A Phase I Study of RO , a γ-secretase Inhibitor of Notch Signaling, in Patients with Refractory Metastatic or Locally Advanced Solid Tumors
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1 A Phase I Study of RO , a γ-secretase Inhibitor of Notch Signaling, in Patients with Refractory Metastatic or Locally Advanced Solid Tumors Tolcher, et al Data Supplement 1
2 ONLINE APPENDIX Appendix Table A1. Summary of Mean RO AUC 0-24 (ng*h/ml) With Percent Coefficient of Variation (%CV) and Subject Number (n) in Each Dosing Cohort (Fasted) of the Dose Escalation Study Dose Day 1 cycle 1 Day 10/17 cycle 1 Day 1 cycle 2 Schedule (mg) AUC % no. AUC % no. AUC % no. A B Abbreviation: AUC 0 24, area under the plasma concentration versus time curve during 24-hour interval. 2
3 Appendix Table A2. Clinical benefit summary No. of patients (%) Schedule Schedule Schedule Total Duration of stable disease A B C (n=96*) (n=48*) (n=44*) (n=4*) 4 cycles (3 months) 7 (15) 8 (18) 0 15 (15) 6 cycles (4 months) 4 (8) 5 (11) 0 9 (9) 8 cycles (6 months) 1 (2) 3 (7) 0 4 (4) *Evaluable patients. Tumor types most commonly among clinical benefit population: Melanoma (4 of 24 patients) Sarcoma (4 of 12 patients) Ovarian (3 of 9 patients). Twelve patients (11%) had FDG-PET response (European Organization for Research and Treatment of Cancer criteria) in cycle 1 or 2. 3
4 Appendix Fig A1. Mean ± standard deviation exposure (AUC inf ) of midazolam with error bars at baseline and after RO treatment for the original Schedule A, modified Schedule A, and Schedule C Abbreviation: AUC, area under the concentration versus time curve 4
5 Appendix Fig A2. Mean ± standard deviation exposure (AUC inf ) ratios of 1- hydroxy-midazolam:midazolam at baseline and after RO treatment for patients with date for original Schedule A, modified Schedule A, and Schedule C Abbreviation: AUC, area under the plasma concentration versus time curve. 5
6 Appendix Fig A3. Relationship between RO AUC 0-24 and VEGFR-2 % change from baseline Percentage change from baseline presented by open circles for individual patient data and linear regression line. Blood plasma samples were collected baseline and then serially, and analyzed for VEGFR-2 at RBM, USA, using enzyme-linked immunosorbent assay from R&D systems. Abbreviations: AUC, area under the plasma concentration versus time curve; VEGFR-2, vascular endothelial growth factor receptor-2. 6
7 Appendix Fig A4. Relationship between RO plasma concentration and Hes-1 expression changes in hair follicles Changes in hair follicles presented with open circles as individual patient data and linear regression line. Hair follicles in the anagen phase were collected at baseline and at 4 and 8 hours after dosing on day 1 of cycle 1 (C1D1). Hes-1 mrna expression was assessed by reverse transcriptase polymerase chain reaction and normalized against a housekeeping gene (Alas-1) using the Ct method. Increasing values demonstrate a down-regulation of Hes-1 upon treatment with RO
8 Appendix Fig A5. Partial response in a patient with colorectal adenocarcinoma with neuroendocrine features: Computed Tomography scans at baseline and post-treatment (inset depicts nodal regression following 4 treatment cycles) Jan 6, 2009 April 7,
9 Appendix Fig A6. Case study, metastatic melanoma: baseline and posttreatment PET scan 9
10 Supplementary text Pretreatment evaluations Pretreatment evaluations included a complete history, physical examination, and routine laboratory studies including a complete blood count (CBC), white blood cell count, chemistry (electrolytes, blood urea nitrogen, creatinine, uric acid, glucose, alkaline phosphatase, lactate dehydrogenase, ALT, AST, total bilirubin, calcium, total protein, albumin, cholesterol, and triglycerides), urinalysis, electrocardiogram, relevant radiologic studies, and tumor markers. Pre and post treatment 24-hour urine samples were scheduled for patients enrolled following the observation of dose limiting hypophosphatemia. Electrocardiogram Evaluations Triplicate cardiograms were obtained at study baseline, and before and after drug administration on the first and last dosing days of cycles 1 to 3, and at the offstudy visit. Electrocardiogram data were verified by centralized reading. For this study, alert cardiograms were classified as those with a 30 to 60 ms increase in QTc interval relative to baseline, or QTc interval of 470 to 499 ms. QTc signal electrocardiograms were defined as those with a >60 ms increase relative to baseline, or an interval >500 ms (considered a grade 3 AE). PK/PD Correlations The observed PK/PD trends were in accordance with previous reports. γ-secretase proteolytic activity is required for the generation of Aß-40 and other γ-secretase inhibitors have been shown to inhibit plasma Aß-40 production. 1,2 Notch signaling in endothelial cells was reported to down-regulate VEGFR-2 expression 3 and treatment with a γ-secretase inhibitor was shown to reconstitute VEGFR-2 expression in endothelial cells. 4 A role for Notch signaling has been described in hair follicles, 5,6 inhibition of the Notch pathway through RO should therefore lead to down-regulation of Notch target genes such as HES-1 10
11 which already has been demonstrated in pre-clinical experiments [Luistro et al., Cancer Res, 2009]. Other angiogenic factors and cytokines were analyzed at RBM, USA (amphiregulin, ANG-2, BTC, epidermal growth factor, epidermal growth factor receptor, epiregulin, fibroblast growth factor receptor-ß, heparin-binding epidermal growth factor, IL-6, IL-8, platelet-derived growth factor-bb, placental growth factor, tenascin-c, transforming growth factor-α, TSLP, VEGF-A) using Multi-Analyte Profiling technology and VEGFR-1 using enzyme-linked immunosorbent assay from R&D systems. Flow cytometry analysis (FCA) was used to assess peripheral blood T cell subsets. Available paired tumor biopsies were collected in the expanded cohorts to assess changes of microvessel density using CD31 immunohistochemistry (PECAM1, Clone 1A10 on Ventana benchmark XT, performed at HistoGeneX, Belgium), and to analyze gene expression by quantitative reverse transcription polymerase chain reaction (qrt- PCR) with a special attention to genes shown to be affected by Notch signaling (e.g. HES-1). Hes-1 and c-myc expression were assessed by qrt-pcr in peripheral blood mononuclear cells in the expanded cohorts of the study. Hair follicle mrna was also used to measure c-myc expression changes. All RT-PCR assays were developed by Roche Applied Science, Germany. No consistent effects could be detected for these angiogenic biomarkers and cytokines. PBMC and FCA analyses did not reveal any consistent trends, maybe due to low samples numbers. Expression of c-myc in hair follicles decreased upon treatment in higher dose cohorts. RNA could be isolated from 5 tumor biopsy pairs of which 4 showed slight decreases in Hes-1 and c-myc expression during treatment as determined by RT-PCR. In two biopsy pairs that were evaluable for microvessel density assessment, a moderate increase of vessel number was observed in the on-treatment biopsy. The tumor RT-PCR and immunohistochemistry results would need to be confirmed with more samples. 11
12 Online references 1. Henley DB, May PC, Dean RA, Siemers ER: Development of semagacestat (LY450139), a functional gamma-secretase inhibitor, for the treatment of Alzheimer's disease. Expert Opin Pharmacother 10: , Steiner H, Fluhrer R, Haass C: Intramembrane proteolysis by gammasecretase. J Biol Chem 283: , Taylor KL, Henderson AM, Hughes CC: Notch activation during endothelial cell network formation in vitro targets the basic HLH transcription factor HESR-1 and downregulates VEGFR-2/KDR expression. Microvasc Res 64:372-83, Williams CK, Li JL, Murga M, Harris AL, Tosato G: Up-regulation of the Notch ligand Delta-like 4 inhibits VEGF-induced endothelial cell function. Blood 107:931-9, Demehri S, Kopan R: Notch signaling in bulge stem cells is not required for selection of hair follicle fate. Development 136:891-6, Pan Y, Lin MH, Tian X, et al: Gamma-secretase functions through Notch signaling to maintain skin appendages but is not required for their patterning or initial morphogenesis. Dev Cell 7:731-43,
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