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The search for thalidomide analogues with increased immunomodulatory activity and an improved safety profile led to the testing of amino-phthaloyl-substituted thalidomide analogues. These 4-amino analogues, in which an amino group is added to the fourth carbon of the phthaloyl ring of thalidomide  Fig.(2), brought about the class termed “IMiDs”. The bioactivities of the IMiDs follow the parent drug thalidomide closely, but with some increase in potency. Fig.(3) summarizes the multifaceted effects of thalidomide and its analogues.

4.1.1. LENALIDOMIDE (CC-5013)
CC-5013, lenalidomide (α -(3-aminophthalimido) glutarimide; Revlimid®)) is an immunomodulatory analogue that has demonstrated higher potency than thalidomide in the HUVEC (human umbilical vein endothelial cells) proliferation and tube formation assays [19]. The proliferation inhibition responded in a dose-dependent manner with increasing concentrations of the drug. Anti-migratory effects as well as tumor growth inhibition in vivo have also been demonstrated [20].

Fig. (2) . Chemical structure of thalidomide, lenalidomide, and CC-4047

4.1.2. ACTIMID (CC-4047)
CC-4047 is a costimulatory thalidomide analogue that can prime protective, long-lasting, tumor-specific, Th1-type responses in vivo [21].

4.1.3. ENMD-0995 (S-3-Amino-phthalimido-glutarimide, S-3APG)
ENMD-0995 is a small molecule analogue of thalidomide that is the S(-) enantiomer 3-amino thalidomide. Thalidomide is a racemic glutamic acid analogue, consisting of S(-) and R(+) enantiomers that interconvert under physiological conditions [22]. The S(-) form potently inhibits release of TNF-alpha from peripheral blood mononuclear blood cells[23], whereas the R(+) form seems to act as a sedative[24]. This 3-amino derivative of thalidomide was demonstrated to have improved angiogenesis inhibitor activity that in animal models has shown no evidence of the toxic side effects as reported for the thalidomide molecule.

Fig (3). Mechanism of anti-angiogenic and immunomodulatory actions of IMiDsMechanisms of anti-angiogenic and immunomodulatory functions of IMiDs.Immunomodulatory drugs (IMiDs), like thalidomide metabolites upon oxidation, inhibits interleukin 1 β or TNF-alpha – induced activation of IκK, which prevents dissociation of IκBα from NFκB , precluding its nuclear translocation and induction of genes that function in metastasis, angiogenesis, cellular proliferation, inflammation, and protection from apoptosis.IMiDs and thalidomide metabolites also function in T cell activation as its T cell costimulatory function, enhancing T cell proliferation. The activated T cells release interleukin-2 (IL-2) and interferon-gamma (IFN-γ), which activate the Natural Killer cells (NK cells).

The precise mechanism(s) of thalidomide and the IMID®s efficacious activities are unknown. This class of compounds, however, has numerous immunomodulatory and non-immunomodulatory properties, which are probably working in concert to produce the observed efficacy.

Thalidomide and the IMiDs Have Potent Immunodulatory Properties Inhibition of TNF-α, IL-1β, IL-6, IL-12, GM-CSF, and Stimulation of IL-10 in Peripheral Blood Mononuclear Cells Cytokines are soluble glycoproteins released by cells of the immune system that act non-enzymatically through specific receptors to regulate immune responses. TNF-α is a proinflammatory cytokine produced by monocytes, macrophages, lymphocytes, and NK cells. It plays an important role in host immune and inflammatory response to viral, parasitic, fungal, and bacterial infections. TNF-α has been implicated in the pathogenesis of infections and autoimmune diseases. Elevated levels of TNF-α have been associated with various inflammatory and immune disorders such as rheumatoid arthritis, Crohn’s disease, tuberculosis, cancer cachexia, and ENL. Thalidomide’s ameliorative effects on ENL have been particularly striking. Thalidomide and its analogs are potent inhibitors of TNF-α production by lipopolysaccharide-stimulated human monocytes. This inhibition is due to the increased degradation of TNF-α mRNA by thalidomide. Levels of other cytokines, IL-1β, IL-6, and granulocyte macrophage-colony stimulating factor (GMCSF), are also inhibited by thalidomide, whereas IL-10 is stimulated. Lenalidomide and CC-4047 also had similar effects on these cytokines, although with varying degrees of potency compared with thalidomide. The effects of these findings on various diseases are still being investigated.[3]

T Cell Costimulators
Thalidomide and the immunomodulatory drugs induced proliferation of MM patients’ T cells and interleukin-2 and interferon-gamma production in vitro, but were not cytotoxic to MM cells[25]. These drugs enhanced natural killer cell-mediated lysis of autologous tumor cells [26]. The increase in IL2 and IFN-gamma also upregulates the CD40L expression on T cells when IMiDs were added to anti-CD3 stimulated T cells[25]. Although a greater costimulatory effect on the CD8+ T cell subset compared to the CD4+ T cell subset has been observed in one study [27], another showed similar co-stimulation for CD4+ and CD8+ T lymphocytes which correlated with TNFR2 inhibition[28]. This T cell costimulatory effect of the IMiDs may be paradoxical to the suppression of ligand-stimulated release of apoptotic and inflammatory cytokines (i.e., TNF-alpha), and the effects of the IMiDs therefore, similar to thalidomide, results in the clinically divergent effects with regard tospecific pathways involved during an inflammatory response or clinical condition [29].

Pro-Apoptotic Agents
Hideshima et. al. first demonstrated that thalidomide and its analogues induce tumor cell apoptosis, evidenced by increased sub-G1cells or induction of p21 and related G1growth arrest [30]. The IMiDs inhibited the proliferation of chemoresistant MM cells by 20% to 35%, and of Dexamethasone-resistant MM cells by 50%[29]. Enhanced caspase-8 activation, increased sensitivity to Fas induction, reduced expression of cellular inhibitor of apoptosis protein-2, and potentiation of the activities of other apoptosis inducers such as TNF-related apoptosis inducing  ligand (TRAIL), has been demonstrated with the IMiDs[31]. This antiproliferative effect was demonstrated in a chromosome 5 deleted (5q-) deletion cell line of Myelodysplasia (MDS) and other hematologic malignancies where the inhibitory concentration of 50% (IC50) was higher for the IMiDs as compared to thalidomide, with induced G0/G1 cell cycle arrest, inhibited Akt and Gab1 phosphorylation, and inhibited the ability of Gab1 to associate with a receptor tyrosine kinase [32].

Anti-Angiogenic Activity
Angiogenesis is the development of new blood vessels. In cancer this can nurture the growth and metastasis of tumors and tumor cells respectively. Thalidomide and IMiDs has been shown to have antiangiogenic properties that are independent of their immunomodulatory effects.This activity is thought to play a role in the apparent efficacy seen with various cancers. In the rat aorta assay, the IMiDs were found to be 2 to 3 times more potent in their antiangiogenic activity compared with thalidomide. Lenalidomide but not thalidomide and CC-4047 significantly inhibited the migration of endothelial cells. CC-4047 on the other hand inhibited VEGF but not basic fibroblast growth factor (bFGF). The IMiDs’ anti-TNF-α activity had no effect on antiangiogenic activity. In multiple myeloma, the close proximity interaction between the indigenous bone marrow stromal cells and patient multiple myeloma cells significantly increased levels of the pro-angiogenic factors VEGF and IL-6 (a multiple myeloma growth and survival factor). Thalidomide and CC-4047 significantly decreased expression of these factors thereby reducing the production and growth of new blood vessels feeding the multiple myeloma cells. These findings underscore the importance of stromal and multiple myeloma cell interaction in the bone marrow microenvironment for the maintenance and progression of the disease and provide another target for thalidomide and its analogs.[3]

Thalidomide was serendipitously found to have anti-myeloma activity when it was thought its anti-angiogenic activity could slow the disease by inhibiting the formation of new blood vessels in this highly vascularized cancer. There is now ample evidence to show that the anticancer activity of thalidomide and its analogs in multiple myeloma is through different mechanisms and sites in the bone marrow.[33,34] Fig(4) shows the bone marrow microenvironment in multiple myeloma, which contains aberrations in various cellular processes, immunology, and cell interactions. Thalidomide and the IMiDs’ immunomodulatory activities consist of inhibiting the expression of IL-6 and TNF-α by bone marrow stromal cells that in turn inhibit the growth of multiple myeloma cells. The compounds also enhance T cell stimulation and proliferation with the activated cells, then releasing IL-2 and IFN-γ. These cytokines activate NK cells causing lysis of the multiple myeloma cells.[34] The combination of immunomodulatory and non-immunomodulatory anticancer activities in the bone marrow is thought to produce the significant anti-tumor responses observed in some multiple myeloma patients. This combination activity has significant implications for other blood and solid tumor cancers and is currently being investigated in numerous clinical trials.

Fig (4).

Sites of activity of thalidomide and IMiDÒs in the bone marrow of multiple myeloma patients.



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