Cancer Therapy: Clinical
`
`A Phase I and Pharmacokinetic Study of Temsirolimus (CCI-779)
`Administered Intravenously Daily for 5 Days Every 2 Weeks
`to Patients with Advanced Cancer
`Manuel Hidalgo,1,2 Jan C. Buckner,3 Charles Erlichman,3 Marilyn S. Pollack,2 Joseph P. Boni,4
`Gary Dukart,4 Bonnie Marshall,4 Lisa Speicher,4 Laurence Moore,5 and Eric K. Rowinsky1,2
`
`Abstract Purpose: Patients with advanced cancer received temsirolimus (Torisel, CCI-779), a novel inhib-
`itor of mammalian target of rapamycin, i.v. once daily for 5 days every 2 weeks to determine the
`maximum tolerated dose, toxicity profile, pharmacokinetics, and preliminary antitumor efficacy.
`Experimental Design: Doses were escalated in successive cohorts of patients using a conven-
`tional phase I clinical trial design. Samples of whole blood and plasma were collected to determine
`the pharmacokinetics of temsirolimus and sirolimus, its principal metabolite.
`Results: Sixty-three patients were treated with temsirolimus (0.75-24 mg/m2/d). The most
`common drug-related toxicities were asthenia, mucositis, nausea, and cutaneous toxicity. The
`maximum tolerated dose was 15 mg/m2/d for patients with extensive prior treatment because,
`in the 19 mg/m2/d cohort, two patients had dose-limiting toxicities (one with grade 3 vomiting,
`diarrhea, and asthenia and one with elevated transaminases) and three patients required dose
`reductions. For minimally pretreated patients, in the 24 mg/m2/d cohort, one patient developed
`a dose-limiting toxicity of grade 3 stomatitis and two patients required dose reductions, establish-
`ing 19 mg/m2/d as the maximum acceptable dose. Immunologic studies did not show any
`consistent trend toward immunosuppression.Temsirolimus exposure increased with dose in a less
`than proportional manner. Terminal half-life was 13 to 25 hours. Sirolimus-to-temsirolimus expo-
`sure ratios were 0.6 to 1.8. A patient with non ^ small cell lung cancer achieved a confirmed partial
`response, which lasted for 12.7 months. Three patients had unconfirmed partial responses; two
`patients had stable disease for z24 weeks.
`Conclusion:Temsirolimus was generally well tolerated on this intermittent schedule. Encouraging
`preliminary antitumor activity was observed.
`
`The mammalian target of rapamycin (mTOR) is a serine/
`threonine kinase and a member of the phosphatidylinositol
`family of kinases, which is involved in the response of
`eukaryotic cells to proliferative and nutritional stimuli (1 – 4).
`mTOR is downstream of Akt
`in the phosphatidylinositol
`3-kinase (PI3K)/Akt signaling pathway and regulates the
`ribosomal protein S6 kinase (p70 S6 kinase) and eukaryotic
`translation initiation factor 4E-binding protein-1. Activation
`of these proteins increases the translation of mRNAs with a
`
`Authors’ Affiliations: 1Institute for Drug Development, Cancer Therapy and
`Research Center, Brook Army Medical Center and 2The University of Texas Health
`Science Center, San Antonio, Texas; 3Mayo Clinic, Rochester, Minnesota; 4Wyeth
`Research, Collegeville, Pennsylvania; and 5Wyeth Research, Cambridge,
`Massachusetts
`Received 1/18/06; revised 5/17/06; accepted 6/15/06.
`Grant support: Wyeth Research (Collegeville, PA).
`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.
`Requests for reprints: Manuel Hidalgo, The Sidney Kimmel Comprehensive
`Cancer Center at Johns Hopkins, 1650 Orleans Street, Room 1M89, Baltimore, MD
`21231-1000. Phone: 410-502-9746; Fax: 410-614-9006; E-mail: mhidalg1@
`jhmi.edu.
`F 2006 American Association for Cancer Research.
`doi:10.1158/1078-0432.CCR-06-0118
`
`5¶-terminal oligopyrimidine tract or 5¶-cap structure, which
`encode for proteins involved in G1-S cell cycle regulation (5, 6).
`The PI3K/Akt pathway is activated in cancer by growth factor
`and/or hormone receptor activation or by mutations in genes,
`such as PI3K or PTEN, or byAkt amplification (7 – 21).
`its
`The discovery of mTOR and the understanding of
`biological functions have been greatly facilitated by studies
`with sirolimus (rapamycin), a naturally occurring macrolide
`that inhibits mTOR (2, 22). Sirolimus binds to the intracellular
`immunophilin FKBP12 and this complex inhibits mTOR,
`which results in inhibition of p70 S6 kinase and 4E-binding
`protein-1 functions, followed by a decrease in cyclin D1 levels,
`increase in p27 levels, and cell cycle arrest (23). In certain
`preclinical models, sirolimus induces apoptosis (24). Siro-
`limus also has antiangiogenesis effects by decreasing hypoxia-
`inducible factor-1a – induced secretion of vascular endothelial
`growth factor (25). Recently, sirolimus has been shown to
`inhibit the transforming capabilities of PI3K mutants (26),
`which supports the notion that mTOR inhibitors may be useful
`for the treatment of tumors with these mutations.
`Temsirolimus (Torisel, CCI-779) is an ester of sirolimus
`(Fig. 1) selected for clinical development based on a favorable
`pharmacologic and toxicity profile. Temsirolimus inhibited the
`growth of a variety of tumor cells and was particularly effective
`in tumors with a defective PTEN gene (27 – 33). Temsirolimus
`
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`Research.
`
`NOVARTIS EXHIBIT 2056
`Par v. Novartis, IPR 2016-01479
`Page 1 of 9
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`

`Cancer Therapy: Clinical
`
`Fig. 1. Structure of temsirolimus and sirolimus, its principal metabolite.
`
`also was effective in reversing resistance to conventional
`chemotherapy and hormone therapy conferred by PTEN defects
`(30, 34).
`Because of the immunosuppressive effects of sirolimus, an
`expected metabolite of temsirolimus, temsirolimus was evalu-
`ated for inhibition of T lymphocyte function in euthymic mice
`(CCI-779 Investigator’s Brochure). Although i.v. temsirolimus
`inhibited T lymphocyte activity, its effects were reversible and
`T lymphocyte activity returned to normal within 24 hours
`after drug treatment was stopped. Multiple cycles of temsir-
`olimus treatment did not result in cumulative deterioration of
`T lymphocyte function. Further studies in mice indicated that
`antitumor activity could be achieved with different intermittent
`dosing regimens, including a daily 5-day regimen given every
`2 weeks. Accordingly, this intermittent schedule was used in a
`phase I study to minimize the immunosuppressive effects of
`temsirolimus while maintaining antitumor activity.
`Based on the data summarized above, temsirolimus was
`selected for clinical development. Three phase I single-agent
`studies have been conducted with this drug based on different
`administration regimens, including i.v. weekly (35), i.v. once
`daily for 5 days every 2 weeks (this study), and oral once daily
`for 5 days every 2 weeks (36). In this study, patients with
`advanced cancer were treated with temsirolimus to evaluate
`safety, determine the maximum tolerated dose (MTD), charac-
`terize pharmacokinetics, and seek preliminary evidence of
`antitumor activity.
`
`Materials and Methods
`
`In this phase I, dose escalation study, temsirolimus
`Trial design.
`was administered as a 30-minute i.v. infusion once daily on days 1 to
`5 of each treatment cycle of f2 weeks. Patients were observed at least
`9 days after their day 5 dose of temsirolimus before receiving the next
`cycle of drug. Patients could remain on study as long as temsirolimus
`was well tolerated and there was no evidence of disease progression.
`The primary objectives of the study were to determine the safety and
`tolerability and to identify the MTD of temsirolimus given i.v. once
`daily for 5 days every 2 weeks in patients with advanced solid tumors.
`The secondary objectives were to determine the pharmacokinetics of
`temsirolimus on this schedule and to obtain preliminary information
`on antitumor activity.
`Patient selection. Patients with histologically confirmed advanced
`cancer (solid tumors or lymphomas) who failed to respond to standard
`therapy or for whom standard therapy was not available were eligible
`for this study. Eligibility criteria also included age z18 years; an Eastern
`Cooperative Oncology Group performance status V2 (ambulatory and
`capable of self-care); life expectancy z12 weeks; no prior chemother-
`apy, radiation therapy, or immunosuppressive therapy (except cortico-
`
`steroids for management of emesis or peritumoral edema) within
`3 weeks of starting study treatment; no treatment with investigational
`agents within 30 days before commencing study treatment; adequate
`hematopoietic (hemoglobin level z9 g/dL, absolute neutrophil
`count z1,500/AL, platelet count z100,000/AL), hepatic [bilirubin
`<1.5 mg/dL, aspartate and alanine aminotransaminases <3 times
`institutional normal upper limit (<5 times institutional normal
`upper limit for patients with liver metastases)], and renal (creatinine
`<2 mg/dL) functions; measurable or evaluable disease; and no active
`infections or history of hypersensitivity to macrolide antibiotics,
`unstable angina, or myocardial infarction within 6 months or coexist-
`ing medical problems of sufficient severity to limit compliance in the
`study. Due to the known toxicities of sirolimus, patients who entered
`the trial were also required to have serum levels of cholesterol
`and triglycerides V350 and V300 mg/dL, respectively. Patients with
`clinically and radiologically stable brain tumors were eligible. Patients
`receiving hepatic enzyme-inducing anticonvulsants or antiarrhythmic
`agents were ineligible. Before treatment, patients were required to give
`written informed consent according to federal and institutional guide-
`lines.
`Because patients who have received extensive anticancer therapy tend
`to have greater drug-related toxicity than those who have received less
`extensive therapy, patients treated with higher dose levels of temsir-
`olimus were classified as being minimally pretreated or heavily
`pretreated. Heavily pretreated patients were defined as having received
`radiotherapy to z25% of bone marrow – producing areas, more than six
`cycles of an alkylating agent (except low-dose cisplatin), more than four
`courses of a carboplatin-containing regimen, or more than two courses
`of carmustine or mitomycin C (37).
`Drug dosage and administration. The starting dose of temsirolimus
`was 0.75 mg/m2 based on animal toxicology studies and prior clinical
`experience with sirolimus. A modified version of the Continual Reas-
`sessment Method (38, 39) was to be used to guide dose escalation.
`
`Table 1. Patient characteristics
`
`Characteristics
`
`n
`
`63
`60
`39/24
`
`Patients
`Fully assessable patients
`Sex (men/women)
`Age (y)
`56
`Median
`19-79
`Range
`Eastern Cooperative Oncology Group performance status, patients
`0
`20
`1
`30
`2
`13
`Prior therapy, patients
`Chemotherapy alone
`Radiotherapy alone
`Chemotherapy and radiotherapy
`Tumor type, patients
`Renal
`Colorectal
`Non – small cell lung cancer
`Soft-tissue sarcoma
`Endometrial
`Ovarian
`Sarcoma
`Other*
`
`58
`2
`28
`
`16
`10
`9
`7
`3
`2
`2
`14
`
`*One each of anaplastic astrocytoma, cervical, esophageal,
`gastric, head and neck-adenoid cystic carcinoma, hepatocellular,
`non-Hodgkin’s lymphoma, nasopharyngeal, osteosarcoma, pan-
`creatic, prostate, squamous cell carcinoma of the skin, thyroid,
`and unknown.
`
`Clin Cancer Res 2006;12(19) October 1, 2006
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`

`Phase I Study of Temsirolimus
`
`Table 2. Dose escalation and toxicity experience
`
`Temsirolimus
`dose (mg/m2/d  5)
`
`No. patients
`entered (inevaluable*)
`
`DLT (cycle 1)
`
`No. patients
`
`Toxicity and grade
`
`No. patients
`reduced to dosec
`
`Total at dose
`
`No. evaluable
`patients
`
`No.
`cycles
`
`0.75
`1.25
`1.5
`1.8
`2.16
`2.6
`3.12
`3.74
`4.5
`5.4
`6.5
`7.8
`9.4
`11.3
`Minimally pretreated
`15
`19
`24
`Heavily pretreated
`15
`19
`
`3
`4 (1)
`1
`1
`6
`1
`2
`2
`4
`2
`2
`3
`1
`4 (1)
`
`3
`6
`6
`
`6 (1)
`6
`
`Total
`
`63 (3)
`
`0
`0
`0
`0
`1
`0
`0
`0
`0
`0
`0
`0
`0
`0
`
`1
`0
`1
`
`0
`2
`
`4
`
`Grade 3 hypocalcemia
`
`Grade 3 hyperglycemia
`
`Grade 3 stomatitis
`
`Grade 3 aspartate and alanine
`aminotransaminase elevations
`Grade 3 vomiting, diarrhea,
`and asthenia
`
`0
`0
`0
`1
`0
`0
`0
`0
`0
`0
`0
`0
`0
`1
`
`2
`3
`0
`
`5b
`0
`
`3
`3
`1
`2
`6
`1
`2
`2
`4
`2
`2
`3
`1
`4
`
`5
`9
`6
`
`10
`6
`
`10
`7
`2
`15
`32
`4
`12
`24
`50
`5
`10
`29
`3
`17
`
`33
`45
`24
`
`31
`7
`
`361
`
`*Reasons inevaluable for determining dose escalation: two disease progression (1.25 and 15 mg/m2/d) during cycle 1 and one hypersensitivity
`reaction (11.3 mg/m2/d) during the first 24 hours after the first temsirolimus dose.
`cIncludes all patients reduced from the next higher dose level at any subsequent cycle.
`bOne patient required a second dose reduction to 11.3 mg/m2/d.
`
`However, because of adverse events observed at the first two dose
`levels and after discussions with the U.S. Food and Drug Administration,
`the protocol was amended and a fixed 20% dose escalation was used.
`A later amendment allowed fixed dose escalation increments of up
`to 30%.
`The National Cancer Institute Common Toxicity Criteria version 2.0
`was used to grade toxicity. Unacceptable toxicities included temsir-
`
`olimus-related (a) grade 3/4 nonhematologic toxicity (excluding
`nausea or vomiting in patients on suboptimal antiemetic prophylaxis
`or serum triglycerides <1,500 mg/dL if recovery occurred by the next
`cycle), (b) grade 4 thrombocytopenia, or (c) grade 4 neutropenia
`lasting >5 days. If a patient had an unacceptable toxicity, dose reduction
`by one to two levels and/or a delay in treatment could occur. If a grade
`3 toxicity was observed in a patient at a given dose level, the cohort was
`
`Fig. 2. Frequently occurring toxicities of
`temsirolimus included, for all 63 patients,
`asthenia (35 patients, 56%), mucositis
`(34 patients, 54%), nausea (26 patients,
`41%), cutaneous toxicity (26 patients, 41%),
`hypertriglyceridemia (23 patients, 37%),
`thrombocytopenia (21patients, 33%),
`hypercholesterolemia (14 patients, 22%),
`elevated transaminases (12 patients, 19%),
`and hyperglycemia (11patients, 17%).
`Temsirolimus doses: 0.75 to 11.3, 15, 19, and
`24 mg/m2/d. MP, minimally pretreated; HP,
`heavily pretreated.
`
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`Research.
`
`on August 24, 2015. © 2006 American Association for Cancerclincancerres.aacrjournals.org
`
`NOVARTIS EXHIBIT 2056
`Par v. Novartis, IPR 2016-01479
`Page 3 of 9
`
`

`

`Cancer Therapy: Clinical
`
`Table 3. Pharmacokinetic variables of temsirolimus on day 5, mean F SD (no. patients)
`
`Dose group (mg/m2)
`
`0.75
`1.25
`2.16
`4.5
`15
`19
`
`C max (ng/mL)
`72 F 16 (3)
`133 F 64 (3)
`186 F 51 (5)
`331 F 72 (4)
`503 F 293 (5)
`796 F 226 (12)
`
`t max (h)
`0.67 F 0.29 (3)
`0.46 F 0.28 (30)
`0.59 F 0.24 (5)
`0.46 F 0.11 (4)
`0.26 F 0.18 (5)
`0.41 F 0.13 (12)
`
`t 1/2 (h)
`24.8 F 7.5 (2)
`12.6 F 5.1 (3)
`16.4 F 6.9 (5)
`13.9 F 2.6 (4)
`20.0 F 22.0 (5)
`15.4 F 15.6 (12)
`
`AUC (cycle 1; ng h/mL)
`
`1,355 F 732 (2)
`2,502 F 1,531 (3)
`3,896 F 986 (5)
`5,350 F 792 (4)
`8,619 F 2,188 (5)
`9,838 F 3,504 (12)
`
`Abbreviations: Cmax, peak observed concentration; t max, time to C max; t1/2, terminal half-life; AUC, area under the concentration versus time
`curve; CLc, central clearance; Vdss, steady-state volume of distribution; AR, accumulation ratio of day 5 to day 1; B/Pratio, blood-to-plasma
`concentration ratio.
`
`expanded to three patients. If an unacceptable toxicity was observed
`in a patient at a given dose level in cycle 1, a dose-limiting toxicity
`(DLT) occurred and that cohort was expanded to six patients. The MTD
`was defined as the highest dose for which two or fewer patients had a
`DLT. However, the combination of DLTs and dose reductions that
`occurred at a given dose level were taken into account in identifying
`the MTD.
`Temsirolimus (25 mg/mL in 100% ethanol, Wyeth Research,
`Collegeville, PA) is a light-sensitive drug and was protected from
`sunlight and unshielded fluorescent
`light during preparation and
`administration. The drug-ethanolic concentrate was diluted 10-fold in a
`polyethylene glycol/polysorbate diluent and then further diluted with
`0.9% saline solution to a total volume of 50 to 100 mL, which was
`administered for f30 minutes using glass or polyolefin infusion kits
`and an automatic dispensing pump.
`Evaluation of patients. Physical examination and routine laboratory
`evaluations were done before treatment and weekly.
`For assessment of
`immunologic activity, whole blood samples
`were collected before treatment, on days 1 and 5 of cycles 1 to 3, and
`on day 8 of cycle 1. Three assays were done. (a) WBC counts and
`differentials were monitored to check for changes in lymphocyte
`numbers. (b) Proliferative responses (uptake of tritiated thymidine) of
`patient’s lymphocytes to pokeweed mitogen, phytohemagglutinin, and
`concanavalin A and to pooled allogeneic cells were monitored as
`standard indicators of altered lymphocyte function (40). (c) Lympho-
`cyte subsets (cell surface phenotypes CD4/CD3, CD8/CD3, CD14, and
`CD45 and the CD4/CD3:CD8/CD3 ratio) were monitored using
`standard methods (41). Measured variables were graphically depicted
`and visually analyzed.
`Radiologic studies for disease assessment were repeated after
`alternate cycles or as needed. A complete response was reported if
`there was disappearance of all active disease. A partial response was
`reported if there was at least a 50% reduction in total tumor size (the
`sum of
`the product of
`the bidimensional measurements of all
`lesions). A confirmed response was reported if two measurements
`separated by a minimum of 4 weeks indicated a response and an
`unconfirmed response was reported if a response occurred but did not
`meet the criteria required for a confirmed response. Stable disease was
`scored if there was <50% reduction in total tumor size or <25%
`increase in the size of one or more measurable lesions. An increase in
`the size of one or more measurable lesions by at least 25% or the
`appearance of any new lesion was considered disease progression
`(42). Clinical benefit included the number of patients with confirmed
`and unconfirmed complete and partial responses and the number of
`patients with stable disease for at least 24 weeks. Time to tumor
`progression was measured from day 1 of temsirolimus treatment until
`documented disease progression.
`Pharmacokinetic analyses. Whole blood samples for the determina-
`tion of temsirolimus and sirolimus concentrations were collected in
`sodium EDTA tubes (3 mL each) in cycles 1 and 3: on days 1 and 5 at
`0 (before treatment), 0.25, 0.50, 1, 2, 4, and 6 hours; on days 2 to 4 at
`
`0 hours; and on days 8, 10, and 12. The samples were frozen at 70jC
`until assayed. To determine the blood to plasma partitioning of
`temsirolimus, 6 mL blood samples were collected in cycle 1 on days
`1 and 5 at 0.5 hour after drug administration and in cycle 2 on day 1
`before drug administration. These samples were centrifuged immedi-
`ately and the plasma was stored at 70jC until assayed.
`Temsirolimus and sirolimus concentrations in whole blood were
`measured using a liquid chromatography-tandem mass spectrometry
`procedure (Taylor Technology,
`Inc., Princeton, NJ) as described
`(35). Both temsirolimus and sirolimus concentration data were
`analyzed by noncompartmental methods. A compartmental model
`was also used to fit temsirolimus concentration data. Pharmacoki-
`netic analyses were based on concentrations derived in whole blood
`due to the limited stability of
`temsirolimus in plasma. A two-
`compartment open model was fit to the concentration data with
`dose administration and elimination from the central compartment.
`Variable estimation for each patient and treatment period was
`individually derived using the maximum likelihood estimation
`algorithm in the ADAPTII software, release 4, March 1997 (Bio-
`medical Simulations Resource, University of Southern California,
`Los Angeles, CA).
`Dose-dependent variables were normalized and all pharmacokinetic
`variables were log transformed before performing ANOVA. The ANOVA
`assessed variability factors for course (j) and patient (k) using the
`model: yjk = l + coursej + patientk + ejk, in which l is the overall mean
`and e is the within-patient random error in variable y. Statistical
`
`Fig. 3. Representative pustular skin rash in a patient treated with temsirolimus.
`
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`Downloaded from
`clincancerres.aacrjournals.org
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`5758
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`NOVARTIS EXHIBIT 2056
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`Page 4 of 9
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`

`Phase I Study of Temsirolimus
`
`Table 3. Pharmacokinetic variables of temsirolimus on day 5, mean F SD (no. patients) (Cont’d)
`
`AUC (cycle 3; ng h/mL)
`
`—
`2,812 (1)
`4,705 F 1,781 (3)
`5,439 F 2,223 (3)
`7,756 (1)
`9,353 F 1,053 (4)
`
`CLc (L/h)
`6.2 F 3.3 (2)
`5.2 F 2.7 (3)
`5.5 F 1.4 (5)
`7.6 F 1.5 (4)
`16.5 F 5.3 (5)
`19.9 F 7.5 (12)
`
`Vdss (L)
`132.1 F 14.2 (2)
`57.1 F 21.7 (3)
`81.5 F 23.7 (5)
`111.1 F 8.7 (4)
`232.5 F 110.9 (5)
`239.2 F 116.0 (12)
`
`AR
`
`2.2 F 1.1 (3)
`1.0 F 0.6 (3)
`1.3 F 0.4 (5)
`1.1 F 0.2 (4)
`0.7 F 0.4 (4)
`0.8 F 0.1 (11)
`
`B/Pratio (day 1)
`10.9 F 13.9 (2)
`10.4 F 3.2 (4)
`9.1 F 6.3 (6)
`3.7 F 1.7 (4)
`1.5 F 0.9 (6)
`1.5 F 0.6 (12)
`
`differences with P < 0.05 were considered significant. Before statistical
`analysis, C max was normalized to the daily temsirolimus dose, and
`AUC and AUCsum were normalized to the cumulative dose adminis-
`tered over the respective 2-week cycle. All available data were included
`in the statistical analysis. To assess the proportionality of exposure
`with dose, C max, AUC, and AUCsum were analyzed using the power
`b
`model Y = aDOSE
`, in which Y is the pharmacokinetic variable of
`interest, b is the variable estimate for slope, and a is the intercept. For
`this analysis, the null hypothesis, Ho: b = 1 was tested. Rejection of
`the relationship between Y and DOSE is not
`Ho indicates that
`proportional.
`
`Results
`
`General. A total of 63 patients, whose relevant character-
`istics are shown in Table 1, were enrolled on this study from
`August 1998 to May 2000. The last patient completed the study
`in February 2002. Patients received a total of 361 2-week cycles
`of temsirolimus. The median number of cycles administered
`per patient was 4 (range, 1-21). Fifty-eight patients had received
`prior treatment with chemotherapy alone and 30 had received
`prior treatment with radiation therapy either alone (2) or
`combined with chemotherapy (28).
`the temsirolimus dose
`Dose escalation. The results of
`escalation are shown in Table 2. The first patient
`in the
`0.75 mg/m2/d cohort experienced grade 3 neutropenia.
`Because of this grade 3 toxicity, the cohort was expanded to
`three patients as dictated by the protocol. The two additional
`patients who were treated at this dose developed no adverse
`events. The first patient in the next cohort (1.25 mg/m2/d)
`also experienced grade 3 neutropenia and three additional
`patients were treated at this dose and developed no adverse
`the 2.16 mg/m2/d
`events. No DLTs were observed until
`cohort. In this cohort, one patient had a DLT of grade 3
`hypocalcemia; five additional patients were treated and had
`no DLTs. Dose escalation continued without additional DLTs
`until the 15 mg/m2/d cohort. In this cohort, one patient had a
`DLT of grade 3 hyperglycemia; two additional patients were
`treated and had no DLTs. In the 19 mg/m2/d cohort, one
`patient had DLTs of grade 3 elevations in transaminases; five
`additional patients were treated and one of these had grade 3
`thrombocytopenia. To further evaluate this dose level, six
`additional patients were treated and one patient had DLTs of
`grade 3 vomiting, diarrhea, and asthenia and two had grade 3
`thrombocytopenia, which required dose reductions. The two
`patients with the DLTs and the three with the dose reductions
`in the 19 mg/m2/d cohort were heavily pretreated. Thus, the
`decision was made to classify patients based on whether they
`had been heavily pretreated or minimally pretreated for the
`remainder of the dose escalation.
`
`Five additional heavily pretreated patients were treated
`with 15 mg/m2/d temsirolimus for a total of six in the
`heavily pretreated cohort and no DLTs were observed. Of
`the six heavily pretreated patients who had been treated with
`19 mg/m2/d temsirolimus, two had DLTs and three required
`dose reductions. Based only on DLTs, the MTD would have
`been 19 mg/m2/d but, because of the dose reductions, the dose
`of 15 mg/m2/d was considered the MTD for heavily pretreated
`patients.
`Six minimally pretreated patients had been treated with
`19 mg/m2/d temsirolimus and none had DLTs. Thus, six
`minimally pretreated patients were treated with 24 mg/m2/d
`temsirolimus. One had a DLT of grade 3 stomatitis and two
`required dose reductions, one because of grade 2 thrombo-
`cytopenia and the other because of grade 2 erythema
`nodosum. Based on the DLT and the two dose reductions, a
`MTD was not formally identified but the dose of 19 mg/m2/d
`was considered the maximum acceptable dose in minimally
`pretreated patients.
`Toxicity. Selected temsirolimus-related toxicities as a func-
`tion of dose that occurred in at least 10% of patients in any
`treatment cycle are summarized in Fig. 2. The most common
`drug-related adverse events observed across all dose levels were
`asthenia (56%), mucositis (54%), nausea (41%), and cutane-
`ous toxicity (41%). The two most frequent drug-related grades
`3 to 4 adverse events were hypophosphatemia and hypergly-
`cemia in 11% and 8% of patients, respectively. Overall, 10
`patients required dose reductions; 7 of these and 20 additional
`patients required dose delays.
`Hematologic toxicity consisted mainly of thrombocytopenia
`(33%) and leukopenia (27%). Grade 3 thrombocytopenia
`occurred in five patients, including three heavily pretreated
`patients treated at the 19 mg/m2/d dose (Fig. 2). Thus, this
`incidence seemed to be dose related. Thrombocytopenia
`was the most common cause for dose reductions and delays
`(four patients with both and seven with only delays). Five
`patients developed grade 3 neutropenia; three were treated
`with <15 mg/m2/d temsirolimus, suggesting that severe
`neutropenia was not dose related. Neutropenia contributed
`to dose reduction and delay in one patient. Seventeen (27%)
`patients developed temsirolimus-related grades 1 to 2 epistaxis,
`which resolved rapidly; 10 were treated with doses of at least
`15 mg/m2/d.
`Treatment with temsirolimus resulted in few severe non-
`hematologic toxicities. Although 54% of patients developed
`mucositis, only one patient who was treated with 24 mg/m2/d
`temsirolimus developed grade 3 mucositis, a DLT (Table 2).
`Drug-related cutaneous toxicity was commonly observed in
`patients treated with temsirolimus over a wide range of doses
`
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`5759
`Clin Cancer Res 2006;12(19) October 1, 2006
`
`Research.
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`
`
`NOVARTIS EXHIBIT 2056
`Par v. Novartis, IPR 2016-01479
`Page 5 of 9
`
`

`

`Cancer Therapy: Clinical
`
`Table 4. Pharmacokinetic variables of sirolimus on day 5, mean F SD (no. patients)
`
`Dose group (mg/m2)
`
`0.75
`1.25
`2.16
`4.5
`15.0
`19.1
`
`C max (ng/mL)
`9.3 F 4.1 (3)
`27.3 F 7.6 (3)
`34.3 F 21.2 (5)
`48.7 F 16.7 (4)
`57.8 F 24.3 (5)
`133.9 F 70.3 (12)
`
`t max (h)
`25.9* F 37.5 (3)
`3.6 F 2.2 (3)
`3.6 F 2.1 (5)
`2.8 F 2.3 (4)
`1.8 F 1.5 (5)
`2.0 F 1.5 (12)
`
`t 1/2 (h)
`102.0 F 46.3 (3)
`80.3 F 15.5 (3)
`49.4 F 27.2 (6)
`50.3 F 6.8 (4)
`39.9 F 1.3 (2)
`52.3 F 14.5 (11)
`
`AUC (cycle 1; ng h/mL)
`1,866 F 1,324 (3)
`4,186 F 1,293 (3)
`3,240 F 2,463 (6)
`5,583 F 2,728 (4)
`4,729 F 545 (2)
`15,503 F 8,573 (11)
`
`Dose gr
`
`Abbreviations: AUCratio, uncorrected ratio of sirolimus to temsirolimus AUC; AUCsum, arithmetic sum of temsirolimus and sirolimus AUC.
`*One of three patients exhibited an abnormally prolonged t max.
`
`(26 total patients, 41%; Fig. 2). These included maculopapular
`rashes, acne, pustular rashes (Fig. 3), and pruritus. All skin
`reactions were grades 1 to 2 in severity and reversible. One
`patient treated with 24 mg/m2/d temsirolimus presented on
`day 94 with a clinical picture consistent with erythema
`nodosum (grade 2), which was considered to be possibly
`related to study medication, resulted in a dose reduction, and
`resolved with prednisone treatment. Three patients developed
`symptoms consistent with an allergic drug reaction, which
`began shortly after the start of the first i.v.
`infusion and
`ended after stopping the infusion. One patient treated with
`11.3 mg/m2/d temsirolimus developed a grade 4 allergic
`reaction and discontinued treatment. The other two patients
`were treated with 24 mg/m2/d temsirolimus, developed grades
`1 and 3 allergic reactions, and had dose delays.
`Some laboratory abnormalities were frequently reported as
`adverse events. Temsirolimus-related hyperglycemia was
`reported in 11 (17%) patients and was grades 3 to 4 in 5
`(8%) patients (Fig. 2). Hyperglycemia was a DLT for one
`patient treated with the 15 mg/m2/d dose (Table 2). Temsir-
`olimus-related elevations in plasma triglyceride and choles-
`terol levels occurred in 23 (37%) and 14 (22%) patients,
`respectively, and reached grades 3 to 4 levels in 3 (5%) and
`2 (3%) patients, respectively (Fig. 2). One minimally pretreated
`patient treated with the 15 mg/m2/d dose developed grade 3
`hypertriglyceridemia and discontinued treatment. Other tem-
`sirolimus-related grades 3 to 4 laboratory abnormalities
`included hypophosphatemia (7 patients, 11%), hypokalemia
`(3 patients, 5%), and hypocalcemia (2 patients, 3%).
`Hypocalcemia was a DLT for one patient treated with the
`2.16 mg/m2/d dose. Elevations of aspartate and alanine
`aminotransaminases occurred in 9 (14%) and 8 (13%)
`patients, respectively. Elevations in aspartate and alanine
`aminotransaminases were DLTs in one heavily pretreated
`patient treated with the 19 mg/m2/d dose.
`Immunologic studies. There were no episodes of infections
`or any other clinical manifestation that indicated an opportu-
`nistic infection or immunosuppressed state. Lymphocyte cell
`surface phenotype analysis and mitogen proliferation assays
`did not show any consistent trend toward immunosuppression
`(data not shown). Although intersubject variability was
`considerable, results for individual patients were consistent.
`Proliferative responses to mitogens and pooled allogeneic cells
`were within the control ranges, with two exceptions that
`recovered to the reference range within the study period.
`Pharmacokinetic analysis. Whole blood and plasma samples
`were available from 62 patients receiving doses of 0.75 to
`
`24 mg/m2 (1.42-55.2 mg). Following drug administration,
`temsirolimus concentrations decreased with time in a poly-
`exponential manner. A summary of relevant pharmacokinetic
`variables measured for temsirolimus and sirolimus is provided
`in Tables 3 and 4, respectively. Figures 4 and 5 show the
`relationship between temsirolimus exposure (C max and AUC,
`respectively) and dose. Over the wide range of doses evaluated,
`temsirolimus exposure increased with dose in a less than pro-
`portional manner. Steady-state volume of distribution (Vdss)
`was extensive,
`increased with dose, and exhibited values
`typically exceeding total body weight. At <15 mg/m2, temsir-
`olimus exhibited preferential partitioning into RBC, with mean
`blood-to-plasma ratios of 3.7 to 10.9 (coefficient of variation,
`31-128%), whereas at z15 mg/m2 the ratios approached unity.
`Mean clearance from whole blood increased with increasing
`dose from 5.2 to 19.9 L/h and was associated with modest
`to moderate intersubject variability (coefficient of variation,
`20-54%). Mean terminal half-life was 13 to 25 hours.
`Sirolimus was a major metabolite that was observed early
`(after 15 minutes of infusion) and decreased with time in an
`apparent monoexponential or biexponential fashion. Sirolimus
`exposure was generally comparable with temsirolimus expo-
`sure, with sirolimus-to-temsirolimus AUC ratios of 0.6 to 1.8.
`No statistically significant differences in pharmacokinetic
`variables were apparent when cycle was a factor in ANOVA
`analysis, a finding consistent with limited degrees of drug
`accumulation observed with multiple cycles of treatment. No
`age-related (Figs. 4 and 5) or sex-related effects were apparent.
`Analysis of toxicity events as a function of exposure revealed
`a positive correlation between temsirolimus C max and the
`severity of thrombocytopenia (P = 0.014). Temsirolimus AUC
`showed a positive correlation with the severity grades of
`aspartate aminotransaminase elevation and hypocalcemia
`(P = 0.012 and 0.030, respectively). Sirolimus AUC showed a
`positive correlation with the severity grades of hypophospha-
`temia and thrombocytopenia (P = 0.003 and 0.011, respec-
`tively). AUCsum were positively r