OSIP related AACR abstracts.
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Abstract Number: 677
Presentation Title:
Antitumor activity of OSI-930, a novel multi-targeted tyrosine kinase inhibitor, in combination with standard
chemotherapeutics in preclinical cancer models.
Presentation Start/End Time:
Sunday, Apr 17, 2005, 8:00 AM -12:00 PM
Category:
ET06-09 Tyrosine kinase and phosphatase inhibitors
Author Block:
Mary Srebernak, Shannon Winski, David Emerson, Blake Tomkinson, Andrew Stephens, Andrew Crew, Andrew Flood, Paul Briner. OSI Pharmaceuticals, Boulder, CO, OSI Pharmaceuticals, Farmingdale, NY
The full potential and optimal utilization of targeted kinase inhibitors in combination with standard chemotherapeutic regimens has yet to be realized. OSI-930 potently inhibited the closely-related kinases Kit, KDR, and PDGFRß in cell-based assays (IC50 values < 10nM), and has demonstrated antitumor efficacy across a wide range of xenograft models, including small cell lung cancer (SCLC) and colorectal carcinoma (CRC). Cisplatin and etoposide, standard agents used in the treatment of SCLC, were selected for combination studies with OSI-930 in the NCI-H526 SCLC model. These agents were administered at 6 mg/kg IV on Day 1 and 25 mg/kg IP Day 1-3, respectively, followed by maintenance therapy of either vehicle (Day 8-36) or OSI-930 200 mg/kg PO (Day 8-59). The combination of cisplatin/ etoposide induced tumor regression, and the addition of maintenance OSI-930 was effective in delaying tumor growth by 29 days over vehicle maintenance. Additionally, the log cell kill index was three times greater with one durable cure generated in the group receiving OSI-930. The combination of 5-fluorouracil (5-FU), oxaliplatin, and leucovorin, known collectively as FOLFOX, has recently been approved for first-line use in colon cancer. A FOLFOX-like regimen (5-FU/ oxaliplatin) was selected for combination studies with OSI-930 in two human CRC models, SW48 and COLO 205. 5-FU was administered at 20 mg/kg IV Day 1-5, oxaliplatin at 10 mg/kg IP on Day 1, and vehicle or OSI-930 200 mg/kg PO administered as maintenance therapy Day 8-21. Treatment with maintenance OSI-930 resulted in enhanced tumor growth inhibition (TGI) and growth delay (GD) compared to vehicle maintenance in both tumor models (SW48: TGI=84.4% compared to 68.9%, GD=6.8 days over vehicle; COLO 205: TGI=70.8% compared to 28.4%, GD=12.4 days over vehicle). In a separate COLO 205 study, OSI-930 administered at 100 mg/kg PO either concomitantly (Day 1-14) or as maintenance therapy (Day 8-21) improved tumor growth delay by 2-3 fold and increased TGI by 15-20% compared to vehicle maintenance. Both dosing schedules were well-tolerated with body weight loss in both groups <5%. These studies demonstrate that OSI-930 can be administered safely and effectively with current standard therapeutic regimens in preclinical models of SCLC and CRC, and suggest the potential for use of OSI-930 in combination with chemotherapeutics or as maintenance therapy in the clinic.
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Abstract Number: 410
Presentation Title:
Time-dependent modulation of phosphotyrosine signaling in mast cell leukemia expressing constitutively active c-kit kinase measured by isobaric peptide labeling
Presentation Start/End Time:
Sunday, Apr 17, 2005, 8:00 AM -12:00 PM
Category:
CH03-08 Proteomics and systems biology
Author Block:
Filippo Petti, April Thelemann, Siobhan Mc Cormack, Linda Castaldo, Herbert Haack, Laura Sullivan, Andrew Garton, John D. Haley. OSI Pharmaceuticals, Inc., Farmingdale, NY, Cell Signaling Technology, Beverly, MA
Disregulation of the c-kit tyrosine kinase has been observed in mast cell leukemia, in gastrointestinal stromal tumors (GIST) and in SCLC. OSI-930, a novel small molecule inhibitor of c-kit and KDR kinases, has shown growth inhibitory and proapoptic effects at nM concentrations in mast cell and SCLC lines in vitro and shows tumor growth inhibitory activity in corresponding xenograft models in vivo. Here we have used a novel quantitation technique that rapidly identify and quantitate target proteins altered in phosphorylation in response to c-kit inhibition in vitro in the mast cell leukemia line HMC1. Anti-phosphotyrosine affinity chromatography and multiplex isobaric peptide derivitization were coupled to multi-dimensional LC-MS/MS to identify signaling networks pharmacological blockade. To improve sensitivity and dynamic range of these measurements, we have used a novel tagging method with greatly improved peptide fragment ion generation and quantitation. The labeling method employed a multiplexed set of isobaric reagents which yield amine-derivatized peptides that are chromatographically identical and indistinguishable in MS, but which produce strong low-mass MS/MS signature ions following collision induced dissociation that permit quantitation. The use of these labels therefore permits simultaneous measurement of relative and/or absolute protein abundance of multiple, complex samples. This approach allowed for identification and temporal measurement of >300 phosphotyrosine-containing and associated proteins at 0, 1, 4 and 24 hours of c-kit kinase inhibition. Proteins were hierarchically clustered according to temporal expression patterns allowing facile identification of protein groups and quantitation of time-dependent changes in receptor signaling pathways associated with blockade by the small molecule c-kit/KDR inhibitor OSI-930. Protein changes were verified and explored by extensive use of cell pellet tissue microarray and immunoblot approaches, allowing a detailed temporal map of c-kit signaling inhibition in mast cell leukemia to be constructed.
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Abstract Number: 154
Presentation Title:
PGE2-stimulated proliferation of NSCLC cells is epidermal growth factor receptor-independent and resistant to erlotinib
Presentation Start/End Time:
Sunday, Apr 17, 2005, 8:00 AM -12:00 PM
Category:
CB01-07 Protein kinases
Author Block:
Kostyantyn Krysan, Karen L. Reckamp, Brian Escuadro, Joanne M. Bando, Steven M. Dubinett. David Geffen School of Medicine at UCLA, Los Angeles, CA, Cedars-Sinai Medical Center, Los Angeles, CA
The Epidermal Growth Factor Receptor (EGFR) is frequently overexpressed in a variety of malignancies and plays an important role in tumor cell growth, proliferation and metastasis. Because of its important role in tumor progression, EGFR is a promising target for cancer therapy. However, only a limited population of cancer patients responds to EGFR inhibitor therapy. Recent studies have linked sensitivity to EGFR TK inhibitors to activating somatic mutations in the TK domain observed in some non-small cell lung cancer (NSCLC) patients. Other studies have focused on the cross-talk between the downstream components of the EGFR pathway and other signal transduction pathways that can overcome pharmacological inhibition of EGFR. Cyclooxygenase 2 (COX-2) is often upregulated in a wide variety of human cancers including lung cancer and is linked to all stages of tumorigenesis. We have found that prostaglandin E2 (PGE2), the major COX-2 metabolite, is able to cross-activate the EGFR pathway in a subset of NSCLC cell lines through its G-protein coupled receptors in an EGFR-independent manner. This cross-activation is resistant to EGFR inhibitors including erlotinib (Tarceva®) and is not evident in bronchial epithelial cells. The functional manifestation of such trans-activation was an enhanced NSCLC cell proliferation in response to PGE2 treatment that was resistant to EGFR inhibition, suggesting that COX-2 overexpression may contribute to EGFR inhibitor resistance in NSCLC. We tested a panel of NSCLC cell lines for sensitivity to erlotinib and celecoxib (Celecoxib®) alone or in combination. We have found that combination treatment of NSCLC cells with COX-2 and EGFR inhibitors significantly reduces cell proliferation compared to either drug alone. These results provide a strong rationale for the combined use of COX-2 and EGFR inhibitors in NSCLC therapy.
Erlotinib was generously provided by OSI Pharmaceuticals (Farmingdale, NY).
This study was supported by the UCLA SPORE In Lung Cancer NIH P50 CA90388, Tobacco-Related Disease Research Program grant #12FT-0061 (K.K.) and VA Career Development Award, ASCO Young Investigator Award, STOP Cancer Memorial Award (K.L.R.).
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Abstract Number: 1706
Presentation Title:
Cell screens with siRNA for cancer target identification
Presentation Start/End Time:
Sunday, Apr 17, 2005, 3:30 PM - 3:45 PM
Category:
ET02-01 Identification of molecular targets
Author Block:
Julie LC Kan, Feng-Lei Sun, David M. Keane, Suzanne Russo, Nancy Ng, Neil W. Gibson, Lee Arnold. OSI Pharmaceuticals, Farmingdale, NY
The use of RNA interference (RNAi) is becoming an increasingly important tool for the study of mammalian gene function. This has certainly come to bear for the identification of new targets for cancer drug discovery. RNAi methodology provides a means to perform genetic screens in mammalian cells similar to those routinely performed in model organisms such as yeast, Drosophila, zebrafish and Caenorhabditis elegans. Using siRNAs and with the ability to perform high throughput cell screens, the entire human genome can potentially be screened for drug targets. This requires that appropriate phenotypic readouts be chosen for the cellular screens.
We report here the use of siRNAs in phenotypic readouts for decrease of proliferation and apoptosis induction to identify new cancer drug targets. The screens were performed with siRNA alone or in combination with Tarceva™. Through this effort, we have identified drug targets that act alone or are additive in the presence of Tarceva™. As an example, the combination of IGF-1R with EGFR was shown to be additive. Using an siRNA to IGF-1R and Tarceva™ for inhibition of EGFR, the non-small cell lung cancer lines, H358 and H441 gave additivity for both induction of apoptosis and a concomitant decrease in proliferation. In H358 cells, the combination of siRNA to IGF-1R and Tarceva™ resulted in an 8 fold induction of caspase-3/7 with greater than 60% decrease in cell proliferation compared to siRNA to IGF-1R or Tarceva™ alone having less than 2 fold induction of caspase 3/7 and a 25% decrease in proliferation. This data supports the notion of using inhibitors of EGFR and IGF-1R in combination as a useful strategy in lung cancer. Moreover, mammalian cell screens with RNAi have proven to be a valuable tool for the identification of new targets and/or new drug combinations.
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Abstract Number: 2284
Presentation Title:
Cell cycle effects of the EGFR TK inhibitor erlotinib in NSCLC cell lines
Presentation Start/End Time:
Monday, Apr 18, 2005, 8:00 AM -12:00 PM
Category:
ET03-01 Cell cycle mechanisms of anticancer drug action
Author Block:
Yi-He Ling, Roman Perez-Soler. Albert Einstein College of Medicine, Bronx, NY
p27KIP, a cyclin-dependent kinase (cdk) inhibitor and a putative tumor suppressor, plays a critical role in the regulation of cell proliferation and cell cycle progression. Erlotinib, an orally bioavailble EGFR tyrosine kinase inhibitor, has been shown a potential activity against cancer cell growth in vitro and in vivo. In previous work, we have reported that erlotinib exerts the inhibitory effects on EGFR high expressed human non-small cell lung cancer cell H322 through the disruption of G1 -related cell cycle regulators and blocking cell cycle progression from G1 to S phase transition. The present study shows that erlotinib arrests H322 cells at G1 phase by accumulating the p27KIP protein, and initiating a chain of events by decreasing the amounts of cyclin A and cyclin E and the suppression of cyclin A- or cyclin E-associated cdk-2 kianse activity. Erlotinib induced G1 arrest and p27KIP up-regulation in the tested sensitive cell lines (H322, H358, and A431), but to lesser degree in the resistant cell lines (A549, H596, and H1299). The G1 arrest and p27KIP up-regulation by erlotinib was sustained within 8 h of removal of drug exposure, and completely relieved at 24 h. The immunocytochemical and immunoblot studies revealed that erlotinib treatment resulted in p27KIP localization in nucleus, whereas; the p27KIP was predominately detected at cytoplasm in control cells. In addition, erlotinib treatment led to a dramatic inhibition of p27KIP phosphorylation at Thr 187, and down-regulation of Skp2 expression. As a result, the half-life of p27KIP was significantly increased by erlotinib treatment. Furthermore, the reduction of p27KIP expression using p27KIP small interfering RNA abrogated erlotinib-induced G1 phase arrest and cell growth inhibition. Taken together, the results of this study demonstrated that p27KIP as a key mediator of cell cycle plays an implant role in erlotinib-induced cell growth inhibition at G1 phase arrest. This work was supported in part by NIH grant CA50270 and OSI Pharmaceuticals.
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Abstract Number: 2330
Presentation Title:
Down-regulation of the cGMP/PKG pathway in primary human colon cancers and cancer cell lines.
Presentation Start/End Time:
Monday, Apr 18, 2005, 8:00 AM -12:00 PM
Category:
ET02-01 Identification of molecular targets
Author Block:
Atsuko Deguchi, Koushik K. Das, Steven W. Xing, Bert Oehlen, I. Bernard Weinstein. Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY, OSI Phamaceuticals, Inc., Farmingdale, NY
There is increasing evidence that activation of the cGMP-dependent enzyme protein kinase G (PKG) can play an important role in inhibiting cell proliferation and inducing apoptosis. The intracellular level of cGMP is regulated through a dynamic balance between its rate of synthesis by guanylyl cyclases (GCs) and its degradation by specific cGMP-phosphodiesterases (PDEs), especially PDEs 2 and 5. In previous studies we found that cGMP-PDE inhibitors, a GC activator, and constitutively activated mutants of PKG Iß induced apoptosis in SW480 human colon cancer cells (Deguchi et al., Mol. Cancer Ther. 1, 803-809, 2002, and Cancer Res. 64, 3966-3973, 2004). However, the actual profile of components of the cGMP/PKG signaling pathway in human cancers has not been previously studied in detail. In this study, we examined by western blot analysis cellular levels of PDE5, PKG Iß and phospho-VASP (vasodilator-stimulated phosphoprotein), a marker of PKG activation, in 19 pairs of primary human colon carcinomas and paired adjacent normal colonic mucosa. PKG Iß was decreased in 54%, PDE5 was increased in 58%, and phospho-VASP was decreased in 89% of the colon tumors. We also did similar assays in several cell lines. When compared to the normal human fetal colon cell line CCD841CoN cells, PDE5 was increased and phospho-VASP was decreased in the colon cancer cell lines SW480, SW620, and DLD1. When compared to MCF-10F normal human mammary epithelial cells, MCF7 human breast cancer cells also displayed an increase in expression of PDE5 and decreased levels of PKG Iß and phospho-VASP. Furthermore, ras- and raf-transformed Rat6 fibroblasts displayed decreased levels of PKG Iß and increased levels of PDE5 when compared with the parental Rat6 cells. Taken together, these findings suggest that down-regulation of cGMP/PKG-mediated signaling pathways often occurs during tumorigenesis and cell transformation. These results are consistent with previous evidence indicating that agents that increase the activation of PKG can inhibit growth and induce apoptosis in cancer cells, and provide a rationale for the further development of such agents as a novel strategy for cancer therapy.
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Abstract Number: 2313
Presentation Title:
Characterization of the molecular determinants of Erlotinib sensitivity in NSCLC cell lines.
Presentation Start/End Time:
Monday, Apr 18, 2005, 8:00 AM -12:00 PM
Category:
ET03-02 Cellular responses to anticancer drugs
Author Block:
Graeme Griffin, April Thelemann, Siobhan McCormack, Filippo Petti, Eric Brown, Victoria Drewett, Iain Mulford, Stuart Thomson, Feng-lei Sun, Suzanne Russo, David Keane, Julie Kan, Andrew Garton, David Emerson, John Haley, Lee Arnold, Neil Gibson. OSI Pharmaceuticals, Farmingdale, NY
Erlotinib (TarcevaTM) is a quinazoline-based epidermal growth factor (EGFR) inhibitor recently approved by the FDA for the treatment of NSCLC. Clinical studies have demonstrated that erlotinib is the first EGFR targeted therapeutic to demonstrate a survival benefit in a clinical setting. However, the treatment benefit can vary from individual to individual. The aim of this study was to investigate some of the molecular mechanisms behind erlotinib sensitivity in pre-clinical models toward the goal of understanding the factors behind the variable responses in the clinic.
The activity of erlotinib in in vitro cell viability and in vivo xenograft models was compared in a panel of NSCLC cell lines that were demonstrated to express only wild-type EGFR. A good correlation was observed between erlotinib sensitivity in in vitro and in vivo models. NSCLC cells that represented examples of erlotinib-sensitive and relatively insensitive lines, as assessed by cell viability and tumor growth inhibition, were then selected and their EGFR signaling pathways investigated by Western blot. Although significant similarities exist in EGFR signaling pathways, in erlotinib sensitive and insensitive cell lines, differences were observed that primarily related to the role of EGFR in regulation of the PI3K/Akt pathway. In erlotinib sensitive cell lines, the ability of EGF to stimulate, and of erlotinib to block, activation of the Akt pathway was apparent but in the insensitive lines, erlotinib had little effect on Akt pathway proteins. These findings were extended in pharmacodynamic models in tumor samples taken from mice treated with erlotinib or a vehicle control.
In conclusion, pre-clinical data demonstrate that a range of NSCLC cell lines, all containing wild-type EGFR, exhibit a similar range of erlotinib responsiveness to that seen in recent clinical trials. It has previously been suggested that mutations of the EGF receptor may play a role in predicting clinical responsiveness to EGFR inhibitors in NSCLC. Our data indicate that in wild-type EGFR containing NSCLC cells, differences in the downstream signaling pathways impacted by EGFR inhibition are major determinants of responsiveness to erlotinib.
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Abstract Number: 3108
Presentation Title:
Large scale profiling of phospho-proteins in cell and tissue microarrays using phospho specific antibodies: Identification of biomarkers and pharmacodynamic activity for a c-Kit inhibitor.
Presentation Start/End Time:
Monday, Apr 18, 2005, 1:00 PM - 5:00 PM
Category:
CH03-02 High throughput proteomics
Author Block:
Herbert Haack, Laura C. Sullivan, Andrew Garton, John D. Haley, Bradley L. Smith. Cell Signaling Technology, Beverly, MA, OSI Pharmaceuticals Inc., Farmingdale, NY
The successful development and use of targeted therapeutics may be improved by the use of biomarkers to characterize drug biological activity and allow patient selection. OSI-930, a small molecule inhibitor of Kit and KDR kinases, has shown growth inhibitory and pro-apoptotic effects in mast cell and SCLC lines in vitro and shows tumor growth inhibitory activity in corresponding xenograft models in vivo. We have used model cell lines and xenografts in large-scale tissue microarray screens with phospho-specific antibodies to identify potential biomarkers for the c-Kit inhibitor and other drug candidates. Cells in culture and xenografts were exposed to the compound and then fixed, embedded and arrayed for IHC analysis. Over 130 phospho-specific and total antibodies were systematically validated for IHC before use on the test samples. The screens successfully identified multiple markers whose expression or phosphorylation was altered by the drug treatment. Specific profiles of expression and inhibition by the compound were observed for mutant and wild-type c-Kit in cell lines. Many of the known downstream targets of c-Kit were identified along with novel markers that have not been previously linked to the drug target. The profile observed for mutant c-Kit using IHC corresponds well with the drug response profile obtained using multi-dimensional LC-MS/MS. In addition, specific tumor and stromal activities of the compound were identified in xenograft arrays, demonstrating the advantage of cellular resolution that can be achieved using tissue microarray profiling. These markers may now be moved forward into validation studies using patient material to determine their clinical usefulness.
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Abstract Number: 3398
Presentation Title:
Anti-tumor activity of EGFR/TK inhibitor erlotinib (Tarceva™, OSI-774 ) alone and in combination with CPT-11 (irinotecan) in human colorectal cancer xenograft models.
Presentation Start/End Time:
Monday, Apr 18, 2005, 1:00 PM - 5:00 PM
Category:
ET06-09 Tyrosine kinase and phosphatase inhibitors
Author Block:
Jianping Chen, Melissa Smith, Kenneth Kolinsky, Violeta Adames, Nila Mehta, Bupesh Desai, Mohammad Rashed, Eric Wheeldon, Michael Linn, Brian Higgins. Hoffman La-Roche Inc, Nutley, NJ
Erlotinib (Tarceva™, OSI-774) is a potent, orally bioavailable, small molecule inhibitor of EGFR (HER1, erbB1) tyrosine kinase (TK). Erlotinib inhibits phosphorylation of the EGFR tyrosine kinase domain, thereby blocking key signal transduction molecules downstream from the receptor. Erlotinib has demonstrated improved overall survival in previously treated NSCLC patients and is also being tested in other types of solid tumors. CPT-11 is used in the first line management of patients with advanced CRC. In this study, the anti-tumor activity of erlotinib was evaluated in two human colorectal tumor xenograft models (LoVo and HCT116) in athymic mice. Both cell types express EGFR and have a similar doubling time in vitro and in vivo. When erlotinib was administered as monotherapy, significant tumor growth inhibition (TGI) was seen in the LoVo model at both 100 mg/kg (TGI=98%, 4/10 partial regressions (PRs), p<0.001), and 25 mg/kg (TGI=53%, p=0.010). However, the HCT116 xenograft model was not responsive to any dose of erlotinib tested. Based on the single agent activity in LoVo, a combination study with CPT-11 was performed. Single agent arms of erlotinib at 100 mg/kg and 25 mg/kg rendered reproducible efficacy of TGI>100%, 6/10 PRs (p<0.001) and TGI=79% (p<0.001), respectively. LoVo bearing mice treated with CPT-11 at the optimal dose of 60 mg/kg or a lower dose of 15 mg/kg also resulted in significant tumor growth inhibition (TGI>100%, p<0.001; TGI=93%, p<0.001). Combination treatment with erlotinib (25 mg/kg) and CPT-11 (15 mg/kg) caused significantly increased anti-tumor activity than either agent alone (TGI>100%, 10/10 PRs, p<0.001) with no enhanced toxicity. This enhanced anti-tumor activity was statistically significant compared with suboptimal monotherapy activity of erlotinib or CPT-11. Thus, anti-tumor activity of CPT-11 was enhanced by co-administration of erlotinib in the LoVo model. The data support the conclusion that erlotinib can enhance the anti-tumor activity of CPT-11, without enhanced toxicity, in the LoVo human colorectal tumor xenograft model.
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Abstract Number: 3396
Presentation Title:
Pancreatic tumors show variable sensitivity to Erlotinib: characterization of the response of cell lines in vitro and patient derived xenografts grown in SCID mice.
Presentation Start/End Time:
Monday, Apr 18, 2005, 1:00 PM - 5:00 PM
Category:
ET06-09 Tyrosine kinase and phosphatase inhibitors
Author Block:
Bonnie L. Hylander, Elizabeth A. Repasky, John Gibbs, Jeffrey Senfield, Christine Mantis, Kenneth K. Iwata. Roswell Park Cancer Inst., Buffalo, NY, OSI Pharmaceuticals, Boulder, CO
EGFR/HER1 overexpression has been observed in 30 to 50% of pancreatic adenocarcinomas. Coexpression of this receptor and at least one of its ligands has been associated with large tumor size, advanced clinical staging, and decreased survival period. This suggests that therapies targeting EGFR/HER1 could be applicable to the treatment of pancreatic cancer. We therefore are evaluating the response of both pancreatic tumor cell lines in in vitro studies and patient derived tumor xenografts to erlotinib, a potent EGFR/HER1 tyrosine kinase inhibitor. In vitro, the pancreatic tumor cell lines BxPC3, CFPAC-1, and HPAC responded well to erlotinib with IC50<9 uM, while ASPC1, MiaPaca and PANC1 were nonresponsive. In order to evaluate the reponsiveness of patient derived tumors, we are using a patient tumor/ SCID mouse xenograft model. The value of this model is that these xenografts maintain the histological features and characteristic heterogeneity of the original patient tumors. Tumor bearing SCID mice were treated with 100mg/kg erlotinib for 21-35 days and tumor growth was evaluated. Of the 5 patient tumors evaluated to date, 2 showed statistically significant growth inhibition as a result of erlotinib treatment, while the growth of 3 was not significantly inhibited. We are currently characterizing these tumors for EGFR expression and the effect of erlotinib on tumor cell proliferation. Some characteristics of these responsive and non-responsive xenografts will be presented. In addition to examining erlotinib as a single agent, combination studies have been initiated to identify agents that can enhance the anti-tumor activity of erlotinib. In in vitro combination studies, some of the pancreatic cell lines showed additive inhibition when combined with the mTOR inhibitor, rapamycin. The responsive and non-responsive pancreatic tumor cell lines and patient tumor xenografts provide material for further study of biomarkers of responsive and non-response to erlotinib. A randomized Phase III clinical study of Tarceva(TM) (erlotinib), in combination with gemcitabine demonstrated a 23.5 percent improvement in overall survival for patients with locally advanced or metastatic pancreatic cancer when compared to patients receiving gemcitabine plus placebo. The results of these preclinical studies will provide valuable information for the design of future clinical studies.
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Abstract Number: 4182
Presentation Title:
Investigation of metabolism and efficacy of OSI-461 in murine and rat preclinical models
Presentation Start/End Time:
Tuesday, Apr 19, 2005, 8:00 AM -12:00 PM
Category:
ET05-05 Pharmacokinetics and pharmacodynamics
Author Block:
Andy Cooke, Mike Vrkljan, Jo Dunseath, Eric Brown, Darla Landfair, Carol Bell, Carrie Pirritt, Marc Wiles, Alexandra Eyzaguirre, Nancy Ng, Chienling Ma, Shripad Bhagwat, An-Hu Li, Bert Oehlen, Michael Boisclair, Lee Arnold, Neil Gibson. OSI Pharmaceuticals, Boulder, CO, OSI Pharmaceuticals, Farmingdale, NY
OSI-461 is a compound that is currently in clinical trials to determine whether it has anticancer activity in hormone refractory prostate cancer as well as several other tumors. It is a known inhibitor of the cGMP family of phosphodiesterases and induces apoptosis in a variety of cancer cell lines in vitro by a process that may involve activation of PKG and the MEKK1/JNK signaling pathway. Twice daily oral administration of OSI-461 in mice (up to 200 mg/kg) and in female rats (up to 1000 mg/kg) did not show significant anti-tumor activity in animals implanted with rat AT6.1 or human DU-145 prostate cancer xenografts. The current studies were designed to determine whether the pharmacokinetic and or metabolic profile of OSI-461 in rodents could help explain the lack of anticancer activity in these model systems. A detailed analysis of the in vitro metabolism of OSI-461 in murine, rat and human liver microsomes shows that it can be converted to three major metabolites: (I) an N-oxide metabolite (N-oxidation of pyridinyl moiety); (II) a debenzyl metabolite (cleavage of benzyl amide) and (III) a hydroxyl metabolite (hydroxylation at the 2-methyl position). The formation of I and II represent metabolic inactivation, whereas the formation of III does not affect activity, as determined by induction of apoptosis in cancer cell lines. The various metabolites are also formed upon oral administration in mice and rats, with the N-oxide and debenzyl metabolites being the most abundant in plasma from these rodents. The overall OSI-461 exposure in female rats was much greater than in male rats and in mice. To estimate drug plasma levels expected to be required for anti-tumor efficacy in rodent models, the in vitro anti-proliferative activity of OSI-461 was tested in the presence of physiological levels of plasma proteins. When tested in the presence of 10% serum, OSI-461 shows growth inhibitory activity in vitro with an IC50 of 1-3 µM against several prostate cancer cell lines (DU145, PC3, 22Rv1). However, it was more than 10 fold less potent when the protein concentrations were raised to physiological levels by adding 0.05% alpha acid glycoprotein and 3 % human serum albumin. The OSI-461 plasma levels achieved upon repeat dosing in rodents were not sustained at concentrations that are efficacious in vitro in the presence of such protein concentrations. The plasma exposure levels achieved, in combination with extensive conversion of OSI-461 to metabolites with reduced activity may help explain the lack of tumor growth inhibitory efficacy in the murine and rat models.
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Abstract Number: 4124
Presentation Title:
Evaluation of molecular effects of KIT/KDR inhibitor (OSI-930) in H526 and WBA small cell lung xenografts
Presentation Start/End Time:
Tuesday, Apr 19, 2005, 8:00 AM -12:00 PM
Category:
ET05-05 Pharmacokinetics and pharmacodynamics
Author Block:
Karen L. Hart, Gina M. Sennello, David Emerson, Shannon Winski, Mary Srebernak, Greg Biesecker, Linda Castaldo, Andrew J. Garton, Blake E. Tomkinson, Frank C. Richardson. OSI Pharmaceuticals, Boulder, CO, OSI Pharmaceuticals, New York, NY
OSI-930 is a Receptor Tyrosine Kinase (RTKs) inhibitor that is highly effective in treating an array of human xenograft tumors with greater efficacy against non-small cell lung WBA compared to H526 tumors. In an effort to find a useful pharmacodynamic (PD) tumor marker for use in clinical trials, we examined changes in several phenotypic and molecular endpoints in these tumors following treatment with OSI-930. Mice bearing either H526 or WBA tumors (100-200 mg) were treated orally for three days with 200 mg OSI-930/kg/day and were given BrdU IP 16 hours prior to tumor collection. Tumors were collected 4 hours after the last dose and separate portions of each tumor were either frozen in liquid nitrogen or fixed in 10% neutral buffered formalin (4oC) for 24 hours. Morphologic endpoints that were evaluated either visually or using image analysis included: BrdU labeling, apoptosis, mitosis, pAKT, pS6, S6, p70S6, and CD-31. BrdU labeling was greater in H526 tumors prior to treatment, while mitotic index and apoptosis was greater in WBA tumors. Following treatment, BrdU labeling was decreased approximately 50% in WBA tumors while no change was observed in H526 tumors. Treatment-induced increases in apoptosis were greater in H526 tumors while decreases in mitotic index were greater for WBA tumors. Tumor markers phospho-AKT (p-AKT) and p-S6 were inhibited 52% and 75% respectively, while total S6 and total AKT remained unchanged in WBA tumors. In addition, the vascular markers CD-31 and p-KDR were inhibited 52% and 25%, respectively. In contrast, these markers were unchanged in the H526 xenograft. Western blot results confirmed the IHC results, and in addition showed inhibition of p-70S6K in WBA xenografts. In vitro analysis of SCF-stimulated WBA and H526 cells revealed that inhibition of Kit by OSI-930 was associated with a reduction in the phosphorylation status of AKT, S6, and p70S6 kinase, paralleling in vivo observations. The increased efficacy in WBA tumors is therefore associated with a greater effect on BrdU labeling and mitosis, CD-31, pS6, and pAKT. These results show that phosphorylation markers can be utilized to demonstrate tumor response to OSI-930 in vivo and suggest possible PD endpoints that could be used in future clinical
trials.
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Abstract Number: 4134
Presentation Title:
Relationships between irinotecan pharmacokinetics and UGT1A1 and MDR1 polymorphisms in a Phase I trial of irinotecan and erlotinib
Presentation Start/End Time:
Tuesday, Apr 19, 2005, 8:00 AM -12:00 PM
Category:
CL02-03 Pharmacogenetics and therapeutic responses
Author Block:
Joel M. Reid, Matthew P. Goetz, Ravi D. Rao, Stephanie L. Safgren, Renee M. McGovern, Alfred F. Furth, Sara J. Felten, Sumithra J. Mandrekar, Alex A. Adjei, Charles Erlichman, Henry C. Pitot, Matthew M. Ames. Mayo Clinic College of Medicine, Rochester, MN
Background: Irinotecan (CPT-11) disposition and toxicity in cancer patients is affected by genetic variation of several drug metabolizing enzymes and transport proteins. We conducted a phase I study of CPT-11 and erlotinib (OSI-774) and previously reported (ASCO 2004) that patients with the UGT1A1*28 polymorphism (either 6/7 or 7/7 TA repeats), had a significantly higher incidence of either gastrointestinal or hematologic toxicity (p=.047). Here we report the results of the CPT-11 pharmacokinetics (pk) performed to evaluate a possible pk interaction with OSI-774, as well as the effect of genetic variation within UGT1A1 and MDR1 on CPT-11 pk.
Methods: Advanced cancer patients with tumors known to over-express EGFR, ECOG performance scores 0-2, adequate hematologic, renal and hepatic functions were treated with daily oral OSI-774 and intravenous CPT-11 every 3 weeks (started on day 7 of OSI-774 in cycle 1). DLTs were defined as grade 4 neutropenia, grade 3 thrombocytopenia or = grade 3 non-hematologic toxicities despite maximal supportive care. Genotype analysis was performed by GeneScan (UGT1A1 TA indel), or sequencing (MDR1 exons (E) E12, E21 and E26 and UGT1A1 -3156). CPT-11, SN-38, and SN-38G pk were determined by non-compartmental analysis.
Results: CPT-11 pk were similar to those found in previous single agent studies (CPT-11 CL= 14.5 ± 5.1 L/h/m2). Mean (range) SN-38/CPT-11, SN-38G/CPT-11 and SN38G/SN-38 AUC ratios were 0.047 (0.016-0.094), 0.115 (0.058-0.262) and 3.086 (0.884-16.0), respectively. At a fixed dose of 120 mg/m2 CPT-11, OSI-774 dose reduction from 75 mg/day to 50 mg/day was associated with an increased SN-38G/SN-38 AUC ratio (p=0.048, rank sum). At a fixed dose of 240 mg/m2 CPT-11, OSI-774 dose reduction from 100 mg/day to 75 mg/day did not alter the SN-38G/SN-38 AUC ratio. Allele frequencies for UGT1A1*28, UGT1A1-3156, MDR1 E12, E21, and E26 were 0.33, 0.28, 0.38, 0.40, and 0.43, respectively. Spearman correlations between genotype and AUC ratios were determined. The UGT1A1*28 variant (6/7 or 7/7 genotype) was significantly correlated with increased SN-38/CPT-11 (p=0.0004) and decreased SN-38G/SN-38 (p=0.01). Similarly, the UGT1A1 -3156 variant was associated with increased SN-38/CPT-11 (p=0.03) and decreased SN-38G/SN-38 (p=0.01). There was no correlation between MDR1 variants and any pk parameter.
Conclusions: Patients receiving OSI-774 and CPT-11 who carry the UGT1A1*28 polymorphism have significantly increased SN-38/CPT-11 and decreased SN-38G/SN-38G AUC ratios. OSI-774 may inhibit SN-38G formation. Based on these results, we are continuing to study this combination by stratifying patients into UGT1A1 genotype specific cohorts to determine whether UGT1A1 genotype affects the maximally tolerated dose. Supported by CA69912-10, P30-CA15083, MM01-RR00585 and the Commonwealth Cancer Res. Fdn.
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Abstract Number: 3831
Presentation Title:
Autofluorescent mouse models of human medulloblasoma
Presentation Start/End Time:
Tuesday, Apr 19, 2005, 8:00 AM -12:00 PM
Category:
TB01-01 Mouse models of human cancer
Author Block:
Christopher R. Calabrese, M. Waleed Gaber, John Killmar, Christine Fuller, Meredith Allen, Richard J. Gilbertson. St. Jude Children's Research Hospital, Memphis, TN
We have developed a new in vivo model of medulloblastoma that recapitulates the behavior of classic and large cell anaplastic (LCA) forms of the disease and allows for serial measurement of tumor growth. To do this we generated green fluorescent human medulloblastoma cells (Daoy, MEB-MED-8A and MHH-MED-1) by in vitro infection using an MSCV-Green Fluorescent Protein (GFP) retrovirus. Under stereotactic control, 106 GFP-tagged medulloblastoma cells are inoculated into the superficial cerebral cortex of CD1 nude mice and the cranium is sealed with a glass plate. Serial tumor measurements can then be made through the cranial window using intravital fluorescence microscopy. By comparing in vivo total fluorescence measurements with 3D tumor reconstruction we show that the measurement of in vivo fluorescence provides a highly accurate measure of tumor burden. MEB-MED-8A tumors grow very rapidly and result in the death of animals within two weeks: histopathologically these tumors are LCA, invasive and contain an isochromosome of 17q and amplification of MYCC. In contrast, MHH-MED-1 and Daoy tumors display classic morphology with focal anaplasia and are much less aggressive in behavior taking more than 7 weeks to become symptomatic. We are now using these models to test a number of molecular targeted therapies for medulloblastoma. We previously reported ERBB2 to be an independent marker of poor prognosis in medulloblastoma. Daoy.2 and MEB-MED-8A tumors express high levels of the ERBB2 receptor while MHH-MED-1 cells express ERBB4 and low levels of ERBB2. Using our models we now show that well tolerated doses (50mg/kg/bd) of the oral ERBB kinase inhibitor erlotinib (Tarceva™, OSI-774) abolish ERBB2 signaling, induce cell cycle arrest and generate marked inhibition of medulloblastoma growth in the CNS. Our model system provides a useful new tool for studying the biology of signal transduction systems in medulloblastomas in the brain, and affords an efficient system for pre-clinical testing of novel therapies for this disease.
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Abstract Number: 4360
Presentation Title:
Proteomic view of NSCLC cell sensitivity to wt and mutant EGFR inhibition
Presentation Start/End Time:
Tuesday, Apr 19, 2005, 12:10 PM -12:25 PM
Category:
CH03-06 Proteomic analysis
Author Block:
John D. Haley, Filippo Petti, April Thelemann, Graeme Griffin, Neil Gibson. OSI Pharmaceuticals, Inc., Farmingdale, NY
Epidermal growth factor receptor (EGFR) inhibitors have shown single agent activity in the treatment of non-small cell lung cancer (NSCLC). While mutant EGFRs more frequently show enhanced response to EGFR inhibition by erlotinib, the blockade of wt EGFRs also can promote long term survival in NSCLC patients. Here we have examined the mechanisms that may control response to EGFR inhibitors in NSCLC cell lines and xenografts that express either wild type EGFR or mutant EGFR. The molecular determinants which confer cell sensitivity in wild type (wt) EGFR tumors remain unclear. Using anti-phosphotyrosine affinity chromatography, quantitative isobaric peptide labeling and uLC-MSMS mass spectrometry methods, signaling networks associated in NSCLC lines sensitive (H292, H358), moderately sensitive (H441, A549) and refractory (H460, H1703, Calu6) to the downsteam effects of EGFR kinase inhibition were identified and protein components quantified. Over 200 phosphoproteins and proteins complexed therewith were unequivocally identified for each cell line, allowing cell line comparisons to be made. Comparison was made to two EGFR mutant lines, the erlotinib sensitive line H1650 (?746-750) and the refractory line H1975 (L858R). Proteins and complexes associated with the differential temporal sensitivity to EGFR pharmacological inhibition in NSCLC were identified using a multiplex isobaric peptide tagging approach allowing for quantitation of protein phosphorylation at several time points within a single experiment. In wt EGFR tumors, kinase blockade markedly disrupted complexes containing immediate signaling proteins, membrane adhesion complexes and internalization complexes in a manner correlating with sensitivity to erlotinib. The importance of an EGFR-dependent PI3 kinase pathway to erlotinib sensitivity was reinforced. The refractory nature of the EGFR L858R mutant line H1975 may be explained through alternative non-EGFR mechanisms leading to constitutive activation of Akt.
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Abstract Number: 4499
Presentation Title:
Identification of OSGPR78 and OSGPR114 as two novel lysophosphatidic acid G-protein coupled receptors.
Presentation Start/End Time:
Tuesday, Apr 19, 2005, 1:00 PM - 5:00 PM
Category:
CB01-02 Receptors
Author Block:
Graeme Griffin, April Thelemann, Gina Radosinic, Diane Triebe, Alexandra Eyzaguirre, Kathryn Weinslaw, Jianping Song, Graham Craggs, Utsav Bali, Salam Shaaban, Martin McKinney, Neil Gibson, Bert Oehlen, John Haley. OSI Pharmaceuticals, Farmingdale, NY
Lysophosphatidic acid (LPA) is a well characterized growth and survival factor for cancer cells in culture. This study details the identification of two G-protein coupled receptors, OSGPR78 and OSGPR114 as novel lysophosphatidic acid (LPA) receptors. OSGPR78 and OSGPR114, when expressed in a yeast-based reporter assay, are activated by multiple analogs of LPA, including myristoyl, palmitoyl, oleoyl, stearoyl and polyunsaturated LPA analogs. Similarly, when expressed in mammalian cells, the receptors also respond to the same LPA analogs. Pertussis toxin sensitivity of the effects of LPA activation of OSGPR114 expressed in CHO cells suggests a coupling to Gi/o proteins. Amino acid homologies of OSGPR114 and OSGPR78 demonstrate no obvious similarity with the known LPA G-protein coupled receptors LPA1-3. The expression of these novel LPA receptors in normal human tissues, cancer cell lines and in tumor samples has been characterized using fluorogenic real-time PCR. In normal tissues, OSGPR114 was expressed at the highest levels in areas such as the lung, kidney, leukocytes and ovary, whereas OSGPR78 was found to be expressed at high levels in almost every tissue studied. Several cancer cell lines have been identified which express OSGPR78 or OSGPR114 as the predominant LPA receptor and both receptors are widely expressed in human tumor samples. Furthermore, in cell lines shown to express OSGPR114 or OSGPR78, small interfering RNA oligonucleotides (siRNA) designed to specifically knockdown OSGPR114 and OSGPR78 expression induced a growth-inhibitory and pro-apoptotic phenotype in cells maintained in full serum. In summary, we demonstrate that OSGPR78 and OSGPR114 are novel LPA activated G-protein coupled receptors. Initial evidence suggests that OSGPR114 and OSGPR78, like the known LPA receptors (LPA1-3 and gpr23), are involved in mediating the proliferation and survival effects of LPA on cancer cells in culture.
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Abstract Number: 5284
Presentation Title:
Erlotinib sensitivity of mutant forms of EGFR in vitro and in vivo
Presentation Start/End Time:
Tuesday, Apr 19, 2005, 3:10 PM - 3:25 PM
Category:
ET06-09 Tyrosine kinase and phosphatase inhibitors
Author Block:
Andrew J. Garton, Jennifer Kahler, Qun-Sheng Ji, Gilda Mak, Matthew O'Connor, Yan Yao, Graeme Griffin, Eric Brown, Ken Foreman, April Thelemann, Filippo Petti, Barbara C. Leon, Frances E. Park, John Haley, David Emerson, Lee Arnold, Neil Gibson. OSI Pharmaceuticals, Farmingdale, NY, OSI Pharmaceuticals, Boulder, CO, Structural GenomiX, Inc., San Diego, CA
Erlotinib (Tarceva™) is a quinazoline-based epidermal growth factor receptor (EGFR) inhibitor, and recent Phase III clinical trial evaluation has revealed that this agent elicits a significant survival benefit in non-small cell lung cancer (NSCLC) patients. Recently, a number of different somatic mutations in the EGFR gene were identified, which occur in approximately 10% of NSCLC patients. Furthermore, it has been reported that patients expressing mutant forms of EGFR exhibit a clinical benefit relative to those expressing wild-type EGFR when treated with EGFR inhibitors. These observations have led to the suggestion that the presence of EGFR mutations may play a role in determining the response to EGFR inhibitors. The aim of the present study was to investigate the potential influence of EGFR mutations on erlotinib sensitivity in NSCLC. Comparison of the properties of a recombinant wild-type EGFR kinase domain with those of mutant kinase domains containing the two most prevalent types of mutation found in NSCLC patients (i.e. L858R point mutant and deletion mutant (del746-750)) revealed that in both cases the isolated kinase domains are catalytically competent, and furthermore display enhanced sensitivity to inhibition by erlotinib, particularly at high ATP concentrations (100µM). Interestingly, a second deletion mutant construct (del747-753) failed to demonstrate significant kinase activity in vitro, although the reason for this apparent difference is currently unclear. Analysis of cell lines that express different forms of EGFR revealed that their response to erlotinib in vitro varied considerably, even among cell lines expressing exclusively wild-type EGFR, including differential effects on signaling pathways downstream of EGFR as well as on the phenotypic consequences of incubation with erlotinib. The mutant EGFR-expressing lines examined also differed in their response to erlotinib treatment when grown as xenografts in vivo, in that growth of a cell line expressing an EGFR deletion mutant (H1650) was inhibited by erlotinib when dosed daily at 100 mg/kg, as was the wild-type EGFR-expressing line (H292), whereas in vivo growth of a cell line expressing the L858R point mutant (H1975) was not affected at the same dose level. The data therefore demonstrates that NSCLC cell lines expressing different mutant forms of EGFR are capable of responding differently to erlotinib, and that the presence of EGFR mutations in a subset of NSCLC patients is unlikely to be sufficient to explain the broad clinical anti-tumor activity of erlotinib in NSCLC.
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Abstract Number: 6140
Presentation Title:
Short course of EGF receptor tyrosine kinase inhibitor erlotinib (OSI-774, ‘Tarceva’) reduces tumor cell proliferation and active MAP kinase in situ in untreated operable breast cancers: A strategy for patient selection into phase II trials with signaling inhibitors
Presentation Start/End Time:
Wednesday, Apr 20, 2005, 8:40 AM - 8:55 AM
Category:
ET07-04 Growth factor receptors and other surface antigens as targets for therapy
Author Block:
Marta Guix, Mark S. Kelley, Michelle L. Reyzer, Joan Zhang, Yu Shyr, Bernadette K. McLaren, Kim Newsome-Johnson, Wendy Lipscomb, Teresa C. Dugger, Carlos L. Arteaga. Vanderbilt University, Nashville, TN
EGFR inhibitors are in clinical development for the treatment of solid tumors but, so far, they have shown modest clinical activity in unselected patients (pts) with breast cancer. These drugs might be active only in a small subpopulation of pts, but currently there are no known predictors of response. This study was designed: 1) to determine the biological activity of the drug in breast tumors, and 2) to identify a profile suggestive of EGFR dependence.
After the initial diagnostic biopsy, pts are treated with 150mg/day erlotinib for 10-14 days until surgery. The cellular activity of the drug is measured by changes in tumor cell proliferation (determined by Ki67 IHC) and apoptosis (by TUNEL) in the pre- versus post-treatment tissue samples. Two types of analysis are being conducted to identify predictive markers of response: 1) IHC studies of total EGFR, P-EGFR, P-HER2, HER2, Akt, P-Akt, MAPK, P-MAPK; and 2) protein profiling of whole tissue sections using matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS).
14 pts have been analyzed. Overall, treatment was well tolerated with no delays in surgery. Two pts stopped treatment due to grade 2 skin rash which was reversible upon discontinuation of therapy. 5/14 pts exhibited a =75% inhibition of tumor cell proliferation in the surgical biopsy (p=0.008, paired T-test). A marked reduction in P-MAPK (=85%) was seen in 8/14 pts (p=0.0002, paired T-test). EGFR protein was detected in 4/14 specimens but it did not correlate with changes in cell proliferation. Erlotinib did not induce apoptosis. Consistent with this finding, there were no significant changes in the levels of P-Akt, an important survival molecule in cancer cells. Cluster analysis of the protein profiles identified by MALDI-MS showed that pts can be correctly classified accordingly to their change in Ki67 using a small subset of protein peaks. Work is now ongoing to identify some of these peaks both in the patient tumor samples and in human breast cancer cell lines, where similar changes have been identified in response to erlotinib treatment. This trial represents a novel exploratory approach to define a molecular signature in a cohort of breast cancers in which EGFR inhibitors may have clinical activity. This profile could be then used to identify candidates for enrollment into phase II clinical trials with EGFR inhibitors. This approach is applicable to exploratory studies with other signaling inhibitors for which the molecular phenotype of appropriate pts is not clearly recognizable. More importantly, this strategy may allow for the exclusion of pts in which novel drugs are unlikely to exhibit an anti-cancer effect and, in turn, potentially accelerate drug approval.
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Abstract Number: 6165
Presentation Title:
Tarceva™ (erlotinib) exposure/effects (EE) analysis from a Phase III study in advanced NSCLC: Effect of smoking on the PK of erlotinib.
Presentation Start/End Time:
Wednesday, Apr 20, 2005, 8:55 AM - 9:10 AM
Category:
ET05-05 Pharmacokinetics and pharmacodynamics
Author Block:
Marta Hamilton, Julie L. Wolf, Denni Zborowski, Jianfeng Lu, Bertram L. Lum, Keyue Ding, Gary M. Clark, Ashok Rakhit, Lesley Seymour, Ann M. Ptaszynski, Jason Rusk, Frances Shepherd. OSI Pharmaceuticals Inc., Boulder, CO, Genentech, Inc., South San Francisco, CA, National Cancer Institute of Canada Clinical Trials Group, Kingston, ON, Canada, F. Hoffman LaRoche, Nutley, NJ, Princess Margaret Hospital, Toronto, ON, Canada
NCIC CTG BR21 was a randomized, double-blind, placebo-controlled study of erlotinib conducted in 731 NSCLC patients (pts) after at least 1 prior chemotherapy regimen. Pts were randomized 2:1 to daily oral erlotinib 150 mg tablets (n=485) or placebo. Baseline plasma samples and 1 predose sample/month/pt were specified for determination of erlotinib and its metabolite, OSI-420. Documented samples appropriate for Pop PK analysis were obtained from 342 pts; 311 pts had at least 1 steady state confirmed predose sample; and 133 patients had 3 or more steady state confirmed predose samples. Post-hoc estimates of PK parameters for individual pts were obtained from Pop PK, and an exploratory analysis using median trough plasma concentrations (where n=3) was also conducted. Wilcoxon rank sum and Kruskal-Wallis tests were used to compare erlotinib Cmax, C24h, and apparent clearance (CL/F) with baseline characteristics and safety outcomes (p<0.05 was considered significant). Pt characteristics included demographics, PS, prior chemotherapy (# regimens, best response, prior platinum), serum creatinine, bilirubin, stage and EGFR status. These subsets were not representative of the treated population, for example, median survival was 10, 14 and 6.7 months, respectively, for the Pop PK, median trough level, and overall erlotinib treatment group. Within the subsets analyzed, there were no consistent relationships between erlotinib exposure and safety outcomes. Post-hoc estimates of Cmax, Cl/F, and C24h differed significantly among pts categorized at baseline by age, prior wt. loss, PS, and smoking status. Of these, smoking status was associated with the greatest effect, with “never/former” (N/F) smokers having greater exposure to erlotinib than current smokers. In the Pop PK subset (n=342), current smokers (n=47) had median Cmax, C24h, and Cl/F values that were 79%, 60%, and 143% of that in the N/F smoking group (n=270). In the subset with = 3 trough samples, the median in current smokers (n=16) was 50% of those in N/F smokers (n=108). Erlotinib is metabolized in part by CYP1A2 and CYP1A1, which are inducible by cigarette smoke and may therefore account for these differences. The clinical relevance of this magnitude of reduced erlotinib exposure is not known. A healthy volunteer study is underway to further investigate the PK of erlotinib in smokers and nonsmokers. Results of the BR.21 EE analysis and the subsequent PK study will be presented.
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