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`CP-346086: an MTP inhibitor that lowers plasma cholesterol
`and triglycerides in experimental animals and in humans
`
`Charles E. Chandler, Donald E. Wilder, Judith L. Pettini, Yvette E. Savoy, Stephen F. Petras,
`1
`George Chang, John Vincent, and H. James Harwood, Jr.
`Department of Cardiovascular and Metabolic Diseases, Pfizer Global Research and Development, Groton
`Laboratories, Pfizer, Inc., Eastern Point Road, Groton, CT 06340
`
`Abstract A microsomal triglyceride transfer protein (MTP)
`inhibitor, CP-346086, was identified that inhibited both hu-
`man and rodent MTP activity [concentration giving half-max-
`imal inhibition (IC
`) 2.0 nM]. In Hep-G2 cells, CP-346086
`50
`inhibited apolipoprotein B (apoB) and triglyceride secretion
`(IC
` 2.6 nM) without affecting apoA-I secretion or lipid syn-
`50
`thesis. When administered orally to rats or mice, CP-346086
`lowered plasma triglycerides [dose giving 30% triglyceride
`lowering (ED
`) 1.3 mg/kg] 2 h after a single dose. Coadmin-
`30
`istration with Tyloxapol demonstrated that triglyceride lower-
`ing was due to inhibition of hepatic and intestinal triglyceride
`secretion. A 2 week treatment with CP-346086 lowered total,
`VLDL, and LDL cholesterol and triglycerides dose depen-
`dently with 23%, 33%, 75%, and 62% reductions at 10 mg/
`kg/day. In these animals, MTP inhibition resulted in in-
`creased liver and intestinal triglycerides when CP-346086 was
`administered with food. When dosed away from meals, how-
`ever, only hepatic triglycerides were increased. When admin-
`istered as a single oral dose to healthy human volunteers, CP-
`346086 reduced plasma triglycerides and VLDL cholesterol
`dose dependently with ED
`s of 10 mg and 3 mg, and maxi-
`50
`mal inhibition (100 mg) of 66% and 87% when measured 4 h
`after treatment. After a 2 week treatment (30 mg/day), CP-
`346086 reduced total and LDL cholesterol and triglycerides
`by 47%, 72%, and 75%, relative to either individual baselines
`or placebo, with little change in HDL cholesterol.
` Together,
`these data support further evaluation of CP-346086 in hyper-
`lipidemia.
`—Chandler, C. E., D. E. Wilder, J. L. Pettini, Y. E.
`Savoy, S. F. Petras, G. Chang, J. Vincent, and H. J. Harwood, Jr.
`CP-346086: an MTP inhibitor that lowers plasma cholesterol
`and triglycerides in experimental animals and in humans.
`J.
`
`Lipid Res. 2003.
` 1887–1901.
`44:
`
`Supplementary key words
`microsomal triglyceride transfer protein
`•
`lipid transfer inhibition
` very low density lipoprotein
` low density li-
`•
`•
`poprotein
` apolipoprotein B
` apolipoprotein A-I
` Hep-G2 cells
`•
`•
`•
`
`Cardiovascular disease remains the leading cause of death
`in industrialized nations and accounted for 950,000, or
`
`41%, of all deaths in the United States in 1998 (1). As a
`consequence of atherosclerosis, coronary heart disease
`(CHD) is the most common cause of cardiovascular mor-
`bidity and mortality, with an estimated 12 million people
`suffering from CHD in the United States alone (1). Ele-
`vated total and LDL cholesterol are both accepted pri-
`mary risk factors for atherosclerosis (1–3). An estimated
`101 million United States adults have elevated blood cho-
`⬎
`lesterol (
`200 mg/dl) and are candidates for LDL choles-
`terol lowering through dietary intervention (1, 4, 5). Of
`these, 41 million are considered high risk, having blood
`cholesterol greater than 240 mg/dl, and drug therapy is
`recommended (1, 4, 5).
`Epidemiological studies have shown that elevated tri-
`glycerides and reduced HDL cholesterol are also contrib-
`uting factors for the development of CHD (2, 3, 6–8).
`Among the adult United States population, 19% of people
`⬍
`have low HDL cholesterol (
`40 mg/dl) (3, 9, 10) and
`⬎
`21% have hypertriglyceridemia (
`150 mg/dl) (3, 10).
`Thus, as important as elevated LDL cholesterol is as a risk
`factor for CHD, it is important to recognize that the most
`common spectrum of lipid abnormalities is atherogenic
`dyslipidemia, which is present in 45–50% of men with
`CHD (11, 12) and includes borderline high-risk LDL cho-
`lesterol (e.g., 130–159 mg/dl), elevated triglycerides, small
`dense LDL particles, and low HDL cholesterol.
`The HMG-CoA reductase inhibitors (statins) are very
`effective in lowering LDL cholesterol and somewhat effec-
`tive in reducing triglycerides, but they have only minimal
`effects on HDL cholesterol (2, 5, 13–15). Indeed, al-
`though numerous clinical trials have demonstrated that
`LDL cholesterol reduction can significantly reduce CHD
`risk, a great number of treated subjects who achieve sub-
`stantial LDL cholesterol reduction still experience a clini-
`cal event (2, 3, 13–18). Therefore, with the goal of develop-
`
`Manuscript received 2 February 2003 and in revised form 3 June 2003.
`Published, JLR Papers in Press, July 1, 2003.
`DOI 10.1194/jlr.M300094-JLR200
`
`Abbreviations: CETP, cholesterol ester transfer protein; CHD, cor-
`onary heart disease; ER, endoplasmic reticulum; MTP, microsomal tri-
`glyceride transfer protein.
`1
` To whom correspondence should be addressed.
`e-mail: h_james_harwood@groton.pfizer.com
`
`Copyright © 2003 by the American Society for Biochemistry and Molecular Biology, Inc.
`
`This article is available online at http://www.jlr.org
`
`Journal of Lipid Research
`
`Volume 44, 2003
`
`1887
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`Wilder, and Moberly (34). In the second stage of the pro-
`tocol, confirmed apoB secretion inhibitors were evaluated
`for their ability to inhibit the MTP-mediated transfer of ra-
`diolabeled triolein from synthetic phospholipid donor li-
`posomes to acceptor liposomes (34). Using this two-stage
`screening protocol, we identified CP-94792, a potent in-
`hibitor of apoB, but not apoA-I, secretion (33). Inhibition
`of apoB secretion was subsequently determined to be
`through inhibition of MTP activity (33, 35). However, al-
`though CP-94792 inhibited Hep-G2 cell apoB secretion with
`an half-maximal inhibition (IC
`) of 200 nM and inhib-
`50
`ited MTP-mediated triglyceride transfer (rat MTP) with an
`IC
` of 250 nM, the compound displayed only weak triglyc-
`50
`eride lowering activity when administered orally to rats (33).
`The potent and orally efficacious MTP inhibitor, CP-
`⬘
`346086 (4
`-trifluoromethyl-biphenyl-2-carboxylic acid [2-(2H-
`[1,2,4]triazol-3-ylmethyl)-1,2,3,4-tetrahydro-isoquinolin-6-yl]
`Fig. 1
`amide);
`, inset), was ultimately identified (33, 35–
`1
`37) by
`) employing a robotics-assisted parallel synthesis
`strategy as a means of developing structure-activity rela-
`2
`tionships and improving in vitro potency, and
`) using in
`vitro hepatic microsomal clearance and in vivo triglycer-
`ide lowering as guides for improving pharmacokinetic
`properties. In this report, we describe the biochemical
`
`Fig. 1.
`Inhibition of human microsomal triglyceride transfer pro-
`tein (MTP)-mediated neutral lipid transfer by CP-346086. Aliquots
`of solubilized human liver MTP, 150 ␮l, were incubated at 37⬚C for
`45 min with 50 ␮l donor liposomes, 100 ␮l acceptor liposomes, and
`200 ␮l assay buffer containing either 5% BSA (control) or 5% BSA
`plus sufficient CP-346086 to produce the indicated final concentra-
`tions of CP-346086, as described in Experimental Procedures. After
`incubation, triglyceride transfer was terminated by addition of 300
`␮l of a 50% (w/v) DEAE cellulose suspension in assay buffer. After
`a 4 min agitation, the donor liposomes, bound to DEAE cellulose,
`were selectively sedimented by low speed centrifugation (3,000 g, 5
`min). An aliquot of the supernatant containing the acceptor lipo-
`somes was assessed for radioactivity as outlined in Experimental
`Procedures. Shown is the percentage of control [14C]triolein or
`[14C]cholesterol oleate transfer as a function of CP-346086 concen-
`tration. Control triolein and cholesterol oleate transfer during
`the 45 min assay averaged 42% and 13%, respectively. Inset: The
`structure of CP-346086 (4⬘-trifluoromethyl-biphenyl-2-carboxylic
`acid [2-(2H-[1,2,4]triazol-3-ylmethyl)-1,2,3,4-tetrahydro-isoquinolin-
`6-yl]amide).
`
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`ing a therapy for treating patients with dyslipidemias that
`extend beyond primary hypercholesterolemia, we targeted
`inhibition of microsomal triglyceride transfer protein
`(MTP) as a mechanism for preventing triglyceride-rich li-
`poprotein assembly in the liver and intestine.
`MTP, which is located within the lumen of the endo-
`plasmic reticulum (ER) in hepatocytes and absorptive en-
`terocytes, is a heterodimeric protein consisting of a 97
`kDa subunit, which confers all of the lipid transfer activity
`of the heterodimer, and the 58 kDa multifunctional pro-
`tein disulfide isomerase (19). MTP plays a pivotal, if not
`obligatory role, in the assembly and secretion of triglycer-
`ide-rich, apolipoprotein B (apoB)-containing lipoproteins
`(VLDL and chylomicrons) from the liver and intestine
`and also catalyzes the transport of triglycerides, choles-
`teryl esters, and phospholipids between membranes (19–
`21). Although the exact role of MTP in the assembly of
`apoB-containing lipoproteins is still under investigation
`(21–23), MTP is proposed to transport lipids from the ER
`membrane to the growing apoB polypeptide chain in the
`ER lumen, thereby allowing proper translocation and
`folding of apoB to occur (19–24). MTP has also been pro-
`posed to mediate bulk triglyceride transfer into the ER lu-
`men for incorporation into poorly lipidated apoB-con-
`taining lipoprotein particles during the process of VLDL
`and chylomicron assembly (25, 26). Recent studies have
`also suggested a role for MTP in the movement of choles-
`terol ester into the ER lumen for inclusion into nascent
`apoB-containing lipoprotein particles (27).
`The initial suggestion that MTP inhibition could be a vi-
`able lipid-lowering therapy came with the discovery that
`functional MTP is absent in individuals with abetalipopro-
`teinemia, a genetic disorder characterized by low plasma
`cholesterol and triglycerides due to a defect in the assem-
`bly and secretion of apoB-containing lipoproteins (28,
`29). A similar phenotype is observed in MTP knockout
`mice (23, 30). Abetalipoproteinemia, however, represents
`an extreme example of MTP inhibition and is not without
`its clinical sequelae, all of which presumably are related
`directly or indirectly to fat malabsorption (steatorrhea),
`vitamin malabsorption, and hepatic and intestinal steato-
`sis (29, 31). A less severe, and probably more relevant, ex-
`ample of the consequences of therapeutic MTP inhibition
`is a related genetic disease, hypobetalipoproteinemia,
`caused by mutations in apoB (32). Heterozygous individu-
`als with this disease possess half of the normal levels of
`apoB-containing lipoproteins, lack the clinical signs and
`symptoms of abetalipoproteinemia, and have a normal
`lifespan (24).
`We devised a two-stage empirical screening protocol for
`compound evaluation (33) to identify potent MTP inhibi-
`tors with the potential to inhibit hepatic and intestinal
`apoB-containing lipoprotein assembly and consequently
`lower plasma total, LDL, and VLDL cholesterol and tri-
`glycerides in experimental animals and in humans. In the
`first stage of the protocol, compounds were evaluated for
`their ability to inhibit apoB, but not apoA-I secretion,
`from Hep-G2 cells in a high-throughput, 96-well multi-
`plexed format, essentially as described by Haghpassand,
`
`1888
`
`Journal of Lipid Research
`
`Volume 44, 2003
`
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`mechanism of action of CP-346086 that leads to its LDL
`cholesterol-, VLDL cholesterol-, and triglyceride-lowering
`efficacy in experimental animals and in humans.
`
`EXPERIMENTAL PROCEDURES
`
`Materials
`14
`14
`Sodium [2-
`C]acetate (56 mCi/mmol), [
`C]triolein (110
`14
`3
`mCi/mmol), cholesteryl [1-
`C]oleate (55 mCi/mmol), [
`H]tri-
`3
`olein (25 Ci/mmol), [
`H]egg phosphatidylcholine (50 mCi/
`mmol), and Aquasol-2 were from New England Nuclear (Boston,
`3
`MA). [
`H]glycerol (20 Ci/mmol) was from American Radiochemi-
`cals (St. Louis, MO). Ready Safe™ liquid scintillation cocktail was
`from Beckman Instruments (Fullerton, CA). Dulbecco’s modified
`Eagle’s medium (DMEM),
`-glutamine, and gentamicin were from
`l
`GIBCO Laboratories (Grand Island, NY). Heat-inactivated fetal bo-
`vine serum was from HyClone Laboratories (Logan, UT). DEAE
`cellulose was from Whatman International (Maidstone, England).
`Silica gel 60C TLC plates were from Eastman Kodak (Rochester,
`NY). BCA protein assay reagent was from Pierce (Rockford, IL).
`Cholesterol/HP reagent (Cat. 1127578), TG/GPO reagent (Cat.
`1128027), Preciset Cholesterol Calibrator Set (Cat. 125512), and
`Precitrol-N serum (Cat. 620212) were from Boehringer Mannheim
`(Indianapolis, IN). Cholesterol CII reagent kit (Cat. 276-64909),
`Triglyceride E reagent kit (Cat. 432-40201), Standard Cholesterol
`CII Solution (Cat. 277-64939), and Standard Triglyceride G Solu-
`tion (Cat. 998-69831) were from Waco Chemicals USA (Richmond,
`VA). Hep-G2 cells were from the American Type Culture Collec-
`tion (Rockville, MD). Mouse anti-human apoB monoclonal anti-
`bodies (MoAB-012), goat anti-human apoB polyclonal antibodies
`(AB-742), mouse anti-human apoA-I monoclonal antibodies
`(MAB-011), goat anti-human apoA-I polyclonal antibodies (AB-
`740), and human apoA-I purified standard (ALP10) were from
`Chemicon (Temecula, CA). B6CBAF1J mice were from Jackson
`Laboratory (Bar Harbor, ME). Transgenic huA1/CIII/cholesteryl
`ester transfer protein (CETP) mice, originally obtained from
`Charles River (Boston, MA), were bred on site. Sprague Dawley rats
`were from Charles River. RMH 3200 laboratory meal was from Ag-
`way, Inc. (Syracuse, NY). AIN76A semipurified diet was from US
`Biochemicals (Cleveland, OH). F0739 liquid diet powder was from
`Bio-Serve, Inc. (Frenchtown, NJ). Fast protein liquid chromatogra-
`phy (FPLC) instrumentation was from Gilson, Inc. (Middletown,
`WI). Superose-6 gel filtration columns were from Pharmacia (Pis-
`cataway, NJ). Postcolumn reaction (PCX) instrumentation was
`from Pickering Laboratories (Mountain View, CA). All other chem-
`icals and reagents were from previously listed sources (38, 39).
`
`Preparation of human MTP
`The procedure for isolating and solubilizing human MTP
`from frozen hepatic tissue is based on the method of Wetterau
`and Zilversmit (40) and was conducted essentially as described
`by Haghpassand, Wilder, and Moberly (34). Solubilized human
`⬚
`liver MTP was stored at 4
`C and was diluted 1:5 with assay buffer just
`before use. MTP preparations showed no notable loss of transfer
`activity with storage up to 30 days. Rat and mouse MTP were pre-
`pared similarly, except that freshly isolated liver tissue was used.
`
`Preparation of phospholipid vesicles for use in lipid
`transfer assays
`Donor and acceptor liposomes were prepared essentially as
`described by Wetterau et al. (28). Donor liposomes were pre-
`pared under nitrogen by room temperature bath sonication of a
`␮
`␮
`dispersion containing 447
`M egg phosphatidylcholine, 83
`M
`
`␮
`14
`bovine heart cardiolipin, and 0.91
`M [
`C]triolein (110 Ci/mol).
`Acceptor liposomes were prepared under nitrogen by room tem-
`perature bath sonication of a dispersion containing 1.3 mM egg
`␮
`3
`phosphatidylcholine, 2.6
`M triolein, and 0.5 nM [
`H]egg phos-
`phatidylcholine in assay buffer. The donor and acceptor lipo-
`⬚
`g
`somes were centrifuged at 160,000
` for 2 h at 7
`C. The upper
`80% of the supernatant, containing small unilamellar liposomes,
`⬚
`was carefully removed and stored at 4
`C until use.
`
`Measurement of MTP inhibitory activity
`MTP inhibitory activity as outlined by Haghpassand, Wilder,
`␮
`and Moberly (34) was determined by adding 200
`l of a buffer
`containing either 5% BSA (control) or 5% BSA plus CP-346086 to
`␮
`␮
`a mixture containing 50
`l donor liposomes, 100
`l acceptor lipo-
`␮
`somes, and 150
`l of solubilized, dialyzed MTP protein diluted in
`⬚
`assay buffer as outlined above. After incubation at 37
`C for 45 min,
`␮
`triglyceride transfer was terminated by addition of 300
`l of a 50%
`(w/v) DEAE cellulose suspension in assay buffer. After 4 min of ag-
`itation, the donor liposomes, bound to DEAE cellulose, were selec-
`
`g; 5 min). An
`tively sedimented by low speed centrifugation (3,000
`aliquot of the supernatant containing the acceptor liposomes was
`14
`3
`assessed for radioactivity, and the
`C and
`H counts obtained were
`used to calculate the percent recovery of acceptor liposomes and
`the percent triglyceride transfer using first order kinetics.
`
`Measurement of Hep-G2 cell apoB and apoA-I
`secretion inhibition
`HepG2 cells were grown in DMEM containing 10% fetal bo-
`vine serum in 96-well plates in a humidified, 5% CO
` atmo-
`2
`ⵑ
`⬚
`sphere at 37
`C until
`70% confluent. CP-346086 was dissolved
`␮
`in DMSO, diluted to 1
`M in growth medium, serially diluted in
`growth medium to the desired concentration range, and added
`␮
`in 100
`l aliquots to separate wells of 96-well culture plates con-
`taining HepG2 cells. Twenty-four hours later, growth medium
`was collected and assessed for apoB and apoA-I concentrations as
`described by Haghpassand and Moberly (41).
`
`Measurement of Hep-G2 cell cholesterol and fatty
`acid synthesis
`Cholesterol and fatty acid synthesis were evaluated in Hep-G2
`14
`cells by measuring incorporation of [2-
`C]acetate into cellular
`lipids as previously described (38, 39), with the following modifi-
`cations to allow simultaneous assessment of both cholesterol and
`fatty acid synthesis. After collection and assessment of the hexane
`fraction containing cholesterol and nonsaponifiable lipids as pre-
`viously described (38, 39), the remaining aqueous phase (con-
`⬍
`taining fatty acid sodium salts) was acidified to pH
`2 by addi-
`tion of 0.5 ml of 12 M HCl. The resulting mixtures were then
`transferred to glass conical tubes and extracted three times with
`4.5 ml hexane. The pooled organic fractions were dried under ni-
`␮
`trogen, resuspended in 50
`l of chloroform-methanol (1:1; v/v),
`⫻
`and applied to 1
` 20 cm channels of Silica Gel 60C TLC plates.
`Channels containing nonradioactive fatty acids were included on
`selected TLC plates as separation markers. TLC plates were devel-
`oped in hexane-diethyl ether-acetic acid (70:30:2; v/v/v) and air
`dried, and the region of chromatograms corresponding to fatty
`acid mobility was removed and assessed for radioactivity in Aqua-
`sol-2 using a Beckmann LS6500 liquid scintillation counter. Cho-
`14
`lesterol and fatty acid synthesis are expressed as dpm [2-
`C]ace-
`tate incorporated into either cholesterol or saponifiable lipids
`⬚
`during the 6 h incubation at 37
`C per mg cellular protein.
`
`Measurement of HepG2 cell triglyceride synthesis
`and secretion
`HepG2 cells, grown in T-75 flasks as previously described (38,
`⫻
`5
`39), were seeded into 24-well plates at 4–6
`10
` cells/well and
`
`Chandler et al.
`
`Lipid-lowering efficacy of the MTP inhibitor CP-346086
`
`1889
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`model 715-system controller software running under Microsoft
`Windows. The mobile phase was 154 mM NaCl, 1 mM EDTA,
`0.02% sodium azide (pH 8.1) at a flow rate of 0.36 ml/min. The
`⫻
`cholesterol-PCX reagent, Cholesterol/HP, was prepared at 2
`concentration by adding deionized water. The triglyceride-PCX
`⫻
`reagent, TG/GPO, was prepared at 2
` concentration by adding
`two bottles of enzyme to one bottle of buffer. The sterile filtered
`PCX reagents were stored and used under nitrogen in the dark
`⬚
`at 4
`C at a flow rate of 0.06 ml/min. Preciset Cholesterol Calibra-
`tors, Standard Cholesterol CII, Standard Triglyceride G, Pre-
`citrol-N serum, fresh human plasma (EDTA), and unknown sam-
`⬚
`ples were serially diluted in mobile phase and placed in a 2
`C
`␮
`refrigerated rack for FPLC sampling. Aliquots of 50–500
`l were
`injected automatically. Plasma standards were included to com-
`pare peak areas obtained via direct PCX injection (e.g., Super-
`ose-6 column bypassed), with peak areas obtained after column
`fractionation via FPLC-PCX and as an intra/inter-assay control
`sample, which was used before, interdispersed with, and after the
`unknown samples. The split-column eluent was combined with
`the cholesterol or triglyceride reagent in the PCX module form-
`ing a reaction product that was measured spectrophotometri-
`⬚
`cally at 500 nm after an 11 min pass through the 37
`C reaction
`coil. The Gilson 715 software performed the analysis of the spec-
`trophotometer output. The peak start, peak end, and baselines
`were checked visually and adjusted as necessary for each stan-
`dard, control plasma, and sample prior to integrating the peak
`areas. In the case of unresolved peaks, a perpendicular was
`drawn from each peak valley to the horizontal baseline for deter-
`mining the peak area. A plot of peak area versus quantity of cho-
`lesterol injected was made for the cholesterol and triglyceride
`standards, and a least squares regression analysis was performed
`and then used to convert chromatographic peaks from unknown
`samples into cholesterol and triglyceride concentrations in units
`of mg/dl.
`
`Studies using experimental animals
`The Institutional Animal Care and Use Procedures Review Board
`approved all procedures using experimental animals. B6CBAF1J
`mice, mice hemizygous for the human genes encoding apoA-I,
`apoC-III, and CETP (huA1/CIII/CETP mice), or Sprague Daw-
`ley rats were given food (RMH3200 laboratory meal or AIN76A
`semipurified diet) and water ad libitum and treated orally at a
`volume of 1.0 ml/200 g body weight (rats) or 0.25 ml/25 g body
`weight (mice) with either an aqueous solution containing 0.5%
`methyl cellulose (vehicle) or an aqueous solution containing
`0.5% methyl cellulose plus CP-346086. In experiments in which
`CP-346086 was administered in the feed, animals were fed pow-
`dered RMH3200 laboratory meal for 1 week prior to commenc-
`ing administration of CP-346086 as a dietary supplement to pow-
`dered chow. In studies in which rates of hepatic and intestinal
`triglyceride secretion were determined after Tyloxapol treat-
`ment, Tyloxapol (1.0 ml; 125 mg/ml) was administered intrave-
`nously 60 min after oral administration of CP-346086. In experi-
`ments in which rates of intestinal triglyceride absorption were
`14
`determined in the absence of Tyloxapol, [
`C]triolein mixed in
`Bioserve F0739 liquid diet [17.7% (w/w) fat] at a concentration
`␮
`of 0.5
`Ci/ml was administered orally to fasted mice as a 0.5 ml
`aliquot.
`
`Human subjects and study design
`Protocols involving human subjects were approved by the In-
`stitutional Protocol Review Board of Pfizer, Inc., and by the Pro-
`tocol Review Board of the Food and Drug Administration. Forty-
`eight healthy male adults, aged 18–45 years, participated in the
`randomized, double-blind, placebo-controlled, escalating single-
`dose study, and 30 healthy male adults, aged 18–45 years, partici-
`
`⬚
`C for
` atmosphere at 37
`maintained in a humidified, 5% CO
`2
`48 h prior to use. At the beginning of each experiment, media
`were removed and replaced with fresh media containing 0.2%
`⫾
`␮
`DMSO
` CP-346086. One hour after compound addition, 25
`l of
`␮
`3
`media containing 50
`Ci of [
`H]glycerol was added to each incuba-
`tion well. Plates were then sealed with Parafilm to avoid evapora-
`⬚
`tion, and cells were incubated at 37
`C for 6 h with gentle shak-
`ing. After incubation, the media were removed, and the cells
`were washed three times with PBS and scraped from wells into
`PBS. Lipids were extracted from the media and the cell lysate
`⫻
`with chloroform-methanol (2:1; v/v) and applied to 1
` 20 cm
`channels of Silica Gel 60C TLC plates. Channels containing non-
`radioactive triglycerides were included on selected TLC plates as
`separation markers. TLC plates were developed in petroleum
`ether-diethyl ether-acetic acid (75:25:1; v/v/v) and air dried. The
`region of chromatograms corresponding to triglyceride mobility
`was removed and assessed for radioactivity in Aquasol-2 using a
`Beckman LS6500 liquid scintillation counter. Triglyceride syn-
`3
`thesis and triglyceride secretion are expressed as dpm [
`H]glyc-
`erol incorporated into cellular triglycerides or secreted into the
`⬚
`culture medium during the 6 h incubation at 37
`C per mg cellu-
`lar protein.
`
`Measurement of plasma cholesterol and triglyceride levels
`Plasma triglyceride and total cholesterol concentrations were
`determined using Cholesterol CII and Triglyceride E reagent kits
`and employing Standard Cholesterol CII Solution and Standard
`Triglyceride G Solution as calibration standards.
`
`Measurement of hepatic and intestinal cholesterol and
`triglyceride levels
`Hepatic and intestinal cholesterol and triglyceride levels were
`measured as previously described (42) with the following modifi-
`cations. Sections, 0.5 g, of hepatic or intestinal tissue were ho-
`⬚
`mogenized at 4
`C in 3.0 ml PBS using a Polytron tissue homoge-
`␮
`nizer. Aliquots, 200
`l, of the homogenates were transferred to
`15 ml screw-capped glass tubes containing 7.5 ml of a mixture of
`CHCl
`-MeOH (2:1; v/v) and mixed vigorously for 20 s. One mil-
`3
`liliter of ddH
`O was then added, and the resulting suspension
`2
`was mixed vigorously for 15 s then centrifuged at 3,000 rpm for 5
`min at room temperature in a Sorvall RT-6000 bench-top centri-
`fuge. The chloroform-methanol layer was removed, placed in a
`⫻
`13
` 100 mm test tube, and evaporated to dryness under nitro-
`⬚
`␮
`gen at 60
`C. The lipid residue was resuspended in 200
`l 1% Tri-
`ton X-100 in absolute EtOH, and the cholesterol and triglyceride
`concentrations were determined using Cholesterol CII and Tri-
`glyceride E reagent kits, adapted for colorimetric analysis in 96-
`well plate format.
`
`Plasma lipoprotein subfractionation and visualization by
`FPLC and PCX
`Plasma lipoproteins were separated on a single Superose-6 col-
`umn by FPLC, and the effluent was split into two equal streams.
`One stream was reacted postcolumn with cholesterol enzymatic
`assay reagents for the determination of lipoprotein cholesterol.
`The second stream was reacted postcolumn with triglyceride en-
`zymatic assay reagents for the determination of lipoprotein tri-
`glyceride. Lipoprotein FPLC utilized a single Gilson autoinjector
`and dual PCX setups, each consisting of a Pickering nitrogen
`pressurized reagent delivery system, a Gilson reagent pump, a
`Pickering CRX 400 PCX with a custom 2.8 ml knitted reaction
`coil, and a Gilson spectrophotometer set at 500 nm. The stream
`splitting method used one Gilson mobile phase pump, one Phar-
`⫻
`macia Superose-6 10
` 300 mm column, and multiple switching
`valves for controlling effluent stream splitting and PCX reagent
`flow. The entire system was computer controlled by Gilson
`
`1890
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`Volume 44, 2003
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`III concentrations by ELISA, CP-346086 inhibited Hep-G2
`cell apoB secretion with an IC50 of 2.0 nM (Fig. 2A). Un-
`der these conditions, neither apoA-I secretion (Fig. 2A)
`nor apoC-III secretion (not shown) were inhibited by CP-
`
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`
`Fig. 2.
`Inhibition of Hep-G2 cell apolipoprotein B (apoB) and
`triglyceride secretion by CP-346086. A: Hep-G2 cells seeded and
`maintained in culture as described in Experimental Procedures
`were incubated for 24 h at 37⬚C in supplemented DMEM plus the
`indicated concentrations of CP-346086. After incubation, the me-
`dium was removed and assessed for apoB and apoA-I concentra-
`tions by apolipoprotein-specific ELISAs as outlined in Experimen-
`tal Procedures. Data for apoB and apoA-I secretion are the mean of
`triplicate determinations ⫾ SD and are expressed as a percentage
`of control apolipoprotein secretion as a function of CP-346086 con-
`centration. B: Hep-G2 cells seeded and maintained in culture as de-
`scribed in Experimental Procedures were incubated for 6 h at 37⬚C
`in supplemented DMEM containing either 1% DMSO (control) or
`1% DMSO containing CP-346086 sufficient to produce the indi-
`cated final inhibitor concentrations. For measurement of triglycer-
`ide synthesis and secretion, cells also received 50 ␮Ci of [3H]glyc-
`erol (closed symbols). After incubation, the media was removed,
`the cells were washed three times with PBS, and the secreted (me-
`dia) and cellular triglycerides were quantitated as described in Ex-
`perimental Procedures. For measurement of cholesterol and fatty
`acid synthesis, cells also received 4 ␮Ci of [2-14C]acetate (open sym-
`bols). After incubation, newly synthesized cholesterol and fatty ac-
`ids were separated and quantitated as described in Experimental
`Procedures. Data for triglyceride secretion (closed circles), triglyc-
`eride synthesis (closed triangles), cholesterol synthesis (open dia-
`monds), and fatty acid synthesis (open triangles) are the mean of
`triplicate determinations ⫾ SD and are expressed as a percentage
`of control synthesis and secretion as a function of CP-346086 con-
`centration.
`
`pated in the 2 week randomized, double-blind, placebo-con-
`trolled, parallel-group multidose study. Inclusion criteria for
`both studies were body weight within 10% of ideal based on 1983
`Metropolitan Life Insurance Height and Weight Tables (43), fast-
`ing plasma cholesterol levels in the upper 50% of the normal
`range for the American population based on age, sex, and race
`(e.g., 180–250 mg/dl) (1), fasting plasma triglyceride levels
`⬎
`⬍
`150 mg/dl and
`400 mg/dl, no evidence of clinically signifi-
`cant disease based on complete medical history, full physical ex-
`amination, 12-lead ECG and clinical laboratory testing, and no
`prescription, OTC, or other drug usage for at least 2 weeks prior
`to beginning the study. Subjects were confined to the Clinical Re-
`search Unit and fed standardized meals (30% of daily calories
`from fat).
`In the single-dose study, after randomization (six subjects/
`group; eight groups) each group was assigned a CP-346086 dose
`ranging in half-log intervals from 0.1 mg to 300 mg. At 7 AM on
`the day of study, after fasting for at least 8 h, four subjects from
`each group received their respective doses of CP-346086, and two
`subjects received placebo. Plasma samples were obtained at vari-
`ous times over the next 72 h for use in determining total and li-
`poprotein cholesterol and triglyceride levels by FPLC (see above).
`In the multidose study, subjects were randomized with eight
`receiving 30 mg CP-346086 and six receiving placebo at 10 PM
`(bedtime) for 14 days. Plasma samples were obtained daily pre-
`⫺
`⬚
`dose, frozen at
`20
`C, and forwarded to Medical Research Labs
`(39 Excelsiorlaan Zaventem, Brussels, Belgium) for determina-
`tion of total and lipoprotein cholesterol and triglyceride levels
`using standardized autoanalyzer technology.
`
`RESULTS
`
`Inhibition of MTP-mediated lipid transfer by CP-346086
`When donor and acceptor liposomes, prepared as de-
`scribed in Experimental Procedures, were incubated with
`varying concentrations of inhibitor, CP-346086 dose depen-
`dently inhibited human MTP-mediated triglyceride trans-
`fer between vesicles with a concentration giving an IC
`50
`equal to 2.0 nM (Fig. 1). Similarly, when the radiolabeled
`triolein of donor vesicles was replaced by radiolabeled
`cholesteryl oleate, CP-346086 inhibited transfer of choles-
`teryl oleate between vesicles with an IC50 of 1.9 nM (Fig.
`1), demonstrating the compound’s ability to equally in-
`hibit transfer of both neutral lipids. Similar IC50 values
`were also noted for inhibition of rat (1.7 nM) and mouse
`(6.7 nM) MTP activity.
`Inhibition of neutral lipid transfer by CP-346086 was
`specific to MTP-mediated lipid transfer, however, as in an
`identical assay in which MTP was replaced by CETP, no in-
`hibition of CETP-mediated lipid transfer was observed at
`concentrations of CP-346086 up to 10 ␮M (data not
`shown), indicating the compound’s specificity for inhibi-
`tion of MTP-mediated lipid transfer and demonstrating
`the lack of an effect of CP-346086 on the physicochemical
`properties of the donor and acceptor vesicles.
`
`Inhibition of apoB and triglyceride secretion from
`Hep-G2 cells by CP-346086
`When Hep-G2 cells were incubated with various con-
`centrations of CP-346086 for 24 h at 37⬚C, and the culture
`media was evaluated for human apoB, apoA-I, and apoC-
`
`Chandler et al.
`
`Lipid-lowering efficacy of the MTP inhibitor CP-346086
`
`1891
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`Fig. 3.
`Inhibition of hepatic and intestinal triglyceride secretion
`by CP-346086. Sprague Dawley rats given RMH3200 laboratory meal
`and water ad libitum were fasted overnight and then