V O L U M E 2 6 䡠 N U M B E R 1 0 䡠 A P R I L 1 2 0 0 8
`
`JOURNAL OF CLINICAL ONCOLOGY
`
`O R I G I N A L R E P O R T
`
`From the Medical Oncology and
`Pathology Departments, Vall d’Hebron
`University Hospital, Barcelona, Spain;
`Medical Oncology Department, Sarah
`Cannon Cancer Center, Nashville, TN;
`Medical Oncology Department, Royal
`Marsden Hospital, London, United
`Kingdom; Novartis Oncology, Basel,
`Switzerland; and Novartis Oncology,
`Florham Park, NJ.
`
`Submitted September 20, 2007;
`accepted November 16, 2007;
`published online ahead of print at
`www.jco.org on March 10, 2008.
`
`Presented in part at the 41st Annual
`Meeting of the American Society of
`Clinical Oncology, Orlando, FL, May
`13-17, 2005; and at the 13th European
`Cancer Conference, Paris, France,
`October 30 to November 3, 2005.
`
`Authors’ disclosures of potential con-
`flicts of interest and author contribu-
`tions are found at the end of this
`article.
`
`Corresponding author: Jose´ Baselga,
`MD, Medical Oncology Department,
`Vall d’Hebron University Hospital, P.
`Vall d’Hebron, 119-129, 08035, Barce-
`lona, Spain; e-mail:
`jbaselga@vhebron.net.
`
`© 2008 by American Society of Clinical
`Oncology
`
`0732-183X/08/2610-1603/$20.00
`
`DOI: 10.1200/JCO.2007.14.5482
`
`Dose- and Schedule-Dependent Inhibition of the
`Mammalian Target of Rapamycin Pathway With
`Everolimus: A Phase I Tumor Pharmacodynamic Study in
`Patients With Advanced Solid Tumors
`Josep Tabernero, Federico Rojo, Emiliano Calvo, Howard Burris, Ian Judson, Katharine Hazell,
`Erika Martinelli, Santiago Ramon y Cajal, Suzanne Jones, Laura Vidal, Nicholas Shand, Teresa Macarulla,
`Francisco Javier Ramos, Sasa Dimitrijevic, Ulrike Zoellner, Pui Tang, Michael Stumm, Heidi A. Lane,
`David Lebwohl, and Jose´ Baselga
`
`A
`
`B
`
`S
`
`T
`
`R
`
`A
`
`C
`
`T
`
`Purpose
`Everolimus is a selective mammalian target of rapamycin (mTOR) inhibitor with promising anticancer
`activity. In order to identify a rationally based dose and schedule for cancer treatment, we have
`conducted a tumor pharmacodynamic phase I study in patients with advanced solid tumors.
`Patients and Methods
`Fifty-five patients were treated with everolimus in cohorts of 20, 50, and 70 mg weekly or 5 and
`10 mg daily. Dose escalation depended on dose limiting toxicity (DLT) rate during the first 4-week
`period. Pre- and on-treatment steady-state tumor and skin biopsies were evaluated for total and
`phosphorylated (p) protein S6 kinase 1, eukaryotic initiation factor 4E (elF-4E) binding protein 1
`(4E-BP1), eukaryotic initiation factor 4G (eIF-4G), AKT, and Ki-67 expression. Plasma trough levels
`of everolimus were determined on a weekly basis before dosing during the first 4 weeks.
`Results
`We observed a dose- and schedule-dependent inhibition of the mTOR pathway with a near complete
`inhibition of pS6 and peIF-4G at 10 mg/d and ⱖ 50 mg/wk. In addition, pAKT was upregulated in 50%
`of the treated tumors. In the daily schedule, there was a correlation between everolimus plasma
`trough concentrations and inhibition of peIF4G and p4E-BP1. There was good concordance of mTOR
`pathway inhibition between skin and tumor. Clinical benefit was observed in four patients including
`one patient with advanced colorectal cancer achieving a partial response. DLTs occurred in five
`patients: one patient at 10 mg/d (grade 3 stomatitis) and four patients at 70 mg/wk (two with grade 3
`stomatitis, one with grade 3 neutropenia, and one with grade 3 hyperglycemia).
`Conclusion
`Everolimus achieved mTOR signaling inhibition at doses below the DLT. A dosage of 10 mg/d or
`50 mg/wk is recommended for further development.
`
`J Clin Oncol 26:1603-1610. © 2008 by American Society of Clinical Oncology
`
`INTRODUCTION
`
`The mammalian target of rapamycin (mTOR) is a
`serine/threonine kinase, downstream of the phos-
`phatidyl inositol 3⬘-kinase (PI3K)-AKT signaling
`pathway.1,2 mTOR is activated in response to differ-
`ent stimuli such as nutrients and growth factor re-
`ceptors.3 With the involvement of the PI3K-AKT
`pathway, mTOR relays a signal to translational reg-
`ulators, specifically enhancing the translation of
`mRNAs encoding proteins essential for cell growth
`and cell cycle progression through G1 to S phase.2,4,5
`As a result of its central position within this signal
`transduction pathway, mTOR has been considered
`
`an important target for new anticancer drug devel-
`opment.2,6,7 In support of its role in cancer, the
`mTOR pathway is aberrantly activated in around
`half of human tumors1,4,8 and plays a critical role
`in angiogenesis.9-14
`mTOR signals to at least two downstream ef-
`fectors, the translational repressor protein eukary-
`otic initiation factor 4E (elF-4E) binding protein 1
`(4E-BP1) and the ribosomal protein S6 kinase 1
`(S6K1).15-17 Binding of 4E-BP1 to the translational
`activator eIF-4E is modulated by mTOR-dependent
`phosphorylation of multiple specific serine and
`threonine residues.18,19 After a final phosphoryla-
`tion at Ser65, 4E-BP1 dissociates from eIF4E,
`
`© 2008 by American Society of Clinical Oncology
`
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`Copyright © 2016 American Society of Clinical Oncology. All rights reserved.
`
`NOVARTIS EXHIBIT 2052
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`Tabernero et al
`
`thereby allowing for the reconstitution of a translationally competent
`initiation factor complex with the involvement of eIF-4F20 and eIF-
`4G.21-23 eIF-4F activation results in the translation of a subset of
`capped mRNAs containing highly structured 5⬘-untranslated regions
`and encoding proteins involved in the G1 to S transition.24,25
`Everolimus (RAD001), an oral rapamycin derivative, has dem-
`onstrated potent antiproliferative effects against a variety of mamma-
`lian cell
`lines. Everolimus inhibits cytokine-driven lymphocyte
`proliferation,26 as well as the proliferation of human tumor-derived
`cells, both in vitro in cell culture and in vivo in animal xenograft
`models.27-31 As a result of these properties, everolimus has been devel-
`oped as an immunosuppressant32-34 and is now being developed as an
`anticancer agent. In the syngenic CA20948 rat pancreatic tumor
`model, everolimus has been shown to inhibit 4E-BP1 phosphoryla-
`tion and S6K1 signaling in tumor and normal tissue.29,35,36 Treatment
`with everolimus demonstrated equivalent activity with daily and in-
`termittent schedules, this activity being dose-dependent for the two
`administration schedules.29
`Based on these considerations, the objectives of this trial were to
`assess the optimal dose and schedule of orally everolimus, adminis-
`tered weekly or daily, based on the safety profile and pharmacody-
`namic (PD) effects on mTOR dependent pathways in sequential
`tumor and skin biopsies. In addition, the effects of everolimus on
`tumor and skin specimens were correlated to plasma trough concen-
`trations of the drug.
`
`PATIENTS AND METHODS
`
`Patient Population
`Main inclusion criteria were histologically confirmed advanced tumors,
`unresponsive to standard therapy; presence of disease accessible to repetitive
`biopsies; age ⱖ 18 years; life expectancy ⱖ 12 weeks; WHO performance status
`of 0 to 2; and adequate bone marrow, hepatic, and renal function. All patients
`gave informed consent, and approval was obtained from the ethics committees
`at the participating institutions and regulatory authorities. The study followed
`the Declaration of Helsinki and good clinical practice guidelines.
`
`Treatment and Dose Escalation Criteria
`Everolimus was administered either as a single weekly oral dose (20, 50,
`and 70 mg) or as a continuous daily oral dose (5 and 10 mg) until progression.
`Initially, six to eight patients were to be enrolled to each dose level to have a
`minimum of four fully assessable patients for the PD end points of the study.
`Dose escalation proceeded in the absence of more than one of six patients with
`dose-limiting toxicity (DLT) in the first 28 days of treatment. If two or more
`patients presented DLT at a dose level, enrollment of patients to that dose level
`was discontinued and the immediately preceding dose level was considered the
`maximum tolerated dose for a given schedule. DLT was defined as any one of
`the following drug-suspected toxicities: grade 3 or higher National Cancer
`Institute Common Toxicity Criteria version 3.0 hematologic toxicity and
`grade 3 or higher nonhematologic toxicity despite the use of adequate/maxi-
`mal medical intervention and/or prophylaxis.
`
`Safety and Response Assessments
`Routine clinical and laboratory assessments were conducted on a weekly
`basis during the first 4 weeks of treatment and thereafter every 2 weeks; after 6
`months, assessments were conducted once per month. ECG monitoring was
`performed at baseline and at the fourth week of treatment after dosing. Ad-
`verse events were recorded, graded using the National Cancer Institute Com-
`mon Toxicity Criteria version 3.0, and assessed by the investigator for any
`relationship with everolimus treatment. Objective measurement of tumor
`mass was assessed in accordance with the Response Evaluation Criteria in Solid
`Tumors criteria37 after 8 weeks on treatment, and thereafter every 8 weeks.
`
`Pharmacokinetic Analysis
`A significant linear correlation between steady-state trough concentrations
`and overall exposure (area under the curve) to everolimus had been previously
`found when the drug was administered daily.38 In this context, steady-state
`trough blood levels were chosen as a convenient monitoring pharmacokinetic
`(PK) parameter for this trial. Plasma levels of everolimus were determined on
`a weekly basis before dosing during the first 4 weeks of treatment.
`The concentration of everolimus in whole blood was determined by
`liquid chromatography–mass spectrometry after liquid-liquid extraction. This
`method for blood sample has a lower limit of quantification of 0.3 ng/mL.
`Trough concentrations were reported as mean and standard deviation.
`
`Pharmacodynamic Assessments
`The PD effects by everolimus in tumor and skin were determined in all the
`patients included in the study. The timing of tumor and skin tissue biopsies was
`different in the two schedules: in the daily schedule at baseline and before dosing at
`day 2 in week 4; and in the weekly schedule at baseline, 24 hours and at day 6
`postdose administration in week 4. The aim of the third biopsy in the weekly
`schedule was to assess whether everolimus-related inhibition was sustained be-
`tween dosing. Processing of the samples, immunohistochemistry, and statistical
`analysis were performed as previously described (online-only Appendix A1).39,40
`Briefly, immunohistochemical analysis of total AKT, phosphorylated
`AKT at Ser473 (pAkt), total 4E-BP1, phosphorylated 4E-BP1 at Thr70 (p4E-
`BP1), phosphorylated eIF-4G at Ser1108 (peIF-4G), total S6, phosphorylated
`S6 at Ser235/236 and at Ser240/244 (pS6), and proliferation marker Ki-67 were
`performed in formalin-fixed paraffin-embedded sections from tumor and skin
`samples. Qualitative changes in the expression of markers were assessed in a
`blinded fashion. For quantitative analysis, the histochemical score (Hscore) was
`calculated to evaluate complete tumor sections and epidermis on skin samples at
`high magnification using a light microscopy, as previously described. Paired pre-
`therapy and on-therapy samples were analyzed using the Wilcoxon rank test by
`SPSS Data Analysis Program version 10.0 (SPSS Inc, Chicago, IL). Statistical tests
`wereconductedatthetwo-sided.05levelofsignificance.Pearsonlinearcorrelation
`was employed to examine the potential relationships between trough concentra-
`tion values of everolimus and PD effects in tumor and skin samples.
`
`RESULTS
`
`Characteristics of the 55 patients included in the weekly schedule
`(n ⫽ 31) and the daily schedule (n ⫽ 24) are listed in Table 1. The
`distribution of patients across dose levels is present in Table 2.
`
`Clinical Toxicities
`The numbers of patients reported with suspected toxicities were
`similar in the weekly and daily schedule (Table 2). Hematologic ab-
`normalities were uncommon with only 12 patients (22%) presenting
`grade 1 to 3 neutropenia (only one patient presented grade 3) and 17
`(31%) patients presenting grade 1 to 2 thrombocytopenia. Of these,
`only the grade 3 neutropenia was reported as drug related toxicity. The
`most frequent nonhematologic toxicities were skin rash/erythema
`(42%), stomatitis/oral mucositis (38%), headache (36%), and fatigue
`(29%). There were no grade 4 toxicities while grade 3 toxicities were
`reported in nine patients (16%), including stomatitis/oral mucositis
`(9%), hyperglycemia (4%), and fatigue (2%). DLT occurred in five
`patients: one patient at 10 mg daily (grade 3 stomatitis) and four
`patients at 70 mg weekly (two with grade 3 stomatitis, one with grade
`3 neutropenia, and one with grade 3 hyperglycemia). Hence, at the
`dose of 70 mg weekly four of seven patients presented DLTs and this
`dose was considered too toxic for further study. The cumulative tol-
`erance of everolimus was acceptable with only four additional patients
`presenting grade 3 toxicities after the first 28-day period: stomatitis/
`oral mucositis,2 hyperglycemia,1 and fatigue.1
`
`1604
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`© 2008 by American Society of Clinical Oncology
`
`JOURNAL OF CLINICAL ONCOLOGY
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`Copyright © 2016 American Society of Clinical Oncology. All rights reserved.
`
`NOVARTIS EXHIBIT 2052
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`Page 2 of 8
`
`

`

`Phase I Tumor PD Study of Weekly and Daily Everolimus
`
`Table 1. Patient Characteristics
`
`Characteristic
`
`Total No. of patients
`Sex
`Male
`Female
`Median age, years
`Range
`WHO PS
`0
`1
`2
`Primary diagnosis
`Breast
`Colorectal
`Pancreas
`NET
`Renal
`NSCLC
`Gallbladder
`Melanoma
`Other
`
`59
`27-85
`
`No.
`
`55
`
`21
`34
`
`34
`20
`1
`
`19
`16
`4
`2
`2
`2
`2
`2
`6
`
`%
`
`38
`62
`
`62
`36
`2
`
`35
`29
`7
`4
`4
`4
`4
`4
`11
`
`Abbreviations: PS, performance status; NET, neuroendocrine tumors; NSCLC,
`non–small-cell lung cancer.
`
`Antitumor Activity
`There was one partial response in a patient with a heavily pre-
`treated metastatic colorectal cancer treated at 20 mg weekly that lasted
`
`5.3 months (disease control during 9 months). Three patients presented
`stabilization of their disease for more than 5 months: one patient with
`an advanced renal cell cancer treated at 50 mg weekly lasting 14.6⫹
`months and two patients with advanced breast cancer treated at 70
`and 20 mg weekly lasting 10.7 and 5.6⫹ months, respectively.
`
`PK
`
`Table 3 depicts the trough concentrations of everolimus accord-
`ing to the different schedules and doses. Unweighted linear regression
`analysis of the relationship between dose and pharmacologic exposure
`was performed. Everolimus exhibited a linear dose-trough concentra-
`tion relationship in the daily schedule (regression slope 0.96; 90% CI,
`0.25 to 1.66), whereas this relationship could not be found in the
`weekly schedule due to insufficient serum sampling.
`
`Tumor and Skin mTOR Pathway Signaling PD Studies
`Inhibition of mTOR signaling was observed at all dose levels and
`schedules. Figure 1 shows the box-plots of the collated PD effect in
`tumor and skin in all the patients with paired pre- and on-therapy
`biopsies (30 patients with tumor and 43 with skin paired samples). As
`a whole, treatment with everolimus resulted in an almost complete
`inhibition of pS6 (P ⬍ .001) and peIF-4G (P ⬍ .001). p4E-BP1
`(Thr70) was profoundly reduced in skin (P ⬍ .001), this reduction
`being of less magnitude in the tumor (P ⫽ .058). There was also an
`overall increase in AKT phosphorylation (Ser473) both in tumors
`(P ⫽ .006) and skin (P ⬍ .001). The observed effects on protein
`phosphorylation were not due to changes in protein expression, as
`total AKT, 4E-BP1, and S6 protein levels were unmodified as a result
`
`Table 2. Frequency Distribution of Drug-Related AEs in the Daily and in the Weekly Schedule (reported in three or more patients)
`
`Schedule
`
`Parameter
`
`Daily
`
`Dose, mg
`No. of patients
`Treated
`With drug-related AEs
`AE
`Skin rash and erythema
`Stomatitis/oral mucositis
`Headache
`Fatigue
`Anorexia
`Vomiting
`Hypercholesterolemia
`Nausea
`Anemia
`Upper abdominal pain/dyspepsia
`Diarrhea
`Dry mouth
`Abdominal distension
`Pruritus
`Dysguesia
`Hyperglycemia
`Constipation
`Nail disorders
`Epistaxis
`
`NOTE. All grades (grade 3 in parentheses).
`Abbreviation: AEs, adverse events.
`
`5
`
`12
`11
`
`7
`4 (1)
`6
`1
`2
`0
`5
`1
`1
`2
`0
`1
`1
`2
`3
`0
`1
`0
`0
`
`10
`
`12
`12
`
`7
`6 (1)
`4
`5 (1)
`4
`5
`1
`4
`1
`3
`2
`2
`1
`0
`1
`1
`2
`0
`0
`
`20
`
`12
`11
`
`1
`2
`4
`3
`1
`1
`1
`1
`2
`1
`0
`2
`0
`1
`1
`2 (1)
`0
`1
`1
`
`Weekly
`
`50
`
`12
`11
`
`5
`5 (1)
`3
`4
`4
`4
`2
`2
`0
`1
`3
`0
`2
`1
`0
`0
`1
`1
`1
`
`70
`
`7
`7
`
`3
`4 (2)
`3
`3
`1
`0
`0
`0
`3
`0
`1
`1
`1
`1
`0
`1 (1)
`0
`1
`1
`
`Total
`
`—
`
`55
`52
`
`23
`21 (5)
`20
`16 (1)
`12
`10
`9
`8
`7
`7
`6
`6
`5
`5
`5
`4 (2)
`4
`3
`3
`
`www.jco.org
`
`© 2008 by American Society of Clinical Oncology
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`NOVARTIS EXHIBIT 2052
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`
`

`

`Tabernero et al
`
`Table 3. Trough Concentrations According to the Different Schedules
`and Doses
`
`Trough Concentrations (ng/mL)
`
`Schedule and Dose (mg)
`
`No.
`
`Mean
`
`Dailyⴱ
`5
`10
`Weekly
`20
`50
`70
`
`12
`11
`
`10
`12
`7
`
`8.5
`17.0
`
`0.7
`1.0
`4.2
`
`SD
`
`5.5
`12.4
`
`0.5
`1.5
`4.4
`
`Abbreviation: SD, standard deviation.
`ⴱDose-trough concentration relationship: regression slope ⫽ 0.96 (90% CI,
`0.25 to 1.66).
`
`of everolimus administration (data not shown). Cellular proliferation
`was reduced both in tumor (P ⫽ .014) and skin (P ⫽ .008).
`In order to dissect the effects of the studied doses and schedules of
`everolimus, we analyzed mTOR-dependent PD changes in individual
`patients (Fig 2). In the daily schedule, inhibition of pS6 was near
`complete at both dose levels, while inhibition of peIF-4G was only
`partial at 5 mg, and complete at 10 mg. Reductions in p4E-BP1 were
`also more profound at 10 mg, albeit with a greater interpatient vari-
`ability. At both dose levels, the majority of patients presented a reduc-
`tion in the proliferation index whereas pAKT increased in around half
`of the patients. In the weekly schedule, inhibition of tumor mTOR–
`dependent signaling was evaluated 24 hours after drug administration
`(early effect) and at 24 hours before the next weekly administration, in
`order to assess whether any effects persisted until the next dose (sus-
`tained or trough effect). Inhibition of pS6, both early and sustained,
`
`A
`
`Tumor samples
`
`was almost complete at all doses. Early inhibition of peIF-4G was
`complete at all dose levels, but sustained inhibition was only observed
`at doses ⱖ 50 mg. As with the daily schedule, p4E-BP1 inhibition was
`not observed in all patients, but in those patients achieving inhibition
`it was sustained. Increased tumor pAKT was greater at doses ⱖ 50 mg
`than at 20 mg but it was not sustained. Proliferation was reduced in
`most of the patients 24 hours after the weekly dosing but, in most cases,
`it was not sustained. Because the number of patients that achieved
`clinical benefit is small, it is not possible to analyze the predictiveness
`of the PD markers. Representative biomarker expression changes in
`tumor and skin from two selected patients with advanced breast car-
`cinoma treated at 10 mg/d and 50 mg/wk are shown in Figure 3.
`We explored the potential relationships between plasma trough
`concentrations of everolimus and tumor and skin PD effects, only in
`patients on daily therapy as, for this schedule, a correlation is proven
`between trough concentrations and overall exposure.33,34,38 The al-
`most complete inhibition shown for pS6 in tumors from patients treated
`at the two daily dose levels did not permit any correlation analysis with
`respect to this biomarker. However, a trend could be observed in the
`relationship between everolimus trough values and tumor peIF-4G
`inhibition (r ⫽ ⫺0.49; P ⫽ .17). Trough concentrations also correlated
`significantly with inhibition of tumor p4E-BP1 (r ⫽ ⫺0.6; P ⫽ .049).
`Nevertheless, no trends in PD correlations between trough values and
`treatment-related upregulation of tumor pAkt were evident.
`
`DISCUSSION
`
`This phase I study was aimed at identifying a recommended dose
`and schedule of everolimus in patients with cancer defined by the
`achievement of a complete and sustained inhibition of mTOR
`
`pS6Ser235/6
`
`pS6Ser240/4
`
`peIF4GSer1108
`
`p4EBP1Thr70
`
`pAktSer473
`
`Ki67
`
`350
`300
`250
`200
`150
`100
`50
`0
`-50
`
`350
`300
`250
`200
`150
`100
`50
`0
`-50
`
`350
`300
`250
`200
`150
`100
`50
`0
`-50
`
`350
`300
`250
`200
`150
`100
`50
`0
`-50
`
`350
`300
`250
`200
`150
`100
`50
`0
`-50
`
`350
`300
`250
`200
`150
`100
`50
`0
`-50
`
`Day 0 Day 22
`
`Day 0 Day 22
`
`Day 0 Day 22
`
`Day 0 Day 22
`
`Day 0 Day 22
`
`Day 0 Day 22
`
`B
`
`Skin samples
`
`pS6Ser235/6
`
`pS6Ser240/4
`
`peIF4GSer1108
`
`p4EBP1Thr70
`
`pAktSer473
`
`Ki67
`
`350
`300
`250
`200
`150
`100
`50
`0
`-50
`
`350
`300
`250
`200
`150
`100
`50
`0
`-50
`
`350
`300
`250
`200
`150
`100
`50
`0
`-50
`
`350
`300
`250
`200
`150
`100
`50
`0
`-50
`
`350
`300
`250
`200
`150
`100
`50
`0
`-50
`
`350
`300
`250
`200
`150
`100
`50
`0
`-50
`
`Day 0 Day 22
`
`Day 0 Day 22
`
`Day 0 Day 22
`
`Day 0 Day 22
`
`Day 0 Day 22
`
`Day 0 Day 22
`
`Fig 1. Pharmacodynamic effects after everolimus treatment in all the patients included in the study with (A) assessable paired tumor (n ⫽ 30) and (B) skin samples
`(n ⫽ 43). Box-plots showing the expression at baseline and on-treatment for the following markers: pS6Ser235/6, pS6Ser240/4, peIF4GSer1108, p4E-BP1Thr70, pAktSer473
`and Ki-67. Boxes indicate 90% of the values. Bold lines indicate the mean of the values. External lines indicate the complete range when beyond 90% of the values.
`Day 0: baseline sample; day 22: sample obtained at fourth week 24 hours after dosing.
`
`1606
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`© 2008 by American Society of Clinical Oncology
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`JOURNAL OF CLINICAL ONCOLOGY
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`Downloaded from ascopubs.org by Reprints Desk on November 28, 2016 from 216.185.156.028
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`Copyright © 2016 American Society of Clinical Oncology. All rights reserved.
`
`NOVARTIS EXHIBIT 2052
`Breckenridge v. Novartis, IPR 2017-01592
`Page 4 of 8
`
`

`

`Phase I Tumor PD Study of Weekly and Daily Everolimus
`
`A
`
`300
`250
`200
`150
`100
`50
`0
`-50
`
`B
`
`300
`250
`200
`150
`100
`50
`0
`-50
`
`C
`
`300
`250
`200
`150
`100
`50
`0
`-50
`
`Daily - 5 mg
`pS6Ser235/6
`
`pS6Ser240/4
`
`peIF4GSer1108
`
`p4EBP1Thr70
`
`pAktSer473
`
`Ki67
`
`300
`250
`200
`150
`100
`50
`0
`-50
`
`300
`250
`200
`150
`100
`50
`0
`-50
`
`300
`250
`200
`150
`100
`50
`0
`-50
`
`300
`250
`200
`150
`100
`50
`0
`-50
`
`300
`250
`200
`150
`100
`50
`0
`-50
`
`Day 0 Day 22
`
`Day 0 Day 22
`
`Day 0 Day 22
`
`Day 0 Day 22
`
`Day 0 Day 22
`
`Day 0 Day 22
`
`Daily - 10 mg
`pS6Ser235/6
`
`pS6Ser240/4
`
`peIF4GSer1108
`
`p4EBP1Thr70
`
`pAktSer473
`
`Ki67
`
`300
`250
`200
`150
`100
`50
`0
`-50
`
`300
`250
`200
`150
`100
`50
`0
`-50
`
`300
`250
`200
`150
`100
`50
`0
`-50
`
`300
`250
`200
`150
`100
`50
`0
`-50
`
`300
`250
`200
`150
`100
`50
`0
`-50
`
`Day 0 Day 22
`
`Day 0 Day 22
`
`Day 0 Day 22
`
`Day 0 Day 22
`
`Day 0 Day 22
`
`Day 0 Day 22
`
`Weekly - 20 mg
`pS6Ser235/6
`
`300
`250
`200
`150
`100
`50
`0
`-50
`
`Day
`27
`
`pS6Ser240/4
`
`peIF4GSer1108
`
`p4EBP1Thr70
`
`pAktSer473
`
`Ki67
`
`300
`250
`200
`150
`100
`50
`0
`-50
`
`Day
`0
`
`Day
`22
`
`Day
`27
`
`300
`250
`200
`150
`100
`50
`0
`-50
`
`Day
`0
`
`Day
`22
`
`Day
`27
`
`300
`250
`200
`150
`100
`50
`0
`-50
`
`Day
`0
`
`Day
`22
`
`Day
`27
`
`300
`250
`200
`150
`100
`50
`0
`-50
`
`Day
`0
`
`Day
`22
`
`Day
`27
`
`Day
`0
`
`Day
`22
`
`Day
`27
`
`~ ~ ~ ~ l! ~
`~ l1_ ~ ~ ~ ~
`~ lh lk ~ ~ ~
`~ lh ll lg ~ lk
`b_~l_~t£b
`
`300
`250
`200
`150
`100
`50
`0
`-50
`
`300
`250
`200
`150
`100
`50
`0
`-50
`
`300
`250
`200
`150
`100
`50
`0
`-50
`
`300
`250
`200
`150
`100
`50
`0
`-50
`
`Day
`0
`
`Day
`22
`
`Day
`27
`
`Day
`0
`
`Day
`22
`
`Day
`27
`
`Day
`0
`
`Day
`22
`
`Day
`27
`
`Day
`0
`
`Day
`22
`
`Day
`27
`
`300
`250
`200
`150
`100
`50
`0
`-50
`
`300
`250
`200
`150
`100
`50
`0
`-50
`
`Day
`0
`
`Day
`22
`
`Day
`27
`
`Ki67
`
`Day
`0
`
`Day
`22
`
`Day
`27
`
`pS6Ser240/4
`
`peIF4GSer1108
`
`p4EBP1Thr70
`
`pAktSer473
`
`Ki67
`
`D
`
`300
`250
`200
`150
`100
`50
`0
`-50
`
`E
`
`300
`250
`200
`150
`100
`50
`0
`-50
`
`Day
`0
`
`Day
`22
`
`Weekly - 50 mg
`pS6Ser235/6
`
`Day
`Day
`Day
`0
`22
`27
`Weekly - 70 mg
`pS6Ser235/6
`
`Day
`0
`
`Day
`22
`
`Day
`27
`
`300
`250
`200
`150
`100
`50
`0
`-50
`
`300
`250
`200
`150
`100
`50
`0
`-50
`
`300
`250
`200
`150
`100
`50
`0
`-50
`
`300
`250
`200
`150
`100
`50
`0
`-50
`
`Day
`0
`
`Day
`22
`
`Day
`27
`
`Day
`0
`
`Day
`22
`
`Day
`27
`
`Day
`0
`
`Day
`22
`
`Day
`27
`
`Day
`0
`
`Day
`22
`
`Day
`27
`
`pS6Ser240/4
`
`peIF4GSer1108
`
`p4EBP1Thr70
`
`pAktSer473
`
`Fig 2. (A-E) Tumor pharmacodynamic effects in patients treated in the daily (treated at 5 and 10 mg) and in the weekly schedules (treated at 20, 50, and 70 mg). The
`lines show the individual evolution of the expression of the biomarkers (pS6Ser235/6, pS6Ser240/4, peIF4GSer1108, p4E-BP1Thr70, pAktSer473, and Ki-67) at baseline and
`on-treatment. In the daily schedule samples were obtained on day 0 (baseline) and on day 22 (on-treatment). In the weekly schedules samples were obtained on day
`0 (baseline), on day 22 (on-treatment, 24 hours after dosing) and on day 27 (on-treatment, 144 hours after dosing). The aim of this third biopsy was to assess the
`persistent effect of everolimus administered on a weekly basis.
`
`dependent–signaling pathways on tumor and skin. Our approach
`was based on the existing correlation in preclinical models between
`antitumor activity and mTOR-signaling inhibition. In a CA20948
`syngenic rat pancreatic tumor model, doses of everolimus that
`
`inhibited tumor growth also dramatically inhibited mTOR signal-
`ing in tumor, skin, and peripheral blood mononuclear cells (PB-
`MCs).29 In this model, a decrease in p4E-BP1 (Thr70) and an
`increase in eIF-4E and 4E-BP1 association were consistently
`
`www.jco.org
`
`© 2008 by American Society of Clinical Oncology
`
`1607
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`Copyright © 2016 American Society of Clinical Oncology. All rights reserved.
`
`NOVARTIS EXHIBIT 2052
`Breckenridge v. Novartis, IPR 2017-01592
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`

`

`Fig 3. Biomarker expression changes in
`(pS6Ser235/6, peIF4GSer1108,
`the tumor
`p4E-BP1Thr70, and pAktSer473) and in the
`skin (pS6Ser235/6, peIF4GSer1108, and p4E-
`BP1Thr70) between baseline (day 0) and
`on-treatment (day 22) in two selected pa-
`tients with advanced breast cancer treated
`at 10 mg daily and 50 mg weekly.
`
`Tabernero et al
`
`Tumor
`
`Skin
`
`10 mg Daily
`pS6Ser235/6
`
`peIF4GSer1108
`
`p4EBP1Thr70
`
`pAktSer473
`
`pS6Ser235/6
`
`peIF4GSer1108
`
`p4EBP1Thr70
`
`On-therapyPre-therapy
`
`50 mg Weekly
`pS6Ser235/6
`
`peIF4GSer1108
`
`p4EBP1Thr70
`
`pAktSer473
`
`pS6Ser235/6
`
`peIF4GSer1108
`
`p4EBP1Thr70
`
`On-therapyPre-therapy
`
`observed in all three tissues. Striking reductions in pS6 (Ser240/
`244) were demonstrated only in tumor, as baseline pS6 levels in
`skin and PBMCs were too low for immunoblot detection. By
`contrast, in vitro kinase assay, using 40S ribosomal subunits as a
`substrate, revealed a significant and consistent inhibition of S6K1
`in tumor, skin, and PBMCs.29 The relationship between S6K1
`inhibition in tumor and PBMCs has also been demonstrated in
`vivo with temsirolimus.41 Taken together, these data demonstrate
`that both 4E-BP1 and S6K1 pathways are affected in tumor, skin,
`and PBMC samples obtained from preclinical models after treat-
`ment with mTOR inhibitors. We, therefore, included PD evalua-
`tion of molecular markers exploring the 4E-BP1 pathway (total
`and p4E-BP1, peIF-4G) and the S6K1 pathway (total and pS6) in
`the tumor and in the skin. In addition, it has been shown that
`mTOR inhibition can induce upstream insulin-like growth factor 1
`receptor (IGF-1R) signaling resulting in AKT activation in cancer
`cells.39,42 This phenomenon has been suggested to play a role in the
`attenuation of cellular responses to mTOR inhibition.2,43 In order
`to study whether AKT activation was also observed in vivo, we
`incorporated an assessment of total and pAKT as well as the effects
`of treatment on proliferation.
`There are several key findings in our study. We observed a
`tight correlation in the degree of everolimus-induced inhibition of
`mTOR signaling in the skin and in the tumors. Hence, skin may be
`useful as a surrogate tissue for PD evaluation with mTOR inhibi-
`tors. This has implications for future studies with novel agents
`interfering with the mTOR pathway. We identified a daily and a
`weekly dose level that resulted in maximal inhibition of the path-
`way. In the daily schedule, pS6 was completely inhibited at the 5-
`and 10-mg doses. However, inhibition of peIF-4G was only com-
`plete at the 10-mg dose level. Although p4E-BP1 expression was
`also lower at the higher dose level, it is felt to be a marker less
`reproducible of mTOR inhibition (W. Sellers, personal communi-
`cation, January 2006).42 In the weekly schedule, complete pS6
`
`inhibition was again seen at all the studied dose levels. However,
`complete and prolonged inhibition of peIF-4G was observed only
`at doses ⱖ 50 mg. Because peIF-4G, together with pS6, have been
`suggested as the best indicators of mTOR blockade,35,36 we consid-
`ered optimal dose levels of everolimus those that inhibited both
`markers in full. The upregulation of tumor AKT phosphorylation
`that was observed with both daily and weekly everolimus schedules
`raises the question on whether it may attenuate the clinical activity
`of this agent. It should be noted that the increase in pAKT is not
`tumor specific, as we observed it also in skin. In addition, it did not
`occur in the tumors of all patients (approximately 50% in this
`study) and it was not always sustained in between doses in patients
`treated on the weekly schedule. Upregulation of AKT signaling occurs
`in experimental tumor models known to be very sensitive to mTOR
`inhibition, including the CA20948 model described earlier.29 Never-
`theless, it is likely that strategies aimed at preventing the activation of
`this IGF-1R mediated feed loop could result in an enhancement of the
`antitumor activity of mTOR inhibitors. We have observed that inhi-
`bition of the IGF-1R pathway with an anti-IGF-1R antibody given in
`combination with mTOR inhibitors is highly synergistic39,42 and we
`are planning to explore this combination in the clinic.
`Oral everolimus is characterized by a rapid and a moderate oral
`absorption (oral availability of approximately 30%), and the terminal
`half-life is around 30 hours.32-34,38,44,45 We have evaluated potential
`relationships between trough levels and treatment-related changes in
`the activated signaling markers in both tumor and skin samples in
`patients treated in the daily schedule. There was a trend for more
`profound decreases in tumor peIF-4G and p4E-BP1 levels in those
`patients with higher plasma exposure to everolimus. These data, to-
`gether with the more profound dose effect in tumor discussed earlier,
`suggest that monitoring the 4E-BP1/eIF-4G downstream module of
`mTOR signaling, rather than the S6K1 pathway, may be more valuable
`for dose evaluation in the clinic.
`
`1608
`
`© 2008 by American Society of Clinical Oncology
`
`JOURNAL OF CLINICAL ONCOLOGY
`
`Downloaded from ascopubs.org by Reprints Desk on November 28, 2016 from 216.185.156.028
`
`Copyright © 2016 American Society of Clinical Oncology. All rights reserved.
`
`NOVARTIS EXHIBIT 2052
`Breckenridge v. Novartis, IPR 2017-01592
`Page 6 of 8
`
`

`

`Phase I Tumor PD Study of Weekly and Daily Everolimus
`
`Oral everolimus was satisfactorily safe and well tolerated, with
`the exception of the weekly dose of 70 mg which had high fre-
`quency of grade 3 toxicities. Differences in reported toxicity be-
`tween the daily and the weekly schedules were marginal and of little
`clinical relevance. The observed toxicity profile revealed the occur-
`rence of well-known drug class effects of rapamycin and its deriv-
`atives, including stomatitis/oral mucositis, skin rash/erythema,
`and metabolic abnormalities.46,47
`In summary, this phase I study with oral everolimus has shown
`that this agent can be safely administered with the two different sched-
`ules. Based on PK/PD modeling efforts associated with another
`everolimus phase I trial in patients with advanced cancer,48 where
`PBMC-derived S6K1 activity was used to establish a concentration-
`effect model, as well as the clinical safety profile and the tumor PD
`analysis presented herein, we recommend everolimus treatment at
`either 10 mg/d or 50 mg/wk. Moreover, also consistent with previous
`PK/PD modeling,48 our tumor PD analysis shows a different pattern
`for the two schedules, with mTOR-pathway inhibition being more
`profound (and maintained) with the daily schedule. Whether these
`PD properties are critical enough to suggest a preference for the daily
`rather than the weekly schedule cannot be answered in the context of
`this phase I study. One point to consider is that flexibility of dosing
`could be extremely valuable in the context of drug combination sce-
`narios which are being aggressively followed for mTOR inhibi-
`tors.7,49,50 To our knowledge, this study is the first comprehensive
`study with an mTOR inhibitor, using a detailed tumor and skin PD
`analysis to provide evidence to support dosage and regimen in subse-
`quent phase II-III studies. This study provides assurance of primary
`drug activity at dosages not necessarily limited by toxicity, providing
`direction for the further development of mTOR inhibitors like
`everol