Hematol Oncol Clin N Am
`16 (2002) 1101 – 1114
`
`Clinical development of mammalian target of
`rapamycin inhibitors
`
`Janet E. Dancey, MD
`Cancer Treatment Evaluation Program, Division of Cancer Treatment and Diagnosis,
`Investigational Drug Branch/CTEP/DCTD/NCI, 6130 Executive Boulevard,
`EPN 7131, Rockville, MD 20854, USA
`
`Rapamycin, a natural product, has antimicrobial, immunosuppressant, and
`antitumor activities that result from modulating signal transduction pathways that
`link mitogenic stimuli to the synthesis of specific proteins needed for cell cycle
`progression from G1 to S phase [1]. Today, rapamycin (sirolimus, RapamuneTM) is
`approved as an immunosuppressive drug for renal transplant recipients. Two
`related compounds are in under development: SDZ RAD as an immunosuppressant
`and the ester CCI-779 as a cancer therapeutic. The immunosuppressant effects of
`rapamycin are due to its inhibition of the biochemical events required for IL-2
`stimulated T cells to progress from G1 to S phase of the cell cycle [2]. However, the
`growth-inhibitory actions of rapamycin and its related compounds are not restricted
`to lymphoid cells; these agents have cytostatic or cytotoxic activities against solid
`and lymphoid tumor cell lines. This article focuses on recent advances in the
`understanding of the mechanisms of cell growth inhibition by rapamycin and
`the issues surrounding the development of this class of agent as a potential can-
`cer therapy.
`
`Target of rapamycin
`
`The phosphoprotein kinase, target of rapamycin (TOR), was first described in
`the yeast Saccharomyces cerevisiae as the functional target of rapamycin. Two
`distinct genes have been identified in yeast, but only a mammalian homolog
`(mTOR) of TOR2 has been described. In mammalian cells, mTOR is a large
`polypeptide kinase of 290 kDA [3] (also known as FRAP [4], RAFT1 [5], and
`RAPT1 [6]). The yeast TOR proteins exhibit a high degree of overall sequence
`identity ( > 40%) to mTOR, with even greater identity (>65%) observed in their
`carboxy-terminal catalytic domains [7].
`
`E-mail address: danceyj@ctep.nci.nih.gov
`
`0889-8588/02/$ – see front matter D 2002, Elsevier Science (USA). All rights reserved.
`PII: S 0 8 8 9 - 8 5 8 8 ( 0 2 ) 0 0 0 5 1 - 5
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`Fig. 1. Rapamycin-sensitive signaling pathways. Receptor-ligand binding activates the PI3K/Akt/
`mTOR pathway. The TOR regulates the activities of the translational regulators 4E-BP1 and p70S6
`kinase. Rapamycin binds to FKBP12 and the complex inhibits mTOR. Rapamycin may cause G1 block
`by inhibiting translation of proteins important for G1/S transition although other mechanisms may be
`involved in the drug’s antiproliferative effect. RTK, receptor tyrosine kinase; GPR, G-protein receptor.
`
`mTOR, a downstream component in the phosphoisinositol-3 kinase (PI3K)/
`Akt pathway (Fig. 1), acts as a nutrient sensor and regulator of translation [8,9].
`In the presence of mitogen stimulation of the PI3K/Akt pathway and sufficient
`nutrients, mTOR participates in the activation of p70S6 kinase (p70s6k) and in the
`inactivation of 4E-binding protein-1 (4E-BP1). These events and possibly signals
`to other kinases result in the activation of the translation of specific mRNA
`subpopulations important for cell proliferation and survival. Although mutations
`of mTOR have not been reported in human cancers, mTOR is a component of the
`PI3K/Akt pathway, which is of considerable interest to cancer therapeutics
`development because of the high frequency of mutations in components of the
`pathway seen in human malignancies (Table 1).
`Yeast and mammalian TOR proteins are members of phosphoinositide 3 kinase
`(PI3K)-related kinases (PIKK) family [10]. Among these PIKK family members
`are the cell cycle regulatory protein kinases ataxia-telangiectasia – mediated,
`ataxia-telangiectasia – related, and DNA-dependent protein kinase catalytic sub-
`unit. The PIKK family members share a carboxyl-terminal catalytic domain that
`bears significant sequence homology to the lipid kinase domains of PI3Ks,
`although no intrinsic lipid kinase activity has been described for mTOR. Members
`of this family are highly conserved throughout evolution and are involved in a
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`Table 1
`Abnormalities in the phosphatidyl-inositol 3 kinase/Akt-mTOR pathway in human cancers
`
`Abnormality
`
`Growth factor receptors
`(eg, EGFR, PDGFR,
`IGF-R, IL-2)
`PI3 kinase
`PTEN
`
`Function
`
`Oncogene
`
`Oncogene
`Tumor suppressor gene
`
`Akt
`
`eIF-4E
`
`Cyclin D
`
`P16
`
`Oncogene
`
`Oncogene
`
`Oncogene
`
`Tumor suppressor gene
`
`Tumors
`
`Lung, bladder, ovary, endometrium,
`cervix, prostate carcinomas,
`glioma, lymphoma
`Ovary
`Prostate, endometrium,
`breast carcinomas, melanoma
`Breast, gastric, ovary, pancreas,
`prostate carcinomas
`Breast, bladder, and head,
`and neck carcinomas; lymphoma
`Mantle cell lymphoma; breast, head
`and neck carcinomas
`Familial melanoma, pancreas carcinomas
`
`Abbreviations: EGFR, epidermal growth factor receptor; PDGFR, platelet-derived growth factor
`receptor; IGF-R, insulin-like growth factor receptor; IL-2, interleukin-2 PI3, phosphoisinositol-3;
`eIF-4E, eukaryotic initiation factor-4E.
`
`range of essential cellular functions, including cell cycle progression, cell cycle
`checkpoints, DNA repair, and DNA recombination [11,12].
`The upstream signaling pathway that couples growth factor receptor occu-
`pancy to mTOR protein activation is only partially understood. mTOR is a
`phosphoprotein, and its phosphorylation state and catalytic activity have been
`reported to be modulated by the mitogen-activated PI3K/Akt [13,14]. PI3 kinase
`and Akt are considered to be proto-oncogenes, and the pathway is inhibited by
`the tumor suppressor gene PTEN [15]. Activation of the pathway through over-
`expression of PI3K, Akt, or loss of PTEN augments the activity of mTOR and
`may increase the importance of this pathway in tumor cell survival and cell
`sensitivity to rapamycin compounds [16 – 18].
`The downstream actions of mTOR on translation are better understood than its
`upstream effectors. For the subset of mRNAs that contain regulatory elements
`0
`-untranslated regions, the binding of the mRNA to the ribosomal
`located in the 5
`subunit and the efficient initiation of translation is mediated by the multi-subunit
`eukaryotic initiation factor-4 (eIF-4) complex [19]. 4E-BP1 is a low-molecular-
`weight protein that inhibits the initiation of translation through its association with
`eIF-4E, the mRNA cap binding subunit of the eIF-4F complex [4]. Binding of
`4E-BP to eIF-4E is dependent on the phosphorylation status of 4E-BP1. In qui-
`escent cells, 4E-BP1 is relatively underphosphorylated and binds tightly to eIF-4E
`[19]. Stimulation of cells by hormones, mitogens, growth factors, cytokines, and
`G-protein – coupled agonists results in 4E-BP1phosphorylation through the action
`of mTOR and possibly other kinases, which promotes the dissociation of the
`4E-BP1/eIF-4E complex. The eIF-4E can then bind to the eIF-4F complex, and
`this interaction leads to an increase in translation rates of a subset of mRNAs.
`The second downstream target of mTOR is p70s6k, the kinase that phosphoryl-
`ates the 40S ribosomal protein S6. In response to mitogenic stimuli, p70s6k
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`phosphorylates S6 on multiple sites, and these modifications favor the recruit-
`ment of the 40S subunit into actively translating polysomes [20] and enhance the
`0
`terminal oligopolypyrimidine tracts. Although
`translation of mRNAs bearing 5
`these transcripts represent only 100 to 200 genes, they can encode up to 20% of
`the cell’s mRNA [21].
`In summary, under appropriate physiologic conditions, mTOR activation
`results in the transduction of signals that initiate the translation of specific
`subsets of mRNA transcripts and the activation of cyclin-dependent kinases,
`promoting progression through the cell cycle. This results in the activation and
`proliferation of T and B cells and such nonimmune cells as fibroblasts,
`endothelial cells, hepatocytes, and smooth muscle cells [22]. In tumor cells,
`activation of the pathway through inappropriate growth factor stimulation, over-
`expression of PI3K, Akt, or loss of PTEN augments the activity of mTOR and
`may increase the importance of this pathway in tumor cell survival and cell
`sensitivity to rapamycin compounds.
`Agents that specifically inhibit mTOR are limited to rapamycin and the
`structurally related compounds CCI-779 and SDZ RAD. Wortmannin and
`LY294002 are structurally unrelated molecules that, at low concentrations, are
`relatively specific, cell-permeable PI3K inhibitors [10]. However, wortmannin
`also directly inhibits mTOR autokinase activity with an IC50 that is  100-fold
`higher than that required for PI3K inhibition ( 200 nM in vitro and 300 nM in
`vivo) [10]. LY294002 inhibits mTOR autokinase activity in vitro, with an IC50 of
`5 mM [10]. Rapamycin and SDZ RAD are being developed as immunosuppres-
`sants, and CCI-779 is being developed as a cancer therapy.
`
`The discovery of rapamycin and its antiproliferative activity
`
`identified as product of the fungus
`Rapamycin, a macrolide, was first
`Streptomyces hygroscopicus, an organism isolated from the soil samples from
`Easter Island [23,24]. Although it was originally identified as an antifungal agent,
`subsequent studies demonstrated impressive anti-tumor and immunosuppressant
`activities. The National Cancer Institute (NCI) originally evaluated rapamycin in
`the late 1970s. It was found to have antiproliferative activity in a variety of
`murine tumor systems, including B16 melanoma and P388 leukemia models
`[25,26]. Rapamycin has since been shown to inhibit
`the growth of B-cell
`lymphoma cell lines [27], small-cell lung cancer cell lines [28], rhabdomyosar-
`coma cell lines [29], and MiaPaCa-2 and Panc-1 human pancreatic cancer cell
`lines [30]. Rapamycin also augmented cisplatin-induced apoptosis in murine
`T-cell lines, the human promyelocytic cell line HL-60, and human ovarian cancer
`cell line SKOV3 [68]. These data suggest that rapamycin has intrinsic anti-
`proliferative activity and may enhance the efficacy of selected cytotoxic agents.
`Similar to other natural immunosuppressants, such as cyclosporin A and
`FK-506, rapamycin binds to a member of the ubiquitous immunophilin family of
`FK-506 binding proteins (FKBP), termed FKBP-12, inhibiting its enzymatic
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`isomerase [31,32]. Although this enzymatic function is
`activity as a prolyl
`important for altering protein conformation, it is not relevant to the action of
`rapamycin [33]. However, rapamycin must complex with FKBP-12 to inhibit
`mTOR. Thus, rapamycin may be considered a ‘‘prodrug’’ for the active agent at
`the cellular level, the FKBP12-rapamycin complex.
`Because inhibiting mTOR-mediated p70S6K and 4E-BP1 phosphorylation by
`rapamycin are coupled to growth arrest in G1, rapamycin’s anti-proliferative
`properties may be due to its effects on the regulation of protein translation [34 –
`36]. Inhibiting these key signaling pathways results in inefficient translation of
`mRNAs of proteins, such as cyclin D1 [69] and ornithine decarboxylase [37],
`which are important for cell cycle progression through the G1 phase. However, in
`addition to its actions on p70s6k and 4E-BP1, rapamycin prevents cyclin-
`dependent kinase (cdk) activation and retinoblastoma protein (pRb) phosphoryl-
`ation [38 – 41]. Rapamycin also seems to accelerate the turnover of cyclin D1 at
`the mRNA and protein levels, resulting in a deficiency of active cdk4/cyclin D1
`complexes required for pRB phosphorylation and release of E2F transcription
`factor, and to increase association of p27kip1 with cyclin E/cdk2. These two
`events, decreased cdk4/cyclin D and increased p27kip1 with cyclin E/cdk2, along
`with the inhibition of translation of other mRNAs, can explain the observed
`inhibition at the G1/S-phase transition [34,42]. However, cells derived from mice
`in which the p27 gene has been disrupted by homologous recombination are only
`partially rapamycin resistant, which indicates that rapamycin can inhibit cell
`cycle progression by p27-independent mechanisms [43]. In addition, there are
`data showing that proliferation can proceed despite rapamycin-induced inhibition
`of 4E-BP1 and S6 kinase phosphorylation [43,44]. Thus, although the target of
`rapamycin has been identified, the downstream pathway from target to inhibition
`of cell cycle progression is uncertain.
`
`Clinical development
`
`Although rapamycin, RAD, and CCI-779 share many biochemical and
`physiologic properties [22,45,46], they are being developed for different indica-
`tions, in part because of their different pharmacologic features. Rapamycin and
`RAD are available in oral formulations and are being developed as immunosup-
`pressants, whereas an intravenous formulation of CCI-779 is being evaluated as
`an anti-cancer therapeutic. Preclinical studies of rapamycin and the 40-O-
`(2-hydroxyethyl)-sirolimus derivation, RAD, showed that both compounds are
`effective in preventing and treating acute allograft rejection in a variety of
`transplant models as single agents and function synergistically with standard
`immunosuppressants [22]. Both agents are orally administered, and the efficiency
`of absorption is modulated by p-glycoproteins. Rapamycin has a terminal half-
`life of 62 hours in stable renal transplant recipients treated with cyclosporine, and
`its steady state is usually reached within 7 to 14 days. RAD has slightly increased
`bioavailability and a shorter half-life of 30 hours. Rapamycin and RAD are
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`metabolized by liver and intestinal cytochrome P-450 enzyme CYP3A4, and
`metabolites are excreted predominantly through the gastrointestinal tract. The
`safety and efficacy of rapamycin for the prevention of renal transplantation graft
`rejection were evaluated in two studies with a total of 1295 patients. Sirolimus 2
`or 5 mg/day was compared with 2 to 3 mg/kg/day of azathioprine [47] or placebo
`[48]. In all groups, the immunosuppressive regimen included cyclosporin and
`corticosteroids. In both studies, there was a significant reduction in graft failure at
`6 months compared with control arms, with the degree of benefit favoring the
`higher dose of rapamycin. Reduction in graft failure was associated with low
`rates of infection, myelosuppression, and hyperlipidemia. Randomized trials with
`RAD are on going.
`In addition to the favorable reductions in graft rejections rates seen with
`rapamycin, it is possible that clinical studies rapamycin and RAD may show a
`reduction in post-transplantation lymphoproliferative disorders (PTLD). In labor-
`atory studies, rapamycin caused not only profound growth inhibition but also
`apoptosis in a series of B-cell lymphoma cell lines [27]. In contrast, neither
`FK506 nor CsA affected the normal growth of these cells. Similarly, RAD had a
`profound inhibitory effect on in vitro growth of six different PTLD-like Epstein-
`Barr virus+ lymphoblastoid B cell lines. The drug also had a profound inhibitory
`effect on the growth of PTLD-like Epstein-Barr virus+ B cells xenografts in mice
`[49,50]. RAD markedly delayed growth or induced regression of the established
`tumors. When RAD treatment was initiated before tumor cell injection, a marked
`inhibition of tumor growth was seen [49,50]. Thus, follow-up on the incidence of
`post-transplantation lymphoproliferative disorders seen in patients treated with
`rapamycin is of interest.
`Although rapamycin induces therapeutic immunosuppression on chronic oral
`dosing, prolonged immunosuppression is not a desirable effect for a cancer
`therapeutic. In preclinical models, intermittent dosing schedules of rapamycin
`and CCI-779 were effective in delaying tumor growth without causing prolonged
`immunosuppression [51]. However, the pharmacologic properties of variable
`intestinal absorption, prolonged terminal half-life, and poor aqueous solubility in
`intravenous formulations coupled with the preference for an intermittent schedule
`to minimize immunosuppression compromised the evaluation of rapamycin as an
`antiproliferative agent in cancer patients. Wyeth-Ayerst, in collaboration with
`NCI, examined several derivatives of rapamycin and selected one agent, CCI-
`779, for further development based on its mechanism of action and favorable in
`vitro and in vivo efficacy, toxicity, and pharmacologic data.
`CCI-779 is a soluble ester of rapamycin with impressive in vitro and in vivo
`cytostatic activity. Results from the NCI human tumor cell line screen showed
`that CCI-779 and rapamycin share a mechanism of action that is distinct from
`other cancer therapeutics. The two agents are similar in activity: The Pearson
`correlation coefficient of the in vitro anti-proliferative activities and potencies of
`the two agents across the 60-cell line screen is 0.86. In vitro, human prostate and
`breast cancer lines; CNS, melanoma, and small-cell lung carcinoma; and T-cell
`leukemia human tumor lines were among the most sensitive to CCI-779 with
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` 8 M [44]. Platelet-derived growth factor stimulation of the human
`IC50 < 10
`glioblastoma line T98G was markedly inhibited (IC50  1 pM), which is
`consistent with its proposed mechanism of action as an inhibitor of signal
`transduction pathways. Growth-inhibited cells were arrested in G1 phase, and
`growth inhibitory effects were blocked by the FKBP inhibitory molecule
`ascomycin, suggesting that the mechanism of action of CCI-779 is similar to
`rapamycin [51].
`In most in vivo human tumor xenograft studies, CCI-779 caused significant
`tumor growth inhibition rather than tumor regression [44,51,52]. For example,
`CCI-779 delayed medulloblastoma cell line DAOY xenograft growth by 160%
`after 1 week and 240% after 2 weeks of systemic treatment compared with
`controls [52]. Growth inhibition of DAOY xenografts was 1.3 times greater after
`simultaneous treatment with CCI-779 and cisplatin than after cisplatin alone [52],
`suggesting that CCI-779 should be developed as a cytostatic rather than cytotoxic
`agent. Several intermittent dosing regimens of CCI-779 were effective in these
`animal models [51]. These findings are important because preclinical studies
`have shown that when CCI-779 is given intermittently, its immunosuppressive
`effects resolve within 24 hours of the last dose. Given its proposed properties as a
`cytostatic agent, CCI-779 may be of value in delaying time to tumor progression
`and increasing survival in patients when used alone or in combination with other
`anticancer agents.
`Preliminary results from two phase I studies evaluating increasing doses of
`CCI-779 on different schedules have been reported. The first study [53,54]
`evaluated the feasibility, pharmacokinetics, and biologic effects of escalating
`doses of CCI-779 administered as a 30-minute intravenous infusion daily for
`5 days every 2 weeks to patients with solid neoplasms. In this trial, 51 patients
`received 262 cycles at doses ranging from 0.75 to 24 mg/m2/day. Grade 3
`toxicities included hypocalcemia, elevation in hepatic transaminases, vomiting,
`and thrombocytopenia. In heavily pretreated patients, the recommended phase II
`dose was 15 mg/m2/day; thrombocytopenia caused treatment delays at 19.1 mg/-
`m2/day. The maximum tolerated dose (MTD) in minimally pretreated patients is
`19.1 mg/m2/day. Other toxicities were generally mild to moderate and included
`neutropenia, rash, mucositis, diarrhea, asthenia, fever, and hyperlipidemia.
`Hypersensitivity phenomena,
`including chest discomfort, dyspnea, flushing,
`and urticaria, during CCI-779 infusions were observed. Pharmacokinetic data
`were reported on the initial 17 patients receiving doses of 0.75 to 3.12 mg/m2/day.
`In this limited dataset, CCI-779 exhibited increasing peak concentrations with
`increasing dose, preferential red blood cell partitioning, and a median terminal
`half-life of 32.6 hours. One patient with non-small cell lung carcinoma achieved a
`partial response, and minor antitumor responses or prolonged (>4 months) stable
`disease were noted in patients with soft-tissue sarcoma and cervical, uterine, and
`renal cell carcinomas [53,54].
`In the second study, CCI-779 was given as a weekly 30-minute infusion over a
`dose range of 7.5 to 220 mg/m2/week [55,56]. The MTD of CCI-779 had not been
`defined at the time of this trial report. Mild to moderate toxicities reported on this
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`trial included skin toxicity, which was variously described as dryness with mild
`puritis, eczema-like lesions, urticaria, and aseptic folliculitis. Skin biopsies from
`some patients with folliculitis showed superficial peri-capillar dermatitis. Although
`the frequency of infections was not noted to be high, five patients experienced
`reactivation of peri-oral herpes lesions. However, immunologic analysis of blood
`cells did not show evidence of immunosuppression. Mild to moderate mucositis,
`nails changes, thrombocytopenia, leukopenia and anemia, asymptomatic hyper-
`lipidemia, and decrease in serum testosterone were also reported. Preliminary
`pharmacokinetic analysis of doses up to 60 mg/m2 indicated that peak plasma
`concentration, area-under-curve, clearance, and volume of distribution at steady
`state of CCI-779 increased with dose. The mean clearance was 22 L/hour and mean
`half-life was about 20 hours. Three patients had partial responses (one each with
`renal cell, neuro-endocrine, and breast carcinomas) [55,56].
`From these phase I studies, it seems that CCI-779 is well tolerated and has anti-
`tumor activity over a broad dose range. The most common toxicities of CCI-779—
`skin reactions and stomatitis, hyperlipidemia, and myelosuppression—are tran-
`sient, are generally mild to moderate in severity, and are similar to those reported
`for rapamycin and RAD. Rapamycin has been reported to cause pneumonitis, and
`this toxicity may be seen with CCI-779 treatment as the agent enters broader
`clinical development [57,58]. In addition, infections have not occurred with
`alarming frequency in phase I clinical trials of CCI-779; however, laboratory
`studies have shown that rapamycin stimulated viral protein synthesis and aug-
`mented the shutoff of host protein synthesis upon infection encephalomyocarditis
`and polio picornavirus [67]. Thus, the continued monitoring for unusual infections
`in patients treated on early clinical trials of CCI-779 is warranted.
`The observations that CCI-779 induced tumor regressions in patients treated at
`relatively nontoxic doses on the phase I study are particularly noteworthy. If CCI-
`779 is biologically active at lower doses, it may not be necessary to treat patients
`with higher and likely more toxic doses of this agent. Defining and limiting drug
`concentrations to the biologically effective range would prevent cross-reactions
`with other molecules that cause toxicity at higher drug concentrations. The tra-
`ditional phase I and II studies to determine MTD and response rates in unselected
`patients may not be the most appropriate development strategy for therapies
`that have specific molecular targets. Rather, the efficient clinical development
`of CCI-779 and other molecular targeted agents requires careful consideration
`of novel trial designs that incorporate biological studies to select patients for
`enrollment, to define a biologically active dose, and surrogate endpoints of anti-
`tumor activity.
`To define a biologically active dose, a target plasma concentration effective in
`laboratory models could be used as the target endpoint of a phase I dose escalation
`study. Pharmacokinetic studies in patients can then define the dose range that
`results in plasma concentrations/exposures that are above the target thought be
`active based on preclinical studies. Randomized phase II or III trials could then be
`designed to define clinical activity over a range of doses. Such trials require many
`more patients than traditional phase II and II studies because more than one dose
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`needs to be evaluated. However, incorporating pharmacodynamic assays to assess
`target modulation and pharmacokinetic assays to evaluate drug concentrations
`could more efficiently identify a biologically active dose range in early clinical
`trials of molecularly targeted agents. This approach requires that the change in
`target is directly (or indirectly but closely) related to drug pharmacokinetics and
`that the pharmacokinetic and pharmacodynamic assays are sensitive, reliable, and
`quantifiable so that the threshold, slope, and plateau in the dose-response curve can
`be identified. Changes in target to determine an active dose may not, however,
`directly correlate with anti-tumor effect. To predict efficacy, the changes in target
`should correlate with changes in cell survival or rate of proliferation and correlate to
`patient benefit.
`For CCI-779, assays to determine the degree of inhibition of mTOR by
`assessing the phosphorylation state of its downstream targets 4E-BP1 or p70s6k
`may be helpful in defining a pharmacologically active dose. For example, assays
`for measuring decreases in phosphorylation of threonine-70 of 4E-BP1 in tumor
`tissue [8] and p70S6 kinase activity [59] and 3H-thymidine incorporation [60,61] in
`peripheral blood mononuclear cells have been developed and may be a useful
`surrogate for determining the inhibition of mTOR activity with CCI-779.
`However, rapamycin-induced hypophosphorylation of these molecular targets
`may not predict anti-proliferative effects in all patients because there is evidence
`that cell cycle progression and translation can proceed despite hypophosphoryl-
`ation of 4E-BP1 and p70s6k by rapamycin [29,62]. Thus, assessing drug effects
`using these targets may assist in determining a pharmacologically active dose but
`may not predict anti-tumor activity of CCI-779 because the assays assess targets
`that are not related to drug effects on proliferation or, more likely, because
`signaling pathways parallel or downstream of mTOR are rendering the cells
`resistant to the agent.
`Choosing the appropriate efficacy endpoint for phase II studies of CCI-779
`requires careful consideration. Preclinical data suggested that CCI-779 would
`delay the growth of tumors rather than induce tumor regressions. Based solely
`on these preclinical results, efficacy endpoints other than response should be
`used in phase II trials of CCI-779. Possible surrogate phase II endpoints that
`have been proposed for evaluating other cytostatic agents include time to pro-
`gression, changes in tumor markers, target inhibition, and PET scan assayed
`indices of cell proliferation [63]. None of these proposed endpoints has been
`shown to correlate with patient benefit. However, the objective responses seen in
`the phase I studies of the agent suggest that CCI-779 may induce apoptosis in
`certain tumors, and it is possible that molecule profile of the tumor may predict
`for drug activity.
`Given our understanding of the mechanism of action of rapamycin, a number
`of hypotheses regarding the molecular abnormalities that may correlate with
`efficacy of CCI-779 can be generated. Based on preclinical results in glioma [51],
`small-cell carcinoma [28] and rhabdomyosarcoma [29,64], tumors that rely on
`paracrine or autocrine stimulation of receptors that trigger the PI3K/Akt/mTOR
`pathway or tumors with mutations causing constitutive activation of the PI3/Akt
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`pathway may depend on rapamycin-sensitive pathways for growth. In fact,
`abnormal activation of this pathway is relatively common. For example,
`mutations of the tumor suppressor gene PTEN, which encodes for a lipid
`phosphatase that inhibits PI3K-dependent activation of Akt, occur in multiple
`tumor types with a frequency approaching that of p53 [15] and have been linked
`to more aggressive tumor phenotypes [65]. Deletion or inactivation of PTEN
`results in unregulated Akt activity. Thus, the presence PTEN mutations may also
`predict for activity of CCI-779. In fact, in vitro and in vivo studies of isogenic
`PTEN(+/+) and PTEN( / ) mouse embryonal stem cells and human cancer
`cell lines with defined PTEN status showed that the growth of PTEN null cells
`was preferentially sensitive to CCI-779 [16 – 18]. The data suggest that mTOR
`may be a good target for cancer therapy in tumors with Akt activation resulting
`from growth factor dependency or loss of PTEN function.
`Abnormalities of the G1 checkpoint regulators p53, pRB, p16, p27, and cyclin
`D also occur frequently in cancer and may be a determinate of tumor-cell sen-
`sitivity to CCI-779. The parent compound, rapamycin, affects the efficiency with
`which cdk are activated by altering the expression of the cyclin D subunit [34].
`Because p16 inhibits the cyclin D-cdk4/6 phosphorylation of pRb required for
`progression through G1, loss of p16 results in unregulated cyclin D/cdk activity.
`Decreasing cyclin D might re-introduce the so-called ‘‘cdk-inhibitory’’ effect and
`arrest the cell cycle. Last, Huang et al demonstrated that in normal wild type, p53
`cooperates in enforcing G1 cell cycle arrest, leading to a cytostatic response to
`rapamycin. In contrast,
`tumor cells or mouse embryonic fibroblasts having
`deficient p53 function treated with rapamycin led to cell cycle progression fol-
`lowed by apoptosis [66].
`By defining the molecular characteristics of tumors that correlate with activity
`or inactivity of targeted agents in carefully designed clinical trials, we may be
`able to determine which patients are most likely to benefit from treatment with a
`specific agent [63]. However, the mechanisms of tumor suppressor gene in-
`activation and oncogene activation are complex, as are the various combinations
`and permutations of molecular abnormalities in the mTOR signaling pathway and
`parallel pathways that may determine cell sensitivity to the agent. As a result of
`these complexities, a broad molecular diagnostic approach assessing pathway
`activation may be required to identify the subset of patients most likely to benefit
`from treatment with a specific targeted agent.
`
`Summary
`
`Rapamycin and CCI-779 have significant in vitro and in vivo anti-proliferative
`activity against a broad range of human tumor cell lines, justifying the clinical
`evaluation of this class of agent in cancer patients. Preliminary results from phase
`I studies of CCI-779 suggest that the agent is well tolerated and has anti-tumor
`activity. The challenge to investigators is to efficiently determine what role this
`class of agent will play in the treatment of cancer patients.
`
`West-Ward Pharm.
`Exhibit 1009
`Page 010
`
`

`

`J.E. Dancey / Hematol Oncol Clin N Am 16 (2002) 1101–1114
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`1111
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