BMC Cardiovascular Disorders
`
`Cl
`
`()
`BioMed Central
`
`Research article
`O~nAccess
`JTT-130, a microsomal triglyceride transfer protein (MTP) inhibitor
`lowers plasma triglycerides and LDL cholesterol concentrations
`without increasing hepatic triglycerides in guinea pigs
`Dimple Aggarwal1, Kristy L West1, Tosca L Zern 1, Sudeep Shrestha1,
`Marcela Vergara-Jimenez2 and Maria Luz Fernandez* 1
`
`Address: 1 Department of Nutritional Sciences, University of Connecticut, Storrs, CT, USA and 2Department of Nutritional Sciences, University of
`Sinaloa, Culiacan, Mexico
`
`Email: Dimple Aggarwal - dimple.aggarwal@uconn.edu; Kristy L West - kristy_west@hotmail.com; Tosca L Zern - toscal9@hotmail.com;
`Sudeep Shrestha - sudeep.shrestha@uconn.edu; Marcela Vergara-Jimenez - marveji@uas.uasnet.mx; Maria Luz Fernandez• - maria(cid:173)
`Iuz.fernandez@uconn.edu
`• Corresponding author
`
`Published: 27 September 2005
`
`BMC Cardiovascular Disorders 2005, 5:30 doi: I 0.118611471-2261-5-30
`
`This article is available from: http://www.biomedcentral.com/l 471-2261/5/30
`
`Received: 22 June 2005
`Accepted: 27 September 2005
`
`© 2005 Aggarwal et al; licensee BioMed Central Ltd.
`This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http'i/creatjvecommons orgllicenses/by/2 0),
`which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
`
`Abstract
`Background: Microsomal transfer protein inhibitors (MTPi} have the potential to be used as a
`drug to lower plasma lipids, mainly plasma triglycerides (TG}. However, studies with animal models
`have indicated that MTPi treatment results in the accumulation of hepatic TG. The purpose of this
`study was to evaluate whether JTT-130, a unique MTPi, targeted to the intestine, would effectively
`reduce plasma lipids without inducing a fatty liver.
`Methods: Male guinea pigs (n = I 0 per group) were used for this experiment. Initially all guinea
`pigs were fed a hypercholesterolemic diet containing 0.08 g/ I 00 g dietary cholesterol for 3 wk.
`After this period, animals were randomly assigned to diets containing 0 (control), 0.0005 or 0.0015
`g/ I 00 g of MTPi for 4 wk. A diet containing 0.05 g/ I 00 g of atorvastatin, an HMG-CoA reductase
`inhibitor was used as the positive control. At the end of the ]th week, guinea pigs were sacrificed
`to assess drug effects on plasma and hepatic lipids, composition of LDL and VLDL, hepatic
`cholesterol and lipoprotein metabolism.
`
`Results: Plasma LDL cholesterol and TG were 25 and 30% lower in guinea pigs treated with MTPi
`compared to controls (P < 0.05). Atorvastatin had the most pronounced hypolipidemic effects with
`a 35% reduction in LDL cholesterol and 40% reduction in TG. JTT-130 did not induce hepatic lipid
`accumulation compared to controls. Cholesteryl ester transfer protein (CETP) activity was
`reduced in a dose dependent manner by increasing doses of MTPi and guinea pigs treated with
`atorvastatin had the lowest CETP activity (P < 0.0 I). In addition the number of molecules of
`cholesteryl ester in LDL and LDL diameter were lower in guinea pigs treated with atorvastatin. In
`contrast, hepatic enzymes involved in maintaining cholesterol homeostasis were not affected by
`drug treatment.
`
`Conclusion: These results suggest that JTT-130 could have potential clinical applications due to
`its plasma lipid lowering effects with no alterations in hepatic lipid concentrations.
`
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`
`Background
`Microsomal triglyceride transfer protein (MTP) is a resi(cid:173)
`dent protein in the lumen of endoplasmic reticulum and
`is primarily responsible for transfer of triglycerides (TG)
`and other lipids from their site of synthesis in the endo(cid:173)
`plasmic reticulum into the lumen during the assembly of
`very low density lipoprotein (VLDL) [l]. VLDL produced
`by the liver are the major source ofLDL in plasma and ele(cid:173)
`vated levels ofLDL are associated with the development of
`atherosclerosis and cardiovascular disease
`(CVD).
`Increased total cholesterol and LDL cholesterol (LDL-C)
`are both considered primary risk factors for atherosclero(cid:173)
`sis [2,3]. To reduce CHD risk factors improvements in diet
`and exercise are primary recommendations however
`when plasma cholesterol concentrations reach a certain
`limit drug intervention is necessary. Statins, which are tar(cid:173)
`geted to 3-hydroxy-3-methylglutaryl coenzyme A (HMG(cid:173)
`CoA) reductase and are used extensively, are effective in
`lowering LDL-C, and somewhat effective in reducing
`plasma TG (4,5]. A number of studies done in the past
`have indicated that reduction in LDL-C values by using
`statins can significantly reduce the risk of CHD however a
`large population of patients still experience a clinical
`event (2,4,5]. Therefore, pharmaceutical companies are
`continuing to research other drug options to control
`hypercholesterolemia with the goal of developing a ther(cid:173)
`apy for treating patients with dyslipidemias. Microsomal
`triglyceride transfer protein inhibitor (MTPi) is one such
`option. It is believed that blocking MTP will not only
`reduce plasma total and LDL cholesterol (LDL-C) but also
`plasma VLDL and TG by affecting the packaging and secre(cid:173)
`tion of VLDL and chylomicrons. Certain animal and
`human studies [ 6, 7] have shown that the inhibition of
`MTP blocks the hepatic secretion ofVLDL and the intesti(cid:173)
`nal secretion of chylomicrons. Consequently, this mecha(cid:173)
`nism provides a highly efficacious pharmacological target
`for the lowering of LDL-C and reduction of postprandial
`lipemia. These effects could afford unprecedented benefit
`in the treatment of atherosclerosis and consequent cardi(cid:173)
`ovascular disease. The promise of this therapeutic target
`has attracted widespread interest in the pharmaceutical
`industry.
`
`This research study had a primary goal to evaluate
`whether (fIT-130), an MTPi reduces plasma cholesterol
`and triglyceride concentrations in male Hartley guinea'
`pigs. Since fIT-130 is mainly targeted to the intestine,
`another main objective of this study was to evaluate
`whether this MPfi resulted in less hepatic lipid accumula(cid:173)
`tion compared to other inhibitors [ 6, 7]. Guinea pigs were
`used as the animal model for this study because of their
`similarities to humans in terms of hepatic cholesterol and
`lipoprotein metabolism. Previous studies done in our lab(cid:173)
`oratory report that guinea pig serve as a good model for
`evaluating cholesterol lowering drugs [8-10].
`
`Methods
`Materials
`Reagents were obtained from the following sources. fIT-
`130, the MTPi tested was provided by Akros Pharma Inc
`(Princeton, NJ). Enzymatic cholesterol and TG kits, cho(cid:173)
`lesterol oxidase, cholesterol esterase and peroxidase were
`purchased from Roche-Diagnostics (Indianapolis, IN).
`Phospholipid and free cholesterol enzymatic kits were
`obtained from Wako Pure Chemical (Osaka, Japan).
`Quick-seal ultracentrifuge tubes were from Beckman
`(Palo Alto, CA). DL-hydroxy- [3-14CJ methyl glutaryl
`coenzyme A (1.81 GBq/mmol), DL- [5-3HJ mevalonic
`acid (370 GBq/mmol), cholesteryl- [1,2,6,7-3H] oleate
`(370 GBq/mmol), Aquasol, Liquiflor (toluene concen(cid:173)
`trate) and [1 4CJ cholesterol were purchased from DuPont
`NEN (Boston, MA). Oleoyl- [1-I4CJ coenzyme A (1.8
`GBq/mmol) and DL-3-hydroxy-3-methyl glutaryl coen(cid:173)
`zyme A were obtained from Amersham (Clearbrook, IL).
`Cholesteryl oleate, glucose-6-phosphate, glucose-6-phos(cid:173)
`phate dehydrogenase, nicotinamide adenine dinucleotide
`phosphate (NADP), sodium fluoride, Triton, bovine
`serum albumin and sucrose were obtained from Sigma
`Chemical (St. Louis, MO). Aluminum and glass silica gel
`plates were purchased from EM Science (Gibbstown, NJ).
`
`Diets
`Diets were prepared and pelleted by Research Diets (New
`Brunswick, NJ). Isocaloric diets were designed to meet all
`the nutritional requirements for guinea pigs. The four
`diets had identical composition except for the type and
`dose of tested drug as indicated in Table 1. The amount of
`cholesterol in the diets was adjusted to be 0.08 g/100 g, an
`amount equivalent to 600 mg/day in the human diet [ 11].
`
`Animals
`Forty male guinea pigs (Harlan Sprague-Dawley, Hills),
`weighing 250-300 g, were randomly assigned to either a
`control, low dose of MTPi (LDI), high dose of MTPi (HDI)
`or an atorvastatin (AT) treatment (n = 10/group) for 4
`weeks. Initially, all guinea pigs were fed the control diet
`for 3 weeks to raise plasma cholesterol concentrations.
`Two animals were housed per metal cage in a light cycle
`room (light from 0700-1900 h) and had free access to
`diets and water. Non-fasted guinea pigs were sacrificed by
`heart puncture after isoflurane anesthesia. Blood and liv(cid:173)
`ers were harvested for analysis and were stored at -80 ° C
`for further analysis. All animal experiments were con(cid:173)
`ducted in accordance with U.S. Public Health Service/U.S.
`Department of Agriculture guidelines. Experimental pro(cid:173)
`tocols were approved by the University of Connecticut
`Institutional Care and Use Committee.
`
`Lipoprotein isolation
`Plasma samples were collected from blood obtained by
`heart puncture from guinea pigs under anesthesia. A
`
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`Table I: Composition of Control, low dose of the inhibitor (LOI), high dose of the inhibitor (HDI} and atorvastatin diets
`
`Components
`
`Control%
`
`Soybean protein
`Methionine
`Sucrose
`Corn Starch
`Fat mixl
`Cellulose
`Guar gum
`Mineral Mix2
`Vitamin Mix2
`Cholesterol
`JTT-130
`Statin
`
`22.S
`o.s
`2S
`IS
`IS. I
`10
`2.S
`8.2
`I.I
`0.08
`0
`0
`
`LOI%
`
`22.S
`o.s
`2S
`IS
`IS.I
`10
`2.S
`8.2
`I.I
`0.08
`o.ooos
`0
`
`HDI%
`
`Atorvastatin %
`
`22.S
`o.s
`2S
`IS
`IS.I
`10
`2.S
`8.2
`I.I
`0.08
`O.OOIS
`0
`
`22.S
`o.s
`2S
`IS
`IS.I
`10
`2.S
`8.2
`I.I
`0.08
`0
`o.os
`
`I Fat mix for the diet contains olive oil-palm kernel oil-safflower oil (I :2: 1.8), high in lauric and myristic acids.
`2 Mineral and vitamin mix adjusted to meet NRC requirements for guinea pigs. Detailed composition of the vitamin and mineral mix has been
`reported elsewhere (Fernandez et al. I 992b).
`
`preservation cocktail of aprotonin, phenyl methyl sulfo(cid:173)
`nyl fluoride and sodium azide was added to plasma sam(cid:173)
`ples to minimize changes in lipoprotein composition
`during isolation. Plasma was aliquoted for LCAT and
`CEfP determinations, plasma lipid analysis and lipopro;
`tein isolation.
`''
`
`Lipoproteins were isolated by sequential ultracentrifuga(cid:173)
`tion [12] in a LE-SOK ultracentrifuge (Beckman Instru(cid:173)
`ments, Palo Alto, CA). VLDL was isolated at d = 1.006 kg/
`Lat 125,000 g at 15 °C for 19 h in a Ti-50 rotor. LDL was
`isolated at d = 1.019-1.09 kg/Lin quick-seal tubes at 15 ° C
`for 3 hat 200,000 gin a vertical Ti-65 rotor [13). LDLsam(cid:173)
`ples were dialyzed in 0.9 g/L sodium chloride-0.1 g/L eth(cid:173)
`ylene diamine tetra acetic acid (EDTA), pH 7.2, for 12 h
`and stored at 4 ° C for further analysis.
`
`Plasma and hepatic lipids
`Plasma samples were analyzed for cholesterol and TG by
`enzymatic methods [14]. Hepatic total and free choles(cid:173)
`terol and TG were determined according to the method by
`Carr et al. [15] following extraction of hepatic lipids with
`chloroform-methanol 2: 1. Cholesteryl ester concentra(cid:173)
`tions were calculated by subtracting free from total
`cholesterol.
`
`Lipoprotein characterization
`VLDL and LDL composition was calculated by determin(cid:173)
`ing free and esterified cholesterol [14], protein by a modr.
`ified Lowry method [16], and TG and phospholipids by
`enzymatic kits. VLDL apo B was selectively precipitated
`with isopropanol [17). The number of constituent mole(cid:173)
`cules of LDL was calculated on the basis of one apo B per
`particle with a molecular mass of 412000 kD[l8]. The
`molecular weights were 885.4, 386.6, 645 and 734 forTG>
`free and esterified cholesterol, and phospholipids, respec-
`
`tively [19]. LDL diameters were calculated according to
`Van Heek et al (20). HDL cholesterol was also determined
`according to Warnick et al, with a modification, which
`consisted of using 2 mol/L MgC12 for precipitation of apo(cid:173)
`B containing lipoproteins [ 13).
`
`Lecithin Cholesterol Acyltransferase (LCAT) and
`Cholesterol Ester Transfer Protein (CETP) determinations
`in plasma
`LCAT and CETP activities were determined according to
`Ogawa & Fielding [21). Physiological CETP activity was
`determined without inhibiting LCAT activity by measur(cid:173)
`ing the mass transfer of cholesterol ester between HDL
`and apo B containing lipoproteins. Samples were incu(cid:173)
`bated at 3 7 ° C for 6 h in a shaking water bath and total
`and free plasma cholesterol and HDL cholesterol were
`measured. LCAT activity was determined by mass analysis
`of the decrease in plasma free cholesterol between 0 and
`6 h at 37°C. Assays were carried out concurrently with
`measurements of CETP. Both of these methods have been
`well-standardized for guinea pig plasma (22].
`
`Hepatic microsome isolation
`Hepatic microsomes were isolated as described previously
`[8]. Briefly a microsomal fraction was isolated by two 25-
`min centrifugations at 10,000 g (JA-20 rotor, J2-2 l) fol(cid:173)
`lowed by ultracentrifugation at 100,000 gin a Ti-50 rotor
`at 4 ° C for 1 hour. Microsomes were resuspended in the
`homogenization buffer and centrifuged for an additional
`hour at 100,000 g. After centrifugation, microsomal pel(cid:173)
`lets were homogenized and stored at-70°C.
`
`Hepatic HMG-CoA reductase assay
`The activity of microsomal HMG-CoA reductase (E.C.
`1.1.1.34) was measured
`in hepatic microsomes as
`described by Shapiro et al. (23]. HMG-CoA reductase
`
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`activity was expressed as pmol of [1 4C] mevalonate pro(cid:173)
`duced per min per mg microsomal protein. Recoveries of
`[3H] mevalonate ranged from 60-90%.
`
`Hepatic Acy/ CoA Cholesteryl Acyltransferase (ACAT)
`activity
`Hepatic ACAT (E.C. 2.3.1.26) activity was measured by
`the incorporation of [ 14C] oleoyl CoA in cholesteryl ester
`in hepatic microsomes by preincubating 0.8-1 mg of
`microsomal protein per assay with 84 g/L albumin and
`buffer for microsomal isolation [24]. Recoveries of (3H]
`cholesteryl oleate were between 70-90%.
`
`Hepatic cholesterol 7 a-hydroxylase activity
`Cholesterol 7a-hydroxylase (E.C. 1.14.13. 7) activity was
`measured according to the method modified by Jelinik et
`al [25]. (14C] cholesterol was used as a substrate and deliv(cid:173)
`ered as cholesterol-phosphatidylcholine liposomes (1:8
`by weight) prepared by sonication. An NADPH-regenerat(cid:173)
`ing system (glucose-6-phosphate dehydrogenase, NADP,
`and glucose-6-phosphate) was included in the assay as a
`source of NADPH.
`
`700
`
`600
`
`c;
`:::- 400
`.c
`"" a; 300
`;;::
`
`100
`
`'
`'!
`
`Time (weeks)
`
`Figure I
`Weight gain of guinea pigs treated with control, low dose,
`high dose of JTT-130 and atorvastatin.
`
`Statistical analysis
`One-way analysis of variance (ANOVA) (SSPS for Win(cid:173)
`dows version 12) was used to evaluate significant differ(cid:173)
`ences among groups in regards to plasma and hepatic
`lipids, LDL composition, hepatic enzyme activities and
`LCAT and CEfP activities. The LSD post hoc test was used
`to evaluate the differences among groups. Data are pre(cid:173)
`sented as the mean± SD. Differences were considered sig(cid:173)
`nificant at P < 0.05.
`
`The changes in plasma TC values were mostly due to
`decreases in the cholesterol carried by LDL. LDL-C was
`also significantly decreased (24. 7% & 26.9%) by the MTPi
`diets tested. There were no major differences between
`these two doses for plasma lipid parameters except for
`VLDL-C, which were significantly higher when compared
`to the low dose of the drug. No significant differences
`were observed for HDL-C values with MTPi or with AT
`(Table 2).
`
`Results
`Plasma lipids and lipoproteins
`No difference in weight gain overtime was observed in
`guinea pigs fed the different test diets (Fig 1 ), indicating
`that animals consumed comparable amounts of their
`respective test diets. After feeding the test diets for a period
`of four weeks, blood was isolated and plasma was ana(cid:173)
`lyzed for cholesterol and TG concentrations. The two
`doses of MTPi evaluated, low dose (LDI) and high dose
`(HDI) decreased plasma total cholesterol values signifi(cid:173)
`cantly by 19.2% (P < 0.01) as compared to the control ani(cid:173)
`mals (Table 2). There was no significant difference
`between the two doses of MTPi tested. Atorvastatin, which
`was used as a positive control, led to a significantly robust
`decrease in plasma total cholesterol values of 46%, which
`was significantly different (P < 0.01) from the two MTPi
`doses used. Plasma TG values were also significantly lower
`in LDI (50.8%) and HDI ( 45.3%) when compared to their
`control counterparts whereas atorvastatin (AT) treatment
`resulted in the maximum decrease of plasma TG (Table
`2).
`
`LDL size and composition
`No significant effect of MTPi on the number of CE mole(cid:173)
`cules or on the size of LDL particle was found with any of
`the two doses tested as compared to their control counter(cid:173)
`parts (Table 3). ATtreatment reduced the number of ester(cid:173)
`ified cholesterol molecules ( 45%) as well as decreased the
`size ofLDL particle (30%) (Table 3).
`
`LCAT and CETP activities
`Table 3 also summarizes the activities of these two pro(cid:173)
`teins, which play a major role in the intravascular process(cid:173)
`ing of plasma cholesterol. There were no significant
`differences in LCAT activity when comparing MTPi or sta(cid:173)
`tin groups to the control group. However, HDI decreased
`the activity of CEfP, which was comparable to the atorv(cid:173)
`astatin treated group (P < 0.01).
`
`Hepatic lipids and enzymes
`No significant changes were found in hepatic total choles(cid:173)
`terol, free cholesterol, cholesteyl ester or TG values in any
`of the four treatments (Table 4). Results suggest that MTPi
`did not lead to lipid accumulation in the liver, as there
`
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`Table 2: Plasma total cholesterol (TC), triglycerides (TG), VLOL-C, LOL-C and HOL-C of guinea pigs fed a control diet, low dose MTPi
`(LOI), high dose MTPi (HOI) or atorvastatin
`
`Diets
`
`TC
`
`TG
`
`VLDL-C
`
`LDL-C
`
`HDL-C
`
`Control (I 0)
`LDI (10)
`HDI (9)
`Atorvastatin (9)
`
`146.9 ± 42.2•
`116.9± 29.7b
`116.1 ± 23.3b
`76.5 ± 29.8<
`
`135.8 ± I 18.9•
`66.7 ± 29.6b
`74.3 ± 31.?b
`49.4 ± 32.2<
`
`(mg/dL}
`8.1 ± 5.S•b
`3.7 ± 3.0•
`9.0 ± 8.6b
`2.9 ± 2.6•
`
`123.9 ± 43.5•
`93.3 ± 26.2b
`90.5 ± 28.8b
`59.4 ± 29.3<
`
`13.6 ± 4.5•
`18.5 ± 8.0•
`I 5.6 ± 7.1•
`14.1 ± 5.6•
`
`1 Data are presented as mean ± SD for the number of guinea pigs indicated in parenthesis. Numbers in a column with different superscripts are
`considered significantly different (P < 0.0 I) as determined by one way AN OVA and LSD as a post-hoc test
`
`Table 3: Number of molecules of cholesteryl ester (CE), LOL diameter and LCAT and CETP activities of guinea pigs fed a control diet,
`low dose MTPi (LOI), high dose MTPi (HOI) or atorvastatin.
`
`Diets
`
`CE molecules
`
`LDL diameter
`
`LCAT
`
`CETP
`
`Control (n = I 0)
`LDI (n= 10)
`HDI (n = 9)
`Atorvastatin ( n = 9)
`
`1993 ± 422•
`2072 ± 536•
`2026 ± 132.5•
`1080 ± 1092b
`
`nanometers
`16.47 ± 3.63•
`16.67 ± 3.05•
`16.58 ± 7.85•
`I 1.52 ± 2.69b
`
`(pmol/min.mg)
`
`19.6 ± 11.0•
`14.4 ± 6.4•
`14.6 ± 8.5•
`13.4 ± 10.9•
`
`36.I ± 12.5•
`31.0 ± 21.I•
`19.4 ± 13.0•b
`12.8 ± 4.2b
`
`1 Data are presented as mean ± SD for the number of guinea pigs indicated in parenthesis. Numbers in a column with different superscripts are
`considered significantly different (P < 0.01) as determined by one way ANOVA and LSD as post hoc test.
`
`Table 4: Hepatic total cholesterol (TC), free cholesterol (FC) cholesteryl ester (CE) and TG of guinea pigs fed a control diet, low dose
`MTPi (LOI), high dose MTPi (HOI) or atorvastatinl,
`
`Diets
`
`TC
`
`FC
`
`CE
`
`TG
`
`Control (n = I 0)
`LDI (n = 10)
`HDI (n = 9)
`Atorvastatin (n = 9)
`
`1.20 ± 0.53
`1.47 ± 0.40
`1.42 ± 0.66
`1.71±0.76
`
`(mg/g)
`
`1.00 ± 0.44
`1.24 ± 0.35
`0.98 ± 0.39
`1.31 ± 0.56
`
`0.20 ± 0.23
`0.23 ± 0.11
`0.44 ± 0.58
`0.40 ± 0.39
`
`16.2 ± 2.8
`13.9 ± 9.4
`13.5 ± 7.8
`18.8 ± 10.8
`
`1 Data are presented as mean ± SD for n = the number of guinea pigs indicated in parenthesis.
`
`was no significant difference between the two doses of
`MTPi tested with control or with atorvastatin group. Sim(cid:173)
`ilarly no significant changes were observed in any of the
`three regulatory hepatic enzymes involved, namely CYP7, .·
`ACAT and HMG-CoA Reductase (Table 5).
`
`Discussion
`In this study we were able to demonstrate that JTI-130,
`the MTPi tested has the potential to decrease the prime
`risk factors of cardiovascular disease, namely plasma TG
`and LDL-C concentrations in guinea pigs. The novelty of
`the drug
`tested
`is
`that
`there was no significant
`accumulation of lipid in the liver as seen in some other
`studies done with other MTPi (26,27].
`
`Drug treatment and hepatic lipids
`Previous studies evaluating MTPi have shown increase in
`the lipid content of the liver (7,26,27]. Chandler et al (7]
`treated Hep-G2 cells with CP-346086, another MTPi, for a
`period of two weeks. They reported that in addition to
`decreasing plasma TC, LDL-C, VLDL-C and TG values, this
`treatment also increased hepatic and intestinal TG when
`the MTPi was administered with food and when it was
`dosed away from meals, only hepatic TG were influenced.
`In contrast, the major finding of this study is that the MTPi
`tested did not lead to any fat accumulation in the liver as
`confirmed by no significant changes found in the hepatic
`lipid content as compared to their control counterparts.
`The main reason for these differences between MTPi could
`
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`
`Table 5: Hepatic HMG-CoA reductase, ACAT and cholesterol 7a-hydroxylase activities (CYP7} guinea pigs fed a control diet, low dose
`MTPi (LOI}, high dose MTPi (HDI} or atorvastatin'
`
`Diets
`
`HMG-CoA Reductase
`
`ACAT
`
`Control
`LDI
`HDI
`Atorvastatin
`
`1.8 ± 0.8
`2.7 ± 0.8
`2.5 ± 1.4
`3.4 ± 2.9
`
`(pmol/min.mg)
`0.8 ± 0.4
`2.2 ± 0.9
`3.5 ± 2.4
`4.4 ± 3.1
`
`I Data are presented as mean ± SD for n = 3-7 guinea pigs per group.
`
`CYP7
`
`1.7 ± 1.8
`1.2 ± 0.5
`0.6 ± 0.7
`0.7 ± 0.4
`
`be that the main target of JTI-130 was the intestine.
`Because of this, we speculate that due to MTP inhibition,
`less TG were transferred to the chylomicron particle being
`packaged in the intestine. As a result a lower concentra(cid:173)
`tion of TG was taken up by the hepatocytes through the
`chylomicron remnant. Thus the VLDL particles secreted
`from the liver had lower concentrations ofTG molecules
`due to the major inhibitory effect of the MTPi in the intes(cid:173)
`tine. Because there were no significant changes in hepatic
`cholesterol concentrations, we did not find any significant
`differences in hepatic enzyme activities. Similar to the
`study by Conde et al. [9] in atorvastatin treated guinea
`pigs with 0.015% atorvastatin, there were no significant
`differences in hepatic cholesterol concentrations when
`compared with a control group. However, significant dif(cid:173)
`ferences in hepatic esterified cholesterol were observed
`when guinea pigs were treated with a higher dose of the
`statin (0.05%) (9).
`
`Drug treatment and plasma lipids and lipoproteins
`Abetalipoproteinemia, a genetic disorder characterized by
`low plasma cholesterol and TG levels, is caused by a func(cid:173)
`tional deficiency of MTP. Absence of lipid transfer activity
`in the microsomes of abetalipoproteinemia patients
`established its pivotal function in lipoprotein assem(cid:173)
`bly[ 1). This finding led to the suggestion that MTP inhibi(cid:173)
`tion could be used as a possible lipid lowering therapy.
`Further evidence was obtained from a cell culture study in
`which researchers (28] proved that MTP is limiting in the
`production of apo B containing lipoproteins. Another
`study [6) further confirmed this finding using hetero(cid:173)
`zygous MTP knockout mice which had 20% less plasma
`total cholesterol levels compared to wild type mice fed
`high fat diets; however, they did not find any significant
`differences in plasma TG concentrations. In our study, wfi
`have demonstrated that animals treated with MTPi had
`not only lower plasma TC and LDL-C but also significant
`reductions in plasma TG. It is possible that the VLDL par(cid:173)
`ticle secreted by the liver was more readily catabolized and
`therefore there was less conversion to LDL, which contrib(cid:173)
`uted to the hypocholesterolemic effects of the MTPL
`Conde et al. (9) demonstrated that there was a significant
`
`reduction in plasma TG in guinea pigs treated with atorv(cid:173)
`astatin when compared to controls. This was partly
`explained by lower secretion of VLDL particles and by
`increases in the LDL receptor [9), which could have con(cid:173)
`tributed to the faster removal ofVLDL particles. A similar
`mechanism may have taken place with the MTPi. By
`blocking MTP, JTI-130 reduced the secretion ofVLDL par(cid:173)
`ticles, and therefore, the formation ofLDL in the plasma.
`
`There was a dose response in CETP activity with ITT-130,
`and in addition, guinea pigs treated with atorvastatin
`exhibited decreased activity of this transfer protein. The
`main function of CETP is to contribute to the reverse cho(cid:173)
`lesterol pathway by transferring cholesteryl ester from
`HDL to the apo B containing lipoproteins [29]. However,
`this action prolongs the residence time of CE in LDL and
`increases the possibility of its deposition in the arterial
`wall. Thus lower CETP activity has been associated with
`decreased atherogenesis in animal studies [30). Therefore
`the lowering of CETP activity by drug treatment can be
`considered beneficial.
`
`Results from this study indicate that JTI-130 has the
`potential to reduce the primary risk factors for coronary
`heart disease. While these results are quite promising,
`more studies are needed to clarify the possibility of
`adverse effects including steatorhea, fat malabsorption
`and fat-soluble vitamin absorption. Although the lipid
`lowering effects were not as pronounced as
`those
`observed with atorvastain, the doses of MTPi used in the
`current study were lower than the doses of atorvastatin. It
`is possible that using JTI-130 in combination with statins
`could reduce the wide array of adverse effects associated
`with reductase inhibitors (31). This study also demon(cid:173)
`strates that MTP inhibitor which is mainly targeted
`towards the intestine may open a new avenue for treat(cid:173)
`ment of hyperlipidemic patients who are at high risk for
`cardiovascular diseases.
`
`List of abbreviations used
`ACAT: acyl CoA cholesteryl acyl transferase; AT: atorvasta(cid:173)
`tin; CETP: cholesterol ester transfer protein; CHD: coro-
`
`Page 6 of 8
`(page number not for citation purposes)
`
`6 of 8
`
`PENN EX. 2264
`CFAD V. UPENN
`IPR2015-01836
`
`

`
`BMC Cardiovascular Disorders 2005, 5:30
`
`http://www.biomedcentral.com/14 71-2261 /5/30
`
`nary heart disease; CYP7: cholesterol 7a-hydroxylase;
`HDi: high dose of the inhibitor; HDL-C: HDL cholesterol;
`HMG-CoA; 3-hydroxy-3methyl glutaryl Conezyme A;
`LCAT: lecithin cholesterol acyltransferase; LDi: low dose
`of the inhibitor; LDL-C: LDL cholesterol; MTP: micro(cid:173)
`somal transfer protein; MTPi: microsomal transfer protein
`inhibitor; NADP: nicotinamide adenenine dinucleotide
`phosphate; TG: triglycerides; VLDL: very low density
`lipoprotein.
`
`Competing interests
`Authors received funding from Akros Pharma Inc. (Princ(cid:173)
`eton, NJ) to carry out the studies presented in this
`manuscript.
`
`Authors' contributions
`DA did the assays, wrote the manuscript and participated
`in the interpretation of data; KLW: assisted in the assays
`for plasma lipids, CETP and LCAT; TLZ: assisted in the
`determination of ACAT activity and participated in data
`interpretation, SS: assisted in taking care of guinea pigs,
`isolation of microsomes and data interpretation; MVJ
`assisted in the determination of CYP7 and in data inter(cid:173)
`pretation and MLF designed the experiment, evaluated the
`results, interpreted the data and participated in manu(cid:173)
`script preparation.
`
`Acknowledgements
`These studies were supported by Akros Pharma Inc, Princeton, NJ
`
`4.
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