562
`
`Microsomal triglyceride transfer protein (MTP) inhibitors: Discovery of
`clinically active inhibitors using high-throughput screening and parallel
`synthesis paradigms
`George Chang*, Roger B'Ruggeri & H James Harwood Jr
`
`Address
`Departments of Metabolic Diseases and Medicinal Chemistry
`Pfizer Global Research and Development
`Eastern Point Road
`Groton
`CT06340
`USA
`Email:! george_c_chang@ groton.pfizer.com
`
`*To whom correspondence should be addressed
`
`Current Opinion in Drug Discovery & Development 2002 5(4):562-570
`© PharmaPress Ltd ISSN 1367-6733
`
`The inhibition of microsomal triglyceride transfer protein (MTP)
`blocks the hepatic secretion of very low density lipoproteins
`(VLDL)
`and
`the
`intestinal
`secretion of chylomicrons.
`Consequently,
`this mechanism provides a IJighly efficacious
`pharmacological target for the lowering of low density lipoprotein
`(LDL) cholesterol and reduction of postprandial lipemia. The
`combination of these effects could afford unprecedented benefit in
`the treatment of atherosclerosis and consequent cardiovascular
`disease. The promise of this therapeutic target has attracted
`widespread interest in the pharmaceutical industry. Independent
`efforts have yielded strikingly similar series of lipophilic amide
`inhibitors. The way in which the evolutionary paths of distinct
`inhibitor series have tended to converge through the course of
`robotics-assisted synthesis efforts is illustrated with candidates
`from Bristol-Myers Squibb and Pfizer. Hanging in the balance
`with the exceptional potency of the compounds presented are the
`potential adverse effects due to blockage of intestinal fat absorption
`and hepatic lipid secretion. Finding a degree of efficacy that can be
`safely tolerated defines the dilemma surrounding the advancement
`of these compounds to clinical practice.
`
`Keywords Apolipoprotein A1, apolipoprotein B, cholesterol,
`lipid
`transfer
`inhibition, MTP,
`HepG2 cells, LDL,
`triglycerides, VLDL
`
`Abbreviations
`AST
`Aspartate aminotransferase
`AL T
`Alanine aminotransferase
`apo
`Apolioprotein
`CETP
`Cholesteryl ester transfer protein
`CHD
`Coronary heart disease
`ER
`Endoplasmic reticulum
`FH
`Familial hypercholesterolemia
`HDL
`High density lipoprotein
`HTS
`High-throughput screening
`LDL
`Low density lipoprotein
`MTP
`Microsomal triglyceride transfer protein
`VLDL
`Very low density lipoprotein
`WHHL
`Watanabe heritable hyperlipedemic
`
`Introduction
`Cardiovascular disease remains the leading cause of death
`in industrialized nations and, as such, accounted for 950,000
`or 41% of all deaths in the US in 1998 [1•]. Coronary heart
`
`disease (CHD), as a consequence of atherosclerosis, is the
`most common cause of cardiovascular morbidity and
`mortality, with an estimated 12 million people suffering
`from CHD in the US alone [1•]. Elevated total and low
`density lipoprotein (LDL) cholesterol are both accepted
`primary risk
`factors
`for atherosclerosis
`[1•,2•,3]. An
`estimated 101 million US adults have elevated blood
`cholesterol (> 200 mg/dl) and are candidates for LDL
`cholesterol lowering through dietary intervention [1•,4,5•].
`Of these, 41 million are considered high risk, having blood
`cholesterol > 240 mg/ dl, and drug therapy is recommended
`[1•,4,5•].
`
`that elevated
`shown
`studies have
`Epidemiological
`triglycerides and reduced high density lipoprotein (HDL)
`cholesterol are also contributing factors for the development
`of CHD [2•,3,6-8]. Among the adult US population, 19%
`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, one which is present in 45
`to 50% of men with CHD, is atherogenic dyslipidemia
`[11,12]. which includes borderline high-risk LDL cholesterol
`(eg, 130 to 159 mg/dl), elevated triglycerides, small dense
`LDL particles and low HDL cholesterol.
`
`Although the HMG-CoA reductase inhibitors (statins) are
`effective in lowering LDL cholesterol, and somewhat
`effective in reducing triglycerides, they have only minimal
`effects on HDL cholesterol [2•,5•,13 .. ,14••.15]. Indeed,
`although numerous clinical trials have demonstrated that
`LDL cholesterol reduction can significantly reduce the risk
`of CHD, a great number of treated subjects who achieve
`substantial LDL cholesterol reduction still experience a
`clinical event [2•,3,13 .. ,14 .. ,15,16•.17•,18]. Therefore, with
`the goal of developing a therapy for treating patients
`with dyslipidemias
`that extends beyond primary
`hypercholesterolemia, the pharmaceutical industry has
`targeted inhibition of microsomal triglyceride transfer
`protein (MTP) as a mechanism for red!.JCing not only
`plasma total and LDL cholesterol, but also plasma very
`low
`density
`lipoprotein
`(VLDL)
`cholesterol
`and
`triglycerides.
`
`MTP, which is located within the lumen of the endoplasmic
`reticulum (ER) in hepatocytes and absorptive enterocytes, is
`a heterodimeric protein consisting of a 97-kDa subunit,
`which confers all of the lipid transfer activity of the
`heterodimer, and a 58-kDa multifunctional protein disulfide
`isomerase [19••]. MTP plays a pivotal, if not obligatory role
`in
`the assembly and secretion of
`triglyceride-rich,
`apolioprotein B (apoB)-containing lipoproteins (VLDL and
`chylomicrons) from
`the
`liver and intestine, and also
`catalyzes the transport of triglycerides, cholesteryl ester and
`phospholipids between membranes [19 .. ,20,21]. Although
`
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`
`

`
`MTP inhibitors Chang et a/563
`
`the exact role of MTP in the assembly of apoB-containing
`lipoproteins is still under investigation [21,22,23•], MTP is
`proposed to transport lipids from the ER membrane to the
`growing apoB polypeptide chain in the lumen of the ER.
`allowing proper translocation and folding of apoB to occur
`[19 ... 20-22,23•,24 .. ]. Hence,
`inhibition of MTP should
`reduce plasma lipids by preventing triglyceride-rich, apoB(cid:173)
`containing lipoprotein assembly in the liver and intestine.
`
`The initial suggestion that MTP inhibition could be a viable
`lipid-lowering therapy came with the discovery that
`functional MTP
`is
`absent
`in
`individuals with
`abetalipoproteinemia, a genetic disorder characterized by
`low plasma cholesterol and triglycerides, due to a defect in
`the assembly and secretion of apoB-containing lipoproteins
`[25 ... 26 .. ]. A similar phenotype is observed in MTP
`knockout mice [23•,27 .. ]. However, abetalipoproteinemia
`represents an extreme example of MTP inhibition and is
`not without its clinical sequalae, all of which presumably
`are
`related directly or
`indirectly
`to fat malabsorption
`(steatorrhea), vitamin malabsorption, and hepatic and intestinal
`steatosis [26 .. ,28]. A less severe, and probably more relevant
`example of the consequences of therapeutic MTP inhibition is a
`related genetic disease, hypobetalipoproteinemia, caused by
`mutations in apoB [29]. Heterozygous individuals with this
`disease possess half of the normal levels of apoB-containing
`lipoproteins, lack the clinical signs and symptoms of
`abetalipoproteinemia and have a prolonged lifespan [24 .. ].
`
`robotics(cid:173)
`High-throughput screening and
`assisted parallel synthesis for identification of
`potent developmental candidates
`To identify potent MTP inhibitors, various members of the
`pharmaceutical industry have devised relatively similar
`two-stage, empirical screening protocols for compound
`evaluation. The primary difference is the order of in vitro
`versus cell culture evaluation. At Pfizer, for example, in the
`first stage of the protocol, compounds were evaluated for
`their ability to inhibit apoB but not apoA1 secretion from
`HepG2 cells in a high-throughput, 96-well multiplexed
`format [30 .. ]. In the second stage of the protocol, confirmed
`apoB secretion inhibitors were evaluated for their ability to
`inhibit the MTP-mediated transfer of radiolabeled triolein
`from synthetic phospholipid donor liposomes to acceptor
`liposomes [30,..]. Using this two-stage screening protocol,
`Pfizer scientists identified compound 1 (Figure 1) as a potent
`inhibitor of apoB secretion (IC50 = 200 nM) but not of apoA1
`secretion
`[31 .. ].
`Inhibition of apoB
`secretion was
`determined to be through the action of inhibiting MTP (rat
`MTP IC50 = 250 nM).
`
`With the rather simple structure of compound 1, Pfizer
`scientists pursued a robotics-assisted parallel synthesis
`strategy as a means of exploring alternatives for the 4-toluidine
`moiety, with the goal of improving potency. Unfortunately,
`the biphenyl carboxylic acid moiety of compound 1 was not
`suitable for automated organic synthesis. Attempts to
`activate the acid group for amide formation resulted in
`exclusive formation of fluorenone 2 (Figure 1). With the
`intent of deactivating
`the aromatic
`ring and,
`thus,
`suppressing fluorenone formation, the 4'-trifluoromethyl-2-
`biphenyl carboxylic acid 3 (Figure 1) was utilized. In this
`case, amide formation was uneventful and the resulting
`
`compound 4 (Figure 1) remained a potent inhibitor of apoB
`secretion (IC 50 = 170 nM) and MTP activity (rat MTP IC 50 = 90
`nM). Using a novel parallel synthesis paradigm, compound
`5 (Figure 1) was identified (rat MTP IC 50 = 30 nM). Removal
`of the Boc protecting group resulted in a tetrahydro(cid:173)
`isoquinoline, which served as an advanced template for
`further parallel synthesis. Using
`this paradigm, 500
`compounds were prepared in a period of 4 months, leading
`to the discovery of amide 6 (Figure 1; rat MTP IC 50 = 7 nM).
`
`Despite improved inhibitory activity in vitro, compound 6
`displayed only weak triglyceride lowering activity when
`administered orally to rats. This lack of efficacy was
`presumed to be due to poor solubility and rapid
`clearance. Using in vitro hepatic microsomal clearance as
`a guide, followed by screening for triglyceride lowering
`in vivo, CP-346086 (7, Pfizer Inc; Figure 1) was ultimately
`identified as a potent (IC 50 = 2.0 nM) and orally efficacious
`MTP inhibitor [31••.32 ... 33,101].
`
`Bristol-Myers Squibb also reported success in identifying
`potent and efficacious MTP inhibitors via a similar high(cid:173)
`throughput screening
`(HTS) and parallel synthesis
`paradigm [24 .. ]. HTS of their chemical library yielded
`compound 8 (Figure 2). which inhibited MTP activity
`(IC 50 = 2.2 f.1M) and HepG2 cell apoB secretion (IC 50 = 1.8
`f.1M) with no effect on apoA1 secretion (IC 50 > 30 f.1M). The
`fluorene analog 9 (Figure 2) was also identified by HTS,
`but its MTP inhibitory activity was much weaker (IC 50 =
`36 f.1M). Proposing that the fluorenyl moiety overlaps
`with the diphenylmethyl moiety of compound 8, 'hybrid'
`analogs of 8 and 9 were prepared, yielding compounds
`with significantly improved MTP inhibitory activity, eg,
`compound 10 (Figure 2; MTP IC 50 = 36 nM). Modification
`of the isoindolone moiety of compound 10 revealed that
`benzamide 11 (Figure 2; MTP IC 50 = 23 nM) was a suitable
`replacement. Optimization of compound 11 was carried
`out by an automated organic synthesis paradigm to yield
`BMS-201038 (12, Figure 2; MTP IC 50 = 0.5 nM) [24•,102].
`Of particular interest is the similarity of this molecule to
`the Pfizer series
`in which
`the 4'-trifluoromethyl-2-
`biphenyl carboxylic acid moiety was also found to aid
`optimal MTP inhibitory activity.
`
`Convergent development of structure-activity
`relationships across the industry
`Inhibition of MTP as a means of treating dyslipidemia is being
`pursued by many other pharmaceutical companies, including
`Bayer [34,35••,103], Boehringer Ingelheim [104], Glaxo Group
`Ltd [105], Janssen Pharmaceutica [106], Japan Tobacco [107],
`Meiji Seika Kaisha Ltd [108]. Norvartis [36 .. ,109] and
`Wakunaga Pharmaceutical Co Ltd [110]. Representative
`structures from some of these pharmaceutical companies are
`It is worth noting
`shown
`in Figure 3.
`that
`the 4'(cid:173)
`trifluoromethyl-2-biphenyl carboxylic acid moiety is a re(cid:173)
`occurring group in many of these structures.
`
`In vitro efficacy and mechanism of action
`studies
`Mechanistic studies, using a variety ofMTP inhibitors that have
`entered
`into development, have provided
`consistent
`information regarding the mechanism of action of this class
`of lipid lowering agents. Developmental prototypes, such as
`
`CFAD Ex. 1015 (2 of 9)
`
`

`
`564 Current Opinion in Drug Discovery & Development 2002 Vol 5 No 4
`
`Figure 1. Strategy followed by Pfizer, leading to the synthesis of CP-346086.
`
`+
`
`H2N-R
`
`OH
`
`+
`
`OH
`
`3
`
`Robotics-assisted
`synthesis
`
`O CH3
`
`.&
`
`H2N
`
`------~
`
`Robotics-assisted
`synthesis
`
`~CH3
`
`N
`
`N
`H
`
`4
`
`Rn
`~~~s;>
`N~
`
`H
`
`6
`
`_.R
`N
`H
`
`0
`
`2
`
`5
`
`7 CP-346086
`(Pfizer)
`
`(13,
`BAY-13-9952
`and
`BMS-201038
`CP-346086,
`implitapide, Bayer AG; BMS-201038 and BAY-13-9952 {13,
`implitapide, Bayer AG; Figure 3), all potently and dose(cid:173)
`dependently
`inhibited human and/ or rodent MTP(cid:173)
`mediated triglyceride transfer between synthetic donor
`and acceptor liposomes with IC 50 values of 2 to 10 nM
`[24••,31 .. ,32 .. ,33,35 .. ]. CP-346086 also inhibited human
`MTP-mediated transfer of cholesteryl oleate between
`donor and acceptor liposomes with an IC 50 value of 1.9
`nM, indicating the ability of this compound to equally
`inhibit transfer of both neutral lipids [32 .. ,33]. However,
`CP-346086 did not inhibit cholesteryl ester
`transfer
`protein (CETP) activity at concentrations of up to 10 J.lM.
`Not only does this indicate this compound's specificity
`for inhibition of MTP-mediated neutral lipid transfer, but
`it also demonstrates
`its
`lack of effect on
`the
`physicochemical properties of the donor and acceptor
`vesicles [32 .. ,33].
`
`As a consequence of their inhibition of MTP-mediated
`triglyceride transfer, CP-346086, BMS-201038 and BAY-
`13-9952 also inhibited HepG2 cell apoB secretion and
`
`triglyceride secretion with IC50 values of 1 to 3 nM
`[24 .. ,32 .. ,33,35 .. ,37] without concomitant inhibition of
`apoA1 secretion
`[24 .. ,32 .. ,33,35 .. ,37] or cholesterol,
`fatty acid or triglyceride synthesis [32 .. ,33].
`
`In vivo efficacy and mechanism of action
`summary
`The lipid lowering and anti-atherosclerosis effects of MTP
`inhibitors have been consistently observed and broadly
`demonstrated across all series evaluated using a wide
`variety
`of
`representative
`animal models. The
`pharmacological effects observed with all MTP inhibitors
`evaluated are consistent with their mechanism of action.
`
`The effect of MTP inhibition on plasma triglyceride levels
`is rapid. For example, a single dose of CP-346086
`administered orally to rats or mice lowered plasma
`triglycerides in a dose-dependent manner, exhibiting an
`ED 30 of 1.3 mg/kg in both species 2 h after administration
`[32 .. ,33]. Consistent with its mechanism, CP-346086 did
`not simultaneously lower plasma cholesterol levels after
`single dose administration [32 .. ,33].
`
`CFAD Ex. 1015 (3 of 9)
`
`

`
`Figure 2. Synthesis of BMS-201038.
`
`MTP inhibitors Chang et a/565
`
`9
`
`Robotics-assisted
`synthesis
`
`10
`
`l
`
`12 BMS-201 038
`(Bristol-Myers Squibb)
`
`11
`
`The reduction of plasma triglycerides in animal models
`by MTP inhibitors is due to inhibition of secretion of
`apoB-containing lipoproteins. For example, in studies in
`which mice were treated with Triton WR-1339 to prevent
`lipoprotein lipase-mediated triglyceride hydrolysis and,
`hence, VLDL and chylomicron clearance from plasma,
`single-dose administration of CP-346086
`inhibited
`triglyceride secretion from the liver in fasted animals,
`indicating that the acute triglyceride lowering described
`above was an effect of the compound on hepatic
`triglyceride secretion
`rather
`than on clearance of
`triglyceride-rich particles from the circulation [32 .. ,33).
`Similar conclusions were also drawn from studies in rats
`with BMS-201038
`[24 .. ] and
`in Watanabe heritable
`hyperlipidemic
`(WHHL)
`rabbits with BAY-13-9952
`[34,35••). BMS-201038 and CP-346086 also
`inhibited
`lipoprotein secretion in fed rats, indicating their ability to
`inhibit both hepatic and intestinal lipoprotein secretion
`[24 .. ,32 ... 33).
`
`Dose-dependent cholesterol lowering was observed in
`several animal species after multiple-day administration.
`For example, after 2-week oral administration to rats, CP-
`346086 (10 mg/kg/day) lowered plasma triglycerides
`{62%), total cholesterol (23%), VLDL cholesterol (33%)
`and LDL cholesterol (75%)
`[32 ... 33]. Similarly, oral
`administration of BAY-13-9952 to obese fa/fa Zucker rats
`for 4 weeks at a dose of 0.5 mg/kg/day lowered plasma
`triglycerides (84%) and cholesterol {30%), with 80%
`reduction in plasma cholesterol noted at a dose of 5
`mg/kg/day
`[35 .. ].
`In other studies, 1-week oral
`administration of BMS-201038 to golden Syrian hamsters
`dose-dependently decreased plasma triglycerides (35%).
`
`total cholesterol {59%), VLDL + LDL cholesterol (51%),
`and HDL cholesterol (66%) at a dose of 3 mg/kg [24 .. ].
`
`triglycerides and
`BAY-13-9952 also reduced plasma
`cholesterol in normolipidemic dogs, with 4-week oral
`treatment at a dose of 4 mg/kg reducing plasma
`cholesterol levels by 60% [35 .. ]. Similar efficacy in dogs
`was also noted with the Novartis MTP inhibitor 17
`(Novartis; Figure 3) [36 .. ] and with CP-346086 [32 .. ,33].
`Studies conducted with orally administered BMS-201038
`(2 mg/kg)
`in
`cynomolgus macaque monkeys
`demonstrated 50% lowering of total cholesterol after 7
`days of treatment [37].
`
`The effects of MTP inhibitors were also investigated in
`homozygous WHHL rabbits, an animal model in which
`statins have minimal effects. These rabbits have hepatic
`LDL receptor activity that is < 5% that of normal rabbits,
`levels of LDL
`resulting
`in dramatically elevated
`cholesterol [24••]. Hence, these rabbits are a model for
`human homozygous familial hypercholesterolemia (FH),
`where levels of LDL cholesterol are also very high due to
`a non-functional or missing LDL receptor [24 .. ]. Studies
`with BAY-13-9952 administered at 12 mg/kg/day for 4
`weeks led to plasma total cholesterol and triglyceride
`reductions of 70 and 45%, respectively, conditions under
`which the hepatic VLDL secretion rate was decreased by
`80% [34). BMS-201038 also showed efficacy in the WHHL
`rabbit, demonstrating an ED 50 value for total plasma
`cholesterol and triglyceride lowering of 1.9 mg/kg and a
`complete normalization of atherogenic apoB-containing
`lipoprotein particles at a dose of 10 mg/kg [24 .. ].
`
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`
`566 Current Opinion in Drug Discovery & Development 2002 Vol 5 No 4
`
`Figure 3. MTP inhibitors.
`
`F F
`
`F
`
`F F
`
`~.~~~~0
`
`H3C
`
`13 BAY-13-9952
`(Bayer)
`
`14
`(Boehringer lngelheim)
`
`(GiaxoSmithKiine)
`
`F F
`
`F
`
`F F
`
`?xaV~o
`
`H3C
`
`0
`
`0
`
`16
`(Janssen)
`
`F F
`
`F
`
`::::,.,_
`
`0
`
`~slf~
`~ N-N
`CH 3
`CH3
`
`Y( CH
`
`3
`
`17
`(Novartis)
`
`18
`(Wakunaga Pharmaceuticals)
`
`Recently, the anti-atherosclerotic and plaque stabilizing
`potential of MTP
`inhibitors were studied by Bayer
`[35 .. ,38,39,40••]. In apoE knockout mice fed a Western-type
`diet containing BAY-13-9952 for 14 weeks at doses ranging
`from 1 to 15 mg/kg/day, in addition to reductions in
`plasma total cholesterol and triglycerides of up to 68 and
`61%, respectively, significant reductions in atherosclerotic
`lesions and
`lesion
`lipid content were also observed
`[35••,38,40 .. ]. The average cross-sectional plaque areas of
`the
`aortic
`root,
`determined
`by
`computer-aided
`morphometric analysis, were reduced by up to 93%, and
`lipid content was reduced by up to 99% [35 .. ,38,40 .. ]. This
`reduction in lesion development and lesion lipid content
`translated to a dose-related increase in survival time such
`that, while only one of 25 untreated mice was still alive after
`18 months, up to 24 of 25 mice were still alive after 18
`months of treatment with BAY-13-9952 [35 .. ,40 .. ]. It is also
`interesting to note that significant reductions in plaque area
`(66%) and lipid moieties within the plaque (55%) were
`observed at the 1 mg/kg-dose, where plasma cholesterol
`and triglycerides were unaffected [40 .. ]. In addition, orally
`administered BAY-13-9952 (12 mg/kg/day) reduced fatty
`streak formation to control levels in New Zealand White
`rabbits fed a 0.5% cholesterol-enriched diet for 3 months
`[39].
`
`that delayed
`Finally, because of increasing evidence
`lipemia
`is an
`clearance of postprandial
`important
`contributing factor to the development of atherosclerosis
`[41], the effect of the MTP inhibitors on postprandial lipemia
`was studied. In both rats and dogs, compound 17 (I mg/kg)
`administered just prior to an oral fat load, effectively
`prevented the elevation of plasma triglycerides [36 .. ]. This
`that MTP
`observation provides convincing evidence
`
`inhibition in the intestinal mucosa can result in substantial
`reduction of apoB-containing lipoprotein particle assembly
`and/or release, and can effectively attenuate postprandial
`lipemia.
`
`Clinical efficacy of MTP inhibitors
`CP-346086 showed evidence of activity consistent with its
`mechanism of action. When administered as a single oral
`dose to healthy human volunteers, CP-346086 reduced
`plasma triglycerides and VLDL cholesterol in a dose(cid:173)
`dependent manner, with ED 50 values of 10 and 3 mg.
`respectively, and maximal inhibition (100 mg) of 66 and 87%
`when measured 4 h after treatment [32 .. ,33]. In a 2-week,
`multiple-dose, safety and
`toleration study in healthy
`volunteers, CP-346086 (30 mg) administered at bedtime,
`produced an average decrease in plasma total and LDL
`cholesterol of 47 and 68%, respectively, relative to either
`individual baseline values or placebo, with little change in
`HDL cholesterol [32••.33]. Plasma triglycerides were also
`decreased by up
`to 75%
`immediately after dose
`administration, but the reduction was transient [32••.33].
`
`Similar efficacy was reported for BAY-13-9952, which
`produced a dose-dependent decrease in total cholesterol
`(45%), LDL cholesterol (55%) and triglycerides (29%) after 4
`weeks of treatment at an oral dose of 160 mg/day [42••].
`BMS-201038 also showed similar efficacy in phase I and
`phase II clinical trials [43].
`
`In addition to direct effects on plasma lipid levels, MTP
`inhibitors have the potential to attenuate postprandial
`lipemia and, thus, favorably affect atherogenesis over and
`above their effects mediated through reduced steady state
`
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`
`MTP inhibitors Chang et al567
`
`lipid levels [36 .. ]. Indeed, BAY-13-9952 administered to
`healthy volunteers at 160 mg/day resulted in almost total
`inhibition of the postprandial hyperlipemia following a high-fat
`meal [35 .. ,44•]. thus providing convincing evidence that MTP
`inhibition in the intestinal mucosa results in substantial
`reduction of apoB-containing lipoprotein assembly and release.
`
`Concerns regarding potential for adverse
`effects associated with MTP inhibition
`The major developmental issues confronting MTP inhibition
`relate to the potential for adverse effects associated with
`mechanism-based steatorrhea,
`fat-soluble vitamin
`(eg,
`vitamin A. vitamin E and vitamin K) malabsorption, and fat
`accumulation in the liver and intestine [28]. Such adverse
`findings are observed in patients with abetalipoproteinemia
`[26••.28] and, thus, have the potential to occur with MTP
`inhibition, especially if the degree of inhibition is marked or
`the exposure to inhibitor is prolonged. In general, however,
`the preclinical adverse effect profiles of MTP inhibitors have
`been promising and related mainly to gastrointestinal
`disturbances
`(presumably associated with steatorrhea
`and/or intestinal mucosal lipid accumulation) and liver
`function abnormalities (estimated by elevations of plasma
`aspartate aminotransferase (AST) and alanine aminotransferase
`(ALT) levels), which are presumably related to hepatic lipid
`accumulation.
`
`For example, after 2-week oral administration ofCP-346086 (10
`mg/kg/ day) to rats, which lowered plasma triglycerides
`and LDL cholesterol by 62 and 75'fo, respectively. MTP
`inhibition led to increases in both liver and intestinal
`triglyceride content when administered in close temporal
`proximity to eating [32 ... 33]. However, when dosed away
`from meals, only hepatic triglyceride levels were increased
`[32 ... 33]. Similarly, in rats, inhibitor 17 increased hepatic
`cholesterol and triglyceride content in a dose-dependent
`manner (0.5 to 5 mg/kg) that was correlated with plasma
`lipid lowering and was reversed after treatment withdrawal
`[36 .. ]. Furthermore, hamsters treated for 1 week with up to
`6 mg/kg/day of BMS-201038 did not have steatorrhea, as
`indicated by the absence of fat in their stools [24••].
`However, visual and biochemical
`evidence of
`fat
`accumulation in enterocytes and liver, which returned to
`control levels after termination of treatment, were reported
`[24 .. ]. Similarly, WHHL rabbits treated with BAY-13-9952 (6
`mg/kg/ day) did not exhibit steatorrhea, but did show
`accumulation of both triglycerides and cholesterol in the
`liver after 4 weeks of treatment [34]. In this study, whereas
`hepatic cholesterol levels reached a plateau with increasing
`dose, hepatic triglyceride levels continued to increase in a
`dose-dependent manner
`throughout
`the dose
`range
`evaluated [34]. Likewise, histopathological examination of
`the small intestines of apoE knockout mice treated for 14
`weeks with BAY-13-9952
`to 15 mg/kg/day) also
`(1
`displayed a dose-dependent
`increase of cytoplasmic
`vacuoles in the enterocytes [35 ... 40 .. ].
`
`It is important to note, however, that although hepatic lipid
`levels were markedly increased with treatment, hamsters
`administered BMS-20 1038
`(6 mg/kg/ day) for 3 weeks
`showed minimal change in liver weight, and plasma AL T
`and AST
`levels did not rise significantly during the
`treatment period [24••]. Similarly, WHHL rabbits treated
`with BMS-201038 (10 mg/kg/day) for 2 weeks showed no
`
`in plasma AST or ALT
`alteration
`normalization of plasma lipids [24 .. ].
`
`levels, despite
`
`inhibitors,
`In clinical studies of a variety of MTP
`gastrointestinal toleration issues associated with steatorrhea
`were observed in phase I multi-dose studies [35 .. ,39,42••].
`particularly when the inhibitors were administered together
`with meals [32 .. ,33). For example, in clinical trials, BAY-13-
`9952 at doses of 80 and 160 mg/day, resulted in a high
`incidence of digestive adverse effects (mainly diarrhea)
`(35••.42 .. ]. However, using a regimen of dosing CP-346086
`away from meals circumvented similar issues [32 .. ,33).
`
`To date, no evidence for vitamin deficiencies have been
`noted in subjects treated with MTP inhibitors. Clinical
`studies with BAY-13-9952 at doses of up to 160 mg/day for 4
`weeks have demonstrated only minor changes in circulating
`vitamin A levels [45]. Changes in vitamin E levels paralleled
`LDL cholesterol lowering, with no significant changes in
`vitamin E/LDL or vitamin E/HDL ratio. Such maintenance
`of vitamin E/LDL and vitamin E/HDL ratios are consistent
`with a minimal effect of MTP inhibition on whole body
`vitamin E status in these studies [45].
`
`The impact of fat accumulation in the liver and intesti~e
`remains to be evaluated in the clinical setting, particularly m
`hepato-compromised patients and in patients suffering from
`diabetes or gastrointestinal abnormalities. In this regard, it is
`important to note that plasma AL T and AST levels were
`increased 3-fold above normal in 12 to 27% of patients
`receiving 80 mg/ day and 160 mg/ day doses of BAY -13-9952
`[42 .. ]. Similar AST and ALT elevations, of a magnitude
`sufficient to halt the development of BMS-201038, were also
`reported [43]. Whether these transaminase elevations are a
`consequence of hepatic lipid accumulation, as had been
`observed in experimental animals, or are structure-specific
`remains to be determined experimentally.
`
`Future directions
`Although MTP inhibitors have demonstrated impressive lipid
`lowering efficacy in clinical studies, potentially significant
`adverse effects surround this mechanism. While those related to
`steatorrhea or fat-soluble vitamin malabsorption are readily
`addressed by either drug administration away from meals or
`by co-administration of a vitamin admix, the effects of long(cid:173)
`term hepatic lipid accumulation must be evaluated more
`thoroughly prior to broad use of MTP inhibitors. Given their
`safety, efficacy, tolerability and wide acceptance, statins have
`raised the hurdles for successfully marketing MTP inhibitors, or
`any other future lipid lowering approach. Clearly, the ability to
`demonstrate a readily managed therapeutic index will be
`critical for the progression of inhibiting MTP as a viable chronic
`lipid lowering therapy.
`
`References to primary literature
`
`••
`•
`
`of outstanding interest
`of special interest
`
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`
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`•• Most recent summary of the recommendations of NCEP for
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`
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`•• A large clinical trial that demonstrated lipid lowering therapy
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`
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`•• A primary prevention trial showing that LDL lowering therapy reduces
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