Supplementary Data from Antitumor Activity and Pharmacology of a Selective Focal Adhesion Kinase Inhibitor, PF-562,271

Highly efficient photo-cross-linking reactions enable numerous applications in biomaterials. Here, a photopatternable biodegradable aliphatic polyester with benzophenone pendent groups was synthesized by copper-catalyzed alkyne-azide cycloaddition, affording polyesters that undergo UV-induced cross-linking to yield photopatterned films. Using this material, a self-folding multilayer structure containing polyester/hydrogel bilayer hinges was fabricated. Upon swelling of the hydrogel layer, the construct folds into a triangular tube, which subsequently unfolds due to lipase-catalyzed degradation of the polyester layer. The ability to precisely design such degradation-induced structural changes offers potential for biomaterials and medical applications, such as evolving and responsive 2D and 3D tissue engineering scaffolds.

Iron (Fe) uptake and transfer to the fetus is mediated by the placenta. The objectives of this study were to examine the degree of variability in neonatal Fe status between siblings born to women carrying twins or triplets and to investigate determinants of neonatal Fe status in this group. A total of 67 women carrying multiples aged 30.6 ± 5.2 were enrolled between the 1st and 3rd trimester of pregnancy. Maternal blood across pregnancy and umbilical cord blood at birth were utilized to assess neonatal Fe status including Hb, serum ferritin (SF), transferrin receptor (TfR), erythropoietin (EPO), and hepcidin. Neonates were born at week 34.5 ± 2.9 of gestation and weighed 2188 ± 613 g; 69% of neonates were preterm and 72% had low birth weight (LBW). Approximately 19% of neonates were anemic (Hb<13 g/dL) at birth, 27.3% had SF < 76 µg/L and 18% had TfR > 8.5 mg/L. Mean cord SF was 120 ± 82 µg/L (n=70); a value comparable to the 50th percentile using normative data. Total body iron (TBI) in neonates was calculated using birth weight (kg), cord Hb (g/dL) and functional Fe in tissues as described by Georgieff et al with adjustment for gestational age at birth. TBI averaged 171 ± 27.3 mg per neonate at birth (n = 65) was significantly correlated to amnionicity (p = 0.0005). Ratio between maternal and neonatal TBI was 0.61 ± 0.1. Significant variability was evident in cord SF (n = 70, p = 0.0002), TfR (n = 74, P=0.0001) and neonatal birth weight (p < 0.0001) among siblings. High rates of preterm birth/LBW and low TBI stores at birth suggest the need for more data on determinants of Fe status and anemia in this group.Grant Funding Source: Supported by the Gerber Foundation

Pregnant teens may be at increased risk for pregnancy‐induced bone loss. To assess the impact of dietary Ca and vitamin D status on bone loss, 171 teens (13–18 yrs) were followed from 22.3±5.9 weeks of gestation through delivery. Bone quality was assessed up to three times across pregnancy using calcaneus heel ultrasound (US) measures. Vitamin D, calcitropic hormones, and osteoprotegerin (OPG) were assessed at mid‐gestation and delivery. Speed of sound (SOS) at the heel decreased as pregnancy progressed (p<0.001, after controlling for multiple measures). SOS decreased by 8.6±17.0 m/s (over an 11.7±5.6 week interval), which is equivalent to a 0.28±0.54 decrease in T‐score. The change in SOS/week was positively associated with pre‐pregnancy weight (p=0.012) and BMI (p=0.007), such that heavier teens experienced less bone loss. Bone loss did not differ by race, age, gynecological age, or diet (caloric, protein, or Ca intake). The decrease in SOS across pregnancy was not influenced by mid‐gestation or delivery 25(OH)D, calcitriol, or PTH concentrations. Teens who did not lose bone SOS exhibited a trend for higher OPG at delivery (p=0.089). Maternal bone loss was not associated with infant weight, ponderal index, or gestational age at birth. These findings suggest that teens are at risk for pregnancy‐induced bone loss and BMI pre‐conception plays a role in adolescents’ skeletal response to pregnancy.USDA: 2005‐35200

The NASA Glenn Research Center Auditory Demonstration Laboratory (ADL) is a dual purpose facility, constructed in 2007 to support hearing conservation programs across the agency. Configured as a reverberant room, the ADL is an appropriate space for evaluating the performance of personal hearing protectors, using either human subjects or a test fixture. Hearing protector evaluations are conducted using NASA REATMASTER software, developed in partnership with the National Institute for Occupational Safety and Health. This software is available free on request to qualified laboratories, which are encouraged to participate in a collaborative program to fund continued software development. The ADL can also be configured as a free-field room to support the development of auditory demonstrations, widely used for a variety of training purposes within NASA and externally. The ADL provides an environment, sound system, and audio engineering tools for presenting and developing calibrated demonstrations of various acoustical and auditory phenomena that include fundamental acoustical and concepts, noise control principles, and simulations of hearing loss. Current work at the ADL will establish the capability of making three-dimensional surround sound recordings, which will expand the scope of the laboratory’s educational products into additional areas of psychoacoustics such as binaural hearing and localization.

AbstractCancer cells are characterized by the ability to grow in an anchorage-independent manner. The activity of the nonreceptor tyrosine kinase, focal adhesion kinase (FAK), is thought to contribute to this phenotype. FAK localizes in focal adhesion plaques and has a role as a scaffolding and signaling protein for other adhesion molecules. Recent studies show a strong correlation between increased FAK expression and phosphorylation status and the invasive phenotype of aggressive human tumors. PF-562,271 is a potent, ATP-competitive, reversible inhibitor of FAK and Pyk2 catalytic activity with a IC50 of 1.5 and 14 nmol/L, respectively. Additionally, PF-562,271 displayed robust inhibition in an inducible cell-based assay measuring phospho-FAK with an IC50 of 5 nmol/L. PF-562,271 was evaluated against multiple kinases and displays >100× selectivity against a long list of nontarget kinases. PF-562,271 inhibits FAK phosphorylation in vivo in a dose-dependent fashion (calculated EC50 of 93 ng/mL, total) after p.o. administration to tumor-bearing mice. In vivo inhibition of FAK phosphorylation (>50%) was sustained for >4 hours with a single p.o. dose of 33 mg/kg. Antitumor efficacy and regressions were observed in multiple human s.c. xenograft models. No weight loss, morbidity, or mortality were observed in any in vivo experiment. Tumor growth inhibition was dose and drug exposure dependent. Taken together, these data show that kinase inhibition with an ATP-competitive small molecule inhibitor of FAK decreases the phospho-status in vivo, resulting in robust antitumor activity. [Cancer Res 2008;68(6):1935–44]

Polymer-protein conjugation was performed using N-hydroxysuccinimide and aldehyde-terminated zwitterionic polymers, and the resulting polymer-protein conjugates were characterized by gel electrophoresis and fast protein liquid chromatography. Methacryloyloxyethyl phosphorylcholine (MPC) polymers were prepared by atom transfer radical polymerization in which the requisite functional end-groups for protein conjugation were embedded within the polymerization initiators. These phosphorylcholine polymers were conjugated to lysozyme as a model protein, as well as two therapeutic proteins, granulocyte colony stimulating factor (G-CSF) and erythropoietin (EPO). These MPC polymer-protein conjugates represent alternatives to PEGylated proteins, with the potential to provide improved efficacy in a therapeutic treatment relative to the protein itself.

The synthesis and SAR for a series of diaminopyrimidines as PYK2 inhibitors are described. Using a combination of library and traditional medicinal chemistry techniques, a FAK-selective chemical series was transformed into compounds possessing good PYK2 potency and 10- to 20-fold selectivity against FAK. Subsequent studies found that the majority of the compounds were positive in a reactive metabolite assay, an indicator for potential toxicological liabilities. Based on the proposed mechanism for bioactivation, as well as a combination of structure-based drug design and traditional medicinal chemistry techniques, a follow-up series of PYK2 inhibitors was identified that maintained PYK2 potency, FAK selectivity and HLM stability, yet were negative in the RM assay.

Focal adhesion kinase (FAK) is a member of a family of nonreceptor protein-tyrosine kinases that regulates integrin and growth factor signaling pathways involved in cell migration, proliferation, and survival. FAK expression is increased in many cancers, including breast and prostate cancer. Here we describe perturbation of adhesion-mediated signaling with a FAK inhibitor, PF-573,228. In vitro, this compound inhibited purified recombinant catalytic fragment of FAK with an IC50 of 4 nM. In cultured cells, PF-573,228 inhibited FAK phosphorylation on Tyr(397) with an IC50 of 30-100 nM. Treatment of cells with concentrations of PF-573,228 that significantly decreased FAK Tyr(397) phosphorylation failed to inhibit cell growth or induce apoptosis. In contrast, treatment with PF-573,228 inhibited both chemotactic and haptotactic migration concomitant with the inhibition of focal adhesion turnover. These studies show that PF-573,228 serves as a useful tool to dissect the functions of FAK in integrin-dependent signaling pathways in normal and cancer cells and forms the basis for the generation of compounds amenable for preclinical and patient trials.

ND-4 Cancer cells are characterized by the ability to grow in an anchorage-independent manner. This characteristic phenotype is due, in large part, to the activity of the non-receptor tyrosine kinase, focal adhesion kinase. FAK localizes in focal adhesion plaques and has a role as a scaffolding and signaling protein for other adhesion molecules. As such, FAK acts as a signaling molecule for many integrins which do not have intrinsic kinase activity, as well as, complimenting signal transduction through other RTKs (e.g. EGFR, VEGFR).Recent studies demonstrate a strong correlation between increased FAK expression and the invasive phenotype of aggressive human tumors. There has been some controversy regarding the importance of the kinase activity relative to the scaffolding functions of FAK.Potent inhibitors of FAK have been identified based upon a discovery approach that combined compound screening, structure-based drug design and traditional medicinal chemistry. Two different series of compounds, 2,4-di-anilino pyrimidines and 3,5-di-substituted indoles, were found to be modest inhibitors of focal adhesion kinase. Modeling and co-crystal structures of these inhibitors with FAK led to specifically substituted 2,4-diamino pyrimidines, which were found to inhibit FAK in both kinase and cell assays (1 nM-900 nM). Within this particular class of molecules, the anchoring hydrogen bond donor-acceptor motif for kinase activity was identified. Small, seemingly minor changes to inhibitor structure caused major conformational changes in the way the inhibitor bound and co-crystallized with FAK. Structure-based drug design (SBDD) paved the way for design of novel inhibitors with optimal ADME, selectivity, and potency properties by specific substitution at the C2, C4, and C5 positions of the 2,4-diamino pyrimidine core. Replacement of the 3,5-di-substituted indole moiety with a 5-amino oxindole at the pyrimidine C2 position allowed us to retain a key hydrogen bond interaction to Arg 426, while reducing MW and removing a potential metabolic liability in the original dehydropiperidine. Co-crystal structures of C4 derivatives demonstrated the plasticity of the kinase active site in this region, and furthermore suggested a possible route to achieve selectivity over other kinases by regioselective substitution off of the amino-methyl aryl ring with a RSO2R substituent. Finally, SBDD led to the replacement of the original (pyrimidine C5) Br atom with a CF3 group. PF-562,271 is a potent ATP competitive, reversible inhibitor of FAK and Pyk2 kinase with IC50 of 1.5 and 14 nM, respectively. PF-562,271 is potent in an inducible cell based assay* measuring phospho-FAK with an IC50 of 5 nM. PF-562,271 was evaluated in a number of kinase screens and panels and displays [[Unsupported Character - Codename s]]>100x selectivity against a long list of non-target kinases. PF-562,271 inhibits FAK phosphorylation in vivo in a dose dependent fashion (calculated EC50 35 ng/mL, free, 0.5-4 hrs post dose) following oral administration to female athymic (nu/nu) mice bearing human glioblastoma, U87MG subcutaneous tumors. In vivo inhibition of FAK phosphorylation was sustained (56%) for over 4 hours with a single oral dose of 33 mg/kg. The antitumor efficacy of PF-562,271 was evaluated in the following human s.c. xenograft models: PC-3M (prostate), BT474 (breast), BxPc3 (pancreatic), LoVo (colon), U87MG (glioblastoma), and H460 (lung). Regressions were observed in PC-3M, BT474, BxPc3, and LoVo models at doses of 25-50 mg/kg, BID corresponding to Cmax (free) ranges of 77-885 ng/ml, Cave (free) of 14-40 ng/ml, and inhibition of phospho-FAK of 31-76% for >4 hours. Maximum tumor growth inhibition in the U87MG tumor was 51% using an osmotic mini-pump corresponding to a Css of 0.7 ng/mL (free) with a concomitant decrease in phospho-FAK of 45%. No weight loss, morbidity, or mortality were observed in any TGI experiment (up to 50 mg/kg BID x 28 days or 100 mg/kg QD x 25 days). Tumor growth inhibition was dose and drug exposure dependent. BID dosing and mini-pump experiments (Css) resulted in greater tumor growth inhibition in multiple models compared to QD dosing (equivalent total daily dose) suggesting Cave and the time above the Cave are more relevant to efficacy than Cmax. Taken together, these data demonstrate that kinase inhibition with an ATP competitive small molecule inhibitor of FAK results in robust anti-tumor activity. This compound is presently in Phase I clinical trials (clinical data to be presented at 2007 ASCO).

5711 Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that localizes to focal adhesion plaques and is phosphorylated in response to integrin β1, β3, β5 binding to cellular ligands. FAK is the kinase signal transducer for integrins which when constitutively active inhibits anoikis allowing anchorage independent cell growth. The continuing growing body of FAK biology suggests that FAK is an important target for anticancer chemotherapy and necessitated the need to further investigate the development of FAK inhibitors. We previously reported that PF-941222, a 2,4-dianilino pyrimidine and related analogs demonstrated potent FAK kinase and cell activity and that these molecules have potential for further lead optimization (2005 AACR NCI EORTC, Philadelphia, PA). Chemistry efforts reported herein focused on increasing analog selectivity for FAK and improving ADME while maintaining or improving potency. SAR efforts focused on the C-2 indole, C-4 linker, C-4 aryl ring coupled with replacement of the aryl ring with heteroaryl rings combined with additional functional group(s) and the C-5 position. At the pyrimidine C-2 position, the indole was replaced with other heteroaromatic rings that were additionally substituted with groups to improve binding interactions with the Arg and Glu at the periphery of the ATP pocket. At the C-4 position, the 2-amino pyridine was replaced with different anilines, heteroaryl amines, aryl/heteroaryl methylamines and aryl/heteroaryl 2-phenyl ethylamines. Additional functional group(s) on the aryl/heteroaryl ring further optimized inhibitor binding interactions with FAK in the ribose phosphate binding region in the ATP pocket. Detailed SAR studies allowed us to obtain >100 fold selectivity for FAK over other structurally related kinases including PYK-2 as well as all other kinases with closely related sequence homology. The pyrimidine C-5 position interacts with a flexible lipophilic pocket. We undertook SAR studies at C-5 to determine the impact of replacing the bromine of PF-941222 with other groups. A seemingly minor modification to the C-5 position had a pronounced effect on kinase activity. In summary, detailed SAR studies were executed at the pyrimidine C-2, C-4 and C-5 positions of PF-941222. Taking all the SAR together, novel analogs have been produced that are potent inhibitors of FAK [kinase (1–10 nM), cell (10–100 nM)] and are >100X selective against other related kinases. A number of these potent and selective inhibitors have been co-crystallized with FAK. Subsets of these analogs demonstrated excellent in vitro selectivity against all CYPs screened with a low susceptibility to microsome and hepatocyte clearance and are suitable for further in vitro and in vivo evaluation. Design, synthesis, inhibitor kinase and cell activity, kinase selectivity profile, ADME as well as inhibitor chemical structure and inhibitor-FAK co-crystal structure data will be presented.

CP-673,451 is a potent inhibitor of platelet-derived growth factor beta-receptor (PDGFR-beta) kinase- and PDGF-BB-stimulated autophosphorylation of PDGFR-beta in cells (IC(50) = 1 nmol/L) being more than 450-fold selective for PDGFR-beta versus other angiogenic receptors (e.g., vascular endothelial growth factor receptor 2, TIE-2, and fibroblast growth factor receptor 2). Multiple models have been used to evaluate in vivo activity of CP-673,451 and to understand the pharmacology of PDGFR-beta inhibition and the effect on tumor growth. These models include an ex vivo measure of PDGFR-beta phosphorylation in glioblastoma tumors, a sponge model to measure inhibition of angiogenesis, and multiple models of tumor growth inhibition. Inhibition of PDGFR-beta phosphorylation in tumors correlates with plasma and tumor levels of CP-673,451. A dose of 33 mg/kg was adequate to provide >50% inhibition of receptor for 4 hours corresponding to an EC(50) of 120 ng/mL in plasma at C(max). In a sponge angiogenesis model, CP-673,451 inhibited 70% of PDGF-BB-stimulated angiogenesis at a dose of 3 mg/kg (q.d. x 5, p.o., corresponding to 5.5 ng/mL at C(max)). The compound did not inhibit vascular endothelial growth factor- or basic fibroblast growth factor-induced angiogenesis at concentrations which inhibited tumor growth. The antitumor efficacy of CP-673,451 was evaluated in a number of human tumor xenografts grown s.c. in athymic mice, including H460 human lung carcinoma, Colo205 and LS174T human colon carcinomas, and U87MG human glioblastoma multiforme. Once-daily p.o. x 10 days dosing routinely inhibited tumor growth (ED(50) < or = 33 mg/kg). These data show that CP-673,451 is a pharmacologically selective PDGFR inhibitor, inhibits tumor PDGFR-beta phosphorylation, selectively inhibits PDGF-BB-stimulated angiogenesis in vivo, and causes significant tumor growth inhibition in multiple human xenograft models.