`© John Libbey Eurotext
`
`La rapamycine et Ie CCI-779
`
`Rapamycin and CCI-779
`
`Bull Cancer 1999; 86 (10): 808·11
`
`Jerome ALExANDRE
`~ric RAYMOND
`Jean-Pierre ARMAND
`
`Dc!partement de mc!decine. Institut
`Gustave-Roussy. 94805 Villejuif
`Cedcx.
`
`Article rer;u Ie 15 juin 1999, accepte apces
`revision Ie 9 aout 1999.
`Tires a pact: J. Alexandre.
`
`Resume - La rapamycine (sirolimus) est un macrolide proche de la cidosporine
`possedant des proprietes immunosuppressives et une activite antiproliferative in
`vitro sur plusieurs lignees tumorales humaines et sur des modeIes de tumeurs trans(cid:173)
`plan tees. La kinase cytoplasmique mTOR. qui contrDIe l'iniciation de Ia traduction
`des ARN messagers en reponse a des facteurs de croissance, est Ia principale cible
`cellulaire connue de Ia rapamycine. Au cours des essais diniques, la rapamycine uti(cid:173)
`Iisee par voie orale comme agent immunosuppresseur ne presentait pas de toxicite
`!imitanre et etait responsable uniquement de thrombopenies et d'hyperlipidemies
`asymptomatiques. Dans des modeIes murins, une meilleure activite antitumorale
`etait observee en utilisant une administration parenterale. Le CCI-779, un analogue
`con~u pour ulle administration intraveineuse, possede chez Ia souris une activite
`tumoraIe sans propriete immunosuppressive significative et fait actuellement I'objet
`d' essais de phase I chez l'homme. A
`Mots des: rapamycine, mTOR, CCI·779.
`
`Abstract - Rapamycin (sirolimUJ) is a macrolide, related to eydosporine with immuno(cid:173)
`suppressive properties and antiproliferative activity in various human tumor cells lines
`and tumor xenograft models. The eytosolic kinase mTOR which controls the initiation
`of the translation of messenger RNA is the main known target of rapamycin. During cli(cid:173)
`nical studies, rapamycin given by oral route as immunosuppressant did not show dose(cid:173)
`limited toxicity and only asymptomatic thrombopenia and hyperlipemia were observed.
`In murine models. best antitumoral activity was observed using parental routes. CCI-
`779. an analog formulated for intravenous use, has antitumor activity without signifi(cid:173)
`cant immurlosuppressive property in mice and is currently in phase I trials in man. A
`Key words: rapamycin, mTOR, CCf·779.
`
`La rapamycine (figure 1) ou sirolimus est un macrolide
`
`produit par Streptomyces hygroscopicus, proche de la
`cic/osporine et du FK506 (tacrolimus). Elle fut initiale(cid:173)
`ment identifiee eomme un agent antifongique il ya mainte·
`nant trente ans [1]. Secondairement, elle a surtout ete deve(cid:173)
`loppee comme un agent immunosuppresseur (Rapamune'8>,
`Wyeth~Leder/e). Plusieurs essais c1iniques ont montre son
`interet dans Ie traitement du rejet de greffe d'organe (2]. Ses
`proprietes antitumorales sur differents modelescellulaires
`sont egalement connues depuis plusieurs annees, mais n'ont
`jamais fait f'objet de publications en C/inique humaine (3].
`L'originalite de fa rapamycine tient a son mecanisme d'ae(cid:173)
`tion. Elle inhibe specifiquement une proteine kinase eyto(cid:173)
`plasmique, mTOR (pour mammalian target of rapamycin)
`qui intervient dans une voie de signalisation mitogenique
`reglant I'initiation de la traduction (4].
`L'interet pour la rapamycine a recemment etc relance par
`Ie developpement d'ana'logues tels que Ie ((1-779
`(figure 1) temoignant, sur des modeles preC/iniques, d'une
`
`___ 808
`
`aetivite antiproliferative et d'un faible effet immunosup(cid:173)
`presseur [5].
`
`Figure 1. La rapamycine et Ie CCI·779.
`
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`Alexandre 1999
`Page 001
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`
`La rapamycine
`
`Activite antitumorale
`
`/n vitro, la rapamycine inhibe la croissance de plusieurs
`lignees tumorales humaines, en particulier d'osteosarcome
`[6]' de rhabdomyosarcomes Rhl et Rh30 [7), de cancers
`bronchiques a petites cellules H69, H345 et H51 0 [S]' d'he(cid:173)
`patome H4 (9) et de cancer du sein hormonodependant
`MCF7 [10). La gamme des concentrations efficaces va de
`0,3 a 50 nM. Dans plusieurs de ces lignees, il a ete montre
`que la rapamycine bloque les cellules en phase Gl du cyele
`cellulaire. Cet effet cytostatique a ete confirme in vivo sur
`des modeles de xenogreffes de tumeurs humaines chez la
`souris nude [3). Un effet proapoptotique a egalement ete
`observe sur des lignees de rhabdomyosarcome cultivees
`sans facteur de croissance (11) et en association avec Ie cis(cid:173)
`platine sur une I ignee de carcinome ovarien [12).
`
`Mecanisme d'action moltkulaire
`
`La rapamycine peut etre consideree comme une « pro(cid:173)
`drogue» dans la mesure OU son action intracellulaire neces(cid:173)
`site sa fixation a une immunophiline denommee FKBP12
`(FKS06 binding protein, car elle fixe egalement Ie FK506)
`[13). Le complexe ainsi forme est tres stable (temps de
`demi-dissociation de 17,5 h) et autorise un effet biologique
`prolonge de la rapamycine (7 jours apres une heure d'expo(cid:173)
`sition in vitro) [11 J, suggerant que celle-ci puisse etre admi(cid:173)
`nistree de fa~on discontinue.
`La seule cible actuellement connue du complexe rapamy(cid:173)
`cinelFKBP12 est la proteine mTOR. Celle-ci est une serine(cid:173)
`threonine kinase de la famille des phosphatidyl inositol
`kinases [4). Elle est activee par un grand nombre de facteurs
`de croissance, en particulier les interleukines2, 4 et 6, I'in(cid:173)
`suline, et J'insulin-like growth factor 1. En reponse a ces sti(cid:173)
`muli mitogeniques, mTOR va activer I'initiation de la tra(cid:173)
`duction d'un grand nombre d' ARNm par deux voies
`paralleles [4, 14) (figure2):
`- Ie facteur d'initiation de la traduction 4E (eIF4E) est a I'etat
`basal sequestre et inhibe par la proteine 4E-BP1. mTOR, en
`phosphoryl ant cette derniere, va permettre la liberation de
`elF4E et I'initiation de la traduction. Les proteines ainsi syn(cid:173)
`thetisees sont supposees induire, directement ou indirecte(cid:173)
`ment, la transition GlIS;
`- mTOR active la p70/S6 kinase qui active alors a son tour
`la proteine ribosomique 56. Sous sa forme phosphorylee,
`cette derniere contrale selectivement la traduction d' ARNm
`possedant un domaine riche en pyrimidine a leur
`extremite 5'. Ces ARNm codent pour des proteines riboso(cid:173)
`miqucs et des facteurs d'elongation.
`Les facteurs proteiques actives en amont de mTOR apres
`fixation du facteur de croissance sur son recepteur sont
`moins bien connus. Une isoforme de la pS5/PI3 kinase serait
`en particulier impliquee [15}.
`L'inhibition de la fonction biologique de mTOR par la rapa(cid:173)
`mycine semble jouer un role essentiel dans son action cyto(cid:173)
`statique. Cependant, il reste encore beaucoup a apprendre
`sur, d'une part, les mecanismes exacts par lesquels mTOR
`c.:ontrale la transition G 115 et, d'autre part, d'eventuelles· autres
`cibles cellulaires de la rapamycine. Ainsi, iI a ete recemment
`montre sur des fibroblastes murins 3T3 stimules par du serum
`
`La rapamycine et Ie CCl-779
`
`Recepteur
`
`;1;1
`••
`
`I
`I
`\
`
`--."J.
`
`-::; elF4E:"'~
`.; -. ". '
`4E-BPl
`
`P
`
`P
`
`Initiation de la traduction
`
`Figure 2. Voie de transduction impliquant mTOR. Fc: facteur de
`croissance; PllKase: phosphatidyl inositol 3 kinase; mTOR: mam(cid:173)
`ma/ian target of rapamycin; rapa: rapamycine; FKBP: FKS06 bin(cid:173)
`ding protein; p70/56 Kase : kinase de la proteine ribosomique 56;
`eIF4E: facteur d'initiation eucaryote 4E; 4E-BPl : elF4E binding
`protein.
`
`que la rapamycine diminuait Ie taux de cyeline 01. Cepen(cid:173)
`dant, contrairement a ce que I'on pouvait croire, cela n'etait
`pas dO a un defaut de synthese mais a une degradation acce(cid:173)
`leree de la proteine (16). Sur d'aulres modeles, la rapamycine
`induit I'accumulation de I'inhibiteur de cyeline p27kip1 (17).
`L'action proapoptotique de la rapamycine est egalement
`mal connue. Elle semble dependante de I'inhibition de
`mTOR, mais pas de p53 (11), ni de bel-2 (18).
`Une meilleure connaissance des mecanismes d'action de la
`rapamycine au niveau moleculaire permettrait idealement
`de predire la sensibilite des tumeurs a cette molecule en
`fonction de I'expression ou de la non-expression de tel ou
`tel gene. 1/ a ainsi deja ete montre que des tumeurs surex(cid:173)
`primant c-myc etaient resistantes a la rapamycine [19], de
`meme que celles issues de patients atteints d'ataxie-telan(cid:173)
`giectasie (20). De fat;on interessante, Ie gene ATM, deficient
`au cours de I'ataxie-telangiectasie, code pour une phospha(cid:173)
`tidyl inositol kinase proche de mTOR.
`
`Experience clinique
`
`Elle concerne essentiellement I'utilisation de la rapamycine
`comme agent immunosuppresseur, seule ou en association
`
`West-Ward Exhibit 1030
`Alexandre 1999
`Page 002
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`
`
`J. Alexandre et al.
`
`avec la cielosporine. La rapamycine est administree par
`voie orale et est metabolisee par Ie cytochrome P450 3A en
`I?lusieurs metabolites inactifs (21).
`A la dose recommandee pour les phases II de 7 mglm2/j, la
`toxicite porte essentiellement sur des parametres biologiques
`[2]. II existe une toxicite hematologique dose-dependante
`predominante sur les plaquettes, et generalement asympto(cid:173)
`matique et ne mkessitant pas de transfusion. Cette hemato(cid:173)
`toxicite est d'origine centrale et pourrait etre liee a I'inhibition
`par la rapamycine des signaux mitogenes transmis par les
`cytokines. La rapamycine augmente Ie taux de triglycerides et
`de cholesterol parfois de fa~on considerable. Cette hyperlipe(cid:173)
`mie pourrait surtout poser des problemes lors d'une adminis(cid:173)
`tration au long cours du fait de Vaugmentation du risque car(cid:173)
`diovasculaire. II n'a jamais ete observe de pancreatite aigue
`liee a I'hypertriglyceridemie, ni d'autre manifestation eli(cid:173)
`nique. Chez ces patients deja lourdement immunodeprimes,
`la rapamycine ne semble pas augmenter Ie risque d'infec(cid:173)
`tions, sauf peut-etre celles liees au virus herpes simplex. Cette
`constatation est a rapprocher du fait que la rapamycine aug(cid:173)
`mente la traduction des ARNm de certains virus (22).
`
`Un nouvel analogue de la rapamycine:
`Ie CCI-779
`
`Dans plusieurs modeles murins, I'action antitumorale de la
`rapamycine est plus importante lorsqu'une administration
`parenterale est utilisee. Cependant, celle-ci est difficilement
`realisable en pratique elinique courante du fait d'une faible
`solubilite de la molecule (5]. Des analogues de la rapamy(cid:173)
`cine ont donc ete developpes dont les proprietes physico-
`
`chimiques permettent une administration par voie intravei(cid:173)
`neuse ·aisee.
`Le CCI-779 est I'un de ces analogues. II presente une actio
`vite cytostatique sur plusieurs modeles de xenogreffes de
`tumeurs humaines a des souris nude, en particulier dans des
`glioblastomes, carcinomes de prostate, du pancreas et du
`sein (23) et des medulloblastomes (24).
`Tout comme la rapamycine, la fixation du CCI-779 a la
`FKBP12 est une etape indispensable a son action (23). II reste
`cependant a determiner si Ie meqnisme d'action du CCI-779
`est entierement superposable a celui de la rapamycine.
`De fat;on interessante, I'activite antitumorale decrite chez la
`souris nude est maintenue pendant 14 jours apres une admi(cid:173)
`nistration quotidienne de 5 jours alors que I'effet immuno(cid:173)
`suppresseur disparait au bout de 24 h (23).
`Des essais therapeutiques de phase I sont en cours en
`France et aux Etats-Unis.
`
`Conclusion
`
`La rapamycine est Ie premier representant d'une nou(cid:173)
`velle classe d'anticancereux au mecanisme d'action tout
`a fait original et au profil de toxitite favorable. Les
`etudes precliniques suggerent pour ces molecules une
`action essentiellement cytostatique. Le CCI-779, premier
`analogue administrable par voie parenterale, fait actuel(cid:173)
`lement I'objet d'essais c1iniques de phase I. Parallele(cid:173)
`ment, une meilleure connaissance des cibles molecu(cid:173)
`laires de la rapamycine et de ses analogues pourrait
`gUider la realisation de futurs essais c1iniques de
`phase II. T
`
`REFERENCES
`
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`14. Beretta L. Grolleau A. La rapamycine: identification d'une nouvelle
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`16. Hashemolhosseini S. Nagamine Y. Morley SJ. Desrivieres S,
`Mercep L, Ferrari S. Rapamycin inhibition of the G 1 to S transition is
`mediated by effects on eyelin D 1 mRNA and protein stabiliry. ] Bioi
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`17. Nourse j, Firpo E, Flanagan WM, Coars S, Polyak K, Lee MH, ~t al
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`18. Veverka KA. Germain GS, Dilling MB. Houghton P]. Overexpres(cid:173)
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`induced apoptosis. Proc Am Ass Canc~r Res 1998; 39: 571 (abstr. 3880).
`19. Dilling MB. Hosoi H. Liu LN. Germain GS, Houghton PJ.lntrinsic
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`protein levels. Proc Am Ass Canctr R~s 1999; 40: 428 (abstr. 2827).
`20. Beamish H. Williams R, Chen p. Khanna KK. Hobson K, Watters D.
`Rapamycin resistance in ataxia-telangiectasia. Oncogm; 1996; 13: 963-70.
`21. Trepanier OJ. Gallant H, Legan OF, Yarscoff RW. Rapamycin: dis-
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`tribution, pharmacokinetics and therapeutic range investigations: an
`update. Clin Bioch~ 1998; 31: 345-51.
`22. Beretta L. Svitkin YV. Sonenberg N. Rapamycin stimulates viral pro(cid:173)
`tein synthesis and augmenrs the shutoff of host protein synthesis upon
`picornavirus infection. J Virol 1996; 70: 8993-6.
`23. Gibbons JJ. Discafani C. Peterson R. Hernandez R. Skotnicki J.
`Frost P. The effect of CCI-779. a novel macrolide anti-tumor agent. on
`the growth of human tumor cells in vitro and in nude mouse xenografrs
`in vivo. Proc Am Ass CanuT R~s 1999; 40: 301 (abStr. 2000).
`24. Geoerger B. Kerr K. Janss AJ. Sutton LN. Phillips PC. Rapamycin
`analog CCI-779 inhibirs growth of human medulloblastoma xenografrs.
`Proc Am Ass Can," R~s 1999; 40: 603 (abstr. 3978).
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`Rapamycin and CCI-779
`
`
`
`Jérôme ALEXANDRE
`
`Éric RAYMOND
`
`Jean-Pierre ARMAND
`
`
`
`Department of Medicine,
`
`Gustave-Roussy Institute, 94805 Villejuif
`
`Cedex.
`
`
`
`
`
`Article received on June 15, 1999,
`
`accepted after revision
`
`on August 9, 1999.
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`
`
`Reprints: J. Alexandre.
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`Rapamycin (Figure 1) or sirolimus is a macrolide produced by Streptomyces hygroscopicus
`
`that is similar to cyclosporine and FK506 (tacrolimus). Now it has been thirty years since it was
`
`initially identified as an antifungal agent [1]. Secondarily, it has been developed as an
`
`immunosuppressant (Rapamune®, Wyeth-Léderlé). Several clinical trials have demonstrated its
`
`usefulness in the treatment of organ graft rejection [2]. Its antitumor properties on different cell
`
`models have also been known for several years, but they have never been the subject of publications
`
`in human clinical medicine [3]. The originality of rapamycin is due to its mechanism of action. It
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`specifically inhibits a cytoplasmic protein kinase mTOR (mammalian target of rapamycin) which
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`intervenes in a mitogenic signaling pathway regulating the initiation of translation [4].
`
`
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`The interest in rapamycin has recently been rekindled by the development of analogs such as
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`CCI-779 (Figure 1) which have demonstrated an antiproliferative activity and a slight
`
`immunosuppressive effect in preclinical models [5].
`
`
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`Rapamycin
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`CCI-779
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`
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`Figure 1. Rapamycin and CCI-779.
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`Rapamycin and CCI-779
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`Rapamycin
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`Antitumor activity
`
`
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`In vitro, rapamycin inhibits the growth of several human tumor lines, in particular
`
`osteosarcoma [6], rhabdomyosarcomas Rh1 and Rh30 [7], small-cell bronchial cancers H69, H345
`
`and H510 [8], hepatoma H4 [9] and hormone-dependent breast cancer MCF7 [10]. The range of
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`effective concentrations is from 0.3 to 50 nM. In several of these lines, it has been shown that
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`rapamycin blocks the cells in the G1 phase of the cell cycle. This cytostatic effect was confirmed in
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`vivo on xenograft models of human tumors in nude mice [3]. A proapoptotic effect has also been
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`observed on rhabdomyosarcoma lines cultured without growth factor [11] and in combination with
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`cisplatin on an ovarian carcinoma line [12].
`
`
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`Mechanism of molecular action
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`
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`Rapamycin can be considered a "prodrug" to the extent that its intracellular action requires
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`that it be bound to an immunophilin called FKBP12 (FK506 binding protein, because it also binds
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`FK506) [13]. The complex so formed is very stable (half-dissociation time of 17.5 h) and allows a
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`prolonged biological effect of rapamycin (7 days after an hour of exposure in vitro) [11], suggesting
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`that it can be administered discontinuously.
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`
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`The only currently known target of the rapamycin/FKBP12 complex is the protein mTOR.
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`The latter is a serine-threonine kinase of the family of the phosphatidyl inositol kinases [4]. It is
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`activated by a large number of growth factors, in particular the interleukins 2, 4 and 6, insulin, and
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`insulin-like growth factor 1. In response to these mitogenic stimuli, mTOR will activate the initiation
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`of the translation of a large number of mRNA by two parallel pathways [4, 14] (Figure 2):
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`– the translation initiation factor 4E (elF4E) is sequestered in the basal state and inhibited by
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`the protein 4E-BP1. mTOR, by phosphorylating the latter, will allow the release of elF4E and the
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`initiation of the translation. The resulting synthesized proteins are assumed to induce, directly or
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`indirectly, the G1/S transition;
`
`
`
`– mTOR activates the p70/S6 kinase, which in turn activates the ribosomal protein S6. In its
`
`phosphorylated form, the latter selectively controls the translation of mRNA having a pyrimidine-
`
`rich domain at their 5' end. These mRNA code for ribosomal proteins and elongation factors.
`
`
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`The protein factors activated upstream of mTOR after the binding of the growth factor to its
`
`receptor are less well known. An isoform of p85/PI3 kinase is thought to be involved in particular
`
`[15].
`
`
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`The inhibition of the biological function of mTOR by rapamycin seems to play an essential
`
`role in its cytostatic action. However, much remains to be learned about, on the one hand, the exact
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`mechanisms by which mTOR controls the G1/S transition, and, on the other hand, other possible
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`target cells of rapamycin. Thus, it has been demonstrated recently on murine fibroblasts 3T3
`
`stimulated with serum that rapamycin decreases the cyclin D1 level. However, in contrast to what
`
`one might believe, this was not due to a synthesis defect, but to an accelerated degradation of the
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`protein [16]. On other models, rapamycin induces the accumulation of the inhibitor of cyclin P27kip1
`
`[17]. The proapoptotic action of rapamycin is also poorly known. It seems to be dependent on the
`
`inhibition of mTOR, but not of p53 [11] or bcl-2 [18].
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`Receptor
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`Initiation of the translation
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`
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`Figure 2. Translation pathway involving mTOR. Fc: growth factor; Pl3Kase: phosphatidyl inositol 3
`
`kinase; mTOR: mammalian target of rapamycin; rapa: rapamycin; FKBP: FK506 binding protein;
`
`p70/S6Kase: ribosomal protein S6 kinase; elF4E: eukaryotic initiation factor 4E; 4E-BP1: elF4E
`
`binding protein.
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`A better knowledge of the mechanism of action of rapamycin on the molecular level would
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`ideally make it possible to predict the sensitivity of the tumors to this molecule as a function of the
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`expression or non-expression of a given gene. Thus, it was demonstrated that c-myc over-expressing
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`tumors were resistant to rapamycin [19], as was the case for those originating from patients with
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`ataxia-telangiectasia [20]. Interestingly, the ATM gene, which is deficient in ataxia-telangiectasia,
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`codes for a phosphatidyl inositol kinase similar to mTOR.
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`Clinical experimentation
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`The clinical experimentation relates essentially to the use of rapamycin as
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`immunosuppressant, alone or in combination with cyclosporine. Rapamycin is administered by the
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`oral route and is metabolized by cytochrome P450 3A into several inactive metabolites [21].
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`
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`At the dose recommended for the phases II of 7 mg/m2/day, the toxicity relates essentially to
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`biological parameters [2]. Dose-dependent hematological toxicity affecting mainly the platelets
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`exists and, in general, it is asymptomatic and does not require transfusion. This hematotoxicity is of
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`central origin and could be associated with the inhibition by rapamycin of the mitogenic signals
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`transmitted by the cytokines. Rapamycin increases the level of triglycerides and cholesterol,
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`sometimes considerably. This hyperlipidemia could pose problems during long-term administration
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`due to the increase in cardiovascular risk. No acute pancreatitis associated with hypertriglyceridemia
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`or other clinical manifestations have ever been observed. In these already severely immunodepressed
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`patients, rapamycin does not seem to increase the risk of infections, except perhaps infections
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`associated with the herpes simplex virus. This observation should be considered in light of the fact
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`that rapamycin increases the translation of the mRNA of certain viruses [22].
`
` A
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`
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` new analog of rapamycin: CCI-779
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`In several murine models, the antitumor action of rapamycin is greater when a parenteral
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`administration is used. However, this is difficult to carry out in routine clinical practice due to low
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`solubility of the molecule [5]. Analogs of rapamycin have therefore been developed, the
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`physicochemical properties of which allow an easy administration by the intravenous route.
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`CCI-779 is one of these analogs. It has a cytostatic activity on several models of xenografts
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`of human tumors in nude mice, in particular in glioblastomas, carcinomas of the prostate, the
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`pancreas and the breast [23], and medulloblastomas [24].
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`
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`As in the case of rapamycin, the binding of CCI-779 to FKBP12 is an indispensable step for
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`its action [23]. However, it remains to be determined whether the mechanism of action of CCI-779
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`and that of rapamycin entirely overlap.
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`
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`Interestingly, the antitumor activity described in nude mice is maintained for 14 days after
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`daily administration for 5 days, while the immunosuppressive effect disappears after 24 h [23].
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`Phase I therapeutic trials are in progress in France and in the United States.
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`Conclusion
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`
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`Rapamycin is the first representative of a new class of anticancer agents with an entirely
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`original mechanism of action and a favorable toxicity profile. The preclinical studies suggest that
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`these molecules have an essentially cytostatic action. CCI-779, the first analog that can be
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`administered by the parenteral route, is currently undergoing phase I clinical trials. In parallel, a
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`better understanding of the molecular targets of rapamycin and of its analogs could guide the
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`performance of future phase II clinical trials.
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`REFERENCES
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`May l l, 2017
`
`Certification
`
`Park IP Translations
`
`TRANSLATOR'S DECLARATION:
`
`I, Francois Lux, hereby declare:
`
`That I possess advanced knowledge of the French and English languages. The
`attached French into English translation has been translated by me and to the
`best of my knowledge and belief, it is a true and accurate translation of the
`article titled "La rapamycine et le CCl-779" in French and "Rapamycin and CCl-
`779" in English, written by Jerome Alexandre, Eric Raymond, and Jean-Pierre
`Armand at the Department of Medicine, Gustave-Roussy Institute, 94805 Villejuif
`Ced ex.
`
`Francois Lux
`
`Project Number: MEGOP _ 1705_004
`
`15 W. 37th Street 8th Floor
`New York, NY 10018
`212.581.8870
`ParklP.com
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