Design of chimeric GLP-1A using oligomeric bile acids to utilize transporter-mediated endocytosis for oral delivery

Background Despite the effectiveness of glucagon-like peptide-1 agonist (GLP-1A) in the treatment of diabetes, its large molecular weight and high hydrophilicity result in poor cellular permeability, thus limiting its oral bioavailability. To address this, we developed a chimeric GLP-1A that targets transporter-mediated endocytosis to enhance cellular permeability to GLP-1A by utilizing the transporters available in the intestine, particularly the apical sodium-dependent bile acid transporter (ASBT). Methods In silico molecular docking and molecular dynamics simulations were used to investigate the binding interactions of mono-, bis-, and tetra-deoxycholic acid (DOCA) (monoDOCA, bisDOCA, and tetraDOCA) with ASBT. After synthesizing the chimeric GLP-1A-conjugated oligomeric DOCAs (mD-G1A, bD-G1A, and tD-G1A) using a maleimide reaction, in vitro cellular permeability and insulinotropic effects were assessed. Furthermore, in vivo oral absorption in rats and hypoglycemic effect on diabetic db/db mice model were evaluated. Results In silico results showed that tetraDOCA had the lowest interaction energy, indicating high binding affinity to ASBT. Insulinotropic effects of GLP-1A-conjugated oligomeric DOCAs were not different from those of GLP-1A-Cys or exenatide. Moreover, bD-G1A and tD-G1A exhibited improved in vitro Caco-2 cellular permeability and showed higher in vivo bioavailability (7.58% and 8.63%) after oral administration. Regarding hypoglycemic effects on db/db mice, tD-G1A (50 μg/kg) lowered the glucose level more than bD-G1A (50 μg/kg) compared with the control (35.5% vs. 26.4%). Conclusion GLP-1A was conjugated with oligomeric DOCAs, and the resulting chimeric compound showed the potential not only for glucagon-like peptide-1 receptor agonist activity but also for oral delivery. These findings suggest that oligomeric DOCAs can be used as effective carriers for oral delivery of GLP-1A, offering a promising solution for enhancing its oral bioavailability and improving diabetes treatment.This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (grant nos. 2020R1A2C1102831, 2022R1A5A8033794, 2022R1A4A3034038). This study was also supported by Regional Innovation Strategy (RIS) through the NRF funded by the Ministry of Education (MOE) (2021RIS-001

Enhancement of the anticancer effect of atorvastatin-loaded nanoemulsions by improving oral absorption via multivalent intestinal transporter-targeting lipids

Atorvastatin (ATV) has attracted considerable attention as a potential therapeutic agent for cancer because it inhibits cancer cell proliferation by suppressing the mevalonate pathway. However, because of its low oral absorption, high doses of ATV are required for chemotherapeutic applications. In this study, we constructed ATV-loaded nanoemulsions (ATV-NEs) containing multivalent intestinal transporter-targeting lipids to improve the oral bioavailability of ATV. ATV-NEs were prepared via oil-in-water emulsification for transporter-targeted delivery, and contained the following anchors: an ionic complex of deoxycholic acid (DOCA) with the cationic lipid 1,2-dioleyl-3-trimethylammonium propane (DOTAP) (DOCA-DOTAP), a biotin-conjugated lipid (Biotinyl PE), and d-alpha-tocopherol polyethylene glycol succinate (TPGS) to allow bile acid- and multivitamin transporter-mediated permeation of ATV without P-glycoprotein (P-gp)-mediated efflux. The optimized formulation (ATV-NE#6) had 1,091% higher oral bioavailability than free ATV. Finally, treatment of 4T1 cell-bearing mice with oral ATV-NE#6 (equivalent to 40 mg/kg ATV) significantly suppressed tumor growth; the maximum tumor growth reduction was 2.44-fold that of the control group. The results thus suggest that ATV-NEs allow for effective oral chemotherapy by enhancing the oral bioavailability of ATV.Y

Metronomic dose-finding approach in oral chemotherapy by experimentally-driven integrative mathematical modeling: Mathematical modeling for oral metronomic therapy of doxorubicin

© 2022 Elsevier LtdIn 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.N

Enhanced oral bioavailability of an etoposide multiple nanoemulsion incorporating a deoxycholic acid derivative–lipid complex

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

Absorption Mechanism of a Physical Complex of Monomeric Insulin and Deoxycholyl‑l‑lysyl-methylester in the Small Intestine

Currently, oral administration of insulin still remains the best option to avoid the burden of repeated subcutaneous injections and to improve its pharmacokinetics. The objective of the present investigation was to demonstrate the absorption mechanism of insulin in the physical complexation of deoxycholyl-l-lysyl-methylester (DCK) for oral delivery. The oral insulin/DCK complex was prepared by making a physical complex of insulin aspart with DCK through ion-pair interaction in water. For the cellular uptake study, fluorescein-labeled insulin or DCK were prepared according to a standard protocol and applied to Caco-2 or MDCK cell lines. For the PK/PD studies, we performed intrajejunal administration of different formulation of insulin/DCK complex to diabetic rats. The resulting insulin and DCK complex demonstrated greatly enhanced lipophilicity as well as increased permeation across Caco-2 monolayers. The immunofluorescence study revealed the distribution of the complex in the cytoplasm of Caco-2 cells. Moreover, in the apical sodium bile acid transporter (ASBT) transfected MDCK, the insulin/DCK complex showed interaction with ASBT, and also demonstrated absorption through passive diffusion. We could not find that any evidence of endocytosis in relation to the uptake of insulin complex in vitro. In the rat intestine model, the highest absorption of insulin complex was observed in the jejunum at 1 h and then in the ileum at 2–4 h. In PK/PD study, the complex showed a similar PK profile to that of SC insulin. Overall, the study showed that the effect of DCK on enhancing the absorption of insulin resulted from transcellular processes as well as bile acid transporter activity

Enhancement of the anticancer effect of atorvastatin-loaded nanoemulsions by improving oral absorption via multivalent intestinal transporter-targeting lipids

Atorvastatin (ATV) has attracted considerable attention as a potential therapeutic agent for cancer because it inhibits cancer cell proliferation by suppressing the mevalonate pathway. However, because of its low oral absorption, high doses of ATV are required for chemotherapeutic applications. In this study, we constructed ATV-loaded nanoemulsions (ATV-NEs) containing multivalent intestinal transporter-targeting lipids to improve the oral bioavailability of ATV. ATV-NEs were prepared via oil-in-water emulsification for transporter-targeted delivery, and contained the following anchors: an ionic complex of deoxycholic acid (DOCA) with the cationic lipid 1,2-dioleyl-3-trimethylammonium propane (DOTAP) (DOCA-DOTAP), a biotin-conjugated lipid (Biotinyl PE), and d-alpha-tocopherol polyethylene glycol succinate (TPGS) to allow bile acid- and multivitamin transporter-mediated permeation of ATV without P-glycoprotein (P-gp)-mediated efflux. The optimized formulation (ATV-NE#6) had 1,091% higher oral bioavailability than free ATV. Finally, treatment of 4T1 cell-bearing mice with oral ATV-NE#6 (equivalent to 40 mg/kg ATV) significantly suppressed tumor growth; the maximum tumor growth reduction was 2.44-fold that of the control group. The results thus suggest that ATV-NEs allow for effective oral chemotherapy by enhancing the oral bioavailability of ATV.</p

The Risk Factors, Incidence and Prognosis of Postpartum Breast Cancer: A Nationwide Study by the SMARTSHIP Group

The term &apos;pregnancy-associated breast cancer&apos; is no longer used as it has been consistently reported that breast cancer during pregnancy and breast cancer after delivery (postpartum breast cancer) have different characteristics and prognosis. The purpose of this study is to define postpartum breast cancer by analyzing the incidence rate, related factors, and prognosis according to the timing of breast cancer. Data from the Korean National Health Insurance Service were used to analyze 1,292,727 women aged 20-49 years who birthed their first child between 2007 and 2012. The annual incidence rate of breast cancer after delivery increased every year (7.7 per 10,000 person-years after 5 years, 19.36 per 10,000 person-years after 10 years). The risk of breast cancer was significantly higher (hazard ratio 1.15, 95% CI 1.05-1.27, P=0.0037) in women diagnosed with gestational diabetes, but that was not associated with overall survival (OS). Patients diagnosed with breast cancer within 5 years of delivery had a poorer prognosis than those diagnosed later (5-year OS, &lt;5 years: 91.1% vs. 5-10 years: 96.0%). In multivariate analysis of OS, the hazard ratio of patients diagnosed within 5 years after delivery was twice as high as of patients diagnosed between 5 and 10 years. Women diagnosed with gestational diabetes had an increased risk of breast cancer. Breast cancer patients diagnosed within 5 years of delivery had a poorer prognosis than those diagnosed later. In this regard, careful screening for early diagnosis of high-risk patients and intensive research on new treatment strategies are needed.N

Anticoagulation therapy promotes the tumor immune-microenvironment and potentiates the efficacy of immunotherapy by alleviating hypoxia

Purpose Here, 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 design A 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. Results CAT 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 alpha PD-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. Conclusions Collectively, 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.Y