Michelle Hemkens

Effects of chronic PPAR-agonist treatment on cardiac structure and function, blood pressure, and kidney in healthy sprague-dawley rats.

PPAR-γ agonists have been associated with heart failure (HF) in diabetic patients. These incidences have been reported mostly in patient populations who were at high risk for HF or had pre-existing impaired cardiovascular function. However, whether there are similar effects of these agents in subjects with no or reduced cardiovascular pathophysiology is not clear. In this study, the effects of chronic treatment with PD168, a potent peroxisome proliferator activated receptor (PPAR) subtype-γ agonist with weak activity at PPAR-α, and rosiglitazone (RGZ), a less potent PPAR-γ agonist with no PPAR-α activity, were evaluated on the cardiovascular-renal system in healthy male Sprague-Dawley (SD) rats by serial echocardiography and radiotelemetry. Rats were treated with vehicle (VEH), PD168, @ 10 or 50 mg/kg·bw/day (PD-10 or PD-50, resp.) or RGZ @ 180 mg/kg·bw/day for 28 days (n = 10/group). Relative to VEH, RGZ, and both doses of PD168 resulted in a significant fall in blood pressure. Furthermore, RGZ and PD168 increased plasma volume (% increase from baseline) 18%, 22%, and 48% for RGZ, PD-10, and PD-50, respectively. PD168 and RGZ significantly increased urinary aldosterone excretion and heart-to-body weight ratio relative to VEH. In addition, PD168 significantly decreased (10–16%) cardiac ejection fraction (EF) and increased left ventricular area (LVA) in systole (s) and diastole (d) in PD-10 and -50 rats. RGZ significantly increased LVAd; however, it did not affect EF relative to VEH. In conclusion, chronic PPAR-γ therapy may predispose the cardiorenal system to a potential sequela of structural and/or functional changes that may be deleterious with regard to morbidity and mortality.

Discovery of N-((3R,4R)-4-Fluoro-1-(6-((3-methoxy-1-methyl-1H-pyrazol-4-yl)amino)-9-methyl-9H-purin-2-yl)pyrrolidine-3-yl)acrylamide (PF-06747775) through Structure-Based Drug Design: A High Affinity Irreversible Inhibitor Targeting Oncogenic EGFR Mutants with Selectivity over Wild-Type EGFR.

Mutant epidermal growth factor receptor (EGFR) is a major driver of non-small-cell lung cancer (NSCLC). Marketed first generation inhibitors, such as erlotinib, effect a transient beneficial response in EGFR mutant NSCLC patients before resistance mechanisms render these inhibitors ineffective. Secondary oncogenic EGFR mutations account for approximately 50% of relapses, the most common being the gatekeeper T790M substitution that renders existing therapies ineffective. The discovery of PF-06459988 (1), an irreversible pyrrolopyrimidine inhibitor of EGFR T790M mutants, was recently disclosed.1 Herein, we describe our continued efforts to achieve potency across EGFR oncogenic mutations and improved kinome selectivity, resulting in the discovery of clinical candidate PF-06747775 (21), which provides potent EGFR activity against the four common mutants (exon 19 deletion (Del), L858R, and double mutants T790M/L858R and T790M/Del), selectivity over wild-type EGFR, and desirable ADME properties. Compound 21 is currently being evaluated in phase-I clinical trials of mutant EGFR driven NSCLC.

Abstract LB-203: Development of a nonclinical model for the assessment of oncology therapy-induced cardiomyopathy

With the increasing number of people surviving cancer, patients and doctors are becoming equally focused on the quality of life in survivorship as they are on fighting cancer. Consequently, cardio-oncology has emerged as a new clinical discipline with focus on both the stratification of patients at risk and therapies that pose a cardiovascular risk, eg molecularly targeted therapies including tyrosine kinase inhibitors (TKI). Therefore, nonclinical research should strive not only to detect preclinical drug-induced changes in cardiac structure or function, but also to understand the relevance of these cardiotoxic effects to humans. As is typical for new disciplines, there is an unmet need for early and predictive tools to identify both of these factors. Currently, preclinical assessment of cardiovascular toxicity consists of repeat-dose in vivo toxicity studies focused primarily on histopathological endpoints; additional measures, such as cardiac function and biomarkers of cardiac injury, are not routinely assessed in these studies. Furthermore, analysis of these studies suggests that using young and healthy, drug-naive animals do not adequately translate the TKI-induced cardiomyopathy phenotype; therefore, new nonclinical models are needed to address this gap. The developed model would ideally be used to assess the preclinical cardiotoxicity of proposed oncology therapies, but may also have utility in assessing cardioprotective therapies for use in cardio-oncology. This poster will propose a new model for studying cardio-oncology nonclinically that includes myocardial transcriptional signatures, strain echocardiography imaging, and pharmacologic (dobutamine) stress challenge. In general, rats with radiotelemetry were treated with vehicle or low dose doxorubicin (2 mg/kg/week IP) for 1 month. Cardiac structure and function at rest and during stress were assessed using strain echocardiography in concert with a weekly dobutamine infusion. Circulating blood biomarkers of cardiac toxicity (eg, NTproBNP, NTproANP, cTropin, and miR-208) were collected weekly and at necropsy. At the end of the study, samples of cardiac tissue were taken from the ventricular apex and were submitted for transcriptional profiling along with standard histopathology. These data as well as recent publications give high confidence in developing a rodent model predictive of drug-induced cardiomyopathy. Citation Format: Michelle Hemkens, Samantha Cardenas, Jessica Frey, Brad Hirakawa, Petra Koza-Taylor, Jianying Wang, Paul Butler. Development of a nonclinical model for the assessment of oncology therapy-induced cardiomyopathy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-203.

Abstract 2594: Characterization of a novel irreversible third generation EGFR TKI that targets T790M-mediated resistant EGFR-mutant NSCLC while sparing wild type EGFR

Activating mutations in EGFR confer constitutive activity providing the oncogenic drive in EGFR-mutant NSCLC. First and 2nd generation EGFR tyrosine kinase inhibitors (TKIs) are effective drugs in this setting, but are constrained by dose-limiting toxicities attributed to inhibition of wild type (WT) EGFR and by drug resistance caused, in the majority of cases, via a T790M secondary mutation in EGFR. We report the pharmacology of a novel irreversible 3rd generation EGFR TKI active against EGFR with activating and T790M mutations, but sparing WT EGFR. Our novel 3rd generation EGFR TKI was studied in a variety of in vitro and in vivo models to determine its inhibitory potencies on different EGFR variants, pharmacokinetics (PK), antitumor efficacy, exposure-response relationships, mechanism of action, and predicted human efficacious dose. In enzyme and cell assays, our compound is a highly potent inhibitor of EGFR double mutants (L858R/T790M and Del/T790M) and EGFR activating mutants (L858R and Del), but a weak inhibitor of WT EGFR (26-fold margin over mutant target potencies). Effects on downstream signaling and function indicate the underlying mechanism of the compound is direct inhibition of EGFR, with subsequent inhibition of downstream signaling that results in apoptosis and viable cell decline. In xenograft mouse models, the compound demonstrates tumor growth inhibition and regression at well-tolerated doses in models driven by EGFR double mutants and EGFR activating mutants. The antitumor efficacy is dose-dependent and strongly correlates with inhibition of EGFR phosphorylation and EGFR-mediated downstream signaling, and induction of apoptosis. Plasma concentrations assumed to be sufficient for efficacy (Ceff) were defined using a mathematical model incorporating the plasma levels of the compound, the associated inhibitory effects on EGFR phosphorylation, and the antitumor efficacy in the double and activating mutant xenograft models. Ceff was in agreement across several models and was used with in vitro human PK properties to calculate required human dose. While our compound possesses a similar profile as other recently disclosed 3rd generation EGFR TKIs, this molecule is distinguished by better potency on the activating mutants and by the widest potency margin on WT EGFR. Given that the target potencies and WT margins of 3rd generation EGFR TKIs have been sufficient for tolerated clinical efficacy in preliminary results, it can be inferred that our compound will have similar promise in the clinic. These results support our compound as a novel EGFR TKI with an inhibitory profile and favorable drug-like properties that suggest utility for treating patients with NSCLC with EGFR activating and resistance mutations. Citation Format: Mike Zientek, Sangita Baxi, Henry Cheng, Valeria Fantin, Jun Li Feng, Allison Given, Zelanna Goldberg, Jie Guo, Michelle Hemkens, John Kath, Jennifer Lafontaine, Gary Li, Pramod Mehta, Brion Murray, Sajiv Nair, Simon Planken, Chad Ray, Yuli Wang, Manli Shi, Anand Sistla, Tod Smeal, Greg Stevens, Wei Tan, Paolo Vicini, Marlena Walls, Liu Yang, Min-Jean Yin, Scott L. Weinrich. Characterization of a novel irreversible third generation EGFR TKI that targets T790M-mediated resistant EGFR-mutant NSCLC while sparing wild type EGFR. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2594. doi:10.1158/1538-7445.AM2015-2594

Abstract 4479: PF-04691502, a potent and selective PI3K/mTOR dual inhibitor with antitumor activity

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The PI3K pathway, which regulates cell growth, proliferation and survival, is activated in many types of human tumors by mutational activation of PI3Kα, loss of function of PTEN or activation of receptor tyrosine kinases. Inhibition of key signaling proteins in the pathway, such as PI3K, AKT and mTOR, therefore represents a high value targeting strategy for diverse cancers. PF-04691502 is a dual-specificity inhibitor of PI3K and mTOR which shows potent and selective activity in in vitro biochemical, cell and xenograft models. In in vitro biochemical assays PF-04691502 inhibited recombinant PI3Kα, β, γ and δ isoforms with Kis of 1.2-2.2 nM and recombinant mTOR with a Ki of 9.1 nM. PF-04691502 demonstrated a high degree of selectivity for inhibition of PI3K family kinases as shown by lack of activity against a panel of >75 protein kinases, including the Class III PI3K hVps34. PF-04691502 also inhibited transformation of avian fibroblasts mediated by PI3K γ, δ, mutant PI3Kα E545K or membrane-localized AKT with IC50s of ∼100nM. In cell assays PF-04691502 inhibited PI3K/mTOR signaling in SKOV3 ovarian cancer cells with PI3Kα mutations and in U87MG glioblastoma cells with PTEN alteration, as indicated by reduced levels of phosphorylation of AKT(T308), AKT(S473) and S6 ribosomal proteins. Functional studies for anti-proliferative effects suggest PF-04691502 has broad efficacy across tumor types. In SKOV3 and U87MG xenograft models PF-04691502 treatment resulted in dose-dependent tumor growth inhibition (TGI) with maximum TGI of ∼70% at the maximum tolerated dose of 10 mg/kg, by once daily oral dosing. Inhibition of AKT(S473) phosphorylation and S6RP(S235/236)/PRAS40(T246)/4EBP1(T37/46) phosphorylation were used as quantitative and qualitative pharmacodynamic (PD) endpoints, respectively; a clear pharmacokinetic (PK)/PD relationship was established in both models after multiple dose oral administration. In the U87MG xenograft model AKT(S473) phosphorylation was inhibited with an estimated EC50 of 5.7 nM (free plasma concentration) based on PK/PD modeling. The free plasma Area Under Curve was estimated to be 850 nM*hr for 70% TGI at 10mg/kg and was found to be similar in the SKOV3 model. The projected human efficacious dose of 10 mg once daily oral dosing provides Caverage steady state exposure of 22.4 nM (free plasma concentration) which is sufficient for 50-80% inhibition of pAKT S473, and corresponds to 74% TGI. Phase 1 clinical trials of PF-04691502 as a single agent are planned. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4479.

Abstract 5043: Effects of a novel PI3 kinase/mTOR inhibitor on proliferation and pAKT signaling in canine lymphoma

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC PF-04691503 is a dual inhibitor of both PI3K and mTOR, inhibits PI3K signaling in cancer cell lines, and exhibits in vitro and in vivo anti-proliferative activity in PI3K-pathway driven cell lines. It is a nanomolar inhibitor of all 4 isoforms of the catalytic subunit of PI3K and of both TORC1 and TORC2. Anti-cancer activity of the inhibitor is hypothesized to be through inhibition of survival, proliferative, and anti-apoptotic processes. Phosphorylation of the protein kinase, Akt, is associated with activation of the phosphatidyl 3-kinase (PI-3K)/mammalian target of rapamycin (mTOR) signaling pathway, which plays a role in cell proliferation (Witzig and Kaufmann 2006). Recent studies by Gulmann et al (2005) showed increased pAKT in human lymphoma samples, and those of Rassidakis et al (2005) suggested that inhibition of Akt phosphorylation (pAKT) may be of value in the treatment of lymphoma. Using flow cytometry we report that cells obtained from 10 of 11 lymph node biopsies of dogs with lymphoma exhibit detectable pAKT using a phospho-Akt (Ser473) antibody, compared to an IgG isotype control or a competative phospho-Akt (Ser473) blocking peptide. Cells from normal canine lymph nodes do not exhibit detectable pAKT. The majority of the pAKT signal was generated from lymphoblasts present in the malignant, but not in the normal, lymph nodes. In separate studies, lymph node cells obtained from healthy dogs and dogs with lymphoma were stimulated in vitro with the mitogen, Con A. The novel PI3K/mTOR dual inhibitor, PF-04691503, produced dose dependent inhibition of proliferation as exemplified in a dog with T-cell lymphoma (EC50 = 18 nM)) and in a normal dog (EC50 = 53 nM)). No pAKT signal could be detected in peripheral blood mononuclear cells, from normal or lymphoma patients stimulated with hu-IGF-1 (the endogenous ligand for PI3), Con-A or LPS. These data suggest that PI-3 kinase and pAKT (1) are activated in canine lymphoma, (2) play a role in the lymphoproliferation associated with this disease, (3) represent legitimate targets for therapeutic intervention in lymphoma, and (4) can be studied ‘translationally’ in dogs as a model for humans. Gulmann C, Espina V, Petricoin E, et al. Proteomic analysis of apoptotic pathways reveals prognostic factors in follicular lymphoma. Clin Cancer Res 11:5847-5855, 2005. Rassidakis GZ, Feretzaki M, Atwell C, et al. Inhibition of Akt increases p27 Kip1 levels and induces cell cycle arrest in anaplastic large cell lymphoma. Blood 105:827-829, 2005. Witzig TE and Kaufmann SH. Inhibition of phosphatidyl 3-kinase (PI-3K)/mammalian target of rapamycin (mTOR) pathway in hematologic malignancies. Current Treatment Options in Oncology 7:285-294, 2006. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5043.