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The selective cytotoxicity of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) to cancer cells but not to normal cells makes it an attractive candidate for cancer therapeutics. However, the disadvantages of TRAIL such as physicochemical instability and short half-life limit its further clinical applications. In this study, TRAIL was encapsulated into a novel anti-angiogenic nanocomplex for both improved drug distribution at the tumor site and enhanced anti-tumor efficacy. A nanocomplex was prepared firstly by entrapping TRAIL into PEG-low molecular weight heparin-taurocholate conjugate (LHT7), which is previously known as a potent angiogenesis inhibitor. Then, protamine was added to make a stable form of nanocomplex (PEG-LHT7/TRAIL/Protamine) by exerting electrostatic interactions. We found that entrapping TRAIL into the nanocomplex significantly improved both pharmacokinetic properties and tumor accumulation rate without affecting the tumor selective cytotoxicity of TRAIL. Furthermore, the anti-tumor efficacy of nanocomplex was highly augmented (73.77±4.86%) compared to treating with only TRAIL (18.49 ± 19.75%), PEG-LHT7/Protamine (47.84 ± 14.20%) and co-injection of TRAIL and PEG-LHT7/Protamine (56.26 ± 9.98%). Histological analysis revealed that treatment with the nanocomplex showed both anti-angiogenic efficacy and homogenously induced cancer cell apoptosis, which suggests that accumulated TRAIL and LHT7 in tumor tissue exerted their anti-tumor effects synergistically. Based on this study, we suggest that PEG-LHT7/Protamine complex is an effective nanocarrier of TRAIL for enhancing drug distribution as well as improving anti-tumor efficacy by exploiting the synergistic mechanism of anti-angiogenesis.

The development of anticancer therapeutics has two main objectives: enhancing efficacy and lowering toxicity. Given these goals, an antibody-drug conjugate (ADC), which delivers highly cytotoxic payloads to tumor tissue specifically, is one of the most favorable treatments. ADC is a growing drug delivery technology, with twelve FDA-approved ADCs as of 2022. Most of ADCs in use today target particular molecules for hematologic malignancies. ADCs for solid tumors are relatively in the early stages of development due to the lack of well-defined solid tumor-specific target molecules. To put it another way, the market has an enormous unmet demand for solid cancer-specific ADCs. As seen by recently approved ADCs and ADCs in clinical trials, finding novel targets for solid cancer has drawn a lot of attention in current development trends. Doppel, a prion-like protein, is transiently expressed in the neonatal brain endothelium; in adults, it is exclusively present in testicular cells. In our previous study, we found that Doppel is specifically expressed only in tumor tissues and tumor blood vessels, but not in normal adult tissues; its expression promoted cancerous blood vessel formation. Then, we generated several monoclonal antibodies which target Doppel; the most effective clone was selected. We observed that angiogenesis-related factors were suppressed in the tumor endothelial cells treated with the Doppel-targeting antibody. In addition, we injected the antibody into mouse models and visualized the tumor-targeting efficacy using in vivo imaging system (IVIS); the antibody accumulates in the tumor significantly higher compared to non-targeting IgG. We suggest what we believe to be a novel strategy to target a molecular marker that is altered and overexpressed specifically in tumor tissues and tumor endothelial cells. Based on the above discoveries, we have developed the Doppel-targeting ADC; this drug, according to our hypothesis, will target only cancer cells that express Doppel and result in apoptotic cell death. The ADC has demonstrated remarkable anticancer efficacy in both cell and mouse models, although it is still in the early stages of research. We believe that the enhanced specificity for targeting tumors, the high tumor-to-organ ratio, and the bystander killing effect would make this Doppel-targeting ADC a successful “solid cancer-specific” one. Citation Format: Ha Kyeong Lee, Byoungmo Kim, Seho Kweon, Jeong Uk Choi, Sang Yoon Kim, Youngro Byun. Developing a novel doppel-targeting antibody-drug conjugate (ADC) for solid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2710.

In this study, we developed oral pemetrexed (PMX) for metronomic dosing to enhance antitumor immunity. PMX was electrostatically complexed with positively charged lysine-linked deoxycholic acid (DL) as an intestinal permeation enhancer, forming PMX/DL, to enhance its intestinal permeability. PMX/DL was also incorporated into a colloidal dispersion (CD) comprised of the block copolymer of poly(ethylene oxide) and poly(propylene oxide), and caprylocaproyl macrogol-8 glycerides (PMX/DL-CD). CD-containing PMX/DL complex in a 1:1 molar ratio [PMX/DL(1:1)-CD] showed 4.66- and 7.19-fold greater permeability than free PMX through the Caco-2 cell monolayer and rat intestine, respectively. This resulted in a 282% improvement in oral bioavailability in rats. In addition, low-dose metronomic PMX led to more immunogenic cell death in CT26.CL25 cells compared to high PMX concentrations at the maximum tolerated dose. In CT26.CL25 tumor-bearing mice, oral metronomic PMX/DL-CD elicited greater antitumor immunity not only by enhancing the number of tumor-infiltrating lymphocytes but also by suppressing T cell functions. Oral PMX/DL-CD substantially increased programmed cell death protein ligand-1 (PD-L1) expression on tumor cells compared to the control and PMX-IV groups. This increased antitumor efficacy in combination with anti-programmed cell death protein-1 (aPD-1) antibody in terms of tumor rejection and immunological memory compared to the combination of PMX-IV and aPD-1. These results suggest that oral metronomic scheduling of PMX/DL-CD in combination with immunotherapy has synergistic antitumor effects.

In this study, a system for oral delivery of docetaxel (DTX) was prepared to enhance the oral absorption and anticancer efficacy of DTX via metronomic chemotherapy. DTX was complexed with low-molecular-weight methylcellulose (LMC) and loaded into a nanoemulsion (NE), yielding DTX/LMC-NE (DLNE). To further enhance the oral bioavailability, d -alpha-tocopherol polyethylene glycol succinate and sodium deoxycholate (DOCA) complexed with cationic lipid 1,2-dioleyl-3-trimethylammonium propane (DOTAP) (DOCA-DOTAP [DA-TAP] complex) was incorporated into DLNE, yielding the formulation DLNE#10. As expected, DLNE#10 showed 11.3- and 5.81-fold increases in artificial membrane (Pe) and Caco-2 permeability (Papp), respectively, resulting in 249% greater oral bioavailability, compared to free DTX. In contrast, inhibition of clathrin- and caveola-mediated endocytosis, macropinocytosis, and bile acid transporters by chlorpromazine, genistein, amiloride, and actinomycin D in the Caco-2 monolayer reduced the Papp by 55.3%, 44.2%, 35.9%, and 36.5%, respectively; these findings suggest that these routes play important roles in enhancing the oral absorption of DLNE#10. In addition, our mechanistic study suggested that P-glycoprotein (P-gp) did not have an inhibitory effect on the permeation of DLNE#10. Notably, the half-maximal inhibitory concentrations (IC50) of DLNE#10 were 43.5% and 16.8% greater than those of Taxotere® in MCF-7 and 4T1 cells, respectively. Finally, the tumor inhibitory rates in 4T1 cell tumor-bearing mice after oral metronomic dosing of DLNE#10 (20 mg/kg DTX) were 5.02- and 1.65-fold greater than the rates in the untreated control group and intravenously injected DTX (10 mg/kg) group, respectively. These observations support the improved oral absorption and enhanced chemotherapeutic efficacy of DTX in DLNE#10 via metronomic chemotherapy, suggesting that it is a better alternative than intravenous Taxotere®.

While conventional approaches for PTEN-loss cancers mainly focus on turning off growth promoting process through modulation of PI3K/AKT pathways, no effective therapeutic treatments that target PTEN-loss cancer cells have yielded results. Moreover, conventional targeted therapies, which are potent against only a subset of cancer cells with limited specificity, bring on temporary response. Here, we report the development of albumin-binding caspase-3 cleavable peptide-drug conjugate (PDC), which utilizes the enhanced albumin metabolism pathway in PTEN-loss cancer cells to enhance the intracellular delivery of chemotherapeutic payload that could exert a bystander killing effect. Albumin metabolism-mediated apoptosis triggered expression of caspase-3 allows the continuous activation of the PDC, accumulation of payloads, sustained upregulation of tumoral caspase-3, and intensified in-situ apoptosis. Importantly, PDC strategy exerts potent therapeutic efficacy against PTEN-loss metastatic triple-negative breast cancer, the highly aggressive and heterogenous nature of which remains a challenge conventional targeted therapies need to overcome. This study thus presents a conceptually novel approach to treat PTEN-loss cancer and creates new translational perspectives of exploiting PTEN-loss for providing an avenue to advance current targeted therapy.

Based on the severity of the disease progression, the treatment of ulcerative colitis involves administration of single or multiple therapeutic agents. In refractory cases of ulcerative colitis, FK506 (FK) is the drug of choice in clinics. To maintain a long-term stable blood concentration of FK and to reduce its dosing frequency, we propose single-dose injection of FK-loaded biodegradable microspheres (FK-Ms). FK-Ms were intraperitoneally-injected into mice fed with dextran-sulfate sodium (DSS). Pharmacokinetics study revealed presence of FK in blood for over 20 days. The single intraperitoneal injection of the drug-loaded microspheres effectively alleviated DSS-induced murine colitis. Mechanistically, the single injection of FK-Ms inhibited the infiltration of T cells into colon and attenuated differentiation of T cells into interferon-γ secreting Th1 and interleukin-17A secreting Th17 cells in colon-draining mesenteric lymph nodes. Therefore, administration of prolonged-release type microspheres can be considered as an alternative for the effective treatment of inflammatory bowel diseases.

Rudra Pangeni,1,* Laxman Subedi,2,* Saurav Kumar Jha,2 Seho Kweon,3 Seo-Hee Kang,4 Kwan-Young Chang,4 Jeong Uk Choi,5 Youngro Byun,3 Jin Woo Park1,2 1College of Pharmacy and Natural Medicine Research Institute, Mokpo National University, Muan-gun, Jeonnam, 58554, Republic of Korea; 2Department of Biomedicine, Health & Life Convergence Sciences, BK21 Four, Mokpo National University, Muan-gun, Jeonnam, 58554, Republic of Korea; 3Department of Molecular Medicine and Biopharmaceutical Science, Graduate School of Convergence Science and Technology, College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea; 4Global R&D Center, IcureBNP, Seoul 08511, Republic of Korea; 5College of Pharmacy, Chonnam National University, Gwangju 61186, Republic of Korea*These authors contributed equally to this workCorrespondence: Youngro ByunCollege of Pharmacy, Seoul National University, 1 Gwanak-Ro, Seoul 08826, Republic of KoreaTel +82 2 880 7866Fax +82 2 872 7864Email yrbyun@snu.ac.krJin Woo ParkCollege of Pharmacy and Natural Medicine Research Institute, Mokpo National University, 1666 Youngsan-Ro, Muan-gun, Jeonnam 58554, Republic of KoreaTel +82 61 450 2704Fax +82 61 450 2689Email jwpark@mokpo.ac.krObjective: The anticancer efficacy of orally administered chemotherapeutics is often constrained by low intestinal membrane permeability and oral bioavailability. In this context, we designed a solid oral formulation of oxaliplatin (OP), a third-generation cisplatin analog, to improve oral bioavailability and investigate its application in metronomic chemotherapy.Methods: An ion-pairing complex of OP with a permeation enhancer, Nα-deoxycholyl-l-lysyl-methylester (DLM), was successfully prepared and then mixed with dispersing agents (including poloxamer 188 and Labrasol) to form the solid, amorphous oral formulation OP/DLM (OP/DLM-SF; hereafter, ODSF).Results: The optimized powder formulation was sized in the nanoscale range (133± 1.47 nm). The effective permeability of OP increased by 12.4-fold after ionic complex formation with DLM and was further increased by 24.0-fold after incorporation into ODSF. ODSF exhibited respective increases of 128% and 1010% in apparent permeability across a Caco-2 monolayer, compared to OP/DLM and OP. Furthermore, inhibition of bile acid transporters by actinomycin D and caveola-mediated uptake by brefeldin in Caco-2 cell monolayers reduced the apparent permeability values of ODSF by 58.4% and 51.1%, respectively, suggesting predominant roles for bile acid transporters and caveola-mediated transport in intestinal absorption of ODSF. In addition, macropinocytosis and paracellular and transcellular passive transport significantly influenced the intestinal permeation of ODSF. The oral bioavailabilities of ODSF in rats and monkeys were 68.2% and 277% higher, respectively, than the oral bioavailability of free OP. In vivo analyses of anticancer efficacy in CT26 and HCT116 cell-bearing mice treated with ODSF demonstrated significant suppression of tumor growth, with respective maximal tumor volume reductions of 7.77-fold and 4.07-fold, compared to controls.Conclusion: ODSF exhibits therapeutic potential, constituting an effective delivery system that increases oral bioavailability, with applications to metronomic chemotherapy.Keywords: oxaliplatin, bile acid transporter-mediated permeation, oral bioavailability, metronomic dosing, colorectal cancer

Triple-negative breast cancer (TNBC) is characterized by its highly heterogeneous microenvironment and propensity for aggressive behavior, both of which represent, along with poor prognosis and high incidence of relapse, the main challenges of curing the disease. Although recent progress in targeted chemotherapy combinations has shown promising outcomes, conventional targeted chemotherapeutic approaches have relied on exploiting the expression of certain molecules or proteins overexpressed on cancer cells as drug targets, which have demonstrated limited clinical benefit against metastatic cancers. Here, we describe a tumoral caspase-3 mediated peptide-doxorubicin conjugates (PDC) switch therapy that adopts two different caspase-3 cleavable PDCs, RGDEVD-DOX (TPD1) and EMC-KGDEVD-DOX (MPD1), for targeting metastatic triple-negative breast cancer (mTNBC). First, using TPD1, an integrin αVβ

Mesenchymal stem cells (MSCs) therapy has brought a great enthusiasm to the treatment of various immune disorders, tissue regeneration and transplantation therapy. MSCs are being extensively investigated for their immunomodulatory actions. MSCs can deliver immunomodulatory signals to inhibit allogeneic T cell immune responses by downregulating pro-inflammatory cytokines and increasing regulatory cytokines and growth factors. Islet transplantation is a therapeutic alternative to the insulin therapy for the treatment of type 1 diabetes mellitus (T1DM). However, the acute loss of islets due to the lack of vasculature and hypoxic milieu in the immediate post-transplantation period may lead to treatment failure. Moreover, despite the use of potent immunosuppressive drugs, graft failure persists because of immunological rejection. Many in vitro and in vivo researches have demonstrated the multipotency of MSCs as a mediator of immunomodulation and a great approach for enhancement of islet engraftment. MSCs can interact with immune cells of the innate and adaptive immune systems via direct cell-cell contact or through secretomes containing numerous soluble growth and immunomodulatory factors or mitochondrial transfer. This review highlights the interactions between MSCs and different immune cells to mediate immunomodulatory functions along with the importance of MSCs therapy for the successful islet transplantation.

Pancreatic islet transplantation holds great potential as a curative therapy for treating type 1 diabetes. However, the need for lifelong systemic immunosuppression with inevitable side effects is an obstacle to clinical success. Here we devised a strategy for the site-specific delivery of an immunosuppressant (tacrolimus) using layer-by-layer assembly of polymeric particles and collagen on the islet surface. This approach aims to provide a continuous and sustained supply of tacrolimus in the vicinity of transplanted cells while avoiding systemic drug exposure. The dose and release rate of tacrolimus can be tunable to achieve therapeutic windows by varying layer-by-layer construction and chemistry of polymers. Transplanting 400 IEQ of pancreatic islets coated with particles containing ∼3 μg of TAC per recipient provided controlled drug release and rectified diabetes for up to 5 months in a xenogeneic rodent model of type 1 diabetes. We anticipate that the findings of this study will be found useful by those developing local immunomodulation strategies aimed at improving the outcomes and safety of cell therapies for curing type 1 diabetes.

PurposeHere, this study verifies that cancer-associated thrombosis (CAT) accelerates hypoxia, which is detrimental to the tumor immune microenvironment by limiting tumor perfusion. Therefore, we designed an oral anticoagulant therapy to improve the immunosuppressive tumor microenvironment and potentiate the efficacy of immunotherapy by alleviating tumor hypoxia.Experimental designA novel oral anticoagulant (STP3725) was developed to consistently prevent CAT formation. Tumor perfusion and hypoxia were analyzed with or without treating STP3725 in wild-type and P selectin knockout mice. Immunosuppressive cytokines and cells were analyzed to evaluate the alteration of the tumor microenvironment. Effector lymphocyte infiltration in tumor tissue was assessed by congenic CD45.1 mouse lymphocyte transfer model with or without anticoagulant therapy. Finally, various tumor models including K-Ras mutant spontaneous cancer model were employed to validate the role of the anticoagulation therapy in enhancing the efficacy of immunotherapy.ResultsCAT was demonstrated to be one of the perfusion barriers, which fosters immunosuppressive microenvironment by accelerating tumor hypoxia. Consistent treatment of oral anticoagulation therapy was proved to promote tumor immunity by alleviating hypoxia. Furthermore, this resulted in decrease of both hypoxia-related immunosuppressive cytokines and myeloid-derived suppressor cells while improving the spatial distribution of effector lymphocytes and their activity. The anticancer efficacy of αPD-1 antibody was potentiated by co-treatment with STP3725, also confirmed in various tumor models including the K-Ras mutant mouse model, which is highly thrombotic.ConclusionsCollectively, these findings establish a rationale for a new and translational combination strategy of oral anticoagulation therapy with immunotherapy, especially for treating highly thrombotic cancers. The combination therapy of anticoagulants with immunotherapies can lead to substantial improvements of current approaches in the clinic.

Rudra Pangeni,1,* Saurav Kumar Jha,1,* Ruby Maharjan,2 Jeong Uk Choi,2 Kwan-Young Chang,3 Young Kweon Choi,3 Youngro Byun,4 Jin Woo Park11Department of Pharmacy, College of Pharmacy and Natural Medicine Research Institute, Mokpo National University, Jeonnam 58554, Republic of Korea; 2Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea; 3Global R&D Center, Icure BNP, Seoul 06170, Republic of Korea; 4Department of Molecular Medicine and Biopharmaceutical Science, Graduate School of Convergence Science and Technology, College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea*These authors contributed equally to this workObjective: The rational combination of immunotherapy with standard chemotherapy shows synergistic clinical activities in cancer treatment. In the present study, an oral powder formulation of pemetrexed (PMX) was developed to enhance intestinal membrane permeability and investigate its application in metronomic chemotherapy in combination with immunotherapy.Methods: PMX was ionically complexed with a bile acid derivative (Nα-deoxycholyl-l-lysyl-methylester; DCK) as a permeation enhancer and mixed with dispersing agents, such as poloxamer 188 (P188) and Labrasol, to form an amorphous oral powder formulation of PMX/DCK (PMX/DCK-OP).Results: The apparent permeability (Papp) of PMX/DCK-OP across a Caco-2 cell monolayer was 2.46- and 8.26-fold greater than that of PMX/DCK and free PMX, respectively, which may have been due to the specific interaction of DCK with bile acid transporters, as well as the alteration of membrane fluidity due to Labrasol and P188. Furthermore, inhibition of bile acid transporters by actinomycin D in Caco-2 cell monolayers decreased the Papp of PMX/DCK-OP by 75.4%, suggesting a predominant role of bile acid transporters in the intestinal absorption of PMX/DCK-OP. In addition, caveola/lipid raft-dependent endocytosis, macropinocytosis, passive diffusion, and paracellular transport mechanisms significantly influenced the permeation of PMX/DCK-OP through the intestinal membrane. Therefore, the oral bioavailability of PMX/DCK-OP in rats was 19.8%±6.93%, which was 294% higher than that of oral PMX. Moreover, an in vivo anticancer efficacy study in B16F10 cell-bearing mice treated with a combination of oral PMX/DCK-OP and intraperitoneal anti-PD1 exhibited significant suppression of tumor growth, and the tumor volume was maximally inhibited by 2.03- and 3.16-fold compared to the oral PMX/DCK-OP and control groups, respectively.Conclusion: These findings indicated the therapeutic potential of a combination of low-dose oral chemotherapy and immunotherapy for synergistic anticancer efficacy.Keywords: pemetrexed, bile acid derivative, oral powder formulation, intestinal membrane permeability, oral delivery, oral metronomic chemotherapy &nbsp

In conventional chemotherapy, maximum tolerated dose approach is considered as a first-line medication for cancer treatment in clinics. In contrast to the conventional chemotherapy which has heavy tumor burdens arising from high dose treatment, metronomic chemotherapy (MCT) engages relatively low dose without drug-free breaks, and is recognized as a promising strategy for a long-term management of the disease. Although doxorubicin (DOX), an anthracycline anti-cancer drug, showed a potential of maintenance effect in vitro, further study on in vivo-relevant concentration to achieve tumor suppression with no toxicity is required to apply the MCT in clinicals. Therefore, the objective of this study was to identify an optimal MCT regimen of DOX by determining concentration-response relationships of tumor suppression (pharmacodynamic; PD) and cardiac toxicity (toxicodynamic; TD). Utilizing an oral DOX formulation complexed with deoxycholic acid (DOX/DOCA complex) which has enhanced bioavailability, physiologically-based pharmacokinetic (PBPK) model was linked to TD and PD models to generate drug profiles from the combined PK, TD, and PD parameters. The integrated model was validated for various scenarios of administration route, formulation, dose, and frequency. The established mathematical model facilitated calculations of adequate in vivo-relevant dosages and intervals, suggesting the optimum oral metronomic regimen of DOX. It is expected to serve as a useful guideline for the design and evaluation of oral DOX formulations in future preclinical/clinical studies.

Introduction: Approved angiogenesis inhibitors generally target certain growth factors or their receptors, which exist in both normal and cancerous cells; this results in suppression of cell-signaling pathways throughout the body and causes adverse effects. For this reason, there is a need for a new angiogenesis inhibitor that can specifically target the tumor vasculature. In our previous study, we revealed that doppel, a prion-like protein, was overexpressed specifically in the tumor vasculatures, but not in normal endothelium, and its expression enhanced blood vessel formation. To develop our new anti-angiogenic agent, we produced monoclonal antibodies which target doppel as the antigen. Methods: We evaluated whether the doppel antibody inhibits angiogenesis by a spheroid assay. We cultured human colonic tumor-associated endothelial cells (HCTEC) and collected the cells by a hanging drop method. After producing compact aggregates, the spheroids were collected and embedded into collagen-based 3D cultures. The culture media/growth factor/doppel antibody mixture was added to each well that contained 3D spheroid cultures. After 24 hours, the number of sprouts was counted and analyzed. Further, HCTECs were incubated with culture media, growth factor, and doppel antibodies for investigating the underlying mechanisms. Each well was lysed, and we conducted western blot and microarray experiments using the lysates. In addition, we injected fluorescent dye-conjugated doppel antibody into tumor xenograft mouse models and visualized the tumor-targeting efficacy using in vivo imaging system (IVIS). Results: In spheroid assays, we found that doppel antibody SNU-H01 and SNU-H02 inhibit angiogenesis (by 32% and 29%, respectively) compared to control. In anti-phosphorylation assays using western blot, the amount of phosphorylated VEGFR2 and phosphorylated FGFR1 was decreased in doppel antibody SNU-H01 and SNU-H02-treated groups. Also, when SNU-H02-treated lysate was added to the microarray kit, STAT5A and beta-catenin were inhibited. Inhibition of STAT5A may promote apoptosis and increase sensitivity to anticancer drugs, and inhibition of beta-catenin may further increase suppression of angiogenesis. In addition, doppel antibody SNU-H04 accumulates in the tumor significantly higher compared to IgG injected mouse group. Conclusion: Doppel antibodies in development, SNU-H01, -H02, -H03, -H04, will be screened and selected for their anti-angiogenic efficacy and favorable pharmacodynamic features. We hope that doppel antibody is expected to be a new and superior tumor vasculature-specific angiogenesis inhibitor that can overcome the limitations of existing anti-angiogenic agents. Citation Format: Ha Kyeong Lee, Ruby Maharjan, Yeojin Jeon, Jeong Uk Choi, Youngro Byun. Anti-doppel monoclonal antibody as a tumor endothelial cell-specific angiogenesis inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 339.

In this study, we investigated the immune-modulating effects of a novel metronomic chemotherapy (MCT) featuring combined oral oxaliplatin (OXA) and pemetrexed (PMX) for colon cancer. OXA and PMX were ionically complexed with lysine derivative of deoxycholic acid (DCK), and incorporated into nanoemulsions or colloidal dispersions, yielding OXA/DCK-NE and PMX/DCK-OP, respectively, to improve their oral bioavailabilities. MCT was not associated with significant lymphotoxicity whereas the maximum tolerated dose (MTD) afforded systemic immunosuppression. MCT was associated with more immunogenic cell death and tumor cell MHC-class I expression than was MTD. MCT improved the tumor antigen presentation of dendritic cells and increased the number of functional T cells in the tumor. MCT also helped to enhance antigen-specific memory responses both locally and systemically. By combining MCT with anti-programmed cell death protein-1 (αPD-1) therapy, the tumor volume was suppressed by 97.85 ± 84.88% compared to the control, resulting in a 95% complete response rate. Upon re-challenge, all tumor-free mice rejected secondary tumors, indicating the induction of a tumor specific memory response. Thus, MCT using an OXA and PMX combination, together with αPD-1, successfully treated colon cancer by activating both innate and adaptive immune cells and elicited tumor-specific long-term immune memory while avoiding toxicity caused by MTD treatment.

Inflammation is a defense mechanism that protects the body from infections. However, chronic inflammation causes damage to body tissues. Thus, controlling inflammation and investigating anti-inflammatory mechanisms are keys to preventing and treating inflammatory diseases, such as sepsis and rheumatoid arthritis. In continuation with our work related to the discovery of bioactive natural products, a polyphenol, catechin-7,4′-O-digallate (CDG), was isolated from Woodfordia uniflora, which has been used as a sedative and remedy for skin infections in the Dhofar region of Oman. Thus far, no study has reported the anti-inflammatory compounds derived from W. uniflora and the mechanisms underlying their action. To investigate the effects of CDG on the regulation of inflammation, we measured the reduction in nitric oxide (NO) production following CDG treatment in immortalized mouse Kupffer cells (ImKCs). CDG treatment inhibited NO production through the downregulation of inducible nitric oxide synthase expression in lipopolysaccharide (LPS)-stimulated ImKCs. The anti-inflammatory effects of CDG were mediated via the inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation, an important inflammatory-response-associated signaling pathway. Moreover, CDG treatment has regulated the expression of pro-inflammatory cytokines, such as IL-6 and IL-1β. These results suggested the anti-inflammatory action of CDG in LPS-stimulated ImKCs.

Here we report a novel combination of a caspase-cleavable peptide-doxorubicin conjugate (MPD-1) with CD47-antagonizing nanocage therapeutics for the treatment of microsatellite-stable (MSS) colorectal cancer (CRC). MPD-1 (i) upregulated markers of immunogenic cell death (ICD) in tumor, and increased co-stimulatory markers on dendritic cells (DCs), (ii) enhanced CD8+ T cell infiltration and antigen presenting cell (APC) activation, and (iii) showed negligible off-target immune-related toxicity compared to free dox. Then, the CD47 antagonist FS nanocage, a SIRPα-expressing ferritin nanocage, was co-administered with MPD-1 that resulted in 95.2% (p < 0.001) tumor growth inhibition in an established CRC model. T cell-mediated elimination of tumors was also confirmed by the tumor-specific activation of T cells detected by IFNγ and tumor-free mice were observed (95%) that bared a memory response when re-challenged. The strategically developed MPD-1 is an ideal adjuvant to immunotherapy and the combination with FS nanocage triggers potent immunity against MSS CRC. In summary, we present an approach to initiate and stimulate immune-mediated eradication of cancer cells using synergistic immunogenic agents targeting the MSS CRC.

In this study, a system for oral delivery of etoposide (ETP) was designed to avoid the problems associated with low and variable bioavailability of a commercially available ETP emulsion comprised of polyethylene glycol, glycerol, and citric acid anhydrous. ETP was complexed with low-molecular-weight methylcellulose (ETP/LMC) and loaded into a water-in-oil-in-water multiple nanoemulsion to formulate an ETP/LMC-nanoemulsion (ELNE). To further enhance the oral bioavailability, an ionic complex formed by anionic lipid 1,2-didecanoyl-sn-glycero-3-phosphate (sodium salt) and cationic Nα-deoxycholyl-l-lysyl-methylester was incorporated into ELNE, yielding ELNE#7. As expected, ELNE#7 showed 4.07- and 2.25-fold increases in artificial membrane and Caco-2/HT29-MTX-E12 permeability (Papp), respectively, resulting in 224% greater oral bioavailability compared with the commercially available ETP emulsion. In contrast, inhibition of clathrin- and caveola-mediated endocytosis, macropinocytosis, and bile acid transporters by chlorpromazine, genistein, amiloride, and actinomycin D in Caco-2/HT-29-MTX-E12 monolayers reduced the Papp by 45.0%, 20.5%, 28.8%, and 31.1%, respectively. These findings suggest that these routes play important roles in enhancing the oral absorption of ELNE#7. In addition, our mechanistic study suggested that P-glycoprotein did not have an inhibitory effect on the permeation of ELNE#7. Notably, ELNE#7 showed significantly enhanced toxicity in LLC and A549 cells compared with ETP-E. These observations support the improved oral absorption of ETP in ELNE#7, suggesting that it is a better alternative than ETP emulsion.

Metronomic chemotherapy, which is defined as a low-dose and frequent administration of cytotoxic drugs without drug-free breaks, has been recently emerged as an alternative to traditional MTD therapy and has shown therapeutic benefit in breast cancer patients in numbers of clinical studies. Unlike MTD, metronomic chemotherapy acts by multiple mechanisms including antiangiogenic effect and immunomodulation, but the direct cytotoxic effect only playing a minor role due to the lowered dose. In this light, within the limits of p53-deficient breast cancer, we demonstrate the enhanced anticancer effect of metronomic chemotherapy using doxorubicin when combined with Chk1 inhibitor MK-8776 by specifically augmenting the direct cytotoxic effect on cancer cells. Since the oral drug is greatly favored in metronomic chemotherapy due to the frequent and potential long-term administration, we prepared an oral doxorubicin by producing an ionic complex with deoxycholic acid, which showed sufficient bioavailability and anticancer effect when administered orally. MK-8776 selectively enhanced the cytotoxic effect of low-concentration doxorubicin in p53-deficient breast cancer cells by abrogating the Chk1-dependent cell cycle arrest in vitro. Consistently, combining MK-8776 significantly improved the anticancer effect of the daily administered oral doxorubicin in p53-deficient breast cancer xenografts especially in a lower dose of doxorubicin without evident systemic toxicities. Combination therapy of MK-8776 and metronomic oral doxorubicin would be thus promising in the treatment of p53-deficient breast cancer benefited from the augmented direct cytotoxic effect and low risk of toxicities.

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.

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.

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.

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.

In this study, a system for oral delivery of oxaliplatin (OXA) was prepared for metronomic chemotherapy to enhance antitumor efficacy and modulate tumor immunity. OXA was complexed with Nα-deoxycholyl- l -lysyl-methylester (DCK) (OXA/DCK) and formulated as a nanoemulsion (OXA/DCK-NE). OXA/DCK-NE showed 3.35-fold increased permeability across a Caco-2 cell monolayer, resulting in 1.73-fold higher oral bioavailability than free OXA. In addition, treatment of the B16F10.OVA cell line with OXA/DCK-NE resulted in successful upregulation of immunogenic cell death (ICD) markers both in vitro and in vivo. In a B16F10.OVA tumor-bearing mouse model, treatment with OXA/DCK-NE substantially impeded tumor growth by 63.9 ± 13.3% compared to the control group, which was also greater than the intravenous (IV) OXA group. Moreover, treatment with a combination of oral OXA/DCK-NE and anti-programmed cell death protein-1 (αPD-1) antibody resulted in 78.3 ± 9.67% greater inhibition compared to controls. More important, OXA/DCK-NE alone had immunomodulatory effects, such as enhancement of tumor antigen uptake, activation of dendritic cells in tumor-draining lymph nodes, and augmentation of both the population and function of immune effector cells in tumor tissue as well as in the spleen; no such effects were seen in the OXA IV group. These observations provide a rationale for combining oral metronomic OXA with immunotherapy to elicit synergistic antitumor effects.

Metronomic chemotherapy (MCT) is defined as the frequent administration of low-dose chemotherapeutics, without long drug-free periods, with the exertion of antitumor activity exclusively through anti-angiogenic mechanisms. In this study, we have developed an orally available formulation of pemetrexed (PMX) for MCT. PMX was first complexed ionically with N&alpha, deoxycholyl-l-lysyl-methylester (DCK) as the permeation enhancer. This was followed by dispersion with poloxamer 188 and Labrasol to form the solid oral formulation of PMX (PMX/DCK-OP). PMX/DCK-OP exhibited a 10.6-fold increase in permeability across a Caco-2 cell monolayer compared to PMX alone. This resulted in a 70-fold increase in the oral bioavailability of PMX/DCK-OP in mice over oral PMX alone. In the A549 xenograft model, tumor volume was reduced by 51.1% in the PMX/DCK-OP treated group compared to only 32.8% in the maximum tolerated dose (MTD)-treated group. Furthermore, PMX/DCK-OP exhibited a significant anti-angiogenic effect on the A549 xenograft mice when compared to the MTD-treated group, as indicated by microvessel density quantification for CD-31. In addition, PMX/DCK-OP enhanced the release of an endogenous angiogenesis inhibitor, thrombospondin-1 (TSP-1), into both the blood circulation and the tumor microenvironment. Therefore, due to its oral route of administration, PMX/DCK-OP appears to be a better alternative to the conventional treatment of PMX.

Existence of the genomically and epigenomically diverse subclones in a tumor severely limits the therapeutic efficacy of targeted agents. To overcome such a limitation, we prepared a novel targeted prodrug, EMC-DEVD-S-DOX, which comprises two important features: radiation-induced apoptosis targeting and albumin-binding properties. In particular, the prodrug binds circulating albumin after intravenous administration and then activated by caspase-3, which is upregulated from apoptotic cells that responded to radiotherapy. The prodrug was designed to bind circulating albumin to extend half-life and facilitate tumor accumulation in order to increase the possibility of contacting caspase-3, which is only transiently upregulated during apoptosis. Our results showed that EMC-DEVD-S-DOX had a prolonged half-life with enhanced tumor accumulation, which clearly benefited the therapeutic effect of the prodrug. Also, agreeing with the in vitro studies that showed ignorable cytotoxic effect in the absence of caspase-3, the prodrug was effective only when combined with radiotherapy without any noticeable systemic toxicity in vivo. Due to the highly selective action of EMC-DEVD-S-DOX independent to the complex genomic profiles of tumor, the prodrug would overcome the limitation of current targeted therapy and potentiate radiotherapy in the clinical oncology.

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.

The selective targeting of cytotoxic agents to a tumor has shown limited success by difficulties in identifying the appropriate target molecules, and more importantly, by the phenotypically dynamic nature of the tumor cells and intratumoral heterogeneity. In an attempt to overcome these issues and efficiently deliver cytotoxic drugs to the tumor, we previously reported a strategy termed radiation-induced apoptosis-targeted chemotherapy (RIATC), which utilizes the radiotherapy for intentionally triggering the caspase-3 and in situ amplification of tumor apoptosis by caspase-3 activated prodrug. Herein, we propose an advanced form of RIATC prodrug, AP1-DEVD-S-DOX, that could more actively target to the ligands of radiation-induced tumor cells, which could accumulate more prodrugs, thereby allowing more effective in situ activation and amplification of tumor apoptosis, comparing to RIATC. Indeed, AP1-DEVD-S-DOX was able to exert improved doxorubicin (DOX) delivery to the tumor and anticancer effect than the RIATC prodrug that lacks apoptotic cell-binding property but having a similar degree of off-target distribution in the other organs. Accordingly, AP1-DEVD-S-DOX could be an efficient prodrug for concurrent chemoradiotherapy by selectively delivering doxorubicin to the tumor with less systemic cytotoxicity.

Despite the emergence of advanced therapeutics such as targeted therapy and immunotherapy in the modern oncology, cytotoxic chemotherapy still remains as the first-line treatment option in a wide range of cancers attributing to its potency. Many endeavors have been made to overcome the toxicity issues of cytotoxic chemotherapy by improving the specific delivery to the tumor, with active tumor targeting being one of the most popular approaches. However, such an approach has been challenged by the intratumor heterogeneity and the lack of valid molecular target in many types of cancer. Here, we introduce a novel albumin-binding prodrug MPD02 that could specifically deliver highly potent cytotoxin monomethyl auristatin E (MMAE) to the tumor as an important component of chemoradiotherapy for the treatment of triple-negative breast cancer (TNBC). MPD02 was synthesized by conjugating MMAE to the C-terminus of the KGDEVD peptide via self-eliminating linker and introducing a maleimide group to the Lys side chain of the peptide. MPD02 was able to bind albumin after administration via maleimide group for an extended circulation time and metabolized into MMAE in tumor-specific manner by reacting with the caspase-3 upregulated in tumor by radiotherapy, exerting a highly potent anticancer effect with good safety profile in two different TNBC xenograft models.

Rudra Pangeni,1,* Jeong Uk Choi,2,* Vijay Kumar Panthi,1 Youngro Byun,3 Jin Woo Park1 1College of Pharmacy and Natural Medicine Research Institute, Mokpo National University, Muan-gun, Jeonnam, Republic of Korea; 2College of Pharmacy, Seoul National University, Seoul, Republic of Korea; 3Department of Molecular Medicine and Biopharmaceutical Science, Graduate School of Convergence Science and Technology, College of Pharmacy, Seoul National University, Seoul, Republic of Korea *These authors contributed equally to this work Objective: The current study sought to design an oral delivery system of pemetrexed (PMX), a multitargeted antifolate antimetabolite, by enhancing its intestinal membrane permeability. Materials and methods: PMX was ionically complexed with a permeation enhancer such as Nα-deoxycholyl-l-lysyl-methylester (DCK) and prepared as an amorphous solid dispersion by mixing with dispersants such as 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD) and poloxamer 188 (P188), forming an HP-beta-CD/PMX/DCK/P188; the complex was incorporated into multiple water-in-oil-in-water nanoemulsions in a supersaturated state (HP-beta-CD/PMX/DCK/P188-NE). Results: After complex formation, the partition coefficient and in vitro membrane permeability of PMX were markedly increased, but it showed similar cytotoxic and inhibitory effects on cancer cell proliferation/migration. Furthermore, the intestinal membrane permeability and epithelial cell uptake of PMX were synergistically improved after HP-beta-CD/PMX/DCK/P188 was incorporated into a nanoemulsion with a size of 14.5±0.45nm. The in vitro permeability of HP-beta-CD/PMX/DCK/P188-NE across a Caco-2 cell monolayer was 9.82-fold greater than that of free PMX, which might be attributable to the partitioning of PMX to the epithelial cells being facilitated via specific interaction of DCK with bile acid transporters, as well as the enhanced lipophilicity accompanied by surfactant-induced changes in the intestinal membrane structure and fluidity. Therefore, the oral bioavailability of HP-beta-CD/PMX/DCK/P188-NE in rats was evaluated as 26.8%±2.98% which was 223% higher than that of oral PMX. Moreover, oral HP-beta-CD/PMX/DCK/P188-NE significantly suppressed tumor growth in Lewis lung carcinoma cell-bearing mice, and the tumor volume was maximally inhibited by 61% compared with that in the control group. Conclusion: These results imply that HP-beta-CD/PMX/DCK/P188-NE is an effective and promising delivery system for enhancing the oral absorption of PMX. Thus, there is the potential for new medical applications, including applications in metronomic cancer treatment. Keywords: pemetrexed, deoxycholic acid derivative, multiple nanoemulsions, permeability, oral absorption, oral anticancer therapy

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.

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.

Heparin-like sulfated polysaccharides are potential drug candidates owing to their ability to interact with angiogenic factors and inhibit angiogenesis, tumor growth, and metastasis. This study aimed to improve the delivery of heparin-like anticancer polysaccharides for accumulation at the tumor site. We designed a nanocarrier system using protamine attached to polyethylene glycol (PEG) and evaluated the stability, tumor targeting, and tumor growth inhibition of the nanocarrier loaded with heparin derivatives. When mixed with various polyanionic heparin derivatives, the polycationic PEG-protamine formed stable self-assembled nanocomplexes via ionic interactions, with flexible PEG chains located on the outside. Among the complexes, a nanocomplex loaded with a low-molecular-weight heparin-suramin conjugate (LHsura) had the most suitable average size (101.9nm) for the enhanced permeability and retention effect and allowed accumulation of LHsura at the tumor site for up to 48h. In a tumor-bearing mouse model, the PEG-protamine and LHsura nanocomplex (10mg/kg/3days, intravenously), which could be extravasated through the tumor vasculature, significantly inhibited tumor growth, more than LHsura alone did. Overall, the self-assembled nanocomplexation of PEG-protamine and LHsura helped control the release and extravasation of LHsura, which resulted in an antitumor effect on the target tumor cells.

Clinical studies have found that the incidence of cancer metastasis through the lymphatic vessels are 3-5 times higher than that through the blood vessels. These findings suggest the potency of anti-lymphangiogenic therapy in reducing the incidence of cancer metastasis. Previously, we reported LHbisD4, which is the conjugate of low molecular weight heparin (LMWH) and four bis-deoxycholates as a potent anti-angiogenic drug with less toxicity and orally active property. Here, we show that LHbisD4 could also suppress the formation of new lymphatic vessels and attenuate the incidence of metastasis by blocking VEGF-C signaling pathway. LHbisD4 significantly enhanced binding affinity with VEGF-C when compared with LMWH, which enables LHbisD4 to suppress the proliferation, migration and formation of tubular structures of human dermal lymphatic endothelial cells(HDLECs) in in vitro condition even in the presence of excessive amounts of VEGF-C. Similarly, we found that the density of lymphatic vessels in the primary tumor tissue in breast cancer bearing mice was significantly diminished when LHbisD4 was administered compared with the control group. Also, the incidence of axillary lymph nodes and distant organ metastasis was significantly reduced in the LHbisD4 administered group, which demonstrates that LHbisD4 could successfully lower the incidence of metastasis through blocking VEGF-C induced lymphangiogenesis. Based on these results, we propose LHbisD4 as a potent anti-cancer drug that can reduce the incidence of metastasis by suppressing lymphangiogenesis through blocking VEGF-C signaling pathway.

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 ± 0.757 nm in diameter and a zeta potential of 56.5 ± 1.13 mV. 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.

Clinical efficacy of immune checkpoint blockade is often enhanced by their co-administration with immunogenic chemotherapy. Here a novel chemo-immunotherapy combination is reported that successfully overcomes the immunosuppressive tumor microenvironment to elicit an effective anti-tumor immune response. The CD47-SIRPα innate immune checkpoint was blocked by surface engineering human ferritin with a SIRPα variant, and this FHSIRPα efficiently inhibited the “don’t-eat-me” signal of cancer cells, thereby enhancing cancer cell phagocytosis in a colon carcinoma (CT26) model in immunocompetent mice. Doxorubicin prodrug, EMC-DEVD-S-DOX, that binds to circulating albumin following intravenous administration showed enhanced half-life, and the active drug was released via caspase-3 at tumor site following radiation treatment. Radiation-induced apoptosis of cancer cells resulted in release of active drug doxorubicin and upregulation of CD47 on cancer cell surface, sensitizing cancer cells to FHSIRPα treatment. The induction of immunogenic cell death (ICD) by the prodrug was confirmed in vitro by upregulation of calreticulin, translocation of HMGB1 and increased release of chaperone proteins in CT26.CL25 mouse colon cancer cells by flow cytometric analysis and confocal microscopy. The ICD effect of the prodrug correlated well in vivo and increased phagocytosis of tumor cells by antigen-presenting cells (APCs) in immunocompetent mice as analyzed by flow cytometry of tumor tissue and draining lymph nodes. Tumor infiltrates analyzed by flow cytometry also showed enrichment of APCs; and T cell-mediated elimination of immunogenic tumors was confirmed by the tumor-specific activation of T cells ex vivo as detected by secretion of IFNγ. Whereas FHSIRPα monotherapy showed modest tumor growth inhibition, combination with a doxorubicin prodrug resulted in effective inhibition of tumor growth by 95.2% (p Citation Format: Na Kyeong Lee, Jeong Uk Choi, Jung Je Park, Ji-Hyun Seo, Mi Ra Kim, Hyo Won Chang, Sang Yoon Kim, In-San Kim, Youngro Byun. Nanocage therapeutics FHSIRPα mediating immune checkpoint blockade awakens immunity against cancer with an albumin-binding caspase-cleavable doxorubicin prodrug EMC-DEVD-S-DOX [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2251.

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.