AN”l'AT|ON
`
`Roxane Labs., Inc.
`Exhibit 1031
`Page 001
`
`

`
`This material may be protected by Copyright law (Tftle.17 U.S. Gode)
`
`Rapamyc. ins: An... tifungal1 Antitumor1
`Antiproliferative1 and
`Immunosuppressive Macrolides
`Randall Ellis Morris
`
`Hlhat in-loow ira drop. H'liritwtrion_-} bwwiI 1111 «tim.
`bMcNrultm
`
`P rogress in rapamycin (RPNI) research has been
`
`rapid and is poised to accelerate even more
`dram<t;tically. An Investigational Nf!\V Drug applica(cid:173)
`tion (IND) for phase I tfials of RPNI as a treatn1ent
`for prospective graft recipients was approved less
`than 2 years after the first published reports1.1 and
`Pl!hlic disclosure' of the al>ility of RPNI to. prO!ong
`graft sUniival in experimental animals. RP~I is _a
`macro~de fermentation prcxluct th.it h<¥> antifungal
`and anlitumor activity. However, its effects' on the
`immune system have generated the most interest
`because RPM is structurally similar to another new
`lrnmunosuppressive macrolide, l<'K506. RP1'1 is par(cid:173)
`ticularly intriguing because it inhibits the activation
`of hnmune cells by unique, relatjvely selective. and
`extremely potent and highly effective mechanisnu.
`For example, o~c half microgram of "R.P1'1 ad minis~
`tered daily to mouse recipients of cotnpletely 'n1is-
`01atched heart aUografts prolongs graft survival.
`When these mice arc t_reated for only 2 weeks ·with
`higher doses of RP1'1, or when a siqglc dose of RP~! is
`administered to rat heart aJlograft recipients, strain(cid:173)
`spe<;ific unresponsiveness is induced, .and grafts sur(cid:173)
`vive in'r;lefinitelyin both spedes.
`'The r·csearch on EPN! is rcprcscntath•c of a
`signiflca_nt shift in e1nphasis in transplantationfrom
`the 1nacrocostnic world in which innovative surgical
`techniques predominated fron1 the 1950s through
`the 1970s to our current focus on the microcosm of
`cellular and 1nolecular immunopharmacologr. A re\'(cid:173)
`olution in the discovery, development, and clinical
`use of new strategics to control the immune response
`is dearly upon us: it took more than 35 years to
`
`P-10;,1 tM i.AhxaltA.J fr Trarupla11/a/ioo IW11U1~lf, Dtpirtrr.n_:J ef
`birrliolharndr Su,,;ay_. StntJ!Wl lllntt-~i!r Srf.col ef.\/trfid>ir, St111!_/01rJ,
`CA
`.4'Jdrrss rrfxinl rtqunlf /ti Ran&i/l E/Us .lfooir, MD, Lnbxo.twy j.v-
`1ia111µanlatiw. lm1mm@g, lkparlwnl rd lim/Wlhmxfr Sm~ny, Sla11-
`farf U11frmi!J Sdiwl oj.\foiidu, S1aefxd, CA 94105-5217.
`Cof:Jrr(F/11o1992hy IVJJ. SaumkrrCw1/xHIJ'
`0955-./ 70.YJ 921{)(Jl)/.OOOJS5.f)O/0
`
`arcrue the four imperfect mainsta}'S of immunosup(cid:173)
`prcssion for tra.nsplan~ation-~teTQids1 azathioprine,
`anti-T-cetl antibodies, and Cydosporin A (CsA). In
`1992, six new xcnobiotic immunosupprcssantswill be
`in clinical trials (Fig I).
`This new Crain in1munosupptcssion can be traced
`to the convergence of several lines of rt'seiirch: (I)
`the discovery and suct&ful ~linical use of CsAi (2)
`an increased understanding of the fundamental biol(cid:173)
`ogy of i'mmune c'cUs that enables th.e actions of
`different in1munosuppressants to be better J,Jnder(cid:173)
`stood and thus lay the foundation foi mofc rational
`means to discover, develop, and use improved drugs;
`and (3) orgiti-i1ied preclinical- fC!i_earcl1 proSrams
`designed to id en tif y potentially yalu_abie irilmunosup(cid:173)
`pressants and to gcfic~te the knowledge needed for
`these agents to be used intelligently in t_he clinic.
`Figure 2 shows the research pr~S"ram used for several
`years in the Labor8.tory forTranspl311tadonI~munol­
`ogy at Stan[ord Un!~·~rs:lty that enab_fed us tO identify
`RPNI:s- 11 and the morpholinomethyI este"r"of m)'cophc(cid:173)
`nolic acid (1-£PA) 1~·16 . as immunosupprcssants for
`graft rejection. The mi;chanisms of action andimmu(cid:173)
`nopham1acology of th'cse two cmnpounds, as well as
`19 deoxyspcrgualin (DSG).~ 1 a,nd brequinar
`FK5Q6,11
`•
`sodium (BQR)n _have _<Uso been studied and com(cid:173)
`pared with one another in ~u1· Jakratory.
`Our spectrum of experimental ~terns begins
`with in vivo mouse models that are so rapid, quantita(cid:173)
`tive, and inexpensive that we have been able to
`evaluate hundreds of molecules for suppression of
`alloimmunity. 'fhc vast majority of these drug candi(cid:173)
`dates fail during testing in rodents because they lack
`efficacy or safety, and they are diScarded quitj\ly so
`that our resoun:cs can be concentrated on com(cid:173)
`pounds with the greatest potential. Co1npounds that
`sho\V promise are evaluated further in rodent models
`to identify those with the following ideal Characteris(cid:173)
`tics: (I) uniqtie _mode of action; (2) high efficacy for
`the prevention Or treatment df acute, acrclerated, or
`chronic rejection; and (3) low toxicity. This DarA>in(cid:173)
`ian selection process acromplishes h\'O tasks: first, it
`insures that only the agents \vi.th the greatest po ten~
`
`Trmupla11tatio11 Rnini;s, Vol 6,A'o I (Janum;~, 1992: pp39-87
`
`39
`
`Roxane Labs., Inc.
`Exhibit 1031
`Page 002
`
`

`
`40
`
`R11.ndall Ellis .\fonis
`
`M!ZORIBINE
`DEOXYSPERGUALIN
`FK506
`MYCOPHENOLJC ACID
`AAPAMYCJN
`BREQUINAA SODIUM
`
`CYCLOSPOAINE
`OKT3 l.tAb & OTHER MAbs
`
`CORTISONE
`AZATHIOPRINE
`ANT!-T CELL Abs
`
`1950
`
`1970
`
`1980
`
`1eao
`Figure 1. History of the use ofdn1gs used lo control_ graft rejection. AH of the foJJowingxenobiodcs recentlydhcm·ered to
`suppress gtaft rejection in prediniral models. have ndvnnced to clinkal trials: the nntimetaOOlites. such as mi7.oribinr.(cid:173)
`(i\JZR), i\IPA in its prodrug form ofRS-6H43, and BQlt; the cydosfK>rine-likr chug FK.506, and drugs that define two new
`c!assesnfimmuno:iuppn•.<iS.1nts, fi')G ancl RP.\{.
`
`1990
`
`2000
`
`tial are advanced to the expensive nonhutnan pri(cid:173)
`mate transplant model; and second, it prepares. us to
`be able to use these compounds. intelligently in
`nonhuman pd1nates. TI1e nonhuman primate n1odcl
`is important because it is highly prcdicti,·e of the
`safety and efficar..)'of a test drug in humans. The suni
`of all knowledge produced from well-planned preclin-
`
`kal studies is the essential foundation fro111 which
`succcssCul clinira.I trials arc designed and executed.
`New drugclcvclopment is a highlycon1plcx, 1nultidis(cid:173)
`ciplinal)' task, and our contribution to the develop(cid:173)
`ment and clinical use of new ilnnnmosuppressants
`depends on \'cry close collaboration with scicn.tists
`and clinicians in the ph<trmm:eutical industt)'.
`,
`
`FUNDAMENTAL ~
`IMMUNOLOGY /~
`
`/ • ""'
`. 4~~-~-~-
`n~ .....
`@ &
`
`' - - - - - - IN VIVO------'
`
`'-------DISCOVERY------~
`
`'------ DEVELOPMENT---~
`
`TRANSPLANTATION
`
`AUTOIMMUNE
`DISEASES
`L CLINICAL TRIALS _J
`Figure 2. &::hrmatic teprr~entation of the program used ;lC the Lahornto1yurTransp!anlacion Inununnlogy at .S1anford
`Uni\'ersily to idrntffycompounds \dth immunusuppressive activities fur 1111.mplantation J.lld to dew·!np these compounds
`for dinil."J.I ust: for the prcVi!ntion and trratmcnt of rejecti011. Fund;.uncnta! knmdedgr of Lhc immune system couplrd with
`an nppre_ciation of thr d1ararteristics of the clnig c-.mdidatr is usrd to dt·Sig"ll't>XJ>criments lo profile the activity nr the
`~ompoundanddt:fine- its rnrdmnismsofaction. Hcterntopic transplantation ofnronatnl muu.~e heart <1llug-rnrts into the enr
`pinrr.1e or moust' recipients and alhJantigcnk and mitugt:nic stimuli ur pop!iteal !)1nph nrnle hrperplasia an: used as rapid
`and quanlitalil-e bioas'ltly:s beforr proceeding lo the more laborious technique~ uf primarily \"U.Scula.rize<l heteralopic
`(abdominal) and secomlarily\nscularize<l h!!tt:rotopic (subrcnal capsule) heart al!ngrnft nnd xcnograft transplnntion in the
`ral. As.'ir,'Qmrnt nr the dlicnty and tht' safety oCLhe mmpnurul in cynomulgus monkey n•ripicnts ur hete.nitopir r1!1ograJts
`pn~crdt's phase I c!inil·.iJ trials in lransplmll pa.dents and µati1·11Lo; with autoimmune disr:i~es.
`
`Roxane Labs., Inc.
`Exhibit 1031
`Page 003
`
`

`
`41
`
`~t fj\
`
`~ + .... ~ ~1FULLV
`
`ca2•.DEPEHDENT ca2t.1NDEPENOENT
`
`UGNlOS
`
`LIGAND
`
`TCR-COO CDOOOS
`
`0028
`
`•
`•
`f
`f C<>2 LFM
`IA\
`.
`L
`ti~K..·~ ~~!Vl.._
`~ ,.. CITO~>"S
`>L·•
`
`-
`
`-
`
`-
`
`-
`
`OlffER-
`
`MZR '@ @ ='°
`
`FK50B
`
`RPM
`{B CELLJ
`
`DSD
`(Bml}
`
`~;:
`{B CEll}
`0 0-01--------------s ... az+ld
`Fi~~e 3. ~hernatic representation ol'lhe possible sites of action of the follm1ing irimnmosupprcssants 1m activated T
`re)!s: CsA and FK.506 PIT\'l'nl the trm_l~niptiun ofeadr t1huse c:ytnkine genes; RP11 inhibits the signal transduction ofIL.2
`bound to its receptor and mar hm·e other antiproliforali\'e effects unrelated to lymphokinesigimls; ~lZR, ~WA, and BQR
`all inhibit purine (:\IZR, ~IPA) or p)limldhie (BQR) nucleotide S}1llhesis; DSG seerris to inhibit late stages of T·cell
`maturation. RPAf, :\lZR, ~fPA, BQR, :mO DS,G also tn:t _oi1 ncti\'atcd B cells at the sites shm\n,
`
`Even fuore importan~ than the relatively large
`number ofncw'imrpunosuppressants th<lt have been
`<lisCovered _js their variety. Each pf these. new molc(cid:173)
`c:ules su.ppresses ·the in1n1une system by blocking
`distin~tlydiffcrent bjoch~mical reactions that initiate
`the <\_ctivation ofjmmune cells that cause the ma,ny
`f<?rf!lS .of graft rejection (Fig 3) .. Bfieqy. 9sJ\ an.cl
`F_K5Q6 Uct soon after CaH-dePendent ·T-cell activa(cid:173)
`tion to prevent the syntheSis of_cytokincS impcirtaJ11
`for the ~rpctU<ltion a~d an1plification of the i111-
`mun~ re!ipotj:se."-13 RP~f acts 1ater t9 block multiple
`effects-. of c}1okincs rin in11nunc cells including the
`inhibi.tion ofinterleukin-2-(IL-2-)triggered T-cell pro(cid:173)
`lif erati9n,')-)..1t but its antiproliferative effects are not
`restricted solely to T and B tells. RPM·alsd selectively
`inhibits.the proliferation or growth factor-dependent
`and grmvth fhctor-indepcndent -nonimn1une cells.
`i\·fizoribine (1·1ZR)/! ~IPA,1 1 and BQR:u arc antin1e(cid:173)
`tabolites that inhibit DNA synthesis pli1na:ril)· in
`l~phc>cytcs. These n·ew antimetabolites are more
`selective than azathioprine because these con1-
`pounds block the activity of CO:l.)TI1Cs restricted only
`to the de nova purine or pyrimidine biosynth~tic
`pathways. LymphOC)1csare inore dependent on these
`pathv,iays for nucleotide synthesis than other cells.
`Recent re\'iews1
`>-"5ll discuss these and other in1mu(cid:173)
`nosuppressants. RP1'1 has recently been the subjec;t
`nf four brie-f re\'icws,'.:n-11 a long revicw,-1-~ and 'has
`been included in reviews thn.t have pri1narily focused
`on FK506. un.This rc\<ie.\v pro\~des a co1nplcte profile
`of RP?i.-1 from work published through the end of
`August 1991. Despite the progress 111ade in under(cid:173)
`standing RP~-1 since the first publication on this
`compound in 1975,l'i the pescription of its ability to
`suppress graft rejrction has stimulated renrwed
`
`interest by a \\idc variety of investigators whose work
`has not yet been published. As a result, research on
`macrolide immunosupprcssant.s has become fluid
`and extremely fast-paced. Bc~ause unpublished data
`generally are not available-for evaltiation, I have i16t
`referred to unpublished work or personal cdfhtnuni(cid:173)
`cations. Hm\·evCr, I have reiied on ·many studies of
`RPi\f from the Laboratoryo(Tran.spl_antatiori Imrnu·
`nol_ogy at.Stanford Uniyersity th.\\t have yet to _be
`publiShed in full. In niost of these. qi.scs,, I h~ve
`supplied the data f~mn \vhich ~onclusioh~ in the 't~xt
`are drawn.
`Because this review· is being written relatively
`early in the researdi lire of RP.~1, and because the
`majority Of the ,\urk on this corilplex moleCule has
`yet to be published, the 1nateri51l ~bsequently pre(cid:173)
`sented should be regarded, more a.s a )1re\iew rather
`than as a review. A_t the very lea~t. this a'rtide \viii
`pro\~dc a logical framework th.1.t o~hcx investigators
`can use to organize and to evaluate 'new information
`on RPi\·I as it is published. For n1any investigators
`"1th highly specialized interests, only selected sec·
`lions \\-ill be of use. For 9the'ts, it is essential to
`understand all that is knowfl about a new and unique
`1nolcculc s_uch as RPi\-I. Without an understanding of
`RFl\.[ that is both decp11nd broad, it 'viii be difficult to
`1neet the challenging tasks of using-RP!\{ as ·a tool to
`learn more about the in1n1unc S)'Sten1, ma.,.imizing
`its thcrapc.utic potential, and discoycring new and
`in1pro\•cc! 1ncmbcrs of this class of i1nmunosuppres(cid:173)
`sant. Jfwe strive to understand thoroughly the little
`that is now known about RP1\'f, \\'C \\1ll 1nake more
`efficient and rapid progress toward our goal of
`understanding all ur the imr.or1ant biological effects
`of this 1nolecllle.
`.
`
`Roxane Labs., Inc.
`Exhibit 1031
`Page 004
`
`

`
`42
`
`Randall EHis .lloni1
`
`~'' ~~~~~~~~"'cruc''~~~~~~~~~~
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`
`Figure 4. Evol.utionaiy path ofRP.i\'f as an immunosup(cid:173)
`pres5anl for transplantation.
`
`In addition to reviewing the inforn1ation on RPli.-1,
`this article warns of the danger of inductive reason(cid:173)
`ing in which, in an adolescent field like immunolot,')',
`arguing from highly specific cases to general laws
`
`often promotes the illusion of knowledge rather than
`its true acquisition. Howc\'cr, br interrelating infor-
`1nation conccrningthe structure, the 1uu!ccu/ar n1ech(cid:173)
`anisrns, and the actions ofRP!vl on drfined cdl t)ves
`in vitro, its effects in vivo, as well as its disposition in
`the body and its toxicity, new and in1portant insights
`into the actions of RPi\·I can be gained. In general,
`the conceptual tools used in this review to analyze the
`data from experin1cnts on RPll..Ican be applied to the
`study of many other itnmunosuppressants, <"specially
`other xcnobiotics.
`Before dissecting and exntnining every aspect of
`RPi\,[ in detail, it is worth reviewing the events that
`led to the attention RPi\·f is now -receiving. Figure +
`shows the relationship of the.evolution of RPN1 as an
`in1n1unosuppressant to the develop1nent of CsA and
`FK.506 as im1nunosuppressants. Table I proddcs a
`n1ore detailed outline of the sequence of the inain
`events that have defined progress in RP1•1rescarch in
`its firsl 15 years. 1421'~i&i.! The ancestors of RPri.'1 are
`CsA and FK.506. As shown in Fig 4, CsA stin1ulatcd
`the organi7.ation of a rational screening progratu
`designed to discover other fcnncntation products
`\\1th mechanisn1s ofin1munosuppressive action iden(cid:173)
`tical to CsA. The discovcryofFK506was the product
`of this program,i1 and "'hen the structure ofFK506
`was defined, its similarity to the structure of RP~.f
`was immediately recognizcd."1 Years before, the
`structure of RPi\I had been cletern1i11cd as a consc-
`
`Table 1. HistoryofRP~f Drug Desdopmenl: Thr First ]j Yt>ar.o;
`
`TsolntiOJJ from Ea~ter Island (Ra1xi Nui) soil
`.sample and characterization ofantimicro(cid:173)
`bin! nctidty
`Jn \'h'O uSe:
`Tuxicil\'
`Pharm;cokinetics
`Bioa\·.iik1bilitv
`Antifungal a~tivity
`Immunosuppression or autoimmune dis·
`ease
`Elucidntiun of structure
`Antilumol' activltv deseri!xd
`Immunnsuppression of a!logrnft rejection
`RP.~1 alone
`
`RP}.l in combination with Csr\
`Diffrn:nti<ttiu11 uf effects ufRP}.f and FKSOG
`on immune rdl~ in vitro
`
`Diffrren\intion of effects of RP~f and FlGOfi
`on immune S}Stem in vhu
`Demon~tratiun urliimliug: urRP}.I tu FK501i
`binding prntein
`
`1975
`
`1978
`
`1977
`
`1980
`191ll
`
`1989
`
`] 99{)
`1989
`1990
`
`19'XJ
`
`1989
`
`Vt!zina, Km-lel$ki, and Sehgal.ii;
`Sehg•li, Haker, and Vezina 1
`;
`
`Baker, Sidorm~icr, Sehgal, et al''.
`
`l\fartel, Klicius, nntl Ga!etH
`
`Findlay and Rad ks'"
`Duuros nm! Suffness1'
`
`i\Iorris and i\-feiser 1
`C:alne, Collier, Lim, et ilf
`i\[t"i~f'r, \Vang, and }..{orrisJ
`'ford, l\Jatkovich, Collier, el a!'1
`:\Ietralfe nnd Richards.!!
`Dumont, Staruch, Koprak, rt al:r'
`i\Jonis, \Vu, ant! Shorthnu~e 1
`
`;
`
`Harding, Cn!nt, Uehling, et al"'
`
`Roxane Labs., Inc.
`Exhibit 1031
`Page 005
`
`

`
`Rnpan~Jri11r
`
`43
`
`quence of the identification of RPM as an antifungal
`antibiotic (Table 1). Shortly after the antibiotic
`activities of RPM \\'ere described, it was found to
`have imn1unosupprcssive activity.1'his was oid}' a few
`}'cars after. the immunosuppressive activity of CsA
`was discovered, hut ironically, RPh-1 was not de..•cl(cid:173)
`opcd as an immunosuppressant at that time. In a
`review;;; of immunosupprcssivc agent.s published in
`1988, Devlin· and Hargrave encouraged "ii detailed
`comparison of the biologica1 profile of these ma:c(cid:173)
`rolides [FK.506 and RPM]." 1bcse inmtigators sug(cid:173)
`gestion was based on the structural sin1ilarityofboth
`compounds and their knov.n immunosuppres.si\•c
`activity.
`Sehgal was aware that investigators at the L'lbora(cid:173)
`tory for Transplantation Immunology at Stanford
`University had developed a_quantal bioassay for the
`evaluat_ion of immunosuppressant potency and ef(cid:173)
`ficacy, had validated the assay 'vith Cst\/1 and.had
`µsed it .to study FJ{506." In 1988, he offered to
`provide us with.enough RPl\{ to.enable us to deter(cid:173)
`mine whether .its activity differed fro1n 1'1\.506 in
`mouse as well .. as rat heart transplant recipients. As
`subscquendy discussed,- the·activity·of RP~·f is ex(cid:173)
`tremely dependent on the vehicle in whici1 it is
`sus~nded and the route b)•which it is ad1ninistered,
`Had ourfin;:t e~perimcnt u.sedsuboptimal conditions
`: for the ~dministration ofRPNI, we would have found
`no difference in potency 'or efficacy between· RPA-1
`and FK506 and. might not have pursued our shidy of
`RP~·I. IO retrospect, the mode of admini.'>tration used
`at.the outset was optirrial and, under thosC condi(cid:173)
`tio1fs, RPM· was clearly more potent and effective
`than Fl\.506. This clear difference in p~1arn1acological
`effect betv;cen these t\vo structuni,lly related mac(cid:173)
`rolides prompted our continued investigations Qf the
`activity of.RPM At the same time as these stµdies
`were being conducted, investigators at the Uqiversity
`ofCambricfge, England, were testing the innnunosup(cid:173)
`_pressive activity of RP~'1 in rodents, dogs, and pigs. 1
`Siinl!ltaneous studies11
`·"l'J perfonncd at Cambridge
`and by various groups of investigators at }..ferck
`Sharp and Dah111e Research ~boratories, United
`States showed that RPM and FK506 affect imn1une
`cells quite diffcrcn.tly in vitro, .
`
`oo •.
`
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`'
`I
`R,... 1 .... oJ......."-.. o-1,ro;-t
`
`~N·O£TlM.JJl..tlO;POO!'IOOATE ll'l"MOCHl..OFIOEW..T
`'
`!\.. J .... oJ....,...-...,.;...... oo
`'-
`
`~~,SUJl'RATE ll'l~CRUESALT
`
`,,.,_~o.~
`
`Figure5. Chemiatl structures OfRP1·f, 29-demethoxyra-
`p.arpydn, FK5CH;; a.nd the prudrugs ofRP~f.
`·
`
`tj~s. jn the; m.iddJe 1970s.ffi.H The aerial m}i:elium of
`this b~ctcriun1 is. monopodally branchcQ (Fig 6),
`contains sporophores terminated py. short,, coiled
`spore chains, and absorbs \\'ater. It was ultimately
`identified ns belonging tO the! species Strrplon!)'<ts
`hygTlJS(opicus, designated by Arcrst Research as strain
`AY B-W4, and deposited in both thir ARS cqlturc
`collection of the United States Department of Agri(cid:173)
`culture (assigned n1;1mPcr NRRL 5191) and the
`American Type Culture Collection (ATC029253).A
`structurall}' related compound,y; 29-demctho.'\.-yra(cid:173)
`pam)t:in (AY~24,66R [Fig 5]) is coproduced \\1th
`RP1t Another culture isolated from the sa~e soil
`sample and designated AY B·I206 produces higher
`levels of RP.NI than AY B-994 and little or no
`29-dcmethoxyrapamycin.~1
`
`Fermentation, Purification, and
`In Vitro Aritiniicrobial Activity
`ofRPMs
`
`Origin and Characterization of the
`BacteriumProducing RPMs
`RP~l (AY-22,989 [Fig 5]) is made by a filamentous
`bactcriun1 frotn the strcpton1}i::etc group that 'vas
`isolated fron1 an Easter Island soil san1ple by Vezina
`et al and Sehgal ct al at Ayerst Research Laborato-
`
`Fermentatiori Of RPM
`Soun after the availability of a pure strain of S
`h;irosropicu.1, fr·rmcntation conditions (type of media,
`media pH, and tc1nperature) were varied to define
`its cultural charactCrislics.-lli.~ Although this microbe
`grows and sporulates in a wide range of culture
`
`Roxane Labs., Inc.
`Exhibit 1031
`Page 006
`
`

`
`44
`
`Randall EJliJ illorri1
`
`Figure 6. (A) Photomicro(cid:173)
`graph or the lilamentnus bac.
`te1ium, S hygrosropi(us. that
`produces RP;\f (magnification
`X-!53). (B) Electron micro·
`graphofSqygrrutopkw(magn.ifi·
`cation X2,500). (Reprinted
`with permission."')
`
`produce about 500 gm of oily residue from a J6Q.Jiter
`fennentation run. After extracting the residue with
`methanol, the extracts are evaporated to yield approx(cid:173)
`i1nately 50 gm of residue that is thenclissoked in 15%
`acetone in hexane and mixed \\1th silica gel, 'Ille
`dissolved RP!vf is adsorbed to the silica gel and
`remains bound to the gel after the mixture has been
`filtered and washed onto a column from which RP1,1
`is eluted with an acetone:hexane mixture. After
`e\'aporating the column eluate to dryness, the resi(cid:173)
`due is dissolved in ether from which pure crystals of
`RPI\i are obtained. In this initial purification process,
`recoveries of RPM are on the order of 40%; 10 L of
`broth produce 300 mg pure RPM. A more recent
`.
`method of purification has been reported.';;
`Except for minor n1odifications, the methods
`described for the isolation of 29-dcmcthm,·yrapan1y(cid:173)
`dn are the same as those used for RPNl ;(;
`
`In Vitro Antimicrobial Activity of RPJ\fs and
`J\feehanisms of Their Antimicrobial Actions
`The antimicrobial screening program at Ayerst Re(cid:173)
`search Laboratories identified RPI\{ for its antifungal
`acti\1ty. RP1·l inhibits the> gro\vth of yeasts and
`filamentous fungi including the dermatophytcsJ1icm(cid:173)
`JjJomm gypuum and Trid1oph_}'lo11 granuloswn. «'H The
`1ninih1um inhibitoIJ' concentrations (~1IC) of RP~i
`against ten strains of C albica11J were in the range of
`less than 0.02 to 0.2 µg/n1I, representing greater
`potency than that of amphotericin B, nystatln, or
`candicidin in this assay. RP~{ has no antibacterial
`activity. The spectrum of antimicrobial activity of
`29-demethoxyrapatnycin is similar lo RPNI, but its
`potency is only about 25% that of RP1[ although
`nearly as potent as a1nphotericin B.:ri
`One study ha'l investigated the n1echanis1ns br
`which RP~f mediates its antifungal r:ffcct'>,'"" and the
`results of this study are sum1narizecl in Tab!c 2.
`Approxin1atd}' 90 1ninutcs after adding RPl\I to C
`albicaiu cultures, growth is inhibited and subsc-
`
`conditions, more narrowly defined conditions are
`nccessaty for the optimun1 production ofRP!-.f. RP1'1
`has been produced by aerobic subn1erged fennenta(cid:173)
`tion similar to that used for tnost antibiotics. Inocu(cid:173)
`Ju111 is prcpa_red in two stages in a medium contain(cid:173)
`ing soybean 1neal) glucose, (l'U-I1)1'50l> and CaCO,
`and used at 2%, For the fermentation in stirred
`vessels, the starting 1neditnn was S0}1Jenn n1eal,
`glucose, (NHf)i)01, and KH1PO+· Glucose is fed
`continuous}}' after the 2nd day and the pH was
`controlled at 6.0 _\vith NH+Oll i\fa.,..irnum titers of
`RP1'[ arc _reached in 96 hours. Paper disc-agar diffu(cid:173)
`sion assays \vi.th Candida alhicmtr are used to deter-
`111inc the antibiotic titer.
`The formcntation n1etbods required to produce
`29-deniethoxyrapamydn are the same as ihose de(cid:173)
`scribed for RP~f_:.r;
`
`Purification of RP Ms
`
`The purification schetne (Fig 7} adopted for the
`production of RPM was developed shortly after the
`identification of the anti fungal activity ofRPlv! and is
`subsequently sun1n1arizcd.n After fermentation, the
`pH of the beer is adjusted to 'l,O. The 1nyccliun1,
`extracted with trichloroethane, is filtered offancl the
`extract is d1ied v.rith anhydrous sodiu111 sulfate to
`
`FERMENT STREPTOMYCES HYGROSCOPICUS
`
`EXTRACT MYCEUUM WITH ORGANIC SOL VEITTS
`
`APPLY CONCENTRATED EXTRACT TO SILICA OEL COLUMN
`
`I
`I
`I
`I
`
`ELUTE Wlnl. ACETONE
`
`RAPAMYClNS
`
`Figure 7. Ferm~ntatiun and isolation ofRP~-is.
`
`Roxane Labs., Inc.
`Exhibit 1031
`Page 007
`
`

`
`Tab~e 2. :iafechauisms of Antifungal Actions orRP~I.
`
`1'..Jftct ofRPJl
`Trta/111<nl
`
`Not inhibited
`Not i11cT\'asetl
`
`Not inhibited
`
`Not inhibited
`
`Not inhibited
`
`Inhibited
`
`Inliibited
`
`Inh(bited
`
`· Inhibited more for
`Ri.~A than QXA
`
`Increased
`
`Sorbose retention byC alhkmu.
`lm:reased hrmnl~1iis of rat t'l'd
`blood cells, effitiX ofK+,Pi,
`UV·absorbing material fmm
`Cafhicam.
`C albimns anerobic glyrol~1iis,
`:ierqbic respinttion.
`Pmlein synthesis by cell-free
`preparations: nfC alhicmis, E
`roii, rat lin:r, nncf mitochon~
`drial pn•i»irations ofCalhicmu.
`A1ilino add metabolism by glu(cid:173)
`tamic-oxaloacetic transami(cid:173)
`nase, g!ut;imic-P}TU\'ilte
`transaminase in C nlbit:a11s.
`Glurosamine and N-acet}1-glu(cid:173)
`rosamine incutporatiun into
`whole C albirmu.
`Oxidative deal'oination of glu(cid:173)
`li!-mkµnd nspartic ad~s in C
`·
`olhiauu.
`In<:orpor.ltion Ofglucost: inlo
`tria1~nan in C alhicah1.
`Incorporatioh ofNaacetate and
`methionine into total lipid of
`c albi<OJJI.
`Iilrorpun1lion of adenine and
`phosphate into RJ"{A and DNA
`ore alhicans.
`Degtacla:tion of"'Zp_labelled intra(cid:173)
`!Ccl!ular macromolecules and
`leakage through C a!hirnn.r
`membrane.
`
`qucntlyycastcells lose viabilitrand begin to lrsc. The
`candiddal actions ofRPi'rl differ fro1n polycnc antibi(cid:173)
`otics that increase yea.'lit cell penucability by binding
`tosterols in the cytoplas1nicmc:mbrane, thus causing
`leakage of cellular con1pot!cnts. Not only do stcrols
`not reverse the actions of RP~'1, but RP~-I docs not
`increase the leakage of sorbose or the efflux of
`potassium, phosphate, or UV-absorbing 1naterials
`front yeast cells.
`The effects ofRP~l on other metalx1lic systen1s of
`C albicaru have also been investigated.-"1 For exa1nplc,
`RP~l does not inhibit anaerobic glycolysis or aerobic
`respiration, nor cloc!i it inhibit the incorporation of
`glurosaminc or N-acetyl-glucosamine. RPn·I does not
`inhibit protein synthesis in cell-free preparations of C
`a!hirans, rat liver, or mitochondria l"rom C albicmir.
`Although RPJ\·I inhibits the incorporation of glu(cid:173)
`cose into 1nannan and acetate into lipids, the synthc·
`sis of glucan is 1ninin1ally affected, indicating that
`
`45
`
`inhibition of cell \\'all synthesis is not thC [}ri111ary site(cid:173)
`of the antifungal action ot'RP1i.'.I
`TI1e 1nost profound effects of RP~[ on C alhicans
`ma)' also pmvidr clues to its actions on mainmalian
`. I
`cl'lls. For exainple, vl'r)' low conccntnitiol'ls ~.02 -
`µg/1nL) ofRPlvI inhibit the inc:orporntiori of adenine
`and phosph0.1e· into RNA and DNA. At the 1·IIC for
`RPh·I, phosphatc-roittqining ntolccuies leak out of
`the yeast cell rilcmbran~. The degradation nf these
`nuilecules, pre:Sumabl)• including nucleic acicl~, see ins
`to be pron1otrd in so1ne way hr RPJ\'1. ~1
`
`Phy~ieP·C:hemical P~opertjes of
`RPM~
`
`Structure ofRPMS
`Althougii tht: initial analrsis of the siructurc of RPM
`by infrarccl a°-d ,nuclear ~agnetic resonance (MvIR)
`spcc.troscopydid not provide tlie con1plete picture or
`its structure/; these techniques indicated that RP~,i
`was a completely nc''' type of_ macr_olide antibiotic.
`Ultimately, :-;:-ray crystallographic data clarified the
`structure ofRi>M. 71 RPn·I is__a 31 ·men1bered 1nacrqcy(cid:173)
`clc lactone coptajning-an, afnidc with a C 15 carbonyl
`and a lacton_c \\ith a C21 carqonyl (Fig 5). Additional
`analyses of th~ 110 ~nd 1H NNfR spectra of Jl.Pl\'f
`confi.r~i~cl the x·.rarcrystaJ stn1c~~1:c o~RP~I.~1 X-ray
`studies shoW~d that Rf~{ in its so_li9 crystal fonn is
`conformationally hon1ogeneous; in solution however,
`RP~! exis.ts as a mixture of two conformatinnal
`isomers caused bytraiu tocis amide isornerizatiqn vi.1.
`hindCrcd rotation about the piJlecolic add N-CO
`bond. Tile ratio.oftranr tod.r rotamcrs in chlorofonu
`solutions is 31 to 4: I ."_'1
`1
`.J.
`Illustrations of the stntc:turc ofRPl\·I wen; initially
`inconsistent; different enantiomerS were dra\\'11 15'1 a
`novel nun1bering S)'Stcnl was used,:.i :intl incorrect
`stercochcmistry at C28 was .reprc'sented."1 Ulti(cid:173)
`~ately, thcc:orrcctstructure was published,"' and the
`coordinates are deposited in the crystal data bank.
`Using advanced 2-<liinensinnal filfR spectroscopic
`ntethods, new assigmnents of the proton and carbon
`spectra fnr the ntajor rotamer of RP~{ ha\·e been
`1nade and a nc"' numbering S)'Ste1n suggested.01
`The closest stluctural relati\'c to RPJ\I is the
`antifi1ngal and i1nn1unosupprcssivc macrulidcFK.506,
`which is also produced by a strcpto1nycete.u FK.106 is
`a 23-1nen1bered macnxyde lactonc that shares a
`nniquc be1nikctal n1askcd a,f>-Oikctopipecolic add
`mnide substructure with RP~i:,'1 but lacks the Cl-C6
`triene segment nfRPn..f.
`The results of 11C-labdlcd acetate and propionate
`
`Roxane Labs., Inc.
`Exhibit 1031
`Page 008
`
`

`
`46
`
`Randall EJIU ,\loni.r
`
`and 11C-labcl!ed methionine incorporation studies of
`the bios)11thesis of RPNI were consistent with the
`proposed poly kc tide pathway in which the carbons of
`the lactonc ring ofRP~.f arc drriYc<l fron1 condensa(cid:173)
`tion of acetate and propionate units in a 1nanner
`shnilar to that responsible for fatty add synthesis.
`The methyl group of methionine is an efficient
`source for the three 1nethoxy carbons of RPWL
`Because none of the labelled precursors .was incorpo-(cid:173)
`rated into either the cyclohexane or hctcrocydic
`ring, these n1oieties probably orginate fron1
`the
`shikimatc pathway and !)'Sine, respectively.(,]
`VVhen 1H and "C Nl\·IR, infrared, lJV, mass
`spectroscopy, and optical rota1y dispersion/circular
`dichroism (ORD/CD) analyses were used to con1·
`pare the structures ofRP1''1 and 29-dctncthoA-yrnpa·
`mydn, these 1noleculeswercshown to bcconfiguration(cid:173)
`ally identical at all chiral centers and to have identical
`structural features at all but C29. Like RP1'.J, approx·
`in1ately 20% of 29-clernethoxyrapamycin in solution
`exists as thecU rota1ner IOrm.fi.l
`In addition to the naturally occurring 29·
`demethoxyraparnydn, amino acid ester analogues of
`RP1·I have been synthcsizCd to produce three water
`soluble prodrugs ofRP'l\.f4 (Fig 5). The amine fi.Inc·
`tions of the appended esters can be converted to
`water soluble salts that are enzyn1atically hydrolyzed
`in the plasma to produce RPi\L 1\lthough RP~1 forn1s
`both monocstcr and diester adducts depending on
`the reaction conditions, only monocster salts are
`
`Table 3. Physiml and Chemical Properties ofRPl\1
`
`discussed because these arc suffidently water soluble
`to ob\~ate the need for the disubstitutcd forn1s. The
`28·h)'Clroi'1.-yl group ofRP::i..f hus been proposed as the
`site of e.'licrification for each of these prodrugs, bul
`this remains to be confirnied.
`
`Physical Properties ofRP~fs
`Table 3 lists the physical properties of RPJ..t10
`W
`Although 29·dcmethoxyrapamp::in is also a white
`crystalline solid, it has a lower melting point (107" to
`JOWC) than RP~J.y, Bolh RP.~dand its 29.demethO:\.)'
`fonn are lipophilic and only n1inimally soluble in
`water. The water solubilities of both the 010110-N,N(cid:173)
`dilnethylg!ycinate methanesulfonic acid salt and thr.
`mono-N,N-dicthylpropionate hydrochloride salt pro(cid:173)
`drugs ufRPi\.I arc more than 50 n1g/mL The \~atcr
`solubility of the 111ono-+-(pyrroli<lino)butyrate hydro(cid:173)
`chloride salt prodrugis 15 n1g/mL.ri-J
`Because i\-IICs for the antifungal activity ofRP1·1
`in vitro vaiy depending on the 1nedium used and the
`length of the assay, it was suggested that RPi\'{ is
`unstable.~~ Subsequent studies showed that 5 µg/mL
`of RPt-.·I in uninoculated broth loses 80% of its
`antin1icrobial activity after 7 days of incubation at
`37"C.n Later analysis showed that 5iJ'Ai of the antimi(cid:173)
`crobial activity of 1 or 5 µg/mL concentrations of
`RP?\:f are lost after only 24- hours of incubation in
`culture mediun1.'1
`I-Iigh-prcssure liquid chromatography (I-IP