ANNALS of THE NEW YORK
`'
`· .. --~
`
`ACADEMY OF SCIENCES
`
`VOLUME
`1 241
`
`V.
`Galderma Laboratories, Inc.
`IPR2015-__
`Exhibit 1054
`
`Exh. 1054
`
`

`
`Ann. NY Acad . Sei. ISSN 0077-8923
`
`ANNALS OF THE NEW YORK ACADEMY OF SCIENCES
`lssue: AntimicroiJia/ Themp eutics l leviews
`
`The history of the tetracyclines
`Mark L. Nelson 1 and Stuart B. Levy 1 2
`' Paralek Pll urmaceu licnls. lnc .. Boston. Massacllusells. :·lul ts Univcrsily Scl1ool ol Mcdicinc. Boston. Massacllusclls
`
`Address lor corrcs pondence Mark L. Nelson. l0 ;rralel\ F'llurn lilcculicals, lnc .. 7':J l<ncc lancJ Sirec l. Boston. MA 02 111
`mnelson@pa ra1ekpllarrn.com
`
`The histo ry of th e tetracyclines invo lves the collective contributions of thousands of dedicated researchers, sci-
`entists, clinicians, and business executives over the course of more than 60 years. Discovered as natural products
`from actinomyceles soil bacteria, the tel racycl ines werc first reported in the scientific Iiterature in 1948. They were
`noted for their broad spectrum antibacterial activity and were commercialized with clinical success beginning in
`the late 1940s to the early 1950s. The second-generation semisynthetic analogs and more recent third-generation
`compounds show the continued evolution of the tetracycline scaffold toward derivatives with increased potency
`as weil as efficacy against tetracycline-resistant bacteria, with improved pharmacokinetic and chemical properties.
`Their biologic activity against a wide spectrum of mi crobial pathogens and their uses in mammalian modcls of
`intlammation, neurodegeneration, and other biological systc ms indicate thatthe tetracycl ineswill continue tobe
`successfulthcrapcutics in infectious diseases and as potent ialthcrapeutics aga inst intlammation-based mammalian
`ccll d iseases.
`
`Keywords: ll'lr;lcycl in cs; history: hio <lLii v il ~'; mcchanisrn; rcs istan cc; uscs
`
`The antibiotic era
`ln ea rl y 194i-\, f-ive -year-old Toby Hocket t ( Fig. I) was
`rushcd hy his parents to ]ohns Hopkins C:hildren's
`Hospital in Washington, DC, with severc abdomi -
`nal pain, and diagnosed with a rup tured appendi x.
`/\ lthough emcrgency su rgny was successf'ul , <1 sc-
`rious inf'cction ;1nd complications sd
`in , and the
`kw antibiol ies clinicall y usef'ul at the time proved
`ineffective, lcaving hin1 ti1cing imminent de;1th. 1
`1-loweve r, <1 new l'X ~lcrim c ntal antibiot ic h<1d re-
`cent ly arrived on camp us fi1r clinical use, a com -
`pound that h;1d h<1rdl y Ileen used in humans <1nd
`wa s still bein g cva luated <1S a new chenwthnapeu -
`ti c agent. ln this post- YVW II l'l'<l, antibiolies werc
`con sidered novel th erapcut ics, with pcnicillin heing
`heralcl ed ;1s a "wo nclcr drug" h<1v in g saved count -
`lcss li Vl's on th e hattlcfields. lt IV<1S also <1 ~lLTiml oi'
`chem ic d d iscovery, where t he seien t i lic 111et hods of
`microbiology and organic chemistry werc mcrging,
`and thc promises of ini'ell ious discase ch emother-
`apy becarnc <1 111<ljor d rive of' mcdical 1'l'Se<1rch in
`<1Cadem ia a ml the chem ic 1l i nd ust ry.
`
`'lbby
`Since thc antibiolies o f' thc da y fi1i lecl
`Hocket I, his parcnts in desperat ion consentcd !in
`him to be treatcd with thc yellow-colorcd com -
`llüu nd rccently sent hy thc Lederle l.ahoratories
`Division o f' American Cy<ln;llnid, under the n;1111c
`/\urcon1ycinT'\1. lt was a risk wit hout choicc; " I rc-
`ill L'Illher hcing pul on thc oper;1ting 1<1blc, scrca ming
`and crying, and sccing thc gas lll<lsk corning down
`on my fi1ce and being in th;1t hospit;J ]I·in a vcr y long
`time <1t"tcr that, " hc rccallcd. VVithinmonths he i'ull y
`recowrcd <11lcl was onc of' th c first of' 111<111)' Jll'oplc
`whose li ves wcre savcd hy /\urcomycin. 1
`/\ ureomycin had hee n discovcred ;dmost f-ive
`)'l'<l rs earlin in th c C<lrly 1940s hy l.l'licrk, whosc
`mission to generate ncw C0111poumls and drugs,
`parti cularly <lntihiotics, h<ld hegun L'VCll carlier in
`thc lat c 1930s.2 lndustri <11 ehemi e d producers in
`t h is cr;1 wne <1W<1 re of' t he d iscovny <ll HI com 1nercia I
`valuc o( jll'll icill i 11, ;111d i t ch;1nged t he course of' I hci r
`business. Non11 <111 )' th cy wcrc resigncd to j>roducing
`consum n ~>rodu c t s, hut now they heg<1 ll hi1·ing sc i-
`cntists i'rom 111 <111)' mcdic d d iscipl in cs and sta rll'd
`sn ee ning chemica ls, hiologics, imllll1nc-ser<l, and
`
`li•Jr 10 111 1/J I?;I!J-GG:32 20 11 OG3b4 x
`I?:! I {?Oll ) 17 1? f 20 11 hJ uw Ynr k/\C d tl r· r Hyu i Sr·
`A rlfl I~Y /\ctrrl Sr;r
`
`r t· flr_ ·p ~;
`
`17
`
`Exh. 1054
`
`

`
`Thc lltslory ollll c telracyciiiJes
`
`1\lc lson & f_c vY
`
`~
`
`~o
`
`and methocls ol· microbiologist Re ne Dubos·1 and
`the ehem ie d d iversity der ived rro m the suil <Jct i-
`nolll )'Cctes shovvn by Sclm<Jn W<~ ks m<~n <Jild col-
`leagues.·1 it w<Js ev id ent that the microhi<JI world
`pn~d u ced <1 we<J ith oi" ll<l!Ur<l l products and antibi -
`otic co tnpounds C<lpa blc o i" fi ghting rnicrohial dis-
`e~lses. 1\ut it w<Js their medi cal <Jnd lin<Jnci<J I potenti<d
`th <J t drove th e cx pa nsion ol· lll<lllY phar m<Jceutie<d
`co mp<~ni es with in the United States, with ;\mericafl
`Cya namid <JS on e ol"th e lirst to commit !'o <Jntihio tiC
`resea rch "nd developmen!.
`C)'<J namid huilt new I<Jbo r<Jtories in Pe<Jrl River.
`NJ under the clirect ion ol" ge ncr<~ l man<lger VVilbur
`M<~lco l m <Jnd their hc<1d of rcsea rch, Ye l l <~ pr<~ g<~ d <'
`Suhh<Jrow. They thcn began a se<J rch fü r <111 a ntibi -
`otic th cy telt sho ulcl ri va l W<Jks m<~n's streptomycin .
`enlistin g <JS co n s ult <~n t 7 1-ye<Jr-old Benj<Jmin Mingc
`i)U" "'". (F i<>. 2)," retired nrofcssor o l" J-llant physi-
`b
`.
`ology <J ild econom ic bot<~n y rrom the Unive rsil)' of
`Wisconsin, to h e<~d their soil scree ning deJ-lartment.
`Du gg<~r W<lS world renowned for his ex tensive
`knowlcdge <J nd stud y of so il fu ngi; he cnllccted so il
`s<J mplcs l"ro m <J II over the wo rlcl sent to him b)'
`l"ricnd s l"rorn sit es hc in stru cted wo ulcl yield ~!C t in o­
`mycetes so ii ha c t c ri <~, o r "uit ra -moicls" as he called
`thcm, th ose with ground cove rings Iei"! unclisturbcd
`<Jnd n<Jtur<JI. Thc sa m~'l es were suhjected to culturc
`<Jnd hro th di lu tion <Jssays ~K· rli mn ed hy his tcchni -
`ci<Jn s, in whi ch th e microorg<~ni s m s were p l <~ t ed, <J nd
`the colon ies <Jss<Jyed fo r <Jntibiotic <Jctivity <~ g<~inst ''
`p<~n c l of C r<l!Tl-positi vc ~111d C r<Jill - tH.:gative b<Jcte-
`ria.2 /\ lthough many soi l o rg~1 ni s m s wcrc known 1"0
`producc <~nt i hioti cs, tnost we rc toxic o r h<Jd unde-
`sir<J hlc pro pertics, ~l!ld the te <Jill encountercd m<J il)'
`f<1 lse Iead s.
`O ne samplc, howevcr, d rcw their attentio n carl)'
`0 11. 1t was m<Jrkcd A-:)77 <Jnd sent by VVilli<J m /\ 1-
`hrecht , clug fron1 Plot 23 o n Sanborn il cld , a dor-
`lll<Jnt tim ot hy h<lyl-icld o n th e Un ivcrsit yo i" M isso uri
`Gllllpus, ou tsiele C :olumhi<l, Misso uri . it yielclcd ~111
`unusual yellow-colo red co lo ny that in hih ited thc
`growth of <1 1i
`their strains in an in iti al pan el of
`bacteria, and produccd remarkably largc zo ncs of
`grow th inhih it ion in ~1 gar. This w<Js an unhca rd -of
`prope rl y ~~ t t h is poi nt , ~1 s com pa red to t he i"cw a 11 ti bi -
`o tics av<lilah le fo r compa riso n. Th cy further i"ou nd
`tiJ <Jt eVCll cr udc CX!rac!S of th e COIOll )' rctain ed re-
`Jilarlzahlc antib<Jct e ri~d activit y aga in st lcth<JI scrub
`typhus and th c rickett si<Js, such as Rocky fVio unt<Jin
`s ~1ott ecl ICver, an in !Cction ii1r wh ich thcrc was no
`
`Figure 1. l'i vc-yca r-old "li>hcy llockctt , o nc ofthc li rst patic nts
`lrl'al ed wilh Aureom yt:in .
`
`ot her promisin g ~1 nd ~'o te nti<JI molen des <~ g<~in s t"
`host ol"disL\lses in ~~ Si.> irit ul"op timi sm, grow th, and
`rnedic d discovny that was uniJrecedentcd in the
`histor y o r th c emcrging ph ;mnace utic d illllustry.
`in i'J:IH, Cyanam id pres ident Wil lian1 1\. ik ll un -
`vcilcd to his exec uti ves a new missio n statement ,
`"You tn<~ y come up with no t hing, but yo u nw y clis-
`covcr a singlc drug that may conq ucr cvc n one major
`di scasc, th en the puh lic will bc wei l scrvcd and o ur
`u mlp<lll)' will prospcr,"2 thus fo rmali1.in g thc U llll -
`pany's cntr y into the are<J o l" antihi otic di scovcry.
`
`The discovery of the tetracyclines
`in th e e~trl y i'J40s antibiot ic discovery was pro-
`grcssing rap idl y, hest exc mplilicd throu gh thc work
`
`18
`
`Exh. 1054
`
`

`
`Nelson & Lcvy
`
`The llistory of tl1c tctracyclir1es
`
`Figure 2. ( l.eft ) Scpttw gcnari;lll Be njamin M in!\<" llll!\gar, who discovc rcd thc " llltra-mold '" Strcpt orll)'n·s 111/rcofirciclls, a soil
`bactcri llrn prod l!Ci ng i\ ureorn yc i n. ( Ri!\h t) "!Cchnicin ns scrccn i ng soil sa rnples in t hc l.cd crl c lnboratorics.
`.
`
`curc. 2 Soon enthusiasm abottt it s hroad rangc of;tc-
`ti vity and potcncy aga inst kth<tl p<tlhogcns kd to
`thc lahcling of thc unkn ow n suhstance as a "hro;td
`spccl" rum" antih iotic, hccomin g on e of thc first in
`mcclic tl histor y to <tttai n this titk. I >ugga r namcd
`thc co mpounclnurcornyciu in refc rcnce to its ycllow
`co lor ancl th c gold -co lored Strcptoruyccs strain (rom
`which it was ex tract"ed. He con tinued the stud y o(
`thc ultra -mold <tnd its med ical microhiology, ta xo n-
`omy, a nd physiology, 11<1 111 i ng i t St rcptorny(ö tlllrco-
`Jilcicns r1111i fi rst publishing his resu lts in 1941> in thL'
`Arnurls oftlic Ncw York Acllllcruy of Scicuccs." This
`estahlished Aureomyc in as <1 new a ncl potent broad-
`spcct rum antibacterial agcnt th<1t wassafe and cffec-
`tive, ;1lthough its ex act chemica l structu re had yet to
`be dctcrm i ned.
`The cfi(lrt s at Cya namid were cx p<1nded, bring ing
`in R.l>. J'vlcC :ormick to produce thecompou nd using
`ad v<1nced k rmcnt ati on meth ods. Soo n th ccompany
`w<1S produ cing Aureomyc in in commcrci<11 qu<1 nt i-
`lies. By lkce mbcr I, 1941>, the cl rug W<1S ap proved
`hy the FUJ\ for clinic;tl usc ancl was an im med iat e
`succcss in thc clin ic, saving countlcss li ves against
`a hro<lli spcct rum of in li:ct ious discascs, and gen-
`LT<1ting notoriet y and proflts f~1 r the comp;1ny. lt
`a ppca red t hat t hc 1ll ission st;1 tetm•n t of C:ya na 1ll id
`by William llcll had come to fruiti o n.2
`Within <1 short tim e, ot her chctnic 1l companics
`werT announcing th cir own discovcrics ofn cw bio-
`prospcc ted an tihiot ics, <11lll by 10:10, ;\ k x;tndcr J.'in -
`lay ancl colleagues at C:h;1rks Pli zer Co., lnc., Croton,
`
`C l", hacl ga th crcd thouS<11His or soil S<1n1plcs from
`around thc world, <lllci iso latcd th c so il hactcrium
`Stnptomyccs rin1osus. 6 Their org;1 nism producccl ;1
`corn pound with similarit y in color to J\urco rn ycin,
`but it W<1S slightl y morc wat cr soluble and had hct -
`tcr hioacti vit y, giving it <1 mcdic tl ;1nd con1pctiti vc
`cdgc ovcr Aureom yc in in th c trcatmcnt of in li:ct ious
`discases. Thc compound W<1S named "il' rrarn ycin in
`reli:rencc to /erm, l.atin for mrt/1, ;1nd fllTil.1flS its
`origin , "il'rrc !laute, lndi;1n <l. lt was <1pprovcd hy thc
`FI>J\ in 19:10, co mpct ing clircct ly with Aureo mycin
`whilc gaining success in thc trcattncnt of a bro;1d
`spcctrum of infect ious dise;1scs. 7
`Th e ehemie d st ruct ur es ofbot h J\ ureomyc i 11 <1rHI
`TerT<1mycin , lw wewr, werc difticult to solve and re-
`lll<1ined elusive f(lr hoth co mpan ies, <11though they
`sh<1rcd thcir rcspcc tivc cotnpounds with e<Kh othn
`in ordcr to detcrmine their comn 1<1 n str uc tur.tl fi..·a-
`turcs <1nd substructures <1 t1 cl sett lc thcir disparate
`mok cular id entitics. In this cra of chcmical c l~<1r­
`actcriza tion ofnatur;1 l products, instru ment al <111;1 1-
`ysis was limi tcd to ultr;wiolet-visihlc spcllroscopy
`(UV-Vis) <1tHI infr;Hcd spectroscopy, anti structtrr;11
`proofs routincly rclied on chcmic;tl modifi c;1tinns
`and degrad;1tion studics th.1t k w L1hor;1torics in th c
`world wcr-c equ ipr1ccl to perf(mn. Scicnt ists ;tt l'fizcr,
`lcd hy K<1 rl llr ut1in gs (l: ig . . >) , aml in L·o1Ldlor<1 -
`ti on with the legc ndary llarv<Hd Univcrsity chcmi st
`Rohcrt VVoodward, raccd to provc thc chcm ic;tl
`structurcs of hoth Clllll flOlllllk J\y Jl):i2 thc prc-
`lilllill<lr)' ehemied structurcs or hoth /\ urco mycin
`
`19
`
`Exh. 1054
`
`

`
`The h1story ol t11e letracyclincs
`
`Nelson & LeVY
`
`Figure 3. pr,zcr g ro up lllCillbers fr o m the stru cturc dete rmination iliHitetracyclin c team (left to ri g ht ): rrede ri ck Pilgrim; Lloyd
`Conovc r, in ventor of telra cyclinc; Kar/ Brunings, dircc tor of ehemie d rcsean:h; Phi I Gordo n; a nd C harl cs Stc phcns, in vcntor of
`doxycyclin e.
`
`<tnd "ICrramycin (Fig. 4) wetT ~ol ved by the Pfi zcr-
`Wooclw<t rd team , postulatin g th <tt both compounds
`possessed a DCHJ\ naphth<tcene co re with similar
`fun ctional groups with on ly mino r differences in
`~ lru c ture . x Thc co t-c scafft>ld f(lr this new htm il y of
`antibiotics became descriptively known <lS thc tetra -
`
`cycl ines; howevcr, -k rramycin possessecl an addi -
`lion<tl C5 posilion hydroxyl group and was dcvoid
`ofa C:7 chlo rinc alom, comp<t recl lo Aureomycin.
`Thc major str uclu r<tl k alu res of th c mol endes
`wcrc puhl ishcd by thc Pflzcr-Wood warcl gro up in a
`i<mcl ma rk pa pcr in I ':154 l i tled "The Structurc o f
`
`4
`7
`6
`5
`8~T~~3
`9~-v~2
`1Dil
`1 Z;o
`11~
`11
`12
`1
`'I)
`
`~~N~
`Y'~0~&fr(lr 'PJ
`OHO OHO
`0
`1
`clllortetracycline
`Aureomycin
`1948
`
`~(C~~
`~- OH
`OH
`--~ :
`~(c~r;"r;l( Yi
`~(l~~('f( 2
`OHO OHO
`0
`
`l
`tetracycline
`TEneyn
`19~
`Figure 4. Th c naphtha ccnc ring sys tem and it s slrud uraii<H . .:ant s of th c firs t-gc ncra tion a n tihio t ics: chl o rt c tracydinc ( I ), o xytc-
`tracyd ine (2 ), and te tracycline (3), fol lo wcd by th c yca r approved hy th r 1'1>1\.
`
`2.
`oxytEtracydine
`·~ 1951
`
`20
`
`1\n r r 1-.J Y 1\r...:;tll Set 1 ;Jt\1 (/0 1 1) 17 J? r;, ?() I 1 1\Juw Yr)r ~~ /\c; ldt;my o l Sc1e n c t~S
`
`Exh. 1054
`
`

`
`Nelson & Levy
`
`Tl1e hislory ol tl1c tclracycilnes
`
`;\urCOill)'Cin ," thus selling the stage for lhe firsl
`semisynthelic deriv<1l ive of this ncw 1;11nily of an -
`libiotics lo emerge.'1
`in this scicnlific era, it was belicvcd that chemi -
`cal modilica tion s of <1nlibiolics dccreased lhcir ac-
`livil y. Bul Pllzer's Lloyd Conovcr Iook the oppo-
`site view, thal th e C7 chlorine in 1\urco rnycin was
`nol rcsponsible fo r aclivity, and lh <1l il co uld he
`renwved o r modificd to producc <1 morc acli vc an;1 -
`log wil h improvcd pharmacologie<1l and <1ntihiotic
`characlcrislics. II)' c11alytic hydrogcnalion of Aure-
`omycin , using palladium rnctal ancl h)'drogen, the
`C7 deschloro deri va tive was synthcsizcd, producing
`a compound o( higher potcncy, a heller solub ilil')'
`profile, ;1nd favorable pharmacologic 1l aclivity; il
`was subseq ucntly namcd tctmcycliuc. 111 This com-
`pound was approved by th e FDA for clini ca l use in
`1954, <1 S thc first novel tel racycl ine by nwclific 1lion
`o( a natural procl ucl, and it was one ofthe firsl com -
`merciall y successfu l semisynthetic <1 11libiol ics used
`i 11 med ici ne.
`13y the mid - 1950s there were now three tclracy-
`clines used clinical ly, their complcx chemic 1l slruc-
`tures h;1d heen solved, ancl lheir ehem ie d 11<1111es
`becamc in ordcr of lheir discovcry, chlortetracy-
`cline (Aureomycin), oxytctracycline (Terramycin ),
`and telr;1cycline (F ig. 4). More importantly, these
`compounds s;1ved tcns of thousands of Ji ves ;1nd
`gene raled much reve nue for both compa nies yearly;
`rccogn il ion was given to
`thc Pfizer-Woodward
`group for their slructur;d elucid<1lion <1Ild lo Lloyd
`Conover f(Jr the invention o f· the n1osl dcscribed
`molccule within ils E1mily, tclracyclinc.
`Second-generation semisynthetic
`tetracyclines
`Slruclural f(:alurcs wit hin thc lclracycli ne nu cleus
`and thc <1pplic 1lion of organi c sy nthctic rc;1ct ions
`scparately by Lcdcrlc and fJfi zcr lcd
`lo further
`modilications oftheir propril'l<1ry lclracyclinc scaf-
`fold s, wilh th c goa ls of gencrating more potent
`;1nd <1Clive tel racydinc <111tibiotics. J>f·izer chcmisls,
`Jcd b)' Robcrl lllackwood, chosc <1 disjunctivc <1p-
`proach, modifying the C- ring of oxylel racyclinc lo
`alford ehemie d slabilit y, wherc ha logcnalion and
`C6-dchydr;ll ion yiclded l hc <1 ntibiotic mcl haq ,cli nc
`( Fig. 5) .11 Wh ilc this new cl;1ss h<1d des ir;1blc phar-
`maco logicd properlies compa rcd lo it s proge nilor,
`il was nevcr suhjected to FDA <1f1Jmn'<li wi thin thc
`Unit ed Stal cs. llowcver, il was uscd as a sl;1rling
`
`mat erial b)' Ch;1rlic Stcphcns (Fig. 3) to produ cc <111
`<1nalog wilh rcmarbblc aclivity, slahi lit y, and phar-
`macological cfhcac y: doxycycline was ;1pprovcd f(~r
`usc by the FI)A in 19<17.12 Doxycycline is slill widely
`usecl loday as an antihiolic with activity againsl a
`bro<1d spect rum of co mmunily-acquired bacterial
`inll:clion s, <1nd a diverse rangc of microbcs, from
`th c Clllsati vc <1gcnl of <1nlhrax infcction s, /lfl( ·il/us
`lllllilmcis, 1·o malari;1 caused hy thc intraccllu l;1r sch-
`izont o( PlnsiiiOrliulll fo/cipnnn/1. u More reccntl y
`doxycyclinc has been show n lo i11h ihil thc orow th
`o( hacleria in the gcnus \1\fo//Jncilio, l·l syn~bionls
`thal hcar closc la xonomi c rclationships wilh the
`lY- proleobactcria, lhe ricJ.:.ettsias, lh c apicopJasl of"
`nwlarial schizonl s,u <111cl animalm il ocho11dri;1.
`!3oth compani cs ;Jiso studied olhcr <1nlibiolic-
`proclu cing so il bactc ria with in the Actinomyccta lcs
`ordcr and crcaled biochemic;d mul;1nts of th cir
`Strcploiii)'CCS strains in a11 clforl lo inducc highcr
`yield s o( produ cls as weil as to discover o~hcr
`novcl tel racycli ncs. One bioengincered sl rai 11 hy
`Lederle scicntists produced <1 new tctracyclinc th cy
`namcd dcmcclocycline (Figs. 5 ;md 6) 1:; <1 lelr<1cy-
`cline lhal posscssed unique C6 a11d C7 fun cliona l
`groups. Whilc hardl y bioaclivc, dcmcclocyclinc was
`cl1em ica ll)' modificd lo yicld <1n int ermediale tlwl
`rct;1incd <1clivily whilc harhor in g thc mosl mini -
`n1<11 slruclurc nceded f·(Jr antib;1cler i<11 aclivit y. Thi s
`new interrncdiate hccame known as S<1ncyclil1c. Fur-
`ther co 11junctivc modili calio11s of thc aroJ11<1l ic ! ) -
`ring by Rohcrt Church produccd novcl C7 and C9
`derivatives of S<1ncyclinc. 11' Onc ;malog posscssi11g a
`C?-di mclhylami11o group was found lo ex hihit t;n
`grealcr an1ih;1cteri;1J <1nd pharmacologie<1l acliv ity
`<1g<1insl a !arger range o( hactcria comparcd lo lhc
`l-irsl -gener<1lion co mpounds <1nd cloxycyc lin c. Thc
`compound was nam cd minocyclin c <1nd W<1S <1Jl-
`provcd f<.Jr clini c<11 usc in I'J7 l. ll hcc1nll'onco flh e
`n1osl widcl y used o( lhc telracyclin cs lo th c f1rCsL'n l
`day, although il wou ld bc thc last of new lclr.lcy-
`clines lo enle r the clinic for lhc nex l :15 YL'<1rs.
`Tetracycline structure versus activity
`i\ILmy of lhc chcmic.1l modilica lions of· hoth lhc
`firsl- and sccond -gcncra tionll'lracyclincs J'rocluccd
`v;1ri;1hly <1Cli vc or inactive co1npo u11lls, which il'd
`lo a ge11cral dcscriptio11 of thc slruclurc- acli vit y
`rclatio11 ships for 1clr<1cyclinc anlihaclnial <JCli vil y.
`/\ 11 active lL'lracyclinc wilh il s minimum pharl11 <1-
`coplw rc musl J'Osscss a I inca rl y aJT<1ngcd I )CJI;\
`
`Ann N Y Ac<HI Sc• I ?'I i (?0 II ) 17 3? "' ?0 I I l~ev.• Yrlll• Ar Cl< lemy 1>1 Sc""'e<'>:
`
`21
`
`Exh. 1054
`
`

`
`Tllo llistory ol lile tc tracyclines
`
`1\lelson & Levy
`
`Lederl e Route
`
`Cl 0H
`
`~~ c
`~~]
`OHO
`
`6
`demeclocymne
`Lederle
`
`8
`lllinocydine
`llinocin
`1971
`Figure 5 S ·11 · ·
`. t>f'· • · ( 'o for doX)'C)'cl inc (5). and
`.
`.
`·
`II . . ·.
`1 1 (' I
`· ' c usyn 1t:Sis ro utcs for the Sl't:ond-generatiOn tclracycl m es chosen >y I ll' _, l as.
`li.CJ
`· ·
`l.cdcrk Lahnrainries l(>r rn inocrclinc (X) . T hc chcmical namc j, followcd h)' lhc ycar approvcd hy lhe I'I>A .
`
`naphthaccnc ring system with ;111 A-ring C i-C::\
`d1keto substructure ancl an cxocycl ic C2 carbon yl
`or amide group. lt ;1 lso requires a C IO-phcnol and
`a C: i i-C I2 kcto-cnol suhstructurc in conjunction
`with <1 12a-0 11 group ( Fig. C1 ) o utlinin g a lower pc-
`riph nal reg ion , whcre chemi L<liln ocl ili c;llion ahol-
`ishes hio<1cti vit y. h1rthn morc, a C4-d imcth ylamino
`group with its n<11Ur<11 4S isomer is rcqu ircd ft) r op-
`tinl<ll antih;1ctn ial <1ct ivity, wh ilc cpimcriza tion to
`its 4R isomer clccrcases Cram -ncgativc <1ct ivit y, as
`li rst rcpml ed by Alhn t Doerschuk <11 J.cdc rlc. 17 1\y
`
`co n t rast, pos i t io ns ( :s to C':J c 1n hc chem ica ll Y nwd -
`ificd to affcc t th eir bioact ivit y as antibiolies and are
`dcsign<1 tcd thc uppcr pcri phcral rcgion, gcncrating
`cln ivativcs with varying antib;1ctcrial activ it y.
`Th c cl in ica Iu t i I i ty oft hc tctracycl i ncs has dcmon -
`slr<1tcd that th cy are acti vc ag;1insl a widc <11T<l)' o f
`infcctious discasc agc nts, from C r<1111-positi ve and
`C ram -ncga tivc p<1thogcns, to mycoplasmas, intra -
`ccl lula r eh Ia myd iac, rickct tsias, and protozoa n pa r-
`asitcs. Th c many primary and scconclary indi catio ns
`f'o r thc tctracyclin cs arc rcprcsc ntecl in ' l ~1hl c I. Th cir
`
`22
`
`/\r II) hJ y f\c;J( I Sr:r I ?11\ (?0 1 1) 17 3? ·C ?0 I 1 \\jew Yr)r k /\C ilci Cill y ol ; ;C IC: IICCS
`
`Exh. 1054
`
`

`
`1\Jelson & Levy
`
`·111e llislory ol 111e tetracyclines
`
`Tetracycline biology
`Thc ea rl y biology of thc tctracyclincs poi nt cd to
`a spccific mcchanism of action that was firsl de-
`scribcd in 1953, when they were shown lo block
`protci n synthcsis in S. !lt.lrt' II S cclls and inhibit cell
`growth in <l baclcriosla lic manncrY Latcr studies
`showed lhal thcy hound to a singlc sitc wi thin th e
`bactcrial 70S riboso mc,2'1 with a dissocialion con -
`stanl dctermin cd to be betwcen I and 30 x 10 "
`M, although wcakcr binding sites were prcsent. 25
`Ccll-frce ri bosome ex perimcnts concluded that the
`ribosomc was indced thc primary site of action of
`thc telracyclines,21' whilc laler photoaf'!-init y labcling
`experi ments ckmonsl ra ted photo-i ncorpora l ion of
`1H [tetracycli ne int o thc 30Ssubunit, 27 ];lbeling mi -
`[
`nor proleins SI H and S4 along with olhers. Co-
`operative bincling dynamics helwecn the rihoso-
`ma I ~not e i ns a nd tel racycli ne werc demonsl ra ted,
`where cxperimenls with 70S ribosomes revcalcd
`a highcr affinit y than with 305 or SOS p<nlicles
`alone. 2x
`Notall telracycli nes actecl sim i];n]y agai nst bactc-
`rial ribosomcs. 'lcsted in a cell-free ribosomc prcpa-
`ration ,2'J a scries or tel racyclines differed in riboso-
`mal inhibitory <lClivit y; minocycline proved to bc
`the mosl polenl' inhibito r of prolein synthesis whilc
`lelracycline was thc least.
`St ructu ral a nd mecha n istic dcta ils of the cl rug-
`riboso me intcraction soon followecl, and many rc-
`porl s were con t-1 icl i ng, bu l i l was soon demonsl rated
`thatthe A-site regionwas involved 30 and thattRNA
`binding lo this sitc was afkctcd. 11 lt was Ccrardo
`Suarcr., howcvcr, who first hypol hesizcd thc bincl-
`ing of letracyclines and aminoacyl-tRNA to the 30S
`ribosome at l hera l'eu l ic concenl ra tions.·12 More dc-
`lailed models ofthe binding ofletracyclincs to RNA
`were subsequcntly describcd, 1·1 and mokcul;n cle-
`tails of the drug- RN A inl eraclion were proposed hy
`Harry Noller, narrowing thc sitc o fhindin g lo spc-
`cific bases with in the I(>S rihosoma l p<lrliclc, the sil e
`of act ion on l he ribosomc l hat is acccptcd today.·\.1
`More rcccnl X- r<l)' studics Iee! by Venki ltllnakrish-
`ll<lll , using ribosomes from 'f'ltcrntus iltcrnwpltilus,
`confinn this modcl, whcrc thc primary binding silc
`of telracycline W<lS detcrmincd al 2.3 ;\ resolution ,
`<lnd found lo occur allhe ribosome ;\ -rcgion, whnc
`th c lowcr pcripheral rcgion of thc molcculc f(mn s
`H-honds and a lll Ci<d bridge wilh ribosomal mag-
`ncsium ;md kcy RNA nuclcotidc bascs. 1
`"'
`
`.
`.
`'
`... ~~ .... ~ ............. .................. ......... .. .................................................... ..
`'
`lower periJileral regon
`Figure 6 . The designated upper and lower peripheral regions
`of the tetracydine mole.:ule.
`
`clini cal elfi cacy and biology havc been thc subjecl ol
`num erous comprehensive rev i cws, 1 ~· 1 '1 books,211·21
`and chapt ers in medicalmicrobiology and pharma -
`co logy tcx ls, 22 rcadi ly detai ling their usclulness in
`chcmol hera py.
`
`Table 1. Uscs of thc tetracyclines against bactcrial and
`microbial infcctions"
`
`1'1·i111ary indica ti ons
`
`Sccondary indiGllions1'
`
`St rcptococcus spccics'
`;\ rn cbiasiso/
`
`/:'. (ti /i'
`I:lllt'rtl!ltlCIIT 1/Crtl~C//CS'
`Sltigcllu spccil's'
`Klcbicllu spccics'
`
`l{ickcttsiac
`tvlycop!ttSIIItl f>II t' llllltltlitlc
`l'si t t;lcosis ;111d Orn it lwsis
`Hurrcliu n'C/1/Tt'lllis and
`ot her spcc"ics
`/lctiiUJ'hili.< tluacyi
`\'a,;i11iu f"'sl is
`Huctcroitlcs spccics
`Vilnit> cltu!au and oth cr
`spcc ics
`Nc isscriu spccics
`'li"t'f'tllll'IIItl spcc ics
`Clt~sl ritlilllll spcc ics
`Hucill11s tllllftmci:>
`Clt!ulll)'tliu spccics
`tvl<ll;ni a prophyb xis
`
`"This currcnt Iist is considerably ;lllcllcd by thc high frc -
`LJLI L' JH.:y oft ctr;Kyciilll' rcsist<liiCC tOLJ.ly, li 111iting thc l\ Sl' of
`tct racyclincs against CO I111110 ll discasc agc nts.
`1'Whcn othcr ant ihi otics, such as pc nicillin , arc CtHl -
`tJ·aind icatcd.
`' Whcn bactnio logic<ll lest ing indicat cs susccptibilit y.
`.I i\djunct to ;un chi cidcs.
`''i\dj un ll to standard thnapy.
`
`Ann I~ Y Acacl Su I ?'II (?0 I I) 17 32 ''' ?0 I I Nt>w Yurl< 1\catlurny ol Sc:~ ences
`
`23
`
`Exh. 1054
`
`

`
`Tllc hislory ol lhc tc tracyclrnes
`
`Other mechanisms of tetracycline activity
`lt was also observed that some tetracyclines hacl
`diverse bioehemie d activities and th<11 the y co uld
`act as bactericidal agc nts, allect ing cellular growth
`through mcmbrane-mediated mechanisms.
`lan
`Chopra showed that lcl r<1cyclines could diffuse
`<JCrnss the out er cell memhrane of C ram -neg<llivc
`bacteria , relying upo n the clcctrochemica l grad ienl
`of energized cclls to pa rt itio n in to I he celltda r cyto-
`plasm, with rnagnesium and other di valc nt metals
`playing a kcy role in antihiotic uptake into the cell
`and antibactcrial activity. Y' Most tctracyclin cs acted
`as bacteriostatic or "typic 1l" agents, as protcin syn-
`thesis inhibitors aga inst bacteria . Bul it was lintnd
`that more li pophilic tetracyclines were "atypica l,"
`with a hactericidal mechanism that relicd on mcm-
`brane perturbation , affect"ing multiple pathways in
`cells, lcad ing to ccll ttlar dysf'un cti o n.·17
`Now biology was show ing the tetracycl ines as a
`family to be chemically and hiologica lly dynamic,
`with mult iple mechanisms of' activity and capahle of
`interacting with multiple targets, eilher ribosorncs
`or cellular rnembranes. '!Ctracycline structural dy-
`namics wetT earlier ev ident and descrihcd in X-ra y
`crys tallograph y studies,1K where it was found thal
`they adaptcd and chan gcd conl(mnat ions through
`II -migrations or tautomcrism that was environ -
`men t dependen t. In aqueous solut ion t he tel racy-
`cl i nes cou ld adopt multi plc con form<llion s, whcre
`bot h hydrophi l ic a nd 1.w i I tcrion ic forms predom -
`inated, while in Iipid cnvirons an un charged and
`chemica ll y neutral form prevai lcd. Modern compu -
`tational analysis of the ioni1.ing behavior and con -
`formations showed that a single tetracycline may
`Iranspose into 64 d ifferent tauto meric substru c-
`lu res, wi 1 h ten o r mo re of 1 hern wi t hin :::: I 0 kca I sep-
`arating their confimllational changes in a n aqueous
`'J Subscquenl UV-Vis spectroscopic
`environment.·1
`studies using Fourier techniqu es furth er cknwn -
`strated that thc meta! bindin g and prolon ioniza -
`tions of th c tetracyclines wet-c equall y complcx,
`whcre t hei r con fimna 1 io ns become dependen I on
`pll and meta! io n co ncentrations in a "chamelcon-
`like" mann cr, with tetracycli ne adapting to its sur-
`rou nd ing envi ronmen 1.'111
`
`Tetracycline resistance
`Within one yea r af'ter the discovery of Aureomycin ,
`the firsl ev idence ofbacterial resistance to the drug
`
`Nelson & Levy
`
`.
`
`.\ t)
`
`·t '(·1 ·11 ( )tJ1cr tclrac)'Ciine-resistant cli ni -
`.
`was tcpot L .
`.
`.
`.
`.
`I . 111 'S .1r1r)•"1r·L'LI esl1ec ial! )' lll l'cstt tl<d n rgan t s m~
`C.l ISO ~ C,
`l t t ..... ,
`' t-
`,
`Ji ke Siliy,cllo dyscntcrioc. Also no ted was t hc coex -
`istcnce o f teiT<H.:yclin e resistancc and res t st~:n ce to
`structurall y dif'!Crent ant1htol 1CS, creat1ng multt -
`d ru <>
`resistant hacteri <L"\2 In
`the
`ILJ70s, 111any
`lahL~·ator ies we re begin ning lo understa nd thc
`molccular hiology a nd meclwnisms ofantih iot rc rc-
`sistance, where transfe rable ext rac hron1osomal R
`1;1ctors were show n lo <Iiter ba cterial phys tology
`when 111ediating resista nce pheno t ypes. O ne <~1 . the
`autho rs, Stuarl Levy, with l.aura McM urry <11 lu hs
`Uni ve rsil )' Sclwol nf Med icine, stud ied the cellular
`properlies of a tetra cycline-res isl<1nl f:. coil be<mng
`R-li1ctor R222. 1.1 They founcl th<rt cells beanng thc
`plasmid showed a cl ccreascd uptakc of t e tra cy~lin e.
`T'his frndi ng suggested that the R-f<rctor spec1hed <1
`tr<r nsporl systcm li1r rcmovi ng thc cl ru g fro rn wt th 1 n
`the cell , thus reneiering the lw sl bactcriurn rcs tslant.
`Th c cnlu x systcm thcy discoverecl was mediated by
`dil'lc rcnt res istan ce determinants and actcd through
`different 111 embra nc-associated ~note ins called 'Iet
`prot eins,'i.\ which they di scovc red <rncl described car-
`lier.'H The protei n <r nd its acti vi ty represcnted thc
`first delllOilSlralion or enlu x <I S <1
`lll CChantSlll o l
`d rug rcsista nce <1 nd t hc Ii rst n1ccha n ism of rcsist<r ncc
`to the telracyclines. ·nld<r y ant d)<1Cien <d clilu x as a
`mechan isrn of drug rcsista ncc is common ly lound
`in all bactcria. Ii may be spcc ili c fnr a singlc drug,
`such as the tct r<1cycl ine el'llu x systl'111, or ro r multipl:
`drugs through a multiprot ci n efflu x mcchanisms: t.'
`A scco nd type of tctracycline resist<ll1 Cl' was sub-
`sequcntly report ed, a protein -hased ribosoma l pro-
`lcct io n mech<rnism, lirstli.ntnd in slrcplococci' 11
`' <111cl
`lat er in anacrohic hacteria .'17 Whcn tctracycl in cs
`bind lo rihosomcs they normall y slop elongation or
`syn t hesizi ng protci ns. Howevcr, rihoso ma I protec-
`1 io n prot ci ns, such as "Iet( lVI ) or CI\.·t ( 0 ), i nlcr<IC I w 1 t h
`t he riboso me ca usi ng t hc Iet racycl i ne to d islodge
`from the rihoso rne, thus prol t'ct ing the hactnial
`cell from telracycli ne's inh ibito ry activit y, result ing
`in cellular grow th.'1x C iven the prcvalcncc of rcs is-
`lance determina nts in clinica lly studicd pathogenic
`slrains, tctra cyclines useful in the l'uturc n1u st have
`act ivit y against strains harhoring both cnlu x <1nd
`ribosomal prot cct ton mechan1sn1s.
`More recentl y, <1 third rcsist<ll1Cl' mcchanism h<rs
`hee n descrihed. Chcn1i cal in act ivation of tctra cy-
`clinc was shown to he duc to an oxygcn -dcpcnd cnt
`llavin -monooxygenasc enzy me cncoded hy
`thc
`
`.._
`
`.
`
`24
`
`f\r111 hJ Y /\cud ~;c 1
`
`\ ~tl\ (?0 11 ) 1/ ]/ 1(: r ?(J I\ I\J ewYor k !\c ;J t lc : n Jy O I Sc H ~ n cos
`
`Exh. 1054
`
`

`
`Nelson & Lcvy
`
`The l1istory ol tl1e tctracyclines
`
`prcviously dcscrihcd lct (X) gcne. 511 Curiously, the
`oxygen-depend<llll dcterminant W<1S first described
`in <1n an ;1erobe /io clcroirlcs, and so its res isl<1nce <1C-
`tivit y was no l noted in th;ll haclerium, hut onl y
`whcn th e pl asmid with the /c/ X gcnc was tr;ms!Crred
`to ~:·. co/i. Still , th e work indi cat es that chemic 1l inac-
`tivation ofthe tctracyclin es is biologically possiblc in
`somc specics of m icrobes, although t his dcgradat ivc
`mech;1nism has not heen report ed to hc of clinic d
`relcvancc.
`Th c stud y and classificalion of tetracycline resis-
`tance mcch;1n isms a nd thci r genet ic hascs, ex prcs-
`sion , and kind ies has resulted in the adoption of
`<1 systcmatic no menclaturc' 1 resulting from world -
`wide compil<1tio ns of tctT<1C)'Ciin e-based rcsist;1nce
`mechanisms th<ll continuc to be recognized. There
`tetracycline res isl<lllCe
`arc currcntl y 46 different
`determinant s.
`The Tet repressor
`l'vl ajor insights into thc mo lendar <ln d structural bi -
`ology of tetracycline resistance hegan in the 1980s
`and were real izcd through the work of Wolfga ng
`I-l ilien and colle;1gues in Gcrmany. They initially
`dcscrihed th e interacti on of the tetracycline reprcs-
`sor protein 'ICtrR ), with its DNA bindin g domain s,
`ancl dctailcd thc mcchanisms of 'll't prot ein cx pres-
`sion at th e hiocherni c;d lcvcl. 52 I-lilien ;dso showcd
`that tran sposons encoding gcncti c control elcments
`co uld con t rol t he efll ux of tel r;1cycl i ne hy sensing t he
`drug via a rcp