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In this study we report that R-etodolac (SDX-101), at clinically relevant concentrations, induces potent cytotoxicity in drug-sensitive multiple myeloma (MM) cell lines, as well as in dexamethasone (MM.1R)-, doxorubicin (Dox40/RPMI8226)-, and bortezomib (DHL4)-resistant cell lines. Immunoblot analysis demonstrates that R-etodolac induces apoptosis characterized by caspase-8, -9, and -3 and PARP (poly-ADP [adenosine diphosphate]-ribose polymerase) cleavage and down-regulation of cyclin D1 expression. Subcytotoxic doses of R-etodolac up-regulate myeloid cell leukemia-1 proapoptotic variant (Mcl-1S), while enhancing dexamethasone (Dex)-induced caspase activation and apoptosis. The combination of R-etodolac with Dex results in a highly synergistic cytotoxic effect. R-etodolac also induces apoptosis against primary cells isolated from patients with MM refractory to chemotherapy. Although interleukin 6 (IL-6) and insulin-like growth factor-1 (IGF-1) abrogate Dex-induced MM cell cytotoxicity, neither IL-6 nor IGF-1 protects against R-etodolac-induced cytotoxicity in MM cells. R-etodolac also inhibits viability of MM cells adherent to bone marrow stromal cells (BMSCs), thereby overcoming a mechanism of drug resistance commonly observed with other conventional chemotherapeutic agents. Our data, therefore, indicate that R-etodolac circumvents drug resistance in MM cells at clinically relevant concentrations, targets Mcl-1, and can be synergistically combined with Dex. (Blood. 2005;106:706-712)

Chronic lymphocytic leukemia (CLL) B cells have defects in apoptosis pathways and therefore accumulate in vivo. However, when removed from the patient and cultured in vitro, these malignant cells rapidly undergo apoptosis. Recent studies suggest that leukemia cell survival is influenced by interactions with nonleukemia cells in the microenvironment of lymph nodes, marrow, and other tissues. To model such cell-cell interactions in vitro, we cultured freshly isolated CLL B cells with a follicular dendritic cell line, HK. CLL B cells cocultured with HK cells were protected from apoptosis, either spontaneous or induced by treatment with anticancer drugs. Protection against spontaneous apoptosis could also be induced by coculturing the CLL B cells with normal dendritic cells (DCs) or with a CD40-ligand (CD154)–expressing fibroblast cell line. Examination of the expression of several apoptosis-regulatory proteins revealed that coculture with HK cells or DCs induced up-regulation of the antiapoptotic Bcl-2 family protein Mcl-1 in CLL B cells, whereas CD40 ligation increased expression of Bcl-XL. Cell-cell contact was required for HK-induced protection, and introducing neutralizing antibodies against various adhesion molecules showed that CD44 was involved in HK-mediated survival, whereas CD40, intercellular adhesion molecule–1 (ICAM-1) and vascular cell adhesion molecule–1 (VCAM-1) were not. Anti-CD44 antibodies also blocked Mcl-1 induction by HK cells. Mcl-1 antisense oligonucleotides reduced leukemia cell expression of Mcl-1, and significantly suppressed HK-induced protection against apoptosis, whereas control oligonucleotides had no effect. Thus, HK cells protect CLL B cells against apoptosis, at least in part through a CD44-dependent mechanism involving up-regulation of Mcl-1, and this mechanism is distinct from that achieved by CD40 ligation. Consequently, the particular antiapoptotic proteins important for CLL survival may vary depending on the microenvironment.

Adenine deoxynucleosides induce apoptosis in quiescent lymphocytes and are thus useful drugs for the treatment of indolent lymphoproliferative diseases. To explain why deoxyadenosine and its analogs are toxic to a cell that is not undergoing replicative DNA synthesis, several mechanisms have been proposed, including the direct binding of dATP to the pro-apoptotic factor Apaf-1 and the activation of the caspase-9 and -3 pathways. In this study it is shown, by means of several assays on whole cells and isolated mitochondria, that 2-chloro-2′-deoxyadenosine (2CdA) and 2-choloro-2′-ara-fluorodeoxyadenosine (CaFdA) disrupt the integrity of mitochondria from primary chronic lymphocytic leukemia (B-CLL) cells. The nucleoside-induced damage leads to the release of the pro-apoptotic mitochondrial proteins cytochrome c and apoptosis-inducing factor. The other adenine deoxynucleosides tested displayed comparable DNA-damaging potency but did not affect mitochondrial function. Interference with mitochondrial integrity, thus, may be a factor in the potent cytotoxic effects of 2CdA and CaFdA toward nondividing lymphocytes.

Multiple myeloma (MM) is characterized by increased osteoclast activity resulting in bone destruction and the development of lytic bone lesions. Current treatment modalities have resulted in an increased overall survival in MM patients and new drugs are required that specifically inhibit bone destruction. Etodolac is a non-steroidal anti-inflammatory drug that is approved for treatment of degenerative joint disease and rheumatoid arthritis. SDX-101, an R-enantiomer of Etodolac, was recently demonstrated to induce cytotoxicity, overcome drug resistance, and enhance the activity of dexamethasone in MM. SDX-308 is a novel and more potent etodolac structural analog with a more favorable safety profile than the racemic etodolac due to a lack of significant COX-inhibitory activity. In this study, we focused on effects of SDX-308 on osteoclastogenesis and MM cells. SDX-308 required a 10-fold lower concentration (5x10−6M) than SDX-101 (50x10−6M) to induce potent inhibition (60–80%) of osteoclast formation using mononuclear bone marrow cells from MM patients and healthy donors. Depending on the MM cell line (MM.1S, RPMI-822, or OPM2), SDX-308 required 10- to 100-fold lower concentration (1–10x10−6M) to inhibit MM cell proliferation compared to SDX-101 (10–100x10−6M). In addition, SDX-308 (7.5x10−6M) completely inhibited bone resorption as determined by dentin-based bone resorption assays. We found that pre-treatment of RAW264.7 osteoclast-like cells with SDX-308 decreased constitutive and RANKL-stimulated NF-κB activation measured by luciferase activity. Further, SDX-308 inhibited phosphorylation of p65, IκBα and p65 nuclear translocation in RAW264.7 cells. In addition, SDX-308 effectively suppressed TNFα-induced IKK-γ and IkB-α phosphorylation and degradation and subsequent NF-κB activation in human MM cells. Even in higher concentrations, SDX-101 was less effective in inhibiting NF-κB signaling. In conclusion, these results indicate that SDX-308 effectively inhibits multiple myeloma cell proliferation and osteoclast activity by specifically targeting the NF-κB signaling pathway. These results show that SDX-308 is a promising therapeutic candidate for inhibiting tumor cell growth and elevated osteoclast activity in MM.

SDX-101, the less toxic R-isomer of the commercially available non-steroidal inflammatory drug R,S-etodolac (Lodine®), lacks COX inhibitory activity and is being investigated in Phase II clinical trials in chronic lymphocytic leukemia. Recently, we reported that R-etodolac, at clinically relevant concentrations, induces potent in vitro cytotoxicity in drug-sensitive and conventional drug-resistant multiple myeloma (MM) cell lines, as well as in primary tumor cells from MM patients. R-etodolac triggers caspase/poly-ADP-ribose polymerase (PARP) cleavage and downregulates of cyclin D1 expression (Yasui et al. Blood 2005). Importantly, R-etodolac at sub-cytotoxic doses upregulates Mcl-1s and synergistically enhances dexamethasone (Dex)-induced caspase-dependent apoptosis in Dex-sensitive MM.1S cells. Combination of R-etodolac with Dex enhances cytotoxicity in Dex resistant OPM1 MM cells and in Dex-resistant patient MM cells in vitro. We further studied the in vivo anti-tumor effect of combined R-etodolac and Dex in SCID mice injected subcutaneously with OPM1 human MM cells. While oral treatment of SCID mice with R-etodolac alone (250 mg/kg/d) or Dex alone (1 mg/kg/d) did not induce any significant reduction of tumor volume compared with control (PBS), the combination of R-etodolac and Dex inhibited tumor growth synergistically (synergism quotient = 1.6) and significantly (p = 0.023), suggesting that R-etodolac may reverse Dex resistance in MM. Finally, we demonstrated that racemic SDX-308, a novel indole-pyran structural analog of etodolac, has 10-fold more potent cytotoxicity than R-etodolac in MM cell lines both sensitive and resistant to conventional therapies, as well as in patient’s MM cells. Moreover, SDX-308, like R-etodolac, can overcome the viability and proliferative enhancing effects of exogeneous IL-6, IGF-1, or bone marrow stroma cells. These combined observations indicate that SDX-308 is a promising more potent second generation analog of R-etodolac for MM therapy. Our data suggest that R-etodolac and its novel analog SDX-308 overcome resistance to some conventional therapeutics used for MM, and provide preclinical rationale to conduct clinical trials of R-etodolac and SDX-308 to treat MM.

Background: SDX-101 (R-etodolac) is the R isomer of the non-steroidal anti-inflammatory drug Etodolac. SDX-101 decreased the in vitro survival of B-cell chronic lymphocytic leukemia (B-CLL) cells and showed synergistic activity with chlorambucil. In multiple myeloma cells, SDX-101 showed cytotoxic activity and displayed a synergistic cytotoxic effect with dexamethasone. Currently, SDX-101 is being tested in phase II clinical trial for treatment of refractory B-CLL in combination with chlorambucil. Aims: To evaluate the ex-vivo cytotoxic effect of SDX-101 in combination with agents proven to be effective first line treatment in B-CLL: fludarabine (FA), cladribine (2-chlordeoxyadenosine, 2-CdA), anti-CD-52 (alemtuzumab, ALT) or anti-CD-20 (rituximab, RTX). Methods: Tumor cells were obtained from 20 untreated patients with B-CLL. Cytotoxicity of SDX-101 and other study drugs was assessed using a propidium iodide-based flow cytometry assay. Apoptosis was assessed measuring activity of caspase-3 and DNA-content (sub-G1 fraction) by flow cytometry, and by measuring protein expression of p53 protein family members (p53 and p73), and BCl-2 family members (pro-apoptotic Bax, Bak and Bid; anti-apoptotic Bcl-2 and Mcl-1). The percentage of apoptotic cells and the expression of apoptosis-regulating proteins were measured at 0 h and 24 h. IC50 values were defined as the concentration of agents that achieved 50% decrease in cell viability. The combination index (CI) was used to estimate sub-additive (CI value 1.2), additive (0.8–1.2) or synergistic ( Results: Dose-ranging experiments indicated that the SDX-101 IC50 in CLL cells was 800 μM, and that 400 μM of SDX-101 reduced CLL viability by 11% on average after 24 hrs. An SDX-101 dose of 400 μM was selected for combination studies with FA (used at 3.5 μM, yielding a viability reduction of 9.9%), 2-CdA (0.175 μM, 10.1%), RTX (20μg/ml, 6.1%), and ALT (20μg/ml, 8.4%). The combination of SDX-101+FA provided a drop of 20.9% in cell viability (p Conclusion: SDX-101 in combination with low doses of either 2-CdA, FA or RTX exerts an additive cytotoxic effect on ex-vivo B-CLL cells, indicating a possible clinical benefit of such a combination treatment. Up-regulation of pro-apoptotic protein expression (p73 and Bax), and down-regulation of anti-apoptotic protein (Mcl-1) seem to play a mechanistic role in these additive cytotoxic effects.

SDX-101 (R-etodolac), which is currently being evaluated in clinical trials for treatment of chronic lymphocytic leukemia, down regulates the activity of the β-catenin pathway and inhibits the growth of non-Hodgkin’s Lymphoma Daudi tumor xenografts in vivo when dosed orally (AACR PROC 2004 Abs# 2061 and #4574). Initial co-immunoprecipitation experiments conducted on cell nuclear fractions identified a heteromeric nuclear protein complex containing β-catenin and PPAR-γ. Furthermore, we have demonstrated that SDX-101 treatment reduces nuclear β-catenin in the immunoprecipitated complex, indicating that this complex may represent a target of SDX-101 (AACR PROC 2004 Abs# 3672). We recently reported evaluation of novel structural analogs of SDX-101 and have shown that these analogs, whose structures were not disclosed, are 5–10 fold more potent in in vitro cytotoxicity assays than SDX-101 and that they are orally efficacious in vivo (NCI/EORTC 2004 Abs #383). Our current studies further characterize the mechanism of action and safety of these analogs and identify the structures of selected analogs. Novel functional assays were developed to test and compare SDX-101 and the analogs at 4 hours post-treatment, a time before appreciable loss of viability was detected. Best results were obtained using a functional assay co-transfecting a β-catenin-dependent reporter construct (TOPFLASH) and β-catenin and RXR expression vectors. The average IC50 of analogs in this β-catenin reporter system ranged from 50 to 160 μM. These values were approximately five- to ten- fold lower than the IC50 for SDX-101 (~700 μM). Similar results were obtained assessing the inhibition of PPAR-γ-mediated transcription, using a PPAR-dependent reporter and co-transfection with PPAR-γ and RXR expression vectors. The average IC50s of the analogs ranged from 50–150 μM in this functional assay, demonstrating an approximately 10-fold increase in potency of the analogs when compared to SDX-101 (~1000 μM). No effect was observed at the 4 hour time point using a constitutive SV40-based control reporter vector. These results suggest that the primary target for these compounds may be a nuclear complex containing β-catenin, PPAR-γ and RXR, supporting a hypothesis developed upon evaluation of earlier results generated with SDX-101. To evaluate the safety of two SDX-101 analogs in vivo, normal mice were administered each analog at 240, 120 and 60 mg/kg/d (M-F) for four weeks. Mortality, morbidity, clinical signs, hematology/chemistry were monitored. There were no mortalities, overt toxicities or abnormal observations at necropsy with either of the analogs at any of the tested dose levels. There was a transient body weight loss ( In conclusion, these data demonstrate that the second generation analogs of SDX-101 display more potent in vitro and in vivo activity while retaining a mechanism of action similar to that of SDX-101.

SDX-101 is an oral antineoplastic agent, has been evaluated in a phase I/II study in B-cell malignancies. It induces cytotoxicity at least in part, by significantly inhibiting expression of Mcl-1, an anti-apoptotic Bcl-2 family protein which is highly expressed in CLL cells. Here, we examined the cytotoxicity of SDX-101 against multiple myeloma (MM) cell lines. SDX-101 significantly inhibited growth in MM.1S, U266, RPMI8226 MM cell lines using MTT assays in a time- and dose-dependent fashion, with IC50s of 0.6mM, 1.0mM, and 0.4mM, respectively. In contrast, SDX-101 did not induce cytotoxicity in normal peripheral mononuclear cells (PBMCs) at these concentrations. Importantly, SDX-101 induced cytotoxicity even in dexamethasone (MM.1R)-, doxorubicin (RPMI-Dox40)-, and melphalan (LR5)- resistant MM cell lines. SDX-101 (0.3–1.25mM) triggered apoptosis associated with pro-caspase-3, pro-caspase-8, and PARP cleavage, as confirmed by immunoblotting. Although, interleukin-6 (IL-6) and insulin-like growth factor (IGF)-1 completely abrogates Dex-induced MM cell apoptosis, neither protects against SDX-101-induced apoptosis in MM.1S and RPMI8226 cells. Moreover, dexamethasone, Melphalan, and AS2O3 augment apoptosis induced by SDX-101. Importantly, SDX-101 downregulated both b-catenin and cyclin D1 expression in RPMI8226 cells. Finally, our recent studies demonstrate that the bone marrow (BM) microenvironment promotes MM cell growth, survival, and drug resistance while SDX-101 inhibits viability even if MM cells adherent to BM stromal cells; an environment in which cytotoxic potency is lost with other anti-neoplastic agents. Our data therefore demonstrate that SDX-101 induces apoptosis in MM cells via a mechanism different from conventional MM drugs, and support clinical trials of this agent, alone or in combination with conventional and/or novel agents to improve therapeutic outcome in MM.

SDX-105 (Treanda™) is an alkylating agent with a distinct mechanism of action that has been shown to be active in clinical trials in NHL and CLL patients refractory to traditional DNA-damaging agents. SDX-105 induces unique changes in gene expression in NHL cells and displays a lack of cross resistance with other 2-chloroethylamine alkylating agents. Quantitative PCR analysis confirmed that the G2/M checkpoint regulators Polo-like kinase 1 (PLK-1) and Aurora A kinase (AurkA) are down-regulated in the NHL cell line SU-DHL-1 after 8 hours of exposure to clinically relevant concentrations of SDX-105. No changes in these same genes were observed when cells were exposed to equi-toxic doses of chlorambucil or an active metabolite of cyclophosphamide. Because our previous studies demonstrated that SDX-105 treatment can activate apoptotic cell death pathways, we examined the ability of SDX-105 to induce cytotoxicity in cells unable to undergo ‘classical’ caspase-mediated apoptosis. Multi-drug resistant MCF-7/ADR cells and p53 deficient RKO-E6 colon adenocarcinoma cells were exposed for two or three days to either 50 μM SDX-105 alone or 50 μM SDX-105 and 20 μM pan-caspase inhibitor zVAD-fmk. Although zVAD-fmk was able to inhibit SDX-105 induced increases in Annexin-V-positive cells, microscopic analysis of nuclear morphology using the DNA stain DAPI in cells treated with either SDX-105 alone or in combination with zVAD-fmk showed increased incidence of micronucleation. Multi/micro-nucleation and abnormal chromatin condensation are both hallmarks of mitotic catastrophe and have been observed in tumor cells exposed to microtubule-binding drugs such as the vinca alkaloids and taxanes. Activation of mitotic catastrophe may amplify the cytotoxicity of SDX-105 and its activity in tumor cells where classical apoptotic pathways are inhibited. This may explain, at least in part, the potent anti-tumor activity of SDX-105 in tumor cells refractory to conventional 2-chloroethylamine DNA-damaging agents. Additional studies are ongoing to further elaborate the role of mitotic catastrophe in SDX-105’s mechanism of action. The capacity to induce mitotic catastrophe may explain the anti-tumor activity of SDX-105 in chemotherapy relapsed and resistant patients.