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`V Morris,Transplantatjormeviéws,1992,6(1):39¢87
`
`*
`
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`
`West-Ward Pharm.
`Exhibit 1022
`Page 001
`
`

`

`West-Ward Pharm.
`Exhibit 1022
`Page 002
`
`West-Ward Pharm.
`Exhibit 1022
`Page 002
`
`

`

`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`Rapamycins: Antifundgal, Antitumor,
`Antiproliferative, an
`Immunosuppressive Macrolides
`
`Randall Ellis Morris
`
`l'i'7ml us’know in: drape Wlmi we don ‘I Avzrm: is an nmnz.
`[mitt rVm;lou
`
`Progress in rapamycin (RPM) research has been
`rapid and is poised to accelerate even more
`dramatically. An Investigational New Drug applica-
`tion (IND) for phase I trials of RPM as a treatment
`for prospective graft recipients was approved less
`than 2 years after the first published reports"? and
`public disclosure3 of the ability of RPM to prolong
`grail survival in e.\;perirnental animals. RPM is 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 structurally similar to another new
`immunosuppressivc: macrolide, FKSOG. RPM is par-
`ticularly intriguing because it inhibits the activation
`of immune cells by unique, relatively selective, and
`extremely potent and highly efl‘eoti\fe mechanisms.
`For example, one half mierogram of RPM adn1inis- ~
`tercd daily to mouse recipients of completely mis-
`matched heart allografts prolongs graft survival.
`When these mice are treated for only 2 weeks witli
`higher doses ofRPM, or when a single dose ofRPM is
`administered to rat heart allograft recipients, strain-
`speeitic unresponsiveness is induced, and grafts sur-
`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 19503 through
`the 19705 to our current focus on the microcosm of
`
`cellular and molecular immunopharmacology. A rev-
`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
`
`’ Fmm (ht Lnboratiqyfir‘ Trmujzlmilritimt InznmImi'Qg1', Dqlmrllmfril of
`Cardin!/mart’: Surgery. .S'!arg’&rd linizverrigy Sr/ml qfzlletiiziiie, Slm_ifi»m’_.
`CA.
`
`Ada’Ir.r.v to/Hint mquzarlr la Randall Elli: Mamlr, MD, Lalirirnlniyjixr
`Tf(lI1.S'[l[l1nI(1Ii0Il1!IlIlllmI)[l{g)g Departmenl {1} Iizrrdmtharant Singer)‘, ‘Slan-
`jiml L'ninerriQ' Sr/molQ/'Me11it'im:, $Iru_ifmfl CA 94305-5247.
`Ca[gi'rright 5? 1992 1;): ll-‘.13. Saumims Cam/mrgr
`09:15--I70X/532/ (K01-000435.00/V’U
`
`accrue the four imperfect mainstays of immunosup-
`pression for transplantation-«steroids, azathioprine,
`anti-T-cell antibodies, and Cyclosporin, A (CSA). In
`1992, six new xenobiotic immunosuppressants will be
`in clinical trials (Fig I).
`This new era in immunosuppression can be traced
`to the convergence of several lines of research: (1)
`the discovery and successful clinical use of ClsA; (2)
`an increased understanding of the fundamental biol-
`ogy of immune cells that enables the actions of
`different immunosuppressants to be better under-
`stood and thus lay the foundation for more rational
`means to discover, develop, and use improved drugs;
`and
`organized preclinical’ research programs
`designed to identify potentiallyvaluable immunosup-
`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 the Laboratory for Transplantation Immunol-
`ogy at Stanford University that enabled us to identify
`RPM3“ and the morpholinomethyl ester'oFrnycophe-
`nolic acid (l\/lIPA)""'"l as immunosuppressants for
`graft rejection. The mechanisms of action and immu-
`nopharmacology of these two compounds, as well as
`FK5O6,"”‘9 deoxyspergualin (DSG),°:"2' and brequinar
`sodium (BQRY2 have also been studied and com-
`pared with one another in our laboratory.
`Our spectrum of experimental systems begins
`with in vivo mouse models that are so rapid, quantita-
`tive, and inexpensive that we have been able to
`evaluate hundreds of molecules for suppression of
`alloimmunity. The vast majority of these drug candi-
`dates fail during testing in rodents because they lack
`efficacy or safety, and they are discarded quickly so
`that our resources can be concentrated on com-
`
`pounds with the greatest potential. Compounds that
`show promise are evaluated further in rodent models
`to identify those with the following ideal characteris-
`tics: (1) unique mode of action; (2) high efficacy for
`the prevention or treatment of acute, accelerated, or
`- chronic rejection; and (3) low toxicity. This Darwin-
`ian selection process accomplishes two tasks: first, it
`insures that only the agents with the greatest poten-
`
`Tmnrlzlmziation Rcrimts, Vol 6, No 1 Ianurlgj, 1992:/1/2 39-57
`
`39
`
`West-Ward Pharm.
`Exhibit 1022
`Page 003
`
`

`

`Rrlmla/I Ellis ;Ifan'i.v
`
`MIZORIBINE
`DEOXYSPERGUALIN
`FK506
`MYCOPHENOLIC ACID
`RAPAMYCIN
`BREQUINAFI SODIUM
`
`CYCLOSPORINE
`OKT3 MA!) 3. omen MAbs
`
`CORTISONE
`AZATHIOPRINE
`ANTI-T cam. Abs
`
`1950
`
`1950
`
`1970
`
`1980
`
`1990
`
`2000
`
`Figure 1. Hisuiry niche us? nlclrugs usecl In coniml grail i‘cjeL‘.li0n. All olthe lbllowing xenuliiutius recent lycliscuxwccl to
`suppress graft I‘(f_jECIiOI1 in preclinical nmtlels lmvc aclvnnitecl In clinical
`lrizils:
`the nminietabolitcs such as rnizmibine
`(MZR), IVIPA in its proclrug form ul RSA6H=1»3, mid BQR; the cyclosporinolilae clrug FK505, and drugs that define two new
`ClElSSE$ of im mu nnsu ppressants, DSG and RIKVI.
`
`tizil are advanced to the expansive nonhuman pri-
`mate transplant model; and second, it prepares us to
`be able to use these compouncls intelligently in
`nonhumzui primates. The nonhuman primate model
`is important because it is highly prcclictivc of the
`sziibty and eflicacy 0l‘zi test drug in humans. The sum
`of all knowledge proclucecl fromwelJ—plm1neclpreclin-
`
`ical studies is the esscnlial l.biin(|atiuxi from which
`
`successful clinical trials are clcsignccl imcl cxccuted.
`New drug dcvclopmcnl: is a highly complex, multidis-
`cipI.ina1'y task, and our contribution to the dcvc:l0p~
`ment and clinical use of" new immimosupprcssants
`clcpcncls on very close colluborzition Mth scientists
`and clinicizms in the pharm ziccutical l11EluSI.'I'}’.
`
`FUNDAMENTAL
`IMMUNOLOGY
`
`TRANSPLANTATION
`
`AUTOIMMUNE
`DISEASES
`
`l-- CLINICAL TRIALS *-l
`'ul"Ti';ms Izmlulion l1]1l111lhOlD”'YZ1l Sluninrcl
`Fi ure 2. Sclimnzitic re )rcsemz1iinn olthe Jru mm used at the LL1l‘JLII”Z1I(')I
`5‘
`a
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`y
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`.
`Uii1\’m‘s|ly to icleiiLil'y min pounds with Immiuiusuppressive i1C[l\'ILI€S Fm‘ tmnsplzimzitiuii and Lu develop these compmmils
`lb!‘ clinical use lbr the 3i*c\’r.=nti0n and ll‘t‘fl1lT](’.n[ Dl\l‘L“(‘,(‘liDn. Funrlamcnlxil l<11m\=lc(l1rnl‘lIii’. immune svsicm mu Jlctl wit h
`.
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`rm zipprcciatiun DI
`the (.‘l]i1l‘ElClET1Sll('S ml the drug c::m(I1clz1I(* is usrrcl in Clt‘SIg1}'L’Xp€l"ln1I:fllS
`Lu prohle the ziclmty ol the
`cuinpmuid and dc'fim3 its n“l(‘L‘lL'1l1iSmS Ollicliuii. Hcterumpic Lrzmspluiilziiiun 0IiiL~c)Ii:LI;il I11ULlSt:llt‘Ell‘l LIllUgl’EIrl5ll]LUll1tfL‘iI.l'
`)innz1(—> of mnusc rcci iiems unrl nllnzimi renic and mini rmic: stimuli iii‘
`:1 ilitral lvm ah nnclc h ‘DC!’
`l1lSiiL arc usccl as F21
`icl
`.
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`I
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`El
`and quumiiriiive bioassnvs I)c*.f0n? pmce.etling Io (hr: more laborious techniques ul p1’iITlL1l‘Ily’\‘fl$CLllQ.l‘i/1BLl
`l‘lBIEI‘Ul0])lC
`(£1l)ClUI11l1lfll) mid Sl:‘CUlILli1l'Il§'\v’iLS(.ILIlilJ'lZl:Ll lIL‘I(.’l'ULU[Ji(.‘ (subrc—u:il capsule) lwurl uliiigiull 21 nd .\cn0gml'i iuxnspluntinn in the
`rat. ASSl‘SSIHL‘IIl, ril llic clhmm; rmcl the safciv ulthc mm muntl in cvnomnl ms mcmkm‘ rvri nicnls of l1(’lt’.!'UiO air zillo i‘zil"ts
`‘
`.
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`precceclcs pliase I (‘lII)lL‘U.l trials in minsplzim patients zuicl p'c1Ut’I'lIS\\'IIl1 ziutoimimime LilSEL1S("S.
`
`West-Ward Pharm.
`Exhibit 1022
`Page 004
`
`

`

`Rn/iu/ngrilo
`
`Oa2"«DEPENDENT Gan?‘-INDEPENDENT
`r"“"“""‘“—"|
`r"““"'—‘1,
`LIGANDS
`LIGAND
`+
`0'
`TCR-CD3 CD4/CD8
`CD28
`
`J
`¥('JD2LFA-1x
`.
`_ _, © .—
`
`CSA
`ntsoa
`
`.
`
`a°.._..c.,
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`it
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`
`_ _,
`
`I@ , © FULLY
`DIFFER-
`
`‘
`
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`
`(6% @\@ __©
`
`RPM
`(8 CELL)
`
`MZR
`MFAnan
`(5 CELL)
`s ..G2..hI
`
`ass
`(3 CELL)
`
`Figure 3. Schematic representation of the possible sites of" action of the following imnmnosuppressants on uctivzttecl T
`tells; CSA and FK506 prevent the transcription ofearly phase cytokine genes; RPM inhibits the signal transduction ol‘IL-2
`bound to its receptor and may have otherniitiprolilierative cilects unrelated to lympholtine. signals; MZR, MPA, and BQR
`all inhibit purine (MZR, MFA) or pyrimidine (BQR) nucleotide synthesis; DSG seems to inhibit late stages of T-cell
`mat urzition. RPM, MZR, MFA, BQR, and DS_G also act on activated B cells at the sites shown.
`
`Even more important than the relatively large
`number of newiimrnunosuppressants that have been
`discovered is their variety. Each of these. new mole-
`culestsuppresses the immune system by blocking
`distinctlydiffercnt biochemical reactions that initiate
`the activation of immune cells that cause the many
`Forms of graft re_jet:tion (Fig 3). (Briefly,
`and
`F_K50_6 act soon after Ca“—depenclent 'T—cell activa-
`tion to prevent the synthesis ofcytokines important
`for the perpetuation and amplification of the im-
`mune response.”""5 RPM acts later to block multiple
`effects ‘of’ C}i"[(‘)liilnCS on immune cells including the
`inhibition of interleukin-2-—(IL—2—)trigge red T—cell pro-
`lil‘c:ration,""“" but its antiproliferative effects are not
`restricted solely to T and B cells. ‘RPM: also selectively
`inhibits the proliferation oligrowth factor—depemlent
`and growth Factor-independentnonimmune cells.
`Mizoribinc (MZR),“ l\/IPA,” and BQR“ are antinuc-
`tabolitfes that inhibit DNA synthesis primarily in
`lyrnpliocytes. These new antimetabolites are more
`selective than ztzathioprinc because these com-
`pounds block the activity of enzymes restricted only
`to the de novo purine or pyrimidine. hinsynthetic
`pathways. Lymphocytes are more dependent on these
`pathways for nucleotide synthesis than other cells.
`Recent revicwsm“ discuss these and other immu-
`nosuppressants. RPM has recently been the subject
`of‘ four brief’ reV'ie\«=s,7'3”““ E1 long review,"’*' and has
`been included in reviews that have primarily focused
`on FK506.‘"”*" This l'CVi(iW provides a complete profile
`of RPM from work published through the end of
`August l99l. Despite the progress made in under-
`standing RPM since the first publication on this
`compound in l975,’“ the description of its ability to
`suppress graft
`rejection has stimulated renewed
`
`interest by a wide variety of investigators whose work
`has not yet been published. As 21 result, research on
`macrolide immunosuppressants has become fluid
`and extremely Fast-pacecl. Because unpublished data
`generally are not available for evaluation, I have not
`referred to unpublished work or personal c.oinmtxni-
`cations. However, I have relied on many studies of
`RPM from the Laboratory oi‘.Transpl_antation Immu-
`nology atgStanford University that have yet to be
`published in full. In most of these cases,’ I have
`supplied the data from which conclusions in the text
`are drawn.
`K
`'
`I
`
`Because this review is being written relatively
`early in the research life of RPM, and because the
`majority of the work on this complex molecule has
`yet to be published, the material subsequently pre-
`sented should be regarded more as a preview rather
`than as a review. At the very least, 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 many investigators
`with highly specialized interests, only selected sec-
`tions will be of use. For otl‘u':rs, it
`is essential
`to
`understand all that is knoxm about a new and unique
`molecule such as RPM. VVithout an understanding of
`RPM that is both deep and broad, itwill be difiicult to
`meet the challenging tasks of using RPM as a tool to
`learn more about the irnmnnc system, maximizing
`its tlierapcutic potential, and discovering new and
`improved members of this class ni‘immunosuppres—
`szmt. Ifwe strive to understand thoroughly the little
`that is now known about RPM, we will make more
`eficient and rapid progress toward our goal of
`understanding all oi’ the important biological effects
`of this molecule.
`
`West-Ward Pharm.
`Exhibit 1022
`Page 005
`
`

`

`Rmzdrzll EL/it A-lam‘ F
`
`———>
`
`FKSDE
`EVENT
`
`CYCLOSPORINE
`
`DATE
`1972- SPECIFIC SUPFIESSIDN
`1975
`OF IMMUNE CELL
`ACTIVAUON SHOWN
`IN VITHO AND IN ‘(NO
`
`1950
`1983
`
`1992
`1963
`
`FDA APPROVAL AND
`COMMON CUNICAL USE
`IN TRANSPLANTATION
`
`I '
`
`SCREEN
`PERMENTATIOM
`PRODUCTS FOR
`NHIBITDRS OF
`THE MGUSE MIXED
`LYMPHOCYTE REACTION
`IIvlMI}NOSUPPfiESS«'VE
`ABTIVITY SHOWN IN
`VITRO AND IN VIVD
`UMDUE, RAPAMVCIN LIKE
`STRUCTURE DEFINED
`
`RAPAMYCIN
`
`'
`
`ANTIFUNGAL ACTIVITY.
`LOW TOXICITY SHOWN
`IMMUNOSUPPHESSION
`Oi’ AUTDIMMUNE
`DISEASE SHOWN
`UNIQUEMAEROLIDE
`STRUCTURE DEFENED
`
`INVESTIGATION
`OF RAPAMYCIN
`AS AN IMMUNOV
`SUPPRESSANT FOR
`TRANSPLANTATION
`
`l
`EAPAMYEIN
`SHOWN TD PROLONG
`ALLOGHAFT SCIFIVIVAL
`
`Figure 4. Evolutionanf path of RPM :15 an immunosup—
`pI‘t1SS’:UIl for ttrmsplrmtztlion.
`
`In acldition to reviewing the informtttioii on RPM,
`this article warns of tho danger of incltictivc reason-
`ing in which, in an zidolcsccnt liclcl like immunology,
`ilI‘g‘I.'IiI”Ig from highly specific cases to general laws
`
`often promotes the illusion ofknowlcclge rather than
`its true ztcquisition. However, by interrclzxting infor-
`mation COI1(.'(’.I'I‘IlI1g‘[l1C stx—‘ucture, the ITIUlBCLIl'c1I‘I1ICCl1~ '
`anisms, and the actions OFRPM on clttliiiccl (‘fill E‘)/'[)C',S
`in vitro, its clllccts in \1'vo, as well as its disposition in
`the body and its toxicity, new and important insights
`into the actions of RPM can he gainccl. In gCl‘iCI‘i1l,
`the conceptual tools used in this rcxricw to analyze the
`data from exper_imc*nts on RPLVI can be applied to the
`stucly oFmz—u1yotl1r:r immnnosiipprnssnnts, nspccially
`other xcnobiotics.
`
`Before dissecting and examining every aspect of
`RPM in cletail, it is worth rtzviewing the evtints that
`led to the attention RPM is now receiving. Figure 4
`shows the relationship of the evolution of RPM as an
`lITII‘l'ILI!‘IOSU,ppI‘CSSEll1C to the clevt-rlopment of GSA and
`FK506 as immunosuppressantsi Table l provicles a
`more detailed outline of the sequence of the main
`events that have clcfinod p1‘ogress in RPM research in
`its first I5 yeai‘s.‘”"”7"”""W The ancestors of RPM are
`CsA and FKBOG. As shown in Fig 4, CsA stimulated
`the orgztnizzltion ol‘ :1 ratioiiztl stzmoniiig program
`clesignecl
`to cliscovcr other lertnentatioii products
`with no cchzinisms ol’imrntmosupprcssive ztction iden-
`tical to CSA. The discovery ol'FK506 was the procluct
`of this program,‘“ and when the strticttire of FK506
`was dcliiied, its similzirity to the sttuctun: of RPM
`was imincdiatcly recognizocl.” Years before,
`the
`structure of RPM had been ClC[CI‘I‘l1lI‘IC(l as 21 consc-
`
`Table 1. Histonj ntRPM Drug De.vr.=lnpmenl: The First l5 Years
`
`Discnvmy
`
`Isolation lirom EastI=,1 Island (Rapn ‘.\lui) soil
`sample and clittrzicterization ol’ antimicio
`blill activity
`In vivo use:
`Toxicity
`Pharmacol<int*tirs
`Biozwztilztlaility
`Alttiluiigttl activity
`lmmunosupprcssion 0ll{ILIl0ll]1l1] Line dis-
`ease.
`ElI.ICl(li1I.lI)II ofst.1"uctuI"e
`Antitumor activity clescrihc-cl
`Imimmosuppression otnllogrilh I‘t‘jt‘:(‘llt7II
`Rl’l\‘l alone
`
`RPM in combinzttion with Cs/\
`r)ll'TI“l‘l’,fl[l21llI,)I”=, f)l‘E‘,l'TI3ClS U!VR1)A;1 and FKSOG
`on immune sells in vitro
`
`DlH['?l‘f’I1lI21llDI1 0liCll‘r“.ClS ol‘l{P;\"l and l7l{f)lIli
`on immune system in vivo
`l)cinoI'l.s'I.i;1tiun llllbllltllllg ul‘Rl’;\/l to FKEOG
`binding protein
`
`Year
`
`i975
`
`I978
`
`1977
`
`I980
`l9H I
`
`I989
`
`l 990
`I989
`l 990
`
`1990
`
`lE)ll9
`
`7
`
`Rtfirmmt
`
`VL*'/.ii1a,Ki1(lclski, and Sfillgllliil
`Sehgzil, Baker, and Vezinzt”
`
`Ht1lm1',Sicloi'o\viit7., Sehgal, et ol M
`
`Martel, Klicius, and Gtxlctlil
`
`Fincllziy zmtl Rilfllflsw
`Donros and §iil‘li1t3ssl'
`
`A\’IUlTlS and hleiserl
`Clalnc,Cnll1cr. Lint, or all
`Mt-ism‘, Wong, and Morris"
`Tomi, Mutltovicli, Gollittr, rt 41”
`i\‘l€lC3lfC and Ricliai‘cls“l‘
`Dumont, Stztruch, Kopmk. at :1!”
`Morris, Wu, and Shortliouscl
`
`I-lztrtling, Gzilnt, Uehling. ct it!“
`
`West-Ward Pharm.
`Exhibit 1022
`Page 006
`
`

`

`R11/Iurigrriru
`
`quence oi‘ the identification ol"RPM as an antifungal
`antibiotic (“able 1). Shortly after the antibiotic
`activities of RPM were described, it was found to
`have immunosuppressive activity. Thiswas only a few
`years after the immunosuppressive activity of CSA
`was discovered, but ironically, RPM was not devel-
`oped as an immunosuppressant at that time. In 21
`review“ of immunosuppressive agents published in
`1988, Devlin and Hargrave encouraged _“.ii detailed
`comparison of the biological profile of these mac-
`rolides [FK5D6 and RPM] .” These investigators sug-
`gestion was based on the structural similarity of both
`compounds and their known immunosuppressive
`activity.
`Sehgal was aware that investigators at the Labora‘
`tory for Transplantation Immunology at Stanford
`University had developed aquantal bioassay for the
`evaluation of immunosuppressant potency and ef—
`fieaey, had validated the assay with CSA,“ andhacl
`used it
`to study FK506." In i988, he offered to
`provide us with enough RPM to enable us to deter-
`mine whetherits activity differed from FKSOB in
`mouse as well as rat heart transplant recipients. As
`subsequently discussed,’ the activity of RPM is ex-
`tremely dependent on the vehicle in which it is
`suspended and the route by which it is administered.
`Had our first experiment used suboptimal conditions
`for the eidniinistration ofRPM, wewould have found
`no difierenee in potency or efficacy between‘ RPM
`and FK506 and might not have pursued our study of
`RPM. In retrospect, the mode of administration used
`at the outset was optimal and, under those condi-
`tions,
`was clearly more potent and effective
`than FK506. This clear difference in pharmacological
`, effect between these two structurally related mae-
`rolides prompted our continued investigations of the
`activity OFRPM. At the same time as these studies
`were being conducted, investigators at the University
`ofCambridge, England, were testing the immunosup-
`pressive activity of RPM in rodents, dogs, and pigs}
`Simultaneous S[l1(llCS‘!w” performed at Cambridge
`and by vaiious groups of investigators at Merck
`Sharp and Dc-hine Research Laboratories, United
`States showed that RPM and FK506 affect immune
`cells quite dilfercntly in vitro.
`
`Origin and Characterization of the
`Bacterium Producing RPMS
`
`RPM (AY-22,989 [Fig 5]) is made by at filamentous
`bacterium from the streptomycete group that was
`isolated from an Easter Island soil sample by Vezina
`et al and Sehgal et al at Ayerst Research Laborato-
`
`fiAPAM‘lClH' R1: OCH: Ha: OH
`DEMETHOXYHAPAMYCIN R‘: H. R1: OH
`
`FKEOB
`
`PRODHUGS OF RAPRMYCIN (H1: DCH3, fig: sad below):
`N.N—DlMETH‘!LGLYClNATE
`METHANE SULFONIC ACID SALT
`O
`l
`i~D/ll\/“x (}I,5o,H
`
`H?‘
`
`3~(N.N—DlETHYLAM|NO)Pl'\ClFlONATE , HYDROCHLORIDE SALT
`O
`
`5:‘
`
`i~¢/ix/~,,/\ g Hex
`K _
`
`4-(FYHHOLIDINQSUTYHATE HYDROCHLORIDE SALT
`0
`‘iv ‘*0/ll\/\/"(\:€l,
`in
`Figure 5. Chemical structures UFRPM, 29-(ZlEl"nCll]D.‘(y‘ti1-
`pzimyein, FK5U6, and the pmdrugs ol'RPM.
`
`ries in the middle 19705.“? The aerial mycelium of
`this bacterium is monopodally branched (Fig 6),
`contains sporophores terminated by short, coiled
`spore chains, and absorbs water. It was ultimately
`identified as belonging to the species Strzptanyicer
`/z_1xgrascupi1;zo‘, designated by Ayeist Research as strain
`AY B994, and deposited in both the ARS culture
`collectionvof the United States Department ol‘Agri-
`culture (assigned number NRRL 5491) and the
`American Type Culture Collection (ATCC 29253). A
`structurally [related compound?" 29-demethoxyrzv
`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 AY B994‘ and little or no
`
`T29-demetlioxyrapainycinffl
`
`Fermentation, Purification, and
`In VitrotAn1:imi('i:robial Activity
`0f'RPMs
`
`Fermentation of RPM
`
`Soon alter the availability of it pure strain ol‘ .5‘
`/gygmtro/Jiczo, Fermentation conditions (type of media,
`media pH, and temperature) were varied to define
`its cultural charactei'istics."”"” Although this microbe
`grows and sporulates in a wide range of culture
`
`West-Ward Pharm.
`Exhibit 1022
`Page 007
`
`

`

`Randall Ellis Mt/ml:
`
`(A) Photomicro-
`Figure 6.
`graph of the filamentous bar-
`teriuni,
`.5‘
`/z_y_grmm/nicur.
`that
`produces RPM (magnification
`X455).
`(B) Electron micro-
`graphofS/iygi-mrafzicur(magnifi-
`cation X1500)‘
`(Reprinted
`with pet'1nission.'"‘)
`
`conditions, more narrowly defined conditions are
`necessary for the optimum production ofRPM. RPM
`has been produced by aerobic submerged fermenta-
`tion similar to that used for most antibiotics. Inocu-
`lum is prepared in two stages in a medium contain-
`ing soybean meal, glucose, (NI-I,)2SO4, and CaCO,
`and used at 2%. For the fermentation in stirred
`
`the starting medium was soybean meal,
`vessels,
`(l\lH,)2SO,, and KI-I9PO,f. Glucose is Fed
`glucose,
`continuously after the 2nd day and the pH was
`controlled at 6.0 with NI-LOH. Maximum titers of
`RPM are reached in 96 hours. Paper disc-agar dilTu-
`sion assays with Caridida albicuzu‘ are used to deter-
`mine the antibiotic titer.
`
`The Fermentation methods required to produce
`29-demethoxyrapamycin are the same as those de-
`scribed for RPl\ 7"
`
`Purification of RPMs
`
`The purification scheme (Fig 7) adopted for the
`production ol" RPM was developed shortly after the
`identification of the antifimgal activity of RPM and is
`subsequently suinmarizcdfu After fermentation, the
`pH of the beer is adjusted to U). The mycclium,
`extracted with trichloroethane, is filtered oFFand the
`extract is dried with anhydrous sodium sulfate to
`FERMENT STREPTOMYCES HYGROSCOPICUS
`
`l
`EXTRACT MYCEUUM WITH ORGANIC SOLVENTS
`
`l
`APPLY CONCENTRATED EXTRACT TO SILICA GEL COLUMN
`
`1
`ELUTE WITH ACETONE
`
`l
`FIAPAMYCINS
`
`Figure 7. Fermentation and isolation of RPMS.
`
`produce about 500 gm ofoily residue from a 160-liter
`Fermentation run. After extracting the residue with
`methanol, the extracts are evaporated to yield approx»
`imately5O gm of residue that is then dissolved in 15%
`acetone in hexane and mixed with silica gel. The
`dissolved RPM 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 RPM
`is eluted with an acetonediexane mixture. After
`
`evaporating the column eluate to dryness, the resi-
`due is dissolved in ether from which pure crystals of
`RPM 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 modifications,
`the methods
`described for the isolation of 29-demethozxyraparny
`do are the same as those used for RPM_“"‘
`
`In Vitro AntimicrobialActiv1'ty of RPMS and
`Mechanisms of Their Antimicrobial Actions
`
`The antimicrobial screening program at Ayerst Re
`search Laboratories identified RPM for its antifungal
`actixity. RPM inhibits the growth of yeasts and
`filamentous Fungi including the dermatophytesMicm-
`sponmz _.g};1J5ezn1z and Trio/20])/yilali granulasttrnllw The
`minimum inhibitory concentrations (MIC) of RPM
`against ten strains of C a/biL'an.s were in the range of
`less than 0.02 to 0.2 ug/ml, representing greater
`potency than that of amphotcricin B, nystatin, or
`candicidin in this assay. RPM has no antibacterial
`activity. The spectrum of antimicrobial activity of
`29—demethoxyrapamycin is similar‘ to RPM, but its
`potency is only about 25% that of RPM although
`nearly as potent as amphotericin BJY’
`V
`One study has investigated the mechanisms by
`which RPM mediates its antifungal effects,” and the
`results of this study are summarized in Table 2.
`Approximately 90 minutes after adding RPM to C’
`albicazzr cultures, growth is
`inhibited and subse-
`
`West-Ward Pharm.
`Exhibit 1022
`Page 008
`
`

`

`Table 2. Mecltanisms oFAntilungal Actions of RPM P
`R
`M
`i
`am: :y'1U~‘.-U
`Test Srslerrl
`Tmm‘zm=n(
`Nut inhibited
`Not increaserl
`
`V
`
`Not inltibilecl
`
`Not inhibited
`
`Not inhibited
`
`Not inhibited
`
`Inhibited
`
`Inhibited
`
`lnlnbitetl
`
`N Inhibited more for
`RNA. than DNA
`
`Increased
`
`7.
`
`C albimm:
`Sorbose retention
`Increased hemolysis of rat red
`blood cells, eliliix ol‘K'*, Pi,
`UV—absnrl)ing material Fmm
`C allrirant.
`C albimm 11116‘ robic glycolysis,
`aerobic respiration.
`Protein synthesis by cell—fre.e
`preparations ol‘C tzII)ictm:, E
`cult, rat liver, and mitochon«.
`drial preparations ol'C albiraltr.
`Amino acid metabolism by glu-
`tamlc-oxaloacetic transami-
`112156, glutamic-pyruvate
`transaminase in C‘ rtwirans.
`Glumsaniine and N-acetyl-glu-
`eosamine incorporation into
`whole Calbimns.
`Oxidative, dcamination alight-
`tamie and aspartic acids in (J
`ttlbiraru.
`V
`_
`i
`Incorporzition ofgltzcose into
`triannan in C alltftaiu.
`Incorporatio_n of.?\’a acetate and
`metliionine. into total lipid of
`C ztlbicam.
`,
`Incorporation of adenine and
`phosphate into RNA and DNA
`ofCnlbirt1ns.
`
`Degradation of“P-labelled-inti'a-
`cellular macromolecules and
`lezikage through C nlbiturtr
`membrane.
`
`Ra]:am_vrin.t
`
`_
`
`45
`
`inhibition ofcell wall synthesis is not the priinary site
`olithc, antifungal action oi'R.PM.5"
`The most profound effects of RPM on C albicanr
`may also provicle clues to its actions on mammalian
`cells. For example, very low concentrations {.02 ~ .1
`ug/mL) of RPM inhibit the incorporation ofaclenine
`and phosphate into RNA and DNA. At the MIC for
`RPM, phosphatmzonttiining molecules leak out of
`the yeast Cell meml)rani*. The (legratlation ol these
`molecules, presumably incluclingnucleitt acids, seems
`to be promotecl in some way by RPi\<‘I.3“
`
`Physoieo-Chemical Properties of
`RPM.s
`
`Structure ofRPMs
`Although the initial analysis of the structure ofRPM
`by infrared and nuclear magnetic resonance (NMR)
`spectroscopy’ did not provide the complete picture of
`its structure,“ these techniques indicated that RPM
`was a completely new type of macrolicle, antibiotic.
`Ultimately, x-ray'* crystallographic data clarified the
`structure ol'RPiVl.5" RPM is a 3 l-memberecl 1nacrqcy—
`ole lacttone containing an amide witha Cl l5 carbonyl
`and a lactone with a C21 carbonyl (Fig 5). Additional
`analyses of the "C and ‘H
`spectra of
`coiifirined the x-ray crystal structure ol:RPM.3" X—ray
`studies showed that RPM in its solid crystal form is
`Conformationallyt homogeneous; in solution however,
`RPM exists as a mixture of two conformational
`isomers caused by trans to (‘if amide isomerization tin
`hindered rotation about
`the pipecolic; acid N-CO
`bond. The ratio oftrans to cis rotamers in chloroform
`solutions is 3; to 4: I .3"""
`Illustrations of the structure ol‘RPM were initially
`inconsistent: different enantiomers were dratm,” a
`novel numbering system: was Lisedf" and incorrect
`stereochemistiy at C28 was represented.” Ulti~
`matcly, the correct structure was published,” and the
`coordinates are deposited in the crystal data bank.
`Using admnced 2~(.llmE3IlSiOI1Ell NMR spectroscopic
`methods, new assignments oi‘ the proton and carbon
`spectra for the niajor rotmner oi‘ RPM have been
`made and a new numbering system suggested.“"
`The closest structural relative to RPM is
`
`the
`
`autifungal and immunosuppressixrt, rnucrolitle FK:3l)b',
`which is also produced by a SlI’C[)i0lTl}'C€tt‘.:fi FK5l,l6 is
`a 23-memberecl macrocycle lactone that shares a
`unique hemiketal masked ot,B—diketopipecolic acid
`amide substructure with RPM,” but lacks the C1-C6
`tricnt: segment oi‘ RPM.
`The results of "C-labelled acetate and propionate
`
`queutly yeast cells lose viability and begin to lyse. The
`candieidal actions ofRPM differ from polyene antibi-
`otics that increase yeast cell permeability by binding
`to sterols in the cytoplasmic membrane, thus causing
`leakage of cellular components. Not only do sterols
`not reverse the actions of RPM, but RPM does not
`increase.
`the leakage of sorbose or the elllux of
`potassium, phosphate, or UV-absorbing materials
`from yeast cells.
`The effects ofRPM on other metabolic systems of
`C.'alln'rzm.t have also been iiivestigatedf“ For example,
`RPM does not inhibit anaerobic glycolysis or aerobic
`respiration, nor does it inhibit the incorporation of
`glucosamine or N—acetyl-glucosamine. RPM does not
`inhibit protein synthesis in cell-free preparations off}
`albimtts, rat live r, or mitochondria Pro in C.‘ albicaru.
`Although RPM inhibits the incorporation of glu-
`cose into mannan and acetate into lipids, the synthe-
`sis of glucan is minimally aFFccted, indicating that
`
`West-Ward Pharm.
`Exhibit 1022
`Page 009
`
`

`

`46
`
`Jtmztlall Ellis .-lflornli
`
`and "'C!-labelled methionine ineorponitioii studies of
`the lniosyntliesis oi‘ RPM were consistent with the
`proposed ])Ol}-'l~'.€[lCltf pzitl1w;1yi.n which the carbons of
`the lactone ring of RPM are clerived from condensa-
`tion of acetate and propionatc units in It mztnnrr
`similar to that responsible for Fatty acid syntlicsis.
`The methyl group of methionine is an efficient
`source For
`the three methoxy carbons oli RPM.
`Because none of the labelled precursors was incorpo-
`rated into either the cyclohexnne. or lieteroeyclic
`ring,
`these moieties probably orginztte [mm the
`shiltiimite pathway and lysine, respeetivelyfg
`\'Vhen 'H and “C Nl\*[R,
`infrared, UV, mass
`spectroscopy, and optical rotaiy dispersion/circular‘
`diehroism (ORD/CID) analyses were used to com-
`pare. the st1‘uctui‘cs oFRPt\I and 29-ClClT1Ctl‘t0)QFl‘£1]JEl-
`mycin, these molecules were shown to be configirzltioir
`ally identical at all chiral centers and to have identical,
`structural fttettu res at all but C29. Like RPM, approx-
`imately 20% of 29-demetlioxyrapamyein in solution
`exists as the ct: rotamer forin."”
`
`In addition to the naturally occurring 29-
`denictlioxyrziparnyciii, amino acid ester analogues 01‘
`RPM have been synthesized to produce three water
`soluble pmclrugs of RPM“ (Fig 5). The ztmine lithe-
`tions of the appenclecl esters can be converted to
`water soluble salts that are enzynmtically hyclrolyzctl
`in the plasma to produce RPM. Although RPM forms
`both monoester and diester nclducts depending on
`the reaction conditions, only monoester salts are
`
`clismssecl beceiuse these are stifliciciitly Wfttcl‘ soluble
`to ohvittte. the need For the tlisubstituted Forms. The
`
`‘.28-hytlrozcyl group of RPM has been pmposccl as the
`site. of esterification lot each of’ these ptodrugs, but
`this !‘t3l1l'c1lnS to be confirmed.
`
`Physical Properties of RPMS
`
`Table 3 lists the physical properties of RPM.’7“""""’
`Although 29-demothoxyrapamyein is also at white
`cnjstalline. solid, it has at lower melting point (107° to
`lO8"C) than RPt\vI.'_“l Both RPM and its 29-demethoxy
`form are lipophilie and only minimally‘ soluble in
`water. The water solubilities of both the mono-N,N—
`climethylglyeinute metlizinesiilfonie acid salt and the
`mo1io—l\’,i\l-cliethylpropionate hyclrochloricle salt pro-
`drugs of RPM are more than 50 mg/mL. The water
`solubility of the inono-*1-(pyrrolidino)butyrate hydro-
`chloride salt prodrug is l5 mg/niL.r"
`Because NLICS for the antifungal activity of‘ RPM
`in vitro vmy clepending on the medium used and the
`length of the assay, it was suggested that RPM is
`tinstalalef“ Subsequent studies sliowecl that 5 tug/mL
`of RPM in uninoculated broth loses 80% of its
`
`antimicrobial activity alter 7 clays of incubation at
`37°C.” Later analysis showed that 50% of the antimi-
`crobial activity of l or 5 pg/mL concentrations ol‘
`RPM are lost alter only 24 hours of incubation in
`Culture tl1€CllLlm.iii
`
`liquid chromzttograpliy (HPLC)
`l”lVlgl1-])l‘f:SSL1l‘C‘,
`has also been used to examine the stability QYRPM in
`
`__é_____
`Table 3. Physical and Chemical Properties ol’RP.\vl
`
`17W = €l1~l.‘2
`3 l-Memberecl m;1croeyt'licluetone C2;,,l'l;,,NO,3
`3-’l:l ratio Oiictil‘-M71115‘ rotnmers nlmut the pipecolie zicitl N-CO bond
`\/Vhite, crystalline solid MP 183-185 C
`
`Solubility:
`' ‘Z0 p.g/ ml. in \~':1tGl