RX-5902, a novel ß-catenin modulator, potentiates the efficacy of immune checkpoint inhibitors in preclinical models of triple-negative breast Cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited systemic treatment options. RX-5902 is a novel anti-cancer agent that inhibits phosphorylated-p68 and thus attenuates nuclear ß-catenin signaling. The purpose of this study was to evaluate the ability of ß-catenin signaling blockade to enhance the efficacy of anti-CTLA-4 and anti-PD-1 immune checkpoint blockade in immunocompetent, preclinical models of TNBC. METHODS: Treatment with RX-5902, anti-PD-1, anti-CTLA-4 or the combination was investigated in BALB/c mice injected with the 4 T1 TNBC cell line. Humanized BALB/c-Rag2nullIl2rγnullSIRPαNOD (hu-CB-BRGS) mice transplanted with a human immune system were implanted with MDA-MB-231 cells. Mice were randomized into treatment groups according to human hematopoietic chimerism and treated with RX-5902, anti-PD-1 or the combination. At sacrifice, bone marrow, lymph nodes, spleen and tumors were harvested for flow cytometry analysis of human immune cells. RESULTS: The addition of RX-5902 to CTLA-4 or PD-1 inhibitors resulted in decreased tumor growth in the 4 T1 and human immune system and MDA-MB-231 xenograft models. Immunologic analyses demonstrated a significant increase in the number of activated T cells in tumor infiltrating lymphocytes (TILs) with RX-5902 treatment compared to vehicle (p < 0.05). In the RX-5902/nivolumab combination group, there was a significant increase in the percentage of CD4+ T cells in TILs and increased systemic granzyme B production (p < 0.01). CONCLUSIONS: Conclusions: RX-5902 enhanced the efficacy of nivolumab in a humanized, preclinical model of TNBC. Several changes in immunologic profiles were noted in mice treated with RX-5902 and the combination, including an increase in activated TILs and a decrease in human myeloid populations, that are often associated with immunosuppression in a tumor microenvironment. RX-5902 also was shown to potentiate the effects of checkpoint inhibitors of CTLA4 and the PD-1 inhibitor in the 4 T-1 murine TNBC model. These findings indicate that RX-5902 may have important immunomodulatory, as well as anti-tumor activity, in TNBC when combined with a checkpoint inhibitor.

First-in-Class Phosphorylated-p68 Inhibitor RX-5902 Inhibits ß-Catenin Signaling and Demonstrates Antitumor Activity in Triple-Negative Breast Cancer.

RX-5902 is a first-in-class anticancer agent targeting phosphorylated-p68 and attenuating nuclear shuttling of ß-catenin. The purpose of this study was to evaluate the efficacy of RX-5902 in preclinical models of triple-negative breast cancer (TNBC) and to explore effects on ß-catenin expression. A panel of 18 TNBC cell lines was exposed to RX-5902, and changes in proliferation, apoptosis, cellular ploidy, and effector protein expression were assessed. Gene expression profiling was used in sensitive and resistant cell lines with pathway analysis to explore pathways associated with sensitivity to RX-5902. The activity of RX-5902 was confirmed in vivo in cell line and patient-derived tumor xenograft (PDX) models. RX-5902 demonstrated potent antiproliferative activity in vitro against TNBC cell lines with an average IC50 of 56 nmol/L in sensitive cell lines. RX-5902 treatment resulted in the induction of apoptosis, G2-M cell-cycle arrest, and aneuploidy in a subset of cell lines. RX-5902 was active in vivo against TNBC PDX models, and treatment resulted in a decrease in nuclear ß-catenin. RX-5902 exhibited dose-proportional pharmacokinetics and plasma and tumor tissue in nude mice. Pathway analysis demonstrated an increase in the epithelial-to-mesenchymal transformation (EMT), TGFß, and Wnt/ß-catenin pathways associated with sensitivity to RX-5902. RX-5902 is active against in vitro and in vivo preclinical models of TNBC. Target engagement was confirmed with decreases in nuclear ß-catenin and MCL-1 observed, confirming the proposed mechanism of action. This study supports the continued investigation of RX-5902 in TNBC and combinations with immunotherapy.

Folate receptor-targeted lipid-albumin nanoparticles (F-LAN) for therapeutic delivery of an Akt1 antisense oligonucleotide.

BACKGROUND: RX-0201 is an antisense oligonucleotide (ASO) against Akt1 currently in clinical trial for metastatic renal cancer. PURPOSE: To improve the delivery of RX-0201 using folate receptor-targeted lipid-albumin nanoparticles (F-LAN). METHODS: F-LAN were synthesized with the composition of DOTAP/soyPC/TPGS/folate-PEG-DSPE (25:70:4:1 m/m), a cationic human serum albumin-pentaethylenehexamine (HSA-PEHA) conjugate and RX-0201. The nanoparticles were evaluated in KB human carcinoma cells in vitro and in a KB murine xenograft tumour model in vivo for pharmacokinetics and antitumor activities. RESULTS: The F-LAN-RX-0201 had a mean particle size of 108.6 ± 5.8 nm, zeta potential of 10.5 ± 3.2 mV and ASO loading efficiency of 71.5 ± 4.5%. In KB cells, uptake and Akt1 inhibition by F-LAN-RX-0201 were greater than those of non-targeted LAN-RX-0201 and could be partially blocked by excess free folate. F-LAN-RX-0201 inhibited cell growth with an IC50 of 11.9 µM. In contrast, LAN-RX-0201 showed lower cytotoxicity with an IC50 of 32.0 µM. No significant cytotoxicity was observed with up to 250 µM of free RX-0201. Pharmacokinetic studies showed that F-LAN-RX-0201 had a longer terminal half-life than free RX-0201 (442 vs. 219 min). In a KB xenograft tumour model, F-LAN-RX-0201 exhibited greater tumour inhibition than LAN-RX-0201 at 16 mg/kg. Moreover, F-LAN-RX-0201 at 16 mg/kg showed comparable tumour inhibition compared to free RX-0201 at a much higher dose of 90 mg/kg. CONCLUSIONS: F-LAN-RX-0201 showed promise as a therapeutic agent for tumours with elevated folate-receptor expression.

Lipid-Albumin Nanoparticles (LAN) for Therapeutic Delivery of Antisense Oligonucleotide against HIF-1α.

Lipid-albumin nanoparticles (LAN) were synthesized for delivery of RX-0047, an antisense oligonucleotide (ASO) against the hypoxia inducible factor-1 alpha (HIF-1α) to solid tumor. These lipid nanoparticles (LNs) incorporated a human serum albumin-pentaethylenehexamine (HSA-PEHA) conjugate, which is cationic and can form electrostatic complexes with negatively charged oligonucleotides. The delivery efficiency of LAN-RX-0047 was investigated in KB cells and a KB murine xenograft model. When KB cells were treated with LAN-RX-0047, significant HIF-1α downregulation and enhanced cellular uptake were observed compared to LN-RX-0047. LN-RX-0047 and LAN-RX-0047 showed similar cytotoxicity against KB cells with IC50 values of 19.3 ± 3.8 and 20.1 ± 4.2 µM, respectively. LAN-RX-0047 was shown to be taken up by the cells via the macropinocytosis and caveolae-mediated endocytosis pathways while LN-RX-0047 was taken up by cells via caveolae-mediated endocytosis. In the KB xenograft tumor model, LAN-RX-0047 exhibited tumor suppressive activity and significantly reduced intratumoral HIF-1α expression compared to LN-RX-0047. Furthermore, LAN-RX-0047 greatly increased survival time of mice bearing KB-1 xenograft tumors at doses of either 3 mg/kg or 16 mg/kg. These results indicated that LAN-RX-0047 is a highly effective vehicle for therapeutic delivery of antisense agents to tumor.

Nuclear receptor Nurr1 agonists enhance its dual functions and improve behavioral deficits in an animal model of Parkinson's disease.

Parkinson's disease (PD), primarily caused by selective degeneration of midbrain dopamine (mDA) neurons, is the most prevalent movement disorder, affecting 1-2% of the global population over the age of 65. Currently available pharmacological treatments are largely symptomatic and lose their efficacy over time with accompanying severe side effects such as dyskinesia. Thus, there is an unmet clinical need to develop mechanism-based and/or disease-modifying treatments. Based on the unique dual role of the nuclear orphan receptor Nurr1 for development and maintenance of mDA neurons and their protection from inflammation-induced death, we hypothesize that Nurr1 can be a molecular target for neuroprotective therapeutic development for PD. Here we show successful identification of Nurr1 agonists sharing an identical chemical scaffold, 4-amino-7-chloroquinoline, suggesting a critical structure-activity relationship. In particular, we found that two antimalarial drugs, amodiaquine and chloroquine stimulate the transcriptional function of Nurr1 through physical interaction with its ligand binding domain (LBD). Remarkably, these compounds were able to enhance the contrasting dual functions of Nurr1 by further increasing transcriptional activation of mDA-specific genes and further enhancing transrepression of neurotoxic proinflammatory gene expression in microglia. Importantly, these compounds significantly improved behavioral deficits in 6-hydroxydopamine lesioned rat model of PD without any detectable signs of dyskinesia-like behavior. These findings offer proof of principle that small molecules targeting the Nurr1 LBD can be used as a mechanism-based and neuroprotective strategy for PD.

A Novel Anti-Cancer Agent, 1-(3,5-Dimethoxyphenyl)-4-[(6-Fluoro-2-Methoxyquinoxalin-3-yl)Aminocarbonyl] Piperazine (RX-5902), Interferes With ß-Catenin Function Through Y593 Phospho-p68 RNA Helicase.

1-(3,5-Dimethoxyphenyl)-4-[(6-fluoro-2-methoxyquinoxalin-3-yl)aminocarbonyl] piperazine (RX-5902) exhibits strong growth inhibition in various human cancer cell lines with IC50 values ranging between 10 and 20 nM. In this study, we demonstrate that p68 RNA helicase is a cellular target of RX-5902 by the drug affinity responsive target stability (DARTS) method, and confirmed the direct binding of (3) H-labeled RX-5902 to Y593 phospho-p68 RNA helicase. We further demonstrated RX-5902 inhibited the ß-catenin dependent ATPase activity of p68 RNA helicase in an in vitro system. Furthermore, we showed that treatment of cancer cells with RX-5902 resulted in the downregulation of the expression of certain genes, which are known to be regulated by the ß-catenin pathway, such as c-Myc, cyclin D1 and p-c-Jun. Therefore, our study indicates that the inhibition of Y593 phospho-p68 helicase - ß-catenin interaction by direct binding of RX-5902 to Y593 phospho-p68 RNA helicase may contribute to the anti-cancer activity of this compound.

A novel cytidine analog, RX-3117, shows potent efficacy in xenograft models, even in tumors that are resistant to gemcitabine.

RX-3117 (fluorocyclopentenylcytosine) is a cytidine analog and this class of drugs, including gemcitabine, has been widely used for the treatment of various types of cancers. However, there is no oral formulation of gemcitabine and drug resistance to gemcitabine is common. In this study, the efficacy of orally-administered RX-3117 was examined in 9 different human tumor xenograft models (colon, non-small cell lung, small cell lung, pancreatic, renal and cervical), grown subcutaneously in athymic nude mice. In the Colo 205, H460, H69 and CaSki models, gemcitabine treatment resulted in 28%, 30%, 25% and 0% tumor growth inhibition (TGI), respectively, whereas oral treatment with RX-3117 induced 100%, 78%, 62% and 66% TGI, respectively. This indicates that RX-3117 may have the potential to be used for the treatment of tumors that do not respond to gemcitabine. RX-3117 was also evaluated in a single primary low-passage human pancreatic Tumorgraft™CTG-0298 (TGI 76%), which is relatively resistant to gemcitabine (TGI 38%) and has a favorable RX-3117-activating enzyme profile. These studies demonstrated the therapeutic potential and anticancer efficacy of RX-3117.

Metabolism, mechanism of action and sensitivity profile of fluorocyclopentenylcytosine (RX-3117; TV-1360).

A novel cytidine analog fluorocyclopentenylcytosine (RX-3117; TV-1360) was characterized for its cytotoxicity in a 59-cell line panel and further characterized for cytotoxicity, metabolism and mechanism of action in 15 additional cancer cell lines, including gemcitabine-resistant variants. In both panels sensitivity varied 75-fold (IC50: 0.4- > 30 µM RX-3117). RX-3117 showed a different sensitivity profile compared to cyclopentenyl-cytosine (CPEC) and azacytidine, substrates for uridine-cytidine-kinase (UCK). Dipyridamole, an inhibitor of the equilibrative-nucleoside-transporter protected against RX-3117. Uridine and cytidine protected against RX-3117, but deoxycytidine (substrate for deoxycytidine-kinase [dCK]) not, although it protected against gemcitabine, demonstrating that RX-3117 is a substrate for UCK and not for dCK. UCK activity was abundant in all cell lines, including the gemcitabine-resistant variants. RX-3117 was a very poor substrate for cytidine deaminase (66,000-fold less than gemcitabine). RX-3117 was rapidly metabolised to its nucleotides predominantly the triphosphate, which was highest in the most sensitive cells (U937, A2780) and lowest in the least sensitive (CCRF-CEM). RX-3117 did not significantly affect cytidine and uridine nucleotide pools. Incorporation of RX-3117 into RNA and DNA was higher in sensitive A2780 and low in insensitive SW1573 cells. In sensitive U937 cells 1 µM RX-3117 resulted in 90% inhibition of RNA synthesis but 100 µM RX-3117 was required in A2780 and CCRF-CEM cells. RX-3117 at IC50 values did not affect the integrity of RNA. DNA synthesis was completely inhibited in sensitive U937 cells at 1 µM, but in other cells even higher concentrations only resulted in a partial inhibition. At IC50 values RX-3117 downregulated the expression of DNA methyltransferase. In conclusion, RX-3117 showed a completely different sensitivity profile compared to gemcitabine and CPEC, its uptake is transporter dependent and is activated by UCK. RX-3117 is incorporated into RNA and DNA, did not affect RNA integrity, depleted DNA methyltransferase and inhibited RNA and DNA synthesis. Nucleotide formation is related with sensitivity.

Clavulanic acid inhibits MPP⁺-induced ROS generation and subsequent loss of dopaminergic cells.

Clavulanic acid is a psychoactive compound that has been shown to modulate central nervous system activity. Importantly, in neurotoxin-induced animal models, clavulanic acid has been shown to improve motor function (Huh et al., 2010) suggesting that it can be neuroprotective; however, the mechanism as how clavulanic acid can induce neuroprotection is not known. We demonstrate here that clavulanic acid abrogates the effects of the neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) which mimics Parkinson's disease (PD) by inducing neurodegeneration. To further establish the mechanism we identified that clavulanic acid inhibits neurotoxin-induced loss of mitochondrial membrane potential and ROS production. Consistent with these results, neurotoxin-induced increase in Bax levels was also decreased in clavulanic acid treated cells. Importantly, neurotoxin-induced release of cytochrome c levels as well as caspase activation was also inhibited in clavulanic acid treated cells. In addition, Bcl-xl levels were also restored and the Bcl-xl/Bax ratio that is critical for inducing apoptosis was increased in clavulanic acid treated cells. Overall, these results suggest that clavulanic acid is intimately involved in inhibiting neurotoxin-induced loss of mitochondrial function and induction of apoptosis that contributes towards neuronal survival.

Synthesis, anticancer activity and pharmacokinetic analysis of 1-[(substituted 2-alkoxyquinoxalin-3-yl)aminocarbonyl]-4-(hetero)arylpiperazine derivatives.

Based on the anticancer activity of novel quinoxalinyl-piperazine compounds, 1-[(5 or 6-substituted alkoxyquinoxalinyl)aminocarbonyl]-4-(hetero)arylpiperazine derivatives published in Bioorg. Med. Chem.2010, 18, 7966, we further explored the synthesis of 7 or 8-substituted quinoxalinyl piperazine derivatives. From in vitro studies of the newly synthesized compounds using human cancer cell lines, we identified some of the 8-substituted compounds, for example 6p, 6q and 6r, which inhibited the proliferation of various human cancer cells at nanomolar concentrations. Compound 6r, in particular, showed the lowest IC(50) values, ranging from 6.1 to 17nM, in inhibition of the growth of cancer cells, which is better than compound 6k (compound 25 in the reference cited above). In order to select and develop a leading compound among the quinoxaline compounds with substitutions on positions 5, 6, 7 or 8, the compounds comparable to compound 6k in in vitro cancer cell growth inhibition were chosen and their pharmacokinetic properties were evaluated in rats. In these studies, compound 6k showed the highest oral bioavailability of 83.4%, and compounds 6j and 6q followed, with 77.8% and 57.6%, respectively. From the results of in vitro growth inhibitory activities and the pharmacokinetic study, compound 6k is suggested for further development as an orally deliverable anticancer drug.

Polymeric LabChip real-time PCR as a point-of-care-potential diagnostic tool for rapid detection of influenza A/H1N1 virus in human clinical specimens.

It is clinically important to be able to detect influenza A/H1N1 virus using a fast, portable, and accurate system that has high specificity and sensitivity. To achieve this goal, it is necessary to develop a highly specific primer set that recognizes only influenza A viral genes and a rapid real-time PCR system that can detect even a single copy of the viral gene. In this study, we developed and validated a novel fluidic chip-type real-time PCR (LabChip real-time PCR) system that is sensitive and specific for the detection of influenza A/H1N1, including the pandemic influenza strain A/H1N1 of 2009. This LabChip real-time PCR system has several remarkable features: (1) It allows rapid quantitative analysis, requiring only 15 min to perform 30 cycles of real-time PCR. (2) It is portable, with a weight of only 5.5 kg. (3) The reaction cost is low, since it uses disposable plastic chips. (4) Its high efficiency is equivalent to that of commercially available tube-type real-time PCR systems. The developed disposable LabChip is an economic, heat-transferable, light-transparent, and easy-to-fabricate polymeric chip compared to conventional silicon- or glass-based labchip. In addition, our LabChip has large surface-to-volume ratios in micro channels that are required for overcoming time consumed for temperature control during real-time PCR. The efficiency of the LabChip real-time PCR system was confirmed using novel primer sets specifically targeted to the hemagglutinin (HA) gene of influenza A/H1N1 and clinical specimens. Eighty-five human clinical swab samples were tested using the LabChip real-time PCR. The results demonstrated 100% sensitivity and specificity, showing 72 positive and 13 negative cases. These results were identical to those from a tube-type real-time PCR system. This indicates that the novel LabChip real-time PCR may be an ultra-fast, quantitative, point-of-care-potential diagnostic tool for influenza A/H1N1 with a high sensitivity and specificity.

Clavulanic acid increases dopamine release in neuronal cells through a mechanism involving enhanced vesicle trafficking.

Clavulanic acid is a CNS-modulating compound with exceptional blood-brain barrier permeability and safety profile. Clavulanic acid has been proposed to have anti-depressant activity and is currently entering Phase IIb clinical trials for the treatment of Major Depressive Disorder (MDD). Studies have also shown that clavulanic acid suppresses anxiety and enhances sexual functions in rodent and primate models by a mechanism involving central nervous system (CNS) modulation, although its detailed mechanism of action has yet to be elucidated. To further examine its potential as a CNS modulating agent as well as its mechanism of action, we investigated the effects of clavulanic acid in neuronal cells. Our results indicate that clavulanic acid enhances dopamine release in PC12 and SH-SY5Y cells without affecting dopamine synthesis. Furthermore, using affinity chromatography we were able to identify two proteins, Munc18-1 and Rab4 that potentially bind to clavulanic acid and play a critical role in neurosecretion and the vesicle trafficking process. Consistent with this result, an increase in the translocation of Munc18-1 and Rab4 from the cytoplasm to the plasma membrane was observed in clavulanic acid treated cells. Overall, these data suggest that clavulanic acid enhances dopamine release in a mechanism involving Munc18-1 and Rab4 modulation and warrants further investigation of its therapeutic use in CNS disorders, such as depression.

Norepinephrine deficiency is caused by combined abnormal mRNA processing and defective protein trafficking of dopamine beta-hydroxylase.

Human norepinephrine (NE) deficiency (or dopamine ß-hydroxylase (DBH) deficiency) is a rare congenital disorder of primary autonomic failure, in which neurotransmitters NE and epinephrine are undetectable. Although potential pathogenic mutations, such as a common splice donor site mutation (IVS1+2T→C) and various missense mutations, in NE deficiency patients were identified, molecular mechanisms underlying this disease remain unknown. Here, we show that the IVS1+2T→C mutation results in a non-detectable level of DBH protein production and that all three missense mutations tested lead to the DBH protein being trapped in the endoplasmic reticulum (ER). Supporting the view that mutant DBH induces an ER stress response, exogenous expression of mutant DBH dramatically induced expression of BiP, a master ER chaperone. Furthermore, we found that a pharmacological chaperone, glycerol, significantly rescued defective trafficking of mutant DBH proteins. Taken together, we propose that NE deficiency is caused by the combined abnormal processing of DBH mRNA and defective protein trafficking and that this disease could be treated by a pharmacological chaperone(s).

Antitumor activity of a novel antisense oligonucleotide against Akt1.

The AKT pathway is an important therapeutic target for cancer drug discovery as it functions as a main point for transducing extracellular and intracellular oncogenic signals. Moreover, alternations of the AKT pathway have been found in a wide range of cancers. In the present study, we found that an Akt1 antisense oligonucleotide (Akt1 AO) significantly downregulated the expression of AKT1 at both the mRNA and protein levels and inhibited cellular growth at nanomolar concentrations in various types of human cancer cells. Combined treatment of Akt1 AO with several cytotoxic drugs resulted in an additive growth inhibition of Caki-1 cells. The in vivo effectiveness of Akt1 AO was determined using two different xenograft nude mouse models. Akt1 AO (30 mg/kg, i.v. every 48 h) significantly inhibited the tumor growth of nude mouse subcutaneously implanted with U251 human glioblastoma cells after 27 days treatment. Akt1 AO (30 mg/kg, i.p continuously via osmotic pump) also significantly inhibited the tumor formation in nude mice implanted with luciferase-expressing MIA human pancreatic cancer cells (MIA-Luc) after 14 days of treatment. The luciferase signals from MIA-Luc cells were reduced or completely abolished after 2 weeks of treatment and the implanted tumors were barely detectable. Our findings suggest that Akt1 AO alone or in combination with other clinically approved anticancer agents should be further explored and progressed into clinical studies as a potential novel therapeutic agent.

Clavulanic acid stimulates sexual behaviour in male rats.

Sexual behaviour in rats can be used to predict putative effects on human sexual behaviour. Anecdotic reports exist, that the beta-lactamase inhibitor, clavulanic acid exerts sexual stimulating activities in monkeys. To characterize these pro-sexual activities, clavulanic acid was tested in three doses and compared to one dose of a sexually inhibitory dose of the selective serotonin reuptake inhibitor, paroxetine, in sexually-experienced male rats, selected for a moderate level of sexual performance in a standard 30-min test with an oestrus female. After acute administration, clavulanic acid had minor sexual stimulating effects at the highest dose in the number of intromissions and in the first ejaculation series. After sub-chronic 7-days treatment, clavulanic acid increased the number of ejaculations at all three doses and reduced the number of intromissions in the 1st series at the highest dose. After chronic 14 days treatment, a similar but stronger pro-sexual profile was observed. The sexual side effects of paroxetine were as expected, including slight sexual inhibitory effects after acute administration, but somewhat stronger overall inhibitory effects after 7 and 14-days pretreatment, particularly notable in the decreasing number of animals contributing to the 2nd ejaculation series, which was even stronger after 14-days treatment. One week after cessation of treatment, the paroxetine group had completely recovered, whereas the highest dose-group of clavulanic acid still showed some pro-sexual effects. This remarkable pro-sexual activity of clavulanic acid cannot readily be explained by its mechanism of action as a beta-lactamase inhibitor but could be due to unexpected central activity of the compound.