Poor Safety and Tolerability Hamper Reaching a Potentially Therapeutic Dose in the Use of Thalidomide for Alzheimer's Disease: Results from a Double-Blind, Placebo-Controlled Trial.

INTRODUCTION: To date there is no cure for Alzheimer's disease (AD). After amyloid beta immunotherapies have failed to meet primary endpoints of slowing cognitive decline in AD subjects, the inhibition of the beta-secretase BACE1 appears as a promising therapeutic approach. Pre-clinical data obtained in APP23 mice suggested that the anti-cancer drug thalidomide decreases brainBACE1 and Aß levels. This prompted us to develop an NIH-supported Phase IIa clinical trial to test the potential of thalidomide for AD. We hypothesized that thalidomide can decrease or stabilize brain amyloid deposits, which would result in slower cognitive decline in drug- versus placebo-treated subjects. METHODS: This was a 24-week, randomized, double-blind, placebo-controlled, parallel group study with escalating dose regimen of thalidomide with a target dose of 400mg daily in patients with mild to moderate AD. The primary outcome measures were tolerability and cognitive performance assessed by a battery of tests. RESULTS: A total of 185 subjects have been pre-screened, out of which25 were randomized. Mean age of the sample at baseline was 73.64 (±7.20) years; mean education was 14.24 (±2.3) years; mean MMSE score was 21.00 (±5.32); and mean GDS score was 2.76 (±2.28).Among the 25 participants, 14 (56%) terminated early due to adverse events, dramatically decreasing the power of the study. In addition, those who completed the study (44%) never reached the estimated therapeutic dose of 400 mg/day thalidomide because of reported adverse events. The cognitive data showed no difference between the treated and placebo groups at the end of the trial. CONCLUSION: This study demonstrates AD patients have poor tolerability for thalidomide, and are unable to reach a therapeutic dose felt to be sufficient to have effects on BACE1. Because of poor tolerability, this study failed to demonstrate a beneficial effect on cognition.

A cellular model of amyloid precursor protein processing and amyloid-ß peptide production.

BACKGROUND: A hallmark pathologic feature of Alzheimer's disease (AD) is accumulation of neuritic senile plaques in the brain parenchyma. Neurotoxic plaque cores are composed predominantly of amyloid-ß (Aß) peptides of 40 and 42 amino acids in length, formed by sequential cleavage of amyloid precursor protein (APP) by ß-, and γ-secretases. There is a great interest in approaches to modulate Aß peptide production and develop therapeutic interventions to reduce Aß levels to halt or slow the progression of neurodegeneration. NEW METHOD: We characterized and present the BE(2)-M17 human neuroblastoma cell line as a novel in vitro model of the APP-cleavage cascade to support future (1) functional studies of molecular regulators in Aß production, and (2) high-throughput screening assays of new pharmacotherapeutics. RESULTS: In BE(2)-M17 cells, both RNA (i.e., RT-PCR, RNA sequencing) and protein analyses (i.e., Western blots, ELISA), show endogenous expression of critical components of the amyloidogenic pathway, APP-cleavage intermediates CTF83 and CTF99, and final cleavage products Aß40 and Aß42. We further report effects of retinoic acid-mediated differentiation on morphology and gene expression in this cell line. COMPARISON WITH EXISTING METHOD(S): In contrast to primary isolates or other cell lines reported in current literature, BE(2)-M17 not only sustains baseline expression of the full contingent of APP-processing components, but also remains stably adherent during culture, facilitating experimental manipulations. CONCLUSIONS: Our evidence supports the use of BE(2)-M17 as a novel, human, cell-based model of the APP processing pathway that offers a potential streamlined approach to dissect molecular functions of endogenous regulatory pathways, and perform mechanistic studies to identify modulators of Aß production.

Inhibition of nuclear factor kappaB activation and cyclooxygenase-2 expression by aqueous extracts of Hispanic medicinal herbs.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are a primary choice of therapy for diseases with a chronic inflammatory component. Unfortunately, long-term NSAID therapy is often accompanied by severe side effects, including cardiovascular and gastrointestinal complications. Because of this, there is critical need for identification of new and safer treatments for chronic inflammation to circumvent these side effects. Inflammatory diseases have been successfully remedied with natural herbs by many cultures. To better understand the potential of natural herbs in treating chronic inflammation and to identify their mechanism of action, we have evaluated the anti-inflammatory activities of 20 medicinal herbs commonly used in the Hispanic culture. We have established a standardized method for preparing aqueous extracts (teas) from the selected medicinal herbs and screened for inhibition of tumor necrosis factor-alpha-induced activation of nuclear factor kappaB (NF-kappaB), which is the central signaling pathway of the inflammatory response. A number of herbal teas were identified that exhibited significant anti-inflammatory activity. In particular, tea from the herb commonly called laurel was found to be an especially potent inhibitor of NF-kappaB-dependent cyclooxygenase-2 gene expression and prostaglandin E(2) production in cultured murine macrophages. These findings indicate that laurel tea extract contains potent anti-inflammatory compounds that function by inhibiting the major signal transduction pathway responsible for inducing an inflammatory event. Based on these results, laurel may represent a new, safe therapeutic agent for managing chronic inflammation.

A Jurkat transcriptional reporter cell line for high-throughput analysis of the nuclear factor-kappaB signaling pathway.

The transcription factor, Nuclear Factor-kappaB (NF-kappaB), regulates many genes involved in host immunity and cell survival. Unregulated NF-kappaB activity has been linked to many chronic inflammatory diseases and is an important target for the identification of inhibitors to better manage these disorders. We present a novel screening system to identify NF-kappaB inhibitors that combines sensitive fluorescence detection with medium- to high-throughput flow cytometry (HyperCyt). To validate this approach, we quantified the activation of NF-kappaB by standard flow cytometry and the HyperCyt platform. Results were comparable with regard to EC(50) values for TNFalpha-mediated activation; however, the HyperCyt platform provided more sensitive signal detection and a greater linear range for detection. To demonstrate the usefulness of this screening tool, we identified a novel inhibitor of NF-kappaB activation from a resveratrol-based chemical library. The inhibition of NF-kappaB activation by analog 6q (IC(50) = 19 microm) showed a 3.7-fold improvement over that of resveratrol (IC(50) approximately 70 microm).

Substituted trans-stilbenes can inhibit or enhance the TPA-induced up-regulation of activator protein-1.

BACKGROUND: The activator protein-1 (AP-1) family of transcription factors contributes to regulation of numerous genes involved in proliferation, apoptosis, and tumorigenesis. A wide array of stimuli can activate AP-1, including pro-inflammatory cytokines, growth factors, tumor promoters and stress. Numerous plant polyphenols have been shown to inhibit the activation of AP-1, which often is ascribed to the anti-oxidant properties of these natural products. METHODS: In the present study, a library of substituted trans-stilbenes, including polyphenols, was screened for activity against the TPA-induced activation of AP-1 using the Panomics AP-1 Reporter 293 Stable Cell Line, which is designed for screening potential inhibitors or activators. RESULTS: Several trans-stilbenes were identified that inhibit TPA-induced activation of AP-1, with IC50 values as low as 0.5 microM. Moreover, some other trans-stilbenes were able to enhance the effects of TPA 2 to 3-fold. Many of the trans-stilbenes identified as inhibitors or enhancers are devoid of anti-oxidant properties. CONCLUSION: The ability of trans-stilbenes to inhibit or enhance the effects of TPA does not depend upon their anti-oxidant properties.

Substituted trans-stilbenes, including analogues of the natural product resveratrol, inhibit the human tumor necrosis factor alpha-induced activation of transcription factor nuclear factor kappaB.

The transcription factor nuclear factor kappaB (NF-kappaB), which regulates expression of numerous antiinflammatory genes as well as genes that promote development of the prosurvival, antiapoptotic state is up-regulated in many cancer cells. The natural product resveratrol, a polyphenolic trans-stilbene, has numerous biological activities and is a known inhibitor of activation of NF-kappaB, which may account for some of its biological activities. Resveratrol exhibits activity against a wide variety of cancer cells and has demonstrated activity as a cancer chemopreventive against all stages, i.e., initiation, promotion, and progression. The biological activities of resveratrol are often ascribed to its antioxidant activity. Both antioxidant activity and biological activities of analogues of resveratrol depend upon the number and location of the hydroxy groups. In the present study, phenolic analogues of resveratrol and a series of substituted trans-stilbenes without hydroxy groups were compared with resveratrol for their abilities to inhibit the human tumor necrosis factor alpha-induced (TNF-alpha) activation of NF-kappaB, using the Panomics NF-kappaB stable reporter cell line 293/NF-kappaB-luc. A series of 75 compounds was screened to identify substituted trans-stilbenes that were more active than resveratrol. Dose-response studies of the most active compounds were carried out to obtain IC50 values. Numerous compounds were identified that were more active than resveratrol, including compounds that were devoid of hydroxy groups and were 100-fold more potent than resveratrol. The substituted trans-stilbenes that were potent inhibitors of the activation of NFkappaB generally did not exhibit antioxidant activity. The results from screening were confirmed using BV-2 microglial cells where resveratrol and analogues were shown to inhibit LPS-induced COX-2 expression.

TPA-induced up-regulation of activator protein-1 can be inhibited or enhanced by analogs of the natural product curcumin.

The activator protein-1 (AP-1) family of transcription factors, including the most common member c-Jun-c-Fos, participates in regulation of expression of numerous genes involved in proliferation, apoptosis, and tumorigenesis in response to a wide array of stimuli including pro-inflammatory cytokines, growth factors, stress, and tumor promoters. A number of plant polyphenols including curcumin, a yellow compound in the spice turmeric, have been shown to inhibit the activation of AP-1. Curcumin is a polyphenolic dienone that is potentially reactive as a Michael acceptor and also is a strong anti-oxidant. Multiple activities reported for curcumin, including inhibition of the stress-induced activation of AP-1, have been suggested to involve the anti-oxidant properties of curcumin. In the present study, a library of analogs of curcumin was screened for activity against the TPA-induced activation of AP-1 using the Panomics AP-1 Reporter 293 stable cell line which is designed for screening potential inhibitors. Numerous analogs were identified that were more active than curcumin, including analogs that were not anti-oxidants and analogs that were not Michael acceptors. Clearly, anti-oxidant activity or reactivity as a Michael acceptor is not an essential feature of active compounds. In addition, a number of analogs were identified that enhanced the TPA-induced activation of AP-1. The results from screening were confirmed using BV-2 microglial cells where curcumin and analogs were shown to inhibit LPS-induced COX-2 expression; analogs identified as more potent than curcumin in the screening assay were also more potent than curcumin in preventing COX-2 expression.