A Novel Selective Peroxisome Proliferator-Activated Receptor α Agonist, 2-Methyl-c-5-[4-[5-methyl-2-(4-methylphenyl)-4-oxazolyl]butyl]-1,3-dioxane-r-2-carboxylic acid (NS-220), Potently Decreases Plasma Triglyceride and Glucose Levels and Modifies Lipoprotein Profiles in KK-A^y Mice

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors and members of the thyroid/steroid hormone nuclear receptor superfamily (Kersten et al., 2000; Berger and Moller, 2002). Three subtypes, α, γ, and δ, with different tissue distributions and physiological roles, are known in mammals. Long-chain fatty acids and their derivatives, such as polyunsaturated fatty acids, oxidized fatty acids, and eicosanoids, have been reported to activate PPARs (Xu et al., 1999; Corton et al., 2000). Therefore, these receptors have been suggested to act as nutrient sensors and to play an important role in the regulation of lipid and carbohydrate metabolism.

PPARα is highly expressed in the liver, heart, kidney, and skeletal muscle, and it regulates fatty acid catabolism. Hypolipidemic fibrate-class drugs (fibrates) activate this receptor. PPARγ is mainly expressed in adipose tissue, the large intestine, and the spleen, and it regulates fatty acid storage. Hypoglycemic glitazone-class drugs (glitazones) activate this receptor. PPARδ is expressed ubiquitously and its physiological role is uncertain. However, it was reported that a potent and selective PPARδ agonist regulated high-density lipoprotein (HDL) metabolism in nonhuman primates (Oliver et al., 2001).

Fibrates, such as gemfibrozil, bezafibrate, and fenofibrate, decrease serum triglyceride levels and moderately increase HDL-cholesterol levels in the patients with hyperlipidemia or type-2 diabetes (Rubins et al., 1999; The BIP Study Group, 2000; Diabetes Atherosclerosis Intervention Study Investigators, 2001) and prevent coronary heart disease or stroke (Rubins et al., 2001; Tanne et al., 2001). They are, however, weak agonists for PPARα with poor subtype selectivity, and their clinical doses are relatively high (200–1200 mg/day). Furthermore, it is uncertain that fibrates directly bind to PPARα. In contrast, glitazones, such as troglitazone, pioglitazone, and rosiglitazone, are potent and selective PPARγ agonists with high-affinity binding to the PPARγ ligand-binding domain (Willson et al., 2000). Although some potent and selective PPARα agonists have already been reported (Brown et al., 1999, 2001; Miyachi et al., 2002; Xu et al., 2003), they are not yet in clinical use. A potent and selective PPARα agonist is expected to be a promising drug for metabolic syndrome and atherosclerosis, because of its potency in ameliorating hyperlipidemia and low HDL cholesterol, as well as its other pleiotropic effects in metabolic disease and inflammation (Torra et al., 1999; Seedorf and Assmann, 2001).

In this study, we investigated the in vitro and in vivo pharmacological properties of NS-220, a novel dioxanecarboxylic acid derivative. NS-220 is one of the most potent and selective human PPARα agonists reported to date, and it markedly ameliorated metabolic disorders in type-2 diabetic mice.

Materials and Methods

Materials. Bezafibrate, ciprofibrate, fenofibrate, and phorbol 12-myristate 13-acetate were from Sigma-Aldrich (St. Louis, MO); carbaprostacyclin was from Cayman Chemical (Ann Arbor, MI); pGL3 and phRLTK were from Promega (Madison, WI); pSG5 was from Stratagene (La Jolla, CA); and pET28a(+) was from Novagen/Merck KGaA (Darmstadt, Germany). NS-220 (WO 01/90087) and pioglitazone were synthesized, and fenofibric acid was prepared by hydrolysis of fenofibrate, in our laboratories. [³H]NS-220 (443 GBq/mmol) was synthesized at Amersham Biosciences Inc. (Piscataway, NJ).

Cell Culture. Cells were obtained from American Type Culture Collection (Manassas, VA) and cultured under humidified 5% CO₂ and 95% air at 37°C with RPMI 1640 medium containing 10% fetal bovine serum (THP-1 cells) or Dulbecco's modified Eagle's minimal essential medium containing 10% fetal bovine serum, 100 unit/ml penicillin G, and 100 μg/ml streptomycin sulfate (CV-1 cells).

Cloning of PPAR cDNAs. PPAR cDNAs were cloned by a reverse transcription-polymerase chain reaction method. Total RNA was prepared by acid guanidinium-phenol-chloroform extraction from macrophage-like THP-1 cells differentiated by 100 nM phorbol 12-myristate 13-acetate and from the liver and testicular adipose tissue of male mice and used for cloning human and mouse PPAR cDNAs, respectively. Polymerase chain reaction primers were synthesized by Sawaday Technology (Tokyo, Japan).

Transactivation Assays. Agonist activity for each PPAR subtype was determined by its transactivation activity in a cell-based reporter-gene assay as previously described (Kliewer et al., 1992), with minor modifications. Briefly, three kinds of expression vectors were cotransfected into CV-1 cells with Tfx-20 (Promega) according to the manufacturer's instructions: 1) pSG5, containing a cDNA of each PPAR subtype; 2) pGL3, containing a tandem three-repeat of a peroxisome proliferator response element derived from the promoter region of the rat acyl CoA oxidase gene and a thymidin kinase promoter sequence (a reporter vector for transactivation activity); and 3) phRLTK, a reporter vector for monitoring the efficiency of transfection. The transfected cells were cultured with or without each drug for 48 h and then the luciferase activity of the cell lysate was measured with a Wallac Arvo SX multiplate reader (Amersham Biosciences Inc.) using the dual luciferase assay system (Promega). Drugs were dissolved in dimethyl sulfoxide, and the final concentration of dimethyl sulfoxide was less than 0.1% (v/v). Each assay was carried out in triplicate.

Binding Assay. Human PPARα ligand-binding domain (hPPARαLBD, amino acid numbers 196–468) was expressed as an N-terminal His₆-tagged fusion protein using pET28a(+) in Escherichia coli and purified on a Ni²⁺ chelating affinity column (His-Bind Fractogel; Novagen/Merck KGaA) and a Q-Sepharose Fast Flow column (Amersham Biosciences Inc.) as described previously (Cronet et al., 2001). Purified His₆-tagged hPPARαLBD was adjusted to 1.25 mg of protein/ml with 20 mM Tris-HCl, pH 8.0, containing 0.15 M NaCl, 10% (v/v) glycerol, and 1 mM Tris-carboxyethylphosphine HCl and stored at -80°C.

Radioligand binding assay was conducted as reported previously by Devchand et al. (1996) and Murakami et al. (1998), with minor modification. Briefly, [³H]NS-220 and 25 ng of His₆-tagged hPPARαLBD were mixed in 50 μl of 10 mM Tris-HCl, pH 8.0, containing 50 mM KCl, 10 mM dithiothreitol, and 0.02 mg/ml bovine serum albumin and incubated for 1 to 2 h at room temperature in the presence or absence of 500 μM NS-220. Bound ligand was separated from free ligand by centrifugation on a Micro Spin G-25 column (Amersham Biosciences Inc.) equilibrated with 25 mM Tris-HCl, pH 7.4, containing 75 mM KCl, 15% glycerol, 0.05% Triton X-100, and 0.5 mM EDTA. The radioactivity was quantified with a Tri-Carb 3100TR liquid scintillation counter (PerkinElmer Life Sciences, Boston, MA). Each assay was carried out in triplicate.

Animals. Male ddY mice and male KK-A^y mice were purchased from SLC (Shizuoka Agriculture Cooperative, Shizuoka, Japan) and Clea Japan (Tokyo, Japan), respectively. PPARα-deficient mice (129S4/SvJae-Ppara^tm1Gonz; Lee et al., 1995) and wild-type mice with the same genetic background (129S3/SvImJ) were purchased from The Jackson Laboratory (Bar Harbor, ME) and bred in our laboratories. All animals were fed on tap water and rodent chow (CE-2; Clea Japan) ad libitum. KK-A^y mice were housed individually in a plastic cage with wood chips, and other animals were housed in groups of four or five in a blanket cage at 23 ± 2°C with a 12-h light/dark cycle. All animal procedures were approved by the Committee for the Institutional Care and Use of Animals at Nippon Shinyaku Co.

Drug Administration. Drugs were dissolved or suspended in 0.5% methylcellulose solution (vehicle) and administered into the stomach with a gastric sonde at 10 ml/kg.

Single-Dose Experiment in ddY Mice. NS-220 (0.3–1 mg/kg), fenofibrate (100 mg/kg), fenofibric acid (100 mg/kg), or ciprofibrate (10 mg/kg) was orally administered to male ddY mice (9 weeks old; N = 5). Plasma was prepared from about 50 μl of blood collected from the tail vein of nonfasted mice in the morning 24 h before and 24, 48, 72, and 144 h after administration, and the plasma triglyceride levels were measured.

Repeated-Dose Experiment in PPARα-Deficient Mice. NS-220 (1 or 10 mg/kg) or fenofibrate (1000 mg/kg) was orally administered to wild-type and PPARα-deficient mice (9–13 weeks old; N = 5) once daily for 10 days. The body weight was measured every day. A day after the last administration, serum was prepared from about 1 ml of blood collected from the abdominal vein of nonfasted animals under sodium pentobarbital anesthesia. The animals were sacrificed by exsanguination, the liver was excised and weighed, and the serum triglyceride levels were measured.

Repeated-Dose Experiments in KK-A^y Mice. NS-220 (0.1–1 mg/kg) or fenofibrate (100–300 mg/kg) was orally administered once daily for 4 or 14 days to male KK-A^y mice (10 weeks old; N = 8). The body weight was measured every day. A day after the last administration, plasma was prepared from about 50 μl of blood collected from the tail vein of nonfasted mice in the morning, and the plasma triglyceride, glucose, free fatty acid, and insulin levels were measured. In the afternoon, serum was prepared from about 1 ml of blood collected from the abdominal vein of nonfasted animals under sodium pentobarbital anesthesia and the animals were sacrificed by exsanguination. The liver and testicular adipose tissue were excised and weighed. The lipoprotein lipid levels in the pooled serum from each group were analyzed by gel-filtration chromatography on a Superose 6HR column (Amersham Biosciences Inc.) eluting with 10 mM phosphate-buffered saline, pH 7.2, containing 0.1% EDTA and 0.01% NaN₃ at a flow rate of 0.25 ml/min/fraction.

Analytical Methods. The triglyceride, glucose, total cholesterol, and free fatty acid levels in each serum or plasma samples were measured with a JCA-BM1250 automatic analyzer (JEOL, Tokyo, Japan). The plasma insulin level was measured with a mouse insulin enzyme-linked immunosorbent assay kit (Shibayagi, Shibukawa, Japan), and the total cholesterol and triglyceride levels of fractions from gel filtration chromatography were measured by enzymatic methods (Wako Pure Chemicals, Osaka, Japan) in 96-well plates, which were read with a Benchmark plate reader (Bio-Rad, Hercules, CA). The assays were carried out in duplicate.

Statistical Analysis. All data were expressed as mean ± S.E., and statistical differences between groups were evaluated by Dunnett's test. The half-maximum effective concentration (EC₅₀) for PPAR agonist activity was evaluated by nonlinear regression analysis. All analysis was done with SAS software, version 6.12 (SAS Institute, Cary, NC).

Results

PPARα-Selective Activation by NS-220. NS-220 activated human PPARα in a dose-dependent manner from 5 × 10^-10 to 5 × 10^-7 M (Fig. 1). The concentration-activity curves for human PPARγ and PPARδ were shifted to the right, revealing the subtype selectivity of NS-220 for PPARα. NS-220 activated human PPARγ partially and its maximum activity by NS-220 at 5 × 10^-5 M or more was 58% of that by pioglitazone at 2.5 × 10^-5 M.

The EC₅₀ value was calculated from each concentration-activity curve. The EC₅₀ values of bezafibrate, fenofibric acid, and pioglitazone for PPARs estimated by our transactivation assays agreed with those estimated by PPAR-GAL4 transactivation assays (Willson et al., 2000). The EC₅₀ value of NS-220 for human PPARα was 19 nM, the lowest among the drugs tested (Table 1). Furthermore, the ratios of EC₅₀ for PPARγ and PPARδ to that for PPARα were much higher for NS-220 than for the other drugs tested.

Binding Assay. [³H]NS-220 showed saturable binding to hPPARαLBD at 10^-8 to 10^-6 M (Fig. 2A). Scatchard plot analysis showed a single high-affinity binding site with a dissociation constant (K_D) of 185 nM (Fig. 2B).

Hypolipidemic Activity of NS-220 in ddY Mice. To evaluate the in vivo pharmacological effects of NS-220 as a potent PPARα agonist, it was orally administered to male ddY mice. A single administration of NS-220 decreased plasma triglyceride levels in a dose-dependent manner for a few days (Fig. 3A). It maximally decreased plasma triglyceride levels (by ca. 40% of the pretreatment value) at 0.3 and 1 mg/kg. Ciprofibrate at 10 mg/kg and fenofibric acid at 100 mg/kg decreased plasma triglyceride levels in the same manner, whereas fenofibrate did not affect plasma triglyceride levels at 100 mg/kg (Fig. 3B). The hypolipidemic effect of NS-220 was more than 1000 times as potent as fenofibrate in normolipidemic mice.

Effects of NS-220 and Fenofibrate in PPARα-Deficient Mice. To elucidate whether the hypolipidemic effect of NS-220 observed in ddY mice was caused by its PPARα-agonist activity, we administered NS-220 to PPARα-deficient mice for 10 days at 1 or 10 mg/kg. PPARα-deficient mice showed slightly higher serum triglyceride levels than age-matched wild-type mice (140.2 ± 3.9 mg/dl in PPARα-deficient mice versus 116.2 ± 5.8 mg/dl in wild-type mice). Although NS-220 markedly decreased serum triglyceride levels and increased liver weight in wild-type mice, these effects were not observed in PPARα-deficient mice (Table 2). Fenofibrate was ineffective in both wild-type and PPARα-deficient mice at 1000 mg/kg. Neither NS-220 nor fenofibrate affected body weight in either wild-type or PPARα-deficient mice. These results strongly suggest that the pharmacological effects of NS-220 were predominantly mediated through the activation of PPARα.

Hypolipidemic and Hypoglycemic Effects of NS-220 in KK-A^y Mice. To evaluate the pharmacological effects of NS-220 in type-2 diabetes, we administered it to 10-week-old KK-A^y mice, which display the typical symptoms of type-2 diabetes, namely, severe hyperglycemia, hyperinsulinemia, hypertriglyceridemia, and obesity (Fujita et al., 1983). After administration of NS-220 for 4 days, plasma triglyceride levels decreased in a dose-dependent manner from 0.3 to 1 mg/kg, and plasma glucose levels significantly decreased at 1 mg/kg (Fig. 4). Although fenofibrate also decreased plasma triglyceride levels at 100 and 300 mg/kg, it was about 1000 times less potent than NS-220.

Subsequently, NS-220 or fenofibrate was administered to KK-A^y mice for 14 days, and the plasma biochemical parameters and lipoprotein lipid levels were examined. NS-220 decreased both the plasma triglyceride and the plasma glucose levels at 1 mg/kg, and the decrease in glucose levels especially was greater than in the 4-day experiment (Fig. 5A). Fenofibrate was ineffective in reducing plasma triglyceride and glucose levels. Plasma insulin and free fatty acid levels tended to decrease after treatment with NS-220, whereas free fatty acid levels increased after treatment with fenofibrate (Fig. 5B).

To assess the hypolipidemic effect of NS-220 in more detail, the lipoprotein lipid levels of pooled serum of animals treated with vehicle (control), 1 mg/kg NS-220 and 100 mg/kg fenofibrate were analyzed. NS-220 decreased triglyceride and cholesterol levels in chylomicron and VLDL, and increased cholesterol levels in HDL and larger size lipoproteins, distributing broadly between low-density lipoprotein and HDL (Fig. 6). Fenofibrate slightly increased triglyceride levels in chylomicron and VLDL and slightly increased cholesterol levels in HDL and the larger size lipoproteins.

Although there were no abnormal body weight changes in any animals treated with drugs compared with control, NS-220 markedly increased the liver weight in a dose-dependent manner and decreased the weight of testicular adipose tissue at 1 mg/kg (Table 3). Fenofibrate also increased the weight of the liver but did not affect the weight of the adipose tissue.

Discussion

In this report, we describe the pharmacological properties of NS-220 and show it to be a novel α-selective PPAR agonist in vitro and in vivo. The EC₅₀ value of NS-220 for human or mouse PPARα was over 510 and 600 times lower than those for human or mouse PPARγ and PPARδ, respectively, so that NS-220 is a highly selective PPARα agonist in humans and mice. Moreover, the PPARα-agonist activity of NS-220 was more than 1000 times as potent as that of fibrates (Table 1). There are many recent reports on compounds affecting PPARs. GW7647, GW9578, KCL1998001079, and LY518674 especially have been reported to be as potent PPARα agonists as NS-220, their respective EC₅₀ values for human PPARα being 6, 50, 40, and 42 nM (Brown et al., 1999, 2001; Miyachi et al., 2002; Xu et al., 2003). However, the ratios of EC₅₀ for human PPARγ to EC₅₀ for human PPARα are only 10 to 180 for the former three agonists, whereas the corresponding ratio for NS-220 is 510. LY518674 seems to be more subtype-selective than these, but its EC₅₀ value for human PPARγ and PPARδ has not been reported. NS-220 is therefore one of the most selective human PPARα agonists reported to date.

A comparison of the chemical structure of NS-220 to those of other PPAR agonists (Fig. 7) reveals the unique dioxanecarboxylic acid moiety of NS-220. Fibrates and glitazones each have a common moiety, namely, 2-phenoxyisobutyric acid and thiazolidinedione, respectively. The other potent PPARα agonists described above are α-substituted carboxylic acids, either α,α-dimethylcarboxylic (GW7647, GW9578, and LY518674) or α-ethylcarboxylic (KCL1998001079) acids. Therefore, we suggest that the dioxanecarboxylic acid moiety of NS-220 is responsible for its high potency and selectivity for the PPARα subtype.

Because the Scatchard plot analysis in the binding assay using purified His₆-tagged hPPARαLBD revealed a single high-affinity binding site, we conclude that NS-220 directly bound to PPARα. There is, however, some discrepancy between K_D in the binding assay (185 nM) and EC₅₀ in the transactivation assay (19 nM) for human PPARα. This discrepancy may be explained by one or both of the following considerations: 1) a difference between the binding affinity of NS-220 to hPPARαLBD and its binding affinity to full-length human PPARα, and 2) intracellular accumulation of NS-220 during the 48-h culture on the transactivation assay.

A single oral administration of NS-220, fenofibric acid, or ciprofibrate lowered plasma triglyceride levels for 2 or 3 days in mice, but fenofibrate was ineffective. It is reported that in rats the plasma half-life of ciprofibrate in the excretion phase is 82 h and that its triglyceride-lowering effect continues for 3 days (Cayen, 1985). Ciprofibrate is expected to have a long plasma half-life in mice also, and this would contribute to the lasting triglyceride-lowering effect observed in the present experiment. On the other hand, the plasma half-life of NS-220 in the excretion phase in mice is about 8 h (A. Nakamura, personal communication) and that of fenofibric acid after oral administration of fenofibrate in rats is also about 8 h (Mogi et al., 1995). Because the liver is considered to be the primary target organ for the hypolipidemic activity of PPARα agonists, the half-life of these drugs in the liver can explain their lasting effects. Although it is unclear why fenofibrate was ineffective at the same dose as fenofibric acid, it has recently been reported that fenofibric acid is a PPARα agonist, whereas fenofibrate is a liver X receptor antagonist (Thomas et al., 2003). This substantial difference between fenofibric acid (free acid) and fenofibrate (ester) probably contributed to the difference in their hypolipidemic effect in mice.

NS-220 was administered to PPARα-deficient and wild-type mice at doses of 1 and 10 mg/kg. These doses were considered to be 10 to 100 times of the effective dose, because NS-220 decreased plasma triglyceride levels at 0.1 mg/kg in ddY mice. There were drastic changes in the serum triglyceride levels and liver weight in wild-type mice treated with NS-220, whereas these effects were not observed at all in PPARα-deficient mice. A very high dose of fenofibrate was also ineffective in PPARα-deficient mice, and the same result has been reported for the PPARα agonists clofibrate and Wy-14643 (Lee et al., 1995). Therefore, it is apparent that NS-220 is a highly specific PPARα agonist and it is probable that its hypolipidemic effect is mediated through the activation of PPARα.

It has been suggested that the activation of PPARα ameliorates metabolic disorders and atherosclerosis by its pleiotropic effects (Barbier et al., 2002), so that PPARα agonists are expected to be a useful medicine for type-2 diabetes, which is usually accompanied by metabolic syndrome or atherosclerosis. KK-A^y mice, which spontaneously develop hypertriglyceridemia, hyperglycemia, hyperinsulinemia, and obesity, are a well known animal model of type-2 diabetes (Fujita et al., 1983). In the present study, NS-220 markedly decreased plasma triglyceride levels in KK-A^y mice to almost the same levels as in normal mice. There are many studies on the hypolipidemic activity of fibrates, and their main mechanisms of action are considered to be an increase in the lipolysis of triglyceride-rich lipoproteins on blood vessels and a decrease in the hepatic secretion of triglyceride-rich lipoproteins. In particular, transcriptional regulation of lipoprotein lipase, apoCIII and apoAV genes is considered to play an important role in lipolysis (Staels et al., 1995; Schoonjans et al., 1996; Vu-Dac et al., 2003). The fact that NS-220 decreased the triglyceride levels of chylomicron and VLDL strongly suggests that its potent hypolipidemic effect also depends on an increase in lipolytic activity.

NS-220 decreased not only plasma triglyceride levels but also plasma glucose levels without increasing insulin levels. The extent of the decrease in plasma glucose levels, however, increased with the duration of the administration (Figs. 4 and 5A). These results suggest that the hypoglycemic effect of NS-220 depends on a more complex mechanism, including an amelioration of insulin resistance, than the hypolipidemic effect. Further research is necessary to clarify its mechanism.

Gel-filtration-chromatography analysis revealed an increase in the cholesterol levels of HDL and larger size lipoproteins distributing broadly between low-density lipoprotein and HDL in KK-A^y mice treated with NS-220. Similar changes were reported in type-2 diabetic db/db mice treated with a PPARγ-selective agonist (Leibowitz et al., 2000) and in human apoAI transgenic mice treated with fenofibrate (Berthou et al., 1996). Although there are marked differences in lipoprotein metabolism between rodents and humans, it seems likely that NS-220 will increase HDL in patients with low HDL-cholesterol levels more potently than do fibrates.

NS-220 increased liver weight in mice except for PPARα-deficient mice. We have observed hypertrophy with peroxisome proliferation and no lipid accumulation in hepatocytes by pathological examination in rats treated with NS-220 (data not shown). And there were no change in hepatic triglyceride content by weight percentage in KK-A^y mice. Therefore, the increase in liver weight caused by NS-220 in this study is probably due to a typical hepatomegary widely known in the rodents treated with PPARα agonists. Several studies have shown that PPARα agonists evidently induce peroxisome proliferation and hepatomegary in rodents but not in humans (Cattley et al., 1998; Hertz and Bar-Tana, 1998; Holden and Tugwood, 1999). The reason of this species-difference is not clear but there are some explanations: 1) expression level of PPARα in human liver is lower than in rodent liver (Palmer et al., 1998), and 2) the peroxisome proliferator response element in the promoter region of acyl CoA oxidase gene in humans lacks response to PPARα agonists (Lambe et al., 1999). Acyl CoA oxidase is an enzyme of peroxisomal fatty acid β-oxidation and typically induced by PPARα agonists associating with peroxisome proliferation in rats and mice. Although fibrates are PPARα agonists and increase liver weight in rats and mice, their pharmacological effects and safety have been proved during a long-term experience of clinical use. NS-220 is also expected tolerable in humans.

In conclusion, NS-220 ameliorated metabolic disorders in type-2 diabetes by its potent PPARα-agonist activity, and it is therefore a promising candidate drug for the treatment of hyperlipidemia, atherosclerosis, and also type-2 diabetes.