RANSPLANETATION
`REVIEWS
`
`1'r
`
`Breckenridge Exhibit 1055
`Morris 1992
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`Page 001
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`Breckenridge Exhibit 1055
`Morris 1992
`Page 001
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`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
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`Morris 1992
`Page 002
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`Rn/in11!Jri111
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`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
`
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`46
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`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:
`20 µ.g/mL in water
`sparingly soluble in ether
`svlublc in methanol, ethanol, acetnnt', chlorol'm m, methrlene dichloride, trichloroethane, dimethyl forrna·
`midc, dimcth)i acctamide, climeth)•I sulli1i.idc
`Stability (drg1-..d:11ion b)' hydrolysis):
`
`Tempera! urt'
`
`2j•c:
`
`37.5°(:
`
`Vehick
`
`nn·t,tlr bulfi·r
`phosphate
`buni>r
`acctatr buffer
`pho~phatc
`hulli:r
`hunmn
`plasma
`rat plasma
`
`pH
`
`3.3
`
`7.1
`3.:l
`
`H
`
`T'li (111 s) by HPLC
`
`J5.H
`
`~7.6
`9.9
`
`10.:l
`
`3
`2.ltl
`
`
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`Morris 1992
`Page 009
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`48
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`Rm1dnl/ Elli< .\Loni.<
`
`cussed previously). Thert•fore, degradation or RPivI
`in scrum may be another explanation for the low in
`\'itro anti-Candida activity or RPi\11 in the presence of
`scru m and may also contribute to the metabolism of
`RPM in vi\·o. Because the stability or RPM in whole
`blood may differ from the stability or RPM in plasma
`or scru m, it is not possible to extrapolate wit h
`certainl)' from the currently a\·ailablc stability s tud(cid:173)
`ies. Studies or the stabilit)' of RPM in the blood or
`different sp<:cics need lo be conducted to understand
`more precisely the fate or RPM in ,;vo.
`
`P harmacokin etics of RPMs Measured by
`HPLC
`A reversed phase HPLC technique has been used to
`monitor the pharmacokinetics or the mono-N,N(cid:173)
`dimethylglycinate methanesulfonic salt of RPtvf and
`its RPM parent after intravenous (IV) injection in
`m ice.''; These studies showed that· 20 minutes aftr r
`administration or I 00 mg/kg of the proclrug, plasma
`levels or RPM exceed those of the p rod rug. This is
`followed by steadily decreasing levels or RPM during
`the first 48 hours arter injection. In addition, the a rea
`undc r the curve is not linear ''~th prodrng dose.
`Using the HPLC m ethod, a pharmacokinctic
`analysis or the prodrug in m ice''; showed that the
`concentration decay of the prod rug in the plasma is
`tricxponential when a dose of' I 00 mg/kg is adminis(cid:173)
`tered, but bicxponential when lower doses arc used.
`Total body clearance ancl vol ume of distribution or
`the prodrug increases with drug dose. The vol ume of
`distribution of the prod rug is 1.74 L /kg and 8.76
`L/kg for doses of 10 and 50 mg/kg, respectively.
`T hese data indicate that plasma binding or the
`prodrug is saturable and that excess prodrug is
`distributed in t he tissues.
`Although another HPLC method has recently
`been developed, it has not been used to detect RPM
`
`in biological Auids.11' Clearly, more sensitive assays for
`RPM and its metabolites arc needed. Until addi(cid:173)
`tional methods that detect pic:ograrn levels or RPM in
`the blood and tissues become a,·ailable, the pharma(cid:173)
`cokinetirs, mrtabolism, distribution, and elimination
`of RPM and its metabolites will remain largely
`unkno\\·11. The doses orRPlVl in experimental ani mal
`grart recipients that are associated \\'it h t he highest
`lherape utic indexes for RPM foil lo produce blow
`levels detectable hr currently available analytic tcch(cid:173)
`niqucs. Consequent ly, wr. have been unable to corre(cid:173)
`late dose and blood level with either immunosu pprcs(cid:173)
`sivc drug efficacy or toxicity. Even though a suitably
`sensitive blood level monitoring techniq ue is not
`
`available, the structural simjlari ty between RP1vf and
`FK.506 suggests that RPM, like FK506,''1 will be
`distributed widely throughout the body (induding
`red blood cells), pri ncipally and completely metabo(cid:173)
`lized by the liver, and excreted in Lhc bile.
`
`Toxicity of RPMs
`
`Limitations of Pr eclinical Models
`
`Fortunately, there is remarkably good agreement in
`results among rodent and large; animal models of
`a llograft rejection concerning lhe relali,·e efficacy
`and indications for use of an immunosuppressant. If
`the appropriate boundary conditions a re sel, the
`results rrom these experiments arc usually highly
`predictive or the efficacy of a compound in human
`grart recipients. However, the ultimate value of a
`new drug is determined not only by its superior
`efficacy, but by whethe r the ratio or itstoxic dose to
`its immunosuppressive dose (therapeutic index) is
`substantially greater t han conventional therapies.
`Unfortunately, preclinical toxicology d ata have
`bcen poor predictors of the toxicity of immunosup(cid:173)
`pressive xenobiotics in humans. Animal studies havl'
`either undcrestimatl:d toxic effects ulli matcly found
`to be significant in humans (CA and FK506 nephro(cid:173)
`roxicity) or certain species of cxperimc11tal animals
`have grossly over estimated drug toxicity never noted
`in humans. For example, now that som e of the newer
`i1nnrnnosuppressivc xenobiotirs have: completed
`pha~c I trials, it is clear that dogs a rc far more
`sensiti\'C to the toxic effects or this class of drugs than
`arc humans. Humans have tolerated FK506, MPA,
`and DSG better than dogs, and the dose-limiting
`toxicities orlhcse drugs in humans are di[fcrcnl from
`those in dogs.
`There is no question tha t the extreme scnsi ti,~ty
`of the dog to the toxidt)• or these xcnobiotics has
`slowed the progress of the development or several of
`these agents. In addition, the incr('asing interest in
`immunosuppressivc monoclonal antib1xlies and cyto(cid:173)
`kincs that arc only effective in ncmhurnan primatr.s
`places additional limitations on the value of dog
`models for the evaluation of these new classes of
`imnrnnosupprcssivc agents. The cle"tcrmination of a
`''no toxic: cAcct" dose of a new clwmical encity may
`be- the prim ar)· value or clogs for irnrnunosupprcssivt•
`drug devcfopment. In \~cw of the sensitivity of lhc
`dog to drug toxicity, it is likely thal this "110 rffcct"
`dose in the dog"~ ll also be a safe lcvel for the humi111s
`LO whom the: drng is being administerc.'cl for the first
`time.
`
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`Breckenridge Exhibit 1055
`Morris 1992
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`Rnpotf!>rinr
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`49
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`Toxicity of RPMs in Rodents
`
`The toxicity of RP~'l in humans is not known.
`Furthermore, none of the published toxic effects of
`RPM in animals has been generated by formal, Good
`LaboratOI]' Practices studies. On the contrary, most
`of the available information on Lhe toxir effects of
`RPM is a byproduct of studies of the efficacy of the
`drug, thus limiting the interpretability of the data.
`Because an IND application for phase I trials ofRP.M
`has been submitted Lo the FDA and because it was
`accompanied by the necessary and complete tox.icol(cid:173)
`ogy results from rodent and large animal experi(cid:173)
`ments, Lhe extensive information on the toxicitv of
`lU1M in animals has been gathered, but has yet t~ be
`published.
`The data that have been reported'&.~• indirale that
`mice and rats arc very re~istant to the acute (single
`dose) toxic effects of RPM (Table 4). The mouse
`intraperitoncal median lethal dose (IP LD.;o) \-aluc is
`considerably higher than the 15 mg/kg IP dose of
`RP.M that produces high scrum le\'Cls of anti.Candida
`acthity in this species. Furthermore, the LDjO values
`for RPM administered IP and brother routes in the
`mouse and rat are also much greater than the doses
`of RPM needed to suppress graft rejection and
`autoimmune diseases in these animals (discussed
`subsequentJ>~ refer to sections headed Effects of
`RPM on Autoimmune Diseases and Effects of RPM
`on Graft and Tissue Rejection). Because RPM is
`administered as multiple doses in most rodent mod(cid:173)
`els designed to evaluate efficacy, the subchronic LD~,
`values for mice and rats that have been treated daily
`for 14 days with RPM would be a more relevant
`estimate of the therapeutic index of RPM than the
`acute LD.111 values shown in Table 4;.
`Unfortunatel)•, no LD;,o values from subchronic
`toxicity studies have been published. However we
`have administered 24 mg/kg of RPM in suspension
`in earboxymcth)'l cellulose IP daily to mice for 2
`weeks and then performed nccropsies, complete
`blood counts, and serum chemistries on day 14orday
`28. None of the mice died during treatment l)r
`during the 2 week rccOVCI]' pcri6cl. The dose b-el
`
`Table 4. Acutr Toi.icil)' of RP'.\ I in Rodents
`
`1l11imn/
`
`Rou/r
`
`Fon111Jnlio11
`
`:\[oUS('
`
`Rat
`
`D'
`PO
`D'
`PO
`
`Suspension in acacia
`Susix-nsion in acacia
`Susix-nsion in acacia
`Suspension in acacia
`
`.-lruttLD,.
`(mg/kg)
`
`59i
`>2,500
`> 1,600
`> 1,600
`
`and schedule of RPM administered b)· this route
`causes thymie involution, lymphoid cell depletion in
`the l)'lllph nodes and spleen, and lowers the white
`blood cell (WBC) count (Zheng B, Morris RE: unpub(cid:173)
`lishc:d obsc1Yation, 1989). In another study (Zheng B,
`Morris RE: unpublish!'d observation, 1990), we
`treated mice IP with 6 mg/kg of RPM for a maxi(cid:173)
`mum of 14 da)'ll and thl'll necropsied the mice 7, 14,
`4-0, or 102 days after the start or treatment. There
`was no evidence of renal, c·arcli<tC:, or liver damage in
`any of the animals, and the marrow ccllularity was
`normal Tcstic:ular atrophy is a clrlaycd drug ~ffect,
`because it is obsc1ved only in mice nccropsied on days
`42 and I 02. ·n1c effects of RPM on the thymus and
`spleen of these animals arc discussed in the section
`headed Effects on the l\lorphology and Function of
`Central Lymphoid Tissues.
`When 2.5 to 10 mg/kg of RPM was administered
`PO dail)' to rats for 7 to 14- days to evaluate the effect
`of the drug on experimental allergic encephalitis, the
`only drug-related adverse eff'ect noted was a depres(cid:173)
`sion of the growth curve. 19 We hm·e also noted that
`the rate of weight gain in rat heart allograft recipi(cid:173)
`ents treated with RPM is lower than normal, but that
`weight gain accelerates after cessation of RPM treat(cid:173)
`ment (l\lorris RE, Wangj: unpublished observation,
`1990). 01hr•rs h:wr :il~o founn that rats treated n~th
`50 mg/kg of RPM in oil administered intramuscu(cid:173)
`larly (IM) dail)' causes a I 0% weight loss.2 The cause
`for the weight loss could be a direct or indirect effect
`oflU1M on the central nervous system or a direct or
`indirect effect on the absorption or metabolism of
`nutrients.
`We have noticed that rats treated \\~th RPM have
`high blood glucose levels that rctum to normal after
`cessation of treatment (Morris RE, Wang]: unpub(cid:173)
`lished observation, 1991). In addition, rats treated
`with RPM once weekly for 50 days often died more
`than a month after the last dose of RP.M of what
`seemed to be pneumonia that was probably second(cid:173)
`ary to prolongrd and irreversible nonspecific immu(cid:173)
`nosuppression (Morris RE, Wang J: unpublished
`obscn-ation, 1990).
`The most complete study"' of the toxicity of a
`l\\O-week course of an immunosuppressi\'e dose (l.5
`mg/kg as suspension, IP dailr) of RPl\1 in rats
`showed that RPM had no effect on: (1) the marrow;
`(2) WBC count or its differential, (3) percentage ofT
`cells, T-ccll subsets, or B cells; or (4) liver function.
`RPl\I treatment docs cause elevations in plasma
`glucose, atroph)' of the thymus medulla, and necrosis
`of the myocardium. When this same immunosupprcs-
`
`
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`50
`
`Handal/ Ellis Munis
`
`sivc dose or RPJ\11 is combined with a high daily dose
`ofCsA ( 15 mg/kg PO), plasma g lucoses arc higher.
`In mice, the LD,,.1 of a single IP dose or 29-
`clemethox)~·apamyci n formulated as a suspension in
`acacia is grcarcr lhan 900 mg/kgi'; which is much
`higher than t he LD.-.. for idcnticall)' formulated and
`ad ministered RPM in this species (Table 4). The
`lower toxicity and lower an tifungal efficacy in vivo
`(pre\·iously discussed) o r 29-demelhoxyrapamyc:in
`compared with RPM could be caused by poor absorp(cid:173)
`tion or increased e limination, or because or its
`metabolis m to less toxic or more rapidl)' excreted
`metaboLitcs. However, these reasons alone ca1mot
`accou nt for the lower in vitro ant ifungal acti\~ty of
`29-dcmcthoxyrapamydn compared with RPi\11 (previ(cid:173)
`ously discussed). Therefore, perhaps the mcthm.)'
`group at C29 contributes d irectly to lhc as yet
`unknown molecular events that are ultimate!)' respon(cid:173)
`sible for the antifungal effects and toxicity in the
`whole animal (discussed in section headed Molecular
`Mechanisms of the Antiru ngal a nd lmmunosuppres(cid:173)
`sive Activities of RPM). There arc no other pu blished
`data on the toxicity of 29-dcmcthox·yrapamycin in
`rats or larger animals; the LD~,s for RPM prodrugs
`;:ilso are not available rrom the litcratl1 re.
`
`Toxicity of RPM in Large Animals
`Long-term toxicity studies of RPM in dogs show that
`RPM causes hypoplasia in lymph nodes, spleen, and
`thymus.'m The d ose, route, and schedule or RPM
`treatment used in these s tu d ies were not stated. In
`separate studies,Vfl 0.25 to 5 mg/kg or RPiVl admi nis(cid:173)
`te red PO daily to dogs also deple ted central lym(cid:173)
`phoid tissues (particularly or B eel.ls) and caused
`vo mi ting, diarrhea, a nd thrombocytopcnia. Ulcer(cid:173)
`ation occurring l"rom t he mouth to t he colon second(cid:173)
`a1y to.necrotizing fibrinoicl vascu litis was also seen.
`Pigs treated with 2 mg/kg of lU'M PO daily
`gained weight normally b ut exhibited m icroscopic
`evidence of colitis without vasculitis. Afte r 50 days or
`treatment with RPM, interstitia l pncumonilis oc(cid:173)
`curred in 50% of the an imals; this was nscri brd to
`nonspecific i111111unosupprcssion.""
`During the last 2 years we have gained experience
`treating cynomolgus (jldaccncnfasiwlmis) reripients of
`heart allografts with RP~·l adminislc red lM and
`forml1hHcd in suspension in carboxymethyl c:ellu(cid:173)
`losc."'' Other ani mals we re treatl".d wit h RJ>M plus
`Ild CsA. No animal was treated for more tlmn 100
`d ays postlransplantntion. RPiVl doses ra nged from
`0.5 mg/kg !".very other day (QOD) to 7 mg/kg once
`per wrt>,k, and doses o r either ·f mg/ kg/cl or 2 mg/kg
`
`QOD of CsA were used. Lethargy <u1d a loss or
`appeti te occurs in animals treated with high-dose
`RPM. Al though animals treated with RPM or CsA
`Iosr similar amounts of weight, these we ight losses
`a rt less severe than in monker heart graft recipicn1·s
`treated with FK506.''' In facl, I mg/kg or FK506 in
`suspension ad ministered IM daily causes significant
`morta li ty in cynomolgus mon keys.1'\ ; i
`In ou r recipients, we have a lso fou nd that high·
`dose RJ>M produces hypoplasia of cen tral ly111phoicl
`tissues in monkeys. Testicular atroph)' with thinning
`o r the seminiferous tu bules occurs in a ll animals
`nccropsied regardless or RPM dose or whether RPM
`is used nlone or combined with CsA. RPM alone or in
`combinat ion wi th CsA is not diabctogcnic in C)'1101110I(cid:173)
`gus monkeys and docs not cause myocardial necrosis.
`Nccrotizingvasculitis was present in only one anima.I,
`but several mon keys showed some microscopic e>i(cid:173)
`cle ncc nf e nterocolitis manifested by Iymphoplasma(cid:173)
`cytic infil trates in the small a nd large intestines. T he
`cellularity or the bone marrow in all RP.M-lreated
`monkeys was normal. It is ne>l known whether the
`le thargy observed in animals treated "ith hig h-dose
`RP!v1 alo11e is a direct or ind irect effect of RPl\·1
`treatment. Despite the administrat ion o r RPM doses
`rhat arc effective for the prolongation or allograft
`survival, all monkeys have remained free of malig(cid:173)
`nancy during treatment and for as long as 100 days
`after the last treatment dose. The incidence or severe
`infection was low in animals treated with only RPM
`•rnd nonexistent in animals treated with low-dose
`RPM plus CsA.
`The toxicity of RPM in baboon rcnnl allograft
`recipien ts is significantly worse than in monkeys
`treated with RPM. These baboons suR'crcd from
`vomiti ng a nd di a rrhea that was probably related to
`the vasculitis present in their intestincs.r!
`
`Nephrotoxicity of RPM
`Although not studied systematically, we have not
`found any blood chemistry or histopathologic c::vi(cid:173)
`dence t ha t RPlVI:causrs impairmrnr o r re nal function
`or damage in rodents treated wit h closes of RPM that
`arc hig hly cl-fcctivc for thr prolongation of heart
`nllograft survivnl (Morris RE, Wang J: unpubl ished
`observation, 1990).
`Also, no indication was lo und rrom repeated blood
`chemistry analyses that treal mrnl \\~th RPi\·I a lone
`or in combination with CsA impairs renal func tion in
`cynomolgus monkeys; mild rena l tubular atropl1r
`was ob:;c1Ycd in animals treated with RPM alonr,."1
`'
`Two studies have investigated the effect of RPl\I
`
`
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`52
`
`Ro11dall Ellis .\/1mi1
`
`Effects of RPM on Tumor Growth in
`Vivo
`
`The National Cancer lnstitute's (NCT) Dc,·elopmrn(cid:173)
`tal Therapeutics Program has screened hundreds of
`thousands or molecules for antineoplastic acthit)
`through its Natural Products Program. Because it
`was well kno\\11 that Strrpto11!1-as organisms arc pro(cid:173)
`lific producers of antimicrobial molecules, the ~rrccn­
`ing of Strr:plo111J'W broths initially dominated thr.
`search for new antitumor antibiotics. Because RP\'.·l
`(NCI designation, NSC 226080) is a product of
`Stnti/0111J'CCS and is structu rally unique, it was ev:llu(cid:173)
`atcd for its ability lO inhibit the growth or lU1110r (TllS
`in mice. 11
`lnitiaUr, RPI\[ mt~ found to be activr against thr
`CDFI mammary tumor, Colon 38 tumor, and
`ependymoblastoma at doses of 200, -lOO, and 25
`mg/ kg, respcrlivclr.11 The acti,·ity of Rl1~1s against
`mouse tumors was further im·cstigated by evaluating
`the response of the P388 l)'l'llphoqtic leukemia, B 16
`mel:mocarcinoma, and Colon 38 tumors to treat(cid:173)
`ment "~th RPl'vl or 29-demet hox-)Tapamycin~";i ad(cid:173)
`ministered IP dail)' for 9 days. RPM lrcalmenL shows
`anti tumor activity against P388 leukemia and BIG
`melanoma at a close range of 12.5 to 100 mg/kg.
`Doses or 200 to -!00 mg/kg or RPM arc required to
`increase the survival or mice "ith Colon 38 tumors.
`lntercstingl)', 29-dcmethoxyrapamycin has no acth·(cid:173)
`ity against the 816 melanoma or the Colon 38
`tumors; slight activity is obsenred against the P388
`leukemia.
`The anlitumor efficacy of RPM is route-depen(cid:173)
`dent.;:. A -WU mg/kg dose of RPM is equally effective
`if injected IP or JM; these two routes a rc superior to
`the SC route tha t is, in turn, more effective than the
`PO route. Other experiments using the mouse tu(cid:173)
`mor model show that RP1"1 treatment is active
`against Colon 31:1 tu11101s that have become estab(cid:173)
`lished in thr host before the start ofRPllJ treatment
`and that this same tumor is more susceptible to the
`antineoplastic activity of RP'.\l + 5-Auorouracil +
`cyclophosphamidc than LO 5-Ruorouracil + adriamy(cid:173)
`cin + c-yclophosphamidc.73
`Jn a separate sllldy, RPl\I was used to t n':\l
`huma n mcdulloblasloma T E-67 I and gliobl:tstnm;i
`multifornw U-25 1 implanted intracranially in nucl1·
`mire.'" lntraprritoncal injcctions of 100 to HOO mg/kg
`closrs nf llP~I dis~olvcd in ethanol and diluted with
`salinr to givr a final concentration or 10% l'th.1nol
`
`incrc;isc the le11g th of survival of mice implanted
`\\'ith the U-251 tumor but have no effect on TE-671.
`RPJ\I is the most recent addition to the follo\\ing
`list of parent compounds that were identified as
`nntitumor agents before their potential or the p<llcn(cid:173)
`tial of their analogues as immunosupprcssants were
`appreciated: 6-mercaptopurinc (and its analogue
`azathioprine), cyclophosphamide, ~CPI\ (and its ana(cid:173)
`logue RS-61-l-l3), spcrgualin (and its analogue DSG),
`n