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.

Oral lymphatic delivery of alpha-galactosylceramide and ovalbumin evokes anti-cancer immunization.

We developed an orally delivered nanoemulsion that induces cancer immunization. It consists of tumor antigen-loaded nano-vesicles carrying the potent invariant natural killer T-cell (iNKT) activator α-galactosylceramide (α-GalCer), to trigger cancer immunity by effectively activating both innate and adaptive immunity. It was validated that adding bile salts to the system boosted intestinal lymphatic transport as well as the oral bioavailability of ovalbumin (OVA) via the chylomicron pathway. To increase intestinal permeability further and amplify the antitumor responses, an ionic complex of cationic lipid 1,2-dioleyl-3-trimethylammonium propane (DTP) with sodium deoxycholate (DA) (DDP) and α-GalCer were anchored onto the outer oil layer to form OVA-NE#3. As expected, OVA-NE#3 exhibited tremendously improved intestinal cell permeability as well as enhanced delivery to mesenteric lymph nodes (MLNs). Subsequent activation of dendritic cells and iNKTs, in MLNs were also observed. Tumor growth in OVA-expressing mice with melanoma was more strongly suppressed (by 71%) after oral administration of OVA-NE#3 than in untreated controls, confirming the strong immune response induced by the system. The serum levels of OVA-specific IgG1 and IgG2a were 3.52- and 6.14-fold higher than in controls. Treating OVA-NE#3 increased the numbers of tumor-infiltrating lymphocytes, including cytotoxic T-cell and M1-like macrophage. Antigen- and α-GalCer-associated enrichment of dendritic cells and iNKTs in tumor tissues also increased after OVA-NE#3 treatment. These observations indicate that our system induces both cellular and humoral immunity by targeting the oral lymphatic system. It may offer a promising oral anti-cancer vaccination strategy that involves the induction of systemic anti-cancer immunization.

Topical Delivery of Atraric Acid Derived from Stereocaulon japonicum with Enhanced Skin Permeation and Hair Regrowth Activity for Androgenic Alopecia.

Atraric acid (AA) is a phenolic compound isolated from Stereocaulon japonicum that has demonstrated anti-androgen properties and was used to design an alternative formulation for the treatment of alopecia. This new topical formulation was designed using a solvent mixture system composed of ethanol as a volatile vehicle, oleic acid as a permeation enhancer, and water for skin hydration. The ideal topical AA formulation (AA-TF#15) exhibited an 8.77-fold higher human skin flux and a 570% increase in dermal drug deposition, compared to 1% (w/w) AA in ethanol. In addition, compared to other formulations, AA-TF#15 (1% [w/w] AA) activated keratinocytes and human dermal papilla cell proliferation at a concentration of 50 µM AA, which is equivalent to 50 µM minoxidil. Moreover, AA-TF#15 treatment produced a significant increase in hair regrowth by 58.0% and 41.9% compared to the 1% (w/w) minoxidil and oral finasteride (1 mg/kg)-treated mice. In addition, AA-TF#15 showed a higher expression level of aldehyde dehydrogenase 1, ß-catenin, cyclin D1, and pyruvate kinase M2 proteins in the skin of AA-TF#15-treated mice compared to that of those treated with minoxidil and oral finasteride. These findings suggest AA-TF#15 is an effective formulation for the treatment of scalp androgenic alopecia.

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.

Enhanced oral absorption of teriparatide with therapeutic potential for management of osteoporosis.

In this study, a system for oral delivery of recombinant human parathyroid hormone [rhPTH(1-34); teriparatide (TRP)] was developed to enhance oral absorption and to demonstrate an equivalent therapeutic effect to that of subcutaneous (SC) TRP injection. The solid oral formulation of TRP was prepared by electrostatic complexation with l-lysine-linked deoxycholic acid (LDA) and deoxycholic acid (DA) at a molar ratio of 1:5:7 in the aqueous dispersion of non-ionic n-dodecyl-ß-d-maltoside (DM) at a 1:15 weight ratio, followed by freeze-drying the dispersal, yielding TRP(1:5:7)-15. As expected, TRP(1:5:7)-15 showed a 414% increase in permeability across the Caco-2/HT29-MTX-E12 cell monolayer, resulting in a 13.0-fold greater oral bioavailability compared with free TRP. In addition, the intestinal transport mechanisms in the presence of specific inhibitors of clathrin-mediated endocytosis, macropinocytosis, and bile acid transporters revealed 44.4%, 28.7%, and 51.2% decreases in transport, respectively, confirming that these routes play crucial roles in the permeation of TRP in TRP(1:5:7)-15. Notably, this formulation showed similar activation of the release of cyclic adenosine monophosphate (cAMP) compared with TRP, suggesting equivalent efficacy in the parathyroid hormone receptor-adenylate cyclase system of osteosarcoma cells. Furthermore, oral TRP(1:5:7)-15 (equivalent to 0.4 mg/kg TRP) demonstrated increases in bone mineral density (36.9%) and trabecular thickness (31.3%) compared with untreated glucocorticoid-induced osteoporotic mice. Moreover, the elevated levels of biomarkers of bone formation, including osteocalcin, were also comparable with those after SC injection of TRP (0.02 mg/kg). These findings suggest that TRP(1:5:7)-15 can be used as an effective oral therapy for the management of osteoporosis.

Preparation of topical bimatoprost with enhanced skin infiltration and in vivo hair regrowth efficacy in androgenic alopecia.

To prepare a topical formulation of bimatoprost (BIM) with high skin permeability, we designed a solvent mixture system composed of ethanol, diethylene glycol monoethyl ether, cyclomethicone, and butylated hydroxyanisole, serving as a volatile solvent, nonvolatile co-solvent, spreading agent, and antioxidant, respectively. The ideal topical BIM formulation (BIM-TF#5) exhibited 4.60-fold higher human skin flux and a 529% increase in dermal drug deposition compared to BIM in ethanol. In addition, compared to the other formulations, BIM-TF#5 maximally activated human dermal papilla cell proliferation at a concentration of 5 µM BIM, equivalent to 10 µM minoxidil. Moreover, BIM-TF#5 (0.3% [w/w] BIM) significantly promoted hair regrowth in the androgenic alopecia mouse model and increased the area covered by hair at 10 days by 585% compared to the vehicle-treated mice, indicating that entire telogen area transitioned into the anagen phase. Furthermore, at day 14, the hair weight of mice treated with BIM-TF#5 (5% [w/w] BIM) was 8.45- and 1.30-fold greater than in the 5% (w/w) BIM in ethanol and 5% (w/v) minoxidil treated groups, respectively. In the histological examination, the number and diameter of hair follicles in the deep subcutis were significantly increased in the BIM-TF#5 (0.3 or 5% [w/w] BIM)-treated mice compared to the mice treated with vehicle or 5% (w/w) BIM in ethanol. Thus, our findings suggest that BIM-TF#5 is an effective formulation to treat scalp alopecia, as part of a novel therapeutic approach involving direct prostamide F2α receptor-mediated stimulation of dermal papilla cells within hair follicles.

Metronomic delivery of orally available pemetrexed-incorporated colloidal dispersions for boosting tumor-specific immunity.

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.

Preparation of Topical Itraconazole with Enhanced Skin/Nail Permeability and In Vivo Antifungal Efficacy against Superficial Mycosis.

In this study, a stable and highly skin-permeable topical delivery system for itraconazole (ITZ) was designed to provide effective treatment against superficial mycosis. Herein, ITZ was incorporated into a solution composed of ethanol, benzyl alcohol, hydrochloric acid, Transcutol P, and cyclomethicone as a delivery vehicle, solubilizer, protonating agent, permeation enhancer, and spreading agent, respectively. At 72 h, the optimal topical ITZ formulation (ITZ-TF#11) exhibited 135% enhanced skin permeability, which led to increases in drug deposition in the stratum corneum, epidermis, and dermis of 479%, 739%, and 2024%, respectively, compared with the deposition of 1% ITZ in ethanol (control). Moreover, on day 7, ITZ-TF#11 demonstrated 2.09- and 2.30-fold enhanced nail flux and drug deposition, compared with the control. At a dose of 40 mg/kg/day, ITZ-TF#11 showed 323% greater lesion recovery, a 165% lower mean erythema severity score, and a 37% lower mean logarithm of viable fungal cells in skin in the treated area, compared with mice that received oral ITZ at the same dose. Overall, the findings imply that ITZ-TF#11 is a superior alternative to oral ITZ for treatment of superficial mycosis.

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 N α-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.

Improvements in the Oral Absorption and Anticancer Efficacy of an Oxaliplatin-Loaded Solid Formulation: Pharmacokinetic Properties in Rats and Nonhuman Primates and the Effects of Oral Metronomic Dosing on Colorectal Cancer.

OBJECTIVE: 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, N α-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.

Enhanced antitumor efficacy of bile acid-lipid complex-anchored docetaxel nanoemulsion via oral metronomic scheduling.

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®.