Deog Joong Kim

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. J. Cell. Biochem. 108: 832–838, 2009.

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.

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 Parkinsons 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.

The Cytidine Analog Fluorocyclopentenylcytosine (RX-3117) Is Activated by Uridine-Cytidine Kinase 2.

Fluorocyclopentenylcytosine (RX-3117) is an orally available cytidine analog, currently in Phase I clinical trial. RX-3117 has promising antitumor activity in various human tumor xenografts including gemcitabine resistant tumors. RX-3117 is activated by uridine-cytidine kinase (UCK). Since UCK exists in two forms, UCK1 and UCK2, we investigated which form is responsible for RX-3117 phosphorylation. For that purpose we transfected A549 and SW1573 cell lines with UCK-siRNAs. Transfection of UCK1-siRNA efficiently downregulated UCK1-mRNA, but not UCK2-mRNA expression, and did not affect sensitivity to RX-3117. However, transfection of UCK2-siRNA completely downregulated UCK2-mRNA and protein and protected both A549 and SW1573 against RX-3117. UCK enzyme activity in two panels of tumor cell lines and xenograft cells correlated only with UCK2-mRNA expression (r = 0.803 and 0.915, respectively), but not with UCK1-mRNA. Moreover, accumulation of RX-3117 nucleotides correlated with UCK2 expression. In conclusion, RX-3117 is activated by UCK2 which may be used to select patients potentially sensitive to RX-3117.

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.

A Novel Anti‐Cancer Agent, 1‐(3,5‐Dimethoxyphenyl)‐4‐[(6‐Fluoro‐2‐Methoxyquinoxalin‐3‐yl)Aminocarbonyl] Piperazine (RX‐5902), Interferes With β‐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 3H‐labeled RX‐5902 to Y593 phospho‐p68 RNA helicase. We further demonstrated RX‐5902 inhibited the β‐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 β‐catenin pathway, such as c‐Myc, cyclin D1 and p‐c‐Jun. Therefore, our study indicates that the inhibition of Y593 phospho‐p68 helicase ‐ β‐catenin interaction by direct binding of RX‐5902 to Y593 phospho‐p68 RNA helicase may contribute to the anti‐cancer activity of this compound. J. Cell. Biochem. 116: 1595–1601, 2015.

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

Abstract 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.

The radiosensitizing effect of fluorocyclopentenyl-cytosine (RX-3117) in ovarian and lung cancer cell lines.

ABSTRACT RX-3117 (fluorocyclopentenyl-cytosine) is a novel cytidine analog currently being evaluated in a Phase Ib clinical trial in cancer patients with solid tumors. The radiosensitizing effect of RX-3117 was studied in A2780 ovarian cancer cells and non-small cell lung cancer cell lines and related to cell survival and the effect on cell cycle and cell cycle proteins. RX-3117 has a schedule-dependent radiosensitizing effect, but only at pre-incubation (dose modifying factors: 1.4–1.8), observed at pulse and fractionated irradiation. Radiosensitizion was also seen in a three-dimensional spheroid model. At the low radiosensitizing concentration, RX-3117 in combination with radiation led to an accumulation of cells in S-phase, which was accompanied with an increase of cell cycle proteins such as p-Chk2 and p-cdc25C. In addition, RX-3117 caused DNA damage and increased apoptosis. In conclusion, our in vitro experiments showed a radiosensitizing effect of RX-3117.

Abstract 1770: Metabolism and mechanism of action of fluorocyclopentenylcytosine (RX-3117)

Cytidine analogs play an important role in the treatment of various types of cancer, both solid tumors and leukemia. A novel cytidine analog fluorocyclopentenylcytosine (RX-3117) was characterized for its cytotoxic effects, its metabolism and its mechanism of action in a panel of 9 solid tumor and leukemic cell lines, as well as 6 variants resistant to gemcitabine, cytarabine and other pyrimidine analogs. Sensitivity in the parent cell lines after 72 hr exposure varied 75 fold with IC50 values from 0.4 to 30 µM RX-3117. The human equilibrative nucleoside transporter mediates transport of RX-3117, since its inhibition protected cells. Uridine and cytidine also protected cells against RX-3117, indicating that activation of RX-3117 is dependent on phosphorylation catalyzed by uridine-cytidine kinase (UCK), which was abundant in all tested cell lines, including the gemcitabine resistant variants. Deoxycytidine did not protect cells against RX-3117. RX-3117 was a very poor substrate for cytidine deaminase (66,000-fold less than gemcitabine). After its uptake in cells, RX-3117 was rapidly metabolised to its nucleotides with the triphosphate being the most prominent form (90% of all nucleotides), while synthesis of the nucleotides was highest in the most sensitive cell lines (U937 and A2780 cells) and lowest in the least sensitive cells (CCRF CEM cells). No difference in nucleotide formation was observed between the SW1573 and its gemcitabine resistant variant SW1573/G. In the AG6000 cells, the dCK- variant of A2780 and resistant to gemcitabine and RX-3117, a normal monophosphate level was found, but no di-and triphosphates were formed, explaining its resistance. Similarly incorporation of RX-3117 into RNA and DNA was higher in the sensitive A2780 and low in the insensitive SW1573 cells, with no difference between the gemcitabine sensitive and resistant variants. The effect of RX-3117 on synthesis of RNA and DNA was quite different; in the sensitive U937 cells 10 µM RX-3117 inhibited RNA synthesis 90%, while in A2780 and CCRF-CEM cells 100 µM RX-3117 was required for 90% inhibition of RNA synthesis. The effect on DNA synthesis was quite different. 1 µM RX-3117 completely inhibited DNA synthesis in the sensitive U937 cells, 80-90% inhibition was achieved with 10 µM in both CCRF CEM variants and with 100 µM in SW1573/G and AG6000, but in A2780 and SW1573 cells 100 µM only resulted in a partial or no inhibition, respectively. In conclusion, RX-3117 showed a completely different sensitivity profile compared to other cytidine analogs. Its uptake is transporter dependent; it is not activated by dCK, but by UCK. RX-3117 is incorporated into RNA and DNA; RX-3117 hardly affected RNA synthesis at IC50 values, but inhibited DNA synthesis. Its metabolism to nucleotides is related with its sensitivity, possibly because they directly inhibit the target presumably DNA methyltransferase. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1770. doi:1538-7445.AM2012-1770