PKC isoforms have been implicated in human cancer proliferation, cell cycle progression, angiogenesis and apoptosis. Altered expression and phosphorylation of PKC has been demonstrated in several NSCLC cell lines versus normal lung epithelia cell lines. Enzastaurin (LY317615), a selective PKC-beta inhibitor, has been shown to inhibit a variety of human tumors in vitro and in vivo. In this report, we evaluated the effects of Enzastaurin on PKC/AKT signaling activation as well as its in vitro and in vivo therapeutic effects against human lung cancers. Western blots were used for PKC/AKT signaling analysis. In vitro antigrowth effects of Enzastaurin were accessed with MTT assay. Athymic nude mice bearing human lung xenografts H2122 adenocarcinoma or H358 bronchial alveolar carcinoma (BAC) were used for in vivo efficacy studies, in conjunction with pharmacodynamic analysis and in vivo imaging studies. By western blot analysis, we found that Enzastaurin significantly reduced phosphorylation of GSK3-beta (Ser9), ribosomal protein S6 (Ser240 /244) but not AKT (Thr308) in both NSCLC and SCLC cell lines. Our MTT assay showed that Enzastaurin inhibited the growth of 14 NSCLC cell lines with average IC50 of 8.2 μM, ranging 1.5 to 20 μM and 14 SCLC cell lines with average IC50 of 7.0 μM, ranging 3 to 10 μM. The sensitivities toward Enzastaurin among the four different NSCLC histologies (adenocarcinomas, squamous, large cell carcinomas or bronchial alveolar carcinoma) are very similar. Using Combination-Index analysis, when Enzastaurin was combined with pemetrexed concurrently or pemetrexed then Enzastaurin sequentially, the interaction is greater than additive effect. Our in vivo studies showed that Enzastaurin by daily BID oral gavages at 38, 75 and 150 mg/kg suppressed human H2122 adenocarcinomas implanted subcutaneously in athymic nude mice, but not in a dose-dependent manner. When treated orally with daily BID at 25, 35 and 75 mg/kg in a xenograft with more sensitive H358 BAC cell line, more impressive therapeutic effects at 35 mg/kg were achieved but again not in a dose-dependent manner. In conjunction with efficacy of Enzastaurin in tumor bearing nude mice, plasma pharmacokinetics were studied on day 3, 10 or 21 following the start of dosing. The mean plasma Enzastaurin levels for these days were approximately 1045±80 ng/mL for 38 mg/kg dose; 1473±436 ng/mL for 75 mg/kg dose; 3435±798 ng/mL for 150 mg/kg respectively. Real time in vivo pharmacodynamics of Enzastaurin was analyzed simultaneously using in vivo fluorescent imaging method in tumor bearing live animals. Enzastaurin has unique fluorescent properties which emits strongly with red fluorescent spectrum giving good tissue penetration when excited with appropriate wavelength. Our in vivo imaging showed the presence of Enzastaurin within the tumor mass and in plasma, in consistent with the observed pharmacodynamic data and supporting the therapeutic effects of Enzastaurin against human lung xenografts. Our preclinical data provided strong evidence that Enzastaurin may be effective for the treatment of human lung cancers.

AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA 5407 PKCβ is a member of the PKC family of serine-threonine protein kinases which have been implicated in human cancer proliferation, cell cycle progression, angiogenesis and apoptosis. Enzastaurin (LY317615), a selective PKCβ inhibitor, has been shown to inhibit a variety of human tumors including glioblastoma, colon, prostate and lung cancer cell lines in vitro. Elevated phosphorylation and altered PKC expression has been demonstrated in several NSCLC cell lines versus normal lung epithelia cell lines. In this presentation we show that enzastaurin inhibited the growth of 14 NSCLC cell lines with average IC50 of 8.2 µM, ranging 1.5 to 20 µM and 14 SCLC cell lines with average IC50 of 7.0 µM, ranging 3 to 10 µM. The sensitivities toward enzastaurin among the four different NSCLC histologies (adenocarcinomas, squamous, large cell carcinomas or bronchial alveolar carcinoma) are very similar. By western blot analysis, we found that enzastaurin significantly reduced phosphorylation of GSK3β (Ser9), ribosomal protein S6 (Ser240 /244) but not in phospho-AKT(Thr308) in both NSCLC and SCLC cell lines. Using Combination-Index analysis, when enzastaurin was combined with pemetrexed concurrently or pemetrexed followed by enzastaurin sequentially, the interaction is greater than additive. Our in vivo studies showed that enzastaurin by daily BID oral gavages at 38, 75 and 150 mg/kg suppressed human lung xenografts H2122 adenocarcinomas in athymic nude mice, but not in a dose-dependent manner. In conjunction with efficacious studies of enzastaurin in tumor bearing nude mice, plasma pharmacokinetics were evaluated on day 3, 10 or 21 following the start of dosing. The plasma enzastaurin levels for these days were found to be 1180±58.6, 931.3±399 and 1018.7±158 ng/ml for 38 mg/kg dose; 636.5±414, 2093.3±432 and 1830±355 ng/ml for 75 mg/kg dose; 2473.3±169, 2906.7±370 and 5020±790 ng/ml for 150 mg/kg respectively, supporting the observed therapeutic effects of enzastaurin against human lung xenografts. Our preclinical data provided strong evidence that enzastaurin may be effective for the treatment of human lung cancers.

Inhibition of the aspartyl protease BACE is of considerable interest as a therapeutic target for the treatment of Alzheimer's disease. This interest stems from its pivotal role in catalyzing the formation of Abeta peptide from the amyloid precursor protein (APP). LY2434074 is a low MW, active–site inhibitor of BACE with an IC50 in whole cell assays of 100 nM. Given the pharmacologic properties of this molecule, a number of in vivo proof–of–concept studies were conducted in young PDAPP mice. Mice were dosed with LY2434074 (100 mg/kg, s.c.) or vehicle (7% Pharmasolve) and tissues collected at multiple time points after dosing. By 3 hours, significant decreases in sAPPbeta, ranging from 25 to 40%, were observed in cortex and hippocampus with levels returning to baseline between 9 and 12 hours post–dose. No significant changes were observed in Abeta levels in cortex or hippocampus. However, CSF Abeta levels were reduced over 50% at the 3 hour time point. A follow–up s.c. dose–response study (10, 30 and 100 mg/kg) confirmed the in vivo efficacy of LY2434074 and demonstrated significant reductions in sAPPbeta of ∼ 15% in hippocampus and cortex of PDAPP mice in the 30 mg/kg dose group, and even greater effects observed at 100 mg/kg. In addition, cortical Abeta levels were statistically reduced at the 100 mg/kg dose, and again, more robust effects (∼40% reduction) were observed in CSF Abeta levels at both of the higher doses. Plasma Abeta levels were reduced 25% to 40% at all doses administered. These studies offer key proof–of–concept data that pharmacologic inhibition of BACE can be achieved by systemic administration of a BACE inhibitor to a PDAPP mouse and that this inhibition results in significant reductions in sAPPbeta and Abeta in central compartments. Of note, the genetic knock out data for BACE suggests that these relatively modest acute changes in Abeta production could translate into significantly larger effects on plaque burden over time. Collectively, these data support the continuing development of BACE inhibitors as a therapeutic approach for Alzheimer's disease.

The azetidinone LY307174 (1) was identified as a screening lead for the vasopressin V1a receptor (IC50 45 nM at the human V1a receptor) based on molecular similarity to ketoconazole (2), a known antagonist of the luteinizing hormone releasing hormone receptor. Structure-activity relationships for the series were explored to optimize receptor affinity and pharmacokinetic properties, resulting in compounds with Ki values <1nM and brain levels after oral dosing approximately 100-fold higher than receptor affinities.

This study describes the integrated application of parallel synthesis and computational chemistry to the design of potent nonpeptide antagonists for the neuropeptide Y-1 (NPY1) receptor. A lead molecule was modeled in the active site of the NPY1 receptor, and a potentially fruitful region for analog construction was identified. Synthesis of suitable scaffolds followed by solution phase generation of a small library of analogs produced a compound with 5-fold improvement in binding over the already potent lead. This new compound was shown to be an unanticipated side product of the parallel synthesis reaction. (C) 1999 Elsevier Science Ltd. All rights reserved.