Rnjiamycim
`
`63
`
`Suppression of Allograft Rejection by
`Combined Treatment With CsA, FK506, and
`MPA
`Until recently, formal pharmacological principles
`had rarely bccn applied to clctrrminc:- whether sinrnl(cid:173)
`tancousl)' administered immunosuppressants pro(cid:173)
`duce a net state of immunosuppression in graft
`recipients that is antagonistic, additive, or ~-ynergistic
`compared with immunosuppression caused b)' the
`individual administration of each agent. For many
`years, Berenbaum has decried the expe1imental
`design and data analysis from studies in transplanta(cid:173)
`tion, among other fields, that have led to incorrect
`conclusions concerning immunosuppressivc drug(cid:173)
`drug interactions.w1 Several years ago, when wf."'.
`converted the standard mouse car-heart transplant
`technique to a quanta! bioassay, we tried to redress
`the inadequacies of pre\~Ous studies of combination
`immunosuppressive therapy. 17
`When we first began investigating the immunosup(cid:173)
`pressivc activity of RPM in vivo, we assumed that the
`structural similarity between RPM and FK506 pre(cid:173)
`dicted that both drugs affected the immune system
`ve11' similarly because at that time there was no in
`vivo or in vitro 'data to the contrary. 111is nssumption,
`combined with our previous finding that treatment
`with FK.506 docs not antagonize CsA immunosup(cid:173)
`pression in vivo,11 led us to treat rat heart allograft
`recipients with minimally effective doses of RPM plus
`CsA.3 This study was not sufficiently rigorous to
`enable us to conclude from our data that these two
`dru~ interact to produce immunosuppression that is
`synergistic. However, we were able to show that this
`combination is not antagonistic and that the immu(cid:173)
`nosupprcssion caused by this combined therapy is at
`lea~t additive. A recent and extensive study! involv(cid:173)
`ing sixteen separatr treatment groups that ex(cid:173)
`panded prc,~ous work!11 clearly showed, that com(cid:173)
`bined treatment with RPM plus CsA produces
`synergistic suppression of rat heart allograft rejec(cid:173)
`tion. A smaller subset of this study showed that
`combined treatment with RPM and CsA is also
`beneficial for the prolongation of rat kidney al(cid:173)
`lografts.
`Using the mouse car-heart bioassa)', we showed
`that multiple combinations of RPM plus CsA or
`RPM plus FK506 cause prolongation of graft sun~val
`that is synergistic as defined by isobologram analysis''
`when the treatment doses of each drug are less than
`their ED,.,s. In addition, probit analysis has been used
`to show that combined treatment or mouse skin
`
`allograft recipients with RPM plus CsA produces
`immunosupprcssion that is syncrgistic.3; Furthcr(cid:173)
`morr, high doses of both RPM and FK506 used in
`combination did not indicate in any way that either
`dnig antagonizes the immunosupprcssive effects of
`the other. The data showing that treatment with
`RPM plus FK506 produces synergistic immunosup(cid:173)
`prcssion at many close levels contradict studies in
`vitro that showed these two drugs antagonize each
`other's effects on immune cells (discussed in section
`ht:aded Effects of RPM on Immune Cells in Vitro).
`Lately, we have extended our interest in combina(cid:173)
`tion immunosuppressive drug therapy to .the com(cid:173)
`bined use of three dn1gs that have dilferent mecha(cid:173)
`nisms of immunosuppressivc action (Fig 3) and
`nonovcrlapping toxicity.9 For example, low-dose CsA,
`RPM, or l\tlPA (administered as its prodrug, RS-
`61443) monotherapy ineffectively prolongs rat heart
`allograft SUr\~val (Table 11 ). When these same doses
`are used, but all three drugs arc administered to(cid:173)
`gether, suppression of graft rejection is not only
`more effective than when each drug is used st:pa(cid:173)
`rately, but it is also more effective than when RPM
`plus .MPA or CsA plus MPA is used. Additional
`follow- up is required to determine whether triple
`drug therapy is superior to treatment with RPM plus
`CsA.
`RPM monotherapy of cynomolgus mopkcy heart
`allograft recipients prolongs graft sU1vival (discussed
`previously). However, unlike the use of RPM in
`rodent graft recipients, monkeys seem more resis(cid:173)
`tant to the immunosuppressive effects ofRPl\·i and
`more sensitive to its toxicity. Because RPM and CsA
`produce immunosupprcssion in rodent graft rec-ipi(cid:173)
`cnts that is ~·ypergistic, and because it is possible that
`the to_xic effects qf each drug are different, we
`treated monkey heart allograft recipients (Morris
`RE, Wangj, Shorthouse R, et al: unpublished obser(cid:173)
`vation, 1991) \\~th low doses of both drugs for the first
`JOO clays posttransplant (Table 10), This treatment
`regimen suppressed r~jection much more dfectively
`than treatment with either CsA or RPM monother(cid:173)
`apy. Only two of the five monkeys treated with
`combination therapy rejected their heart allografts
`during the treatment period; all animals remained
`clinically well. Pharmacokinetic analyses of CsA blood
`levels showed that the 2 mg/kg dose ofCsA produc-es
`CsA levels that arc subtherapcutic: (all <60 ng/mL).
`Concomitant treatment with RPM and CsA docs not
`elevate CsA blood levels compared with levels at(cid:173)
`tained with CsA treatment alone. Thus, the im-
`
`
`
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`Morris 1992
`Page 0026
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`

`64
`
`R1111d11// £/Iii .1/oniJ
`
`Table 11. Ellt-r1 of' Combination Therapy "ith LU>i\1, CsA and ;\l)'Cnphenolk Add (as ils 111orphoincL11yles1cr, RS-6 1-1-11
`un tht' Su1vival of'B1 uwn-:\01way Rat Heternlllpk Abdomi11nl H ~art Grafts Tmnspl::1111cd i11to Le\\ is Rl·cipienls
`
`)
`
`'Drug(s)
`
`GsA
`Cv\
`i\IPA
`IU';\l
`lU':0-1
`+ i\JPA
`CsA
`+l\JPA
`IU'i\1
`+CsA
`CsA
`+ l\lPA
`+RP1'1
`
`DoJf!
`(111g/ l.gJ
`
`Rnu/f!
`
`For11111/ntin11
`
`Sdmlule
`(dt!)I)
`
`.l!rdia11 Grqfi
`(da.J"I)
`
`0.5
`
`0.75
`
`10
`1.5
`1.5
`10
`0.75
`10
`1.5
`0.5
`0.5
`10
`1.5
`
`£P
`
`£P
`PO
`PO
`PO
`PO
`
`II'
`PO
`PO
`IP
`
`IP
`PO
`PO
`
`Solution in C:mnophor EL/ethaool
`Solution in Cremophor EL/ethanol
`
`su~pt'l1>iun in ca1 boxymethrl cellulose
`Smpension in mrboxymcthyl cdlulosc
`Suspension in carbnxpnethrl cellulose
`Suspc11sion in carboxy111e1hyl cellulose
`Snluurm in Crcmuphor l::Uethanol
`Suspension in carboxyme1hyl cellulose
`Suspension in carboxymethyl cellulose
`Solution in Cmnophnr EL/ethanol
`
`Solution in C:remophor EL/ethanol
`Suspension in carboxymethyl cl'lluln.~I'
`Suspt·n~1on in c;u bo.'\)11lcth) I cellulose
`
`I to50
`1 co50
`1 to50
`
`I to.'iO
`
`1 to50
`I 11150
`1 rn51l
`I lu50
`
`I 1050
`I to50
`1 to50
`I to50
`I to50
`
`9
`15
`10
`11
`
`28
`
`67
`
`109
`
`170
`
`proved immunosupprrssive efficacy caused by combi(cid:173)
`nation therapy cannot be explained by high CsA
`blood levels (data not shown). The coadmi11is1ration
`ofCsA could elevate RP/\{ levels, but "~thout a blood
`level assay for RPM, this possibility cannot be exam(cid:173)
`ined. In the raL, coadministration of RPM and CsA
`docs not clcvntc C3A levels-"'
`FK506 is known lo suppress hepatic cy1oduo111c
`NSO and Lhc acth~ties of ethylmorphine 1\-cl<'mcl h(cid:173)
`ylasc and cytochrome c reductase in rats,"'1 and this
`may partly accoun1 for the increased half-life of CsA
`in patients treated \\ith FK506."1 Because we did not
`line! that RJ':-.1J increases tht haJf. Jjfc of CsA in
`monkeys;• the effects of fK.506 and RPJ\I on the
`m etabolism of CsA may differ. The i111 rraC'tinns
`between nonim 111unosuppressive macrolidc antibiot(cid:173)
`ics and CsA have been defined and mar prO\·icle
`additional clues to the interaction between RPM and
`CsA.1111
`·n1csc initial studies of Lhl' lack of effect of
`treatment with RP~L on CsA blood levels sugg<'Sl
`that the combined USC of RPM plus Cst\ ma} anorcl
`the benefits of increased immunosupprcssi\'e efficacy
`\\~thout the penalty of decreased safety. In view or
`the similar mrrhanisms ol' immunosupprcssi\'C ac(cid:173)
`tion or CsA a nd FK5UG, the supt'l'ior cllicac->· and
`potency of FK.506, and lhc synergistic i111 mu11os11p(cid:173)
`prcssion caused by the adminis tration of RPM phis
`FKSOG in mouse car-heart recipients," the combim·d
`usr oflU'M and FK506 may a lso be useful in monkry
`and human graft recipients. H owc,·cr, the incrcilsccl
`nephrotoxicil~ and cliabctogenic dli-ct or eombinccl
`
`high doses of RPM plus CsA in the rat'~ alerts us to
`the possible synergistic toxicit}• that can be caused by
`specific drug combim1lions.
`
`Interactions Between RPM and
`Nonimmunosuppressive Drugs
`RPM is likely to be used in patients receiving compli(cid:173)
`cated treatment involving a \\ide variety of nonimmu(cid:173)
`nosuppressivc drugs. Jn addition to the interactions
`of RPM \\ith other immmunosupprcssants (previ(cid:173)
`ously discussed), coadministration of nonimmunosup(cid:173)
`pressivc drugs may also subslantially inRuence our
`gual or optimizing the dose, route, and schedule or
`aclminis1nuinn uf RPM. Because RJ>l\·I shares some
`physirochemkal characteristics with CsA, and is
`structurally similar to FKSOli, thr extensive experi(cid:173)
`ence of Cs.A. drug interactions"'' and the increasing
`understanding of the pharmacology of FK.506''"""
`may prO\idc lessons that will nor ha,·c to be com(cid:173)
`plctcl)• relearned \\ith RPi\l. If, like CsA and FK506,
`RP~l blood ll'vcls and its pharmacological effects
`\'ary widely among patients, and if' the therapeutic
`index or RJ';\[ is low, the effects of simultaneously
`administered drugs will profoundly affcl·L t he clinical
`use ol'RPi\'L
`As descri bed in a rcccnl review on C:sA drug
`intcractions,"'1 1his potc:nli::tll)' complex problem can
`be simplified, at least cone'' Pl uallr, b)' <rnalyzing how
`drug interactions affect Lil<' absorption, distribu1io11,
`metabolism, and elimination (pharmacokinrtics) of
`drugs and their the biolocial/toxicological effects
`
`
`
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`Page 0027
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`

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`

`

`66
`
`Uandllll Ellis tlloniJ
`
`very high dose (2+ mg/ kg) of RP~l in suspension.
`·n1e WBC counts arc normal on dar I+, but during
`the rec:ovcry period it was found that this dose or
`RPM depresses the \o\'BC counts. llowc1·c1-. artc r 2
`weeks of daily 11' treatment of rat heart rcripirnts
`with 6 mg/kg or RP:\1 (a dosr that produces > 200
`day gr.ift survival for all grafts), the WBC and the
`rotal lymphOC}te counts arc normal. Thus, indefinite
`prolongation of graft survival in rats occurs after
`brief RPl-1 treatmcnt \\ithout depiction of circul<ll(cid:173)
`ing lymphocytes. Although RPM can moderately
`suppress the WBC count in mice, this dTrtt is by nn
`means sufficient lo explain how RP!Vl induces indefi(cid:173)
`nite prolongation of graft su1vi1<1I. The rejection of
`third-party grafts transplnnted into recipients bear(cid:173)
`ing viable primary grafts (discussed in section headed
`Effects of RP~J on Graft and Tissue Rejection) not
`only indicatt's that there arc sufficient numbers of
`cir.culating lymphocytes lo mediate rejection, but
`also indicates that these cells arcsclcctivcl)• immuno(cid:173)
`competent.
`Within 2 weeks of treating monkeys with RPM
`monotherapy or with combination therap)' of RPM
`plus CsA, the absolute lymphocyte count is lowered
`(Morris RE, W:u1g J, Zheng B, et al: unpublished
`obse1vations, 1991). Flowc)'1:ometricanalrsis ofprep(cid:173)
`nrntions of monkey whole blood showed (Mo,.ris RE,
`Wang j , Zheng B, cl al: unpublished obsc1valions,
`1991) that the Lolal numbe rs of both T and B cells
`are lower than pretreatment va.lues. In untreated
`monkeys, the ratio of the number of CD8+:CD4+
`rclls is greater than unity. RPM hcatment causes
`this ratio to become inverted, because there is a
`disproportionate reduction in the number of COil+
`cells compared with CD4+ cells. It docs not seem
`that t he alterations in cell number alone can account
`for the suppression of graft rt'jcction caused b)' RPM.
`More likely, RPM produces immunosupprcssion b)'
`functional!)• inacti\'aling immune cells. Fur example,
`when WC quantitatcd the rcsponsc Of peripheral
`blood mononuclear cells in monkeys treated with
`RPlVf plus CsA to differen t concentrations or con(cid:173)
`canavalen A (ConA) in vitro, we found that mi to(cid:173)
`genic responses arc suppressed al low concentrations
`of ConA but return Lo levels similar to prctreatmenL
`\'alucs a~ the concentration of ConA in culture is
`increased (Morris RE, Wang J, Zheng 13, ct al:
`unpublished obsc"".itions, 1991). Changes in the
`numbers and function of circulating peripheral blood
`B cells, T cells, and T-ccll subsets caused by RJ'l\lf in
`the monkey ma)' also occur in humans Lreated with
`RPM. iJ so, these parameters may provide a more
`
`sensit.ht' index of the effects of RPM on the immune
`system than assessment of graft rejection.
`
`Effects on th e Morphology and Function of
`Central Lymphoid Tissue
`
`Thr only hint uf the rational<' for the firs l investiga(cid:173)
`tion of the immunosupprcssiw rffects of RPM by
`l\fartel ct a1 ~1 was a brief sentence in the Discussion
`section of their article which read, " .. .long-cerm
`toxicity studies in dogs (Hemm RD, Au thicr L:
`pc1 son al communication) have clcmonstratcd that
`rapamyri1i caused hypoplasi;i of lymphatic: tissues
`(l}-mph nodes, splren, and chymus)." This effect of
`RPM has now been confirmed in other species. For
`example, as part of an initial subchronic toxicolog)'
`scudy we treated mice IP daily \\'ilh a dose of RPM
`(24 mg/kg) that far exceeds doses (6 mg/kg) re(cid:173)
`quired to prolong car-heart grafts indefinitely. ~ci.;­
`ropsies (Morris RE: unpublishc;.d obsc1vations, 1989)
`of mice on clay 14 showed the thymus to be dramali·
`caUy involuted, but the lymph nodes and spleen
`seemed normal in size and weight. 1\.Iicroscopic
`analysis of the lymph nodes and spleen did not show
`any abnormalities, but the normal distinction of the
`th)1nic cortex from the medulla was often absenl
`and Lhymic lymphoid depiction was profound. When
`other animals from che same rrcaiment group were
`nccropsicd on clay 25 (2 weeks after the Inst RP~l
`close), thymic involution persisll:d and lymphoid cells
`were decreased in the mcdulla1y cords of lymph
`nodes.
`A more thorough study"" was conducted in mice
`that were treated IP daily with 6 mg or. 75 mg/kg or
`RPM for a maximum of 13 days. These mice and
`aged-matched control mice were necropsied 011 days
`7, If, 21, 4·2, and 102, and thei r thym us and spleen
`weights recorded. Tissues from Lhe thymus and
`spleen \\ere stained with monoclonal antibodies and
`anal}7.t:d by immunohistochemistry and Aowcytomc(cid:173)
`ll) .. Fi nail)•, spleen cells were cultured and stimulated
`\vi th inc1 casing concentrations of either the T-cell
`mitogcn ConA, or the B-ccll 111itogcn Sa/111011ella
`01J11i11111riw11 (STl\>l).
`We found that RPM treatment docs not decrease
`the weight of the spleen. In contrasl, RPi\l treatment
`has complex cffrcts on the weight of the thymus.'"
`111e 6 mg/ kg dose of RPM cuuscs the thmus weight
`to be reduced b)' 80% after I week of treatment. The
`thymus "eight increases, but is still abnormally low
`bydn)'42; by day 102, the weight n·bounds w normal.
`T he lower dose or RPM prolongs sun~val graft less
`effect ivel}' than docs the high dose, but reduces
`
`
`
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`Morris 1992
`Page 0029
`
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`

`

`68
`
`Rnndnlf f.1/ii .\lu1ril
`
`and T issue Rcjeclion), lhc rc was some depictio n o r
`cells in lrmph nodes, but the histology of Lhc spleen
`was essentially normal. Because these monkeys were
`juveniles when their RPi\I treatment began, it was
`difficult to ascribe the lack of th~1llllS !issue al
`necropsy entirely to the effects of RPM.
`The inves tigations of the effects of RP:-.·1 on the
`composition or murine thymus a nd spleen and the
`immune function or cells from the spleen and lymph
`node hnve just barely begun to scratch the surface of
`the complex effects ofRP~l on tissues oft he immune
`system. Despite our present naivete, available data
`show that RPM affects centra l lymphoid tissues
`dilfercntly than CsA and FK506. Both CsA"""" and
`11 cause less thymic involution and affect
`FIG06' 11
`"'
`the thymus more selectively than RPi\l because their
`effects are restTicted to depiction of thymocytes in
`the medulla a nd not the cortex. Brief treatment with
`d thcr CsA or F K506 at doses t hat exceed those
`needed for immunosuppression causes reversible
`thymus weight loss. Prolonged treatment with CsA
`renders the thymus incapable of recovery. Both CsA
`and FK506 seem lo mediate their clfccts directly or
`indirectly by damaging medulla.ry e pitheli um. T his
`effect, perhaps in addition to othe rs, may inrcrru pt
`the maturation of singlc-positi,·e CO.·!-• /CDS", CD+·;
`CDS' th)mOC}tC subsets from their double-positive
`
`precursors, cause· a11 increase in the proportiun of
`double-positive cells, and rcducr thr migration of
`crlls from the cortex to the medulla.
`Although the unusual effects of RPi\I on the
`morphology and th)111ocyre subset composition can(cid:173)
`not br ful ly explained until m ore is k nm1~1 about its
`actions, we can speculate on mechanisms that might
`be rt·sponsiblc !'or the effects of'RPf\I (Fig 8). Ir RPM
`u·catmcm docs not interfere with clonal deletion ol
`double-positive cells b)· apoptosis but docs block the
`rescue of double-positive thymOC)1es from cell death
`(rosith·c srlcction), a net loss or double-positive
`th)mocytcs cells will occur. T reatm e nt with FK506
`(or CsA) will have the opposite effect because FK506
`(or CsA) will prevent ncgati\·e selection of potentially
`autorcactive double-positive cells by programmed
`cell death. This tentati,·c hypothesis may explain why
`animals bric Ay trl'atcd with RPM do no t develop the
`syndrom e or S)'l1gcneic GvH (Zhrng B, Morris RE:
`unpublished obser\'ation, 1991) that has been dr(cid:173)
`scribcd in animals treated \dth CsA.'"I."' Three lines
`or C\idence"'·111 support this hypothesis: (I) RPf\!
`dot's not inhibit acth·ation-induced hybridoma apop(cid:173)
`losis and cell dra th in '~tro; (2) CsA inhibits D NA
`fragme ntation in immature thyi110C)'lt'S, and FK506
`inhibits activation-induced apoptosis of hybridoma
`cells; and (3) recc111 Ir it has been suggested that the
`
`co4-cos-
`FK506 ~ ~ - ~~~ +
`~
`~~~
`
`co4+cos+
`~
`~~~
`~~ ~
`
`co4+co1n co4-cos+
`
`~ ~
`~~
`~
`
`•
`
`RPM ~~~ + ~~ • ~ ~ -
`
`~~
`
`~~~~
`~ ~
`
`~
`
`~~
`
`+
`
`CELL DEATH
`
`+
`
`CELL DEATH
`
`~
`~~~
`~~ ~
`
`Figure 8. Possible crrfclS of trt.·atmtnl with FK506 OJ Rl'.\I Ult (he intrathymic differentiation nf thymncyces. Flow
`q •tornctric analyses ol t h)'m<>cytrs from FKjOG·ltTatt>d min· and R.l'.\1-tri•ated mile show decreast<I proportions of
`si11gk-positivc {CIH+COH- /CD+- CDH+) and doublc-positiw cells (CD·I +C:DIH ), respccti\'d)" 1\l though other
`rxpl:111atio11s a re possible (increased cell dC<llh of single-positive l't0 lls or the ir acl'dcraled migrntiun [lmlrl 1111111 .. j into
`the pc1 ipher)'}, the clfcrl of' FK506 I rrallncnt 1s most likdy rnust'cl hy .111 intt·rrupliun in the maturation of
`douhlt•·positive thymot.ytes. Failure.' to bl· rescued from p11si1iH· selc«lion ri·sulting in incrt':.t.,t·d cell death is the
`m1M hkcl) rxplanation fnr lhl' drrrl'.isr in duublc-positin: th) morytes in mice I rea11·d wi1 h RPl\1; an intaruptiun of
`m,llur:uion from dnublc-nrgath·I' crlls ur an accelerated diffrrentiatinn from double-positive n·lls into ~inglt-­
`pnsith-r t hpnnC) tes could also explain the clfects of RP:'ll u catmcnl.
`
`
`
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`Morris 1992
`Page 0031
`
`

`

`Rofx1119ri11r
`
`69
`
`CsA/FK.506-resistant CD28signal transduction path(cid:173)
`\\':\} in t hytnot')'tCs is necessal)' for positi\'c selection
`in the thymus"" and RP~l inhibits l}mphocytc activa(cid:173)
`11111
`tion in \~tro via this pathway.' '.,.2
`''
`•
`Other phenomena mighL explain the effects of
`RPM treatment on th)1nocytc subpopulations. J:or
`example, RP~! could be directly toxic to double(cid:173)
`positive cells and cause increased cell death. A less
`likely explanation for I hr low percentage of double(cid:173)
`positi\'C cells is that RPM accelerates the differentia(cid:173)
`tion of' this immature population to cells bearing the
`mur.e mature single-positive phcnot)vc. Although
`the percentage of singlc-positi,·c cells increases dur(cid:173)
`ing RPM treatment, the substantial loss of cells in
`the thymus caused br RPl\'l favors a net cell loss of
`thymocytcs rather than their redistribution br accel(cid:173)
`erated maturation from the cortex to the medulla.
`Because the increase in the percent of double(cid:173)
`ncgativc thymocytes in RPM-treated mice is not
`quantitative!)· im'Crsclr proportional to the decrease
`in dnuble-po~itive cells, it is unlikely that RP:\l causes
`a decrease in the percentage of the double-positi\'e
`population solely br arresting the maturation of
`dnuble-negati\-c thymOC)'tCs to the double-positi\·e
`phenotype.
`Our preliminar)' findings on the distinct effects of
`RP.to.I on murine th)1110C)te populations combined
`with our studies in vivo on the acquisition of specific
`unresponsiveness to ear-heart grafts suggests that
`RP.to.I treatm;:nt ma>' Pl"O\ide the appropriate en\iron(cid:173)
`ment for the induction of tolerance. RPM, acting on
`mature circulating T cells, could prevent immediate
`graft rejection. RPM, acting on thymocytes, could
`enable maLuring and potentially alloreactive th)'mo(cid:173)
`C)'lCS in the recovering thymus to be ncgativelr
`sclrctcci when donor m<\jor histocom patibility com(cid:173)
`plex (l'vlHC) peptides are presented by thymic den(cid:173)
`dritic cells in the context of self-l'vlliC. However, we
`have recently found that RPM treatment of adult
`thymectomized recipients of ear-heart allografts also
`causes prolongation of graft survival (Morris RE,
`Shorthousc R: unpublished obsen."ations, 1991). Al(cid:173)
`though prolongation of graft sun.frat in thrse mice is
`not a.s great as in RP:\1-treared euthymic mice, any
`hypothesis or the mechanism of immunosuppression
`of RPM may have tu be expanded to include clonal
`anerm and acti\·c suppression.
`In the future, the pharniaculugical effects ofRPi\l
`on the th)111llS and other primal)· l~111phoid tissues
`may prodde Yaluable dues to define rhc events
`leading lo self and non-self discrimination. Perhaps a
`method will be found to use RP~! tu induce spcriJic
`
`unresponsiveness in human graft recipients and in
`p.1.ticnts ,,;th autoimmune diseases.
`
`Effects of RPM on Immune Cells In
`Vitro
`'llil' structural similarity between FK.506 and RP!\I
`and the pre,fously described immunosuppressi,·e
`effects of RPM in \~vo prompted the initiation of
`studies of the suppression of graft rejection or RPM
`in vivo and its effects on immune cells in ~tro. 111e
`spct:ilic mo tives of the investigators evaluating RPM
`in vivo differed from those who included RPM in
`their in vitro experiments. RPM was the primary
`focus of the in ,;vu studies designed by investigators
`at the Laborator)'ofTransplantation Immunology of
`Stanford Univer'l>ity to define its immunosupprcssi,•e
`cfficac.y and mechanisms of acriun; FK.506 was in(cid:173)
`cluded for comparison. FK.506 was the primar)' focus
`of io vitro studirs designed to define its mechanisms
`ofimmunosupprcssi"e action; RPM was included for
`comparison. Regardless of the difference in motives
`for studying the effects of RPM on the immune
`system in vivo and in vitro, both approaches simulta(cid:173)
`neously showed rhat RPM and FK.506 affect the
`immune system quite differently.
`Despite the role of RPM as a supporting actor in
`most in vitro studies, its effects on immune cells
`would be far less clear had it been gi\'en no role at all.
`Othl·1· immunusupprcssivc drugs such as CsA ha\'C
`been used as tools 10 pry apart the biochemicaJ
`components of T-ct·ll activation in vitro and have
`initiated a self-perpetuating process or immunnsup(cid:173)
`pressive drug discovery and development. For exam(cid:173)
`ple, as t he understanding of immune cell activation
`increases, it bc:cumcs easier lo distinguish among
`differences in the mechanisms of action of new drugs
`like FK506 and RP:\l, and new strategics for immu(cid:173)
`nusupprcssion emerge. It is difficult to pinpoint the
`precise mechanisms ofimmunosuppressive action of
`RPM from in \~Vo experiments because the drug
`effects of RPt\I in vivo art• the net result of many
`unknown interactions among RP:\l, the immnune
`S}stem, and other biological systems. In vitro experi(cid:173)
`ments offer the opportunity to observe the effects of
`unmctabuli7.rd RP~I on defined biochemical events
`during the controlled activation of well-characterized
`immune cells.
`Drspitc thrsr acl\':mtages of working in vitro, it
`can be treacherous to assume that the mechanisms
`of action of an immunosupprcssanl defined in \~tro
`apply equally to its actions in \~\'0.! 1 For example,
`
`
`
`Breckenridge Exhibit 1055
`Morris 1992
`Page 0032
`
`

`

`70
`
`Rn11dnll Eilts J\Jnnis
`
`Table 12. F.ffccts of'Trca1 mcnt \\'ilh FKS06 ur Rl'~ I on T C:dls In Vitro
`
`{11 l'11m. ltliri!1· St111/ml
`
`Cdl 1)t ..
`
`AtlitnlumS/111111/1
`
`Jurl<at
`.Ju1k;u
`HumJnP»I.
`'.\louse splcm, hum.in PDL,
`.Jurkai
`lluma111'131,
`
`NunC'
`lononwcin + PE
`ConA'.t PE
`C' .. onA + PF, l1111n111yl'in + PE,
`anti·C.:03 ±PE
`ConA +PE
`
`Humanl'BL
`
`C:onA +PE
`
`~louse spleen. human PllL, mou:sc
`IJ r2-prntlndng
`hybriduma,.Jurkat
`HumanPl31.
`Human Pl31.
`Human PB!,
`Hum;inPBL
`/\louse spleen, human PBL
`
`Mr1t1M' spleen, humnn 01
`1>0rci11c PBL, muusr 1112
`done
`
`Human primed lpnphoc}'ICs l'rom
`I ranspla 111 biopsies
`Mumn11 or purcinc PBI.
`Pnn.:.inl' PBl-. mowic splCl'll
`
`ConA ± PE. lu11omyd11 + Pf.,
`:uni-C:D3 01 aini-C02 + l'E
`
`Conr\
`ConA
`Co111\
`ConA
`PE+ lonomyc111, ant...C03 or
`anti-CD'l + PE
`ConA or PHA ±PE. lonom)-
`cin +PE, ;uui.(!03 or an1i-
`CD2 + PE, a111i-C:D2
`(T l 1.2) + :1111i-CD2
`(Tl 1.3), rnn
`Allogcneic human 1'13L
`
`Cont\, !'HA, an1i-C:03
`ConA :l: PE
`
`~lou~csplctn, h11m~1111r
`porcim• PBL
`Human or puninc Pill
`~ lnuS<· •plccn
`
`ConA, lo11om110111 + PE. Pl L•\, Con;\
`
`Drug~/fifir
`FK506 Rf'.\/
`
`IJ
`
`0
`n
`11
`
`0
`
`"
`A, l
`
`l
`!
`"
`II
`l
`
`II
`
`II
`No,
`Yes
`
`II
`
`u
`i
`n
`11,n
`
`II
`
`..
`u
`
`11
`0
`0
`II
`0
`
`II
`
`II
`
`ll
`No,
`Yes
`
`l
`
`0
`0
`
`AC11VATION
`Ca' ' -OEP!ll'1DEl''T
`Gene tr:inscnption
`NF-ATac1hit)
`l\T-Xf'.f>:'llA hinrling
`r:fas
`IL-2
`
`llr3, ~f, C~l-C'iF, N!W,
`TNf'-a, IFN·'Y
`IL-2~, T l: R, Tl\'T'-~
`Cytoki11c produciion
`CL-2
`
`u,.1
`IL-Ii
`TGF-~
`IFN·-y
`lL-2R ecll •u• face c•1>rcsJio11
`
`01'\A S)11thc,is
`
`P1 ut1•i11 ~)Ill hc•is
`Rc\'crs.11 or cl rug dfrc1 after
`drug H<"h oul~ P1otci11,
`L>N1\ S\lllhcsis
`Block in c~ll q« lc progrc~•ion
`c . -G,
`
`c,-.s
`lnl11bi1in1111fDNA <mlhc-
`sis iu okadaic acid-
`11cah'd l'c·ll•d111 ing G,
`Apopt'"'i'
`
`~ lou!K' hyl>1 idnma
`
`lonomycin + PE, n11ti.CD3
`
`II
`
`0
`
`Cn' '-11\'DEPENDEl'<T
`Cyl r>kirw produr1 ion
`Yt\C-l lympl1u111a
`IFN"l
`Nom.1 lokin1.:-i11drn·rd JH otdu or Human cir porcine l'BL, alloa1ti\';11ed
`DN!\ 5)11thesis
`hu111:111 l'BL
`Cy1okint·1ml11ccd prot..in 01
`Human l'BL, mouse <pflo(·u
`OXA S)11lh<'"'
`cdb
`Actl\alcd humau l'BL
`CllJ.., po1une IL-2-dcpc:n-
`dcnl line
`Motl!ic 1112 done·
`'.\louse spleen cell•, C:TU.
`IL-6-<lqM·mll'nl linr
`\'A\~l l1 mphum.1
`
`IL-I+ PE
`r\nti-CD211 + PE
`PE
`IL-2 + PE, llr'l,
`
`Jlr:l
`
`IL-I + PE, IL-I + luno111rc111
`~:l:PI;
`IL6
`IFN--y
`
`C.:ytoki1lt'·ind11ccd Li-liE t l'll
`:mr fore· :unigr11 mclu,:1ion
`C:cll 111cdiatt'ci ioxicnr
`Grncr.11 ion ufCTL
`pC11. f1'<'<J11c11cy
`C:ons1it111iv1· UNA ~\nthctiis
`C:un:<1lil111iw prulc·ir; 1')1Hhrsi'\
`CdlviJbility
`
`ll11ma11 Pill.
`Human l'lll.
`~louse hybridoma,.Jurk.\1
`l'ordnc Pill,
`M1tu111c hybaid1u11:.. human
`PBL
`
`i\ ILR
`/vD, R + llr'.!
`Nonl·
`None
`None
`
`Abbn.,i;11irnl!I: Pl IA, phr tcdmnagglutinin; PBL, pcriphrr.11 h~llid ly11111hoa1rs; PE: phorbal r'lrr.
`S~mbul1':-,11ot tt•<itrd;o.nudfl·ct: t .. 1cuvil~ mh1h1tt·d.
`
`!
`II
`II
`!I
`
`II
`u
`II
`II
`II
`
`!
`ti
`u
`i
`u
`
`"
`.,
`"
`
`0
`
`0
`fl
`
`0
`
`0
`
`"
`"
`
`
`
`Breckenridge Exhibit 1055
`Morris 1992
`Page 0033
`
`

`

`Rnpn111Jrins
`
`71
`
`Table 13. Effects ofTreatment "ith FK.506 or RPM on B Cells and Ba50phil~ In Virro
`
`Jn VitmAtlfoi9• Studitd
`
`vll7jpe
`
`Acliunlion Sl1mu/1
`
`Drog Ejfrcts
`FK506
`RPM
`
`BCELLS
`
`ACTIVATION
`Ca1
`' ·DEPENDEi"lff
`Protein synthesis
`DNA synthesis
`
`I? cell surl~ce Ag expression
`Block in cell cycle progression
`G0 -+G1
`
`G1-+S
`s
`
`T c:cll-<.lepcndent lg production
`
`Ca" -1NDEPEJ\1DEJ\1T
`Protein synthesis
`DNAS)nthesis
`
`Viability
`Constitutive DNA synthesis
`
`BASOPHILS
`ACTIVATION
`Histamine release
`
`Mouse spleen
`Mnuse spleen
`
`HumanPBL
`Mouse spleen
`
`Mousespkcn
`HumanPBL
`Mouse spleen
`.Mouse spleen
`HumanPBL
`HumanPBL
`
`Mouse spleen
`Mouse spleen
`
`Mouse spleen
`Daudi, human EB-
`transformed line
`
`Anti·lgM
`Anti-lgi\1, anti-
`IgM + IIA
`PWM
`Anri-lgM
`
`Anri-lgM
`PWM
`Anri-lgM
`Anri-IgM
`PWM
`PWM
`
`LPS
`LPS, 8-mcrcapto-
`guanosine
`Anri·Igl\I
`None
`
`HumanPBL
`
`Anti·lgE
`A23187
`
`it
`u
`
`0
`
`0
`
`l
`l
`!
`
`0
`0
`
`II
`"
`
`u
`u
`
`"
`u
`"
`
`0
`
`0
`l
`l
`0
`0
`l
`
`u
`"
`~ u
`
`.j.
`0
`
`Abbre\'inlion: LPS, lipopol)1accharidc; PWM, pokcwccd mitogcn.
`S)mliols:-, 11011cs1cd; o, no clrcct: l , act hit~· inhibited
`
`concentrations of RPM that arc required to cause
`specific effects in vitro may be well· in excess of the
`maximum tolerated plasma li;vels of RPM. The
`chemical instability of RPM in vitro {previously
`discussed) makes it impossible to know whether the
`parent or a degradation product is responsible for
`the effects observed. Complicating these problems is
`the lack of compll'te dose-response studies in some
`experiments. Furthermore, the contents of the cul(cid:173)
`ture medium (serum, cofactors, growth factors) and
`cell density can affect the observed effects of immu(cid:173)
`nosuppressants in vitro. Finally, the activation signals
`used to stimulate immune cells in vitro may cause
`changes in second messengers along the signal trans(cid:173)
`duction pathway that are quantitativeJy or tempo(cid:173)
`rally different from in vivo stimuli. Similar concerns
`apply to the in \ivo relevance of data derived from
`the exposure of RPM to transformed cell lines.
`Nevertheless, carefully selected information from
`I he study of the effects of RPM on immune ceUs in
`"itro enables us to gain a deep understanding of the
`effects of RPM in vivo. In the final analysis, the sum
`of the knowledge of the in \~Vo and in vitro actions of
`
`RPM is far greater than conclusions derived from
`analyting in vivo or in vitro data separately.
`The subsequent section will review the results of
`published in vitro studies in whicl1 RPM was evalu(cid:173)
`ated, and only the studies in which both FK506 and
`RPM were evaluated simultaneously in the same in
`vitro experimental systems. To date, the in vitro
`effects of RPM have been tested on cells from mice,
`pigs, and humans. These cells have included normal
`lymphocytes, hybridoma cell lines, transformed T
`and B cells, and normal basophils (Tables 12 and 13).
`
`Effects of RPM on T Cells
`Effects on T-cell activation. The effects of RPM on
`the activation of T cells, constitutive protein and
`DNA synthesis, cell-mediated toxicity, and cell viabil(cid:173)
`ity ha\'C been reported (Table 12). The majority of
`these studies have concentrated on defining the
`effects of RP~l on T-cell activation for several rea(cid:173)
`sons: (1) the molecular c\·cnts of this process are
`becoming increasingly clear; (2) FK506, the struc(cid:173)
`tural homologue of RPM, is known to inhibit activa(cid:173)
`Jion; and (3) T-ccll activation is an important compu-
`
`-
`
`
`
`Breckenridge Exhibit 1055
`Morris 1992
`Page 0034
`
`

`

`72
`
`Rn11dnll Eilis .lft11ris
`
`ca2+ ·DEPENDENT
`
`ca2+ ·INDEPENDENT
`
`ACCESSORY MOLECULES
`
`ICAM·1
`+
`LFA·1
`
`87/881
`+
`C028
`
`OTHERS?
`
`Ag - - MHC
`LFA-3
`+
`+
`+
`C045 TCR·C03 C04/C08 C02
`
`P'1SE
`
`t·~K ' /
`
`~
`tPLC
`
`)
`
`)
`
`t
`t
`CYCLIN + p34cdc2 - P'ASE
`t
`t
`- mRNA =::: IL-2 + IL·2A -
`t
`
`JNA
`STABILIZATION
`
`I
`TRAN~6~FPT10N ?
`j
`
`• / / tCYTOKINE
`i ca2+
`
`t PKC
`
`•
`
`~ ~ PAOTEIN(s)X- CsA -CyP(s)
`T ....._ ~ PROTEIN(s)X- FK506 -FKBP(s)
`
`DNA SYNTHESIS
`
`t
`
`TRANSCRIPTION
`FACTOR
`
`Rb-PHOS
`
`cdc2
`
`FKBP(s)
`I
`RPM
`I
`PROTEIN(s) Y
`
`TRANSCRIPTIONAL
`ACTIVATING FAc.;roRS
`
`Go -0 ,
`
`CYTOKINE GENES
`ONCOGENES
`
`PTK
`
`TNF·cz. IFN""(, IL-4,
`GM·CSF, C·myc
`G1 - s
`
`F igure 9. Biochemical pnthways leading to T-ccll prolifcrn1ion a~1ercngagrmrnt of 1he TCR/CD3 complrx with antigt:n
`and the interaction of' nccessoryT-cell 111nlcrnles wiLh their ligands ;md IL-2 with IL-2R. The possible sites of action of lhc
`immunosuppress:mts crnnplexcd lo q•toplnsmic binding ;ind errector proteins a re shown: CsA bound to cyclophi lins, FK.506
`amt RPt-1lx)uncl10 FKJ3P. Although not pro,·en, it has been sugges