triol was used as an internal standard. The efficiency of the extraction was 99% in some studies using [‘4C]deoxycholic acid as an internal standard. Determination of lipids in the gallbladder bile Biliary neutral sterols and squalene were extracted and determined as described above. For the determination of biliary bile acids, the bile was mixed with ethanol, boiled for about 5 min and the mixture filtered. Bile acids were determined by HPLC as described above. Phospholipids were determined by a chemical method using Total Bile Acids Test Wako (Wako Pure Chemical, Osaka, Japan). The lithogenic index was calculated from the following formula: neutral sterols (mol)/ (phospholipids (mol) + total bile acids (mol)).
186 Data analysis Data from these studies were statistically analyzed using Mann-Whitney’s U test [13]. The variations in all mean values in the tables and figures are expressed as standard deviations (SD). Results
Inhibition of squalene epoxidase Figure 2 shows the effect of NB-598 on microsomal squalene epoxidase from dog liver. NB-598 inhibited squalene epoxidase dose-dependently. The concentration required for 50% inhibition (IC,,) was 2.0 nM. Serum lipid levels in dogs NB-598 was administered orally to dogs at dose levels of 10, 3 and 1 mg/kg/day for 21 days and found to decrease serum total cholesterol levels in a dose-dependent manner. The AUC values of NB-598 on days 0, 14, and 21 were relatively constant (data not shown). Fig. 3 shows the time course of relative changes from the initial serum total cholesterol levels. The initial value was the average of 6 pre-treatment values (day - 17 to 0). The cholesterol levels in the control dogs remained essentially constant through the experimental period. In the animals treated with NB-598 at dose levels of 10 and 3 mg/kg/day, there was a rapid decrease in serum total cholesterol. Following 21 days of treatment, NB-598 at dose levels of 10, 3 and 1 mg/kg/day decreased serum total cholesterol levels compared to the initial values by 34%, 14% and 3%, respectively. MK-733 (10
.I
NE-598
Fig. 2. Inhibition NB-598. Squalene
3
I
.3
10
InMl
of microsomal dog squalene epoxidase by epoxidase activity was assayed as described in Materials and Methods.
-50-l>
-20
-10
0
10
20 Days
,
30
40
50
60
Fig. 3. Comparative effects of NB-598 and MK-733 on serum cholesterol in dogs. Beagle dogs were treated orally with NB-598 and MK-733, suspended in 0.58 methylcellulose, 1 h after feeding for 21 days. Each point represents the relative change from the average of pre-treatment values. Each value indicates the mean + SD of 4 dogs. The bar marks the period during which dogs were treated with drugs. T. Control (o), NB-598 10 mg/kg/day P), 3 mg/kg/day (% 1 mg/kg/day MK- 733 10 mg/kg/day (0). Significantly different value in each control group: * P < 0.05.
(A) and from the
mg/kg/day) decreased total cholesterol levels by 32% after 21 days of treatment. Ten mg/kg/day of NB-598 decreased total cholesterol levels as much as, or more than, 10 mg/kg/day of MK-733. After termination of drug treatment, total cholesterol levels returned to the pre-treatment values in each group. No evidence of an overshoot of cholesterol levels was observed in the NB-598 or MK-733 treated groups. As shown in Fig. 4, 10 and 3 mg/kg/day of NB-598 increased serum squalene levels, but squalene levels reached a plateau after 14 days of treatment and were then sustained at relatively constant levels. After termination of treatment, serum squalene levels returned to the control levels. One mg/kg of NB-598 scarcely increased serum squalene levels. On the other hand, MK-733 decreased serum squalene levels during the experimental period (from 0.1 to 0.02 mg/dl). The time course of squalene levels after administration of NB-598 is shown in the inset of Fig. 4. On day 0, serum squalene levels increased after drug administration, and were sustained at relatively constant levels. However, on days 14 and 21, serum squalene levels were decreased until 2 h after drug administration, increased from 3 h after drug ad-
187 NB-598 and MK-733 decreased serum phospholipids and triacylglycerol (Table 3). Serum triacylglycerol levels varied during the experimental period in the control group, but NB-598 at a dose level of 10 mg/kg/day markedly decreased triacylglycerol levels. MK-733 also decreased triacylglycerol levels, but its effect was less than that of NB-598. After treatment with the drugs, phospholipid and triacylglycerol levels returned to control levels. 0
-20
-10
0
10
20
30
40
50
60
Days Fig. 4. Comparative effects of NB-598 and MK-733 on serum squalene in dogs. Experimental conditions are shown in Fig. 3. Each value indicates the mean&SD of 4 dogs. The bar marks the period during which dogs were treated with drugs. T. Control (0), NB-598 10 mg/kg/day (O), 3 mg/kg/day (m), 1 mg/kg/day (A) and MK-733 10 mg/kg/day (0). Inset shows the time course of the effects of oral NB-598 administration (10 mg/kg/day) on plasma squalene on day 0 (v). 14 (A) and 21 (v). Significantly different from the value in each control group: * P i 0.05.
ministration and then decreased to the initial levels at 24 h. Serum lipoprotein cholesterol profiles are shown in Table 1. NB-598 decreased high density lipoprotein (HDL), LDL and very low density lipoprotein (VLDL) cholesterol levels in a dose-dependent manner. MK-733 also decreased all classes of lipoprotein cholesterol. Among the three lipoprotein classes, NB-598 and MK-733 decreased LDL cholesterol levels most potently. After discontinuance of drug treatment, lipoprotein levels returned to control levels. NB-598 (10 mg/kg/day) and MK-733 significantly reduced the atherogenic index, i.e., the LDL/HDL cholesterol ratio. Effects of NB-598 on lipoprotein lipids in each lipoprotein fraction are shown in Table 2. NB-598 at a dose level of 10 mg/kg/day decreased cholesterol, triacylglycerol, phospholipids, and protein in LDL fraction by 69%, 68%, 60%, and 36%, respectively. Lipid composition in each lipoprotein fraction was not remarkably changed by the administration of NB-598, except for an increase of squalene levels. Squalene was distributed in all classes of lipoprotein, but its concentration was highest in the LDL fraction.
Fecal excretion of squalene and other lipids Serum squalene levels were sustained at relatively constant levels. Therefore, fecal excretion of squalene was determined on day 14 in the control and NB-598 (10 mg/kg/day) groups (Table 4). There was no significant difference between the control and NB-598 groups in fecal wet weight. The concentration of fecal squalene in the NB-598 group was about twice as high as in the control group. NB-598 increased the total fecal excretion of squalene by about 150%, while NB-598 did not affect the concentration of cholesterol and total bile acids in the feces. The predominant bile acid in the feces was deoxycholic acid (68%) (Table 5). Cholic acid, lithocholic acid, 12-ketolithocholic acid, and 7-ketodeoxycholic acid were also detected in the feces. NB-598 did not affect fecal bile acid composition. Biliary excretion of squalene and other lipids In a separate experiment, NB-598 was administered to dogs for 30 days and gallbladder bile was collected. NB-598 increased biliary squalene levels, but did not affect neutral sterol, phospholipid and bile acid levels (Table 6). The predominant bile acid was taurocholic acid (77-82%). Taurodeoxycholic acid, taurochenodeoxycholic acid, and glycocholic acid also were detected in gallbladder bile. The composition of bile acids was not affected by administration of NB-598 except for an increase in squalene. NB-598 did not affect the lithogenic index (Table 6). Discussion NB-598 is a mammalian squalene epoxidase inhibitor. It inhibits human squalene epoxidase in a competitive manner [8]. Before the dog experi-
97&20
102+14
108 f 13
107 * 10
3 mg/ kg/day NB-598
1 mg/ kg/day MK-733 95+ (-13+
99f20 (-2kll) 7 4*)
(Ok 4)
102+17
105* 13 (3+ 2) 98f13 (-5f 7*)
4* 1’ (-67*12*)
14k 8 (9ik37)
12+4
1253
8+ 3 (-24k23)
(14+ 29) 3+ 2* (-77f13*)
21+7
27*5
26+5
25_+6
Baseline 7
5)
7
20+ 4 (-31k13)
(-lo+
25+
23+ 7 (-17+12)
17* 5 (-33+10*)
7 8)
(-7f16)
27+
1
24+ 2 (-11*13)
26f (-6k
27k 5 (6+11) 25* 6 (-5f 8)
Day 35
(mg/dl)
IN DOGS
(1 + 22)
26*
Day 14
VLDL-cholesterol
3 29*5 8*)
16+11 (25 f 49)
15+ 9 (26+ 14)
12+ 6 (15 f 30) 12+ 3 (-4*42)
Day 35
102 (-17+
(mg/dl)
11_+ 2
Day 14
12k6
14+6
lOf3
Baseline
LDL-cholesterol
CHOLESTEROL
0.112 0.02
0.12 * 0.03
0.11+0.04
0.13+0.04
0.10_+0.02
Baseline
0.05 + 0.02 *
0.13 + 0.05
0.09+0.03
0.04+0.02*
0.11*0.01
Day 14
0.11* 0.0:
0.15+0.0
0.14+0.0(
0.12+0.0:
0.11 f 0.01
Day 35
Atherogenic index (HDL-C/LDL-C]
Drugs were given orally for 21 days. Experimental conditions are shown in Fig. 3. Each value indicates the mean f SD of 4 dogs. Figures in parentheses represen change from baseline. Significantly different from the value in control group: * P < 0.05.
79k (-27k
6* 2*)
91&18 (-ll+ 6*)
104*15
10 mg/ kg/day NB-598
10 mg/ kg/day
(2f 3) 8Ok19 (-23& 9*)
(-4*10)
105+12
102_+ 13
Day 14
Treated control NB-598
Baseline
Day 35
EFFECTS OF NB-598 AND MK-733 ON SERUM LIPOPROTEIN
HDL-cholesterol (mg/dl)
COMPARATIVE
TABLE 1
189 TABLE
2
EFFECT
OF NB-598
ON LIPOPROTEIN
LIPIDS
IN DOGS
Total cholesterol
Triacylglycerol
Phospholipids
Squalene
Protein
(mg/dI)
(mg/dI)
(mg/dl)
(mg/dI)
(mg/mI)
_
0.55 0.27 51
Chylomicron (d < 0.95) Pre 1.9+ Day 14 1.1* % decrease 42
0.28 0.13 *
1.9 f 0.50 0.6kO.34 * 68
1.3* 1.0* 23
1.12 0.15
0.02 + 0.02
VLDL (0.95 < d < 1.006) Pre 1.4+ Day 14 1.0-t % decrease 29
0.35 0.12 *
2.9+1.14 1.3 +0.56 55
0.9* 0.5+ 44
0.40 0.19
0.12+_0.05
LDL (1.006 < d < 1.063) Pre 16.3+ Day 14 5.1* % decrease 69
6.29 1.41 *
9.6 f 3.80 3.1+0.75 * 68
17.7* 7.0+ 60
5.58 2.48 *
1.08 + 0.35
HDL (I ,063 i d < 1.210) Pre 108.4+ 18.97 Day 14 83.7 + 20.16 % decrease 23
1.4kO.21 0.8+0.13 43
VHDL (1.210 < d) Pre Day 14 % decrease
1.7io.34 1.1*0.33 35
3.0+ 3.3+
0.48 0.08
-10
_
17.0* 16.4* 4
0.046 + 0.008 0.025 rt 0.004 * 46
_
_
223.6 & 28.35 181.8 zh40.29 19
*
0.84kO.17
_
2.97 1.18
+0.14 iO.10
0.25 0.16 36
kO.05 kO.04 *
4.11 3.44 16
+0.58 kO.71
_ _
N.D.
-
Beagle dogs were treated orally with 10 mg/kg/day of NB-598. Experimental conditions are shown in Fig. 3. Each value indicates the mean + SD of 4 dogs. Significantly different from the initial value: * P < 0.05. -, not tested. N.D., not detected.
ments, the inhibitory activity of NB-598 on dog squalene epoxidase was examined. NB-598 inhibited squalene epoxidase dose-dependently and its IC,, value was 2.0 nM. MK-733 has been
TABLE
reported to inhibit HMG-CoA reductase with an IC,, of 0.9 nM [9]. NB-598 was found to inhibit cholesterol synthesis from [i4C]acetate as potently as MK-733 in primary cultured dog hepatocytes
3
COMPARATIVE
EFFECTS
OF NB-598 AND MK-733
Phospholipids
ON SERUM
(mg/dl)
PHOSPHOLIPIDS Triacylglycerol
(mg/dl) Day 14
Baseline
Day 14
Day 35
Baseline
control
325 f 33
8
320 + 28
28+
3
319*39
28+
4
3 mg/kg/day NB-598
317+33
1 mg/Wdw MK-733
338 + 23
325 + 39 (Ok 3) 303 + 20 (-5+ 5) 322k43 (l+ 3) 319k48 (O* 9) 319* 13 (-5* 4)
33i
NB-598 10 mg/kg/day NB-598
309*18 (-4* 5) 233+39 * (-28* 7 *) 285 *48 (-11* 5)
Treated
10 mg/kg/day
306 * 45 (-4+ 6) 250521 * (-265 3*)
AND TRIACYLGLYCEROL
39+13 33*
4
Day 35
31*11 (-9510) 12+ (-59* 15& (-45* 28ill (-30* 17* (-50+
IN DOGS
2* 4 *) 1* 7 *) 9 *) 3 9 *)
30+14 (-lOIt22) 23* 5 (-17k14) 27+ 5 (-4rt 33*
5) 9
(-141 301 (-lo*
7) 2 7)
Drugs were given orally for 21 days. Experimental conditions are shown in Fig. 3. Each value indicates the mean *SD of 4 dogs. Figures in parentheses represent % change from baseline. Significantly different from the value in control group: * P < 0.05.
190 TABLE
4
TABLE
EFFECT OF CHOLESTEROL, IN DOGS
NB-598 ON SQUALENE,
Content
FECAL EXCRETION OF AND TOTAL BILE ACIDS
Control
Wet weight of feces (g) Cholesterol (pmol/g) Squalene (pmol/g) Total Bile Acids ( pmol/g)
171
EFFECT OF NB-598 BILE ACIDS
NB-598
k24
155
k56
1.68k
0.30
2.17&
0.15+
0.02
0.25+_ 0.05 *
1.93&
0.10
1.36+
0.25
ON THE COMPOSITION
OF FECAL
Bile acids (mol W)
Control
NB-598
Deoxycholic acid Cholic acid Lithocholic acid 12-Ketolithocholic 7-Ketodeoxycholic others
68.0 f 10.7 13.1*11.4 7.0+ 2.7 5.8* 4.0 3.8& 3.7 2.3+ 1.4
63.4k7.2 11.4k6.7 8.Ok1.2 11.2& 3.4 3.3+1.6 2.7+ 1.5
acid acid
Beagle dogs were treated orally with 10 mg/kg/day of NB-598. Experimental conditions are shown in Fig. 3. Feces were collected on day 14. Each value indicates the mean f SD of 4 dogs. Significantly different from the value in control group: * P < 0.05.
0.50
Beagle dogs were treated orally with 10 mg/kg/day of NB-598. Experimental conditions are shown in Fig. 3. Feces were collected on day 14. Each value indicates the mean *SD of 4 dogs. Significantly different from the value in control group: * P < 0.05.
of the most potent hypolipidemic drugs at present. Therefore, NB-598 is thought to be a very potent hypolipidemic drug in dogs as are HMG-CoA reductase inhibitors such as MK-733 or lovastatin. The similar potency of NB-598 and MK-733 in dogs may reflect their potency in vitro. Ten and 3 mg/kg/day of NB-598 increased serum squalene levels. After 14 days of treatment, however, squalene levels reached a plateau and were then maintained at relatively constant levels. After the termination of NB-598 treatment, serum squalene
[14]. From these results, NB-598 is thought to be as potent as MK-733 in vitro. Hypolipidemic effects of NB-598 were compared with those of MK-733 in dogs. NB-598 decreased serum total cholesterol levels dose-dependently. It decreased total cholesterol levels as much as or more than MK-733. No overshoot in cholesterol levels was observed either in the NB598 or the MK-733 treated group. MK-733 is one
TABLE
5
6
EFFECT
OF NB-598
ON GALLBLADDER
Treated
Content Dog: Neutral sterols (~mol/ml) cholesterol campesterol p-sitosterol Squalene (~mol/ml) Phospholipids (pmol/ml) Total bile acids (pmol/ml) taurocholic acid taurodeoxycholic acid taurochenodeoxycholic acid glycocholic acid Lithogenic
BILE IN DOGS
index
control
NB-598
1
2
3
4
0.77 0.71 0.03 0.03 N.D. 26.7 251.0 199.6 39.6 10.6 1.2
1.24 1.11 0.07 0.06 N.D. 52.7 366.4 284.4 61.4 18.8 1.8
0.59 0.45 0.09 0.05 0.03 26.9 221.8 180.7 30.0 10.2 0.9
1.36 1.15 0.13 0.08 0.02 41 .o 312.6 247.0 44.9 19.0 1.7
0.0028
0.0030
0.0024
0.0038
Beagle dogs were treated orally with 10 mg/kg/day of NB-598. Experimental conditions are shown in Fig. 3. Gallbladder bile was collected on day 30. The lithogenic index was calculated from the following formula: neutral sterols (mol)/(phospholipids (mol) + total bile acids (mol)). Determination limit of squalene was 0.002 pmol/ml. N.D., not detected.
191 levels returned to the initial levels. NB-598 was found to act as a squalene epoxidase inhibitor in vivo. We have not examined the potential toxicity of elevated squalene caused by squalene epoxidase inhibitor. However, in some animal species such as shark, squalene is the predominant lipid in the liver [15]. Moreover, squalene has been reported to be a safe substance in feeding experiments [16]. To examine the fate of squalene accumulated during treatment with NB-598, squalene levels in the feces were determined. Fecal excretion of squalene was about 25 pmol(10.3 mg) /day /dog in the control group. Squalene was not detected in the gallbladder bile in the control group. Fecal squalene in the control dogs is thought to be derived from diet, because the dogs received squalene 23 pmol/day/dog from the chow pellet (data not shown). NB-598 increased the fecal excretion of squalene to 39 ,umol/day/dog. Cholesterol synthesis has been calculated to be 32 pmol (12.4 mg)/kg/day in a dog fed a cholesterol-free diet [17]. However, the quantity of cholesterol synthesized in the present experiment is unknown. Therefore, we cannot know whether the fecal excretion of squalene is equal to the accumulated squalene caused by the inhibition of squalene epoxidase. NB-598 increased the concentration of squalene in the gallbladder bile, Therefore, an observed increase in the squalene in the gallbladder bile may be one of the major routes of removal from the body. Squalene may also be excreted into sebaceous glands and/or metabolized into unidentified metabolite(s). Fecal excretion of neutral sterols and bile acids in the NB-598 group was almost the same as that in the control group. The predominant bile acid in the feces was deoxycholic acid as described by Parkinson et al. [18]. The composition of neutral sterols and bile acids was not changed by the treatment with NB-598. NB-598 decreased all classes of lipoprotein cholesterol levels in a dose-dependent manner. MK-733 also decreased all classes of lipoprotein cholesterol. NB-598 and MK-733 decreased LDL cholesterol levels most potently among the three lipoprotein classes. NB-598 has been found to induce LDL receptor activity in Hep G2 cells [19]. MK-733 also has been reported to induce LDL
receptor activity in Hep G2 cells [20] and in cholesterol-fed rabbits [lo]. Therefore, the observed reduction in LDL cholesterol may be related to the increased hepatic LDL receptor activity produced by NB-598. NB-598 and MK-733 decreased serum phospholipids in parallel with serum cholesterol levels. NB-598 at a dose level of 10 mg/kg/day markedly decreased triacylglycerol levels. MK-733 also decreased triacylglycerol levels. Neither NB-598 nor MK-733 was found to affect phospholipid or triacylglycerol synthesis in Hep G2 cells [14,20]. Increased uptake of LDL may decrease the levels of phospholipids and triacylglycerol in parallel with cholesterol, and/or decreased cholesterol synthesis may inhibit the maturation of VLDL particles, resulting in a decrease in phospholipids and triacylglycerol. NB-598 did not affect lithogenic index. CS-514, HMG-CoA reductase inhibitor, was reported to reduce lithogenic index [21]. The effect of NB-598 on lipid composition in bile may be different from that of HMG-CoA reductase inhibitor. We have reported that squalene epoxidase is one of the important regulatory steps in the cholesterol synthetic pathway [5,6]. Therefore, NB-598 is thought to decrease serum total cholesterol levels as potently as MK-733. No hypolipidemic agent except HMG-CoA reductase inhibitors shows such a potent hypolipidemic effect in dogs. Therefore, NB-598 is thought to be a potential clinical candidate in the near future. Acknowledgments We are indebted to the many researchers in the Central Research Laboratories in Banyu for help and discussions. Thanks are also due to Drs. M. Yano, S. Iwadare and N. Tanaka for encouraging this study. We express our appreciation to Dr. J.S. Walker for his critical reading of this manuscript. Reference 1 Yamamoto, S. and Bloch, K., Studies on squalene epoxidase of rat liver, .I. Biol. Chem., 245 (1970) 1670-1674. 2 Tai, H. and B&h, K., Squalene epoxidase of rat liver, J. Biol. Chem., 247 (1972) 3767-3773. 3 Ono, T. and Bloch, K., Solubilization and partial characterization of rat liver squalene epoxidase, J. Biol. Chem., 250 (1975) 1571-1579.
192 4 Orto, T., Takahashi, K., Odani, S., Konno, H. and Imai, Y., Purification of squalene epoxidase from rat liver microsome, Biochem. Biophys. Res. Commun., 96 (1980) 522528. 5 Hidaka, Y., Satoh, T. and Kamei, T., Regulation of squalene epoxidase in Hep G2 cells, J. Lipid Res., 31 (1990) 2087-2094. 6 Satoh, T., Hidaka, Y. and Kamei, T., Regulation of squalene epoxidase in rat liver, J. Lipid Res., 31 (1990) 20952101. 7 Eilenberg, H. and Shechter, I., Regulation of squalene epoxidase activity and comparison of catalytic properties of rat liver and Chinese hamster ovary cell-derived enzymes, J. Lipid Res., 28 (1987) 1398-1404. 8 Horie M., Tsuchiya Y., Hayashi M., Iida Y., Iwasawa Z., Nagata Y., Sawasaki Y., H. Fukuzumi, Kitani K. and Kamei T., NB-598: a potent competitive inhibitor of squalene epoxidase, J. Biol. Chem., 265 (1990) 18075-18078. 9 Hoffman, W.F., Alberts, A.W., Anderson, P.S., Chen, J.S., Smith, R.L. and Willard, A.K.J., Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors 4. Side chain ester derivatives of mevinolin, Med. Chem., 29 (1986) 849-852. 10 Ishida, F., Watanabe, K., Sato, A., Taguchi, K., Kakubari, K., Kitani, K. and Kamei, T., Comparative effects of simvastatin (MK-733) and pravastatin (CS-514) on hypercholesterolemia induced by cholesterol feeding in rabbits, Biochim. Biophys. Acta, 1042 (1990) 365-373. 11 Hatch, F.T. and Lees, R.S., Practical methods for plasma lipoprotein analysis, Adv. Lipid. Res., 6 (1968) l-68. 12 Imai, Y., Kawata, S., Inada, M., Miyoshi, S., Minami, Y., Matsuzawa, Y. Uchida, K. and Tarui, S., Effect of cholestyramine on bile acid metabolism in conventional rats, Lipids, 22 (1987) 513-516.
13 Siegel, S. (1956) Nonparametric Statistics, McGraw-Hill Book, New York. 14 Nagata, Y., Horie, M., Hidaka, Y., Yonemoto, M., Hayashi, M., Watanabe, H. and Kamei, T., manuscript in preparation. 15 Deprez, P.P., Volkman, J.K. and Davenport S.R., Squalene content and neutral lipid composition of livers from deepsea sharks caught in Tasmanian waters, Aust. J. Mar. Freshwater Res., 41 (1990) 375-387. 16 Cosmetic, Toiletry and Fragrance Assoc., Final report on the safety assessment of squalane and squalene, J. Am. Coll. Toxicol., 1 (1982) 37-56. 17 Pertsemlidid, D., Kirchman, E.H. and Ahrens, E.H., Regulation of cholesterol metabolism in the dog, J. Clin. Invest., 52 (1973) 2353-2367. 18 Parkinson, T.M., Schneider, J.C., Jr. and Phillips, W.A., Effects of cholestipol hydrochloride (U-26,597A) on serum and fecal lipids in dogs, Atherosclerosis, 17, (1973) 167-179. 19 Hidaka, Y., Hotta, H., Nagata, Y., Iwasawa, Y., Horie, M. and Kamei, T., J. Biol. Chem., (1991) in press. 20 Nagata, Y., Hidaka, Y., Ishida, F. and Kamei, T., Effect of simvastatin (MK-733) on the regulation of cholesterol synthesis in Hep G2 cells, Biochem. Pharmacol., 40 (1990) 843-850. 21 Tsujita, Y., Kuroda, M., Shimada. Y., Tanzawa, K., Arai, M., Kaneko, I., Tanaka, M., Masuda, H., Tarumi, C., Watanabe, Y. and Fujii, S., CS-514, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase: tissue-selective inhibition of sterol synthesis and hypolipidemic effect on various animal species, Biochim. Biophys. Acta, 877 (1986) 50-60.
183 Ireland,
Ltd. 0021-9150/91/$03.50
002191509100122S
ATHERO
04641
Hypolipidemic effects of NB-598 in dogs Masahiro
Horie, Yoshio Sawasaki, Hitoshi Fukuzumi, Keiko Watanabe, Yoshimi Tsuchiya and Toshio Kamei
Central Research Laboratories,
Banyu Pharmaceutical
Co., Ltd., 2-9-3, Shimomeguro,
Meguro-ku,
Yuri Iizuka,
Tokyo 153 (Japan)
(Received 10 December, 1990) (Revised, received 12 February, 1991) (Accepted 15 February, 1991)
Summary
NB-598, a new inhibitor of mammalian squalene epoxidase, was found to be a potent inhibitor of microsomal squalene epoxidase from dog liver. Hypolipidemic effects of NB-598 were compared with those of simvastatin (MK-733, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor) in dogs. NB-598 was found to decrease serum total cholesterol levels and increase serum squalene levels in a dose-dependent manner. MK-733 decreased serum total cholesterol and squalene levels. Both NB-598 and MK-733 decreased all classes of lipoprotein cholesterol, and they decreased low density lipoprotein cholesterol most potently. Both drugs decreased phospholipid levels in parallel with cholesterol levels. NB-598 also decreased triacylglycerol levels. After termination of drug administration, these levels returned to the control levels. The potency of NB-598 is thought to be as great or greater than that of MK-733. Moreover, NB-598 increased squalene concentrations in the feces and gallbladder bile, but it did not affect neutral sterol and bile acid concentrations. NB-598 did not affect the lithogenic index.
Key words: NB-598; Hypolipidemic drug; Squalene epoxidase acids; Lipoprotein; Dog
Introduction
Squalene epoxidase (squalene monooxygenase, EC 1.14.99.7) from rat liver has been extensively studied by Bloch’s group [l-3] and purified to
Correspondence to: Masahiro Horie, Central Research Laboratories, Banyu Pharmaceutical Co., Ltd., 2-9-3 Shimomeguro, Meguro-ku, Tokyo 153, Japan.
inhibitor;
Squalene;
Cholesterol;
Bile
homogeneity by Ono et al. [4]. We have reported that squalene epoxidase is an important site of regulation in the cholesterol synthetic pathway [5,6]. Squalene epoxidase activity is increased by the administration of hypolipidemic drugs such as cholestyramine (a bile acid sequestrant) and lovastatin (MK-803, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor), and it is decreased by the feeding of cholesterol in rats [6,71.
184
NB-598 ((E)-N-ethyl-N-(6,6-dimethyl-2-hepten4-ynyl)-3-[(3, 3’-bithiophen-5yl)methoxy]benzenemethanamine hydrochloride) has been found to be a potent mammalian squalene epoxidase inhibitor [8]. In our previous study [8], we reported that NB-598 inhibited human squalene epoxidase (from Hep G2 cells) in a competitive manner. NB-598 inhibited cholesterol synthesis from [14C]acetate in Hep G2 cells, associated with an increase in intracellular levels of [14C]squalene. A single oral administration of NB-598 suppressed cholesterol synthesis from [14C]acetate in rats. Moreover, multiple oral administrations of NB-598 to dogs decreased serum total and low density lipoprotein (LDL) cholesterol levels. In this experiment, we examined the hypolipidemic effect of NB-598 in dogs. Simvastatin (MK733) [9] was used as a reference drug. Materials and Methods Materials
NB-598 was synthesized in our laboratories. MK-733 was prepared in Merck, Sharp & Dohme Research Laboratories (Rahway, NJ, U.S.A.). The structure of NB-598 is shown in Fig. 1. Chromatographic standards, squalene, cholesterol, campesterol, fi-sitosterol, coprostane, lithocholic acid, deoxycholic acid, and cholic acid were obtained from Sigma (St. Louis, MO, U.S.A.). 7-Ketolithocholic acid, 12-ketolithocholic acid, and Sfi-pregnan3a,17a,20a-triol were purchased from Steraloids (Wilton, NH, U.S.A.). Taurocholic acid sodium salt, taurodeoxycholic acid sodium salt, taurochenodeoxycholic acid sodium salt, and glycocholic acid were obtained from Techno Chemical (Tokyo, Japan). [4,8,12,13,17,21-3H]Squalene (24.6 Ci/ mmol) was purchased from Du Pont - New England Nuclear (Boston, MA, U.S.A.) and [14C]de-
Fig. 1. Structure of NB-598.
oxycholic acid (58 mCi/mol) was obtained from Amersham International (Buckinghamshire, U.K.). All other chemicals used were standard commercial high purity materials. Animals
Male beagle dogs weighing 9-13 kg were purchased from Marshall Farms (North Rose, NY, U.S.A.) and were maintained under the following environmental conditions: room temperature 23 _t 2°C relative humidity 55 f 15%, illumination 12 h from 6 a.m. to 6 p.m. The dogs were housed individually in metal cages and were given a commercial chow pellet (CD-5, Clea, Tokyo, Japan) and water ad libitum. Squalene epoxidase assay
Microsomal squalene epoxidase activity was assayed according to Yamamoto et al. [l] with some modifications. Microsomes were prepared from dog liver and solubilized with 2% Triton X-100 in 0.1 M Tris-HCl buffer, pH 7.5 [3]. The standard assay mixture consisted of a total volume of 0.3 ml, 0.25 ml solubilized microsomes (1.17 mg protein/ml containing 0.3% Triton X-100), FAD (100 PM), NADPH (1 mM), EDTA (1 mM) and squalene (8 PM, about 30 000 dpm) dispersed with the aid of Tween 80. NB-598 was dissolved in dimethyl sulfoxide (Me,SO, final concentration 1%). Incubation was performed at 37°C for 30 min. The reaction was terminated by adding 0.3 ml of 10% methanolic KOH. The mixture was saponified for 1 h at 75°C. Non-saponifiable materials were extracted with petroleum ether and separated on a silica gel TLC plate. The radio-labeled 2,3oxidosqualene was counted by a liquid scintillation counter (2000CA, Packard, IL, U.S.A.). Experimental procedures
Dogs were fed 300 g of the diet at 9 a.m. NB-598 was administered orally at dose levels of 10, 3 and 1 mg/kg/day 1 h after feeding, and MK-733 was administered orally at a dose level of 10 mg/kg/day 1 h after feeding. The drugs were suspended in 0.5 % methylcellulose and given at lo-11 a.m. using a Nelaton catheter (Terumo, Tokyo, Japan). Methylcellulose (0.5%) was given to dogs in the control group. Each group consisted of 4 dogs. Fasting blood samples (3 ml) were
185 collected from a cephalic vein. Feces were collected for 24 h on day 14. In a separate experiment, 2 dogs were administered NB-598 (10 mg/kg/day) and 2 dogs were administered methylcellulose for 30 days and the gallbladder bile was collected for determination of biliary lipids. Preparation of lipoproteins Lipoprotein fractions were separated by ultracentrifugation using a lipoprotein rotor (RPL42T, Hitachi, Tokyo, Japan) and fractionated by a micro-fractionator (DGF-2, Hitachi) as described previously [lo]. For the determination of lipoprotein lipid composition, serum lipoprotein fractions were separated according to Hatch and Lees [ll] using an ultracentrifuge (L8-80M, Beckman, Palo Alto, CA, U.S.A.) equipped with a 40.3 rotor (Beckman). Determination of serum lipids Serum levels of cholesterol, triacylglycerol and phospholipids were measured enzymatically by an autoanalyzer (Centrifichem, Encore, Baker Instruments, Allenton, PA, U.S.A.), using Determiner TC 555, Determiner TG-S 555 and Determiner PL (Kyowa Medex, Tokyo, Japan), respectively. Lipoprotein cholesterol was determined manually using the above enzymatic assay kit. Serum squalene was determined by a high performance liquid chromatograph (HPLC) (LC-800 series, JASCO, Tokyo, Japan). Serum was mixed with isopropanol and centrifuged. The resulting supematant was injected into a Capcell pak Cl8 column (150 x 4.6 mm i.d., Shiseido, Tokyo, Japan) equipped with a pre-column (LiChrospher 100 RP-18e, E. Merck, Darmstadt, Germany). Acetonitrile/isopropanol/ water (70 : 28 : 2, v/v) was used as a mobile phase at a flow rate of 1.0 ml/mm. Squalene was detected by UV absorption at 210 nm. Determination of fecal lipids Feces were homogenized with 5 ~01s. of 50% ethanol, and the homogenate was saponified with KOH for 3 h at 75°C (final concentration: 10% KOH and 70% ethanol). Non-saponified materials were extracted with petroleum ether and dried under a stream of nitrogen. Fecal neutral sterols were determined by a gas liquid chromatograph (GLC) (GC-9A, Shimadzu, Kyoto, Japan) on an
HP-l fused silica megabore column (10 m X 0.53 mm i.d., 2.65 pm film thickness, Hewlett Packard, CA, U.S.A.) equipped with a hydrogen flame ionization detector. Coprostane was used as an internal standard. Fecal squalene was determined by an HPLC method. After applying the sample to HPLC, the column was washed with acetonitrile/ isopropanol/water (72 : 20 : 8, v/v). Squalene was eluted with acetonitrile/isopropanol/ water (82 : 15 : 3, v/v). The other analytical conditions were similar to those described in the determination of serum squalene. Determination of fecal bile acids Fecal bile acids were extracted and hydrolyzed according to the method of Imai et al. [12]. Fecal extracts were separated by a reverse-phase HPLC column (Bilepak II, 125 x 4.6 mm i.d., JASCO). Acetonitrile/methano1/30 mM ammonium acetate (20: 20 : 60 (A) or 30 : 30 : 40 (B), v/v) were used as mobile phases at a flow rate of 1.0 ml/mm. The gradient elution was started from 100% of A, increased linearly to 100% of B for 32 min, and then eluted with B for 38 min. The eluate was mixed with NAD reagent consisting of 0.3 mM NAD, 10 mM KH,PO,, 1 mM EDTA and 0.045% 2-mercaptoethanol, pH 7.8, at a flow rate of 1.0 ml/mm, and passed through an immobilized 3ahydroxysteroid dehydrogenase column (Enzymepak-HSD, 35 X 4.6 mm i.d., JASCO) at 25“C. NADH formed in the column was subsequently detected fluorimetrically (excitation at 345 nm, emission at 475 nm). 5P-Pregnan-3a,17~,20a-triol was used as an internal standard. The efficiency of the extraction was 99% in some studies using [‘4C]deoxycholic acid as an internal standard. Determination of lipids in the gallbladder bile Biliary neutral sterols and squalene were extracted and determined as described above. For the determination of biliary bile acids, the bile was mixed with ethanol, boiled for about 5 min and the mixture filtered. Bile acids were determined by HPLC as described above. Phospholipids were determined by a chemical method using Total Bile Acids Test Wako (Wako Pure Chemical, Osaka, Japan). The lithogenic index was calculated from the following formula: neutral sterols (mol)/ (phospholipids (mol) + total bile acids (mol)).
186 Data analysis Data from these studies were statistically analyzed using Mann-Whitney’s U test [13]. The variations in all mean values in the tables and figures are expressed as standard deviations (SD). Results
Inhibition of squalene epoxidase Figure 2 shows the effect of NB-598 on microsomal squalene epoxidase from dog liver. NB-598 inhibited squalene epoxidase dose-dependently. The concentration required for 50% inhibition (IC,,) was 2.0 nM. Serum lipid levels in dogs NB-598 was administered orally to dogs at dose levels of 10, 3 and 1 mg/kg/day for 21 days and found to decrease serum total cholesterol levels in a dose-dependent manner. The AUC values of NB-598 on days 0, 14, and 21 were relatively constant (data not shown). Fig. 3 shows the time course of relative changes from the initial serum total cholesterol levels. The initial value was the average of 6 pre-treatment values (day - 17 to 0). The cholesterol levels in the control dogs remained essentially constant through the experimental period. In the animals treated with NB-598 at dose levels of 10 and 3 mg/kg/day, there was a rapid decrease in serum total cholesterol. Following 21 days of treatment, NB-598 at dose levels of 10, 3 and 1 mg/kg/day decreased serum total cholesterol levels compared to the initial values by 34%, 14% and 3%, respectively. MK-733 (10
.I
NE-598
Fig. 2. Inhibition NB-598. Squalene
3
I
.3
10
InMl
of microsomal dog squalene epoxidase by epoxidase activity was assayed as described in Materials and Methods.
-50-l>
-20
-10
0
10
20 Days
,
30
40
50
60
Fig. 3. Comparative effects of NB-598 and MK-733 on serum cholesterol in dogs. Beagle dogs were treated orally with NB-598 and MK-733, suspended in 0.58 methylcellulose, 1 h after feeding for 21 days. Each point represents the relative change from the average of pre-treatment values. Each value indicates the mean + SD of 4 dogs. The bar marks the period during which dogs were treated with drugs. T. Control (o), NB-598 10 mg/kg/day P), 3 mg/kg/day (% 1 mg/kg/day MK- 733 10 mg/kg/day (0). Significantly different value in each control group: * P < 0.05.
(A) and from the
mg/kg/day) decreased total cholesterol levels by 32% after 21 days of treatment. Ten mg/kg/day of NB-598 decreased total cholesterol levels as much as, or more than, 10 mg/kg/day of MK-733. After termination of drug treatment, total cholesterol levels returned to the pre-treatment values in each group. No evidence of an overshoot of cholesterol levels was observed in the NB-598 or MK-733 treated groups. As shown in Fig. 4, 10 and 3 mg/kg/day of NB-598 increased serum squalene levels, but squalene levels reached a plateau after 14 days of treatment and were then sustained at relatively constant levels. After termination of treatment, serum squalene levels returned to the control levels. One mg/kg of NB-598 scarcely increased serum squalene levels. On the other hand, MK-733 decreased serum squalene levels during the experimental period (from 0.1 to 0.02 mg/dl). The time course of squalene levels after administration of NB-598 is shown in the inset of Fig. 4. On day 0, serum squalene levels increased after drug administration, and were sustained at relatively constant levels. However, on days 14 and 21, serum squalene levels were decreased until 2 h after drug administration, increased from 3 h after drug ad-
187 NB-598 and MK-733 decreased serum phospholipids and triacylglycerol (Table 3). Serum triacylglycerol levels varied during the experimental period in the control group, but NB-598 at a dose level of 10 mg/kg/day markedly decreased triacylglycerol levels. MK-733 also decreased triacylglycerol levels, but its effect was less than that of NB-598. After treatment with the drugs, phospholipid and triacylglycerol levels returned to control levels. 0
-20
-10
0
10
20
30
40
50
60
Days Fig. 4. Comparative effects of NB-598 and MK-733 on serum squalene in dogs. Experimental conditions are shown in Fig. 3. Each value indicates the mean&SD of 4 dogs. The bar marks the period during which dogs were treated with drugs. T. Control (0), NB-598 10 mg/kg/day (O), 3 mg/kg/day (m), 1 mg/kg/day (A) and MK-733 10 mg/kg/day (0). Inset shows the time course of the effects of oral NB-598 administration (10 mg/kg/day) on plasma squalene on day 0 (v). 14 (A) and 21 (v). Significantly different from the value in each control group: * P i 0.05.
ministration and then decreased to the initial levels at 24 h. Serum lipoprotein cholesterol profiles are shown in Table 1. NB-598 decreased high density lipoprotein (HDL), LDL and very low density lipoprotein (VLDL) cholesterol levels in a dose-dependent manner. MK-733 also decreased all classes of lipoprotein cholesterol. Among the three lipoprotein classes, NB-598 and MK-733 decreased LDL cholesterol levels most potently. After discontinuance of drug treatment, lipoprotein levels returned to control levels. NB-598 (10 mg/kg/day) and MK-733 significantly reduced the atherogenic index, i.e., the LDL/HDL cholesterol ratio. Effects of NB-598 on lipoprotein lipids in each lipoprotein fraction are shown in Table 2. NB-598 at a dose level of 10 mg/kg/day decreased cholesterol, triacylglycerol, phospholipids, and protein in LDL fraction by 69%, 68%, 60%, and 36%, respectively. Lipid composition in each lipoprotein fraction was not remarkably changed by the administration of NB-598, except for an increase of squalene levels. Squalene was distributed in all classes of lipoprotein, but its concentration was highest in the LDL fraction.
Fecal excretion of squalene and other lipids Serum squalene levels were sustained at relatively constant levels. Therefore, fecal excretion of squalene was determined on day 14 in the control and NB-598 (10 mg/kg/day) groups (Table 4). There was no significant difference between the control and NB-598 groups in fecal wet weight. The concentration of fecal squalene in the NB-598 group was about twice as high as in the control group. NB-598 increased the total fecal excretion of squalene by about 150%, while NB-598 did not affect the concentration of cholesterol and total bile acids in the feces. The predominant bile acid in the feces was deoxycholic acid (68%) (Table 5). Cholic acid, lithocholic acid, 12-ketolithocholic acid, and 7-ketodeoxycholic acid were also detected in the feces. NB-598 did not affect fecal bile acid composition. Biliary excretion of squalene and other lipids In a separate experiment, NB-598 was administered to dogs for 30 days and gallbladder bile was collected. NB-598 increased biliary squalene levels, but did not affect neutral sterol, phospholipid and bile acid levels (Table 6). The predominant bile acid was taurocholic acid (77-82%). Taurodeoxycholic acid, taurochenodeoxycholic acid, and glycocholic acid also were detected in gallbladder bile. The composition of bile acids was not affected by administration of NB-598 except for an increase in squalene. NB-598 did not affect the lithogenic index (Table 6). Discussion NB-598 is a mammalian squalene epoxidase inhibitor. It inhibits human squalene epoxidase in a competitive manner [8]. Before the dog experi-
97&20
102+14
108 f 13
107 * 10
3 mg/ kg/day NB-598
1 mg/ kg/day MK-733 95+ (-13+
99f20 (-2kll) 7 4*)
(Ok 4)
102+17
105* 13 (3+ 2) 98f13 (-5f 7*)
4* 1’ (-67*12*)
14k 8 (9ik37)
12+4
1253
8+ 3 (-24k23)
(14+ 29) 3+ 2* (-77f13*)
21+7
27*5
26+5
25_+6
Baseline 7
5)
7
20+ 4 (-31k13)
(-lo+
25+
23+ 7 (-17+12)
17* 5 (-33+10*)
7 8)
(-7f16)
27+
1
24+ 2 (-11*13)
26f (-6k
27k 5 (6+11) 25* 6 (-5f 8)
Day 35
(mg/dl)
IN DOGS
(1 + 22)
26*
Day 14
VLDL-cholesterol
3 29*5 8*)
16+11 (25 f 49)
15+ 9 (26+ 14)
12+ 6 (15 f 30) 12+ 3 (-4*42)
Day 35
102 (-17+
(mg/dl)
11_+ 2
Day 14
12k6
14+6
lOf3
Baseline
LDL-cholesterol
CHOLESTEROL
0.112 0.02
0.12 * 0.03
0.11+0.04
0.13+0.04
0.10_+0.02
Baseline
0.05 + 0.02 *
0.13 + 0.05
0.09+0.03
0.04+0.02*
0.11*0.01
Day 14
0.11* 0.0:
0.15+0.0
0.14+0.0(
0.12+0.0:
0.11 f 0.01
Day 35
Atherogenic index (HDL-C/LDL-C]
Drugs were given orally for 21 days. Experimental conditions are shown in Fig. 3. Each value indicates the mean f SD of 4 dogs. Figures in parentheses represen change from baseline. Significantly different from the value in control group: * P < 0.05.
79k (-27k
6* 2*)
91&18 (-ll+ 6*)
104*15
10 mg/ kg/day NB-598
10 mg/ kg/day
(2f 3) 8Ok19 (-23& 9*)
(-4*10)
105+12
102_+ 13
Day 14
Treated control NB-598
Baseline
Day 35
EFFECTS OF NB-598 AND MK-733 ON SERUM LIPOPROTEIN
HDL-cholesterol (mg/dl)
COMPARATIVE
TABLE 1
189 TABLE
2
EFFECT
OF NB-598
ON LIPOPROTEIN
LIPIDS
IN DOGS
Total cholesterol
Triacylglycerol
Phospholipids
Squalene
Protein
(mg/dI)
(mg/dI)
(mg/dl)
(mg/dI)
(mg/mI)
_
0.55 0.27 51
Chylomicron (d < 0.95) Pre 1.9+ Day 14 1.1* % decrease 42
0.28 0.13 *
1.9 f 0.50 0.6kO.34 * 68
1.3* 1.0* 23
1.12 0.15
0.02 + 0.02
VLDL (0.95 < d < 1.006) Pre 1.4+ Day 14 1.0-t % decrease 29
0.35 0.12 *
2.9+1.14 1.3 +0.56 55
0.9* 0.5+ 44
0.40 0.19
0.12+_0.05
LDL (1.006 < d < 1.063) Pre 16.3+ Day 14 5.1* % decrease 69
6.29 1.41 *
9.6 f 3.80 3.1+0.75 * 68
17.7* 7.0+ 60
5.58 2.48 *
1.08 + 0.35
HDL (I ,063 i d < 1.210) Pre 108.4+ 18.97 Day 14 83.7 + 20.16 % decrease 23
1.4kO.21 0.8+0.13 43
VHDL (1.210 < d) Pre Day 14 % decrease
1.7io.34 1.1*0.33 35
3.0+ 3.3+
0.48 0.08
-10
_
17.0* 16.4* 4
0.046 + 0.008 0.025 rt 0.004 * 46
_
_
223.6 & 28.35 181.8 zh40.29 19
*
0.84kO.17
_
2.97 1.18
+0.14 iO.10
0.25 0.16 36
kO.05 kO.04 *
4.11 3.44 16
+0.58 kO.71
_ _
N.D.
-
Beagle dogs were treated orally with 10 mg/kg/day of NB-598. Experimental conditions are shown in Fig. 3. Each value indicates the mean + SD of 4 dogs. Significantly different from the initial value: * P < 0.05. -, not tested. N.D., not detected.
ments, the inhibitory activity of NB-598 on dog squalene epoxidase was examined. NB-598 inhibited squalene epoxidase dose-dependently and its IC,, value was 2.0 nM. MK-733 has been
TABLE
reported to inhibit HMG-CoA reductase with an IC,, of 0.9 nM [9]. NB-598 was found to inhibit cholesterol synthesis from [i4C]acetate as potently as MK-733 in primary cultured dog hepatocytes
3
COMPARATIVE
EFFECTS
OF NB-598 AND MK-733
Phospholipids
ON SERUM
(mg/dl)
PHOSPHOLIPIDS Triacylglycerol
(mg/dl) Day 14
Baseline
Day 14
Day 35
Baseline
control
325 f 33
8
320 + 28
28+
3
319*39
28+
4
3 mg/kg/day NB-598
317+33
1 mg/Wdw MK-733
338 + 23
325 + 39 (Ok 3) 303 + 20 (-5+ 5) 322k43 (l+ 3) 319k48 (O* 9) 319* 13 (-5* 4)
33i
NB-598 10 mg/kg/day NB-598
309*18 (-4* 5) 233+39 * (-28* 7 *) 285 *48 (-11* 5)
Treated
10 mg/kg/day
306 * 45 (-4+ 6) 250521 * (-265 3*)
AND TRIACYLGLYCEROL
39+13 33*
4
Day 35
31*11 (-9510) 12+ (-59* 15& (-45* 28ill (-30* 17* (-50+
IN DOGS
2* 4 *) 1* 7 *) 9 *) 3 9 *)
30+14 (-lOIt22) 23* 5 (-17k14) 27+ 5 (-4rt 33*
5) 9
(-141 301 (-lo*
7) 2 7)
Drugs were given orally for 21 days. Experimental conditions are shown in Fig. 3. Each value indicates the mean *SD of 4 dogs. Figures in parentheses represent % change from baseline. Significantly different from the value in control group: * P < 0.05.
190 TABLE
4
TABLE
EFFECT OF CHOLESTEROL, IN DOGS
NB-598 ON SQUALENE,
Content
FECAL EXCRETION OF AND TOTAL BILE ACIDS
Control
Wet weight of feces (g) Cholesterol (pmol/g) Squalene (pmol/g) Total Bile Acids ( pmol/g)
171
EFFECT OF NB-598 BILE ACIDS
NB-598
k24
155
k56
1.68k
0.30
2.17&
0.15+
0.02
0.25+_ 0.05 *
1.93&
0.10
1.36+
0.25
ON THE COMPOSITION
OF FECAL
Bile acids (mol W)
Control
NB-598
Deoxycholic acid Cholic acid Lithocholic acid 12-Ketolithocholic 7-Ketodeoxycholic others
68.0 f 10.7 13.1*11.4 7.0+ 2.7 5.8* 4.0 3.8& 3.7 2.3+ 1.4
63.4k7.2 11.4k6.7 8.Ok1.2 11.2& 3.4 3.3+1.6 2.7+ 1.5
acid acid
Beagle dogs were treated orally with 10 mg/kg/day of NB-598. Experimental conditions are shown in Fig. 3. Feces were collected on day 14. Each value indicates the mean f SD of 4 dogs. Significantly different from the value in control group: * P < 0.05.
0.50
Beagle dogs were treated orally with 10 mg/kg/day of NB-598. Experimental conditions are shown in Fig. 3. Feces were collected on day 14. Each value indicates the mean *SD of 4 dogs. Significantly different from the value in control group: * P < 0.05.
of the most potent hypolipidemic drugs at present. Therefore, NB-598 is thought to be a very potent hypolipidemic drug in dogs as are HMG-CoA reductase inhibitors such as MK-733 or lovastatin. The similar potency of NB-598 and MK-733 in dogs may reflect their potency in vitro. Ten and 3 mg/kg/day of NB-598 increased serum squalene levels. After 14 days of treatment, however, squalene levels reached a plateau and were then maintained at relatively constant levels. After the termination of NB-598 treatment, serum squalene
[14]. From these results, NB-598 is thought to be as potent as MK-733 in vitro. Hypolipidemic effects of NB-598 were compared with those of MK-733 in dogs. NB-598 decreased serum total cholesterol levels dose-dependently. It decreased total cholesterol levels as much as or more than MK-733. No overshoot in cholesterol levels was observed either in the NB598 or the MK-733 treated group. MK-733 is one
TABLE
5
6
EFFECT
OF NB-598
ON GALLBLADDER
Treated
Content Dog: Neutral sterols (~mol/ml) cholesterol campesterol p-sitosterol Squalene (~mol/ml) Phospholipids (pmol/ml) Total bile acids (pmol/ml) taurocholic acid taurodeoxycholic acid taurochenodeoxycholic acid glycocholic acid Lithogenic
BILE IN DOGS
index
control
NB-598
1
2
3
4
0.77 0.71 0.03 0.03 N.D. 26.7 251.0 199.6 39.6 10.6 1.2
1.24 1.11 0.07 0.06 N.D. 52.7 366.4 284.4 61.4 18.8 1.8
0.59 0.45 0.09 0.05 0.03 26.9 221.8 180.7 30.0 10.2 0.9
1.36 1.15 0.13 0.08 0.02 41 .o 312.6 247.0 44.9 19.0 1.7
0.0028
0.0030
0.0024
0.0038
Beagle dogs were treated orally with 10 mg/kg/day of NB-598. Experimental conditions are shown in Fig. 3. Gallbladder bile was collected on day 30. The lithogenic index was calculated from the following formula: neutral sterols (mol)/(phospholipids (mol) + total bile acids (mol)). Determination limit of squalene was 0.002 pmol/ml. N.D., not detected.
191 levels returned to the initial levels. NB-598 was found to act as a squalene epoxidase inhibitor in vivo. We have not examined the potential toxicity of elevated squalene caused by squalene epoxidase inhibitor. However, in some animal species such as shark, squalene is the predominant lipid in the liver [15]. Moreover, squalene has been reported to be a safe substance in feeding experiments [16]. To examine the fate of squalene accumulated during treatment with NB-598, squalene levels in the feces were determined. Fecal excretion of squalene was about 25 pmol(10.3 mg) /day /dog in the control group. Squalene was not detected in the gallbladder bile in the control group. Fecal squalene in the control dogs is thought to be derived from diet, because the dogs received squalene 23 pmol/day/dog from the chow pellet (data not shown). NB-598 increased the fecal excretion of squalene to 39 ,umol/day/dog. Cholesterol synthesis has been calculated to be 32 pmol (12.4 mg)/kg/day in a dog fed a cholesterol-free diet [17]. However, the quantity of cholesterol synthesized in the present experiment is unknown. Therefore, we cannot know whether the fecal excretion of squalene is equal to the accumulated squalene caused by the inhibition of squalene epoxidase. NB-598 increased the concentration of squalene in the gallbladder bile, Therefore, an observed increase in the squalene in the gallbladder bile may be one of the major routes of removal from the body. Squalene may also be excreted into sebaceous glands and/or metabolized into unidentified metabolite(s). Fecal excretion of neutral sterols and bile acids in the NB-598 group was almost the same as that in the control group. The predominant bile acid in the feces was deoxycholic acid as described by Parkinson et al. [18]. The composition of neutral sterols and bile acids was not changed by the treatment with NB-598. NB-598 decreased all classes of lipoprotein cholesterol levels in a dose-dependent manner. MK-733 also decreased all classes of lipoprotein cholesterol. NB-598 and MK-733 decreased LDL cholesterol levels most potently among the three lipoprotein classes. NB-598 has been found to induce LDL receptor activity in Hep G2 cells [19]. MK-733 also has been reported to induce LDL
receptor activity in Hep G2 cells [20] and in cholesterol-fed rabbits [lo]. Therefore, the observed reduction in LDL cholesterol may be related to the increased hepatic LDL receptor activity produced by NB-598. NB-598 and MK-733 decreased serum phospholipids in parallel with serum cholesterol levels. NB-598 at a dose level of 10 mg/kg/day markedly decreased triacylglycerol levels. MK-733 also decreased triacylglycerol levels. Neither NB-598 nor MK-733 was found to affect phospholipid or triacylglycerol synthesis in Hep G2 cells [14,20]. Increased uptake of LDL may decrease the levels of phospholipids and triacylglycerol in parallel with cholesterol, and/or decreased cholesterol synthesis may inhibit the maturation of VLDL particles, resulting in a decrease in phospholipids and triacylglycerol. NB-598 did not affect lithogenic index. CS-514, HMG-CoA reductase inhibitor, was reported to reduce lithogenic index [21]. The effect of NB-598 on lipid composition in bile may be different from that of HMG-CoA reductase inhibitor. We have reported that squalene epoxidase is one of the important regulatory steps in the cholesterol synthetic pathway [5,6]. Therefore, NB-598 is thought to decrease serum total cholesterol levels as potently as MK-733. No hypolipidemic agent except HMG-CoA reductase inhibitors shows such a potent hypolipidemic effect in dogs. Therefore, NB-598 is thought to be a potential clinical candidate in the near future. Acknowledgments We are indebted to the many researchers in the Central Research Laboratories in Banyu for help and discussions. Thanks are also due to Drs. M. Yano, S. Iwadare and N. Tanaka for encouraging this study. We express our appreciation to Dr. J.S. Walker for his critical reading of this manuscript. Reference 1 Yamamoto, S. and Bloch, K., Studies on squalene epoxidase of rat liver, .I. Biol. Chem., 245 (1970) 1670-1674. 2 Tai, H. and B&h, K., Squalene epoxidase of rat liver, J. Biol. Chem., 247 (1972) 3767-3773. 3 Ono, T. and Bloch, K., Solubilization and partial characterization of rat liver squalene epoxidase, J. Biol. Chem., 250 (1975) 1571-1579.
192 4 Orto, T., Takahashi, K., Odani, S., Konno, H. and Imai, Y., Purification of squalene epoxidase from rat liver microsome, Biochem. Biophys. Res. Commun., 96 (1980) 522528. 5 Hidaka, Y., Satoh, T. and Kamei, T., Regulation of squalene epoxidase in Hep G2 cells, J. Lipid Res., 31 (1990) 2087-2094. 6 Satoh, T., Hidaka, Y. and Kamei, T., Regulation of squalene epoxidase in rat liver, J. Lipid Res., 31 (1990) 20952101. 7 Eilenberg, H. and Shechter, I., Regulation of squalene epoxidase activity and comparison of catalytic properties of rat liver and Chinese hamster ovary cell-derived enzymes, J. Lipid Res., 28 (1987) 1398-1404. 8 Horie M., Tsuchiya Y., Hayashi M., Iida Y., Iwasawa Z., Nagata Y., Sawasaki Y., H. Fukuzumi, Kitani K. and Kamei T., NB-598: a potent competitive inhibitor of squalene epoxidase, J. Biol. Chem., 265 (1990) 18075-18078. 9 Hoffman, W.F., Alberts, A.W., Anderson, P.S., Chen, J.S., Smith, R.L. and Willard, A.K.J., Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors 4. Side chain ester derivatives of mevinolin, Med. Chem., 29 (1986) 849-852. 10 Ishida, F., Watanabe, K., Sato, A., Taguchi, K., Kakubari, K., Kitani, K. and Kamei, T., Comparative effects of simvastatin (MK-733) and pravastatin (CS-514) on hypercholesterolemia induced by cholesterol feeding in rabbits, Biochim. Biophys. Acta, 1042 (1990) 365-373. 11 Hatch, F.T. and Lees, R.S., Practical methods for plasma lipoprotein analysis, Adv. Lipid. Res., 6 (1968) l-68. 12 Imai, Y., Kawata, S., Inada, M., Miyoshi, S., Minami, Y., Matsuzawa, Y. Uchida, K. and Tarui, S., Effect of cholestyramine on bile acid metabolism in conventional rats, Lipids, 22 (1987) 513-516.
13 Siegel, S. (1956) Nonparametric Statistics, McGraw-Hill Book, New York. 14 Nagata, Y., Horie, M., Hidaka, Y., Yonemoto, M., Hayashi, M., Watanabe, H. and Kamei, T., manuscript in preparation. 15 Deprez, P.P., Volkman, J.K. and Davenport S.R., Squalene content and neutral lipid composition of livers from deepsea sharks caught in Tasmanian waters, Aust. J. Mar. Freshwater Res., 41 (1990) 375-387. 16 Cosmetic, Toiletry and Fragrance Assoc., Final report on the safety assessment of squalane and squalene, J. Am. Coll. Toxicol., 1 (1982) 37-56. 17 Pertsemlidid, D., Kirchman, E.H. and Ahrens, E.H., Regulation of cholesterol metabolism in the dog, J. Clin. Invest., 52 (1973) 2353-2367. 18 Parkinson, T.M., Schneider, J.C., Jr. and Phillips, W.A., Effects of cholestipol hydrochloride (U-26,597A) on serum and fecal lipids in dogs, Atherosclerosis, 17, (1973) 167-179. 19 Hidaka, Y., Hotta, H., Nagata, Y., Iwasawa, Y., Horie, M. and Kamei, T., J. Biol. Chem., (1991) in press. 20 Nagata, Y., Hidaka, Y., Ishida, F. and Kamei, T., Effect of simvastatin (MK-733) on the regulation of cholesterol synthesis in Hep G2 cells, Biochem. Pharmacol., 40 (1990) 843-850. 21 Tsujita, Y., Kuroda, M., Shimada. Y., Tanzawa, K., Arai, M., Kaneko, I., Tanaka, M., Masuda, H., Tarumi, C., Watanabe, Y. and Fujii, S., CS-514, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase: tissue-selective inhibition of sterol synthesis and hypolipidemic effect on various animal species, Biochim. Biophys. Acta, 877 (1986) 50-60.
