50
`
`BBA 52250
`
`Biochimica et Biophysica Acta 877 (1986) 50-60
`Elsevier
`
`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
`
`Y oshio Tsujita a,*, Masao Kuroda a, Y oko Shimada a, Kazuhiko Tanzawa a,
`Mamoru Arai a, I sao Kaneko b, Minoru Tanaka c, Hiroshi Masuda d,
`Chitoshi Tarumi d, Y oshio Watanabe e and Setsuro Fujii r
`a Fermentation Research Laboratories, 6 Bioscience Laboratories, c Analytical and Metabolic Research Laboratories,
`d Laboratory of Animal Science and Toxicology Laboratories, San kyo Co. Ltd., Tokyo, e Institute for Experimental
`Animals, Kobe University School of Medicine, Kobe, and! Osaka University, Osaka (Japan)
`
`(Received November 25th, 1985)
`
`Key words: Sterol synthesis; Hydroxymethylglutaryl-CoA reductase; Hypolipidemic drug; Lipoprotein; (Cell culture)
`
`CS-514 is a tissue-selective inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a key enzyme in
`cholesterol synthesis. For the microsomal enzyme from rat liver, the mode of inhibition is competitive with
`respect to hydroxymethylglutaryi-CoA, and the Ki value is 2.3 • 10- 9 M. CS-514 also strongly inhibited the
`sterol synthesis from 114 C) acetate in cell-free enzyme systems from rat liver and in freshly isolated rat
`hepatocytes; the concentrations required for 50% inhibition were 0.8 ngjml and 2.2 ngjml, respectively. On
`the other hand, the inhibition by CS-514 was much less in the cells from nonhepatic tissues such as freshly
`isolated rat spleen cells, and cultured mouse L cells and human skin fibroblasts. In addition, the cellular
`uptake of 14 C-Iabeled CS-514 by isolated rat spleen cells or mouse L cells was less than one-tenth of that by
`isolated hepatocytes. These differences between hepatic and nonhepatic cells were further confirmed by the
`fact that CS-514 orally administered to rats inhibited sterol synthesis selectively in liver and intestine, the
`major sites of cholesterogenesis. CS-514 markedly reduced serum cholesterol levels in dogs, monkeys and
`rabbits, including Watanabe heritable hyperlipidemic (WHHL) rabbits, an animal model for familial
`hypercholesterolemia in man, but did not reduce those in rats and mice. In the former case, preferential
`lowering of atherogenic lipoproteins was observed in all of the animals tested. The biliary neutral sterols
`significantly decreased, whereas the amount of biliary bile acids was not affected by administration of the
`drug to dogs.
`
`Introduction
`
`the development of atherosclerosis and
`In
`coronary heart disease, which is the major cause of
`death in western and other developed countries, a
`high level of cholesterol in blood is considered to
`be an important risk factor [1]. Since more than
`
`* To whom correspondence should be addressed.
`
`70% of the total input of body cholesterol is
`derived from de novo synthesis in humans [2], it is
`expected that serum cholesterol can be reduced as
`a result of inhibition of cholesterol biosynthesis.
`The :J?lOSt suitable target for this inhibitor is 3-hy(cid:173)
`droxy-3-methylglutaryl coenzyme A
`reductase
`(mevalonate : NADP + oxidoreductase
`(CoA(cid:173)
`acylating), EC 1.1.1.34), the rate-limiting enzyme
`in the pathway of cholesterol biosynthesis [3).
`On the basis of this concept, we carried out
`
`0005-2760j86j$03.50 ® 1986 Elsevier Science Publishers B.V. (Biomedical Division)
`
`Mylan Exhibit 1039, Page 1
`
`
`
`

`
`,,,OH
`
`NaOOC
`HO
`0
`...... )l.o H
`H3C' 1
`~
`
`H3C
`
`Rz
`Fig. 1. Structure of CS-514 and related compounds. CS-514
`(Rt = H, R 2 = OH; molecular weight, 447), ML-236B sodium
`salt (R 1 = R 2 =H) and MB-530B (monacolin K or mevinolin)
`sodium salt (R1 = CH 3 , R 2 =H).
`
`inhibitor of hydroxymethyl(cid:173)
`screening of the
`glutaryl-CoA reductase and found several com(cid:173)
`pounds in microbial products [4-12], including
`ML-236B and MB-530B [12]. The latter is an
`identical compound
`to monacolin K
`[13] or
`mevinolin [14]. Those compounds were potent
`competitive inhibitors of this enzyme.
`their
`Among
`these microbial products and
`trans(cid:173)
`chemically synthesized and microbially
`formed derivatives, CS-514 (Fig. 1) was selected
`because of its potency and tissue selectivity. This
`drug was found at first as a minor urinary
`metabolite of ML-236B in dogs and later was
`obtained by microbial transformation of ML-236B
`[8,9]. In this report, biochemical and pharmaco(cid:173)
`logical characteristics of CS-514 are described.
`
`Materials and Methods
`
`Materials. [1-14 C]Sodium acetate (58 Cijmol),
`DL-[2-14 C]mevalonolactone (27.3 Cijmol) and DL(cid:173)
`[3-14 C]hydroxymethylglutaryl-CoA (55 .1 Cijmol)
`were obtained from New England Nuclear Corp.
`(Boston, MA, U.S.A.) and [carboxyl- 14 C]sodium
`deoxycholate (52 Cijmol) was purchased from
`Amersham International (Amersham, U.K.). CS-
`514 [8,9], ML-236B [5] and 14C-labeled ML-236B
`[7] were prepared as described previously. 14 C(cid:173)
`labeled CS-514 was prepared biosynthetically by
`adding 14 C-labeled ML-236B as a precursor to
`in
`cultures of Nocardia autotrophica grown
`Erlenmeyer flasks, and purified extensively as de(cid:173)
`scribed [8]. MB-530B was prepared from the cul(cid:173)
`ture filtrate of Monascus ruber [12]. As internal
`standards for gas-liquid chromatography, neutral
`sterols, (5a-cholestane, cholesterol, desmosterol,
`campesterol, stigmasterol, ,8-sitosterol and lano-
`
`51
`
`sterol) and bile acids, (methyl esters of cholic,
`deoxycholic, lithocholic, chenodeoxycholic and 7-
`ketolithocholic acid) were obtained from Gasukuro
`Kogyo Co. (Tokyo, Japan) and 9-anthryl di(cid:173)
`azomethane was purchased from the Funakoshi
`Pharmaceutical Co. (Tokyo, Japan).
`Animals. Wistar-Imamichi male rats weighing
`120-140 g were used for usual experiments and
`rats weighing 250-330 g for the experiments of
`Triton-induced hyperlipidemia. The rats were
`maintained on a commercial rat chow (MM 1,
`Funabashi Farm Co, Chiba, Japan) for at least 1
`week prior to use. Pure-bred beagle dogs weighing
`8-10 kg were housed individually and fed a com(cid:173)
`mercial dog food (Type DS, Oriental Yeast Co.,
`Tokyo, Japan) at 200 gjday. Cynomolgus monkeys
`weighing 4.1-6.2 kg (male) and 3.2-4.1 kg (female)
`were housed individually and fed a commercial
`monkey food (Type AB, Oriental Yeast Co.,
`Tokyo, Japan) at 80 gjday. Male Japanese white
`rabbits aged 11-13 months were housed individu(cid:173)
`ally and fed rabbit chow (RC-4, Oriental Yeast
`Co., Tokyo, Japan) at 150 gjday. Watanabe heri(cid:173)
`table hyperlipidemic (WHHL) rabbits weighing
`3-3.5 kg were used for short-term experiments,
`and the rabbits at 3 months of age (weighing
`1.8-2.2 kg) were used for a long-term experiment
`of 8 weeks. WHHL rabbits were housed individu(cid:173)
`ally and fed a commercial rabbit chow (Type GC,
`Oriental Yeast Co., Tokyo, Japan) at 120 gj day.
`In animal experiments with dogs, monkeys and
`rabbits, blood samples were withdrawn between 9
`and 9.30 a.m. on appropriate days before feeding.
`Cells. All cultured cells used were maintained
`at 37°C in a 5% C02 incubator. Mouse L cells
`were seeded at about 2 · 10 5 cells per 60 mm dish
`and cultured for 3 days in Dulbecco's modified
`Eagle's minimum essential medium containing 5%
`fetal calf serum. Human skin fibroblasts from a
`normal subject (GM-442) and a homozygous
`familial hypercholesterolemic patient (GM-486)
`were obtained from the Institute of Medical Re(cid:173)
`search (Camden, NJ, U.S.A.). These cells were
`grown in Dulbecco's modified Eagle's minimum
`essential medium containing 1% non-essential
`amino acids and 10% fetal calf serum. About
`1 · 10 5 cells were seeded onto 60 mm dishes
`according to a standard protocol [15) and were
`cultured for 5 days.
`
`
`
`
`
`Mylan Exhibit 1039, Page 2
`
`

`
`52
`
`Rat hepatocytes were prepared by collagenase
`digestion according to the method of Moldeus et
`al. [16]. For preparation of rat spleen cell suspen(cid:173)
`sion, the spleen was cut into pieces with scissors
`and gently pressed on a stainless filter (100 mesh)
`using a silicon plug. The cells which filtered
`through were further treated twice on a 150 mesh
`filter and isolated by centrifugation at 1000 rpm
`for 5 min. The spleen cells thus obtained were
`completely disaggregated as single cells. Both
`hepatocytes and spleen cells were suspended at
`appropriate cell densities in serum-free Dulbecco's
`modified Eagle's minimum essential medium, and
`used for experiments within 3 h after preparation.
`Rabbit aortic fibroblasts were obtained from
`explants of aortic adventitia and cultured in
`Dulbecco's modified Eagle's minimum essential
`medium containing 5% fetal calf serum. About
`1 · 10 5 cells were seeded onto 60 mm dishes and
`used for experiments after 3 days cultivation.
`Cellular uptake of CS-514. Suspensions of
`freshly isolated rat hepatocytes (10 7 cellsjml) or
`spleen cells (2 ·10 8 cellsjml) were incubated with
`various concentrations of 14 C-labeled CS-514 or
`14 C-labeled ML-236B sodium salt at 37°C for 30
`min in 0 2 jC02 (95: 5) gas with shaking. 80 p,l of
`cell suspension were layered on 250 p,l of the
`solvent mixture (butyl phthalate/ dinonyl phtha(cid:173)
`late, 7 : 3, v jv) in a polyethylene tube (1A tube,
`Sanko Plastic Co., Osaka, Japan) and centrifuged
`at 3000 rpm for 2 min. The bottom of tube
`containing the cells was cut and put into a vial for
`determination of cellular radioactivity.
`Mouse L cells grown to form a monolayer in
`plastic dishes
`received 1 ml of serum-free
`Dulbecco's modified Eagle's minimum essential
`medium containing various concentrations of 14 C(cid:173)
`CS-514 of 14 C-ML-236B sodium salt. After in(cid:173)
`cubation for 30 min at 37°C, cells in the medium
`were scraped with a rubber policeman. After the
`cell suspension was centrifuged at 1000 rpm for 5
`min, about 900 p,l of the medium were removed
`and cells were resuspended with the remaining
`medium. The cell suspension thus obtained was
`assayed for cellular radioactivity by the same pro(cid:173)
`cedure as that employed for rat hepatocytes. After
`measurement of cellular radioactivity, the pellet of
`mouse L cells was taken out from the counting
`vial, washed with phosphate-buffered saline four
`
`times, dissolved with 4 M NaOH at 80°C for 1 h,
`and assessed for protein content.
`Sterol synthesis in the celljree enzyme system
`from rat liver, cultured cells and tissue slices from
`various organs of rats. [1- 14 C]Sodium acetate was
`used as a precursor for sterol synthesis in these
`experiments. For
`the measurement of sterol
`synthesis in the cell-free enzyme system from rat
`liver, the reaction mixture was incubated for 2 h at
`37°C and incorporation of radioactivity into non(cid:173)
`saponifiable lipids was assayed by the method
`described previously [5]. In the experiments with
`cultured cells, monolayers of mouse L cells, hu(cid:173)
`man skin fibroblasts and rabbit aortic fibroblasts
`were incubated with [14 C]sodium acetate at 37°C
`for 2 h. In the experiments with freshly isolated
`
`cells from rats, e4 C]sodium acetate was added to
`
`1 ml of suspension culture containing 2 · 10 6
`hepatocytes or 5 · 107 spleen cells, and the incuba(cid:173)
`tion was carried out at 37°C for 2 or 4 h, respec(cid:173)
`tively. Incorporation of radioactivity into dig(cid:173)
`itonin-precipitable sterols was measured by the
`method described previously [15]. In the experi(cid:173)
`ment using tissue slices, rats were killed by carotid
`puncture and various organs were rapidly re(cid:173)
`moved. The reaction mixture containing tissue
`slices and e4 C]sodium acetate was incubated for 2
`hat 37°C in Krebs-Ringer bicarbonate buffer (pH
`7.4) and incorporation of radioactivity into dig(cid:173)
`itonin-precipitable sterols was measured as de(cid:173)
`scribed previously [7]. All experiments were done
`by duplicate assays, unless stated otherwise.
`Assay of hydroxymethylglutaryl-CoA reductase
`activity. Hydroxymethylglutaryl-CoA reductase
`activities in rat liver microsome [6] and in cultured
`cells [15] were measured as described previously.
`Measurement of hypolipidemic activity of CS-514
`in Triton WR-1339-induced hyperlipidemic rats. The
`experiment was carried out as described previ(cid:173)
`ously [17 ,18] except that the drug was adminis(cid:173)
`tered orally at the time of Triton injection.
`Measurement of lipids and fractionation of serum
`lipoproteins. Serum lipids were measured by en(cid:173)
`zymatic methods using the following assay kits
`unless otherwise stated: cholesterol, Determiner
`TC (Kyowa Hakko Co., Tokyo, Japan);
`tri(cid:173)
`acylglycerol and phospholipid, Triglyceride G-Test
`Wako and Phospholipid B-Test Wako, respec(cid:173)
`tively (Wako Pure Chemical Industries, Osaka,
`
`
`
`Mylan Exhibit 1039, Page 3
`
`

`
`Japan). Serum cholesterol levels in a Triton-in(cid:173)
`duced hyperlipidemic rats were assayed by a
`slightly modified method of Zak [19] and Henly
`[20].
`For the fractionation of serum lipoprotein, 0.5
`ml (WHHL rabbit) or 1 ml (other animals) of
`serum was centrifuged according to the method of
`Hatch and Lees [21] using a Hitachi 65P ultra(cid:173)
`centrifuge equipped with an RPS-56T
`rotor
`(Hitachi Co., Tokyo, Japan). Each lipoprotein was
`fractionated as follows: very-low-density lipopro(cid:173)
`tein (VLDL, d < 1.006);
`intermediate-density
`lipoprotein (IDL, 1.006 < d < 1.019); low-density
`lipoprotein (LDL, 1.019 < d < 1.063); high-density
`lipoprotein (HDL, 1.063 < d < 1.21) and infrana(cid:173)
`tant ( d > 1.21 ).
`Analysis of biliary lipids. Bile samples for analy(cid:173)
`sis were prepared as described previously [19]. For
`correcting recovery of neutral sterols and bile
`acids, 5a-cholestane and [14 C]deoxycholate were
`added to bile samples, respectively. Neutral sterols
`were determined by gas-liquid chromatography
`[22]. Bile acid was measured by high-performance
`liquid chromatography: the samples treated with
`9-anthryl diazomethane were chromatographed on
`a 25 em ODS C-18 column maintained at 35°C in
`a Hitachi Type 655 high-performance liquid chro(cid:173)
`matography (Hitachi Co., Tokyo, Japan) equipped
`with a fluorometric detector FP-110 (Japan Spec(cid:173)
`troscopic Co., Tokyo, Japan). Under these condi(cid:173)
`tions, the overall recovery of bile acid varied be(cid:173)
`tween 65 and 95%.
`The phospholipid in bile was assayed by phos(cid:173)
`phorus analysis using a kit, Phospholipid-Test
`W ako (W ako Pure Chemical Industries, Osaka,
`Japan).
`Other assay. Protein content was determined by
`the method of Lowry et al. [23] with bovine serum
`albumin as standard.
`
`Results
`
`inhibition of sterol
`
`Specific and tissue-selective
`synthesis
`Fig. 2 shows the effect of CS-514 on the incor(cid:173)
`poration of various radiolabeled precursors into
`digitonin-precipitable sterols in the cell-free sys(cid:173)
`
`tem from rat liver. The conversion of e4 C]acetate
`and DL-[ 14C]hydroxymethylglutaryl-CoA was in-
`
`53
`
`_,-ft-
`
`100
`
`~
`
`c 60
`
`0 ·-...
`
`80 /'~
`/.1
`
`..0 40
`:.
`c
`
`- 20
`
`.-"'
`
`-n·
`
`-n·
`
`0.1
`
`1.0
`CS-514 (ng/ml)
`
`10
`
`100
`
`Fig. 2. Effect of CS-514 on cholesterol synthesis from acetate,
`hydroxymethylglutaryl-CoA or mevalonate in a cell-free en(cid:173)
`zyme system from rat liver. The assay method of sterol synthe(cid:173)
`sis is described in Materials and Methods. The concentrations
`of the precursors were; 1 mM, 0.3 mM and 0.5 mM for
`
`[ 14 C]acetate (o), e4 CJhydroxymethylglutaryl-CoA (,t,) and
`e4 CJmevalonate (D), respectively. Each point is plotted as a
`
`percent inhibition to the respective control.
`
`hibited to almost the same extent, i.e., the con(cid:173)
`centrations required for 50% inhibition (1 50 ) were
`0.8 ngjml and 0.9 ngjml, respectively. In the case
`of ML-236B, the mother compound of CS-514, I 50
`for the conversion of e4 C]acetate was 10 ngjml.
`On the other hand, the conversion of e4 CJ(cid:173)
`mevalonate was not affected by CS-514 at con(cid:173)
`centrations up to 100 ngjml, indicating that this
`drug inhibited specifically the enzymatic step of
`the conversion of hydroxymethylglutaryl-CoA to
`mevalonate catalysed by hydroxymethylglutaryl(cid:173)
`CoA reductase. The mode of inhibition of CS-514
`for hydroxymethylglutaryl-CoA
`reductase was
`competitive with
`respect
`to hydroxymethyl(cid:173)
`glutaryl-CoA CoA and non-competitive with re(cid:173)
`spect to NADPH. The Ki values for the reductase
`from rat liver and mouse L cells were 2.3 · 10- 9 M
`and 2.2 · 10- 9 M, respectively.
`Table I summarizes the values of I 50 for incor(cid:173)
`poration of [14 C]acetate into sterols either by CS-
`514 or by ML-236B in freshly isolated and cul(cid:173)
`tured cells. Both CS-514 and ML-236B strongly
`inhibited sterol synthesis in freshly isolated rat
`hepatocytes. The values of I 50 were 2.2 ngjml and
`7.0 ngjml, respectively, both of which were close
`to the values obtained in the cell-free enzyme
`system from rat liver, suggesting that both drugs
`were internalized and reached the target enzyme
`
`
`
`
`
`Mylan Exhibit 1039, Page 4
`
`
`
`
`
`

`
`54
`
`in hepatocytes. In the cells from nonhepatic tis(cid:173)
`sues, such as freshly isolated rat spleen cells and
`various cultured cells, ML-236B exerted the potent
`inhibitory activity as in rat hepatocytes. In con(cid:173)
`trast, the inhibitory activity of CS-514 was much
`less potent in these cells from nonhepatic tissues
`than in rat hepatocytes. In order to verify the
`assumption that the results mentioned above might
`be explained by poor permeability of CS-514 into
`the cells from nonhepatic tissues, cellular uptake
`of 14 C-CS-514 and 14 C-ML-236B sodium salt was
`compared among freshly isolated rat hepatocytes
`and spleen cells, and mouse L cells. As shown in
`the cellular uptake of 14 C-CS-514 in
`Fig. 3,
`hepatocytes occurred to almost the same extent as
`that of 14 C-ML-236B sodium salt. The uptake of
`14 C-CS-514 in spleen cells and mouse L cells,
`however, was less than one-tenth of that of 14 C(cid:173)
`ML-236B sodium salt. From these results, the less
`potent inhibitory activity of CS-514 in the cells
`from nonhepatic tissues can be ascribed to lower
`uptake of the drug by those cells.
`After CS-514, ML-236B or MB-530B was ad(cid:173)
`ministered orally to rats, the activity of sterol
`synthesis in slices of various organs was measured
`in vitro and inhibition in each organ was calcu(cid:173)
`lated (Table II). CS-514 inhibited
`the sterol
`
`TABLE I
`
`EFFECT OF CS-514 AND ML-236B ON STEROL
`SYNTHESIS IN VARIOUS ISOLATED AND CULTURED
`CELLS
`
`The assay methods of sterol synthesis are described in Materi(cid:173)
`als and Methods. The inhibitory activities of CS-514 and
`ML-236B on sterol synthesis are expressed by the concentra(cid:173)
`tions required for 50% inhibition (1 50 ). n.t., not tested.
`
`Cells
`
`Freshly isolated rat hepatocytes
`Freshly isolated rat spleen cells
`MouseL cells
`Human skin fibroblasts
`(normal)
`(homozygous familial
`hypercholesterolemia)
`Rabbit aortic fibroblasts
`
`150 (ngjml)
`CS-514
`ML-236B
`7.0 *
`1.3 *
`1.4
`
`2.2
`70
`600
`
`200
`
`400
`750
`
`18.0
`
`12.0
`n.t.
`
`* Result obtained with sodium salt.
`
`synthesis selectively in liver and ileum (intestine),
`the major sites of cholesterogenesis, but only
`weakly inhibited that in other organs, including
`hormone-producing ones. In the cases of ML-236B
`and MB-530B, although the inhibition of sterol
`synthesis in liver and intestine was most potent,
`that in other organs was significant as well. These
`results indicate that the inhibitory activity of CS-
`514 is tissue selective, compared to those of ML-
`236B and MB-530B, and this observation is con(cid:173)
`sistent with the results obtained with the isolated
`and cultured cells.
`
`A Hepatocytes
`
`c MouseL cells
`
`:e
`e a.
`Q) 20
`01
`E
`' E
`a.
`0 "' 'O
`.:8
`0
`..!!!
`Qi 5.0
`0
`
`10
`
`0 -,!;;
`
`"0 c:: 2.5
`:I
`0
`0.
`E
`0
`0 u
`"'
`.... 0
`-~ 30
`0 20 -0.
`
`0
`
`Q)
`.:.:
`
`::::>
`
`10
`
`5xl05 106
`105
`14C)Compound added (cpm/ml)
`
`(
`
`Fig. 3. Cellular uptake of 14 C-CS-514 and 14 C-ML-236B
`sodium salt into (A) freshly isolated rat hepatocytes, (B) freshly
`isolated spleen cells and (C) mouse L cells. The cells were
`incubated with the indicated concentrations of 14 C-CS-514 (8)
`or 14 C-ML-236B sodium salt (0) at 37°C for 30 min. Uptake
`of radioactivity into cells was determined as described in
`Materials and Methods. Each point expresses the average of
`duplicate (rat hepatocytes and spleen cells) or quadruplicate
`(mouse L cells) incubations.
`
`
`
`Mylan Exhibit 1039, Page 5
`
`

`
`TABLE II
`INHIBITORY ACTIVITY OF CS-514, ML-236B AND MB-
`53oB (MONACOLIN K OR MEVINOLIN) ON STEROL
`SYNTHESIS OF VARIOUS TISSUES IN RATS
`
`CS-514, ML-236B or MB-530B was suspended in 10% gum
`rabic and administered orally at the dose of 25 mgjkg to
`~ale Wistar-Imamichi rats. After 2 h, the rats were killed and
`the sterol synthesis in slices of each organ was measured as
`described in Materials and Methods. The data represent the
`average of values obtained from six rats.
`
`55
`
`as compared to the control value after administra(cid:173)
`tion of CS-514 (20 mgjkg) to the rats.
`The time courses of changes in serum lipid
`levels in beagle dogs treated with CS-514 are
`shown in Fig. 4. When the drug was administered
`orally twice a day at doses of 0.625 and 1.25
`mgjkg per day, serum cholesterol and phospholi(cid:173)
`pid levels were reduced dose and time depen-
`
`Organs
`
`Inhibition(%)
`
`TABLE III
`
`CS-514
`93.1 ***
`94.8 ***
`29.8 *
`2.5
`0
`0
`10.4
`7.6
`7.8
`0
`0
`
`ML-236B
`89.5 ***
`86.5 ***
`80.6 ***
`51.4 **
`54.2 ***
`12.2
`66.2 ***
`54.2 ***
`45.0
`38.5
`27.9
`
`Liver
`Ileum
`Kidney
`Lung
`Spleen
`Cerebrum
`Prostate
`Testis
`Adrenal
`Muscle
`Skin
`Significantly different from control value: * P < 0.05; ** P <
`0.01; *** p < 0.001.
`
`MB-530B
`95.8 ***
`80.1 **
`71.1 **
`30.8
`43.1
`20.8
`70.7 ***
`60.2 ***
`78.7 **
`48.1
`12.2
`
`Hypolipidemic effect of CS-514 on various animal
`species
`As shown in Table III, CS-514 significantly
`decreased serum cholesterol levels in beagle dogs,
`cynomolgus monkeys, Japanese white rabbits and
`WHHL rabbits, an animal model of familial hy(cid:173)
`percholesterolemia in man [24,25], at doses of
`0.625-50 mgjkg per day. Serum phospholipid
`levels also decreased in dogs, rabbits and WHHL
`rabbits, whereas the levels of triacylglycerol were
`not reduced significantly in almost all the animals
`tested. On the other hand, CS-514 had no lower(cid:173)
`ing effect on serum cholesterol levels in rats, even
`at a dose as high as 500 mgjkg per day. The drug
`also showed no effect on serum cholesterol levels
`in mice and spontaneous hyperlipidemic Nagase
`analbuminemia rats [26] (data not shown). Among
`rats and mice, CS-514 exhibited an exceptionally
`hypolipidemic effect on Triton-induced hyper(cid:173)
`lipidemic rats which cannot metabolize serum
`lipoproteins and which show increased cholesterol
`synthetic activity
`in
`liver
`[17]. The serum
`cholesterol levels were reduced by 26% (P < 0.01)
`
`EFFECT OF CS-514 ON SERUM LIPIDS IN VARIOUS
`ANIMAL SPECIES
`
`Each animal group consists of: A and C, 3 males and 3
`females; B, 2 males and 2 females; D and E, all males. CS-514
`was administered twice a day (9.30 a.m. and 4.30 p.m.) at the
`indicated doses, except for rats which received the drug once a
`day (9.30 a.m.). The drug was administered by: A, a gelatin
`capsule; B, nasogastric intubation; C and E, gastric intubation;
`D, oral administration, as described previously [27]. The initial
`values of serum lipids in each animal group were obtained
`from the average value of at least three point assays.
`
`Dose
`Percent of initial value (mean± S.D.)
`(mgjkg per day) total cholesterol phospholipid triacylglycerol
`
`A. Beagle dog
`(18 days, n = 6)
`Control
`0.625
`1.25
`
`96± 5
`88± 5 b
`82± 6 c
`
`96± 4
`88± 6 b
`64± 6 b
`
`103±10
`83± 8 b
`89±14
`
`B. Cynomolgus monkey
`(18 days, n = 4)
`Control
`20
`50
`
`96± 4
`85± 6 a
`69±11 b
`
`C. Japanese white rabbit
`(18 days, n = 6)
`Control
`6.25
`12.5
`
`96± 9
`78± 10 b
`68± 11 b
`
`D. WHHL rabbit
`(12 days, n = 4)
`Control
`12.5
`50
`
`100±20
`82± 5 c
`72± 9 c
`
`E. Wistar-Imamichi rat
`(14 days, n = 8)
`500
`
`118 *
`
`85± 8
`87± 9
`84±11
`
`110±37
`96± 7
`94±15
`
`96± 3
`84± 7 b
`73± 7 b
`
`94±35
`79±18
`77±33
`
`93± 4
`88± 5
`84± 5 b
`
`108±13
`92±11
`107± 10
`
`101 *
`
`69 *
`
`Significantly different from control value: a P < 0.05; b P <
`0.01; c p < 0.001.
`* The values represent percent of control.
`
`
`
`Mylan Exhibit 1039, Page 6
`
`

`
`56
`
`Phospholipid
`
`Triglyceride
`
`:_3 I 00
`:~ ....
`'*-
`
`0
`
`80
`
`70
`
`0 4
`
`II
`
`18 25 32 39
`
`0 4
`
`II
`
`25 32 39
`18
`Days
`
`0 4
`
`II
`
`18
`
`25 32 39
`
`Fig. 4. Effect of CS-514 on serum lipid levels
`in beagle dogs. Experimental conditions are
`described in Table III. CS-514 was adminis(cid:173)
`tered orally at doses of 0.625 (L>.) and 1.25 (0)
`mgjkg per day for 18 days, respectively, and
`control animals (0) received placebo caps(cid:173)
`ules. The data represent average values of
`percent of initial values for six animals at
`each point. Percent of the initial value of
`treated groups is significantly different from
`that of the control group at each point as
`indicated: * P < 0.05; ** P < 0.01; *** P <
`0.001.
`
`dently; the decreases in cholesterol on day 18 were
`12 and 18%, respectively. The reduced levels of
`cholesterol and phospholipid recovered to pre(cid:173)
`treatment levels in about 2 weeks after dicontinua(cid:173)
`tion of drug administration. The effect of CS-514
`on serum triacylglycerol levels was unclear be(cid:173)
`cause of its inconsistently in control animals
`throughout
`the experimental period. In other
`animals, including monkeys, rabbits and WHHL
`rabbits, CS-514 also reduced serum cholesterol
`and phospholipid levels time and dose depen(cid:173)
`dently, and the reduced levels were restored within
`2 weeks after termination of the drug administra(cid:173)
`tion (data not shown).
`Table IV shows the effect of oral administra(cid:173)
`tion of CS-514 on lipoprotein cholesterol in dogs,
`monkeys and WHHL rabbits at the indicated
`doses and periods. In dogs, serum cholesterol levels
`(P < 0.01) and LDL
`were
`lowered by 29%
`cholesterol levels preferentially decreased by 89%
`(P < 0.001) compared with
`those of control
`animals. Although HDL cholesterol levels were
`also reduced, the atherogenic index (A.I.) was
`lowered significantly by 78% as compared with
`that of the control group. In the case of monkeys,
`VLDL and LDL cholesterol levels were signifi(cid:173)
`cantly reduced by 71 and 36%, respectively, while
`HDL cholesterol levels were not affected. A.I. of
`treated monkeys was lowered by 40% (P < 0.01).
`In WHHL rabbits, VLDL,
`IDL and LDL
`cholesterol levels were significantly reduced by 54,
`46 and 27%, respectively, whereas HDL cholesterol
`
`levels were not affected. A.I., which is very high in
`WHHL rabbits, was also reduced by 38%.
`In all the animal experiments described above,
`the effect of CS-514 on the lipid composition of
`lipoproteins was as follows (data not shown): (i)
`the ratio of triacylglycerol to whole lipids in VLDL
`relatively increased by about 10-20%, because the
`decrease of cholesterol and phospholipid was more
`prominent than that of triacylglycerol. The lipid
`composition of other lipoproteins including IDL,
`LDL and HDL was not significantly affected. (ii)
`The ratio of free to total cholesterol in each lipo(cid:173)
`protein fraction was not significantly changed,
`suggesting
`that
`this drug did not alter
`the
`cholesterol esterification.
`
`Effect on biliary lipids in dogs
`Table V shows the effect of CS-514 on the
`biliary lipids in beagle dogs which received the
`drug at doses of 12.5 and 50 mgjkg per day for 5
`weeks. Although the amounts of phospholipid and
`bile acid were not altered significantly, those of
`cholesterol and total neutral sterols were reduced
`by 40% (P < 0.01) and 37% (P < 0.02), respec(cid:173)
`tively.
`The biliary bile acid composition was not
`greatly .affected by the drug treatment except for a
`slight decrease of chenodeoxycholic acid. The lith(cid:173)
`ogenic index was significantly and dose depen(cid:173)
`dently reduced by 21 and 44% at doses of 12.5 and
`50 mgjkg per day, respectively, suggesting that
`the drug had an antilithogenic activity.
`
`
`
`Mylan Exhibit 1039, Page 7
`
`
`
`
`
`

`
`57
`
`TABLE IV
`EFFECT OF CS-514 ON LIPOPROTEIN CHOLESTEROL LEVELS IN VARIOUS ANIMAL SPECIES
`
`The male beagle dogs received CS-514 in· a gelatin capsule by gavage twice a day (9.30 a.m. and 4.30 p.m.). The experimental
`condition of cynomolgus monkeys was the same as in Table III. Three male and three female WHHL rabbits received CS-514 by oral
`administration once a day as described previously [27]. The preparation method of lipoprotein is described in Materials and Methods.
`Each value represents mean± S.D. A.l. (atherogenic index) was calculated from the following formula; ((VLDL cholesterol( C))+ (IDL
`C)+ (LDL C))/((HDL C)+ ( d > 1.21 C)).
`
`Lipoprotein cholesterol mgjdl)
`
`VLDL
`
`IDL
`
`LDL
`
`HDL
`
`d > 1.21
`
`Total
`
`A. I.
`
`Beagle dog
`(20 mgjkg per day, 35 days, 11 = 5)
`1.5 ± 0.3
`control
`0.9± 0.6
`treated
`
`10.1± 1.0
`1.1± 0.9
`(P < 0.001)
`
`77.2±5.6
`59.8±8.0
`(P < 0.01)
`
`4.2±0.8
`4.2±0.4
`
`92.7± 6.1
`66.0± 8.2
`(P < 0.001)
`
`0.143± 0.010
`0.031 ± 0.023
`(P < 0.001)
`
`%decrease
`
`40
`
`89
`
`23
`
`0
`
`29
`
`78
`
`Cynomolgus monkey
`(50 mgjkg per day, 18 days, 11 = 4)
`2.1± 2.0
`control
`0.6± 1.0
`treated
`
`73.1± 13.7
`46.9± 2.8
`(P < 0.01)
`
`40.2±9.1
`42.1±2.6
`
`2.2±0.9
`2.7±0.4
`
`117.6± 20.9
`92.3± 5.3
`(P < 0.05)
`
`1.79 ± 0.25
`1.06 ± 0.06
`(P < 0.001)
`
`%decrease
`
`70
`
`36
`
`-5
`
`-23
`
`22
`
`40
`
`WHHLrabbit
`(50 mgjkg per day, 8 weeks, 11 = 6)
`98±28
`118 ±31
`control
`53±19
`54 ±12
`treated
`(P < 0.01)
`(P < 0.001)
`
`533 ±127
`389 ± 48
`(P < 0.05)
`
`6.4±0.9
`7.0±2.3
`
`5.5 ± 1.2
`6.4±2.1
`
`760 ±152
`509 ± 59
`(P < 0.01)
`
`63
`±13
`39
`±13
`(P < 0.01)
`
`%decrease
`
`54
`
`46
`
`27
`
`-9
`
`-16
`
`33
`
`38
`
`Other effects
`A concentration of more than 100 p,gjml of
`CS-514 was required to cause growth inhibition of
`cultured cells such as mouse L cells and human
`skin fibroblasts. However, this effect was com(cid:173)
`pletely counteracted by addition of a small amount
`of mevalonic acid, a product of hydroxymethyl(cid:173)
`glutaryl-CoA reductase (data not shown). A 20-
`times higher concentration of CS-514 than the I 50
`value of sterol synthesis in human skin fibroblasts
`was required to cause 50% inhibition of coenzyme
`Q synthesis, one of the branched pathway prod(cid:173)
`ucts of mevalonic acid (Shimada, Y. and Tsujita,
`Y., unpublished result). In mouse L cells, CS-514
`had no effect on the synthesis of DNA, RNA,
`protein, triacylglycerol, phospholipid and fatty
`acid even at a 1700-times higher concentration of
`
`I 50 for cholesterol synthesis (data not shown). In
`terms of ketogenesis, the drug neither inhibited
`hydroxymethylglutaryl-CoA lyase from rat liver at
`a 4000-times higher concentration of I 50 for hy(cid:173)
`droxymethylglutaryl-CoA reductase, nor affected
`the blood ketone body levels in rats which were
`given CS-514 at a dose of 500 mgjkg per day for
`14 days (data not shown).
`In the long-term administration of CS-514 to
`dogs (2 years), the appearance of estrus in female
`animals was not affected, in spite of a 30-40%
`decrease in serum cholesterol levels (Masuda et
`al., unpublished result).
`In all animals administered CS-514, body weight
`gain and food intake were not affected during the
`experimental periods.
`
`
`
`Mylan Exhibit 1039, Page 8
`
`

`
`58
`
`TABLE V
`
`EFFECT OF CS-514 ON BILIARY LIPID IN BEAGLE DOGS
`
`The beagle dogs (3 males and 3 females) received CS-514 in a gelatin capsule by gavage once a day (9.30 a.m.) at doses of 12.5 and 50
`mgjkg per day for 5 weeks, respectively. Bile lipids were determined as described in Materials and Methods. The lithogenic index
`was calculated from the following formula: neutral sterol (mol)j(phospholipid (mol)+ bile acid (mol)). n.d., not detected.
`
`Dose (mgjkg)
`
`control
`
`53.3
`(100)
`
`567.5
`(100)
`103.7
`(100)
`
`671.2
`(100)
`
`104.9
`(100)
`
`4.7
`14.0
`5.9
`71.1
`4.3
`
`±
`
`5.3 *
`
`±131.5
`
`± 54.9
`
`±178.8
`
`± 33.3
`
`±
`±
`±
`±
`±
`
`2.1
`6.2
`3.3
`6.4
`0.9
`
`12.5
`
`46.9
`(88)
`
`432.7
`(78)
`104.5
`(101)
`
`538.2
`(80)
`
`107.8
`(103)
`
`±
`
`6.8
`
`± 81.6
`
`± 34.1
`
`± 110.8
`
`± 10.5
`
`6.3
`21.3
`2.8
`63.2
`6.4
`
`2.4
`±
`8.0
`±
`5.9
`±
`± 18.9
`3.8
`±
`
`50
`
`44.4
`(83)
`
`± 7.5
`
`±58.3
`338~1
`(60, p < 0.01)
`82.4
`±30.6
`(80)
`
`420.5
`±85.8
`(63, p < 0.02)
`
`118.4
`(113)
`
`5.2
`14.5
`n.d.
`75.9
`3.9
`
`±12.8
`
`± 1.1
`± 5.6
`
`± 5.3
`± 1.0
`
`0.0057± 0.0002
`(100)
`
`0.0045± 0.001