IMD-0354 targets breast cancer stem cells: a novel approach for an adjuvant to chemotherapy to prevent multidrug resistance in a murine model.

Although early detection of breast cancer improved in recent years, prognosis of patients with late stage breast cancer remains poor, mostly due to development of multidrug resistance (MDR) followed by tumor recurrence. Cancer stem cells (CSCs), with higher drug efflux capability and other stem cell-like properties, are concentrated in a side population (SP) of cells, which were proposed to be responsible for MDR and tumor repopulation that cause patients to succumb to breast cancer. Therefore, targeting of CSCs as an adjuvant to chemotherapy should be able to provide a more effective treatment of this disease. Here, we used IMD-0354, an inhibitor of NF-κB, identified for targeting CSCs, in a combination therapy with doxorubicin encapsulated in targeted nanoparticles. IMD-0354 did target CSCs, evidenced by a decrease in the SP, demonstrated by the inhibition of the following: dye/drug efflux, reduction in ABC transporters as well as in colony formation in soft agar and low attachment plates. Decrease of stem-like gene expression of Oct4, Nanog and Sox2, and apoptosis resistance related to the Survivin gene also was observed after treatment with this compound. In addition, IMD-0354 targeted non-CSCs as indicated by reducing viability and increasing apoptosis. Targeted drug delivery, achieved with a legumain inhibitor, proved to enhance drug delivery under hypoxia, a hallmark of the tumor microenvironment, but not under normoxia. Together, this allowed a safe, non-toxic delivery of both anticancer agents to the tumor microenvironment of mice bearing syngeneic metastatic breast cancer. Targeting both bulk tumor cells with a chemotherapeutic agent and CSCs with IMD-0354 should be able to reduce MDR. This could eventually result in decreasing tumor recurrences and/or improve the outcome of metastatic disease

Combination therapy and tNP drug delivery <i>in vitro</i>.

<p>Viability of MDA-MB-231 cells was assessed by MTT assay after treatment with doxorubicin alone or in combination with IMD-0354 for 48 h (A). Viability of FL4T1 cells was assessed by MTT assay after treatment with doxorubicin (B) or mitoxantrone (C) alone or in combination with IMD-0354 for 48 h. Surface extracellular display of legumain on 4T1 cells assessed by FACS under normoxia (control) and hypoxia (CoCl₂ treatment). Viability of FL4T1 cells was assessed by MTT assay after treatment with free doxorucibin and tNP-doxorubicin for 48 h under normoxia (E) and hypoxia (F); same data as in E and F, plotted to compare tNP-Dox effect on FL4T1 viability under normoxia and hypoxia. Data are shown as mean ± SEM.</p

Combination therapy encapsulated into tNPs for drug delivery <i>in vivo</i>.

<p>Mice bearing experimental metastatic FL4T1 tumors were treated with tNP-Dox, tNP-IMD-0354 or tNP-Dox-IMD-0354. Tumor burden was detected by <i>in vivo</i> non-invasive imaging of the firefly luciferase expressing FL4T1 cells after intraperitoneal luciferin injection. Tumor bioluminescence intensity was plotted in pseudocolor over black/white photographs (A) and quantified as total flux in photons/seconds (p/s). Data are shown as mean ± SEM. *P-value < 0.05.</p

IMD-0354 effects on human and murine breast cancer cells.

<p>Viability of MDA-MB-231 cells was assessed by MTT assay after treatment with IMD-0354 for 24 h (A), 48 h (B) and 72 h (C). Apoptotic cells as Annexin V-FITC+ cells analyzed by FACS of FL4T1 cells treated with IMD-0354 (D). Viability of 4T1 cells was assessed by MTT assay after treatment with IMD-0354 for 24 h (E), 48 h (F) and 72 h (G). Data are shown as mean ± SEM. **P-value < 0.01; ****P-value < 0.001.</p

Effects of IMD-0354 on CSCs.

<p>Number of colony formation in low attachment plates of 4T1 (A) and MDA-MB-231 (B) treated with 1 µM IMD-0354. Number of colony formation in soft agar of 4T1 (C) and MDA-MB-231 (D) treated with 1 µM IMD-0354. Western blot of CSCs gene expression of cytosolic and nuclear extract from 4T1 (F) and MDA-MB-231 (G) cells treated with IMD-0354. Data are shown as mean ± SEM.</p

Murine breast cancer cells are chemoresistant to different chemotherapeutic agents.

<p>Viability of 4T1 or FL4T1 cells was assessed by the MTT assay after treatment with: doxorubicin for 48 h comparing both cell lines (A); doxorubicin for 24 (B) and 48 h (C); mitoxantrone for 24 (D) and 48 h (E); cisplatin for 24 (F) or 48 h (G). Data are shown as mean ± SEM.</p

IMD-0354 inhibits side population of CSCs in human and murine breast cancer cells.

<p>Side population of CSCs as analyzed by FACS and their Hoechst 33342 dye efflux inhibition by reserpine in MDA-MB-231 (A) and 4T1 (B). Dose curve of IMD-0354 on side population in MDA-MB-231 (C) and 4T1 (D). Data are shown as mean ± SEM.</p

Human breast cancer cells are chemoresistant to different chemotherapeutic agents.

<p>Viability of MDA-MB-231 cells was assessed by MTT assay after treatment with doxorubicin for 24 h (A), 48 h (B) and 72 h (C), or cisplatin for 24 h (D), or mitoxantrone for 48 h (E). Data are shown as mean ± SEM.</p

The small molecule alpha-synuclein misfolding inhibitor, NPT200-11, produces multiple benefits in an animal model of Parkinson’s disease

Abstract Accumulation of alpha-synuclein (ASYN) in neurons and other CNS cell types may contribute to the underlying pathology of synucleinopathies including Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and Multiple Systems Atrophy (MSA). In support of this hypothesis for PD, ASYN immunopositive aggregates are a prominent pathological feature of PD, and mutations and gene multiplications of human wild type (WT) ASYN cause rare familial autosomal-dominant forms of PD. Targeted therapeutics that reduce the accumulation of ASYN could prevent or slow the neurodegenerative processes in PD and other synucleinopathies. NPT200-11 is a novel small molecule inhibitor of ASYN misfolding and aggregation. The effects of NPT200-11 on ASYN neuropathology were evaluated in animal models over expressing human alpha synuclein. Longitudinal studies using retinal imaging in mice expressing a hASYN::GFP fusion protein revealed that 2 months of once daily administration of NPT200-11 (5 mg/kg IP) resulted in a time-dependent and progressive reduction in retinal ASYN pathology. The effects of NPT200-11 on ASYN pathology in cerebral cortex and on other disease-relevant endpoints was evaluated in the Line 61 transgenic mouse model overexpressing human wild type ASYN. Results from these studies demonstrated that NPT200-11 reduced alpha-synuclein pathology in cortex, reduced associated neuroinflammation (astrogliosis), normalized striatal levels of the dopamine transporter (DAT) and improved motor function. To gain insight into the relationship between dose, exposure, and therapeutic benefit pharmacokinetic studies were also conducted in mice. These studies demonstrated that NPT200-11 is orally bioavailable and brain penetrating and established target plasma and brain exposures for future studies of potential therapeutic benefit