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`Review
`
`Annals of Oncology 16: 525 – 537, 2005
`
`doi:10.1093/annonc/mdi113
`
`Published online 22 February 2005
`
`mTOR-targeted therapy of cancer with rapamycin derivatives
`
`S. Vignot1†, S. Faivre2†, D. Aguirre2 & E. Raymond1,2*
`
`1Department of Oncology, Hospital Saint Louis, Paris; 2Department of Medical Oncology, Beaujon University Hospital, Clichy, France
`
`Received 6 August 2004; revised 12 November 2004; accepted 15 November 2004
`
`Rapamycin and its derivatives (CCI-779, RAD001 and AP23576) are immunosuppressor macrolides
`that block mTOR (mammalian target of rapamycin) functions and yield antiproliferative activity in a
`variety of malignancies. Molecular characterization of upstream and downstream mTOR signaling
`pathways is thought to allow a better selection of rapamycin-sensitive tumours. For instance, a loss
`of PTEN functions results in Akt phosphorylation, cell growth and proliferation; circumstances that
`can be blocked using rapamycin derivatives. From recent studies, rapamycin derivatives appear to
`display a safe toxicity profile with skin rashes and mucositis being prominent and dose-limiting.
`Sporadic activity with no evidence of dose – effect relationship has been reported. Evidence suggests
`that rapamycin derivatives could induce G1 – S cell cycle delay and eventually apoptosis depending
`on inner cellular characteristics of tumour cells. Surrogate molecular markers that could be used to
`monitor biological effects of rapamycin derivatives and narrow down biologically active doses in
`patients, such as the phosphorylation of P70S6K or expression of cyclin D1 and caspase 3, are cur-
`rently evaluated. Since apoptosis induced by rapamycin is blocked by BCL-2, strategies aimed at
`detecting human tumours that express BCL-2 and other anti-apoptotic proteins might allow identifi-
`cation of rapamycin-resistant tumours. Finally, we discuss current and future placements of rapamy-
`cin derivatives and related translational research into novel therapeutic strategies against cancer.
`Key words: cell signal inhibitors, phase I trial, rapamycin, signal transduction inhibitors, sirolimus
`
`Introduction
`
`Cancer cells need several kinases for cell cycle control, pro-
`liferation, invasion and angiogenesis [1]. Treatments targeted
`against cellular signalling pathways have shown promise in
`the management of solid tumours and hematological malig-
`nancies. mTOR (mammalian target of rapamycin) was shown
`to be a key kinase acting downstream of the activation of the
`phosphatidylinositol 3 kinase (PI3K). Cumulative evidence
`supports the hypothesis that mTOR acts as a ‘master switch’
`of cellular catabolism and anabolism, signalling cells to
`expand, grow and proliferate. Although it is found in virtually
`all mammalian cells, it is particularly important in tumour
`cells that proliferate and invade aggressively. In addition,
`mTOR has recently been found to have profound effects in
`the regulation of apoptotic cell death, mainly dictated by the
`cellular context and downstream targets including P53, BAD,
`BCL-2, P27 and C-MYC.
`Rapamycin (sirolimus) is a macrolide antibiotic produced
`by Streptomyces hygroscopicus, which binds FKBP-12
`
`*Correspondence to: Dr E. Raymond, Department of Medical Oncology,
`Beaujon University Hospital, 100 Boulevard du General Leclerc, 92100
`Clichy cedex, France. Tel: +33-01-4087-5617;
`Fax: +33-01-4087-5487; E-mail: eric.raymond@bjn.ap-hop-paris.fr
`† Ste´phane Vignot and Sandrine Faivre participated equally to this work
`and should be considered as joint first authors.
`
`q 2005 European Society for Medical Oncology
`
`(FK506 binding protein). Thereby, the rapamycin – FKBP12
`complex can inhibit mTOR preventing further phosphorylation
`of P70S6K, 4E-BP1 and, indirectly, other proteins involved in
`transcription and translation and cell cycle control. Rapamycin
`is currently used alone or in combination with cyclosporine as
`an immunosuppressive drug to prevent renal graft rejection.
`Rapamycin analogues currently selected for clinical devel-
`opment are CCI-779 (intravenous formulation currently in
`phase III from Wyeth Ayest), RAD001 (oral formulation cur-
`rently in phase I-II from Novartis Pharma) and AP23573
`(intravenous formulation currently in phase I from Ariad
`Pharma). In clinical settings, using intermittent administration
`of CCI-779, RAD001 and AP23576, no evidence of immuno-
`suppressive effects has been observed. Dose-limiting toxicities
`consist of skin reactions, mucositis and minimal myelosup-
`pression. Evidence of antitumour activity has been reported in
`several patients with renal clear cell carcinoma and breast can-
`cer. Interestingly, rapamycin and its analogues antagonise
`tumour growth induced by loss of the PI3K antagonist, PTEN.
`Selection of patients based on the detection of activated
`P70S6K/AKT and/or loss of PTEN expression might help to
`predict the sensitivity of tumour cells to rapamycin analogues.
`Pharmacodynamic monitoring of the biological activity of
`rapamycin in clinical trials using molecular endpoints such as
`the phosphorylation of AKT, P70S6K and/or 4E-BP1 might
`also help to determine biological relevant dose(s) and plasma
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`Figure 2. Several molecules involved in cell survival, including mTOR,
`are regulated by the PI3K/AKT pathway.
`
`IL-6, insulin-like growth factor (IGF), epidermal growth factor
`(EGF), platelet derived growth factor (PDGF), insulin growth
`factors (IGF-1 and IGF-2) and colony stimulating factor
`(CSF). Activated PI3K phosphorylates inositol lipids at the 30
`position of the ring inositol, generating the lipid products PI3-
`phosphate
`[PI(3)P], PI3,4-biphosphonate
`[PI(3,4)P2]
`and
`PI3,4,5-triphosphate [PI(3,4,5)P3]. These lipid products are
`involved in a number of cellular processes including cell
`proliferation, survival, cytoskeletal reorganisation, membrane
`trafficking, cell adhesion, motility, angiogenesis and insulin
`action [8, 9]. Downstream to PI3K, protein kinase B (PKB),
`also named AKT, impacts on cell survival at multiple levels
`[10]. Substrates of AKT include glycogen synthase kinase
`(GSK3), 6-phosphofructo-2-kinase, the protein BAD, forkhead
`family of
`transcription factors, endothelial nitric oxide
`synthase (eNOS), mTOR, BRCA1 and others (Figure 2).
`GSK3 appears negatively regulated by AKT-dependent phos-
`phorylation. Reduced GSK-3 activity leads to increased levels
`of the growth stimulator beta-catenin [11]. The pro-apoptotic
`protein BAD is also inactivated by AKT-dependent phos-
`phorylation, thus enhancing cell survival. On the contrary,
`AKT indirectly activates mTOR via TSC, which in turn phos-
`phorylates and activates several targets involved in translation
`of specific mRNAs, apoptosis and/or cell cycle, as we will
`discuss later [12].
`Kinase activities are regulated by phosphatases that act in
`opposition to kinases by removing phosphates from the target
`proteins. The phosphatase and tensin homologue gene (PTEN,
`also named MMAC1 or TEP1) is a tumour suppressor gene,
`located on human chromosome 10q23 [13]. PTEN was found
`to be mutated in several human sporadic cancers such as
`breast, endometrial, ovarian (type endometroid), brain, renal
`carcinoma, melanoma and prostate tumour cell
`lines and
`primary tumours. Patients with germline mutations of PTEN
`develop inherited Bannayan Zoanna syndrome characterised
`by multiple hamartomas and Cowden disease, and sub-
`sequently are susceptible to developing breast, thyroid and
`several others cancers [14, 15].
`The PTEN product has a protein tyrosine phosphatase
`domain and extensive homology to tensin (related protein with
`focal adhesions), suggesting that PTEN suppresses tumour cell
`growth by antagonising protein tyrosine kinases, and regulates
`tumour cell
`invasion and metastasis through interactions
`
`526
`
`rapamycin
`treated with
`individuals
`in
`concentration(s)
`analogues. In addition, rapamycin and its analogues may sen-
`sitise cancer cells to apoptosis induction by cisplatin and gem-
`citabine.
`In this review, we will describe the molecular pathways
`involved in rapamycin activity and we will present recent pre-
`clinical and clinical data on rapamycin and its analogues. We
`will then discuss the current and future placement of those
`molecules into current therapeutic strategies against cancer.
`
`PI3K signaling pathway and mTOR
`
`Overview of the PI3K-related kinases (PIKKs)
`
`Following activation of membrane receptors by a variety of
`growth factors, secondary molecular signals are generated to
`transmit the stimulus toward the nucleus and activate a num-
`ber of events. Many of these signals involve the phosphoryl-
`ation of proteins known as kinases (Figure 1). Among those
`kinases, PI3K and PI3K-related kinases (PIKK) belong to a
`family of high molecular mass kinases whose catalytic
`domains show a strong resemblance. This family and the ribo-
`somal protein P70S6K, mTOR, the DNA-dependent protein
`kinase,
`the ataxia telangiectasia mutated gene (ATM),
`the
`ataxia-telangiectasia related (ATR) protein and key com-
`ponents of the histone acetylase complex are involved in
`checkpoint regulation of cell cycle, DNA repair,
`telomere
`length and cell death [2].
`The PI3K pathway is very often activated in cancer and
`contributes to cell cycle progression, to decrease apoptosis
`and to increase metastatic capabilities of cancer cells [3, 4].
`The uncontrolled activation of the PI3K pathway has been
`implicated in cell transformation and tumour progression in
`several tumour types including brain tumours, breast, ovarian
`and renal carcinomas [5 – 7]. Activation of the PI3K pathway
`is mediated by activated RAS or directly by some tyrosine-
`kinase receptors, under the control of several growth factors
`and cytokines including interleukin 1 (IL-1), IL-2, IL-3, IL-4,
`
`Figure 1. Multiple signaling pathways involved in signal transduction
`from tyrosine kinase receptors (TKR).
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`Figure 3. Overview on mTOR main activities in normal cells.
`
`mTOR is a serine/threonine kinase of 289 kDa, highly
`related to yeast TORs that belong to the PIKK family with a
`dual regulation by amino acid availability and by mitogen
`activated PI3K/AKT. TOR proteins in Sacharomyces cerevi-
`sae and the mammalian related proteins (mTOR) are required
`for signalling translational initiation and therefore cell cycle
`progression from the G0/G1 to S phase [22]. Yeast TOR 2
`protein also controls the actin cytoskeleton during cell cycle
`progression but it is not clear whether this function is con-
`served by mTOR [23].
`In humans, mTOR primarily appears to be a nutrient-
`sensing protein: mTOR is constitutively activated in the pre-
`sence of growth factor and nutrients and acts as a master
`switch of cellular catabolism and anabolism [12, 24]. mTOR
`is also regulated by hypoxia and by AMP levels. mTOR is
`inhibited through deacetylated tRNA species accumulating as
`a result of amino acid shortage (but the exact pathway remains
`to be elucidated) and via C-ABL protein tyrosine kinase that
`phosphorylates mTOR and inhibits its action. mTOR is also
`activated by TSC2 mutations or loss of LKB1.
`As discussed above, upregulation of mTOR can be related
`to loss of the tumour suppressor gene PTEN and activation of
`AKT.
`
`Translational control by mTOR
`
`mTOR modulates translation of specific mRNAs via the regu-
`lation of the phosphorylation state of several different trans-
`lation proteins, mainly 4E-BP1, P70S6K and eEF2.
`
`4E-BP1. Protein synthesis is regulated in many instances at
`the initiation phase, when a ribosome is recruited to the 50 end
`of an mRNA. Eukaryotic ribosomes do not have the ability to
`locate and bind to the 50 end of mRNA and need translation
`initiation factors to guide them. The cap structure at the 50 end
`of an mRNA is recognised by the eukaryotic translation
`initiation factor 4E(eIF4E). eIF4E, in association with eIF4G,
`directs the translation machinery to the 50 end of the mRNA.
`The 4E-binding proteins
`(4E-BP)
`are essential
`in the
`regulation of
`the interaction between eIF4E and eIF4G.
`The mTOR signalling
`pathway modulates
`4E-BP1
`phosphorylation and mediates its dissociation from eIF-4E
`[25, 26]. This dissociation is a crucial step toward activating
`translation of mRNAs with specific regulatory elements in
`the 50-untranslated terminal region (50UTR), especially c-myc,
`cyclin D1 and ornithine decarboxylase. In contrast, when
`
`focal adhesions. Davies et al.
`[16] and others, have
`at
`demonstrated that PTEN plays an important role in anchorage-
`dependant cell survival. Additionally, loss of PTEN protects
`cells from apoptosis triggered by matrix detachment (anoikis),
`and the re-expression of PTEN in PTEN-mutated cells causes
`apoptosis in cells in suspension.
`PTEN is involved in the regulation of the PI3K pathway
`[3]. There is evidence that PTEN dephosphorylates phospha-
`tydilinositol 3,4,5-triphosphate while mutated PTEN cannot
`dephosphorylate
`phosphoinositides
`at
`the D3
`position
`(D3-PPI). PTEN ± mice spontaneously develop neoplasia,
`the normal PTEN allele and an
`associated with loss of
`increased activation of AKT, mTOR and P70S6K. In vitro and
`in vivo, the growth of PTEN-deleted human cancer cells and
`PTEN/ mouse cells can be preferentially inhibited by phar-
`macologic mTOR inhibition [17]. This growth inhibition then
`involves both a decrease in proliferation and an increase in
`apoptosis.
`However, although PTEN inactivation might be required, it
`might not be sufficient to explain the sensitivity to rapamycin
`since there is also evidence to show that cancer cells with
`PTEN inactivation might remain resistant to rapamycin. Con-
`versely, a dose dependent tumour growth delay is observed in
`mice bearing PTEN proficient cancer cells. In that case,
`reports have suggested that the effects of rapamycin might be
`related to the inhibitory effects against endothelial cells block-
`ing tumour angiogenesis.
`Several studies have suggested that genomic integrity, tran-
`script and protein levels, phosphorylation and activity of all
`the multiples components of the PI3K pathway, should be
`evaluated to determine whether they predict prognosis or
`response to therapy in several cancers. The members of the
`PIKK family are key components of signals that coordinate
`the activity of the cell cycle and their functional characteris-
`ation gives important insights into cell growth and cell cycle
`checkpoint function. Further, development of molecular thera-
`peutics targeting the PI3K pathway is clearly warranted in
`different types of cancer. Wortmannin inhibits the multiple
`effects of PI3Ks and yield anti-inflammatory, immunosuppres-
`sive, cytotoxic and radio-sensitising properties with potential
`as an anti-neoplastic drug [18, 19]. The multiple molecular
`targets inhibited by this agent (PI3, PI4 and PIKK) raise cau-
`tion about
`its clinical use. Besides, wortmannin presents
`chemical instability and hepatotoxicity, limiting its develop-
`ment. Thus, we need to evaluate more specific molecules in
`this pathway as individual targets.
`
`Focus on mTOR (Figure 3)
`
`mTOR was identified in 1994 by several groups of investi-
`gators as the kinase targeted by rapamycin linked to the cel-
`lular protein FKBP12 (FK506-binding protein).
`It was
`therefore also named FKBP-RAP associated protein (FRAP),
`RAP FKBP12 target
`(RAFT1) and RAP target
`(RAPT1)
`[20, 21].
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`growth factor or nutrients are lacking, or in the presence of
`mTOR inhibitors, 4E-BP1 becomes hypophosphorylated,
`which increases
`its binding with EIF-4E and prevents
`initiation of translation.
`
`involved in amino acid biosynthesis, regulates the activity of
`amino acid permeases and represses autophagy. In the absence
`of the TOR signal, ribosomal biosynthesis is inhibited and
`autophagy is activated.
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`Neuronal function and role in brain development
`
`Recent evidence also suggests that mTOR may be involved in
`neuronal protein synthesis. mTOR could play a role in
`embryonic brain development and in the learning and memory
`process [34]. mTOR could inhibit eEF2 phosphorylation in
`active synapses to locally unrepress translation, whereas some
`studies have reported an increase of eEF2 phosphorylation in
`response to various neurotransmitters.
`
`Rapamycin and analogues
`
`Rapamycin development
`
`Rapamycin, also named sirolimus, is a natural antibiotic pro-
`duced by S. hygroscopicus. This molecule was found 30 years
`ago in the Easter Island Rapa Nui soil from which rapamycin
`was named. Rapamycin was subsequently isolated in Montreal
`by Ayerst Research laboratories in 1972. Rapamycin is a
`macrocyclic lactone developed initially as an anti-fungal drug
`directed against Candida albicans, Cryptococcus neoformans,
`and Aspergillus fumigatus [35 – 38]. It is a white crystalline
`solid insoluble in aqueous solutions, but soluble in organic
`solvents. The chemical structure is shown in Figure 4.
`Recently, rapamycin has been tested by the Developmental
`Therapeutic Branch, National Cancer
`Institute (NCI) and
`identified as a noncytotoxic agent that delays tumour prolifer-
`ation, finding evidence of cytostatic activity against several
`human cancers in vitro and in vivo. However, the development
`program of rapamycin as an anticancer agent was halted in
`1982 and only resumed in 1988 after demonstration of a safe
`toxicological profile in animals.
`In the meantime, rapamycin was developed as an immuno-
`supressive agent and those studies have enabled us to under-
`stand the mechanism of action of this agent. Rapamycin, via
`
`Figure 4. Rapamycin’s chemical structure including FKBP12 and mTOR
`binding domains.
`
`P70S6K. mTOR also phosphorylates and activates P70S6K to
`favour the recruitment of the 40S ribosomal subunit
`into
`actively translating polysomes and enhance the translation of
`mRNAs with 50 terminal oligopyrimidine tracts. These tran-
`scripts can encode up to 20% of the mRNAs [27].
`
`eEF2. Finally, mTOR also acts at the level of the elongation
`phase. The eukaryotic elongation factor 2 (eEF2) promotes
`translocation of the mRNA and mTOR regulates the activity
`of eEF2 kinase, apparently via regulation of a phosphatase
`activity (PP2A) [28].
`
`Anti-apoptotic and pro-apoptotic effects
`
`the downstream target of mTOR,
`There is evidence that
`P70S6K, binds to mitochondrial membranes and phosphory-
`lates the pro-apoptotic molecule BAD [29]. The binding of
`P70S6K to BAD inactivates BAD and increases cell survival.
`In contrast, mTOR might translocate from the cytoplasm to
`the nucleus shortly after the formation of syncitium between
`cells expressing the HIV envelope and CD4 cells. Once in the
`nucleus, it causes phosphorylation of P53, transcriptional acti-
`vation and induction of pro-apoptotic proteins such as BAX,
`and activation of the intrinsic cell death pathway [30].
`
`Cell cycle regulation
`
`mTOR inhibition results in an increase in the turnover of
`cyclin D1, at both mRNA and protein levels [31], and a
`decrease in the elimination of the cyclin dependant kinase
`inhibitor P27. Additionally, mTOR downregulates cyclin-A-
`dependent kinase activity in exponentially growing cells. The
`pharmacological inhibition of mTOR decreases G1 transit in
`the cell cycle [32].
`
`Metabolic modulation
`
`Cells have the ability to adapt to the dynamic pool of nutrients
`in their immediate environment. Mammalian cells respond
`continually to changes in available blood glucose and amino
`acids. mTOR plays an important role in the modulation of
`metabolic pathways, including those related to insulin [12,
`33]. The initiation of translation appears to be the limiting
`phase in protein synthesis. The central role played by mTOR
`in protein translation leads to the control of skeletal muscle
`protein synthesis. Because mTOR inhibition causes cellular
`responses indicating the physiological state of starvation, these
`proteins are thought to be mediators of nutrient-sensing path-
`ways. mTOR can detect nutrients such as carbon and nitrogen,
`signaling cells to grow and proliferate, a fact particularly
`important
`in tumour cells that proliferate aggressively. In
`yeast, TOR functions are well established genetically, with
`somewhat less compelling data in mammalian cells. In yeast,
`TOR signaling modulates the transcription of genes that are
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`its methoxy group, crosslinks the immunophilin FK506 bind-
`ing protein (FKBP12). The rapamycin – FKBP12 complex
`specifically interacts with mTOR to inhibit mTOR signalling
`to downstream targets [38]. Rapamycin inhibits T-cell pro-
`liferation induced by antigen, mitogenic lectins, alloantigen
`and crosslinking of T-cell surface markers with monoclonal
`antibodies. Rapamycin can inhibit proliferative responses
`induced by cytokines, including IL-1, IL-2, IL-3, IL-4 and IL-
`6, IGF, PDGF and CSFs.
`The preclinical development of rapamycin as an immuno-
`suppressor has been extensively reviewed [39, 40]. It has
`demonstrated a high degree of synergy with cyclosporin [41]
`both in vitro and in vivo, lowering the dose of cyclosporin
`necessary for
`immunosuppression, enhancing the rejection
`prevention in renal
`transplantation and minimising cyclo-
`sporin-induced toxicity [42, 43]. There are observations that
`high doses of rapamycin block the proliferative responses to
`cytokines by vascular and smooth muscle cells after mechan-
`ical injury, such as balloon angioplasty or allo-rejection [44,
`45]. In a non-human primate model, supra-therapeutic concen-
`tration of rapamycin stabilised and possibly reversed the inti-
`mal vascular lesion caused by the progression of immune
`injury in aortic allograft [46]. Rapamycin treatment concomi-
`tant with monoclonal antibody blockade of the co-stimulatory
`signal by anti-CD154 in mice induces tolerance, and the com-
`bination of rapamycin with anti-B7 in non-human primates
`seems to facilitate tolerance induction [47]. IC50 values of
`rapamycin as an immunosuppressor are in the range of
`0.1 – 300 nM.
`A relevant point of rapamycin as an immunosuppressor is
`the absence of the vasomotor renal side effects exhibited by
`CsA and tacrolimus. Treatment with rapamycin preserves glo-
`merular filtration and renal blood flow in normal, salt-depleted
`and spontaneously hypertensive rats [48]. The renal
`tissue
`seems to be protected during the rapamycin treatment by an
`inhibition of the intrarenal angiotensin II cascade. However,
`rapamycin does produce a dose-dependent tubular toxicity in
`rats, which is related to the delayed recovery of tubular epi-
`thelial function after injury [49].
`Over the last 8 years, rapamycin has undergone clinical
`trials as an immunosuppressive agent, progressing from phase
`I safety,
`tolerability and pharmacokinetic investigation to
`phase II dose-finding studies and limited sized evaluations of
`drug combination regimens. The completion of phase III trials
`led to approval of rapamycin by the Food and Drug Adminis-
`tration (FDA) of the USA in 1999 to prevent acute rejection
`in combination with cyclosporin and steroids. One year later,
`the drug was approved by the European Agency as an alterna-
`tive to calcineurin antagonists for
`long-term maintenance
`therapy to avoid graft
`rejection.
`Interestingly,
`rapamycin,
`unlike cyclosporin, does not seem to increase the risk of
`malignancy but rather to decrease the risk of post-transplant
`lymphoproliferative disorders.
`Apart from its immunosupressive capacity, rapamycin was
`also recently shown to be capable of preventing coronary
`artery re-stenosis [50, 51]. Growth, migration and differen-
`
`529
`
`tiation of vascular smooth-muscle cells are two major features
`of neointimal proliferation after vascular injury. The proposed
`mechanism of inhibition of proliferation of vascular smooth-
`muscle cells by sirolimus includes binding of the immuno-
`philin FKBP12, blockage of P70S6K, impairment of retino-
`blastoma protein phosphorylation, and prevention of p27
`downregulation. Additionally, rapamycin has been shown to
`be effective in inhibiting PDGF-induced migration of human
`vascular smooth cells in vitro, without affecting the ability of
`these cells to bind collagen and without disrupting their cyto-
`skeletal components [52, 53]. To avoid the systemic effects of
`rapamycin, it has been used locally in an impregnated stent to
`prevent coronary restenosis [51].
`
`Pharmacokinetic and metabolic information
`
`These data were initially obtained from studies that evaluated
`rapamycin as an immunosuppressor [54, 55]. The systemic
`bio-availability of rapamycin is approximately 15%, it has a
`maximal concentration at about 1 h and is widely distributed
`in tissues compared with plasma. The ratio blood cells/plasma
`ranges between 36 in renal transplant cases to 79 in healthy
`volunteers. In vitro experiments using human liver micro-
`somes suggest
`that cytochrome Cyp450 3A4 is the major
`biotransformation system, generating the inactive metabolites,
`hydroxy, dihydroxy, hydroxy-demethyl, didemethyl, 7-0
`demethyl and 41-0 demethyl. More than 90% of the drug is
`recovered in the faeces. Urine represents only 2% of the drug
`elimination. The average elimination half life is variable,
`ranging from 10 h in children to 110 h in patients with hepatic
`impairment.
`Rapamycin exposure is increased by diltiazem and ketoco-
`nazole and decreased by rifamycin and anticonvulsants [56].
`Regarding the interaction between rapamycin and cyclosporin
`(CsA), rapamycin concentrations are increased by concomitant
`administration of Neoral, the microemulsion formulation of
`CsA, and rapamycin increases CsA exposure approximately 2-
`fold, presumably because of competition for metabolism by
`Cyp450 3A4 and, possibly, drug extrusion by P-glycoprotein
`[57].
`Initial clinical studies show that a dose-dependent reversible
`reduction in mean platelet number and, to a far lesser extent,
`leukocyte count, was accompanied by increased serum choles-
`terol and triglyceride values. There were no changes in blood
`pressure, kidney or liver function test results.
`Corroborating preclinical studies, rapamycin does not affect
`glomerular filtration, but hypokalemia and hypophosphatemia
`has been reported as evidence of renal tubular abnormalities
`[58, 59].
`Additionally, rapamycin augments reactions to CsA: hyper-
`tension, acne and hirsutism. It has been associated with minor
`adverse effects such as diarrhea, tachycardia and arthralgia, as
`well as with non-infectious pneumonitis [60].
`
`Rapamycin as an anticancer drug
`
`Rapamycin was shown to inhibit the growth of several murine
`and human cancer cell
`lines in a concentration-dependent
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`manner, both in tissue culture and xenograft models: B16
`melanoma, P388 leukemia, MiaPaCa-2 and Panc-1 human
`pancreatic carcinomas and others [61 – 63]. In the 60 tumour
`cell lines screened at the National Cancer Institute in the USA
`(COMPARE program), the average GI50 obtained for rapamy-
`cin over all cell lines was 8.2 nM when the highest concen-
`tration tested is 1000 nM and 1800 nM when the highest
`concentration tested is 106 nM. In practical terms, they found
`general sensitivity to the drug at doses under 2000 ng/ml,
`more evident
`in leukemia, ovarian, breast, central nervous
`system and small cell lung cancer cell lines.
`Rapamycin induces P53-independent apoptosis in childhood
`rhabdomyosarcoma [64] and enhances the apoptosis induced
`in vitro by cisplatin in murine T-cell and human HL-60 pro-
`myelocytic leukemias and human ovarian SKOV3 carcinoma
`[65]. On the contrary, it inhibits taxol-induced apoptosis in
`human B-cell lines, probably through preventing BCL-2 inac-
`tivation and can inhibit hybridoma cell death in bioreactors,
`thereby increasing the production of monoclonal antibody
`[66].
`In addition, rapamycin inhibits the oncogenic transform-
`ation of human cells induced by either PI3K or AKT and has
`shown metastatic tumour growth inhibition and anti-angio-
`genic effect in in vivo mouse models [67]. Considering this
`effect, there is evidence that the PI3K – P70S6K intracellular
`signaling pathway is required for HIF1 and VEGF expression
`and also for VEGF stimulation of endothelial cells. The
`anti-angiogenic effect of rapamycin seems to be related to
`the inhibition of these effects [68].
`Based on these pre-clinical results, studies with rapamycin
`as an anticancer drug were begun and rapamycin analogues
`were
`developed with more
`favourable
`pharmaceutical
`properties.
`
`Table 1. Doses and schedules of rapamycin derivatives in phase I trials
`
`Rapamycin derivatives (Table 1)
`
`CCI-779, a more water-soluble ester derivative of sirolimus,
`was identified by investigators at Wyeth Ayerst as a noncyto-
`toxic agent that delayed tumour proliferation. CCI-779 was
`designed to increase the solubility of rapamycin making this
`compound readily available for intravenous formulation. At
`several non-toxic doses, CCI-779 demonstrated antitumour
`activity alone or in combination with cytotoxic agents in a
`variety of human cancer models such as gliomas, rhabdomyo-
`sarcoma, primitive neuroectodermal tumour such as medullo-
`blastoma, head and neck, prostate, pancreatic and breast
`cancer cells [69 – 73]. Treatment of mice with CCI-779
`inhibits P70S6K activity and reduces neoplastic proliferation.
`As with sirolimus, PTEN-deficient human tumours are more
`sensitive to CCI-779-mediated growth inhibition than PTEN-
`expressing cells. Specifically, studies in vitro in a panel of
`eight human breast cancer cell lines showed that six of eight
`cancer lines studied were inhibited by CCI-779 with IC50 in
`the low nanomolar range. Two lines, however, were found to
`be resistant with IC50>1 mM. The sensitive cell lines were
`estrogen receptor positive, or overexpressed HER-2/Neu, or
`had lost the tumour suppressor gene product PTEN [74]. Inter-
`estingly, preclinical studies indicate that intermittent adminis-
`tration of CCI-779 reduces its immunosuppressive properties
`while retaining its antitumour activity.
`CCI-779 has recently completed phase I evaluation in cancer
`patients and phase II results are starting to be reported. In phase I
`studies, the drug has been administrated as a single agent on a
`weekly schedule and daily for 5 days every other week. The
`main toxicities of CCI-779 included dermatological toxicities
`[aseptic folliculitis, erythematous macular rashes (Figure 5),
`eczematous reactions, dry skin, herpes-type lesions and nail
`disorders], mild myelosuppression (mainly thrombocytemia at
`
`Drugs
`
`CCI779
`
`Schedules
`
`Range of doses
`
`0.75 – 19.1 mg/m2/day q2 weeks
`
`Limiting toxicities
`
`Thrombocytopenia
`
`MTD
`
`Reached
`
`7.5 – 220 mg/m2/week
`
`Mucositis, asthenia, bipolar disorders
`
`Not reached
`
`25 – 100 mg/m2/day q2 weeks
`
`Mucositis
`
`Reached
`
`RAD001
`
`5 – 30 mg/week
`
`Mucositis, asthenia
`
`Not reached
`
`5 – 10 mg/day
`
`Ongoing
`
`AP23573
`
`3 – 28 mg/day q2 weeks
`
`Mucositis
`
`Ongoing
`
`Ongoing
`
`Ongoing
`
`Reached
`
`Ongoing
`
`Ex. 1102-0006
`
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`
`pharmacokinetic analysis of CCI-779 administrated intra-
`venously, through limited dose ranges has revealed an increase
`in drug exposure with dose and elimination life of 18 – 30 h.
`Based on these results, weekly doses of 25, 75, and 250 mg
`CCI-779, which are not based on classical definitions of maxi-
`mum tolerated doses, were chosen for phase II trials in
`patients with breast and renal cancer [79].
`A recent phase II trial evaluated the safety and efficacy of
`three dose levels of CCI-779 in previously treated patients
`with advanced renal cell carcinoma. The drug was well toler-
`ated, occurrences of partial responses, minor responses and
`prolonged stabilisation of disease in these heavily pretreated
`patients was found, suggesting the evaluation as a single agent
`in phase III trials in renal cell carcinoma [81].
`Another phase II study is currently ongoing in patients with
`advanced breast cancer [82]. The majority of patients in this trial
`received more than two lines of prior chemotherapy containing
`anthracyclines and taxanes. The study was designed to explore
`two doses of weekly intravenous administration of CCI-779 (75
`and 250 mg/m2). Toxicity was significantly higher in patients
`receiving higher doses (requiring dose reduction in 45.1% of
`those patients). At
`the higher dose, psychic disorders were
`reported, including lethargy and depression. Activity was similar
`in the two treatment groups with an overall response rate of
`10%. Immunohistochemistry was performed in samples from
`patients tr