Jeong Uk Choi

Optimization of a Stable Linker Involved DEVD Peptide-Doxorubicin Conjugate That Is Activated upon Radiation-Induced Caspase-3-Mediated Apoptosis.

The current study demonstrates the process of selecting an optimal structure for a caspase-3-cleavable doxorubicin prodrug that could be synthesized by simple chemistry in high yields. The prodrug was intended to activate in the presence of caspase-3, whose expression can be exogenously regulated by inducing apoptosis with radiation therapy at a specific site of interest. For this purpose, doxorubicin was conjugated with a DEVD peptide via a heterobifunctional linker. Since the active form of the prodrug comprises the linker besides doxorubicin, we tested several different linkers and selected EMCS based on the examination of its in vitro biological activities. Consequently, DEVD-cysteamide-EMCS-doxorubicin was synthesized as the final compound. According to the various in vitro and in vivo studies, the synthesized prodrug was highly selective for tumors when coupled with radiation therapy, with the added benefit of ease of production.

Targeting prion-like protein doppel selectively suppresses tumor angiogenesis

Controlled and site-specific regulation of growth factor signaling remains a major challenge for current antiangiogenic therapies, as these antiangiogenic agents target normal vasculature as well tumor vasculature. In this article, we identified the prion-like protein doppel as a potential therapeutic target for tumor angiogenesis. We investigated the interactions between doppel and VEGFR2 and evaluated whether blocking the doppel/VEGFR2 axis suppresses the process of angiogenesis. We discovered that tumor endothelial cells (TECs), but not normal ECs, express doppel; tumors from patients and mouse xenografts expressed doppel in their vasculatures. Induced doppel overexpression in ECs enhanced vascularization, whereas doppel constitutively colocalized and complexed with VEGFR2 in TECs. Doppel inhibition depleted VEGFR2 from the cell membrane, subsequently inducing the internalization and degradation of VEGFR2 and thereby attenuating VEGFR2 signaling. We also synthesized an orally active glycosaminoglycan (LHbisD4) that specifically binds with doppel. We determined that LHbisD4 concentrates over the tumor site and that genetic loss of doppel in TECs decreases LHbisD4 binding and targeting both in vitro and in vivo. Moreover, LHbisD4 eliminated VEGFR2 from the cell membrane, prevented VEGF binding in TECs, and suppressed tumor growth. Together, our results demonstrate that blocking doppel can control VEGF signaling in TECs and selectively inhibit tumor angiogenesis.

Design, Synthesis, and Therapeutic Evaluation of Poly(acrylic acid)-tetraDOCA Conjugate as a Bile Acid Transporter Inhibitor.

Regulation of cholesterol and bile acid homeostasis has been attracting attention as a pharmaceutical target for the treatment of diseases, such as hypercholesterolaemia and type 2 diabetes. In recent years, small bile acid analogues have been developed for the purpose of apical sodium-dependent bile acid transporter (ASBT) inhibition. Here, we designed a novel hydrophilic ASBT inhibitor using oligomeric bile acid with a high affinity with ASBT. Polyacrylic acid-tetraDOCA conjugates (PATD) have the ability to bind to ASBT in order to induce hypocholesterolemic effects. Both the viability and the functionality of PATD were evaluated in vitro, showing that PATDs were effective in inhibiting the increases of cholesterol in the blood and oil in the liver induced by high fat diet (HFD). The results indicated that the newly developed biomaterials with oligomeric bile acids and a hydrophilic polymer are potent therapeutic agents for hyperlipidemia.

Metronomic chemotherapy using orally active carboplatin/deoxycholate complex to maintain drug concentration within a tolerable range for effective cancer management.

&NA; Metronomic chemotherapy has translated into favorable toxicity profile and capable of delaying tumor progression. Despite its promise, conventional injectable chemotherapeutics are not meaningful to use as metronomic due to the necessity of frequent administration for personalized therapy in long‐term cancer treatments. This study aims to exploit the benefits of the oral application of carboplatin as metronomic therapy for non‐small cell lung cancer (NSCLC). We developed an orally active carboplatin by physical complexation with a deoxycholic acid (DOCA). The X‐ray diffraction (XRD) patterns showed the disappearance of crystalline peaks from carboplatin by forming the complex with DOCA. In vivo pharmacokinetic (PK) study confirmed the oral absorption of carboplatin/DOCA complex. The oral bioavailability of carboplatin/DOCA complex and native carboplatin were calculated as 24.33% and 1.16%, respectively, when a single 50 mg/kg oral dose was administered. Further findings of oral bioavailability during a low‐dose daily administration of the complex (10 mg/kg) for 3 weeks were showed 19.17% at day‐0, 30.27% at day‐7, 26.77% at day‐14, and 22.48% at day‐21, demonstrating its potential for metronomic chemotherapy. The dose dependent antitumor effects of oral carboplatin were evaluated in SCC7 and A549 tumor xenograft mice. It was found that the oral carboplatin complex exhibited potent anti‐tumor activity at 10 mg/kg (74.09% vs. control, P < 0.01) and 20 mg/kg dose (86.22% vs. control, P < 0.01) in A549 tumor. The number of TUNEL positive cells in the tumor sections was also significantly increased during oral therapy (3.95% in control, whereas 21.37% and 32.39% in 10 mg/kg and 20 mg/kg dose, respectively; P < 0.001). The enhanced anti‐tumor efficacy of oral metronomic therapy was attributed with its antiangiogenic mechanism where new blood vessel formation was notably decreased. Finally, the safety of oral complex was confirmed by three weeks toxicity studies; there were no significant systemic or local abnormalities found in mice at 10 mg/kg daily oral dose. Our study thus describes an effective and safe oral formulation of carboplatin as a metronomic chemotherapy. Graphical abstract Figure. No caption available.

Bile acid transporter mediated endocytosis of oral bile acid conjugated nanocomplex

The development of highly funtional and orally available nanoparticles is the ultimate goal in nanoparticle delivery. Various functional nanoparticles have been studied to that end but there has yet to be an oral nanoparticle that can be successfully applied. Here, we describe for the first time a novel bile acid conjugated nanoparticle that can be selectively absorbed by bile acid transporters in the small intestine. The bile acid conjugate nanoparticles that were first treated with enterocytes were successfully attached to the cell surface and then internalized inside the cells. We show that bile acid based interaction between a nanoparticle and its transporter induces its endocytosis and cellular uptake. This feature of cellular activity, described here for the first time, could be well utilized in the uptake of nanoparticles or macromolecules inside epithelial cells for oral delivery. In animal studies, bile acid conjugated self-assembling nanocomplexes successfully interacted with bile acid transporters in the ileum and were subsequently taken up into the epithelial cells. Considering the importance of orally deliverable nanoparticles, this nanotechnology using bile acid conjugation and transporter mediated endocytosis could be a crucial method for the successful application of various nanoparticles.

Preparation and in vivo evaluation of cationic elastic liposomes comprising highly skin-permeable growth factors combined with hyaluronic acid for enhanced diabetic wound-healing therapy

To enhance the therapeutic effects of exogenous administration of growth factors (GFs) in the treatment of chronic wounds, we constructed GF combinations of highly skin-permeable epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and platelet-derived growth factor-A (PDGF-A). We genetically conjugated a low-molecular-weight protamine (LMWP) to the N-termini of these GFs to form LMWP-EGF, LMWP-IGF-I, and LMWP-PDGF-A. Subsequently, these molecules were complexed with hyaluronic acid (HA). Combinations of native or LMWP-fused GFs significantly promoted fibroblast proliferation and the synthesis of procollagen, with a magnification of these results observed after the GFs were complexed with HA. The optimal proportions of LMWP-EGF, LMWP-IGF-I, LMWP-PDGF-A, and HA were 1, 1, 0.02, and 200, respectively. After confirming the presence of a synergistic effect, we incorporated the LMWP-fused GFs-HA complex into cationic elastic liposomes (ELs) of 107±0.757nm in diameter and a zeta potential of 56.5±1.13mV. The LMWP-fused GFs had significantly improved skin permeation compared with native GFs. The in vitro wound recovery rate of the LMWP-fused GFs-HA complex was 23% higher than that of cationic ELs composed of LMWP-fused GFs alone. Moreover, the cationic ELs containing the LMWP-fused GFs-HA complex significantly accelerated the wound closure rate in a diabetic mouse model and the wound size was maximally decreased by 65% and 58% compared to cationic ELs loaded with vehicle or native GFs-HA complex, respectively. Thus, topical treatment with cationic ELs loaded with the LMWP-fused GFs-HA complex synergistically enhanced the healing of chronic wounds, exerting both rapid and prolonged effects. STATEMENT OF SIGNIFICANCE We believe that our study makes a significant contribution to the literature, because it demonstrated the potential application of cationic elastic liposomes as topical delivery systems for growth factors (GFs) that have certain limitations in their therapeutic effects (e.g., low percutaneous absorption of GFs at the lesion site and the requirement for various GFs at different healing stages). Topical treatment with cationic elastic liposomes loaded with highly skin-permeable low-molecular-weight protamine (LMWP)-fused GFs-hyaluronic acid (HA) complex synergistically enhanced the healing of diabetic wounds, exerting both rapid and prolonged effects.

Tissue adhesive FK506–loaded polymeric nanoparticles for multi–layered nano–shielding of pancreatic islets to enhance xenograft survival in a diabetic mouse model

This study aims to develop a novel surface modification technology to prolong the survival time of pancreatic islets in a xenogenic transplantation model, using 3,4-dihydroxyphenethylamine (DOPA) conjugated poly(lactide-co-glycolide)-poly(ethylene glycol) (PLGA-PEG) nanoparticles (DOPA-NPs) carrying immunosuppressant FK506 (FK506/DOPA-NPs). The functionalized DOPA-NPs formed a versatile coating layer for antigen camouflage without interfering the viability and functionality of islets. The coating layer effectively preserved the morphology and viability of islets in a co-culture condition with xenogenic lymphocytes for 7 days. Interestingly, the mean survival time of islets coated with FK506/DOPA-NPs was significantly higher as compared with that of islets coated with DOPA-NPs (without FK506) and control. This study demonstrated that the combination of surface camouflage and localized low dose of immunosuppressant could be an effective approach in prolonging the survival of transplanted islets. This newly developed platform might be useful for immobilizing various types of small molecules on therapeutic cells and biomaterial surface to improve the therapeutic efficacy in cell therapy and regenerative medicine.

Radiotherapy-assisted tumor selective metronomic oral chemotherapy

Chemotherapy have commonly been used in maximum tolerated dose to completely eradicate the cancer. However, such treatments often failed due to the complex and dynamic nature of cancer. Therefore, it has been suggested that cancer should be treated as a chronic disease, controlling its growth by providing continuous therapeutic pressure for long‐term. Such an approach, however, requires a therapy that is non‐toxic and orally available with sufficient potency. Herein, we propose a radiotherapy‐assisted orally available metronomic apoptosis‐targeted chemotherapy, which delivers doxorubicin continuously to the irradiated tumor with high selectivity while causing minimal toxicities to the normal tissues. DEVD‐S‐DOX/DCK complex is the anticancer prodrug for our strategy that could selectively release doxorubicin in the irradiated tumor tissue with sufficient oral bioavailability. The prodrug was completely inactive by itself, but displayed potent anticancer activity when coupled with radiotherapy. Consequently, the daily oral administration of DEVD‐S‐DOX/DCK in combination with the low‐dose radiotherapy effectively suppressed the growth of tumor in vivo with no significant systemic toxicities despite that the accumulated dose of doxorubicin exceeded 150 mg/kg. Therefore, the our novel therapy using DEVD‐S‐DOX/DCK complex is considered as an outstanding treatment option for treating cancer for long‐term attributed to its oral availability and low‐toxicity profile as well as the potent anticancer effect.

Abstract B36: Orally active low molecular weight heparin conjugates as a tumoral lymphangiogenesis inhibitor

Heparin has been reported as not only an anticoagulant drug but also as a tumoral angiogenesis inhibitor by blocking tumor growth factors including VEGF, bFGF. Low molecular weight heparin conjugated with 4 pairs of deoxycholic acid (LHbisD4) was newly synthesized to enhance the oral absorption and efficacy. As the tumoral lymphangiogenesis is being considered to play the most important role in tumor metastasis, the purpose of this study is to evaluate whether LHbisD4 could suppress the tumoral lymphangiogenesis as well as the incidence of metastasis. To evaluate the inhibition effect of LHbisD4 on VEGF-C induced lymphangiogenesis, several in vitro experiments using human dermal lymphatic endothelial cell(HDLEC) was carried out. The inhibition effect of LHbisD4 on VEGF-C induced VEGFR-3 phosphorylation was evaluated by western blot. Proliferation assay, tubular formation assay, spheroid based sprouting assay were also done to evaluate the efficacy of LHbisD4 on suppressing lymphagiogenesis using HDLEC. For in vivo test, orthotopic breast cancer metastasis model was used and the incidence of metastasis to distant organs was evaluated. We found that VEGF-C induced phosphorylation of VEGFR3 which is the most important signaling cascade to initiate the lymphangiogenesis was highly inhibited in the presence of LHbisD4. Also through other in vitro experiment such as proliferation assay, tubular formation assay, spheroid based sprouting assay, LHbisD4 was proven to be effective enough to suppress VEGF-C induced lymphangiogenesis. In orthotopic model, 10mg/kg/day dose of LHbisD4 was administered orally and we found the incidence of metastasis is highly decreased compared with control group. Especially, weight and volume of the lymph node is highly decreased in case of drug treated group, which means the lymphatic metastasis is significantly suppressed. Through this study, we demonstrate that our novel heparin conjugate could be a good candidate for targeting VEGF-C induced lymphangiogenesis as well as an effective anti-metastatic agent. Citation Format: Jeong Uk Choi, Youngro Byun. Orally active low molecular weight heparin conjugates as a tumoral lymphangiogenesis inhibitor. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B36.

Oral pemetrexed facilitates low-dose metronomic therapy and enhances antitumor efficacy in lung cancer

ABSTRACT There is a growing interest in preclinical research to consider low‐dose metronomic chemotherapy as antiangiogenic cancer treatment. Oral metronomic therapy, in particular, has shown much promise with its ease of daily administration and higher therapeutics window. In that regard, we developed oral pemetrexed using the physical complex with the bile acid enhancers (DCK). In a caco‐2 permeability study, the oral pemetrexed/DCK complex had significantly higher drug uptake, and inhibited efflux transporter activity as well. We further observed that the mechanism of oral drug uptake was related to transcellular along with bile acid transporter mediated pathways. The oral administration of drug complex in rats revealed high bioavailability (22.37%) and extended mean residence time. Using SCC7 and A549 xenograft models, we demonstrated that antitumor effects from daily oral metronomic pemetrexed significantly reduced tumor in a dose‐dependent manner. The antitumor activity of oral pemetrexed/DCK complex plus cisplatin was superior to both monotherapies. The xenograft study also revealed that oral metronomic therapy markedly reduced microvessel density, proliferation and increased apoptosis in the tumor tissues. Oral metronomic doses were significantly correlated with the elevation of plasma deoxyuridine level, an essential biomarker for pemetrexed therapy. One‐month toxicity study confirmed that daily dosing of oral pemetrexed is safe by investigating apoptosis in the gut tissues from mice. Moreover, we analyzed different biochemical parameters and enzymes from the blood to prove that our newly developed oral pemetrexed complex is well tolerated. Graphical abstract Figure. No caption available.