Roger Shen-Chu Chen

Amino-substituted thalidomide analogs: Potent inhibitors of TNF-α production

Abstract Thalidomide, (1), is a known inhibitor of TNF-α release in LPS stimulated human PBMC. Herein we describe the TNF-α inhibitory activity of amino substituted analogs of thalidomide (1) and its isoindolin-1-one analog, EM-12 (2). The 4-amino substituted analogs were found to be potent inhibitors of TNF-α release in LPS stimulated human PBMC.

The anti-cancer agents lenalidomide and pomalidomide inhibit the proliferation and function of T regulatory cells

Lenalidomide (Revlimid®; CC-5013) and pomalidomide (CC-4047) are IMiDs® proprietary drugs having immunomodulatory properties that have both shown activity in cancer clinical trials; lenalidomide is approved in the United States for a subset of MDS patients and for treatment of patients with multiple myeloma when used in combination with dexamethasone. These drugs exhibit a range of interesting clinical properties, including anti-angiogenic, anti-proliferative, and pro-erythropoietic activities although exact cellular target(s) remain unclear. Also, anti-inflammatory effects on LPS-stimulated monocytes (TNF-α is decreased) and costimulatory effects on anti-CD3 stimulated T cells, (enhanced T cell proliferation and proinflammatory cytokine production) are observed These drugs also cause augmentation of NK-cell cytotoxic activity against tumour-cell targets. Having shown that pomalidomide confers T cell-dependant adjuvant-like protection in a preclinical whole tumour-cell vaccine-model, we now show that lenalidomide and pomalidomide strongly inhibit T-regulatory cell proliferation and suppressor-function. Both drugs inhibit IL-2-mediated generation of FOXP3 positive CTLA-4 positive CD25high CD4+ T regulatory cells from PBMCs by upto 50%. Furthermore, suppressor function of pre-treated T regulatory cells against autologous responder-cells is abolished or markedly inhibited without drug related cytotoxicity. Also, Balb/C mice exhibit 25% reduction of lymph-node T regulatory cells after pomalidomide treatment. Inhibition of T regulatory cell function was not due to changes in TGF-β or IL-10 production but was associated with decreased T regulatory cell FOXP3 expression. In conclusion, our data provide one explanation for adjuvant properties of lenalidomide and pomalidomide and suggest that they may help overcome an important barrier to tumour-specific immunity in cancer patients.

Lenalidomide and CC-4047 Inhibit the Proliferation of Malignant B Cells while Expanding Normal CD34+ Progenitor Cells

Clinical studies involving patients with myelodysplastic syndromes or multiple myeloma have shown the efficacy of lenalidomide by reducing and often eliminating malignant cells while restoring the bone marrow function. To better understand these clinical observations, we investigated and compared the effects of lenalidomide and a structurally related analogue, CC-4047, on the proliferation of two different human hematopoietic cell models: the Namalwa cancer cell line and normal CD34+ progenitor cells. Both compounds had antiproliferative effects on Namalwa cells and pro-proliferative effects on CD34+ cells, whereas p21WAF-1 expression was up-regulated in both cell types. In Namalwa cells, the up-regulation of p21WAF-1 correlated well with the inhibition of cyclin-dependent kinase (CDK) 2, CDK4, and CDK6 activity leading to pRb hypophosphorylation and cell cycle arrest, whereas in CD34+ progenitor cells the increase of p21WAF-1 did not inhibit proliferation. Similarly, antiproliferation results were observed in two B lymphoma cell lines (LP-1 and U266) but interestingly not in normal B cells where a protection of apoptosis was found. Finally, CC-4047 and lenalidomide had synergistic effects with valproic acid [a histone deacetylase (HDAC) inhibitor] by increasing the apoptosis of Namalwa cells and enhancing CD34+ cell expansion. Our results indicate that lenalidomide and CC-4047 have opposite effects in tumor cells versus normal cells and could explain, at least in part, the reduction of malignant cells and the restoration of bone marrow observed in patients undergoing lenalidomide treatment. Moreover, this study provides new insights on the cellular pathways affected by lenalidomide and CC-4047, proposes new potential clinical uses, such as bone marrow regeneration, and suggests that the combination of lenalidomide or CC-4047 with certain HDAC inhibitors may elevate the therapeutic index in the treatment of hematologic malignancies.

Protective antitumor immunity induced by a costimulatory thalidomide analog in conjunction with whole tumor cell vaccination is mediated by increased Th1-type immunity.

Thalidomide and its novel T cell costimulatory analogs (immunomodulatory drugs) are currently being assessed in the treatment of patients with advanced cancer. However, neither tumor-specific T cell costimulation nor effective antitumor activity has been demonstrated in vivo. In this study, we assessed the ability of an immunomodulatory drug (CC-4047/ACTIMID) to prime a tumor-specific immune response following tumor cell vaccination. We found that the presence of CC-4047 during the priming phase strongly enhanced antitumor immunity in the vaccinated group, and this correlated with protection from subsequent live tumor challenge. Protection was associated with tumor-specific production of IFN-γ and was still observed following a second challenge with live tumor cells 60 days later. Furthermore, CD8+ and CD4+ splenocyte fractions from treated groups secreted increased IFN-γ and IL-2 in response to tumor cells in vitro. Coculture of naive splenocytes with anti-CD3 mAb in the presence of CC-4047 directly costimulated T cells and increased Th1-type cytokines. Our results are the first to demonstrate that a costimulatory thalidomide analog can prime protective, long-lasting, tumor-specific, Th1-type responses in vivo and further support their ongoing clinical development as novel anti-cancer agents.

The anti-cancer drug lenalidomide inhibits angiogenesis and metastasis via multiple inhibitory effects on endothelial cell function in normoxic and hypoxic conditions

Lenalidomide (Revlimid) is approved for the treatment of transfusion-dependent patients with anemia due to low- or intermediate-1-risk Myelodysplastic Syndromes (MDS) associated with a del 5q cytogenetic abnormality with or without additional cytogenetic abnormalities, and in combination with dexamethasone for the treatment of multiple myeloma patients who have received at least one prior therapy. Previous reports suggest that lenalidomide is anti-angiogenic and this property appears to be related to efficacy in patients with MDS. We have investigated the effect of lenalidomide on the formation of microvessels in a novel in vitro angiogenesis assay utilizing human umbilical arterial rings and in a capillary-like cord formation assay using cultured primary endothelial cells. We found that lenalidomide consistently inhibits both sprout formation by arterial rings and cord formation by endothelial cells in a dose-dependent manner. We also found an inhibitory effect of lenalidomide on the associations between cadherin 5, beta-catenin and CD31, adherens junction proteins whose interaction is critical for endothelial cell cord formation. Furthermore, lenalidomide inhibited VEGF-induced PI3K-Akt pathway signaling, which is known to regulate adherens junction formation. We also found a strong inhibitory effect of lenalidomide on hypoxia-induced endothelial cell formation of cords and HIF-1 alpha expression, the main mediator of hypoxia-mediated effects and a key driver of angiogenesis and metastasis. Anti-metastatic activity of lenalidomide in vivo was confirmed in the B16-F10 mouse melanoma model by a >40% reduction in melanoma lung colony counts versus untreated mice. Our results suggest that inhibitory effects on microvessel formation, in particular adherens junction formation and inhibition of hypoxia-induced processes support a potential anti-angiogenic and anti-metastatic mechanism for this clinically active drug.

Discovery of (S)-N-{2-[1-(3-Ethoxy-4-methoxyphenyl)-2-methanesulfonylethyl]-1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl}acetamide (Apremilast), a Potent and Orally Active Phosphodiesterase 4 and Tumor Necrosis Factor-α Inhibitor

In this communication, we report the discovery of 1S (apremilast), a novel potent and orally active phosphodiesterase 4 (PDE4) and tumor necrosis factor-alpha inhibitor. The optimization of previously reported 3-(1,3-dioxo-1,3-dihydroisoindol-2-yl)-3-(3,4-dimethoxyphenyl)propionic acid PDE4 inhibitors led to this series of sulfone analogues. Evaluation of the structure-activity relationship of substitutions on the phthalimide group led to the discovery of an acetylamino analogue 1S, which is currently in clinical trials.

The Synthetic Compound CC-5079 Is a Potent Inhibitor of Tubulin Polymerization and Tumor Necrosis Factor-α Production with Antitumor Activity

We have found that the synthetic compound CC-5079 potently inhibits cancer cell growth in vitro and in vivo by a novel combination of molecular mechanisms. CC-5079 inhibits proliferation of cancer cell lines from various organs and tissues at nanomolar concentrations. Its IC(50) value ranges from 4.1 to 50 nmol/L. The effect of CC-5079 on cell growth is associated with cell cycle arrest in G(2)-M phase, increased phosphorylation of G(2)-M checkpoint proteins, and apoptosis. CC-5079 prevents polymerization of purified tubulin in a concentration-dependent manner in vitro and depolymerizes microtubules in cultured cancer cells. In competitive binding assays, CC-5079 competes with [(3)H]colchicine for binding to tubulin; however, it does not compete with [(3)H]paclitaxel (Taxol) or [(3)H]vinblastine. Our data indicate that CC-5079 inhibits cancer cell growth with a mechanism of action similar to that of other tubulin inhibitors. However, CC-5079 remains active against multidrug-resistant cancer cells unlike other tubulin-interacting drugs, such as Taxol and colchicine. Interestingly, CC-5079 also inhibits tumor necrosis factor-alpha (TNF-alpha) secretion from lipopolysaccharide-stimulated human peripheral blood mononuclear cells (IC(50), 270 nmol/L). This inhibitory effect on TNF-alpha production is related to its inhibition of phosphodiesterase type 4 enzymatic activity. Moreover, in a mouse xenograft model using HCT-116 human colorectal tumor cells, CC-5079 significantly inhibits tumor growth in vivo. In conclusion, our data indicate that CC-5079 represents a new chemotype with novel mechanisms of action and that it has the potential to be developed for neoplastic and inflammatory disease therapy.

Isosteric analogs of lenalidomide and pomalidomide: synthesis and biological activity.

A series of analogs of the immunomodulary drugs lenalidomide (1) and pomalidomide (2), in which the amino group is replaced with various isosteres, was prepared and assayed for immunomodulatory activity and activity against cancer cell lines. The 4-methyl and 4-chloro analogs 4 and 15, respectively, displayed potent inhibition of tumor necrosis factor-α (TNF-α) in LPS-stimulated hPBMC, potent stimulation of IL-2 in a human T cell co-stimulation assay, and anti-proliferative activity against the Namalwa lymphoma cell line. Both of these analogs displayed oral bioavailability in rat.