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`

`
`ED1'IDR—IN—CHIEF
`
`SCOTT M. GRU NDY
`
`LEWIS I. GIDEZ, EXECUTIVE EDITOR
`
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`INNERARITY
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`
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`
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`RAY C. PJTTMAN
`A. CATHARINE ROSS
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`GERALD SALEN
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`
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`
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`RANDALL D. WOOD
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`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2122 - 2/19
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`

`
`CONTENTS
`
`JOURNAL OF LIPID RESEARCH
`
`ezzzsra
`NOVEMBER 1992
`
`Amplified Titles
`
`i
`
`Special Article. The discovery and development of
`HMG-COA reductase inhibitors. A. Endo
`1569
`
`Familial apolipoprotein E deficiency and type III
`hypcrlipoproteinemia due to a premature stop
`eodon in the apolipoprotein E gene.
`P. Lohse,
`H. B. Brewer Ill, M. S. Meng, S. l. Skarlatos,
`C.
`l..aRosa, and H. B. Brewer, _]r.
`1533
`
`12ot~Hydroxylase activity in human liver and its
`relation to cholesterol 70¢-hydroxylase activity.
`K.Einarsson, _]-E. Akerluncl, E. Reihnér, and
`I. Bjorkheni
`1591
`
`Peroxisomal I3-oxidation of branched chain fatty
`acids in human skin fibroblasts. H. Singh, M.
`Brogan, D. Johnson, and A. Poulos
`1597
`
`Intestinal lipids and lipoproteins in the human fetus:
`modulation by epidermal growth factor.
`E. Levy,
`L. Thibault, and D. Menard
`1607
`
`Effects of dietary fat on cholesterol movement be-
`tween tissues in CBA{] and C5 7BR..I'cd'J' mice.
`S. I.
`Kuan,
`Stewart. M. K. Dowd, L. Patterson,
`Dupont, and R. C. Scagravc
`1619
`
`Sulfonate analogues of chenodeoxycholic acid:
`metabolism of sodium 30:, 7or-dil'1yd1‘oxy-25-homo-
`5,8-cholane-25-sulfonate and sodium 3oc,7c¢-
`dihydroxy-24-nor-5,3-cholane-23-sulfonate in the
`hamster.
`5. Miki, E. H. Mosbach, B. I. Cohen, M.
`
`Yoshii, N. Ayyad, and C. K. McSherry
`
`1629
`
`Transgenic mice expressing human apolipoprotein
`A-I have sera. with modest trypanolytic activity in
`vitro but
`remain susceptible to infection by
`Trypanosoma brucei bmcei.
`5. Owen, M. P. T.
`Gillett, and T. E. Hughes
`1639
`
`Identification and properties of the proline,—,,«,,-leucine
`mutant LDL receptor in South Africans of Indian
`origin. D. C. Rubinsztein, G. S. Coetzee, A. D.
`Marais, E. Leitersdorf, H. C. Seftel, and D. R. van
`der Wcsthuyzen
`1647
`
`Bisphosphonates used for the treatment of bone
`disorders inhibit squalene synthase and cholesterol
`biosynthesis.
`D. Amin, S. A. Cornell, S. K.
`Gustafson, S.
`Need1e,_]. W. Ullrich, G. E. Bilder,
`and M. H. Pcrrone
`1657
`
`Evidence for a common biliary cholesterol and
`VLDL cholesterol precursor pool
`in rat
`liver.
`B. G. Stone and C. D. Evans
`1665
`
`Effects of apolipoprotein E, .5-very low density lipo-
`proteins, and cholesterol on the extension of
`neurites by rabbit dorsal root ganglion neurons in
`vitro. G. E. Handelmann,
`K. Boyles, K. H.
`Weisgraber, R. W. Mahley, and R. E. Pitas
`1677
`
`Metabolism of high density lipopmtein lipids by the
`rat liver: evidence for participation of hepatic
`lipase in the uptake of cholesteryl ester. H.
`Kadowaki, G. M. Patton, and‘ S.
`Robins
`1689
`
`Effiux of lipid from Fibroblasts to apolipoproteins:
`dependence on elevated levels of cellular un-
`estei-ified cholesterol.
`J. K. Bielicki, W. _]._]ohnson,
`R. B. Weinberg, M. Glick, and G. H. Rothblat
`1699
`
`PAPERS ON METHODOLOGY
`
`High sensitivity negative ion GC—MS method for
`detection of desaturated and chain-elongated prod-
`ucts of deuterated linoleic and linolenic acids.
`
`R.
`1711
`
`Pawlosky, H. W. Sprccher, and N. Salem. Jr.
`
`An in vitro model for essential fatty acid deficiency:
`HepG2 cells permanently maintained in lipid-fnee
`medium.
`E. E. Furth, H. Sprecher, E. A. Fisher,
`H. D. Fleishman, and M. Laposata
`1719
`
`Author Index
`
`1727
`
`Announce ment
`
`172 7
`
`International Conference on HDL-Cholesterol and
`
`Triglycerides
`
`1723
`
`Polyunsaturated Fatty Acids!Eicosanoids and Anti
`oxidants in Biology and Human Diseases
`1728
`
`1st International Congress of the International
`Society for the Study of Fatty Acids and Lipids
`1728
`
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`
`JOURNAL OF LIPID RESEARCH
`
`\r’n|um:': 33 Nunlber ll
`
`Not-'embL-r I993
`
`AMPLIFIED TI'I"L|:".S
`
`Tltis it}! it desfgmd to aid the reader in warming the :'.tstte_for topt'r:'
`qf :'m‘em'!.
`TE: each title are added r’ceg'
`tttarris t'm2’:ltatt'ng topics deal’!
`will:
`in the paper but not sperffirafly raferrcd to in Mt” rifle‘.
`
`'ll-II-I DISCOVERY ASE! DI-;\'[-:I£ir'ME.'|-'T or H.’\/IG-CQA REDIJCTASE INHIBITORS. S1-"ECI/\]'_ AR‘['lt':l.[-'..
`HMG—CoA rerluctase
`'
`clmlcstcml
`-
`I.l')I.—t‘holesttt:rol
`-
`atherosclerosis
`
`A Etta?!
`
`FAMILIAL. APOl.lPl'Jl'-'kCrrI-iI.\'- E DEFICIENCY AND TYPE. Ill HYPERLll'-'tJPRCII'I-;I:‘€IiM|«\ DUE To A
`PRt'.:\m'rI.;RE Smv Clcmcm IN THE APDLIPIJPRUTEIN E Gt-zm-;
`
`atherosclerosis
`olism
`
`- dysbetalipnproteinentia
`
`lipnprolein metab-
`- nonsense mutation -
`1'? Lame, H. B. Hrdtvrf III, M, S. A-i'rr:g_.
`J’. S/rar!a.t‘u.r,
`C. 1.aRw'a, H. B. Bmwrr. fr.
`
`S.
`
`l2t}:-l‘lYDROX\’I_A$i-; ACTIVITY IN HI.=.\im~: LIVER A.\5[!|
`Hvokoxvmsa Activity
`
`ITs RELATION ‘I0 Ct-ior.Es.1't3RtJt_ 7:1-
`
`tcliolcstyramint:
`
`- gallstone disease
`
`intestinal resection
`-
`K. Et'rmr.r.m::, “I-5. Aalwr/ttrid, E. Reialtnéfi
`
`I. B;I't5r/(Item
`
`PI-L|{(JX1SOMAL .8—OxtL:.a.T1on: m-' BRANCI-IE!) CHAIN FM"n' Acms IN Humtm SKIN ]'”|tsRoBL.As'rs
`adrenolcukudystrophy - Zellwegcr syndrome
`-
`rhizome.-lie chondrodysplastia punc-
`tata
`-
`rnit0chon(.l1‘l.'1
`'
`per(Jxisr.mIt.‘.s
`H.
`.S'fn_._gI1, A-I. Brogan, £1 Jofinson. A. H-Jz.tt’n.s
`
`II~"r|3s'1'Ii\‘_Ai_ I.|I=tns AND LIPQPROTEINS IN HIE Hl.'M.-‘\N FE'rL.'s: Mont-=L..-\T[o:\' BY EPIDERMAL
`GROWTH F_atc'rtJR
`
`jejunal trxplants in culture
`
`-
`
`lipid estt:t'ificatiun
`
`-
`
`Iipuprmein secretion
`E. Lug-_. L. Tfttbauft. D .-lrfértard
`
`{'J.N' CHOLESTER('JI_ NlIIJ\.-'l:'..\!ENT BPZTWEEN T'IssL‘-es
`
`1.\: CBA{] AND
`
`F.r\"['
`
`Er-‘I-‘F.(:TS or l)IETAR\’
`CWBR/"calf MICE
`-
`cholesterol synthesis
`3.
`I. Kztwi,
`
`- pulyunsattiratccl fat
`saturated fat
`.S'£.«rmur£, M‘. K. Dowri, L. Pr2tt'm'rm_. ‘ Duprmt, R. C. Skagmur
`
`lViETAfl0l.lSM or S01:-|1.'M 3ot,7or—
`SLILI-'0i\iA'1'1-1 Al\'.A|.DC.i-LIES OF CHI-1|\'ODEOXYC|IOI.IC Acm:
`DII1Yi)R1.’)XY-25-HOMO-53-(‘:1IOLANE-25-SULFON.-‘\'['E AND Son1L‘_\1 Soc,Tat-Dlrtvnaoxv-24-reoR—5.8—
`c:Iaot_.atNe-23—sULF0N.\TE IN "rm-; HAMSTER
`
`-
`intestinal absorption - organic synthesis
`-
`- biliary Fistula
`Me:>5uc"rr'ce.'ttt-aurrtfus
`bacterial 7-dchydrtnxylzition
`°
`l)iI.‘itransforma|'.I0rI
`S.
`It-ftifi, E. H. fiforbarlt,
`B.
`I. Coiim,
`lfia-left", N Ayyari, C. K.
`i‘vf.»:.S‘.r‘iergv
`
`.-‘vi.
`
`I629
`
`.=\—I Haw-1 SERA WITH l.VlOL‘iES'l'
`l‘lL-'-.\IA.\1 Ai=oLtPoPRUn'.Is:
`'l'RA.'~:sc.'I3NIc NIICF. Ext-Iu:ssI.\'t:
`TRYPANOL&"I'lC AC'I'I\’I'I'Y IN VITRO BUT Rem.-u.\t SL-'SCE|-"l'IBI.I~'. T0 Im=E.c1‘Im: av TR't'PAl'l-'0S0.\f.-\
`flR|'J(?EI BRUCE!
`
`higlt density lipnprotein '
`
`trypannlysis
`
`J S. Owen, M.
`
`I’. 7.’ GM.-:t‘!, T E. Hughflj
`
`'fiT="’“"
`n..... .;.II
`I_'_-.
`University
`
`"
`*
`J
`..:I
`V'v"i::.consin
`
`NOV 1 2 1392
`
`1305 Linden Driire
`i Mac-itsson, W:
`.-".:‘.'.-'{‘.=
`
`CFAD v. Anacor, |PR2015-01776 ANACOR EX. 2122 - 4/19
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`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2122 - 4/19
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`

`
`lDI3N'1‘IFlC.-\TIl'JN AND PROPII-‘.R'l‘lES or TIIE PROLINE554-l.EL|G]NE MLI'rAr~t'r LDL RECEPTOR IN
`Sourtt A1=RI(:.atNs or lM3t,\:u ORIGIN
`
`lltmilial
`Sclerusis
`
`corunary l1(.'F1l'l
`llypvrtiholesternlcmia °
`'-
`low clensity lipnproluitt t'::cepIr)I'
`A D.
`/1-Inmrlt. E. L.°.!'f£J'_rrfu:f H. C.
`
`athero-
`''
`chtaluslerol
`°
`cliscase
`D. C. Rnbr'r1.rehrin.
`(3. fl. C0£‘:'If.’E.
`Sag‘/?r.'.l', 11 R. van tier‘
`l'l-';3z5tft:{}'zc':t
`
`lEI4?
`
`l5lSP|IOSl‘H(>NA']'ES USED FOR THF TREATM!-INT or BONE DISIJRLII-_'RS INHIBIT SQL'AI.E.'~.'E SYN'I'H.=tst-;
`A-ND Cll-IOLI-'.$'I‘l-ZROL BIOSYNTHESIS
`
`‘I'M U5 - parnidmnattt:
`cells
`
`'
`
`- _]??-l-
`elntlrunate
`etidrnnatc -
`-
`alenrlennute
`.'V('t?£Ht?
`.4. Cunmfil. S, K. Cmmfsori. 3.
`D. Aritftr, S.
`_ l-‘In’ L-Wri(:‘l. C. E. Bildfl,
`:11. H. Pérmnc
`
`165?
`
`I-1\-IDENCE mu A COMMON BlI.].I!\R\'CHL}J_1~;S'I'EROL AND VLDL CIIGLESTERUI. FRECURSOR Pom.
`IN RAT LIVER
`
`acyl coertzyme Azehctlcsteml
`synthesis
`-
`T at-hyclruxylase
`-
`
`:1cyltrm1sfet‘asr:.
`Iipnpmteins
`
`bile acid
`-- HMG~CnA rct'luctase-
`B. G.
`.‘s‘tam'. C. D. Ema:
`
`1665
`
`EFFECT5 at-' APOL]I'O|'l{t'_‘l'TF.IN 13., I3-VERY Low Dt:Nst‘t'v LIPOPROTEINS, mun C:noLEs'rt~:ItoI. mt 'l'HE
`EXTENSION ut-' NEL'R|Tt=_s BY RABBIT Doasu. R001‘ GANULION NI-‘.URON:-5 IN VITRG
`
`°
`
`apolipopmtein F.
`cltolesterol
`
`-
`
`clorsal root ganglia
`
`- nerve regeneration -
`
`lipuprnteins
`
`C. E Hanni-frnntrrt. _ K. Bqylcr. K. H.
`
`I'I'i'i5grab.4.'r, R. H.’ A-iaitlflli R. E.
`
`P1-(EU
`
`167?
`
`l\v'lE'.'l'AH(){.IS.\-1 or HIGH DENSITY LtPoPRo'rI<;I:s‘ LII-'1D.‘§ BY THE RAT LIVER: EVl1'lEN(.'|-2 FOR
`PARTICIP.-‘\'I'ION or HEPATIC LIFASE.
`IN Tl-IE Uxrram; :3:-‘ CIICILESTERYL lisrt-;k
`HDI.
`-
`liver perfusion -
`triglyrt-1'Idcs
`'
`phospltolipids
`H Kadnzttakf. 0'. M'. Patron, 3.
`
`Ratlzina
`
`I589
`
`EFFLl.‘)i or LIPID mom l-”tuR013L.v\s'rs T0 AI‘()LlPCIF'ROTE.lNS: DEPENDENCE ON EI.F.\a‘A'I'ED LF,\-'I".I.S
`or CEt.LLIL.attt UNESTER1}-‘II-LL) CHDLES'I'ERl’)I.
`
`- plmsphnlipid ctflux
`apnlipopmtcin A-I
`fibroblasts
`-
`revc-|'sc cholesterol transport
`
`-
`
`chulesterol-e|]ric'.hed
`Cl'JlIlll:SlC!‘Ol elllux -
`J’. K. Bt'e{t'nl'i, I-if Jalzmrm.
`l'l'2':'n!mg. j. M’. Gt':'r!r__. G. H. Rambler
`
`R. B.
`
`1699
`
`HIGH SENSITIVITY NF.IZ‘.A‘l'l\'E ION CC.-MS MET:-ton FOR DETECTION OF DES.-‘\TUR.ATED Arm
`CHAIN-ELUNI3.-\'r1~:1J Pnumacrs m'~' DEl.'TERA1‘ED L1.\'0LF.u.:
`.-mu LINCILENIC Atztns
`
`pentafiuorobcnzyl esters
`NCI
`- dcsaturation
`
`- deutcratcti
`
`- GC-MS
`linoleir and linolenir acids
`R. J. Pawiarigy. H.
`I-'1-1’ Sprecfm, N .S‘at’mt, Jr.
`
`ITII
`
`AN IN VITRO MODEL I-‘CIR ESSEN'I'lAl_ F.\'r'n' ACID DF.F1(2lENC\‘I
`MAINTAINED IN LIPID-FREE l\’lEu1t..'.\t
`
`l-{EPG2 CEI..1.S PF.RM:\|\'I-1|\'TI.‘h'
`
`L"SSL'Il[ial Ihtty acids
`
`'
`
`arachidonatc
`
`E, E. Furtlt. H. Sprrctmr,
`- Mead acid
`E. A. Ft‘-jlllff, H. D. Flrfsfinran, AI. Lttpwanla
`
`ITIQ
`
`
`
`COVER: Effect of ,8—VLDL_. chulesterol, and apoE on ncurite outgrowth from
`dorsal root ganglion neurons in vitm. The ,8-VLDL (upper left) and cholesterol
`(upper right) promote neurite extension and branching. Addition of apcIE together
`
`with I3-VLDL (lower left) or cholesterol (lower right) reduces neurite branching but
`
`promotes extension away from the cell body. (See Handelmann et al., p. 16??)
`
`
`
`
`
`
`
`
`
`CFAD v. Anacor, |PR2C_l15-01776 ANACOR EX. 2122 - 5/19
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`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2122 - 5/19
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`

`
`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`special article
`
`The discovery and development of HMG-CoA
`red uctase inhibitors
`
`Akira Endo
`
`D::partt'nL'1'l[ o|' Applietl Biological Science, Tokyo No}-to University. Fuchu.
`
`'liJkyo_.
`
`lflfi japan
`
`lI\lTRODUCTlON
`
`E.‘({L'lt‘~l\-'11‘. epidemiolngic studies performed in many
`[~Qu[][1'1'.§ have shown that
`increased blood cholesterol
`
`[41314-
`increased levels oi’
`levels. or. more specifically,
`cholesi ml. are causally related to an increased risk of
`coronnnv lteart diseztse. Coronary risks rises prt)g1'essi\-‘cly
`with an increase in the cholesterol level. particularly when
`cholesrrnrol levels rise above 200 mg/dl (1, 2). There is also
`substantial evirknrc that
`lowering total and LDL—
`cholesterol
`levels will reduce the incidence oi" coronary
`heart e'i.~;ee1se (2).
`to search for tnicrnbial
`In 19?} we started a project
`metabolites that would inhibit HMG-CoA recluetase, the
`rate—li-niting enzyme in the syntliesis of cholesterol. We
`hoped that the suppression ofde novo cholesterol synthe-
`sis in the bocly by inhibiting HMG-CoA I‘t’.(lL1C[£1.'-EB would
`reduce plasma cholesterol levels in humans. These studies
`led to the discovery of a potent reductasc inhibitor, named
`meva.s|;tt.ii1 (_forn1erly called ML—236B or compactin) (3).
`Subsequently. we elucidated the biochemical mechanisms
`of action ofincvastatiti {_4, 3} and by 1980, had shown that
`[]1t3'\."rlE~I_tilil‘1 marl-zedly lowers the levels of LDL-cholesterol
`in both experiment:-.tl animals and humans (6-8). These
`findiiigs
`stimulated the world-wide development of
`nieva.-:..;itin analogues in the 19805: and. by 1990,
`three
`E'I'LIg~—Iovastatin (l‘orn'ierly called mevinolin), simvasta-
`tin, and pravastatin -had been approved and marketed in
`man} -.'t'=unI'ries (9, 10). These drugs have been well estab-
`lislied as eHi':ctive and sale t'.l10l(:SIt3]‘CIl-lOWc‘1'ln_t_; drugs and
`are used by many patients. Benelicittl eilccts from their
`admit,-istration in patients with coronary heart disease are
`being observcrl (ll).
`
`3|-lppienicnlary key words
`‘3h‘-'lI‘HIn-no]
`- aIhcrosc.lc:-usis
`
`Hi\r[{i-i;Zoi"\ t't.’tlIIt’_‘IiL'a't:
`
`- cholesterol
`
`0
`
`l.I_ll.-
`
`cluding nicotinie acid (12), cholestyramine (I3), clofibrate
`(CPIB) (14). neomycin {.15}, plant sterols (16), triparanol
`[ME-R-'29)
`(IF),
`lJ~tl'1yroxine {I8}. and estrogenic hor-
`mones (19). Of these drugs. nicotinic acid reduces both
`cholesterol and triglyceride (especially the latter) in hu-
`mans. These efli:cts are due to ."-1 decrease in lipoprotcin
`synthesis, resulting in a fall in LDL-cholesterol. The most
`prominent side El'ii'_‘ClIOlIl1iCL3[lI1lC acid is cutaneous vasodi-
`lation. Other adverse eliiects include rash, gastrointestinal
`upset, hyperttricemia, hyperglycemia. and hepatic dys-
`function (20).
`Clholestyramine, an anion—exchange resin, acts by bind-
`ing bile acids within the intestinal lumen, thus interfering
`with their reabsorption and enhancing their fecal e.~:cre-
`tion. As a result, bile acid synthesis is markedly stimu-
`lated. This
`leads
`to an increased requirement
`for
`cholesterol
`in the liver. which causes an elevation of
`
`hepatic HMG-CoA reductasc activity. Cholestyratmine is
`highly ell‘t-.ctive in the treatment of many patients with
`high cholesterol levels, but unfortunately,
`it is not toler-
`ated by all patients. The1'efore, in spite of its proven use-
`fulness.
`the bile acid scqucstrant
`is not
`an ideal
`cholcsterol—lowering agent.
`Clofibrate and its derivatives are the hypolipidemic
`agents most commonly used worldwide. Its major eilcct in
`hyperlipoprotcinemia is to reduce VLDL-cholesterol; in
`most patients the cholesterol-lowering effect is minimal to
`moderate. Clolibrate has several pharmacological actions,
`including stimulation ol’ lipolysis by increasing adipose
`tissue-derived lipoprotein lipase. However, details of its
`actions at the biochemical level are not well understood.
`
`Neomycin is an eilective cholcste1‘ol—lowering agent in
`patients with FH.
`It acts by precipitating cholesterol wi-
`thin the intestinal tract and thus inhibiting its absorption.
`Side elli;'(:ts.
`including nausea and diarrhea.
`limit long-
`tcrm ariininistration.
`
`I-[ISTORICAL BAG KG ROUND
`
`Duting the 19505 and l960s. many cholesterol-lowering
`agent. were reported and introclueed into clinical use. in-
`
`_'\bbrt'\'it|tions: HMi'i, 3-Iiydroxy—3-niethylglutnryl; ‘v'LDI_. very low
`den.-siI_\'
`lipoprotein:
`I.Dl_, hm rlcrisily lipuprotein;
`l[JI.,
`ll'l[t"['lI}l.'L‘ll&li(‘
`llr‘iI.\?ll\_:' lipnprotein'. H[)l.. lligli rlcnrsitv lip:-p:'ot::iri: FH. 1'-alnilial |1ypc:‘-
`('l1I']ll'5It'l‘lIll.‘I'lllI!.
`
`journal of Lipid Research Volurnc 33.
`
`l.‘)9J1
`
`1569
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`
`Plant stcrc-ls, which interfere with the absorption of
`cholesterol
`in the intestinal
`tract, have no effect on
`VLDL-cholesterol, and their effect on l_.Dl_.—eliolesterol is
`
`variable. The long-term eilects ol‘ plant
`unknown.
`
`sterols are
`
`is effective in reducing serum cholesterol.
`Triparanol
`This drug inhibits cholesterol synthesis in the final
`stage(s) in the synthetic pathway, resulting in the accumu-
`lation of other sterols. Because of‘ side cflccts including
`cataracts,
`it was withdrawn from the market early in the
`19603 (21).
`isomer of I.-thyroxine, elTec—
`the optical
`D-Tliyroxine,
`tively lowers LDL-cholesterol
`in both euthyroid and
`hypothyroid patients. The use of D-thyroxinc in a long-
`term trial (Coronary Drug Project) in men with estab-
`lished ischemic heart disease was discontinued because of
`
`increased mortality in patients with arrhythrnias, angina
`pectoris, or multiple infarctions (20).
`Estrogens have been used to treat hyperlipidemia.
`However,
`escrogens are unsuitable as hypolipidemic
`agents in men because of their feminizing ellects and be
`cause they elevate VLDL and triglycerides (20).
`Thus, none of the drugs available by the early 1970s
`could be considered ideal cholesterol-lowering agents.
`However, experience with many drugs suggested that
`drug-induced lowering of plasma cholesterol would be
`effective in the treatment of coronary athcrogenesis and
`heart disease (1, 2).
`In the 19605, cholesterol metabolism in experimental
`animals and human subjects was extensively studied by
`many groups. Cholesterol can be derived either from the
`intestinal absorption of dietary cholesterol or from syn-
`thesis de novo within the body (22, 23). Experiments in
`several animal species showed that when cholesterol is re-
`moved from the diet,
`the liver increases its capacity to
`synthesize cholesterol and that this organ, together with
`the intestine, is able to synthesize sufficient cholesterol to
`meet the needs of all the other cells in the body. Although
`virtually all other tissues also possess the capacity to syn~
`thesize cholesterol,
`their synthetic rates.
`in contrast
`to
`those of liver and intestine. remain low during dietary
`cholesterol deprivation, so that under these circumstances
`the liver and intestine account for 32 and 11%. respec-
`tively. of all detectable sterol synthetic activity found in
`the monkey ('23). On the other hand, when cholesterol is
`added to the diet, cholesterol synthesis is nearly com-
`pletely suppressed in liver; it is partially suppressed in in-
`testine; and it remains low in other body tissues (24, 25).
`Feedback suppression of cholesterol synthesis in the liver
`by dietary cholesterol is mediated through changes in the
`activity of HMG-CoA reductase, a microsomal enzyme
`that catalyzes the conversion of HMG—CoA to mevalonate
`(23, 26).
`In addition, rcductase activity in the liver is
`regulated by many other physiological conditions. Under
`these conditions. changes in reductase activity are closely
`
`1570
`
`journal of Lipid Research
`
`Volumt.-. 33, 199'.’
`
`related to changes in the overall rate of cholesterol synthe-
`sis ('.?.3}. The control mechanism for cholesterol
`nthcsis
`is partially or completely lost when liver cells become
`malignant (23). These findings obtained by lit‘.-'1') sup_
`ported the concept
`that
`the inhibition of I-lT\'lG-CDA
`reductase would be an effective means of lowering plasma
`cholesterol in humans.
`
`DISCOVERY OF MEVASTATIN
`
`I began in 19?], with Dr. Masao Kuroda, to search for
`
`HMG-CoA reductase inhibitors of microbial origin. We
`hoped that certain microorganisms would prorluce such
`compounds as a weapon in the light against other mi-
`crobes that
`required sterols or other
`lS0[_}I\"t'I0ldS for
`growth. Inhibition of}-lMG—CoA reductase would thus be
`lethal to these microbes.
`
`At that time. HMG-CoA reductase was as-_:;::-«ed pi-in.
`cipally by measuring the incorporation of radioactivity
`from ["‘C]HMG—CoA into mevalonate
`('27).
`A5-
`[”C]HMG-CDA was too expensive to use for rl-.'I.r.-rrnining
`the inhibitory activity of thousands of samples, we finit
`searched for microbial culture broths that inhibited d'lE
`
`incorporation oi‘ | "‘C|acetate into nonsaponiliable lipids.
`The active broths were then tested for their ability to in-
`hibit lipid synthesis from [31-Ilrnevalonate. Culture broth!
`that were active in the first assay but not active in the so
`cond determination were suspected to Qontain a com-
`pound (or compounds} that inhibited the c'aIl_\-' stages be-
`tween acetate and mevalonate in the cholesterol synthetic
`pathway. The principal active componentts) from these"
`culture broths were isolated. Rat liver €I12_\_'t"tI:":- were used
`
`for these assays (28).
`Over a 2-year period, approximately l'i,Ul.lU microbial
`strains were tested for their ability to block ll i.-id syntl’1C5l5-
`As a result, the antibiotic citrinin was first isolated as an
`active compound from the mold il-'f'}rttt:'um uttimnm (29l'
`Citrinin was shown to irreversibly inhi't'-i' HMG'G_°l'_\
`rcductase (30). Subsequently. a strain of H':tt'ri’Ht'um 511"’
`mm: was found to produce active CDm[}(1UF'|l.ll_5l- T0 55°13":
`the active component(s), 600 l of culture filtrate was 133‘
`tracted with organic solvents and applied to silica gel
`chromatography, followed by crystallization givmg Cr-35'
`tals (23 n1g)ofmevastatin (formerly called l\-'IL-235B_]. 3)‘
`the end of 1973,
`the structure of rncvastatin was clcfclg
`mined by a combination of spectroscopi-.:
`I licmtcal. 3-“
`X—ray crystallographic methods (Fig. 1'} t".i'}.
`em;-3 sub-
`Mevastatin has a hexahydronaphthalenc skfll
`which can '3‘
`stzituted with a ;3—hydroxy-5-lactone moiet M
`trcatmcfll
`converted into the water-soluble open at-id I3)’
`;.,t3\.'£lSIa1.lI1 was
`with alkali
`(Fig. 2)
`(-1-).
`lnteJ‘estiIi_£_§l_i-3
`I t-t(:j]aCet3ll~"
`shown to inhibit sterol syntheses from limit
`flung. bill
`and ["lC]HMG—CoA at nanomolar r.'='=I1"*‘“”a
`eintfl
`showed no effect on the conversion of ['"'l l
`I "““'al°nm
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`
`petitive with NADPH in the reaction of HMG-CoA
`reductase (33). Kinetic analysis suggr:s1'cr.i
`that HMO-
`CDA first binds to the enzyme, lollowed by the binding of
`NADPH_ Reduction then occurs, with release of NADP.
`
`CoA, and mevalonate from the enzyme. These results
`suggested that
`the lactonc portion of the mevastatin
`molecule is the active center and binds to the HMG bind-
`
`ing site of the reductasc molecule. The structural similar-
`ity between the lactone and HMG portions supports this
`conclusion. Later,
`it was shown that the tight binding of
`mevastatin is the result of its simultaneous interaction
`
`with the I-INIG binding domain ofthe enzyme and the ad-
`jacent hydrophobic pocket. This region is itself not uti-
`lized in substrate binding {34-).
`The structural
`similarity between mevastatin and
`HMG—CoA and the observed competition by these two
`molecules helped to clarify preliminary structurc—activity
`relationships in the inhibition of HMG~CoA reductase.
`The rnevastatin molecule is composed of {our moieties
`(Fig. 2]: :1) the B-hydroxy~§—lactonc (or the 3,5-dihydroxy—
`heptanoie acid portion}, 2:) the moiety bridging the lactone
`and the lipophilic groups,
`c_)
`the hexahydronaphthalene
`nucleus, and rt’)
`the side chain ester. Our preliminary
`study of the structure—activity relationships suggested a
`crucial role for the 3- and 5—hydroxy groups in HMS-
`CoA rcductase inhibition, as activity is abolished by the
`conversion of either of these hydroxyl groups into the
`methyl ester. Replacement of’ the carboxyl group of the
`acid form with carboxamide also ablatcs activity ('32).
`Furthermore, the distance between the lactone and deca-
`
`lin ring influences the inhibitory activity which suggests
`that a certain spatial relationship needs to be maintained
`between the reactive site (lactone) and the putative bind-
`ing site (decalin ring) (32, 35). Another functionally es-
`sential region of mevastatin is its hexahydronaphthalcne
`ring. This is shown by evidence that HMG, which lacks
`a hexahydronaphthalene ring,
`is more than l(l5-fold less
`
`HMG—CoA
`
`Mevastatin (acid fonn)
`
`Fig. 2. HMG—(loA -.1nt'l the acid forrn oli mI:\'.'istatin.
`
`R:H
`
`Mevastatin
`
`{Compactin. ML-236B}
`
`R=CH3 Lovastatin
`(Mevinofin, Monacolin K)
`
`Fig. 1.
`
`Mevastatin and lovaslatin_
`
`5[C]'ols_ The results demonstrated rnevastatin to be a po-
`tent
`inhibitor of I-IMG-CoA reductasc. The same com-
`
`pound {designated compactin} was also isolated as an an-
`tibiotic from Pemrt'£t'z'tm1 brtmirampactuin by Brown et al.
`in
`
`two less active com—
`l9i'o tiil). Along with rnevastatin,
`poun-‘as closely related to tnevastatin were also isolated
`from Pm. rz'!rt'mtm (3. 32}. Subsequently. the search for ad-
`ditional HMG-CoA reductase inhibitors was continued
`
`for ;:mother 10 years, leading to the isolation of several
`compounds of the rnevastatin family (32).
`
`llNHIBITIOt\l OF I-IMG—CoA REDUCTASE. BY
`MEVAS'D’{'l"IN
`
`Tin: water~soluble, open-ring acid of mevastatin (Fig.
`2) g.-..ve more potent and reliable inhibition than the lac-
`tone lorm in the assay of both .sterr_il synthesis from radi-
`ulabt-led substrates and HMG-CoA reductase (4). The in-
`hibition of HMG-CoA rcductasc by mevastatin was
`reversible and competitive with respect
`to HMG-CoA.
`The J’\‘_.- value for the acid form was ~l x 101‘ M (Fig. 3},
`Wl'lllr* under the same conditions, the K”. for HNIG--Ct1A
`was = lfl'5‘ M
`Thus, the aflinity of HMG—C‘.oA reduc-
`tase "or compactin is l{l,0l'}U~liolcl higher than its afiinity
`for the natural substrate I-[MG-CoA, showing nievastatin
`to bl." a highly potent inhibitor. The mechanism by which
`the‘. :statin inhibits rcductase appeared to be ideal For its
`development as :1 drug.
`A. that time, adenosine-2'-munophospho-.5‘-diphosphcr
`Vibe’-c., a synthetic NADP analogue, was found to be com-
`
`Endn HMG-CoA reductase inhibitors
`
`1571
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`
`1:‘V.(nmoles!mint'mg)_l
`
`-60--10 -20 O
`
`20 40 EU
`
`-20
`
`0
`
`20 40
`
`50
`
`1fD.L-HMG-CDA. mm“
`
`1rNADPH,mM"
`
`Fig. 3. Double-rt-.ripI'oca| plots o1‘Iheinl1il)ition of Hl\-1C;-CoA reduc-
`tase by ntevastatiti, Hl\-'[C-CnA I't‘l'lL:I('ljJ5I.‘. was solubilizt-.d from rat liver
`mierosomes and partially purified. Concentrations o1'HMC2'—C0A in (A)
`and NADPH in (B) were varied as indicated. final concentrations of
`rru.-vastatin were 0 (none). 11.6. 23.3. or 69.8 us: (4). {Used with per-
`Inissinn. Ff1‘B.i‘I.r.'t.t.
`1‘.-iTr'fi. 72: 3‘23—3‘2l).l
`
`active
`
`than mevastatin.
`
`On the other hand,
`
`the
`
`sliglttly
`C13-methyl—substituted analogue lovastzttin is
`rnore active than mevastatin (3. 36, 37), while hydroxyla-
`tion at C8 shows reduced activity (38). In addition, the ot-
`tnethylbutyrate ester plays a significant role, since aria-
`logues that lack this moiety {ML—23fiA and ML—236C)
`have one-tenth or less activity, as compared with mevasta-
`tin ('3).
`
`ln 1973. Brown, Dana, and Goldstein (39) I'L'_'.1;r_pr[ed
`that HMG-CoA reduetase ztetivity of cultured mam-
`nialiatn cells is suppressed by LDL, but not by HDL. Sub-
`sequently these investigators discovered a cell
`--urfat;e
`receptor for LDL and elucidated the mechanism bi." which
`this receptor mediates Feetlbaek control of Cbttlesterol syn-
`thesis and I-{MG-COA reduetase (40-42). Fatnilini hype,-_
`tzholesterolernia (FH), a genetic disorder in humans, was
`shown to be caused by inherited defects in the. LDL Tecgp-
`tor; these defects disrupt the normal control ol‘tl-- lesterol
`metabolism. These
`landmark studies of cholt:stet'ol
`metabolism strongly supported our studies in both ex.
`perirne.nta_l techniques and in the general idea ul'.levelop.
`ing I-IMG-COA reductztse inhibitors.
`By using the techniques developed by Goldsnz.-in and
`Brown, we demonstrated that
`tnevastatin strongly in-
`hibited sterol synthesis from [“C]aeetate in a. variety of
`cultured rnammalian cells,
`including L cells ....1d Cells
`
`from patients with homozygous FH. at ctincemmtions as
`low as l0'9 M (Fig. 4) (5. 32. +3). Inhibition ol l”C]acc—
`tate incorporation was 50% at 1 n,\.-1 (0.-'1 n;;_{fr-.2|:u in nor-
`mal human cells and cells from patients with FT-l’. in con-
`trast, LM cells, which grow in a lipid—l'ree.
`synthetic
`medium. were far tnore sensitive, with lI‘Il1ll.)lIl'>I: of sterol
`
`l"l‘lt'V:1S[a[lI1 concentrations of (1.! nM (44).
`synthesis at
`Stem} synthesis from [3H]inc\talonate and t}t:.:._\ acid syn-
`thesis From [“C]acetate in cultured cells wr It.‘ not
`in-
`hibited (5).
`s_t_'nthesis was
`At higher concentrations where sterol
`strongly reduced, tnevastatin inhibited cell grr wth as well.
`
`A. L cells
`
`8. Human fibroblasts
`
`T:
`
`I
`
`/1
`
`./°
`Fatty acids
`
`Fatty acids
`
`[ninja
`“OTC-TO
`
`1Do.'§‘:-—0""""'
`30
`
`O?D
`
`140
`
`120
`
`(%ofcontrol)
`
`
`
`
`
`Sterolandfattyacidsynthesisfrom["'C]acetate
`
`\ ' Sterols
`\
`
`0
`
`0.001
`
`0.01
`
`I11
`
`Ch‘.
`1.0
`10
`
`U
`
`0.001
`
`0.01
`
`D.1
`
`Mevastatin tugfml]
`Mevastatin (pgtmll
`and in Iil:-i'ol'il'rI.x'f-T
`Eflect ol' mevastatin on sterol and fatty acid synthesis lrom |”C]at'etate in L cells
`Fig. 4.
`from at normal subject and from an FH homozygote QB].
`L cells grown in letal calf Sl.T1.iI[1 lor 3 clays were in
`eubated with '["C];u:etate and varying concentrations of mevastatin. Alter inctlbatiun at 3?°CI for '2 l1, sterols and
`fatty acids were extracted and counted. (H) Human fibroblasts grown in fetal calf serum [or 5 clays wen: iI1t.'ulJ;:|-
`'
`with ["C}a<:etate and varying concentrations ol‘ mevastatin at 35°C} for 2 h and assayed liar stern! and Fatty elvitl
`synthesis (5). (Used with permission. Eur
`B:'ar}‘mn_
`IQTB. 37: 313-32].}
`
`1572
`
`Journal of Lipid Research Voltnnt: 33, 1992
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`
`when __"Jll\'\'I1 in the presence of whole serum, growth 0!‘
`1
`,_.L._1]c
`RYE!!!’ completely inhibited by 1.3 ;.t..'\.’l
`(5 pig/ml)
`n-1{-\,';1SE'.ti1D. This inhibition was. however, overcotnc with
`results... nurrnal growth by the addition of a small
`amount of tnevalonatc, the product of I-ll)/IC}—C2oA reduc-
`msc I‘t'.at't'ion (Fig. 5)
`Suh:-...quently, studies by other investigators showed
`that
`jn
`cultured fil)roblast.s high
`concentrations of
`mevast.-itin block passage through the cell cycle (45-47)
`mg al
`--r cell morphology (4:8). Both effects could be
`prevent:--tl by the addition of mevalonate but not by the
`addition of cholesterol. which suggested that either
`mevalt -I are or one (or more) of its nonsterol products was
`involved. In 1984, Schmidt, Schneider. and Glomset (49)
`ineubznetl Swiss