Coordinated ASBT and EGFR Mechanisms for Optimized Liraglutide Nanoformulation Absorption in the GI Tract.

Background: For maintenance therapy in type 2 diabetes, glucagon-like peptide-1 agonist (GLP-1A), which exhibits low cardiovascular risk and high efficacy, is a promising peptide therapeutic. However, developing an oral GLP-1A presents challenges due to the analog's poor cellular permeability and gastrointestinal (GI) stability. Methods: To mitigate such limitations, an oral nanoformulation of liraglutide (LG) was designed and achieved by combining LG with bile acid derivatives using the nanoprecipitation method. This strategy allowed the bile acid moieties to localize at the nanoparticle surface, enhancing the binding affinity for apical sodium-dependent bile acid transporter (ASBT) and improving GI stability. The in vitro characteristics, cellular permeability, and absorption mechanisms of the LG nanoformulation (LG/TD-NF) were thoroughly investigated. Furthermore, the in vivo oral absorption in rats and the glucose-lowering effects in a diabetic (db/db) mouse model were evaluated. Results: The LG/TD-NF produced neutral nanoparticles with a diameter of 58.7 ± 4.3 nm and a zeta potential of 4.9 ± 0.4 mV. Notably, when exposed to simulated gastric fluid, 65.7 ± 3.6% of the LG/TD-NF remained stable over 120 min, while free LG was fully degraded. Relative to unformulated LG, the Caco-2 cellular permeability of the nanoformulation improved, measuring 10.9 ± 2.1 (× 10-6 cm/s). The absorption mechanism prominently featured endocytosis simultaneously mediated by both ASBT and epidermal growth factor receptor (EGFR). The oral bioavailability of the LG/TD-NF was determined to be 3.62% at a dosage of 10 mg/kg, which is 45.3 times greater than that of free LG. In a diabetes model, LG/TD-NF at 10 mg/kg/day exhibited commendable glucose sensitivity and reduced HbA1c levels by 4.13% within 28 days, similar to that of subcutaneously administered LG at a dosage of 0.1 mg/kg/day. Conclusion: The oral LG/TD-NF promotes ASBT/EGFR-mediated transcytosis and assures cellular permeability within the GI tract. This method holds promise for the development of oral GLP-1A peptides as an alternative to injections, potentially enhancing patient adherence to maintenance therapy.

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.

A novel oral metronomic chemotherapy provokes tumor specific immunity resulting in colon cancer eradication in combination with anti-PD-1 therapy.

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.

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.

VGLL1 phosphorylation and activation promotes gastric cancer malignancy via TGF-ß/ERK/RSK2 signaling.

We previously reported that vestigial-like 1 (VGLL1), a cofactor of transcriptional enhanced associate domain 4 (TEAD4), is transcriptionally regulated by PI3K and ß-catenin signaling and is involved in gastric cancer malignancy. However, the precise mechanism underlying the regulation of VGLL1 activation remains unknown. Therefore, we aimed to investigate the molecular mechanism underlying the transforming growth factor-ß (TGF-ß)-mediated activation of VGLL1 and the VGLL1-TEAD4 interaction in gastric cancer cells. We showed that TGF-ß enhanced VGLL1 phosphorylation and that this phosphorylated VGLL1 functioned as a transcription cofactor of TEAD4 in NUGC3 cells. TGF-ß also increased the phosphorylation of ERK and ribosomal S6 kinase 2 (RSK2) in NUGC3 cells, thereby triggering the translocation of phosphorylated RSK2 to the nucleus. Site-directed mutagenesis and immunoprecipitation experiments revealed that RSK2 phosphorylated VGLL1 at S84 in the presence of TGF-ß. Mutation of VGLL1 at S84 suppressed VGLL1-TEAD4 binding and the subsequent transcriptional activation of matrix metalloprotease 9 (MMP9). Moreover, VGLL1 peptide containing S84 suppressed the TGF-ß-induced MMP9 expression and reduced the invasion and proliferation of gastric cancer cells, whereas VGLL1 peptide containing S84A did not. Furthermore, suppression of expression or activation of VGLL1 enhances the therapeutic effects of lapatinib. Collectively, these results indicate that VGLL1 phosphorylation via TGF-ß/ERK/RSK2 signaling plays a crucial role in MMP9-mediated malignancy of gastric cancer. In addition, our study highlights the therapeutic potential of the peptide containing VGLL1 S84 for the treatment of gastric cancer.

The disubstituted adamantyl derivative LW1564 inhibits the growth of cancer cells by targeting mitochondrial respiration and reducing hypoxia-inducible factor (HIF)-1α accumulation.

Targeting cancer metabolism has emerged as an important cancer therapeutic strategy. Here, we describe the synthesis and biological evaluation of a novel class of hypoxia-inducible factor (HIF)-1α inhibitors, disubstituted adamantyl derivatives. One such compound, LW1564, significantly suppressed HIF-1α accumulation and inhibited the growth of various cancer cell lines, including HepG2, A549, and HCT116. Measurements of the oxygen consumption rate (OCR) and ATP production rate revealed that LW1564 suppressed mitochondrial respiration, thereby increasing the intracellular oxygen concentration to stimulate HIF-1α degradation. LW1564 also significantly decreased overall ATP levels by inhibiting mitochondrial electron transport chain (ETC) complex I and downregulated mammalian target of rapamycin (mTOR) signaling by increasing the AMP/ATP ratio, which increased AMP-activated protein kinase (AMPK) phosphorylation. Consequently, LW1564 promoted the phosphorylation of acetyl-CoA carboxylase, which inhibited lipid synthesis. In addition, LW1564 significantly inhibited tumor growth in a HepG2 mouse xenograft model. Taken together, the results indicate that LW1564 inhibits the growth of cancer cells by targeting mitochondrial ETC complex I and impairing cancer cell metabolism. We, therefore, suggest that LW1564 may be a potent therapeutic agent for a subset of cancers that rely on oxidative phosphorylation for ATP generation.

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.

Modulating tumor immunity by metronomic dosing of oxaliplatin incorporated in multiple oral nanoemulsion.

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.

Intestinal transport mechanism and in vivo anticancer efficacy of a solid oral formulation incorporating an ion-pairing complex of pemetrexed with deoxycholic acid derivative.

OBJECTIVE: 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.

Anti-Angiogenic Effect of Orally Available Pemetrexed for Metronomic Chemotherapy.

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 Nα-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.

Interaction of stem cells with nano hydroxyapatite-fucoidan bionanocomposites for bone tissue regeneration.

The combination of bioceramics with biopolymers are playing major role in the construction of artificial bone. Hydroxyapatite (HA) has been extensively studied as a material in bone repair and replacement in last two decades. In the present study, we have prepared the hydroxyapatite-fucoidan (HA-Fucoidan) nanocomposites by in situ chemical method and biologically characterized them for bone graft substitute. Biological results inferred that mineralization effect of HA-F nanocomposites shows significant enhancement compared to HA in adipose derived stem cell (ADSC). It may be due to the addition of fucoidan in the nanocomposites. The important gene expression such as osteocalcin, osteopontin, collagen and runx-2 were checked using ADSC with HA and HA-fucoidan nanocomposites and the results show that the enhancements were found at 7th day. Furthermore, we have performed in vivo study of HA-fucoidan nanocomposites with rabbit model and a slight amount of bone formation was observed in HA-fucoidan nanocomposites. Herewith, we suggest that HA-fucoidan nanocomposites will be good biomaterials for bone repair/replacement in future.