[CANCER RESEARCH 64, 252–261, January 1, 2004]
`
`Antitumor Efficacy of Intermittent Treatment Schedules with the Rapamycin
`Derivative RAD001 Correlates with Prolonged Inactivation of Ribosomal
`Protein S6 Kinase 1 in Peripheral Blood Mononuclear Cells
`
`Anne Boulay,1 Sabine Zumstein-Mecker,1 Christine Stephan,1 Iwan Beuvink,2 Frederic Zilbermann,2 Roland Haller,1
`Sonja Tobler,1 Christoph Heusser,1 Terence O’Reilly,1 Barbara Stolz,1 Andreas Marti,1 George Thomas,2 and
`Heidi A. Lane1
`1Novartis Institutes for BioMedical Research Basel, Novartis Pharma AG, Basel, Switzerland, and 2Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland
`
`ABSTRACT
`
`The orally bioavailable rapamycin derivative RAD001 (everolimus)
`targets the mammalian target of rapamycin pathway and possesses potent
`immunosuppressive and anticancer activities. Here, the antitumor activity
`of RAD001 was evaluated in the CA20948 syngeneic rat pancreatic tumor
`model. RAD001 demonstrated dose-dependent antitumor activity with
`daily and weekly administration schedules; statistically significant antitu-
`mor effects were observed with 2.5 and 0.5 mg/kg RAD001 administered
`daily [treated tumor versus control tumor size (T/C), 23% and 23–30%,
`respectively], with 3–5 mg/kg RAD001 administered once weekly (T/C,
`14 –36%), or with 5 mg/kg RAD001 administered twice weekly (T/C,
`36%). These schedules were well tolerated and exhibited antitumor po-
`tency similar to that of the cytotoxic agent 5-fluorouracil (T/C, 23%).
`Moreover, the efficacy of intermittent treatment schedules suggests a
`therapeutic window allowing differentiation of antitumor activity from
`the immunosuppressive properties of this agent. Detailed biochemical
`profiling of mammalian target of rapamycin signaling in tumors, skin, and
`peripheral blood mononuclear cells (PBMCs), after a single administra-
`tion of 5 mg/kg RAD001,
`indicated that RAD001 treatment blocked
`phosphorylation of the translational repressor eukaryotic initiation factor
`4E-binding protein 1 and inactivated the translational activator ribosomal
`protein S6 kinase 1 (S6K1). The efficacy of intermittent treatment sched-
`ules was associated with prolonged inactivation of S6K1 in tumors and
`surrogate tissues (>72 h). Furthermore, detailed analysis of the dose
`dependency of weekly treatment schedules demonstrated a correlation
`between antitumor efficacy and prolonged effects (>7 days) on PBMC-
`derived S6K1 activity. Analysis of human PBMCs revealed that S6K1 also
`underwent a concentration-dependent inactivation after RAD001 treat-
`ment ex vivo (>95% inactivation with 20 nM RAD001). In contrast,
`human PBMC-derived eukaryotic initiation factor 4E-binding protein 1
`was present predominantly in the hypophosphorylated form and was
`unaffected by RAD001 treatment. Taken together, these results demon-
`strate a correlation between the antitumor efficacy of
`intermittent
`RAD001 treatment schedules and prolonged S6K1 inactivation in PBMCs
`and suggest that long-term monitoring of PBMC-derived S6K1 activity
`levels could be used for assessing RAD001 treatment schedules in cancer
`patients.
`
`INTRODUCTION
`
`RAD001 (everolimus), an orally bioavailable derivative of rapamy-
`cin, is a macrolide antifungal antibiotic that demonstrates potent
`antiproliferative effects against a variety of mammalian cell types.
`Specifically, RAD001 inhibits cytokine-driven lymphocyte prolifera-
`tion (1), as well as the proliferation of human tumor-derived cells
`
`Received 12/18/02; revised 9/10/03; accepted 10/30/03.
`Grant support: Iwan Beuvink, Frederic Zilbermann, and George Thomas were
`supported by Novartis Forschungsstiftung Zweigniederlassung Friedrich Miescher Insti-
`tute for Biomedical Research.
`The costs of publication of this article were defrayed in part by the payment of page
`charges. This article must therefore be hereby marked advertisement in accordance with
`18 U.S.C. Section 1734 solely to indicate this fact.
`Note: Anne Boulay and Sabine Zumstein-Mecker contributed equally to this work.
`Requests for reprints: Heidi A. Lane, Novartis Institutes for BioMedical Research
`Basel, Oncology, Novartis Pharma AG, WKL-125.13.17, CH-4002 Basel, Switzerland.
`Phone: 41-61-696-5438; Fax: 41-61-696-3835; E-mail: heidi.lane@pharma.novartis.com.
`252
`
`grown either in culture or as tumors in animal models (2, 3). As a
`result of these properties, RAD001 is being clinically developed both
`as an immunosuppressant for prevention of allograft rejection (Certi-
`can; Ref. 1) and as a novel therapeutic in the fight against human
`cancer (2– 4).
`RAD001, like rapamycin, binds with high affinity to a ubiquitous
`intracellular receptor, the immunophilin FKBP12. This complex spe-
`cifically interacts with the mammalian target of rapamycin (mTOR)
`protein kinase;
`inhibiting downstream signaling events (5). The
`mTOR kinase is a member of the phosphoinositide kinase-related
`kinase family, which consists of high molecular weight serine/threo-
`nine kinases involved in cell cycle checkpoint control (6). Several
`lines of evidence suggest that mTOR acts as a sensor for stress (7) and
`the availability of amino acids (8 –10) or intracellular ATP (11). In the
`presence of mitogens and sufficient nutrients, mTOR relays a signal to
`translational regulators, specifically enhancing the translation of mR-
`NAs encoding proteins essential for cell growth (12) and progression
`through the G1 to S transition (13, 14). Consistent with targeting the
`mTOR pathway, treatment of mammalian cells with rapamycin has
`been shown to inhibit these signaling events, mimicking a starvation
`phenotype (15) and leading to growth retardation and accumulation of
`cells in G1 phase (16). The mechanism of growth stimulus and
`nutrient level integration by mTOR is, as yet, not fully understood.
`However, an increasing body of evidence suggests the involvement of
`the phosphatidylinositol 3⬘-kinase/Akt/TSC/Rheb pathway (12, 17–
`23). Indeed, it has been suggested that, in tumor cells, the activation
`status of the Akt pathway may be indicative of responsiveness to
`rapamycin or its derivatives (24 –27).
`mTOR is part of a multisubunit complex that contains the regula-
`tory proteins raptor (28, 29) and G␤L (30). The mTOR complex
`signals to at least two downstream effectors, the translational repres-
`sor protein eukaryotic initiation factor 4E (eIF-4E)-binding protein 1
`(4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). These share an
`evolutionary conserved amino acid motif, the TOS motif, that func-
`tions as a docking site for raptor (31–33). Binding of 4E-BP1 to the
`translational activator eIF-4E is modulated by mTOR-dependent
`phosphorylation of specific serine and threonine residues (5). Ser37
`and Ser46 are constitutively phosphorylated, acting as priming sites
`for the mitogen-induced, rapamycin-sensitive phosphorylation of
`Thr70 and Ser65 (34). After a final phosphorylation event at Ser65,
`4E-BP1 dissociates from eIF-4E (35), thereby allowing the reconsti-
`tution of a translationally competent initiation factor complex (eIF-4F;
`Ref. 5). eIF-4F activation results in the translation of a subset of
`capped mRNA containing highly structured 5⬘-untranslated regions
`and encoding proteins involved in G1- to S-phase progression (13,
`14). Mitogen-induced activation of the S6K1 is also dependent on
`mTOR function and has been implicated in the translational regulation
`of mRNAs possessing a 5⬘-terminal oligopyrimidine tract (36 –38).
`5⬘-Terminal oligopyrimidine tract mRNAs are characterized by a
`stretch of 4 –14 pyrimidines located at their extreme 5⬘ terminus and
`typically encode ribosomal proteins as well as components of the
`
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`RAPAMYCIN DERIVATIVE RAD001: EFFICACY AND BIOMARKERS
`
`translational machinery. Activation of S6K1 itself is also tightly
`regulated by hierarchical phosphorylation events, which are depend-
`ent on the activation of various signal transduction pathways and
`culminate in the phosphorylation of the rapamycin-sensitive site
`Thr389, an event closely paralleling kinase activation (12, 39). Im-
`munopurified mTOR has been shown to autophosphorylate on
`Ser2481 (40) and to phosphorylate Ser37, Ser46, and Ser65 on 4E-
`BP1 in vitro (11, 34, 41, 42). However, some of these events have
`been demonstrated to be resistant to antiproliferative concentrations of
`rapamycin (40 – 42). It is therefore unclear what role mTOR kinase
`activity plays per se in rapamycin-sensitive signaling events.
`Because mTOR couples nutrient/growth factor availability to cell
`growth and proliferation in a variety of cell types, there is a potential
`for developing rapamycin derivatives such as RAD001 as novel
`inhibitors of the deregulated cell growth characteristic of human
`cancers. Consistent with this, RAD001 inhibits the proliferation of a
`wide variety of human solid tumor cell lines both in vitro in cell
`culture and in vivo in animal xenograft models (2, 3, 27, 43, 44).
`Furthermore, antiproliferative effects of RAD001 in posttransplant
`lymphoproliferative disorder-like B cell lines have been observed in
`vitro and in vivo (45, 46). In the present study, we have demonstrated
`that RAD001 displays significant antitumor activity in the syngeneic
`CA20948 rat pancreatic tumor model. Equivalent activity was ob-
`served with daily and intermittent treatment schedules, suggesting the
`possibility of a therapeutic window allowing differentiation of anti-
`tumor activity from the immunosuppressive properties of this agent.
`Detailed biochemical analysis of the mTOR effectors 4E-BP1 and
`S6K1 in tumor, skin, and peripheral blood mononuclear cell (PBMC)
`extracts obtained from RAD001-treated rats suggests that modulation
`of 4E-BP1 activity and significant inactivation of S6K1 are associated
`with antitumor activity. Furthermore, the efficacy observed using
`intermittent treatment schedules is paralleled by long-term down-
`regulation of S6K1 activity in all three tissues. We also provide
`evidence that the duration of S6K1 inactivation in PBMCs correlates
`with the dose-dependent suppression of tumor growth observed with
`weekly regimens. Moreover, unlike 4E-BP1 phosphorylation, S6K1
`activity can be reproducibly measured in human PBMCs and repre-
`sents a potentially valuable pharmacodynamic biomarker by which to
`monitor RAD001 treatment schedules in cancer patients.
`
`MATERIALS AND METHODS
`
`Drug Preparation. RAD001 (everolimus) is a derivative of rapamycin
`[40-O-(2-hydroxyethyl)-rapamycin; Ref. 47]. For animal studies, RAD001 was
`formulated at 2% (w/v) in a microemulsion vehicle, which was diluted to the
`appropriate concentration in 5% (w/v) glucose solution just before adminis-
`tration by gavage. For in vitro and ex vivo analyses, RAD001 was prepared in
`DMSO before addition to cell culture or human volunteer blood samples.
`Antitumor Efficacy Studies and Statistical Analyses. Male Lewis rats
`were purchased from Iffa Credo (L’Abresque, France) and allowed food and
`water ad libitum. A suspension of CA20948 tumor cells (obtained from donor
`rats because this line is nonculturable in vitro) in Ham’s F-12 medium
`supplemented with 10% FCS, 0.1 g/100 ml NaHCO3, 1% penicillin, and 1%
`fungizone was injected s.c. into the left flank of rats. Treatment of randomized
`rats started when the tumors reached about 100 mm3. RAD001 was adminis-
`tered p.o. daily at 0.5 or 2.5 mg/kg (⫻6/week), twice weekly at 5 mg/kg, or
`weekly at 0.5, 1, 2, 3, or 5 mg/kg. A volume of vehicle equivalent to the
`highest dose of RAD001 administered in the experiment was used as a
`negative control. As a positive control, the cytotoxic agent 5-fluorouracil
`(5-FU; ICN Pharmaceuticals Inc., Costa Mesa, CA) was administered at a near
`maximum tolerated dose (15 mg/kg, i.v., 4⫻/week, 2 days treatment/2 days
`rest), which gives maximal antitumor effect. Tumors were measured every day
`or every other day with a caliper, and the volumes were calculated by using the
`⫻ d2
`⫻ d3), where d1, d2, and d3 represent
`formula of an ellipsoid [V ⫽ ␲/6 (d1
`the three largest diameters]. Animals were also weighed the same day tumors
`253
`
`were measured. The animals were sacrificed when either their tumor burden
`exceeded 25,000 mm3 or when skin overlaying the tumor exhibited evidence
`of necrosis. All protocols involving animals were approved by the Veteri-
`na¨ramt of Baselstadt, Switzerland.
`Results are presented as mean ⫾ 1 SEM or as percentage of T/C (mean
`increase of tumor volumes of treated animals divided by the mean increase of
`tumor volumes of control animals multiplied by 100). The statistical signifi-
`cance of differences between treatment and control groups were determined by
`ANOVA followed by the Dunnett test. Statistical analyses on body weight
`were performed by ANOVA followed by Tukey’s test, and for comparison
`between weight at start and end of the experiment for individual animals, the
`paired t test was used. The level of significance was set at P ⬍ 0.05. Statistical
`calculations were performed using SigmaStat 2.03 (Jandel Scientific).
`Rat-Derived and Human Volunteer-Derived Tissue/PBMC Protein Ex-
`tract Preparation. CA20948 tumor-bearing rats were given 0.5, 1, 2, or 5
`mg/kg RAD001 or an equivalent volume of vehicle. At the indicated times
`after administration, rats were sacrificed, and tumor and shaved skin samples
`(for 0.5 and 5 mg/kg RAD001 doses) were dissected and weighed. Samples
`were rinsed in ice-cold PBS and immediately extracted in ice-cold extraction
`buffer [50 mM Tris-HCl (pH 8.0), 120 mM NaCl, 20 mM NaF, 1 mM EDTA, 6
`mM EGTA, 15 mM PPi, 30 mM p-nitrophenyl phosphate, 1 mM benzamidine,
`0.2 mM phenylmethylsulfonyl fluoride, and 0.1% NP40] with a constant ratio
`of 45 mg tumor/ml extraction buffer and 90 mg skin/ml extraction buffer, using
`a PT3000 Polytron (probe PT-DA 3012/2S; Kinematica AG) or a hand-held
`PT2100 Polytron (probe PT-DA 2112/2EC), respectively. Lysates were
`cleared by centrifugation for 30 min at 12,000 ⫻ g at 4°C. Supernatants were
`subsequently aliquoted, snap frozen on dry ice, and stored at – 80°C. In the
`case of skin samples, before further analysis, samples were centrifuged for 20
`min at 436,000 ⫻ g at 4°C to remove the fat fraction.
`Blood (for 0.5, 1, 2, and 5 mg/kg RAD001 doses) from tumor-bearing and
`non-tumor-bearing rats was withdrawn into syringes containing EDTA [0.5%
`(w/v) final] and then placed into an ice-cold tube and mixed. Unless otherwise
`stated, the blood from individual animals within the same treatment group was
`analyzed separately. The blood was immediately centrifuged for 20 min at
`430 ⫻ g at 4°C. The PBMCs, deposited at the interface between the RBCs and
`the plasma, were collected and pelleted by centrifugation for 5 min at 3000 ⫻ g
`at 4°C. PBMCs were washed with 10 ml of ice-cold PBS and then repelleted
`by centrifugation for 5 min at 3000 ⫻ g at 4°C. Cell pellets were resuspended
`in ice-cold extraction buffer containing 1% NP40 at the fixed ratio of 500 ␮l
`extraction buffer/10 ml initial blood volume. The cells were sheared by
`vigorous pipetting and then centrifuged for 30 min at 12,000 ⫻ g at 4°C.
`Supernatants were aliquoted, snap frozen on dry ice, and stored at ⫺80°C.
`Human blood from healthy volunteers was collected under medical super-
`vision into tubes containing either sodium citrate (BD Vacutainer 9NC; BD
`Vacutainer Systems, Plymouth, United Kingdom) or EDTA (BD Vacutainer
`K3E) as an anticoagulant. The blood was either immediately processed or, for
`ex vivo treatments, treated with 2, 20, and 200 nM RAD001 or DMSO vehicle
`for 30 min at room temperature. Human PBMCs were isolated and extracted
`as described for rat PBMCs.
`A549 Cell Culture and Protein Extract Preparation. A549 human lung
`carcinoma cells (CCL185) were obtained from the American Type Culture
`Collection (Manassas, VA) and cultured in RPMI 1640 medium (Amimed,
`Allschwil, Switzerland) supplemented with 10% FCS, 2 mM L-glutamine, and
`100 ␮g/ml penicillin/streptomycin at 37°C and 5% CO2. Cell lysates were
`prepared as described previously (48).
`Immunoblot Analysis. Cell lysates (30 – 40 ␮g) were electrophoretically
`resolved on denaturing SDS polyacrylamide gels (SDS-PAGE), transferred to
`polyvinylidene difluoride (Millipore Corp., Bedford, MA), and probed with the
`following primary antibodies: anti-S6 (provided by J. Mestan; Oncology
`Research, Novartis Pharma AG, Basel, Switzerland); anti-4E-BP1 (kindly
`provided by N. Sonenberg; McGill University, Montreal, Quebec, Canada);
`anti-eIF-4E (kindly provided by S. J. Morley; University of Sussex, Brighton,
`United Kingdom); anti-phospho-4E-BP1 Thr70, anti-S6K1, and anti-phos-
`pho-S6 Ser240/Ser244 (all from Cell Signaling Technology Inc., Beverly,
`MA); and anti-␤-tubulin (Tub2.1; Sigma, St. Louis, MO). “Decorated” pro-
`teins were revealed using horseradish peroxidase-conjugated antimouse or
`antirabbit immunoglobulins in conjunction with the enhanced chemilumines-
`cence procedure (Amersham Pharmacia Biotech Inc., Buckinghamshire,
`United Kingdom).
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`
`Affinity Purification of 4E-BP1䡠eIF-4E Complexes with 7-Methyl-GTP-
`Sepharose. Rat tumor (1 mg), skin (0.7 mg), or PBMC (0.25 mg) extracts
`were diluted to a final volume of 500 ␮l in ice-cold extraction buffer and
`adjusted to a final NP40 concentration of 0.1%. The 4E-BP1䡠eIF-4E complexes
`were affinity purified with 20 ␮l of 7-methyl-GTP-Sepharose beads (Amer-
`sham Pharmacia Biotech Inc., Piscataway, NJ) by gentle rotation for 2.5 h at
`4°C. Proteins retained on the beads were washed twice with extraction buffer
`in the absence of NP40 and resuspended in 15 ␮l of Laemmli buffer. Dena-
`tured samples were subjected to 15% SDS-PAGE and transferred to polyvi-
`nylidene difluoride membranes. Membranes were first immunoblotted for
`4E-BP1 protein, followed by stripping as described previously (49) and re-
`probing for eIF-4E protein (see above).
`40S Ribosomal S6 Kinase Assay. Rat tumor (1 mg), skin (0.7 mg), or
`PBMC (0.25 mg) extracts were diluted to a final volume of 1 ml (tumor and
`skin) or 500 ␮l (PBMC) with ice-cold extraction buffer and adjusted to a final
`NP40 concentration of 1%. Human-derived PBMC extracts (0.8 –1 mg) were
`diluted to a final volume of 750 ␮l with ice-cold extraction buffer (final NP40
`concentration, 1%). In some experiments, human-derived PBMC extracts were
`first precleared with 20 ␮l of 50% protein A-Sepharose (Amersham Pharmacia
`Biotech, Uppsala, Sweden) by rotating for 20 min at 4°C. S6K1 was immu-
`noprecipitated from all extracts by addition of 2.5 ␮l of the M5 S6K1-specific
`polyclonal antibody and incubation on ice for 1 h, followed by retrieval of
`immunocomplexes with 20 ␮l of 50% protein A-Sepharose. S6K1 activity was
`measured using rat liver 40S ribosomal subunits as a specific substrate, as
`described previously (50), except that p-nitrophenyl phosphate was omitted in
`the reaction mixture. Phosphorylated S6 was resolved by 12.5% SDS-PAGE
`and analyzed using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
`[␥-32P]phosphate incorporation into S6 was quantified using ImageQuant
`(Molecular Dynamics). Where appropriate, the statistical significance of dif-
`ferences between treatment groups and untreated control groups was deter-
`mined using ANOVA or ANOVA on ranks followed by the Dunnett test. The
`level of significance was set at P ⬍ 0.05. Statistical calculations were per-
`formed using SigmaStat 2.03 (Jandel Scientific). Coefficient of variation is
`defined as SD divided by the mean and multiplied by 100.
`
`RESULTS
`
`Intermittent RAD001 Treatment Schedules Display Antitumor
`Efficacy. Short-term exposure to rapamycin in vitro has long-term
`antiproliferative effects on tumor cell
`lines (51), suggesting that
`intermittent treatment schedules may retain antitumor activity. Fur-
`thermore, daily oral administration of RAD001 is effective in rat
`models of autoimmune disease and allotransplantation (47, 52),
`whereas we have found that weekly (5 mg/kg) RAD001 dosing
`schedules have reduced immunosuppressive properties in rats as com-
`pared with daily treatment (2.5 mg/kg): 66 ⫾ 18% and 98 ⫾ 1%
`inhibition of IgG antibody response after dinitrophenol-coupled key-
`hole limpet hemocyanogen immunization, respectively.3 With these
`observations in mind, we evaluated whether RAD001 treatment
`schedules, with potentially reduced immunosuppressive properties,
`could elicit antitumor responses. Daily versus intermittent RAD001
`administration schedules were compared using the s.c. CA20948 rat
`pancreatic tumor model. Vehicle was used as a negative control, and
`the cytotoxic agent 5-FU was used as a positive control (Fig. 1; Table
`1, Experiment 1). RAD001 treatment at 0.5 or 2.5 mg/kg/day, six
`times a week, resulted in antitumor activity characterized by statisti-
`cally significant inhibition of tumor growth as compared with vehicle
`controls [treated tumor versus control tumor size (T/C), 30% and
`23%, respectively; P ⬍ 0.05 after 10 days of treatment; Fig. 1A; Table
`1, Experiment 1]. Statistically significant tumor growth suppression
`was also observed after intermittent administration of 5 mg/kg
`RAD001 twice a week (T/C, 36%) or once a week (T/C, 36%).
`Moreover, all RAD001 treatment schedules suppressed tumor growth
`to a similar extent as the cytotoxic 5-FU (T/C, 23%). Continued
`
`3 T. O’Reilly, H. A. Lane, and C. Heusser, unpublished data.
`
`Fig. 1. Suppression of tumor growth by daily and intermittent dosing schedules of
`RAD001. Tumors were established in male Lewis rats by s.c. injection of CA20948 tumor
`suspension obtained from donor rats. Treatments started on day 4 after inoculation.
`Formulated RAD001 was diluted in a 5% glucose solution and administered p.o. daily at
`a dose of 0.5 or 2.5 mg/kg (qd ⫻6, 6 times/week) or once (wk ⫻1) or twice (wk ⫻2)
`weekly at 5 mg/kg RAD001. Vehicle and 5-fluorouracil (5-FU ⫻4; 4 times/week) were
`administered as negative and positive controls, respectively. Tumor volumes were meas-
`ured (A), and rats were weighed (B) as described in “Materials and Methods.” Vehicle
`control-treated rats were sacrificed on day 10 due to tumor burden. Data are
`means ⫾ SEM (n ⫽ 7– 8 animals/group). Stars represent P ⬍ 0.05 versus vehicle controls.
`
`treatment with RAD001 after vehicle controls were sacrificed due to
`tumor burden led to a prolonged low tumor growth rate with all
`treatment schedules, resulting in similar tumor burden after 17 days of
`treatment as compared with 5-FU (Fig. 1A). For all treatment sched-
`ules, RAD001 was well tolerated, with no significant body weight loss
`or mortalities observed (Fig. 1B; Table 1, Experiment 1). These results
`demonstrate that RAD001 is a well-tolerated antitumor agent in a rat
`model of pancreatic cancer and indicate a potential for intermittent
`administration schedules that may allow dissociation of antitumor
`from immunosuppressive effects.
`RAD001 Modulates 4E-BP1 and S6K1 Activity in Tumor, Skin,
`and PBMCs Obtained from CA20948 Pancreatic Tumor-Bearing
`Rats. To investigate RAD001-specific effects on mTOR signaling in
`vivo, three CA20948 tumor-bearing rats were treated with vehicle or
`a single efficacious dose of RAD001 (5 mg/kg). Rats were sacrificed
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`Table 1 Effect of daily and intermittent RAD001 administration on CA20948 rat pancreatic tumor-bearing rats
`
`Tumor response
`
`Host response
`
`Schedule
`
`% T/Ca
`
`⌬ Tumor volume (mm3)
`
`⌬ Body weight (g)
`
`%⌬ Body weight
`
`Survival (alive/total)
`
`Compound
`
`Experiment 1
`Vehicle
`5-FU
`RAD001
`RAD001
`RAD001
`RAD001
`
`Experiment 2
`Vehicle
`RAD001
`RAD001
`RAD001
`
`2 ml/kg p.o. daily
`15 mg/kg i.v. 4⫻ weekly
`0.5 mg/kg p.o. daily
`2.5 mg/kg p.o. daily
`5 mg/kg p.o. weekly
`5 mg/kg p.o. twice weekly
`
`2 ml/kg p.o. daily
`0.5 mg/kg p.o. daily
`0.5 mg/kg p.o. weekly
`5 mg/kg p.o. weekly
`
`Experiment 3
`2 ml/kg p.o. weekly
`Vehicle
`0.5 mg/kg p.o. weekly
`RAD001
`1 mg/kg p.o. weekly
`RAD001
`2 mg/kg p.o. weekly
`RAD001
`3 mg/kg p.o. weekly
`RAD001
`5 mg/kg p.o. weekly
`RAD001
`a T/C, treated tumor versus control tumor size.
`b P ⬍ 0.05 versus control, Dunnett test.
`
`100
`23
`30
`23
`36
`36
`
`100
`23
`48
`14
`
`100
`48
`45
`32
`36
`24
`
`12972 ⫾ 2188
`2863 ⫾ 764b
`3904 ⫾ 856b
`2959 ⫾ 624b
`4652 ⫾ 1220b
`4604 ⫾ 928b
`
`12331 ⫾ 1410
`2894 ⫾ 567b
`5951 ⫾ 1739
`1708 ⫾ 339b
`
`19270 ⫾ 3918
`9275 ⫾ 1926
`8617 ⫾ 1704
`6161 ⫾ 1079b
`6869 ⫾ 611b
`4680 ⫾ 1593b
`
`12 ⫾ 8
`18 ⫾ 4
`35 ⫾ 7
`7 ⫾ 2
`22 ⫾ 5
`21 ⫾ 3
`
`29 ⫾ 2
`30 ⫾ 5
`36 ⫾ 2
`32 ⫾ 2
`
`28.3 ⫾ 2.1
`21 ⫾ 2.4
`32.8 ⫾ 2.7
`24.9 ⫾ 1.9
`24.3 ⫾ 3.3
`22.8 ⫾ 1.7
`
`5
`7
`14
`3
`8
`8
`
`14
`17
`15
`15
`
`10
`8
`12
`9
`9
`8
`
`8/8
`7/7
`7/7
`7/7
`7/7
`7/7
`
`8/8
`8/8
`8/8
`8/8
`
`8/8
`8/8
`8/8
`8/8
`6/6
`8/8
`
`matic dephosphorylation of its physiological substrate, 40S ribosomal
`protein S6, in tumor extracts (Fig. 2C). A similar reduction was not
`observed in skin and PBMC extracts because these tissues exhibited
`no detectable S6 phosphorylation in control animals. Interestingly, a
`
`24 h later, and protein extracts were prepared from tumors, skin, and
`PBMCs. By immunoblot analysis, mTOR could be detected in tumor
`and PBMC extracts; however, neither mTOR expression nor phos-
`phorylation on Ser2448 was modified on RAD001 treatment.4 In
`contrast, 4E-BP1 exhibited a decrease in Thr70 phosphorylation in
`tumor, skin, and PBMC extracts (Fig. 2A), a phenomenon associated
`with changes in 4E-BP1 electrophoretic mobility, particularly striking
`in PBMCs. This observation is consistent with previous work dem-
`onstrating dephosphorylation of 4E-BP1 on Thr70 in tumors derived
`from mouse xenograft models after five daily treatments with an ester
`of rapamycin CCI-779 (1 h after last administration; Ref. 53). Inter-
`estingly, the phosphorylation of another rapamycin-sensitive residue
`(Ser65; Refs. 5, 34, and 35) was unaffected by RAD001 treatment,4
`indicating that RAD001-insensitive phosphorylation of this site can
`occur as reported previously (54).
`To determine whether the decreased phosphorylation state of 4E-
`BP1 resulted in a change in functionality, the eIF-4E binding activity
`of 4E-BP1 was assessed using an in vitro 7-methyl-GTP-binding
`assay (Fig. 2B). Whereas similar levels of eIF-4E were recovered in
`the control- and RAD001-treated extracts, in two animals increased
`eIF-4E䡠4E-BP1 complex formation was clearly observed in skin and
`PBMC samples after RAD001 treatment. In tumor samples, two
`electrophoretically distinct forms of 4E-BP1 protein were bound to
`eIF-4E in vehicle control-treated rats (Fig. 2B). After RAD001 treat-
`ment, only the lower migrating form was found bound to eIF-4E, with
`an associated loss of the upper band consistent with reduced 4E-BP1
`phosphorylation levels (Fig. 2A). A similar 4E-BP1 doublet with
`eIF-4E binding activity has been observed previously in proliferating
`cells/tissue (29, 54) and presumably reflects differential 4E-BP1 phos-
`phorylation states within the proliferating tumor.
`To further assess the effect of RAD001 administration on the
`mTOR pathway, S6K1 protein and activity levels were also analyzed
`(Fig. 2, C and D). Whereas S6K1 protein levels were unaffected by
`RAD001 treatment (Fig. 2C), in vitro kinase assay using 40S riboso-
`mal subunits as a substrate revealed a statistically significant reduc-
`tion in S6K1 activity in all extracts [Fig. 2D; 83% (tumors), 80%
`(skin), and 75% (PBMC); all P ⬍ 0.05 versus vehicle-treated con-
`trols]. This reduction in S6K1 activity was associated with the dra-
`
`4 A. Boulay and H. A. Lane, unpublished data.
`
`Fig. 2. RAD001 administration inhibits mammalian target of rapamycin signaling in
`CA20948 tumor-bearing rats. S.c. CA20948 tumor-bearing rats received a single admin-
`istration of an efficacious dose of RAD001 (5 mg/kg) or vehicle and were sacrificed 24 h
`after administration (3 rats/group). Tumors, skin, and PBMCs were individually prepared
`and extracted as described in “Materials and Methods.” Results from individual rats are
`presented. A and C, total protein was subjected to electrophoresis followed by immunoblot
`analysis. Membranes were probed for eukaryotic initiation factor 4E-binding protein 1
`(4E-BP1) and phospho-threonine 70 4E-BP1 [P-4E-BP1 (Thr70)] levels, with eukaryotic
`initiation factor 4E (eIF-4E) and ␤-tubulin levels acting as loading controls (A) or
`ribosomal protein S6 kinase 1 protein, S6 40S ribosomal protein, and phospho-serine
`240/244 S6 [P-S6 (Ser240/Ser244)] levels (C). B, the level of 4E-BP1 bound to eIF-4E was
`measured by purification of 4E-BP1䡠eIF-4E complexes on 7-methyl-GTP-Sepharose, as
`described in “Materials and Methods,” followed by immunoblot analysis. D, ribosomal
`protein S6 kinase 1 was immunoprecipitated from equal amounts of total protein extract,
`and activity was measured by in vitro kinase assay using 40S ribosomal subunits as a
`specific substrate, as described in “Materials and Methods.” Phosphorimages (32P-S6) and
`PhosphorImager quantifications of the kinase assay are presented. Data are means ⫾ SD
`of n ⫽ 3 animals/group. Stars represent P ⬍ 0.05 versus vehicle-treated controls (Dunnett
`test).
`255
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`Roxane Labs., Inc.
`Exhibit 1025
`Page 004
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`RAPAMYCIN DERIVATIVE RAD001: EFFICACY AND BIOMARKERS
`
`reduction in S6 protein expression was observed in RAD001-treated
`skin, but not in tumor or PBMC extracts. A similar phenomenon has
`been reported previously in tumors after treatment of mice bearing
`human prostate cancer xenografts with CCI-779 (24). Moreover, the
`translation of S6 (as a 5⬘-terminal oligopyrimidine tract mRNA) has
`been shown to be specifically inhibited by rapamycin in 3T3 cells
`(36). It is not known why, in this model, RAD001 treatment only has
`effects on S6 expression in skin; however, differential downstream
`effects of mTOR pathway inhibition, depending on the tissue source,
`are a plausible possibility (54). Taken together, these data demonstrate
`that both 4E-BP1 and S6K1 pathways are affected in tumors, skin, and
`PBMC samples obtained from CA20948 tumor-bearing rats after a
`single administration of an efficacious dose of RAD001.
`Prolonged Inactivation of S6K1 in Tumors, Skin, and PBMCs
`Correlates with the Efficacy of Intermittent RAD001 Treatment
`Schedules. To investigate whether the antitumor efficacy of intermit-
`tent RAD001 treatment schedules is associated with prolonged effects
`on the mTOR pathway, CA20948 tumor-bearing rats were treated
`with a single dose of RAD001 (5 mg/kg) or vehicle, and tumor, skin,
`and PBMC extracts were prepared 12, 24, 48, or 72 h after adminis-
`tration. Because S6K1 was significantly inactivated 24 h after a single
`RAD001 administration in all tissues analyzed (Fig. 2D), long-term
`effects on mTOR function were assessed using the 40S kinase assay
`(Fig. 3). Tumor and skin extracts were obtained from each of 3
`rats/treatment group, whereas PBMC extracts were obtained from
`pooled blood from each treatment group. A dramatic reduction in
`S6K1 activity was already observed in tumors, skin, and PBMCs 12 h
`after RAD001 administration (91%, 91%, and 82% inhibition, respec-
`tively; all P ⬍ 0.05 versus untreated controls; Fig. 3). In contrast,
`treatment with vehicle did not significantly modulate S6K1 activity as
`compared with untreated controls (Fig. 3). Moreover, RAD001 treat-
`ment resulted in the sustained inactivation of S6K1 in all tissues. In
`tumors, statistically significant inhibition of S6K1 was maintained up
`to 48 h after administration, with some evidence of recovery after 72 h
`(80% and 62% inhibition at 48 and 72 h, respectively; Fig. 3A). In
`comparison, S6K1 derived from skin samples remained significantly
`inhibited for at least 72 h (72% inhibition at 72 h; Fig. 3B). Although
`a statistical analysis could not be performed on the pooled PBMC
`samples, S6K1 activity was also dramatically inhibited for up to 72 h
`in these samples (82% inhibition at 72 h; Fig. 3C). Thus, consistent
`with the antitumor efficacy of intermittent 5 mg/kg RAD001 treat-
`ment schedules in CA20948 tumor-bearing rats, a single administra-
`tion of 5 mg/kg RAD001 resulted in long-term inactivation of S6K1
`in tumors, skin, and PBMCs.
`The Antitumor Efficacy of Intermittent RAD001 Treatment
`Schedules Is Dose Dependent: Correlation Between Efficacy and
`Prolonged Effects on mTOR Effectors in Rat PBMCs. Following
`the observation that intermittent RAD001 (5 mg/kg) treatment sched-
`and 23%). Because both these schedules involve administration of 3
`ules significantly inhibited tumor growth, we explored the effect of
`mg/kg RAD001 per week, these data indicate that, with the same total
`RAD001 dose on the efficacy of weekly treatment schedules (Table 1,
`RAD001 exposure, intermittent dosing schedules can elicit equivalent
`Experiments 2 and 3). As expected, 5 mg/kg/week RAD001 signifi-
`antitumor responses as daily schedules.
`cantly suppressed CA20948 tumor growth as compared with vehicle
`controls (T/C, 14% and 24% at 7 a