RANSPLAN’WI'ATION
`REVIEWS
`
`1'r
`
`West-Ward Exhibit 1055
`Morris 1992
`
`Page 001
`
`West-Ward Exhibit 1055
`Morris 1992
`Page 001
`
`

`

`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`Rapamycins: Antifungal, Antitumor,
`Antiproliferative, and
`Immunosuppressive Macrolides
`Randall Ellis Morris
`
`Wlial we ~now ir a tlmp. Wlutl wt tla11) foow i• a11 ""'an.
`4nnr. Nr.it•/on
`
`P rogress in rapamycin (RPM) research has been
`
`rapid and is poised to accelerate even more
`dramatically. An Investigational New Drug applica(cid:173)
`tion (IND) for phase I ti-ials of RPM as a treatment
`for prospective graft recipients was approved less
`than 2 years after the first published reports1.2 and
`public disclosure of the ability of RPM to. prolong
`graft survival in experimental animals. RPM L~ a
`macrolide fermentation product that has antifungal
`and antitumor activity. However, its effects on the
`immune system have generated the most interest
`because RPM is structuraUy similar to another new
`immunosuppressive macrolide, FK506. RPM is par(cid:173)
`ticularly intriguing because it inJ1ibits the activation
`of immune cells by unique, relatively selective, and
`e.-.:tremely potent and highly effective mechanisms.
`For example, one half microgram of RPM adminis(cid:173)
`tered daily to mouse recipients of completely mis(cid:173)
`matched heart allografts prolongs graft suniival.
`When these mice are treated for only 2 weeks with
`higher doses of RPM, or when a sil)gle dose of RPM is
`administered to rat heart allograft recipients, strnin(cid:173)
`speciflc unresponsiveness is induced, and grafts sur(cid:173)
`vive indefinitely in both species.
`The research on RPM is representative of a
`significant shift in emphasis in transplantation from
`the macrocosmic world in which innovative surgical
`techniques predominated from the 1950s through
`the 1970s to our current focus on the microcosm of
`cellular and molecular immunopharmacology. A rev(cid:173)
`olution in the discovery, development, and clinical
`use of new strategies to control the immune response
`is clearly upon us: it took more than 35 years to
`
`From lhr LaboralolJ•far Trmuplrmla/1011 !111m1111ology, Dtpnrtment Qf
`Cartliotltorocit Surga;•. Stn'!fortl Univrrsil)• Sdwol ef Mtdicilte, Stm!fortl,
`C.4.
`/ ltldm.r rrprirrl requests lo Rn11tlnll Ellis Alorri<, MD, Lt1/x1mfo1yjor
`"/'rmuplm1lntim1 Jmn11mologr, JJrpartmt11l '!f Gttrdioll1oradc Sm~tlJ', Sln11·
`.ful'fl UniU<ni!J School of Met!irinc, Stni!fnrtl, Cr1 94305-52./7.
`Co19<-ri,~/1/ e 1992 ltJ IV.B. S111111dm Co111pm9'
`0955.-/ 10,\'/ 921()(j(}/.()(}(}4S5.(}(J/0
`
`accrue the four imperfect mainstays ofimmunosup(cid:173)
`pression for transplantation-steroids, azathioprine,
`anti-T-cell antibodies, and Cyclosporin A (CsA). In
`1992, sL-.: new xenobiotic immunosuppressantswill be
`in clinical trials (Fig 1 ).
`This new era in immunosuppression can be traced
`to the convergence of several lines of research: ( 1)
`the discovery and successful clinical use. of CsA; (2)
`an increased understanding of the fundamental biol(cid:173)
`ogy of i'mmune cells that enables the actions of
`different immunosuppressants to be better under(cid:173)
`stood and thus lay the found;i.tion for more rationa l
`means to discover, develop, and use improved drugs;
`and (3) orgariiZed preclinical research programs
`designed to identify potentially valuable irnmunosup(cid:173)
`pressants and to generate the knowledge needed for
`these agents to be used intelligently in the clinic.
`Figure 2 shows the research program used for several
`years in t'he Laboratory for Transplantation Immunol(cid:173)
`ogy at Stanford University that enabled us to identify
`RPM,.11 and the morpholinomethyl ester of mycophe(cid:173)
`nolic acid (:M:PA) 12
`16 as immunosuppressants for
`"
`graft rejection. The mechanisms ofaction and_immu(cid:173)
`nopham1acology of these two compounds, as well as
`FK506,11
`1
`q deoxyspergualin (DSG),:o-21 and brequinar
`'
`sodium (BQR)22 have (!.)s9 been studied and com(cid:173)
`pared with one another in our laboratory.
`Our spectrum of experimental systems 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. The vast majority of these drug candi(cid:173)
`dates fail during testing in rodents because they lack
`effic.acy or safety, and they are discarded quickly so
`that our resources can be concentrated on com(cid:173)
`pounds with the greatest potential. Compounds that
`show promise are evaluated li.irther in rodent models
`to identify those with the following ideal chatactetis(cid:173)
`tics: (1) unique mode of action; (2) high efficacy for
`the prevent.ion or treatment of acute, accelerated, or
`chronic rejcclion; and (3) low toxicity. This Darn~n­
`ian sr.lertion process accomplishes two tasks: first, it
`insures that only the agents with the greatest poten-
`
`Trn11spla11/alio11 Rer.~l'w.s, Vo/ 6, l\'o I (January), 1992: pp39.IJ7
`
`39
`
`' .
`
`
`
`West-Ward Exhibit 1055
`Morris 1992
`Page 002
`
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`

`40
`
`Randoll Ellis ,\Juni.r
`
`MIZORIBINE
`DEOXYSPERGUALIN
`FK506
`MYCOPHENOLIC ACID
`RAPAMYCIN
`BREQUINAR SODIUM
`
`CYCLOSPORINE
`OKT3 MAb & OTHER MAbs
`
`CORTISONE
`AZATHIOPRINE
`ANTl·T CELL Abs
`
`1950
`
`1960
`
`1970
`
`1980
`
`1990
`
`2000
`
`Figure 1. Historyorlhe use or d111gs used to control graft r~jeclion. All or the l'ollowing xenobiotits rcl'en!lydisco\'t"l'ecl to
`suppress grart rejection in precliniml mode ls h~\'C advanced IO clinit'al trials: tlw antime t::ibolites such as mizoribint°
`(MZR), !\IPA in its prodrug form ofRS-6 1-1+3, and BQR; the cyclosporine-like drug FJG06, and drngs Lhat define t11•0 new
`classes of immunosupprcssants, DSG and RPM.
`
`tial are advanced to the e.'l:pensivc nonhuman pri(cid:173)
`mate Lransplan t model; ::md second, it prepares us to
`be able to use these com pounds intelligently in
`non hu man primates. T he nonhuma n primate model
`is important because it is highl)' predictive or the
`safely and efficacy of'a test drug in humans. The sum
`of all knowledge produced from weU-planned prcclin-
`
`ical studies is the essential foundation from which
`successful clinical trials arc designed and executed.
`New d rug dc\'clopment is a highly complex, m ultidis·
`ciplinary task, and our c.ontribution to the dc\'clop·
`mcnt a nd clinical use of new immunosuppressants
`depends on very close collaboration with scicnLisLs
`and elinirians in the pharmacrulical industry.
`
`FUNDAMENTAL ~
`IMMUNOLOGY ,;~
`
`/ +""
`.~~__.. @-__..{!:?' __._
`fi? ~
`
`LINVITRO_J
`
`' - - - - - - - IN VIVO-------'
`
`'--------DISCOVERY-------'
`
`'------ DEVELOPMENT ___ __.
`
`TRANSPLANTATION
`
`AUTOIMMUNE
`DISEASES
`L CLINICAL TRIALS _j
`Figure 2. Sehr ma lit re prrsent;11ion of the progr;un used ar the LahoratC>I)' ofTransplanlalion Immunology a l Stanford
`Unive11'il)' lO identify compounds 11·ith immunusupprrssivc ;1cli\ilies for tntnsplanLalion and Lo d<•wlop these C'Olll[lOUnds
`for dinical use· for the prc1't'.ntion and l rt':llmtnt of rt'jt:ction. Funcbmcnlal knowkdgt nl't he immune S)'litcm conpll•d wit h
`an appreciation of' tlir charactt'ristil's or tht' drug cancliclact' is used w design 1·xpcrim~nls lO proli le tht• activicr or thr
`mm pound and de line ils mechanisms ol'<tctinn. Hcternwpic Lransplantation ofm·on:ual muusi: h1·:1rl :1llugrarls in tu till' 1·;u·
`pinnae of mous1• recipients and alloantigenic and m iLOge.nic stimuli ol' poplitcal lymph node hyperplasia an· used as rapid
`and quantit ative bioassays belore proceeding lo Lhe mor~ laborious techniques uf primarily \'ascnlarized heterulopil'
`(ahclominal) ancJ sernndal'ily 1·:L,cularizc,d hc icrolopic (subn·nul rnpsule) heal'l allogral't and xcnografl transplam ion in the
`rat.. Ass1•ssnwnc or the ,·mcaq• :111d the sa f'l'l~'l•f' 1 he cn1npouncl in cynomolgus monkcr n·C'ipients of hrtr.rulopic a llografts
`pr~ccdl's pha.'~ I dinii:al t rials in trunsph111t patients and p~tirnts with ;n1wirn111un~ diseases.
`
`
`
`West-Ward Exhibit 1055
`Morris 1992
`Page 003
`
`

`

`41
`
`Ca2•.DEPENDIENT Ca2•·1NDEP£NDENT
`LIGANDS
`LIGAND •
`
`TCR-CDa CD4/CDB
`
`CD28
`
`• CD2LFA·1
`
`•
`
`CYTOKINES
`
`CaA
`FKSOG
`
`@k- @"".,_r.t _r.kt •
`ll·2 'r T' 'f\
`
`(jt ~
`
`I:,
`ti~ CELLS
`MZR ~ ~
`
`RPM
`(B CELLJ
`
`M PA
`BQA
`(B CELL)
`
`DSO
`(8 CELL)
`
`Fi~e 3. Schc:malic representation ol'the possible sitrs of action or the follcl\\fog immunosupprrssants on acLivatrd T
`rells: CsA and FK506 prewnl the t r:m..:riptiun ur earl)' phas~ l')'tokinc genes; RP.M inhibits the signal transduction of IL-2
`bound lo its receptor and may ha\'e other antiprolifer:llive eflects unrelated 10 lymphokinr signals; i\IZR, ~IPA, and BQR
`all inhibit purine (i\IZR, MPA) or p}Timidine (BQR) nucleotide sp1thesis; DSG seems to inhibit late st:iges or T-cell
`maturation. RPi\1, i\IZR, i\ll>A, BQR, and DSG also acl on aC'tivated B cells al the sites shown.
`
`Even more important than the relatively large
`number of new immunosuppressants that ha\•e been
`discovered is their variety. Each of these new mole(cid:173)
`cules suppresses the immune S)'Stem by blocking
`distin~tlydiffcrent biochcmital reactions that initiate
`the activation of immune cells that cause the many
`forms of graft rejection (Fig 3). Brie~y, CsA and
`FK5Q6 act soon after C:12
`• -dependent T-cell activa(cid:173)
`tion to prevent the synthesis of cytokines important
`for the perpetuation and amplification of the im(cid:173)
`mune responsc.112s RPM acts later to block multiple
`effects of cytokincs on immune ceUs including the
`inhibition ofintcrlcukin-2-(IL-2- )triggered T-ccll pro(cid:173)
`liferationt"'' but its anliproliferative etfects are not
`restricted solely to T and B cells. RPM also selectively
`inhibits the proliferation of growth factor-dependent
`and growth factor-independent nonimmunc cells.
`: MPA,'' and BQR3-I arc antime(cid:173)
`l\ilizoribinc (MZR),'1
`tabolitcs that inhibit DNA synthesis primaril)' in
`lymphocytes. These new antimetabolitcs arc more
`selective than azathioprine because these com(cid:173)
`pounds block the activity of enzymes restricted only
`to the de novo purine or pyrimidine biOS)11thetic
`pathways. L)'mphOC)tes are more dependent on these
`pathways for nucleotide synthesis than other cells.
`Recent reviews';.J<O discuss these and other imnrn(cid:173)
`nosuppressants. RPM has recently been the subject
`of four brirf re,icws,'"'1
`11 a long review,~! and has
`•
`bl'en included in re\~tws that have primarily focused
`on FK506." i ; This review prO\~des a complete profile
`of RPl\£ from work published through the end of
`August 1991. Despite the progress made in under(cid:173)
`standing RPM since the first publication on this
`compound in 1975,'" the description of its ability to
`suppress graft rcjc·rtion has stimulated renewed
`
`interest by a wide variet)' of investigators whose work
`has not yet been published. A~ a result, research on
`macrolide immunosupprcssants has become Auid
`and cxtn:mcly fast-paced. Because unpublished data
`generally are not available for evaluation, I have not
`referred to unpublished work or personal communi(cid:173)
`cations. However, I have relied on many studies of
`RPM from the LaboratoryofTrnnsplantation Immu(cid:173)
`nology at Stanford Uni\'ersity that have yet to be
`publishrd in full. In most of these cases, I have
`supplied the data from which conclusions in the text
`arc drawn.
`Because this review is being \\Titten relatively
`carlr in the research life of RPM, and because the
`majority or the work on this complex molecule has
`)'Cl to be published, the material subsequently pre(cid:173)
`sented should be regarded more as a preview rather
`than as a review. At the very lea~t, this article will
`provide a logical framework that other investigators
`can use to organize and to evaluate new information
`on RPM as it is published. For man)· investigators
`\\ith highl)' specialized interests, only selected sec(cid:173)
`tions will be of use. For others, it is essential to
`understand all that is known about a new and unique
`molecule such as RPM. Without an understanding of
`RPM that is both deep and broad, it \~ill be difficult to
`meet the challenging tasks of using RPM as a tool to
`learn more about the immune system, maximizing
`ils thrrapcutic potential, and discovering new and
`improved members of this class or immunosupprcs(cid:173)
`s:int. If we strive to understand thoroughly the little
`that is now known about RPM, we \\~II make more
`efficient and rapid progress toward our goal of
`understanding all of the important biological effects
`of this molecule.
`
`
`
`West-Ward Exhibit 1055
`Morris 1992
`Page 004
`
`

`

`Rand(l/l Ellis .\Joni<
`
`CYCLOSPONNE- _.,.
`
`FKSOO
`EVENT
`
`RAPAMYCIH
`
`~~~~~~~~~~~~~~~~~~~
`
`42
`
`OATE
`
`11>72· SPECIFIC SOPAESSION
`1975
`OF IMMUNE-CEl\ •
`.4CTIVA1)QN SHOWN
`tH VITI\() ANO I« vrvo
`
`1975
`
`1978
`
`....
`
`1N3 FOA APPROVAi. AND
`COMMON CUNtc.AL. IJSE
`IN TF\ANS~Nl'ATOO"\'-
`
`\_: FE~~;~ION
`
`PROOUCTS FO.R
`INHIGITORSOF
`THE MOUSE MIXED
`lVMPHOCYT'E AEACTIOf.I
`IMMUNOSUPPRESSlVE
`ACnvnv SHOWN IN
`VITRO NiO If YWO
`
`UN!CUE. RAPAMVCIN LIKE
`SlRUCTURE: OEF'INEO
`
`!m·
`
`.....
`
`1086
`
`....
`
`NffiFUNGA1. ACnVITV.
`WW TOXICITY SHOWN
`11rl.,.l\JMOSUPPRESSK>N
`OF AUTOIA.lt.11.f./E
`Dl~ASE SHOWN
`U..ilOUE MACAOl!OE
`STAUCTUAE OEFINEO
`
`INVESTIGAllON
`OF'RAPAMYCW
`ASAN ,MMUNO.
`SUPPRESSAHT FOR
`TAANSPl.ANTATK>N
`
`RAPAMYQN
`SHO\YH TO PROlONG
`AUOGAAFT SOR\lfYAl
`
`often promotes the illusion of knowledge rather than
`its true acquisition. However, hy interrelating inlor-
`111a1ion com.:t>rning the structure, the mulcclllar mech(cid:173)
`<1nisms, and the actions of RPM on ck fined cell l)lJCS
`in '~ t ro, its effects in vivo, as well as its disposition in
`the body and its toxicil)', new and important insigh ts
`into Lhc actions of RPM can be gained. In general,
`the conceptual tools used in t his re,~cw to analyze the
`data from experiments on RPM can be applied Lo the
`study of many other itnmunosupprcssants, <"specially
`other xcnobiotics.
`Before dissecting and examining cvc1y aspect of
`RPM in detail, it is worth revie''~ng the events that
`led to the attention RPM is now re.cc h~ng. Figure .J.
`shows the relationship or the evolution of RPM as an
`immunosuppressant lo the development of CsA and
`FK.506 as immunosuppressants. Table I prm~dcs a
`more detailed outline of the sequence of t he main
`events that have defined progress in RPM research in
`its first 15 years. 1"'.:!1·~\•rk'1 The ancestors of RPrvr are
`CsA and FK50G. As shown in Fig 4, CsA stimulated
`the organ ization of a rational screening program
`designed to discover other fermentat ion products
`\1~th mechanisms ofimmunosupprcssivc action idcn·
`tical to CsA. T he discove1y ofFK506 was the product
`or this program,' ' and when the structure of FK506
`w:i~ dcfintd, its simi brity 10 die st1·uc1ure of RPI\,f
`wa~ immcdiatelr rccognizcd.'>l Years before, Lhr
`structure of RPM had been determined as a conse·
`
`Figure 4. Evolutionaiy path of R.PiVf as an immunosup·
`pressan l for transplantat ion.
`
`In addition to revie"~ng thi:: information on RPM,
`this article. warns or the danger of inductive reason(cid:173)
`ing in which, in an adolescent field like immunology,
`arguing from highly specific cases to general laws
`
`Table 1. Histnry of RPM Drug Dcvrlopmenl: Thr First 15 Years
`
`Dismlit.'I)'
`
`Isolation rrom E:mer Island (Rapa Nui) soil
`sample and characte rization or ant imicro(cid:173)
`bial :icth~ty
`In vi\'o use:
`Toxicil\'
`Phar m;<:nkinrl ir-s
`Bioavailabilit )'
`Amifu11gal arti1~ty
`Immunosuppression or autoimmune dis·
`ease
`Elucidat ion of structure
`Anti tumor activitv describtd
`l mmunosuppt ession of allngraft rejection
`JU>M a.lone
`
`Rl'l\ l in combinalion 11·ith CsA
`Ditfrrcntiation of effects or RP~l and FIGOO
`on immunt' rdls in vitro
`
`Difrere11tia tion ofcffects ofRP;:VJ a11cl FIGOfi
`on immune syslem in vivo
`Demon,lrntion ufbindi11gofRPI\I lo FK50(j
`bind ing protein
`
`1975
`
`1978
`
`1977
`
`19HO
`!9HI
`
`198!1
`
`1990
`1989
`1990
`
`1990
`
`1989
`
`Rtjerenccs
`
`v~-.i11a, I<udelski, and Sr hgal"'
`Sehgal, Bake r, and Vezina·"
`
`Baker, SidorCll1icz, Sehgal, et al 111
`
`Martel, K.licius, and Ca let 11
`
`Findlay and Radics"'
`Douros and Suffness"
`
`;\forris and Meiser'
`C:~J nr, Collier, Lim, cl •LI'
`~lt-isrr, Wang, and Morris'
`Tocci, 1\fatko\•ich, Collie r, et af"
`:\l~tcalfc and Richards''
`D L1mont, Staruch, Koprak, el al"'
`i\lorris, Wu, and Shorthousc'
`
`Harding, Cal:ll, Urhling, t:.t al"
`
`
`
`West-Ward Exhibit 1055
`Morris 1992
`Page 005
`
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`

`RfljlfJlll)rilLI
`
`43
`
`quence of the identification of RPM as an antifungal
`antibiotic (Table I). Shortly after the antibiotic
`activities or RPM were described, it was found to
`have immunosuppressivc acth~ty. Thiswasonl}ra rcw
`years after the immunosuppressive activity of CsA
`was discovered, but honically, RPM was not devel(cid:173)
`oped as an immunosupprcssant at that time. In a
`rC\~ew"' of immunosuppressivc agents published in
`1988, Devlin and Hargrave encouraged "a. detailed
`comparison of the biological profile of these mac(cid:173)
`rolides [FK506 and RPM]." These investigators sug(cid:173)
`gestion was based on the structural similarity of both
`compounds and their known immunosupprcssive
`activity.
`Sehgal was aware that investigators at the Labora(cid:173)
`tory for Transplantation Immunology at Stanford
`University had developed a quamal bioassay for the
`evaluation of immunosuppressant potency and ef(cid:173)
`ficacy, had validated the assay with C:sA,;..; and had
`used it to study FK506." In 1988, he offered to
`provide us with enough RPM to enable us to deter(cid:173)
`mine whether its activity differed from FK506 in
`mouse as well as rat heart transplant recipients. As
`subsequently discussed, the activity of RPM is ex(cid:173)
`tremely dependent on the vehicle in which it is
`suspended and the route b}' which it is administered.
`Had our first experiment used suboptimal conditions
`for the administration of RPM, we would have found
`no difference in potency or efficacy between RPM
`and FK506 and might not ha\'C pursued our study or
`RPM. In retrospect, the mode of adminL~tration used
`at the outset was optimal and, under those condi(cid:173)
`tions, RPM was clearly more potent and effective
`than FK.506. This clear difference in pharmacological
`effect between these two structurally related mac(cid:173)
`rolides prompted our continued investigations of the
`activity of RPM. At the same time as thesC' studiC's
`were being conducted, investigators at the University
`of Cambridge, England, were testing the immunosup(cid:173)
`pressive activity of RPM in rodents, dogs, and pigs.1
`Simultaneous studics7n ' performed at Cambridge
`and by various groups of investigators at Merck
`Sharp and Dohmc Research Laboratories, United
`States showed that RPM and FK.506 affect immunc
`cells quite differently in vitro.
`
`Origin and Characterization of the
`Bacterium Producing RPMs
`RPM. (AY-22,989 [Fig 5)) is made by a filamentous
`bacterium from the strcptomycele group th:u wa~
`isolated from an Easter Island soil sample by Vczina
`et al and Sehgal ct al at A~erst Research Laborato-
`
`fllAPAMYCIN A11!1 OCtb ff.i:Z: OH
`OlM!lHOXYAAPA~"f'CIN R1a t\ Ri• OH
`
`'K501
`
`PRODRUQS OF RAPAMYCIN (R1• OCH3, R1a '" below)~
`
`N.N·DIMETHVLGLVCINATE
`METHANE SlAFONIC ACID SALT
`I
`o
`R,- 1-.,.A....•, Ot,SO ...
`
`3<( ... N-0£T\MAMINOlf'AOPIC)HATE HYllADCK.DAl>E SALT
`0
`
`II,-''~"- tO
`l..
`
`Figures. Chemical structures of RPM, 29-0cmcthoxyra(cid:173)
`pamycin, FI<.506, and the prodrugs of RPM.
`
`rics in the middle I 970s. 'r,n The aerial mycelium of
`this bacterium is monopodally branched (Fig 6),
`contains sporophores terminated by short, coiled
`spo1 c chains, and absorbs water. It was ultimately
`idrntili.ed as belonging to the species St~plom;'Ces
`lvig1osropic11s, designated by Ayerst Research as strain
`AY B-994, and deposited in both the ARS culture
`collection of the United States Department of Agri(cid:173)
`culture (assigned numpcr NRRL 5491) and the
`American Type Culture Collection (ATCC 29253). A
`structuraUy related compmmd/'r. 29..cfemethoxyra(cid:173)
`pamycin (AY-24,668 [Fig 5)) is coproduced with
`RPM. Another culture isolated from the same soil
`sample and designated AY B-1206 produces higher
`levels of RPM than A Y B-994 and little or no
`29-dcmcthoxyrapamycin."'
`
`Fermentation, Purification, and
`In Vitro Antimicrobial Activity
`ofRPMs
`Fermentation of RPM
`Soon after the availability of a pure strain of S
`ll)'gTOJCV/1icm. li'rmrntation condirions (type of media,
`media pH, and temperature) were \'aried to define
`its cultural characteristics."'"' Although this microbe
`grows and sporulatcs in a wide range of culture
`
`
`
`West-Ward Exhibit 1055
`Morris 1992
`Page 006
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`

`

`44
`
`Randall Ellis Moni.r
`
`Figure 6. (A) Photomicro·
`graph or the fi lamemous bar-
`0t('1 iu111, S ll)'grurropicus.
`that
`produces RPM (magnification
`x.J55). (B) Electron micro·
`graphofSh)'glfl1£npicus(magnili(cid:173)
`cation x'.l,500). (R .. print!"cl
`wiih pt·nnis~ion.'")
`
`conditions, more narrowl>' defined conditions are
`ncccssa1y for th<' optimum production ofR.Pi\1. RPM
`has been produced by aerobic submerged fermenta(cid:173)
`tion similar to that used for most antibiotics. Jnocu(cid:173)
`lum is prepared in two stages in a medium contain(cid:173)
`ing soybean meal, glucose, (l\'H1) 2SO,, and CaC01
`and used at 2%. For the fermentation in stirred
`ves~cls, thr. sta ni11g medium was soybean mc;d,
`glucose, (NH~)2SO,. and KH.zPO,. Glucose is fed
`continuously after the 2nd da>' and the pH was
`controlled at 6.0 with NH.OH. Maximum titers of
`RPM arc readlcd in 96 hours. Paper disc-agar diffu(cid:173)
`sion assays with Candida albicaru are used to deter(cid:173)
`mine the antibiotic titer.
`The fermentation methods required lo produce
`29-demethoxyrapamycin are the same as those de(cid:173)
`scribed for RP.tvL~;
`
`Purification of RPMs
`T he purification scheme (Fig 7) adopted for the
`production of RPM was developed ~hortly after the
`identification of the antifungal activity of RPM and is
`subsequently summarized." After fermentation, the
`pH of the beer is adjusted to 4.0. The mycelium,
`extracted with trichloroetha ne, is filtered olfand the
`extract is dried with anhydrous sodium sulfate to
`
`FERMENT STREPTOMYCES HYGROSCOP/CUS
`
`EXTRACT MYCELIUM WITH ORGANIC SOLVENTS
`
`APPLY CONCENTRATED EXTRACT TO SILICA GEL COLUMN
`
`+
`
`+
`
`ELUTE Willi ACETONE
`
`RAPAMYCINS
`
`Figure 7. Pcrinentatinn and isolation of"lU,Ms.
`
`produce about 500 gm of oily residue from a 160-litcr
`fermentation run. After extracting the residue \1~th
`methanol, the extracts are evdporatcd to yield approx(cid:173)
`imately 50 gm of residue that is then dissoh·ed in 15%
`acetone in hexane and mixed "~th silica gel. The
`dissolved RPM is adsorbed to the silica gel and
`remains bound to the gel after the mi xture has been
`fi ltered and \Vashed onto a colu mn from which RPM
`is r luted with an acetone:hexanc mixture. After
`c\·aporating the column eluate to dryness, the resi(cid:173)
`due is dissolved in ether from which pure crystals of
`RPM arc obtained. In this initial purification process,
`rcco,·eries of RPM are on the order of 40%; 10 L of
`broth produce 300 mg pure RPM. A more recent
`rneli1uu ufpurification has bct:n rcponed."
`·
`Excrpt for m inor modifications, the methuds
`described for the isolation of 29-dcmethoxyrapamy(cid:173)
`cin are the same as those used for RPM:..
`
`In Vitro Antimicrobial Activity of RPMs and
`Mechanisms of Their Antimicrobial Actions
`The antimicrobial screening program a t Ayerst Re(cid:173)
`search Laboratories identified RPtvl for its a ntifungal
`acti\'ity. RPM inhibits the growt h of yeasls and
`filamentous fungi including the dermatophytesMicm(cid:173)
`spom111 .f.Ji/JSlllll! and Tricl1opl!vlo11 granulosum."''1 The
`minimum inhibitory concentrations (MIC) of RPM
`against ten stra.ins of C albicam were in the range of
`less than 0.02 to 0.2 µ.g/m l, representing greater
`potency than that of amphotcricin B, nystatin, or
`candicidin in this assay. RPM has no antibacterial
`activity. The spectru m of a ntimicrobial activity of
`29-demethoxyrapamycin is similar 10 RPM, but its
`potcnc}' is only about 25% that of RPM although
`nearly as potent as amphotcrit:in B. y,
`Onr ~1ucly ha~ investigated the mechanisms by
`which RPM mediates its antifungal cffccts,;11 and the
`rcsulls of this study arc summarized in T a ble 2.
`Approximntcly 90 minutes after adding RPM to C
`albica11s cultures, growth is inhibited and subsc-
`
`
`
`West-Ward Exhibit 1055
`Morris 1992
`Page 007
`
`

`

`Rn/in11!Jri111
`
`45
`
`Table 2. ~lcchanisms of AntiliJngal Actions ofR11lVI
`Effie! of RP.II
`Trea/mml
`
`Trsl ·~)'Jlem
`
`Sut bose relent inn by C nlbirollJ.
`fncn:ased hcmolrsis of rat n·d
`blood cells, tlTiux ofK .. Pi,
`UV-absorbing material fmm
`Colbira11J.
`C alhicnn.1 ancrobic glycoly.;is.
`aerobic respiration.
`Protein syntht"sis b)' ccll-fn·c
`preparutions nf C 11lbicc111s. E
`roli, ral liver, :ind mitudmn,
`drial prt·parations ofC 11/birn11s.
`Amino :icid metabolism b)' glu·
`tamic-oxaloacctic trans:uni·
`nase, glutamit·P>'ruvate
`transanun~ in C olbir111is.
`Glucnsaminc and ~-ac-ctyl-glu·
`cosamim: in<.'rlrporatiun into
`whole C olbic111is.
`Oxidative dearnination or glu·
`tamic and asp.irtic acids in C
`a/bicom.
`Incorporation of glucusr into
`man nan in C albirn11s.
`Incorporation of Na :icetale :u1d
`methionine into tolal lipid of
`C olbico11S.
`Incorporation of adenine and
`phosphate into RNA ;111d D!'\A
`or c:: olbirol!S.
`Degradation of''P-labelled intra(cid:173)
`cellular macromolecules and
`kakagt through C 11/biro11S
`membrane.
`
`~ot inhibited
`;>\ot incn-ased
`
`Nol inhibited
`
`Not inhibited
`
`Knl inhibited
`
`:-;Ill inhibited
`
`Inhibited
`
`Inhibit<"d
`
`lnhibite<.l
`
`Inhibited mnrc for
`R.'\A than D:\A
`
`increased
`
`qucntly yeast cells lo~t· viability and begin to lyse. The
`candicidal actions orRPM differ from polycnc antibi(cid:173)
`otics that incrc:isc yeast cell permeability by binding
`to sterols in the cytaplasmic membrane, thus c:iusing
`leakage of cellular components. :-Jot only do sterols
`not reverse the actions of RPM, but RPM docs not
`increase the leakage of sorbosc or the efflux of
`potassium, phosphate, or W-absorbing materials
`from yeast cells.
`The effects of RPM on other metabolic systems of
`C nlbira11S have also lxcn investigated.:"' For c.'l:ample,
`RPM docs not inhibit anaerobic glycolysis or aerobic
`rrspiration, nor clot·s it inhibit the incorporation of
`glucosamine or N-acdyl-glucosaminc. RPi\•I dues not
`inhibit protein synthesis in cell-free preparations ofC
`n/bica11s, rat liver, or mitochondtia rrom C nlbicans.
`Although RPi\I inhibits the incorporation of glu·
`case into mannan ;111cl aCl'tate into lipids, the S)Tlthe·
`sis of glucan is minimally affected, indicating that
`
`inhibition of cell wall S)'llthcsis is not the primary site
`of the antifungal action of RPM.~'
`'111c mnst profound effects of RPM on C nlbicnns
`may also provide clues to its actions on mammalian
`. I
`cell~. for c.'l:ample, vrry low concentrations (.02 -
`µg/mL) of RPM inhibit the incorporation of adenine
`and phosphate into RNA and DNA. At the i\IlC for
`RPM, phosphate-cont<1ining molecules leak out of
`the yeast cell membrane. The degradation of these
`molecules, presumably including nurlcic acid~, seems
`to be promott'd in some way by RPM. lli
`
`Physico-Chemical Properties of
`RP Ms
`Structure of RPMs
`Although the initial analysis of the structure of RPM
`by infrared and nuclear magnetic resonancl' (l\'1v!R)
`spectroscopy did not provide the complete picture of
`its structure,~; these techniques indicated that RP.M
`was a complete\y new t)'PC of macrolidc antibiotic.
`Ultimately, x-ray crystallographic data clarified the
`structure ofRPM."'RPM is a 31-mcmbcred macrQcy·
`clc laclOnc containing an amide with a C 15 carbonyl
`and a lactonc with a C2 l carbonyl (fig 5). Additional
`analyses of the ,,C and 1H NMR spectra of RPM
`confirmed the x-raycrystal structure of RPM.:'' X-ray
`studies showed that RPM in its solid crystal form is
`conformationall)' homogeneous; in solulion however,
`RPM exists as a mixture of two conformational
`isomer; caused by tram toci.r amide isomcrization via
`hindered rotation about the pipecolic acid N-CO
`bond. The ratio of /rans to t:is rotamers in chloroform
`solutions is 31to 4: 1.~v"
`Iltustmtions of the stnu:turc of RPM were initially
`inconsistent: different enantiomcrs were drawn/' a
`novel numbering systrm was used/'' and incorrect
`stercochcmistrr at C28 was represented.;.' Ulti(cid:173)
`matclr, the correct stmcture was published,''' and the
`coordinates arc deposited in the Cl)"Stal data bank.
`Using ad\-anced 2-dimensional mm spcctrosropir
`methods, new assignments of the proton and carbon
`spectra for thr major rotamer of RPM have been
`made and a new numbering S}"Slcm suggested.'"
`The closest structural relative to RPM is the
`antif11ngal and immunosupprcssive mac.:rolidc FK506,
`which is also produced by a st rcptomycctc. ''1 FK506 is
`a 23-membercd macrocydc lactonc that shares a
`unique hemiketal masked a,13-dikctopipecolic acid
`amide substructure with RPM,11 but larks the Cl-C6
`triene segment of RPM.
`The results of' 'C-labcllccl acetate and propionate
`
`
`
`West-Ward Exhibit 1055
`Morris 1992
`Page 008
`
`

`

`46
`
`Rn111/11/I Elli.f ,\Jmri1
`
`and HG-labelled methionine incorporation studies of
`the biosynthcsis of' RPM were consistent with the
`proposed polykc·tidc pathway in which the carbons of
`the lactonc ring of RPM are dr rived from condensa(cid:173)
`tion of acetate and propionate units in a mannn
`similar to that responsible for fatty acid synthesis.
`The methyl group of methionine is an efficient
`source for I he three mcthoxy carbons of RPi\L
`Because none of the labelled precursors was incorpo(cid:173)
`rated into either the C)'dohcxane: or hcterocrclic
`t hr
`ring, t hese moieties probably orginalc from
`shikimale pathWi.l)' and lysine, respectively.'•!
`When 1H and "C N1v!R, infrared, UV, mass
`spectroscopy, and optical rotary dispersion/circular
`dichroism (ORD/CD) analyses were used to 1:0111-
`parc the structures of RPi\l and 29-demelhoxyrapa(cid:173)
`mycin, these molcculcswcrcshO\m to bcconfigurntion(cid:173)
`ally identical at all chiral centers and to have identical
`structural features at all but C29. Like RPl\l, approx(cid:173)
`imatd)• 20% of 29-demethoxyrapamycin in solution
`exists as the cir rot am er form."-'
`In addition lo the naturally occurring 29-
`demethox)rrapamrcin, amino acid ester analogt1cs of
`RPM have been synthesized ro produce three waler
`soluble prodrugs of RPivf" (Fig 5). The amine f(111c(cid:173)
`tions of the appended cslers t:an be converted to
`water soluble salts that a re enzymatically h)•drolyzccl
`in the plasma to produce RPlvI. Although RPM forms
`both monoestcr and diester adducts depending on
`the reaction conditions, only monocster salts arc
`
`clisrussccl bcc.1usc these arc sulliricnl ly waler soluble
`to ob,~ate the need for the clisubstilutecl forms. Thr
`'.21.:!-hydroll.-yl group of RP~l has been proposed as the
`site of cstcrification for each of these prodrugs, but
`this remains to be confirmed.
`
`Physical Properties of RPMs
`Table 3 lists the phrsical properties of RP~I. ".;o,"
`Although 29-dcmclhox)Tapamrcin is also a white
`crystalline solid, it has a lower melring point (107° to
`I 08°C) than RPM.:.. Both RPM and its 29-demethOll.')'
`form are lipophilic and onl)' minimally soluble in
`water. The water solubilities of both the mono-N,N(cid:173)
`climethylglycinate methancsu lfonic acid salt and the
`mono-N,N-dicthylpropionale hydrochloride salt pro(cid:173)
`drugs or RP~I arc more than 50 mg/mL. The water
`solubility or the mono-4-(p) 1 rolidino)butyratc hydro(cid:173)
`chloride salt prodrug is 15 mg/mL.'"
`Because :vnCs for the antifungal activity of RPM
`in vitro vary depending on the medium used and the
`length of the assay, it was suggested that RPl\1 is
`unstable.<" Subsequent studies showed that 5 µg /mL
`of RPM in uninoculatccl broth loses 80% nf its
`antimicrobial acti,~ty aftrr 7 days of incubation at
`37°C." Lnter anal)'Sis showed that 50% or the antimi·
`crobial activity of I or 5 µg / mL concentrations or
`RPM arc lost after only 2-t hours of incubation in
`culture medium.'"
`High-pressure liquid chromatograph)' (T-.IPLC)
`has nlso been used to examine the stability of RPM i11
`
`Table 3. Ph)sical and Chemical Properties nf'RP1'1
`31-Nlcrnbrn•cl 111am1cydic lactone C11H,,,N011 FW = 91+.2
`3-1: I ratio ol'cis-lrmu rotarncrs about t lic pipcculil' acid :\-CO bond
`White, crystalline solicLvfi> 183- 185 C
`Solubilitr:
`2