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`Mortis, Transplantation Reviews, 1992, 6(1):39-87
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`ANSPLANTATION |
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`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`
`
`Rapamycins: Antifungal, Antitumor,
`Antiproliferative, an
`Immunosuppressive Macrolides
`Randall Ellis Morris
`
`What weknow iva dap. What we don’t know is anacean.
`oo
`:
`Lsiae Newton
`
`pees 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 disclosure’ of the ability of RPM to prolong
`graft survival in experimental animals, RPMis a
`macrolide fermentation product that has antifungal
`and antitumor activity. However, its effects on the
`immune system have generated the most interest
`because RPMis structurally similar to another new
`immunosuppressive macrolide, FK506. RPMis par-
`ticularly intriguing because it inhibits the activation
`of immunecells by unique, relatively selective, and
`extremely potent and highly effective mechanisms.
`For example, one half microgram of RPM adminis: :
`tered daily to mouse recipients of completely mis-
`matched heart allografts prolongs graft survival.
`When these mice are treated for only 2 weeks with
`higher doses of RPM,or whena single dose ofRPMis
`administered to rat heart allograft recipients, strain-
`specific unresponsivenessis 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 1950s through
`the 1970s to our current focus on the microcosm of
`cellular and molecular immunopharmacology.A rev-
`olution inthe discovery, development, and clinical
`use of new strategies.to control the immuneresponse
`is clearly upon us: it took more than 35 years to
`
`° From the Laboratoryjor Transplantation Imnuiialogy, Department of
`Cardiothoracic Surgery, Stanfard Universily Schoal ofMedicine, Stanford,
`CA.
`
`Alddress reprint requests ta Randall Ellis Morris, MLD, Laluratoryfor
`Transplantation Duniunology, Department of Cardiathoracie Surgery, Stan-
`jard University SchaalofMedicine, Siaford, CA 94305-5247.
`Copyright © 199? by WB. Saunders Company
`0955-470N/92) 0601-000485.00/0
`
`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 immunosuppressantswill be
`in clinicaltrials (Fig 1).
`This newcra in imititinosuppression can be traced
`to the convergence of several lines of research: (1)
`the discovery and suctéssful clinical use of CsA; (2)
`an increased understanding of the fundamental biol-
`ogy of immune cells that enables the actions of
`different immunosuppressants to be better under-
`stoodand thus lay the foundation fot more rational
`means to discover, develop, and use improved drugs;
`and (3) organized preclinical research programs
`designed to identify potentially valuable immunosup-
`pressants andto generate the knowledge needed for
`these agents to be. used intelligently in the clinic,
`Figure 2 shows the research program used for several
`yearsin the Laboratory for Transplantation Immunol-
`ogyat Stanford University that enabled usto identify
`RPM™*" and the morpholinomethy! esterofmycophe-
`nolic acid (MPA)'"5. as immunosuppressants for
`graft rejection. The mechanisms of action and immu-
`nopharmacology of these two compounds,as well as
`F506,"deoxyspergualin (DSG),™! and brequinar
`sodium (BOR)” have also been studied and com-
`pared with one another in ourlaboratory.
`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
`showpromise 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-
`
`Transplantation Reviews, Vol 6,No I (January), 1992; pp 39-87
`
`39
`
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`Page 003
`
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`Exhibit 1022
`Page 003
`
`

`

`40
`
`Randall Ellis Maris
`
`MIZORIBINE
`DEOXYSPERGUALIN
`FK506
`MYCOPHENOLIC ACID
`RAPAMYCIN
`BREQUINAR SODIUM
`
`CYCLOSPORINE
`OKTS MAb & OTHER MAbs
`
`CORTISONE
`AZATHIOPRINE
`ANTI-T CELL Abs
`
`1950
`
`1960
`
`1970
`
`1980
`
`1890
`
`2000
`
`Figure 1. Historyof the use of drugs used to control graft rejection. All of the following xenobiotics recentlydiscoveredto
`suppress graft rejection in prechinical moclels have advanced to clinical trials:
`the antimetabolites such as mizortbine
`(MZB), MPA inits prodrug form of RS-61443,and BOR; the eyelosporine-like drug FIK506, and drugs that define two new
`classes of immunosuppressants, DSG and RPM.
`
`tial are advanced to the expensive nonhuman pri-
`mate transplant model; and second, it prepares us to
`be able to use these compounds intelligently in
`nonhumanprimates, The nonhumanprimate model
`is important because it is highly predictive of the
`safety and efficacyof a test drug in humans. The sum
`of all knowledge produced from well-plannedpreclin-
`
`ical stuclies is the essential foundation from which
`successful clinical trials are designed and executed.
`Newdrug development is a highly complex, multidis-
`ciplinary task, and our contribution to the develop-
`ment andclinical use of new immunosuppressants
`depends on very close collaboration with scientists
`and clinicians in the pharmaceutical industry.
`
`FUNDAMENTAL
`
`IMMUNOLOGY
`
`TRANSPLANTATION
`
`AUTOIMMUNE
`DISEASES
`L CLINICAL TRIALS —_
`
`Figure 2, Schematic representation of the program usedat the Laboratory ofTransplantation Immunologyat Stanford
`Universityto identify compounds with immunosuppressive activities for transplantation and (o develop these compounds
`forclinical use for the prevention andtreatment of rejection. Fundamental knowledge ofthe immune system coupled with
`an appreciation of the characteristics of the drug candidateis used to design‘experiments to profile the activity ofthe
`compoundand define its mechanismsofaction. Heterotopic transplantation of neonatal mouse heart allugrafts into the ear
`pinnae of mouse recipients andalloantigenic and mitogenic stimuli ofpopliteal lymph node hyperplasia are used as rapid
`and quantitative bioassays before proceeding to the more laborious techniques of primarily vascularized heterotopic
`(abdominal) and secondarily vascularized heterotopic (subrenal capsule) heart allograft and xenograft transplantionin the
`rat, Assessmentof the efficacy and the safety of the compoundin cynomolgus monkey recipients of heterotopic allografts
`precedes phase I clinical trials in transplant patients andpatients with auloimmunediseases.
`
`West-Ward Pharm.
`Exhibit 1022
`Page 004
`
`West-Ward Pharm.
`Exhibit 1022
`Page 004
`
`

`

`
`
`Rapamycins Al
`
`Ga®*.DEPENDENT Ca?+-INDEPENDENT
`LIGANDS
`LIGAND
`+
`+
`
`\
`TCR-CD3 CH4cDs
`@SouLane =e
`
`|D2LFA4
`CYTOKINES ~2
`
`MZR
`CsA
`:
`MPA
`FK506
`BDSG
`BOR
`(CEL)«=(BCELL)«=(B CELL)
`
`Go—+ Gy
`SeGoeM
`
`.
`
`ot] FULLY
`- CELLS
`
`DIFFER.
`
`ENTIATED
`
`Figure 3. Schematic representation ofthe possible sites ofaction ofthe follawing ithmunosuppressants unactivated T
`cells: GsA and FIK306 prevent the transcription ofearly phase cytokine genes; RPMinhibits the signal transduction ofIL-2
`bound toits receptor and mayhaveother antiproliferative effects unrelatedto lymphokinesignals; MZR, MPA, and BOR
`all inhibit purine (MZR, MPA) or pyrimidine (BQR) nucleotide synthesis; DSG seems to inhibit late stages of T-cell
`maturation. RPM, MZR, MPA, BOR,and DSGalso act‘on activated B cells at the sites shown
`
`Even more important than the relatively large
`number of newimmunosuppressants that have been
`discovered is theit variety. Each of these new mole-
`cules ‘suppresses the immune system by blocking
`distinctly different biochemicalreactionsthatinitiate
`the activation of immunecells that cause the many
`forms of graft rejection (Fig 3): Briefly, CsA and
`FK506 act soon after Ca**sdependent ‘T-cell activa-
`tion to prevent the synthesis ofcytokines iimportant
`for the perpetuation and amplification of the im-
`mune response." RPM actslater to block multiple
`effects ofcytokines on immune cells including the
`inhibition ofinterleukin-2—(IL-2~)triggered T-cell pro-
`liferation,””' but its antiproliferative effects are not
`restricted solely to Tand B eclls.RPMalsa selectively
`inhibits, the proliferation ofgrowth factor-dependent
`and growth factor-independent: nonimmune cells.
`Mizoribine.(MZR),” MPA," and BOR™are antime-
`tabolites that inhibit DNA synthesis primarily in
`lymphocytes. These new antimetabolites are more
`selective than azathioprine because these com-
`pounds block the activity of enzymesrestricted only
`to the de novo purine or pyrimidine biosynthetic
`pathways. Lymphocytes are more dependent on these
`pathwaysfor nucleotide synthesis than othercells.
`Recent reviews” discuss these and other immu-
`nosuppressants, RPMhas recently been the subject
`of four brief reviews," a long review,” and ‘has
`been included in reviews that have primarilyfocused
`on FK506."" This review provides a complete profile
`of RPMfrom work published through the end of
`August 1991, Despite the progress made in under-
`standing RPM since the first publication on this
`compound in 1975," the description ofits ability to
`suppress graft
`rejection has stimulated renewed
`
`interest by a wide varictyof investigators whose work
`has not yet been published. As a result, research on
`macrolide immunosuppressants has become fluid
`and extremelyfast-paced. Because unpublished data
`generally-are not available*for evaltiation, I have uot
`referred to unpublished work or personal comimuni-
`cations. Hawever, I have relied on many studics of
`RPMfrom the Laboratory ofTransplantation Immu-
`nology at Stanford University that have yet to be
`published in full. In miost of these: cases,, I have
`supplied the data from which conclusionsinthe text
`are drawn.
`Because this review is being written -relatively
`early in the research life of RPM, and because the
`majofity of the work on this coniplex molecule has
`yet to be published, the material subsequently. pre-
`sented shouldbe regarded:more as a preview rather
`than as a review. At the very least, this articlewill
`provide a logical framework that otheriinvestigators
`cari use to organize and to evaluate newinformation
`on RPMasit is published, For manyinvestigators
`with highly specialized interests, only selected sec-
`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
`RPMthatis both deep and broad,it will be difficult to
`meet the challenging tasks of usitig RPM as4 tool to
`learn more about the immune system, maximizing
`its therapeutic potential, and discovering new and
`improved membersofthis class of immunosuppres-
`sant. If we strive to understarid thoroughlythelittle
`that is now known about RPM,we will make more
`efficient and rapid progress toward out goal of
`understanding all of the important biological effects
`of this molecule.
`
`West-Ward Pharm.
`Exhibit 1022
`Page 005
`
`West-Ward Pharm.
`Exhibit 1022
`Page 005
`
`

`

`42,
`
`Randall Ellis Moris
`
`
`
`
`
`SCREEN
`FERMENTATION
`PRODUCTS FOR
`INHIBITORS OF
`THE MOUSE MIXED
`LYMPHOCYTE REACTION
`IMMUNOSUPPAESSVE
`ADTIVITY SHOWNIN
`VITRO AND IN VIVO
`UNIQUE, RAPAMYCIN-LIKE
`STRUCTURE DEFINED
`
`
`INVESTIGATION
`
`
`OF RAPAMYCIN
`AS AN IMMUNO-
`
`
`SUPPRESSANT FOR
`TRANSPLANTATION
`
`
`
`
`
`
` CYCLOSPORINE=—> FKS06 —_— RAPAMYCIN
`
`often promotesthe illusion of knowledge rather than
`DATE
`EVENT
`its true acquisition. However, by interrelating infor-
`1972- SPECIFIC SUPACOSION
`4975
`OF IMMUNE CELL
`ACTIVATION SHOWN
`mation concerning the structure, the molecular mech- *
`IN VITRO AND IN YIVO
`anisms, and the actions of RPMondefinedcell types
`in vitro, its effects in vivo, as well as its disposition in
`the bodyandits toxicity, new anc important insights
`into the actions of RPMcan be gainecl. In general,
`the conceptualtools usedin this reviewto analyze the
`datafrom experiments on RPMcanbe applied to the
`study of manyother immunosuppressants, especially
`other xenobiotics,
`Before dissecting and examining every aspect of
`RPMin detail, it is worth reviewing the events that
`led to the attention RPMis nowreceiving. Figure 4
`shows the relationship of the evolution of RPMas an
`immunosuppressant to the development of GsA and
`FK506 as immunosuppressants. Table 1 provides a
`more detailed outline of the sequence of the main
`events that have defined progress in RPMresearch in
`its first 15 years.’"""""” The ancestors of RPMare
`CsA and FK306. As shown in Fig 4, CsA stimulated
`the organization of a rational screening program
`designed to discover other fermentation products
`with mechanisms of immunosuppressive actioniden-
`tical to CsA, The discoveryof FIK506 was the product
`of this program,” and when the structure of FK506
`was defined, its similarity to the structure of RPM
`was immediately recognized.” Years before,
`the
`structure of RPM had been determined as a conse-
`
`ANTIFUNGAL ACTIVITY,
`LOW TOXICITY SHOWN
`IMMUNOSUPPRESSION
`OF AUTOIMMUNE
`DISEASE SHOWN
`UNIQUE MACRGLIDE
`
`STRUCTURE DEFINED
`
`
`
`oe
`
`1975
`1978
`
`1880
`
`1983
`
`1982
`4983
`
`FDA APPROVAL AND
`COMMON CLINICAL USE.
`IN TRANSPLANTATION.
`
`1984-
`4986
`
`1988
`
`1968
`
`FAPAMYCIN
`SHOWNTO PROLONG
`ALLOGRAFT SURVIVAL
`
`Figure 4, Evolutionarypath of RPMas an immunosup-
`pressant fortransplantation.
`
`In addition to reviewing the information on RPM,
`this article warns ofthe dangerof inductive reason-
`ing in which, in an adolescent field like immunology,
`arguing from highly specific cases to general laws
`
`Table 1. History of RPM Drug Development: The First 15 Years
`Year References
`Discovery
`
`
`
`Isolation from Easter Island (Rapa Nui) soil
`sample andcharacterization of antimicro-
`bial activily
`In vivo use:
`Toxicity
`Pharmacokinetics
`Bivavailability
`Antifungal activity
`Immunasuppression ofautonnmune dis-
`ease
`Elucidation of structure
`Antitumoractivity described
`Immunosuppression of allograft rejection
`RPMalone
`
`RPMin combination with GsA
`Dilferentiation of effects of RPM and FK506
`on immunecells in vitro
`
`1975
`
`1978
`
`1977
`
`1980
`1981
`
`1989
`
`1990)
`1989
`1990
`
`/
`
`Differentiation ofeffects of RPMand FIK506
`on immunesystem in vivo
`Demonstration of binding of RPM to FR306
`1989
`
`binding protein
`
`1990
`
`Vezina, Kudelski, and Sehgal”
`Sehgal, Baker, and Vezina”
`
`Baker, Sidorowiez, Sehgal, et al Mt
`
`Martel, Klicius, and Gale”
`
`Findlay and Radics”
`Douros and Suffness”
`
`Morris and Meiser'
`Caine, Collier, Lim, et al?
`Meiser, Wang, and Morris’
`Tocci, Matkovich, Collier, et al”?
`Metealfe and Richards”
`Dumont, Staruch, Koprak, et a?’
`Morris, Wa, and Shorthouse!
`
`Harding, Galat, Uehling, et al”
`
`West-Ward Pharm.
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`Page 006
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`

`

`
`
`Rapainycins 43
`
`quence ofthe identification of RPMas an antifungal
`antibiotic (Table 1). Shortly after the antibiotic
`activities of RPM were described, it was found to
`have immunosuppressiveactivity. This was only afew
`years after the immunosuppressive activity of CsA
`was discovered, but ironically, RPM was not devel-
`oped as an immunosuppressantat that time. In a
`reviewof immunosuppressive agents published in
`1988, Devlin and Hargrave encouraged “a detailed
`comparison of the biological profile of these mac-
`rolides [FK506.and RPM].” These investigators sug-
`gestion was based on the structural similarity of both
`compounds and their known imimunosuppressive
`activity.
`Sehgal was aware that investigators at the Labora-
`tory for Transplantation Immunology at Stanford
`University had developed a.quantal bioassay for the
`evaluation of immunosuppressant potency and ef-
`ficacy, had validated the assay with CsA,” and-had
`used it
`to study FK506." In 1988, he offered to
`provide us with enough RPMto. enable us to deter-
`mine whether its activity differed from F506 in
`mouse as well.as rat heart transplant recipients. As
`subsequently discussed,the activity of RPMis ex-
`tremely dependent on the vehicle in which it:
`is
`suspended and the.route by whichit is administered.
`Hadourfirst experiment used suboptimal conditions
`for the administration ofRPM, we-would have. found
`no difference in potency‘or efficacy between RPM
`and FK506 and might not have pursuedour studyof
`RPM.Inretrospect, the mode of administration used
`at the outset was optimal and, under those condi-
`tioris, RPM: was clearly more potent and effective
`than FK506. This clear difference in pharmacological
`_ effect between these two structurally related mac-
`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 RPMin rodents, dogs, and. pigs.’
`Simultaneous. studies”performed at Cambridge
`and .by various groups of investigators at Merck
`Sharp and Dohme Research Laboratories, United
`States showed that RPMand FK506 affect immune
`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 fromthe streptomycete group that was
`isolated from an Easter Island soil sample by Vezina
`et al and Sehgalet al at Ayerst Research Laborato-
`
` RAPAMYCIN - Ris OCHa FAp=. OH
`
`DEMETHOXYRAPAMYCIN A= H, Ag= GH
`
`PKSO8
`
`PRODRUGS OF RAPAMYCIN (fiz OCHS, Aas see below):
`
`NLN-DIMETHYLGLYCINATE
`METHANE SULFONIC ACID SALT
`6
`!
`Fee rgd, GBH
`
`S+{N,N-DIETHYLAMINO/PROPIONATE . HYDROCHLORIDE SALT
`Ree hehAN HH
`to
`44PYRAQUDINOJBUTYRATE HYOROCHLORIDE SALT
`go
`
`Aye pr ct
`
`Figure 5. Chemical structures ofRPM, 29-demethoxyra-
`pamycin, F506, and the prodrugs of RPM.
`
`ries in the middle 1970s." 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 specics Séeptomyces
`Aygroscopicus, designated by Ayerst Researcli as strain
`AY B-994, and deposited in both the ARS culture
`collectionof the United States Departmentof Agri-
`culture (assigned number NRRL 5491) and the
`American Type Culture Collection (ATCC 29253). A
`structurally related compound,” 29-demethoxyra-
`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 B-994 and little or no
`29-demethoxyrapamycin.”
`
`Fermentation, Purification, and
`In Vitro Antimicrobial Activity
`of RPMs
`
`Fermentation of RPM
`
`Seon alter the availability of a pure strain of §
`Aygroscopicus, fermentation conditions (type of media,
`media pH, and temperature) were varied to define
`its cultural characteristics." Although this microbe
`grows and sporulates in a wide range of culture
`
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`Page 007
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`Page 007
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`

`44
`Randall Ellis Morris
`
`
`
` Figure 6.
`
`(A) Photomicro-
`graph ofthe filamentous bac-
`terium, S Apgrascopicus,
`that
`produces RPM(magnification
`X4355).
`(B) Electron micro-
`grapholSAygraseopicus (magnifi-
`cation X2,500).
`(Reprinted
`with permission.)
`
`conditions, more narrowly defined conditions are
`necessaryfor the optimumproduction 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, (NH,),5O,, and CaCO,
`and used at 2%. For the fermentation in stirred
`vessels,
`the starting medium was soybean meal,
`glucose,
`(NH,),5O,, and KH,PO,. Glucose is
`fed
`continuously after the 2nd day and the pH was
`controlled at 6,0 with NH,OH. Maximum titers of
`RPMare reached in 96 hours. Paperdisc-agar diffu-
`sion assays with Candida albicans are used to deter-
`mine the antibiotic titer.
`The fermentation methods required to produce
`29-demethoxyrapamycin are the same as those de-
`scribed for RPM.”
`
`Purification of RPMs
`
`The purification scheme (Fig 7) adopted for the
`production of RPM was developed shortly after the
`identification of the antifungalactivity ofRPMandis
`subsequently summarized.” After fermentation, the
`pH of the beer is adjusted to 4.0. The mycclium,
`extracted with trichloroethane,is filtered off and the
`extract is dried with anhydrous sodium sulfate to
`FERMENT STREPTOMYCES HYGROSCOPICUS
`
`EXTRACT MYCELIUM WITH ORGANIC SOLVENTS
`{
`APPLY CONCENTRATED EXTRACTTO SILICA GEL COLUMN
`|
`ELUTE WITH ACETONE
`|
`RAPAMYGINS
`
`Figure 7, Fermentation andisolation 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-
`imately50 gm of residue thatis then dissolved in 15%
`acetone in hexane and mixed with silica gel. ‘The
`dissolved RPMis adsorbed to the silica gel and
`remains bound to the gel after the mixture has been
`Altered and washed onto a column from which RPM
`is eluted. with an acetone:hexane mixture. After
`evaporating the column eluate to dryness, theresi-
`due is dissolved in ether from which pure crystals of
`RPMare obtained. In this initia! purification process,
`recoveries of RPMare on the order of 40%; 10 L of
`broth preduce 300 mg pure RPM. A more recent
`methodof purification has been reported.”
`Except
`for minor modclifications,
`the methods
`described for the isolation of 29-demethoxyrapamy-
`cin are the sameas those used for RPM.™
`
`In Vitro Antimicrobial Activity of RPMs and
`Mechanisms of Their Antimicrobial Actions
`
`‘The antimicrobial screening program at Ayerst Re-
`search Laboratories identified RPMforits antifungal
`activity. RPM inhibits the growth of yeasts and
`filamentousfungi including the dermatophytes Micro-
`Sporum gypseum and Trichophyton granulosum.” The
`minimuminhibitory concentrations (MIC) of RPM.
`against ten strains of C albicans were in the range of
`less than 0.02 to 0.2 pg/ml, representing greater
`potency than that of amphotericin 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 RPMalthough
`nearly as potent as amphotericin B.™
`One study has investigated the mechanisms by
`which RPM mediatesits antifungal effects," and the
`results of this study are summarized in Table 2.
`Approximately 90 minutes after adding RPMto C
`albicans cultures, growth is
`inhibited and subse-
`
`West-Ward Pharm.
`Exhibit 1022
`Page 008
`
`West-Ward Pharm.
`Exhibit 1022
`Page 008
`
`

`

`Rapanycins
`
`45
`
`Not inhibited
`
`Notinhibited
`
`Not inhibited
`
`Not inhibited
`
`Inhibited
`
`Inhibited
`
`Inhibited
`
` Table 2. Mechanisms ofAntifungal Actions of RPM
`Evfect ofRPM
`Test System Treatment
`
`Notinhibited
`Sorboase retention byC albicans.
`.
`Notincreased
`Increased hemolysis of rat red
`bloodcells, efflux: of K*, Pi,
`UV-absorbing material from
`Calbicans.
`C albicans anerobic glycolysis,
`aerobic respiration.
`Protein synthesis bycell-free
`preparations ofCalbicans,
`coli, tat liver, and mitachon-
`drial preparations ofG albicans.
`Amino acid metabelismby glu-
`tamic-oxaloacetic transami-
`nase, glutamic-pyruyate
`transaminase in C albicans.
`Glueasamiie and N-acetyl-glu-
`cosamine incorporation into
`whole Calbicans.
`Oxidative deamination ofglu-
`tamic and aspartic acids in C
`albicans. a
`:
`Incorporation of glucose into
`mannanin C albicans,
`Incorporation ofNa acetate and.
`methionine into.total lipid of
`C albicans.
`.
`Incorporation of adenine and
`phosphate into RNA and DNA
`of Galbicans.
`Degradation of*P-labelledintra-
`-cellular macromolecules and
`leakage through C albicans
`membrane.
`
`Inhibited more for
`RNA than: DNA
`
`Increased
`
`quently yeast cells lose viability and beginto lyse. The
`candicidal 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 RPMdoes not
`increase the leakage of sorbose or the efflux of
`potassium, phosphate, or UV-absorbing materials
`fromyeast cells.
`The effects ofRPMon other metabolic systems of
`Galbicans have also been investigated.” For example,
`RPMdoes not inhibit anaerobic glycolysis or aerobic
`respiration, nor does it inhibit the incorporation of
`glucosamineor N-acetyl-glucosamine. RPMdoesnot
`inhibit protein synthesis in cell-free preparations ofC
`albicans, rat liver, or mitochondria fromCalbicans.
`Although RPMinhibits the incorporation of glu-
`cose into mannanandacetate into lipids, the synthe-
`sis of glucan is minimally affected, indicating that
`
`inhibitionofcell wall synthesis is not the primary site
`ofthe antifungal action ofRPM.
`The most profound effects of RPMon C alhicans
`mayalso provide clués to its actions on mammalian
`cells. For exaniple, very law concentrations (02 ~ 1
`jug/mL) of RPMinhibit the incorporation of adenine
`and phosphate into RNA and DNA.At the MIC for
`RPM, phosphate-containing molecules leak out of
`the yeast cell membrant. The’ degradation of these
`molecules, presumably including‘nucleic acids, seems
`to be promoted in some way by RPM.”
`
`Physico-Chemical Properties of
`RPMs
`Structure ofRPMs
`Althoughtheinitial analysis of the structure ofRPM
`by infrared and nuclear magnetic resonance (NMR)
`spectroscopydid not provide the complete picture of
`its structure,” these techniques indicated that RPM
`was a completely newtype of macrolide antibiotic.
`Ultimately, x-ray crystallographic data clarified the
`structure ofRPM.” RPMis a 31-membered macracy-
`cle lactone containhig an amide witha C15 carbonyl
`and a lactone with a C21 carbonyl (Fig 5). Additional
`analyses of the "C and 'H NMR spectra of RPM
`confirmed the x-ray crystal structure of,RPM.” X-ray
`studies showed that RPMin its solid crystal form is
`conformationally homogeneous; in solution however,
`RPM exists as a mixture of two conformational
`isomers caused by trans tacis amide isomerization via
`hindered rotation about
`the pipecelic acid N-CO
`bond. Theratio.of t#raus to cis rotamers in chloroform
`solutionsis 3,to 4:1"
`Iilustrations of the structure ofRPMwereinitially
`inconsistent: different enantiomers were drawn,” a
`novel numbering system was used," aiid incorrect
`stereochemistry at C28 was represented.” Ulti-
`mately, the correct structure was published,” and the
`coordinates are deposited in the crystal data bank.
`Using advanced 2-dimensional NMR spectroscopic
`methods, new assignments ofthe proton and carbon
`spectra for the major rotamer of RPM have been
`made and a new nuinbering system suggested.”
`the
`The closest structural relative to RPM is
`antifungal and immunosuppressive macrolide FE506,
`whichis also producedbya streptomycete.” FK506is
`a 23-inembered macrocycle lactone that shares a
`utiique hemiketal masked a,B-diketopipecolic acid
`amide substructure with RPM,” butlacks the C1-C6
`triene segment of RPM.
`The results of “C-labelled acetate and propionate
`
`West-Ward Pharm.
`Exhibit 1022
`Page 009
`
`West-Ward Pharm.
`Exhibit 1022
`Page 009
`
`

`

`46
`
`Randall Ellis Morris
`
`and "C-labelled methionine incorporation studies of
`the biosynthesis of RPM were consistent with the
`proposedpolyketide pathwayin whichthe carbons of
`the lactone ring of RPMare derived from condensa-
`tion of acetate and propionate units in a manner
`similar to that responsible for fatty acid synthesis.
`The methyl group of methionine is an efficient
`source for
`the three methoxy carbons of RPM.
`Because none ofthe labelled precursors was incorpo-
`rated into either the cyclohexane or heterocyclic
`ring,
`these moieties probably orginate from the
`shikimate pathwayand lysine, respectively.”
`When ‘H and “Ci NMR,
`infrared, UV, mass
`spectroscopy, and optical rotary dispersion/circular
`dichroism (ORD/CD) analyses were used to com-
`pare the structures of RPM and 29-cemethoxyrapa-
`mycin, these molecules were shown tobe configuration-
`allyidenticalatall chiral centers and to have identical
`structural features at all but G29. Like RPM, approx-
`imately 20% of 29-demethoxyrapamycin in solution
`exists as the cis rotamer form.”
`In addition to the naturally occurring 29+
`demethoxyrapamycin, aminoacid ester analoguesof
`RPMhave been synthesized to produce three water
`soluble prodrugs of RPM™ (Fig 5). The amine finc-
`tions of the appended esters can be converted to
`watersoluble salts that are enzymatically hydrolyzed
`in the plasmato produce RPM. Although RPMforms
`both monoester and diester addicts depending on
`the reaction conditions, only monoester salts are
`
`discussed because these are sufficiently water soluble
`to obviate the need for the disubstituted forms. The
`28-hydroxyl group of RPMhas been proposedas the
`site of esterification for each of these prodrugs, but
`this remains to be confirmed.
`
`Physical Properties of RPMs
`Table 3 lists the physical properties of RPM."
`Although 29-demcthoxyrapamycin is also a white
`crystalline solid, it has a lower melting point (107° to
`108°C) than RPM.” Both RPMandits 29-demethoxy
`form are lipophilic and only minimally soluble in
`water, The water solubilities of both the mono-N,N-
`dimethylglycinate methanesulfonic acid salt and the
`mono-N,N-diethylpropionate hydrochloride salt pro-
`drugs of RPMare more than 50 mg/mL. The water
`solubility of the mono-4-(pyrrolidino)butyrate hydro-
`chloride salt prodrug is 15 mg/mL."
`Because MICsfor the antifungal activity of RPM
`in vitro vary depending on the medium used andthe
`length of the assay, it was suggested that RPMis
`unstable.” Subsequentstudies showed that 5 ug/mL
`of RPMin uninoculated broth loses §0% of its
`antimicrobial activity after 7 days of incubation at
`37°C." Later analysis showed that 30% of the antimi-
`crobial activity of | or 5 g/mL concentrations of
`RPMare lost after only 24 hours of incubation in
`culture medium.”
`High-pressure liquid chromatography (HPLC)
`has also been used to examine thestability ofRPMin
`
`Table 3. Physical and Chemical Properties of RPM
`ensernrrt
`
`3l-Membered macrocyclic lactone C;,H,,NO,, FW = 914.2
`3~t:1 ratio ofcis-trans rotamers about the pipecolic acid N-CO bond
`White, crystalline solid MP 183-185 C
`
`Solubility:
`- 20 we/ mLin water
`sparinglysoluble in ether
`soluble in methanol, ethanol, acetone, chloroform, methylene dichloride, trichlofoethane, dimethyl forma-
`mide,dimethyl acetamide, dimethyl sulfoxide
`Stability (degradation byhydrolysis):
`
`Temperature
`
`Vehicle
`
`25°C:
`
`37.39°C
`
`acetate buffer
`phosphate
`buller
`acetate buffer
`phosphate
`buffer
`human
`3
`—_—
`plasma
`2.83
`—
`rat plasma
`
`
`eerstearentninesntnrenemas
`
`pH
`3.3
`
`Fe
`3.3
`7b
`
`T'4 (hrs) by HPLC
`35.8
`
`47.6
`a9
`102
`
`West-Ward Pharm.
`Exhibit 1022
`Page 010
`
`West-Ward Pharm.
`Exhibit 1022
`Page 010
`
`

`

`
`
`Rapanycins 47
`
`different diluents subjected to different tempera-
`tures” (Table 3). RPMis particularly susceptible to
`degraclation in plasma.and in low and neutral pH
`buffers at 37°C. The degradation products of RPM
`have less antifungal activity than RPM and are
`identifiable by HPLC, but their structures are un-
`known. It is known that base-catalyzed hydrolysis of
`RPMatreflux temperature produceswell-character-
`ized neutral and poorly characterized acidic frag-
`ments.”
`HPLChas also been used to determine the in
`vitro half lives of the RPM prodrugs in human and
`rat plasma at 37.9°Cbefore their conversion to RPM.
`_ These half lives varied in length between I and 5
`hours.” There are no published stability data o