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Supplementary data on cell lines

Pharmacokinetics and PK/PD relationships for AZD2014

Pharmacokinetics modelling of AZD2014 intermittent dosing schedule.

HCC1428-eveR and HCC1428-LTED-eveR cell lines resistant to everolimus remain sensitive to dual mTORC1/2 inhibitor AZD2014.

Supplementary table 1: Biochemical and cellular activity of AZD2014. Supplementary table 2: Biochemical activity of AZD2014. Supplementary table 3: Anti proliferative activity of AZD2014 in a panel of ER+, endocrine resistant and LTED cell lines. Supplementary table 4: Activity of AZD2014 in a number of ER+ breast cancer in vivo models.

Supplementary Table 1 from AZD8055 Is a Potent, Selective, and Orally Bioavailable ATP-Competitive Mammalian Target of Rapamycin Kinase Inhibitor with In vitro and In vivo Antitumor Activity

The mammalian target of rapamycin (mTOR) kinase forms two multiprotein complexes, mTORC1 and mTORC2, which regulate cell growth, cell survival, and autophagy. Allosteric inhibitors of mTORC1, such as rapamycin, have been extensively used to study tumor cell growth, proliferation, and autophagy but have shown only limited clinical utility. Here, we describe AZD8055, a novel ATP-competitive inhibitor of mTOR kinase activity, with an IC50 of 0.8 nmol/L. AZD8055 showed excellent selectivity (∼1,000-fold) against all class I phosphatidylinositol 3-kinase (PI3K) isoforms and other members of the PI3K-like kinase family. Furthermore, there was no significant activity against a panel of 260 kinases at concentrations up to 10 μmol/L. AZD8055 inhibits the phosphorylation of mTORC1 substrates p70S6K and 4E-BP1 as well as phosphorylation of the mTORC2 substrate AKT and downstream proteins. The rapamycin-resistant T37/46 phosphorylation sites on 4E-BP1 were fully inhibited by AZD8055, resulting in significant inhibition of cap-dependent translation. In vitro, AZD8055 potently inhibits proliferation and induces autophagy in H838 and A549 cells. In vivo, AZD8055 induces a dose-dependent pharmacodynamic effect on phosphorylated S6 and phosphorylated AKT at plasma concentrations leading to tumor growth inhibition. Notably, AZD8055 results in significant growth inhibition and/or regression in xenografts, representing a broad range of human tumor types. AZD8055 is currently in phase I clinical trials. Cancer Res; 70(1); 288–98

Genetic testing is available and widely used in the neonatal intensive care setting for a broad range of complex presentations arising both antenatally and in the postnatal period. Here we discuss the investigations currently available to clinicians in the neonatal intensive care unit, such as array comparative genomic hybridization (aCGH), FISH (fluorescent in situ hybridization) and quantitative florescent polymerase chain reaction (QF-PCR), later looking to the future of clinical genomic testing, in the form of next generation sequencing panels (NGS panels), whole exome (WE) and whole genome (WG) sequencing. The mainstreaming of genetic medicine requires non-genetic specialists, such as neonatologists and paediatricians, to have confidence and knowledge of the testing process, the technologies available and the ethical and social considerations associated with their use.

mTOR is an atypical serine threonine kinase involved in regulating major cellular functions, such as nutrients sensing, growth, and proliferation. mTOR is part of the multiprotein complexes mTORC1 and mTORC2, which have been shown to play critical yet functionally distinct roles in the regulation of cellular processes. Current clinical mTOR inhibitors only inhibit the mTORC1 complex and are derivatives of the macrolide rapamycin (rapalogs). Encouraging effects have been observed with rapalogs in estrogen receptor–positive (ER+) breast cancer patients in combination with endocrine therapy, such as aromatase inhibitors. AZD2014 is a small-molecule ATP competitive inhibitor of mTOR that inhibits both mTORC1 and mTORC2 complexes and has a greater inhibitory function against mTORC1 than the clinically approved rapalogs. Here, we demonstrate that AZD2014 has broad antiproliferative effects across multiple cell lines, including ER+ breast models with acquired resistance to hormonal therapy and cell lines with acquired resistance to rapalogs. In vivo, AZD2014 induces dose-dependent tumor growth inhibition in several xenograft and primary explant models. The antitumor activity of AZD2014 is associated with modulation of both mTORC1 and mTORC2 substrates, consistent with its mechanism of action. In combination with fulvestrant, AZD2014 induces tumor regressions when dosed continuously or using intermittent dosing schedules. The ability to dose AZD2014 intermittently, together with its ability to block signaling from both mTORC1 and mTORC2 complexes, makes this compound an ideal candidate for combining with endocrine therapies in the clinic. AZD2014 is currently in phase II clinical trials. Mol Cancer Ther; 14(11); 2508–18. ©2015 AACR.

AZD8055 is a small-molecule inhibitor of mTOR (mammalian target of rapamycin) kinase activity. The present review highlights molecular and phenotypic differences between AZD8055 and allosteric inhibitors of mTOR such as rapamycin. Biomarkers, some of which are applicable to clinical studies, as well as biological effects such as autophagy, growth inhibition and cell death are compared between AZD8055 and rapamycin. Potential ways to develop rational combinations with mTOR kinase inhibitors are also discussed. Overall, AZD8055 may provide a better therapeutic strategy than rapamycin and analogues.

This study had 3 major aims: (1) to ascertain the degree to which helicopter emergency medical services (HEMS) administration of antibiotics (Abx) can streamline the time to Abx in open fracture patients, (2) to determine whether any clinical outcome improvements were associated with HEMS Abx therapy, and (3) to calculate the cost-effectiveness of prehospital HEMS Abx. The design of the study was a prospective, nonrandomized, nonintervention, natural study of timing and clinical outcomes for patients with suspected open extremity fracture. There were 138 scene trauma cases transported by 8 participating HEMS programs from July 2009 to June 2010. The participating HEMS programs were both urban and rural. The diagnosis of an open fracture by the HEMS crews had an accuracy rate of 97.8% (95% confidence interval, 90.8%–98.4%). The time from the incident to Abx was 30 minutes shorter ( P = .0001) when Abx were administered by HEMS crews. There was no statistical significance ( P = 1.0) regarding the endpoint of infection or nonunion development in HEMS- versus hospital-administered Abx. In conclusion, the administration of Abx by HEMS crews to patients diagnosed with open extremity fractures is feasible, it may decrease the time to Abx by 30 minutes, and the effect magnitude (40.3% relative risk reduction) was promising.

Vandetanib is a multi-targeted receptor tyrosine kinase inhibitor that is in clinical development for the treatment of solid tumours. This preclinical study examined the inhibition of two key signalling pathways (VEGFR-2, EGFR) at drug concentrations similar to those achieved in the clinic, and their contribution to direct and indirect antitumour effects of vandetanib. For in vitro studies, receptor phosphorylation was assessed by Western blotting and ELISA, cell proliferation was assessed using a cell viability endpoint, and effects on cell cycle determined using flow cytometry. For in vivo studies, Western blotting, ELISA and immunohistochemistry (IHC) were used to assess receptor phosphorylation. Cell culture experiments demonstrated that anti-proliferative effects of vandetanib resulted from inhibition of either EGFR or VEGFR-2 signalling in endothelial cells, but were associated with inhibition of EGFR signalling in tumour cells. Vandetanib inhibited both EGFR and VEGFR-2 signalling in normal lung tissue and in tumour xenografts. In a lung cancer model expressing an activating EGFR mutation, the activity of vandetanib was similar to that of a highly selective EGFR inhibitor (gefitinib), and markedly greater than that of a highly selective VEGFR inhibitor (vatalanib). These data suggest that at the plasma exposures achieved in the clinic, vandetanib will significantly inhibit both VEGFR-2 and EGFR signalling, and that both inhibition of angiogenesis and direct inhibition of tumour cell growth can contribute to treatment response.

The mammalian target of rapamycin (mTOR) kinase forms two multiprotein complexes, mTORC1 and mTORC2, which regulate cell growth, cell survival, and autophagy. Allosteric inhibitors of mTORC1, such as rapamycin, have been extensively used to study tumor cell growth, proliferation, and autophagy but have shown only limited clinical utility. Here, we describe AZD8055, a novel ATP-competitive inhibitor of mTOR kinase activity, with an IC50 of 0.8 nmol/L. AZD8055 showed excellent selectivity (∼1,000-fold) against all class I phosphatidylinositol 3-kinase (PI3K) isoforms and other members of the PI3K-like kinase family. Furthermore, there was no significant activity against a panel of 260 kinases at concentrations up to 10 μmol/L. AZD8055 inhibits the phosphorylation of mTORC1 substrates p70S6K and 4E-BP1 as well as phosphorylation of the mTORC2 substrate AKT and downstream proteins. The rapamycin-resistant T37/46 phosphorylation sites on 4E-BP1 were fully inhibited by AZD8055, resulting in significant inhibition of cap-dependent translation. In vitro, AZD8055 potently inhibits proliferation and induces autophagy in H838 and A549 cells. In vivo, AZD8055 induces a dose-dependent pharmacodynamic effect on phosphorylated S6 and phosphorylated AKT at plasma concentrations leading to tumor growth inhibition. Notably, AZD8055 results in significant growth inhibition and/or regression in xenografts, representing a broad range of human tumor types. AZD8055 is currently in phase I clinical trials. Cancer Res; 70(1); 288–98

The mammalian target of rapamycin (mTOR) is a major sensor of nutrients and energy and is part of two multiprotein complexes mTORC1 and mTORC2. mTOR activation is widely reported in cancer and is associated with poor prognosis and resistance to standard of care treatment. Allosteric inhibitors of mTOR such as rapamycin partially inhibit mTORC1 and do not inhibit mTORC2. AZD2014 is a close analogue of AZD8055 and a selective inhibitor of mTOR kinase. AZD2014 has greater inhibitory activity against mTORC1 compared to rapamycin: AZD2014 decreases p4EBP1 Thr37/46, inhibits the translation initiation complex and decreases overall protein synthesis while rapamycin has no effect. AZD2014 also inhibits the mTORC2 biomarkers pAKTSer473 and pNDRG1Thr346. AZD2014 has broad antiproliferative activity across multiple tumour cell lines. In particular, AZD2014 induces growth inhibition and cell death in breast cancer cell lines, including ER+ cell lines with acquired resistance to hormone therapy. In vitro, the potency of AZD2014 is ∼ 5-fold lower compared to AZD8055. However, AZD2014 has improved pharmacokinetics compared to AZD8055. In vivo, AZD2014 induces tumour growth inhibition against several xenograft models including a human primary explant model of ER+ breast cancer refractory to tamoxifen. The antitumour activity is associated with modulation of both mTORC1 and mTORC2 substrates. Overall, AZD2014 is a potent mTOR kinase inhibitor with an in vitro profile comparable to AZD8055 but with improved pharmacokinetic properties over AZD8055. AZD2014 is currently in phase 1 clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 917. doi:1538-7445.AM2012-917