Minimization of drug-drug interaction risk and candidate selection in a natural product-based class of gamma-secretase modulators.

Early lead compounds in this gamma secretase modulator series were found to potently inhibit CYP3A4 and other human CYP isoforms increasing their risk of causing drug-drug-interactions (DDIs). Using structure-activity relationships and CYP3A4 structural information, analogs were developed that minimized this DDI potential. Three of these new analogs were further characterized by rat PK, rat PK/PD and rat exploratory toxicity studies resulting in selection of SPI-1865 (14) as a preclinical development candidate.

An improved cell-based method for determining the γ-secretase enzyme activity against both Notch and APP substrates.

γ-Secretase modulators (GSM), which reduce amyloidogenic Aß(42) production while maintaining total Aß levels, and Notch-sparing γ-secretase inhibitors (GSIs) are promising therapies for the treatment of Alzheimer's Disease (AD). To have a safety margin for therapeutic use, GSMs and GSIs need to allow Notch intracellular domain (NICD) production, while preventing neurotoxic Aß peptide production. Typically, GSI and GSM effects on these substrates are determined using two different cell lines, one for the measurement of enzyme activity against each substrate. However, predicting selectivity for different substrates across cell systems may reduce the reliability of such ratios such that the in vitro data are not useful for predicting in vivo safety margins. This is especially concerning since the IC(50)'s of some GSIs vary depending upon the level of APP expression in a cell line. To circumvent this problem, we utilized the SUP-T1 cell line which expresses a truncated Notch receptor fragment that does not need sheddase cleavage to be a γ-secretase substrate. When combined with a sensitive method of measuring Aß production, this assay system allows both substrates to be measured simultaneously, reducing the potential to calculate imprecise selectivity margins. To demonstrate the value of this system, known GSIs and GSMs were examined in the SUP-T1 dual substrate assay. IC(50)'s were determined for both substrates and the in vitro selectivity margin was calculated. These data suggest using a single cell line is a more accurate prediction of the fold difference between NICD inhibition and Aß(42) lowering for therapeutically promising GSIs and GSMs.

Optimization of a natural product-based class of γ-secretase modulators.

A series of triterpene-based γ-secretase modulators is optimized. An acetate present at the C24 position of the natural product was replaced with either carbamates or ethers to provide compounds with better metabolic stability. With one of those pharmacophores in place at C24, morpholines or carbamates were installed at the C3 position to refine the physicochemical properties of the analogues. This strategy gave compounds with low clearance and good distribution into the central nervous system (CNS) of CD-1 mice. Two of these compounds, 100 and 120, were tested for a pharmacodynamic effect in the strain and lowered brain Aß42 levels.

Modulation of gamma-secretase for the treatment of Alzheimer's disease.

The Amyloid Hypothesis states that the cascade of events associated with Alzheimer's disease (AD)-formation of amyloid plaques, neurofibrillary tangles, synaptic loss, neurodegeneration, and cognitive decline-are triggered by Aß peptide dysregulation (Kakuda et al., 2006, Sato et al., 2003, Qi-Takahara et al., 2005). Since γ-secretase is critical for Aß production, many in the biopharmaceutical community focused on γ-secretase as a target for therapeutic approaches for Alzheimer's disease. However, pharmacological approaches to control γ-secretase activity are challenging because the enzyme has multiple, physiologically critical protein substrates. To lower amyloidogenic Aß peptides without affecting other γ-secretase substrates, the epsilon (ε) cleavage that is essential for the activity of many substrates must be preserved. Small molecule modulators of γ-secretase activity have been discovered that spare the ε cleavage of APP and other substrates while decreasing the production of Aß(42). Multiple chemical classes of γ-secretase modulators have been identified which differ in the pattern of Aß peptides produced. Ideally, modulators will allow the ε cleavage of all substrates while shifting APP cleavage from Aß(42) and other highly amyloidogenic Aß peptides to shorter and less neurotoxic forms of the peptides without altering the total Aß pool. Here, we compare chemically distinct modulators for effects on APP processing and in vivo activity.

Initial Optimization of a New Series of γ-Secretase Modulators Derived from a Triterpene Glycoside.

The discovery of a new series of γ-secretase modulators is disclosed. Starting from a triterpene glycoside γ-secretase modulator that gave a very low brain-to-plasma ratio, initial SAR and optimization involved replacement of a pendant sugar with a series of morpholines. This modification led to two compounds with significantly improved central nervous system (CNS) exposure.

Transcriptional regulation of vascular endothelial growth factor in cancer.

Vascular endothelial growth factor (VEGF) is critical for vascularization of tissues, including tumors, making it an attractive target for controlling angiogenesis. An important first step towards the goal of effectively blocking tumor angiogenesis is to understand the relationships among tumor-promoting molecules. Whereas little is known about developmental regulation of VEGF, pathological regulation of VEGF during disease states and tumorigenesis is better understood. This review focuses on transcriptional regulation of VEGF expression in cancer. Understanding how VEGF is regulated in tumors cells may provide the basis for future treatments that target both the tumor and its vascular supply.

ErbB2 overexpression in mammary cells upregulates VEGF through the core promoter.

The angiogenic molecule, vascular endothelial growth factor (VEGF), is a critical regulator of normal and pathologic angiogenesis. ErbB2, an epidermal growth factor receptor family member whose overexpression in mammary tumors is correlated with poor patient prognosis, has been implicated as a positive modulator of VEGF expression. Mammary tumor cells overexpressing ErbB2 (NAFA cells) and a normal mouse mammary cell line (HC11) transfected with ErbB2 expression vectors were used to study the effects of ErbB2 overexpression on VEGF regulation. We found that ErbB2 overexpression led to an increase in endogenous VEGF mRNA as well as ErbB3 protein levels in HC11 cells. Additionally, we determined that ErbB2 overexpression-mediated upregulation of VEGF involves at least two distinct promoter elements, one previously identified as the hypoxia responsive element and the other the core promoter region (-161 to -51bp), which is specifically controlled via two adjacent SP1 binding sites (-80 to -60bp).

Identification of genes involved in VEGF-mediated vascular morphogenesis using embryonic stem cell-derived cystic embryoid bodies.

The vasculature forms during development via two processes, vasculogenesis and angiogenesis, in which vessels form de novo from angioblast precursors or as sprouts from pre-existing vessels, respectively. A common and critical aspect of both processes is vascular morphogenesis, which includes branching of endothelial cell cords and lumen formation. Although ample evidence support the central role of vascular endothelial growth factor (VEGF) in both vasculogenesis and angiogenesis, the role of VEGF in vascular morphogenesis is unclear and little is known about the regulation of vascular morphogenesis, in general. We have used the in vitro vessel differentiation system of embryonic stem (ES) cell-derived cystic embryonic bodies (CEB) as a model for studying VEGF-mediated vessel formation. Whereas CEB formed from wild-type ES cells make well-formed vessel-like structures, CEB derived from VEGF-null ES cells contain PECAM-1-positive endothelial cells, but these cells do not participate in vascular morphogenesis. Using gene expression microarray analysis to compare gene expression in these two systems, we have been able to identify many genes and novel ESTs that are downstream of VEGF function, and which may be involved in VEGF-mediated vascular morphogenesis including caveolin-1 and HEY-1. These results support using the CEB model, in combination with gene knockout ES cells, for studying vascular morphogenesis.