`
`St
`Current cancer drug targets.
`~ General Collection
`U709
`Vv. 4, ho. 8
`
`
`
` ES|
`
`
`RESRETRSEELEYHELEUIIISTOTTORLIE
`
`REEEeearias
`[MEDICINE]
`EIN
`
`NATIONAL!
`
`LIBRARY OF}
`
`aTIONAL ENSTATUTEE OF DEALT
`
`
`: igatskp?
` SoedeeecandiSaca
`
`
`CETTeee
`
`
`
`
`
`cul
`
`
`
`
`This material was copied
`
`a
`,
`‘
`West-Ward Pharm.
`Exhibit 1033
`Page 001
`
`West-Ward Pharm.
`Exhibit 1033
`Page 001
`
`

`

`Aims and Scope
`Current Cancer Drug Targets aims to coverall the latest and outstanding developments in
`the medicinal chemistry, pharmacology, molecular biology, genomics and biochemistry of
`contemporary molecular drug targets involved in cancer e.g. disease specific proteins,
`receptors, enzymes, genes.
`Each issue ofthe journal containsaseries of timely in-depth reviews written by leaders in the
`field covering a range of current topics in drug targets involved in cancer.
`As the discovery, identification, characterization and validation of novel human drug targets
`for anti-cancerdrug discovery continues to grow, this journal will be essential reading for all
`pharmaccutical scientists involvedin drug discovery and development.
`i _—
`Multiple Journal Subscriptions & Global Online Licenses
`For multiple journal subscriptions, possible discounts and global online licenses please conlact our special sales
`department at E-mail: subscriptions@bentham.org
`
`Advertising
`To place an advertisement in this journal please contact the advertising department at E-mail: ads@bentham.org
`
`Journal Sample Copies
`A free online sample issue can be viewedat the journal’s internet homepage. Alternatively a free print sample issue may
`be requested, please send your request to E-mail: sample.copy@ybentham.org
`
`Copyright © 2004 Bentham Science Publishers Lid.
`is a condition ofthis publication that manuscripts submitted to this journal have not been published and will not be
`It
`simultancously submitted or published elsewhere, By submitting a manuscript,
`the authors agree that the copyright for
`their article is transferred to the Publisher if and when the article is accepted for publication. The copyright covers the
`exclusive rights to reproduce and distribute the article,
`including reprints, photographic reproductions, microform or
`any other reproduction of similar nature, and translations. Al! rights reserved: no part of this publication may be
`reproduced, stored in a retrieval system, or transmitted in any form or by any means, on-line, mechanical, photocopying,
`recording or otherwise, without the prior written permission ofthe Publisher.
`
`Photocopying Information for Users in the USA
`is
`Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients,
`granted by Bentham Science Publishers Ltd. for libraries and other users registered with the Copyright Clearance Center
`(CCC) Transactional Reporting Services, provided that the appropriate fee of US$ 45.00 per copy perarticle is paid
`directly to Copyright Clearance Center, 222 Rosewood Drive, Danvers MA 01923, USA. Refer also to www.copyright.com
`Photocopying Information for Users Outside the USA
`for private
`Bentham Science Publishers Ltd. grants authorization for
`individuals to photocopy copyright material
`research use, on the sole basis that requests for such use are referred directly to the requestor’s local Reproduction Rights
`Organization (RRO). The copyright fee is USS 45,00 per copyper article exclusive of any charge or fee levied. In order to
`contact your local RRO, please contact the International Federation of Reproduction Rights Organizations (IFRRO), Rue
`du Prince Royal 87, B-1050 Brussels, Belgium; Tel: +32 2 551 08 99, Fax: +32 2 551 08 95; E-mail: secretariat@iftro.be
`This authorization does not extend to any other kind of copying by any means,
`in any form, and for any purpose other
`than privale research use,
`The Item-Fee Code for this publication is: 1568-0096/04 $45.00 + .00
`Online Articles
`This publication is available online from Ingenta at www.ingenta.comIndividual articles are also available for sale online
`via Infotrieve at www.infotricve.com or [rom Ingenta at www.ingenta.com
`
`Permission for Other Use
`The Publisher's consent does not extend to copying for general distribution, for promotion, for creating new works, or for
`resale, Specific permission must be obtained from the Publisher for such copying. Requests must be sent
`to the
`permissions department at E-mail: permission@bentham. org
`
`Disclaimer
`its staff or members of the editorial board for any
`No responsibility is assumed by Bentham Science Publishers Lul.,
`injury and/or damage to persons or property as a maticr of products liability, negligence or otherwise, or from any use or
`operation of any methods, products instruction, advertisements or ideas contained in this publication/journal, Any
`dispute will be governed exclusively by the laws of the U.A.E. and will be settled exclusively by the competent Court at
`the city of Dubai, UA.E.
`
`This material was copied
`atthe NLM and maybe
`Subject US Copyright Laws
`
`West-Ward Pharm.
`Exhibit 1033
`Page 002
`
`West-Ward Pharm.
`Exhibit 1033
`Page 002
`
`

`

`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`Mammalian Target of Rapamycin (mTOR)Inhibitors as Anti-Cancer Agents
`
`Current Cancer Drug Targets, 2004, 4, 621-635
`
`621
`
`Ravi D. Rao, Jan C. Buckner and Jann N. Sarkaria*
`
`Department of Oncology, Mayo Clinic College ofMedicine, 200 First St. SW, Rochester, MN 55905, USA
`
`Abstract: Highly specific signal transduction inhibitors are being developed as anti-cancer agents against an
`array of molecular targets, with the promise of increased selectivity and lower toxicity than classic cytotoxic
`chemotherapy agents. Rapamycin and its analogues are a promising class of novel therapeutics that specifically
`inhibit signaling from the serine-threonine kinase, mammalian target of rapamycin (mTOR). mTORis a key
`intermediary in multiple mitogenic signaling pathways and plays a central role in modulating proliferation and
`angiogenesis in normal tissues and neoplastic processes. Rapamycin potently inhibits T-cell proliferation, and
`is approved for clinical use as an immuno-suppressant following kidney transplantation. Hyperactivation of
`mTORsignaling has been implicated in tumorigenesis, and promising pre-clinical studies in several tumor
`types suggest that the anti-proliferative and anti-angiogenic properties of rapamycin may be useful in cancer
`therapy. These studies have led to several clinical trials evaluating the safety and efficacy of rapamycin analogs
`in cancer therapy. The goal of this article is to review the mechanism of action of rapamycin as an anti-cancer
`agent, and to review the clinical experience with rapamycin and rapamycin analogs as immunosuppressive and
`anti-neoplastic therapeutic agents.
`
`Key words: Rapamycin, CCI-779, temsirolimus, RAD-001, everolimus, AP23573, sirolimus, cancer, cytostatic anti-cancer
`drugs, mTOR,renal transplant, immunosuppressive agents.
`
`INTRODUCTION
`
`Rapamycin and its analogues are novel, molecularly
`targeted drugs that are being developedas anti-cancer agents.
`The parent compound, rapamycin (Sirolimus, Rapamune;
`Wyeth-Ayerst)
`is approved by the Food and Drug
`Administration (FDA) for the prevention of allograft
`rejection following renal
`transplantation, and for
`incorporation into drug-eluting stents to prevent re-stenosis
`following coronary angioplasty. Experience in the transplant
`setting suggests that long-term use of this agent is safe and
`well tolerated. Rapamycin analogues with more favorable
`pharmacokinetic properties are currently being developed as
`anti-cancer drugs. Rapamycin and its analogs inhibit the
`signaling activity of the serine-threonine protein kinase,
`mammalian target of rapamycin (mTOR). mTOR functions
`downstream from multiple growth factor receptor tyrosine
`kinases to promote cell growth and proliferation. Key
`downstream targets of mTOR include p70S6 kinase and
`eukaryotic initiation factor 4E-binding protein (4EBP1),
`which modulate the translation of select mRNA transcripts
`that ultimately impact on cell growth and cell cycle
`progression. More recent data have linked mTORsignaling
`with the cellular response to hypoxia and the expression of
`vascular endothelial growth factor (VEGF), which suggests
`that mTOR may be an important mediator of tumor
`angiogenesis. In tumors that are reliant on mTORsignaling,
`disruption of these key signaling pathways by rapamycin
`results in cell cycle arrest and inhibition of angiogenesis,
`and these effects may account for the anti-neoplastic
`activities of mTOR inhibitors seen in multiple tumortypes.
`Based on promising pre-clinical studies, rapamycin andits
`analogs currently are being tested as anti-neoplastic agents,
`
`*Address correspondence to this author at the Department of Oncology,
`MayoClinic College of Medicine, 200 First St. SW, Rochester, MN 55905,
`USA;E-mail: sarkariajann@mayo.edu
`
`both given alone or in combination with conventional cancer
`therapies. In this review, the biology of mTOR signaling
`and the cellular pharmacology of mTORinhibition will be
`discussed as will
`the clinical development of mTOR
`inhibitors.
`
`History
`
`Rapamycin is a macrocyclic lactone antibiotic that was
`first isolated from the bacterium Streptomyces hygroscopicus
`found in soil samples taken from Easter Island (called ‘Rapa
`Nui’ by its native inhabitants; hence the name ‘Rapamycin’).
`After its isolation and purification, studies revealed that
`rapamycin was a potent anti-fungal agent and an effective
`immunosuppressant[1]. Subsequent studies demonstrated
`that rapamycin inhibited proliferation in several tissues
`including IL-2 stimulated T-cells, vascular endothelium,
`smooth muscle, and tumor cells. These observations
`prompted the development of rapamycin andits analogs as
`immunosuppressive agents,
`inhibitors of vascular re-
`occlusion andas anti-cancer agents.
`
`Rapamycin and three analogs, CCI-779, RAD-001 and
`AP23573, have been developed for human use (Fig. (1)).
`Among these, only rapamycin (Sirolimus, Wyeth
`Pharmaceuticals) is currently approved, for preventing
`kidneyallograft rejection following renal transplantation and
`in drug-eluting stents to reduce the incidence of re-stenosis
`following coronary artery angioplasty. CCI-779
`(Temsirolimus, Wyeth Pharmaceuticals) is an ester of
`rapamycin, with superior oral bioavailability compared to
`the parent compound rapamycin. This drug is available in
`oral and intravenous formulations, and clinical development
`of this drug is well underway with several phase II and III
`trials being conducted. RAD-001 (Everolimus, Novartis
`Pharmaceuticals) is an orally available hydroxyethyl
`derivative of rapamycin developed by Novartis for
`applications in the transplant, cardiovascular and oncological
`
`1568-0096/04 $45.00+.00
`
`© 2004 Bentham Science Publishers Ltd.
`
`West-Ward Pharm.
`Exhibit 1033
`Page 003
`
`West-Ward Pharm.
`Exhibit 1033
`Page 003
`
`

`

`622 Current Cancer Drug Targets, 2004, Vol. 4, No. 8
`
`Fig. (1). Structure of rapamycin and rapamycin derivatives.
`
`Sarkaria et al.
`
`Rapamycin, R = OH
`
`RADOOI,R = OW ~~
`Q
`
`OH
`
`CCI-779, R= O
`
`settings, and clinical testing for all these indications are
`ongoing. The newest mTOR inhibitory agent
`to be
`developed for
`clinical use
`is AP23573
`(Ariad
`Pharmaceuticals). Early phase I clinical trials with this
`agent, which is also an analog of rapamycin, are now
`underway.
`
`BIOLOGY OF mTOR
`
`mTORis a serine-threonine-directed kinase that belongs
`to the family of phosphatidyl-inositol 3-kinase-related
`kinases (PIKK). All members of this PIKK family contain a
`C-terminal kinase domain that shares significant homology
`with that of the phosphatidyl-inositol 3-kinase (PI3K); other
`membersof this family include ataxia telangiectasia mutated
`(ATM), ATM and Rad3related (ATR) and DNA-dependent
`protein kinase (DNA-PK) [2,3]. These latter 3 kinases play
`key roles in orchestrating DNA damage checkpoint responses
`and DNA repair
`[4].
`In contrast, mTOR monitors
`intracellular nutrient and energy availability and promotes
`cell growth and proliferation following mitogenic stimuli,
`dependentupon the availability of requisite nutrients [5].
`
`Rapamycin is a highly specific inhibitor of mTOR
`function. Rapamycin is unable to bind directly to mTOR,
`but forms a complex with the immunophilin, 12 kDa
`FK506-binding protein (FKBP12; FK-506 is an unrelated
`immunosuppressant); it is this drug-protein complex that
`binds to mTOR through an FKBP12-rapamycin binding
`(FRB) domain[6]. The FRB domain is adjacent to the
`kinase domain in mTOR and formation ofthis tri-molecular
`complex markedly attenuates downstream signaling from
`mTOR. Interestingly, rapamycin treatment does not inhibit
`mTOR catalytic kinase
`activity directly,
`since
`autophosphorylation of mTOR is unaffected by rapamycin
`treatment. Instead, binding of the FKBP12/rapamycin
`complex is thought to prevent interaction of mTOR withits
`kinase substrates and thus prevent downstream signaling [7].
`The interaction of the rapamycin/FKBP12 complex with
`mTORis highly specific and is so stable that inhibition of
`mTORby rapamycin is essentially irreversible. The cellular
`and biochemical effects of rapamycin are generally believed
`to result exclusively from inhibition of mTOR signaling
`[8,9].
`
`The mTORsignaling network (Fig. (2)) is important for
`driving cell growth and proliferation in multiple tumor
`types. Several receptor tyrosine kinases (RTKs), including
`the epidermal growth factor receptor (EGFR), platelet-
`derived growth factor receptor (PDGFR)and insulin-like
`growth factor (IGFR) can activate PI-3 kinase activity,
`which, in turn, phosphorylates phosphatidylinositol (PI) on
`the D-3 position [10]. The resulting accumulation of
`phosphatidylinositol-3, 4, 5-triphosphate on the cytoplasmic
`surface of the plasma membrane leads to activation of a
`number of kinase signaling pathways including that
`regulated by protein kinase B (PKB, Akt). Akt stimulates
`mTOR function both through direct phosphorylation of a
`negative regulatory domain within mTOR [11] as well as
`through its effects on the tuberous sclerosis complex-2
`(TSC2) protein [12-15]. TSC2, in a complex with tuberous
`sclerosis complex-1 (TSC1) protein, functions as a GTPase-
`activating protein towards the Rhebl GTPase. Akt-mediated
`phosphorylation of TSC2 disrupts the TSC1/TSC2 complex
`and relieves inhibition of Rheb1 activity; activated Rheb1
`then can stimulate mTOR phosphorylation and signaling
`(see Fig. (2)) [16-19]. The inhibitory effects of TSC2 on
`mTOR activity are stimulated in nutrient deprived
`conditions by activation of LKB-1, which signals through
`AMP-activated protein kinase (AMPK) to enhance TSC2
`activity [20,21]. Signaling from mTORalso is regulated by
`association with Raptor, which probably functions as a
`scaffolding protein to promote transient association and
`phosphorylation of downstream targets
`[22,23].
`Collectively,
`these data highlight the idea that mTOR
`functions within a molecular complex of multiple proteins
`that regulate its activity [24].
`
`PI3K-mediated activation of Akt is normally opposed by
`the lipid phosphatase PTEN (phosphatase and tensin
`analogue), which dephosphorylates phosphatidylinositol at
`the D-3 position. Deletion or mutation of the gene encoding
`this tumor suppressor protein commonly occurs in multiple
`tumortypes andresults in constitutive activation of PI3K-
`dependent signaling pathways that include Akt and activated
`Ras as signaling mediators. Consistent with the potential
`role of the Akt/mTORsignaling pathway in tumorigenesis,
`overexpression of activated Akt and activated Rasin glial
`progenitor cells leads to formation of GBM-like tumors in a
`
`West-Ward Pharm.
`Exhibit 1033
`Page 004
`
`West-Ward Pharm.
`Exhibit 1033
`Page 004
`
`

`

`Mammalian Target ofRapamycin (mTOR) Inhibitors as Anti-Cancer Agents
`
`Current Cancer Drug Targets, 2004, Vol. 4, No.8
`
`623
`
`RTKs
`
`LKB-1
`
`PI3K
`
`AMPK
`
`PTEN —1
`
`i
`
`Akt ——— TSC1/TSC2
`
`.
`j
`amino acid
`
`availability
`
`Ny
`
`iN"h 4E- ™1a
`eats$6 ve
`VEGF,other
`'
`|
`
`Rheb1
`
`eo
`Ow Raptor
`
`hypoxia genes
`
`5'TOP translation
`
`cap-dependent
`translation
`
`Fig. (2). The mTORsignaling network.
`
`[25], and these tumors are
`transgenic mouse model
`exquisitely sensitive to treatment with rapamycin. Germ-line
`inherited deficiencies in PTEN, TSC or LKB-1 result in
`Cowden’s disease, Tuberous Sclerosis and Peutz-Jeghers
`syndrome, respectively, which are all characterized by the
`development of multiple benign hamartomas. Loss-of-
`function in any one of these three proteins results in
`hyperactivation of mTORsignaling, and this presumably
`accounts for
`the development of the characteristic
`hamartomatous lesions. Thus, constitutive activation of
`mTOR signaling can be an important contributor to
`tumorigenesis in both benign and malignant tumors.
`
`mTOR-Dependent Signaling Pathways
`
`mTORsignals downstream to multiple protein targets:
`p70 S6 kinase (p70S6K), eukaryotic translation initiation
`factor 4E binding protein (4E-BP1), and the hypoxia-
`inducible transcription factor, HIF-1a. These 3 well-
`characterized downstream signaling targets have been
`implicated in control of hypoxia- and mitogen-induced
`tumor proliferation and disruption of these pathways may
`play an important role in the anti-tumor effects of
`rapamycin. In the sections below, we will describe the
`potential links between the anti-tumor effects of mTOR
`inhibitors and disruption of downstream signaling to
`p70S6K, 4EBP1 and HIF-1« in more detail.
`
`p70S6K
`
`mTORregulates translation of select mRNA transcripts
`containing 5’-terminal oligopyrimidine (5’TOP) tracts
`
`through phosphorylation of p70S6K. Following PI3K-
`dependent phosphorylation of residues within an auto-
`inhibitory domain, mTORregulates the phosphorylation of
`Thr-389 [26]. Modification of this residue is essential for
`subsequent phosphorylation of other residues within the
`activation loop of the kinase domain, which allows for full
`catalytic activity. After mitogen stimulation, activated
`p70S6K phosphorylates the S6 component of the 40S
`ribosomal subunit, and this promotes translation of mRNA
`containing 5°TOP [27]. Because transcripts for many
`ribosomalproteins and translation elongation factors contain
`this S5’TOP motif, rapamycin-mediated suppression of
`p70S6K activity may inhibit cell growth andproliferation
`by limiting ribosomal biogenesis and restricting protein
`synthesis capacity.
`
`4EBP1
`
`mTOR modulates protein translation initiation by
`regulating the assembly of the eukaryotic initiation factor 4F
`(eIF4F) complex on the 5’-methyl-GTP cap of mRNA
`transcripts. The eIF4F complex is a heterotrimer composed
`of the mRNA cap-binding protein eIF4E, a scaffolding
`protein eI[F4G,and a helicase eIF4A (reviewed in [5]). This
`tripartite complex regulates the rate of cap-dependentprotein
`translation by mediating the rate-limiting step of mRNA
`loading onto the small 40S ribosomal subunit. Formation of
`a functional eIF4F complex is controlled by the
`phosphorylation status of an eIF4E binding protein (4E-
`BP1). In nutrient- or growth factor-deprived cells,
`the
`association of hypophosphorylated 4E-BP1 with eIF4E
`blocks binding of eIF4G to the cap structure and inhibits
`
`West-Ward Pharm.
`Exhibit 1033
`Page 005
`
`West-Ward Pharm.
`Exhibit 1033
`Page 005
`
`

`

`624 Current Cancer Drug Targets, 2004, Vol. 4, No. 8
`
`cap-dependenttranslation. In contrast, nutrient- or mitogen-
`induced phosphorylation of 4E-BP1 disrupts association
`with eIF4E and allows formation of a functional eIF4F
`complex [28,29]. 4E-BP1 is phosphorylated on at least 5
`serine or threonine sites, and these mitogen-induced
`modifications are regulated,
`in part, by mTOR. Several
`studies have suggested that mTOR directly phosphorylates
`all 5 sites on 4E-BP1 [30,31], while others have argued that
`direct mTOR phosphorylation of 2 of the sites (Thr-37 and
`Thr-46) serves as a priming event that allows subsequent
`phosphorylation of the other sites (Ser-65, Thr-70, and Ser-
`83) by other signaling pathways
`[32]. Rapamycin
`diminishes 4E-BP1 phosphorylation, prevents dissociation
`of 4E-BP1 from eIF4E, and results in inhibition of cap-
`dependenttranslation. Because translation is less efficient for
`transcripts with complex secondary structures, rapamycin
`preferentially inhibits translation of mRNA transcripts
`containing complex 5’ untranslated regions (UTR) [33].
`Transcripts with complex 5’UTRs whose translation is
`inhibited by rapamycin include key proteins involved in cell
`proliferation and angiogenesis, such as cyclin D1, ornithine
`decarboxylase and vascular endothelial growth factor
`(VEGF).
`
`HIF-1a
`
`Hypoxia induces the expression of multiple genes
`containing hypoxia response elements (HREs), and
`transcription from this response element
`is regulated
`primarily by the HIF-1 transcription factor. HIF-1 is a
`heterodimer composed of HIF-1a and HIF-1B. Under
`normoxic conditions, HIF-1 heterodimer
`levels are
`undetectable due to rapid degradation of HIF-1a subunit.
`The stability of HIF-la is regulated through post-
`translational modification of an oxygen-dependent
`degradation (ODD) domain. In the presence of oxygen,
`prolyl hydroxylases modify two conserved proline residues
`(Pro-402 and Pro-564) within the ODD [34,35].
`Hydroxylation of these residues promotes HIF-la
`association with the von Hippel Lindau-containing ubiquitin
`ligase complex and subsequent ubiquitin-mediated
`proteosomal degradation. Because molecular oxygen is
`required for catalysis of this reaction, hypoxic conditions
`prevent hydroxylation of the ODD, which results in
`stabilization of HIF-la@. Other transactivating post-
`translational modifications and dimerization with the HIF-1B
`subunit result in formation of an active HIF-1 transcriptional
`complex. HIF-1 drives expression of genes, such as VEGF,
`which contain HREs within their promoter region. The
`repertoire of hypoxia-inducible genes enables tumor or
`normal tissues to adapt to low oxygen environments and
`include genes involved in oxygen and glucose transport,
`glycolysis, growth-factor signaling, immortalization, genetic
`instability,
`invasion and metastasis, apoptosis and pH
`regulation [36].
`
`Signaling through the PI3K/mTORpathway regulates
`HIF-1o expression andactivity [37]. The link between PI3K
`signaling and HIF-1o activity wasfirst established in Ras-
`transformed cells, where hypoxia-induced signaling to HIF-1
`was blocked by genetic or pharmacological inhibition of
`PI3K activity [38]. Subsequent studies have demonstrated
`that restoration of wild-type PTEN function, expression of a
`
`Sarkaria et al.
`
`treatment with
`dominant-negative Akt construct, or
`rapamycin blocks hypoxia and mitogen-induced HIF-1
`signaling [39-41]. Moreover, a recent study demonstrated
`that rapamycin blocks both hypoxia-induced HIF-la
`accumulation and transactivation, and that this effect of
`rapamycin is specifically due to pharmacological inhibition
`of mTOR[42]. Collectively, these data suggest the existence
`of a PI3K/Akt/mTORsignaling pathway that regulates HIF-
`1& expression, stability or activation.
`
`ANTI-TUMOR EFFECTS OF RAPAMYCIN
`
`role for mTOR in modulating cell
`The central
`proliferation in both tumor and normal cells and the
`importance of mTOR signaling for the hypoxic response
`suggests that rapamycin-based therapies may exert anti-
`tumoreffects primarily through either inhibition of tumor
`cell proliferation or suppression of angiogenesis. Pre-clinical
`and early clinical results demonstrate that only a subset of
`tumors will respond to rapamycin-based therapies. In the
`following sections, we will review the pre-clinical efficacy
`data,
`the evidence supporting the anti-angiogenic and
`cytostatic properties of rapamycin, and discuss potential
`mechanismsofresistance to mTORinhibition.
`
`Pre-clinical studies have demonstrated efficacy of
`Tapamycin analogs and the parent compound in multiple
`tumortypes. In the National Cancer Institute 60 tumorcell
`line panel, both rapamycin (NSC 226080) and CCI-779
`(NSC 683864) demonstrated growth inhibitory activity
`against a broad spectrum of tumors with a subset of
`leukemia, lung, brain, prostate, breast, renal and melanoma
`tumor cell
`lines being inhibited at
`low nanomolar
`concentrations (http://dtp.nci.nih.gov/). Early animal studies
`at the NCI and at Ayerst Research Laboratories demonstrated
`modest growth inhibitory properties of rapamycin in murine
`tumor models of B16 melanoma, P388 lymphocytic
`leukemia, EM ependymoblastoma, CD8F1 breast carcinoma,
`Colon 38, CX-1 and 11/A colon cancer models [43].
`Subsequent published studies have documented significant
`tumor growth inhibition with rapamycin or CCI-779
`treatment of DAOY medulloblastoma, U251 or SF295
`glioma, PC-3, DU-145, LAPC4, or LAPC9 prostate
`carcinoma, and Rh-18 rhabdomyosarcoma human tumor
`xenografts [44-47]. These data have provided the impetus for
`development of mTOR inhibitor therapy in a variety of
`tumor types.
`
`Treatment of tumorcells in vitro with rapamycin results
`in an accumulation of cells in the G; phase ofthe cell cycle.
`Similarly, rapamycin treatment in tumor-bearing animals
`results in decreased tumorcell proliferation as indicated by
`BrdU labeling index [48]. At the molecular level, this drug-
`induced cell cycle arrest is associated with an accumulation
`of the cyclin-dependent kinase inhibitor, p27“P!, decreased
`expression of cyclinD1, and a corresponding decrease in
`phosphorylation of the retinoblastoma protein. CyclinD1
`mRNA contains a complex 5’UTR, and reduction in
`cyclinD1 levels presumably results from rapamycin-mediated
`inhibition of 4EBP1 phosphorylation and the resulting
`inhibition of eIF4F function. Likewise, the mRNA encoding
`for multiple oncogenes or proteins involved in DNA
`metabolism and S-phase progression contain complex
`
`West-Ward Pharm.
`Exhibit 1033
`Page 006
`
`West-Ward Pharm.
`Exhibit 1033
`Page 006
`
`

`

`Mammalian Target ofRapamycin (mTOR) Inhibitors as Anti-Cancer Agents
`
`Current Cancer Drug Targets, 2004, Vol. 4, No.8
`
`625
`
`5’UTRs and are regulated by mTOR [49]. Although
`rapamycin can induce apoptosis in select tumor models,
`rapamycin treatment typically slows tumor growth but does
`not induce tumorregression, suggesting that increased tumor
`cell loss through apoptosis or other mechanisms of cell
`death likely are not major contributors to drug effect in most
`tumors. Collectively,
`these data support the idea that
`rapamycin exerts a cytostatic effect on tumorcell growth.
`
`Rapamycin also has cytostatic effects on normal and
`tumor vasculature. As discussed above, mTOR plays an
`important role in modulating the cellular response to
`hypoxia through stabilization and activation of HIF-1a.
`HIF-1 drives expression of hypoxia-responsive genes, and
`these genes include those such as vascular endothelial
`growth factor (VEGF)
`that are important for tumor
`angiogenesis. Translation of VEGF mRNAalso is regulated
`in an mTOR-dependent manner via its complex 5°UTR.
`Consistent with these mechanisms of regulation, treatment
`of tumor-bearing animals with rapamycin results in
`decreased expression of VEGF mRNAin tumors [50] and
`decreased circulating levels of VEGF protein [51]. VEGF
`drives endothelial proliferation through interaction with its
`cognate receptor tyrosine kinases, VEGF receptor 1 and 2,
`and these receptors can signal downstream through the
`PI3K/Akt pathway to mTOR. Thus,proliferation of smooth
`muscle and endothelial cells is inhibited by mTOR
`inhibition [52,53], and this effect is likely related to the
`efficacy of rapamycin-eluting stents in preventing vascular
`
`re-occlusion. In animal models, rapamycin inhibits tumor
`growth and neo-vascularization of CT-26 colon tumors
`grown in a dorsal skin-fold model and suppresses serum
`levels of VEGFin tumor bearing animals[51]. Thus the anti-
`angiogenic effects may contribute to the efficacy of mTOR
`inhibitors in cancer therapy.
`
`The anti-tumoreffects of rapamycin therapies are likely
`secondary to both inhibition of tumorcell proliferation and
`inhibition of tumor angiogenesis. The relative contribution
`of these two effects in any given tumor maybedifficult to
`delineate. However, pre-clinical and clinical data suggest that
`that only a subset of tumors will respond to rapamycin; one
`of the challenges will be to identify patients most likely to
`respond to this class of agents. Future studies delineating
`the relative contributions of the anti-proliferative and anti-
`angiogenic effects to the overall anti-tumor efficacy of
`rapamycin will be invaluable for identifying the relevant
`cellular targets that are responsible for the anti-tumoreffects
`of rapamycin.
`
`COMBINATIONS OF MTOR ANTAGONISTS WITH
`OTHER ANTI-CANCER AGENTS
`
`Angiogenesis and tumorcell proliferation have been
`implicated as important mediators that can influence the
`efficacy of traditional cytotoxic cancer therapies; therefore,
`much research effort has focused on the evaluation of
`
`volume w
`
`Relative
`
`6 -
`
`5 -
`
`1
`0
`
`| —&— Rap
`i
`:
`<«<—— Regrowth delay ———» —- Rap / RT
`
`0
`
`10
`
`20
`
`30
`
`ft
`t
`rtf
`trttttt
`Rap
`
`Days
`,
`
`Fig. (3). Rapamycin enhances the efficacy of radiation in U87 xenografts. Nude mice with established U87 flank xenografts were
`randomized into four treatment groups: 1) placebo, 2) radiation only (4 Gy x 4), 3) rapamycin only (1 mg/kg), or 4) radiation and
`rapamycin. The tumor re-growth for each treatment group is shown. Data points represent the mean relative tumor volume + SE.
`Treatment was initiated on Day 0 with the first injection of rapamycin (Rap). The schedule for Rap and radiation (RT) treatments is
`depicted below the x-axis. Highly similar results were obtained in two independent experiments. (Reproduced with permission,
`Cancer Res., 2002, 62, 7291-7297).
`
`West-Ward Pharm.
`Exhibit 1033
`Page 007
`
`West-Ward Pharm.
`Exhibit 1033
`Page 007
`
`

`

`626 Current Cancer Drug Targets, 2004, Vol. 4, No. 8
`
`combinations of mTOR inhibitors with standard cancer
`therapies. Rapamycin potentiates cisplatin induced apoptosis
`in multiple cell lines including HL-60 leukemia cells and
`SKOV3 ovarian cancer cells [54]. Likewise, insulin-like
`growth factor-induced resistance to cisplatin in Rh30
`thabdomyosarcoma cells can be reversed by rapamycin
`treatment
`[55]. Geoerger et al. demonstrated that
`combinations of rapamycin with either cisplatin or
`camptothecin provides additive growth inhibition in the
`rapamycin-sensitive DAOY medulloblastoma cell line but
`not
`in the rapamycin-resistant D283 cell
`line [46].
`Consistent with this in vitro data, CCI-779 therapy provides
`for additive tumor growth inhibition in animals when
`combined with doxorubicin or cisplatin in PC3 or DAOY
`xenografts, respectively [46]. Several other investigators
`have confirmed the in vitro effect of rapamycin on
`sensitizing cancer cells to chemotherapeutic agents such as
`adriamycin, VP-16, and cisplatin! and hormonaltherapies
`such as tamoxifen? dexamethasone?, and the anti-estrogen
`ERA-9234. Rapamycin also has been combined with other
`molecularly targeted therapeutics. In our laboratory, we
`found that the combination of rapamycin with the EGFR
`inhibitor EKI-785 resulted in synergistic growth inhibition
`(unpublished data), and similar results were reported with
`the combination of RAD-001 and the VEGFR/EGFR
`inhibitor AEE788>. Likewise, rapamycin combined with the
`ber/abl
`inhibitor Imatinib mesylate (Gleevec) provided
`synergistic growth inhibition of chronic myeloid leukemia
`(CML)cell lines®. Taken together, these data suggest that
`mTOR-dependentsignaling may be important for resistance
`to chemotherapy-induced apoptosis and providesa rationale
`for
`the combination of
`rapamycin with specific
`chemotherapy agents.
`
`Rapamycin also can enhance the efficacy of radiation
`therapy. Based on significant pre-clinical and clinical data
`demonstrating that tumorproliferation during fractionated
`radiotherapy contributes to clinical radiation resistance
`
`1 .gavarai, N.; Wu, C.; Wangpaichitr, M.; Lampidis, T.; Robles, C.; Furst,
`A.; Feun, L. Circumvention of drug resistance in small cell lung cancer by
`mTORinhibitor. Proc. Am. Assoc. Cancer Res. 2003, 44, 2nd ed., Abstract
`# 3704.
`
`2. deGraffenried, L.; Friedrichs, W.; Fulcher, L.; Silva, J.; Roth, R.;
`Hidalgo, M. The mTORinhibitor, CCI-779, restores tamoxifen response in
`breast cancer cells with high Akt activity. Eur. J. Cancer 2002, 38(Suppl.
`7), Abstract # 528.
`3-yan, H.; Shi, Y.; Frost, P.; Hoang, B.; Gera, J.; Lichtenstein, A. The
`mTORinhibitor rapamycin sensitizes multiple myelomacells to apoptosis
`induced by dexamethasone. Blood 2003, 102, Abstract # 3453.
`4.7hang, T.; Sadler, T.; Annable, M.; Achilleos, P.; Frost, P.; Greenberger,
`L. Combination therapy for treating breast cancer using the antiestrogen,
`ERA-923 and the mTORinhibitor, CCI-779. Proc. Am. Assoc. Cancer Res.
`2003, 44, 2nd ed., Abstract # 3715.
`5-Goudar, R.; Keir, S.; Hjelmeland, M.; Conrad, C.; Traxler, P.; Lane, H.;
`Wang,X.; Bigner