pH-dependent transport kinetics of the human organic anion-transporting polypeptide 1A2

Organic anion-transporting polypeptide (OATP) 1A2 is expressed on the apical sides of intestinal and renal epithelial cells and considered to be involved in the intestinal absorption and renal reabsorption of drugs. Although the transport activity of OATP1A2 is considered to be pH-dependent, the effects of pH on its kinetic parameters and on the potency of OATP1A2 inhibitors are yet to be elucidated. Some OATP are known to have multiple binding sites (MBS), but it remains unclear whether OATP1A2 has MBS. In the present study, we evaluated the influence of pH on the OATP1A2-mediated uptake of estrone 3-sulfate using OATP1A2-expressing HEK293 cells. The uptake of 0.3 μM estrone 3-sulfate by HEK293-OATP1A2 cells was pH-dependent. OATP1A2 exhibited bimodal saturation kinetics at pH 6.3 and 7.4. Compared with that seen at pH 6.3 (5.62 μM), the K_m value of the high-affinity site was 8-fold higher at pH 7.4 (43.2 μM). In addition, the influence of pH on the potency of inhibitors varied among the examined inhibitors. These results suggest that the transport properties of OATP1A2 under lower pH conditions, such as those found in the microenvironments of the small intestinal mucosa and distal tubules, differ from those seen under neutral pH conditions.     

Investigating a Modified Apparatus to Discriminate the Dissolution Capacity In Vitro and Establish an IVIVC of Mycophenolate Mofetil Tablets in the Fed State

In this study, a modified dissolution apparatus was developed by equipping a USP apparatus Ⅰ with an open-loop system to discriminate the dissolution capacity in vitro and establish an in vitro and in vivo correlation (IVIVC) for mycophenolate mofetil (MMF) tablets. MMF had strong pH-dependent solubility that could influence the dissolution rate in vivo after the meal. Dissolution tests involving reference (Cellcept®) and test formulations (F1 and F2) were conducted using pH 4.5 acetate buffer to simulate gastric fluids in the fed state. The dissolution profiles of the reference and test formulations were distinguished by using the modified dissolution apparatus and compared with those determined using the USP apparatuses Ⅱ and Ⅳ, and the dissolution capacities of the formulations were discriminated at different sampling time-points. The results of human bioequivalence (BE) studies in the fed state were consistent with in vitro evaluations that the maximum concentrations (C_{max, in vivo}) of both F1 and F2 fell below the acceptable range (80.00%). A level A IVIVC between the absorption fraction in vivo and dissolution in vitro, and a level C correlation between C_{max, in vivo} and C_{max, in vitro}, were established to guide the optimization of the tablet formulation containing MMF.     

PEP06 polypeptide 30 is a novel cluster-dissociating agent inhibiting αv integrin/FAK/Src signaling in oral squamous cell carcinoma cells

Collectively migrating tumor cells have been recently implicated in enhanced metastasis of epithelial malignancies. In oral squamous cell carcinoma (OSCC), αv integrin is a crucial mediator of multicellular clustering and collective movement in vitro; however, its contribution to metastatic spread remains to be addressed. According to the emerging therapeutic concept, dissociation of tumor clusters into single cells could significantly suppress metastasis-seeding ability of carcinomas. This study aimed to investigate the anti-OSCC potential of novel endostatin-derived polypeptide PEP06 as a cluster-dissociating therapeutic agent in vitro. Firstly, we found marked enrichment of αv integrin in collectively invading multicellular clusters in human OSCCs. Our study revealed that metastatic progression of OSCC was associated with augmented immunostaining of αv integrin in cancerous lesions. Following PEP06 treatment, cell clustering on fibronectin, migration, multicellular aggregation, anchorage-independent survival and colony formation of OSCC were significantly inhibited. Moreover, PEP06 suppressed αv integrin/FAK/Src signaling in OSCC cells. PEP06-induced loss of active Src and E-cadherin from cell–cell contacts contributed to diminished collective migration of OSCC in vitro. Overall, these results suggest that PEP06 polypeptide 30 inhibiting αv integrin/FAK/Src signaling and disrupting E-cadherin-based intercellular junctions possesses anti-metastatic potential in OSCC by acting as a cluster-dissociating therapeutic agent.     

In vitro and in vivo correlation for lipid-based formulations: Current status and future perspectives

Lipid-based formulations (LBFs) have demonstrated a great potential in enhancing the oral absorption of poorly water-soluble drugs. However, construction of in vitro and in vivo correlations (IVIVCs) for LBFs is quite challenging, owing to a complex in vivo processing of these formulations. In this paper, we start with a brief introduction on the gastrointestinal digestion of lipid/LBFs and its relation to enhanced oral drug absorption; based on the concept of IVIVCs, the current status of in vitro models to establish IVIVCs for LBFs is reviewed, while future perspectives in this field are discussed. In vitro tests, which facilitate the understanding and prediction of the in vivo performance of solid dosage forms, frequently fail to mimic the in vivo processing of LBFs, leading to inconsistent results. In vitro digestion models, which more closely simulate gastrointestinal physiology, are a more promising option. Despite some successes in IVIVC modeling, the accuracy and consistency of these models are yet to be validated, particularly for human data. A reliable IVIVC model can not only reduce the risk, time, and cost of formulation development but can also contribute to the formulation design and optimization, thus promoting the clinical translation of LBFs.     

Antibacterial Collagen Composite Membranes Containing Minocycline

Collagen membranes have been used as bioresorbable barrier membranes in guided tissue/bone regeneration. However, the collagen membranes currently used in clinics lack an active antibacterial function, although infection at surgical sites presents a realistic challenge for guided tissue/bone regeneration. In this study, we successfully prepared novel and advanced collagen composite membranes from collagen and complexes of heparin and chelates of minocycline and Ca²⁺ ions. These membranes were characterized for chemical structures, morphology, elemental compositions and tensile strength. In vitro release studies were conducted to evaluate the release kinetics of minocycline from these membranes. Agar disk diffusion assays were used to assess their sustained antibacterial capability against model pathogenic bacteria Staphylococcus aureus. The chemical and physical characterization confirmed the successful synthesis of minocycline-loaded collagen composite membranes, namely NCCM-1 and NCCM-2. Both membranes had weaker tensile strength as compared with commercial collagen membranes. They achieved sustained release of minocycline for at least 4 weeks in simulated body fluid (pH 7.4) at 37°C. Moreover, both membranes demonstrated potent sustained antibacterial effects against Staphylococcus aureus. These results suggested that the advanced collagen composite membranes containing minocycline can be exploited as novel guided tissue regeneration membranes or wound dressing by providing additional antibacterial functions.     

Design and Optimization of a Novel Strategy for the Local Treatment of Helicobacter pylori Infections

Infections with Helicobacter pylori are a global challenge. Currently, H. pylori infections are treated systemically, but the eradication rates of the different therapy regimens are declining due to the growing number of bacterial strains resistant to major antibiotics. Here, we present a strategy for the local eradication of H. pylori by the use of Penicillin G sodium (PGS). In vitro experiments revealed that PGS shows high antibiotic activity against resistant strains of Helicobacter pylori with a minimum inhibitory concentration (MIC) of 0.125 μg/ml. In order to provide luminal concentrations above the MIC for longer periods of time, an extended release tablet was developed. Alkalizers were included to prevent acidic degradation of PGS within the tablet matrix. Out of the tested alkalizers MgO, l-Lysine, NaHCO3, and Na2CO3 NaHCO3 provided the strongest rise in pH inside the hydrated matrix when tested in simulated gastric fluid. Better PGS stability can mainly reasoned from that, addition of MgO resulted in high pH values within the matrix, causing basic degradation of PGS. This work is a first step towards the use of extended release tablets containing PGS for the local treatment of H. pylori as a safe and cost-effective alternative to common systemic treatment regimens.     

The FAPα-activated prodrug Z-GP-DAVLBH inhibits the growth and pulmonary metastasis of osteosarcoma cells by suppressing the AXL pathway

Osteosarcoma is a kind of bone tumor with highly proliferative and invasive properties, a high incidence of pulmonary metastasis and a poor prognosis. Chemotherapy is the mainstay of treatment for osteosarcoma. Currently, there are no molecular targeted drugs approved for osteosarcoma treatment, particularly effective drugs for osteosarcoma with pulmonary metastases. It has been reported that fibroblast activation protein alpha (FAPα) is upregulated in osteosarcoma and critically associated with osteosarcoma progression and metastasis, demonstrating that FAPα-targeted agents might be a promising therapeutic strategy for osteosarcoma. In the present study, we reported that the FAPα-activated vinblastine prodrug Z-GP-DAVLBH exhibited potent antitumor activities against FAPα-positive osteosarcoma cells in vitro and in vivo. Z-GP-DAVLBH inhibited the growth and induced the apoptosis of osteosarcoma cells. Importantly, it also decreased the migration and invasion capacities and reversed epithelial–mesenchymal transition (EMT) of osteosarcoma cells in vitro and suppressed pulmonary metastasis of osteosarcoma xenografts in vivo. Mechanistically, Z-GP-DAVLBH suppressed the AXL/AKT/GSK-3β/β-catenin pathway, leading to inhibition of the growth and metastatic spread of osteosarcoma cells. These findings demonstrate that Z-GP-DAVLBH is a promising agent for the treatment of FAPα-positive osteosarcoma, particularly osteosarcoma with pulmonary metastases.     

Protective role of berberine on ulcerative colitis through modulating enteric glial cells–intestinal epithelial cells–immune cells interactions

Ulcerative colitis (UC) manifests as an etiologically complicated and relapsing gastrointestinal disease. The enteric nervous system (ENS) plays a pivotal role in rectifying and orchestrating the inflammatory responses in gut tract. Berberine, an isoquinoline alkaloid, is known as its anti-inflammatory and therapeutic effects in experimental colitis. However, little research focused on its regulatory function on ENS. Therefore, we set out to explore the pathological role of neurogenic inflammation in UC and the modulating effects of berberine on neuro–immune interactions. Functional defects of enteric glial cells (EGCs), with decreased glial fibrillary acidic protein (GFAP) and increased substance P expression, were observed in DSS-induced murine UC. Administration of berberine can obviously ameliorate the disease severity and restore the mucosal barrier homeostasis of UC, closely accompanying by maintaining the residence of EGCs and attenuating inflammatory infiltrations and immune cells overactivation. In vitro, berberine showed direct protective effects on monoculture of EGCs, bone marrow-derived dendritic cells (BMDCs), T cells, and intestinal epithelial cells (IECs) in the simulated inflammatory conditions. Furthermore, berberine could modulate gut EGCs–IECs–immune cell interactions in the co-culture systems. In summary, our study indicated the EGCs–IECs–immune cell interactions might function as a crucial paradigm in mucosal inflammation and provided an infusive mechanism of berberine in regulating enteric neurogenic inflammation.     

Chiral mesoporous silica nano-screws as an efficient biomimetic oral drug delivery platform through multiple topological mechanisms

In the microscale, bacteria with helical body shapes have been reported to yield advantages in many bio-processes. In the human society, there are also wisdoms in knowing how to recognize and make use of helical shapes with multi-functionality. Herein, we designed atypical chiral mesoporous silica nano-screws (CMSWs) with ideal topological structures (e.g., small section area, relative rough surface, screw-like body with three-dimension chirality) and demonstrated that CMSWs displayed enhanced bio-adhesion, mucus-penetration and cellular uptake (contributed by the macropinocytosis and caveolae-mediated endocytosis pathways) abilities compared to the chiral mesoporous silica nanospheres (CMSSs) and chiral mesoporous silica nanorods (CMSRs), achieving extended retention duration in the gastrointestinal (GI) tract and superior adsorption in the blood circulation (up to 2.61- and 5.65-times in AUC). After doxorubicin (DOX) loading into CMSs, DOX@CMSWs exhibited controlled drug release manners with pH responsiveness in vitro. Orally administered DOX@CMSWs could efficiently overcome the intestinal epithelium barrier (IEB), and resulted in satisfactory oral bioavailability of DOX (up to 348%). CMSWs were also proved to exhibit good biocompatibility and unique biodegradability. These findings displayed superior ability of CMSWs in crossing IEB through multiple topological mechanisms and would provide useful information on the rational design of nano-drug delivery systems.     

Evaluation of a 3D Human Artificial Lymph Node as Test Model for the Assessment of Immunogenicity of Protein Aggregates

The immunogenicity of protein aggregates has been investigated in numerous studies. Nevertheless, it is still unknown which kind of protein aggregates enhance immunogenicity the most. The ability of the currently used in vitro and in vivo systems regarding their predictability of immunogenicity in humans is often questionable, and results are partially contradictive. In this study, we used a 2D in vitro assay and a complex 3D human artificial lymph node model to predict the immunogenicity of protein aggregates of bevacizumab and adalimumab. The monoclonal antibodies were exposed to different stress conditions such as light, heat, and mechanical stress to trigger the formation of protein aggregates and particles, and samples were analyzed thoroughly. Cells and culture supernatants were harvested and analyzed for dendritic cell marker and cytokines. Our study in the artificial lymph node model revealed that bevacizumab after exposure to heat triggered a TH1- and proinflammatory immune response, whereas no trend of immune responses was seen for adalimumab after exposure to different stress conditions. The human artificial lymph node model represents a new test model for testing the immunogenicity of protein aggregates combining the relevance of a 3D human system with the rather easy handling of an in vitro setup.     

Design of a Novel Nucleus-Targeted NLS-KALA-SA Nanocarrier to Delivery Poorly Water-Soluble Anti-Tumor Drug for Lung Cancer Treatment

In this study, we designed a novel nucleus-targeted nanocarrier (NLS-KALA-SA, NKSN) consisting of Kala peptide (KALA), nuclear localization signal (NLS) and stearic acid (SA) using Fmoc solid phase synthesis method. We chose Curcumin (CUR), Paclitaxel (PTX), Ginsenoside compound K(CK) as models of poorly water-soluble antitumor drugs, The drugs loaded NLS-KALA-SA nanoparticles (CUR/NKSN, PTX/NKSN, CK/NKSN) were obained by the dialysis method, their physicochemical properties were determined and antitumor activity were evaluated. The NLS-KALA-SA nanoparticles were spherical shaped with an average size of 76.4 ± 7.6 mm and a zeta potential of 43.7 ± 5.8 mV. The drug-loaded NLS-KALA-SA nanoparticles were above 86.1% and 17.1% in entrapment efficiency and drug loading capacity, and had sustained drug release behavior. Biodistribution and cellular uptake study exhibited that PTX/NKSN mainly distributed in tumor site of A549-bearing mice, and coumarin-6(C6) loaded NLS-KALA-SA nanoparticle (C6/NKSN) was predominantly accumulated in the nucleus of A549 cells. Western blot analysis indicated that PTX/NKSN could more remarkably inhibit Bcl-2 expression and enhance the expression of Bax and Caspase-3 as compared to the controls in A549 cells. Cell apoptosis and antitumor activity study showed that PXT/NKSN could more obviously induce apoptosis of A549 cells compared with free PXT, the PTX/NKSN administration was more effective than free PTX for lung cancer treatment and displayed mild toxicity in A549-bearing mice. The results demonstrates that the NLS-KALA-SA nanoparticles system could enhance the antitumor effects of the encapsulated drug and reduce tissue toxicity due to its long circulating properties and tumor targeting, which might provide a promising strategy for lung cancer treatment.     

Simultaneous Analysis of Dissolution and Permeation Profiles of Nanosized and Microsized Formulations of Indomethacin Using the In Vitro Dissolution Absorption System 2

The in vitro dissolution absorption system 2 (IDAS2), a recent invention comprised a conventional dissolution vessel containing 2 permeation chambers with Caco-2 cell monolayers mounted with their apical side facing the dissolution media, permits simultaneous measurement of dissolution and permeation of drugs from intact clinical dosage forms. The objectives of this study were (1) to assess the utility of IDAS2 in the determination of the effect of particle size on in vitro performance of indomethacin and (2) to find out whether the behavior in IDAS2 of 2 indomethacin products differing in particle size is correlated with their in vivo behavior. Indomethacin dissolution and permeation across Caco-2 cell monolayers were simultaneously measured in IDAS2; the dissolution and permeation profiles were simultaneously modeled using a simple two-compartment model. Compared to microsized indomethacin, the nanosized formulation increased the dissolution rate constant by fivefold, whereas moderately increasing the permeation rate constant and the kinetic solubility. As a result, the drug amount permeated across the Caco-2 cell monolayers doubled in the nanosized versus microsized formulation. The in vitro results showed a good correlation with in vivo human oral pharmacokinetic parameters, thus emphasizing the physiological relevance of IDAS2 data in predicting in vivo absorption.     

Development and evaluation of febuxostat solid dispersion through screening method

Febuxostat (Febux) is a BCS II drug and has a very low solubility. In order to overcome this shortcoming, the purpose of study is to increase the in vitro dissolution (%) and drug release (%) of Febux by using a screening method. The Febux-SD formulation was prepared by screening solubilizers, pH agents, and carriers using with a solvent evaporation method.The novel Febux SD formulation was successfully developed. The dissolution (%) of Febux of optimal formulation (SD3) was higher than that of Feburic® tab in pH 1.2, distilled water (DW), and pH 6.8 buffer by 6.3-, 2.6-, and 1.1-fold, respectively, at 60 min. The in vitro drug release (%) and permeability (μg/cm²) of SD3 formulation were improved compared to those of Feburic® tab in the pH shifting method and PBS (7.4), respectively. The SD3 formulation was well maintained the stability for 6 months, and that of physicochemical properties were altered. In conclusion, the Febux solubilization study with meglumine was first attempted and successfully performed. Through the improved dissolution (%) of Febux, high bioavailability of SD3 formulation is expected in animal and human studies.     

Vegetable Oil-based Hybrid Submicron Particles Loaded with JMV5038: A Promising Formulation against Melanoma

The aim of this work was to carry out a preformulation study on JMV5038 as a new potent cytotoxic agent, and to develop its formulation within vegetable oil-based hybrid submicron particles (HNP) in order to obtain a versatile dosage form against melanoma. JMV5038 was first characterized through physico-chemical tests and it exhibited high melting point and logP value, an important pH-sensitivity that led to the formation of well-identified degradation products at low pH, as well as a substantial solubility value in silylated castor oil (ICO). Then, JMV5038-loaded HNP were formulated through a thermostabilized emulsion process based on the sol-gel cross-linking of ICO. They showed high loading efficiency and their in vitro release kinetic assessed in a biorelevant PBS/octanol biphasic system showed a constant sustained release over one month. The cytotoxic activity and cytocompatibility of HNP were evaluated on A375 melanoma cells and NIH 3T3 cells, respectively. JMV5038-loaded HNP exhibited a slightly enhanced cytotoxic activity of JMV5038 on melanoma cells while demonstrating their safety on NIH 3T3 cells. In conclusion, JMV5038-loaded HNP proved to be an efficient and safe drug subcutaneous delivery system that will be interesting to evaluate through preclinical studies.     

Aqueous Dissolution and Dispersion Behavior of Polyvinylpyrrolidone Vinyl Acetate-based Amorphous Solid Dispersion of Ritonavir Prepared by Hot-Melt Extrusion with and without Added Surfactants

In this study, the lack of complete drug release from amorphous solid dispersions (ASDs), as observed in most published reports, was investigated. ASDs with 20% ritonavir were prepared by HME using polyvinylpyrrolidone vinyl acetate (PVPVA) alone and in combination with 10% poloxamer 407 or Span 20 as carriers. It was established by the film casting technique that ritonavir was molecularly dispersed in formulations, and accelerated stability testing confirmed that extrudates were physically stable. Dissolution of ASDs (100-mg ritonavir equivalent) was performed in 250 mL 0.01 N HCl (pH 2), pH 6.8 phosphate buffer and FeSSIF-V2. Drug concentrations were measured by filtration through 0.45-μm pores and in unfiltered media; the latter gave total amounts of drug present in dissolution media, both as solution and dispersion. Because of low solubility, ritonavir did not dissolve completely in aqueous media. Rather, it formed supersaturated solutions, and the excess drug dispersed in the oily amorphous form with low particle sizes that could crystallize with time. Due to higher drug solubility, the dissolved drug in FeSSIF-V2 was much higher than that in the phosphate buffer. Complete drug release could be observed by accounting for drug both in solution and as phase-separated dispersion. Thus, the present study provides a complete picture of in vitro drug dissolution and dispersion from ASDs.     

Discovery of the anti-angiogenesis effect of eltrombopag in breast cancer through targeting of HuR protein

HuR (human antigen R), an mRNA-binding protein responsible for poor prognosis in nearly all kinds of malignancies, is a potential anti-tumor target for drug development. While screening HuR inhibitors with a fluorescence polarization (FP) based high-throughput screening (HTS) system, the clinically used drug eltrombopag was identified. Activity of eltrombopag on molecular level was verified with FP, electrophoretic mobility shift assay (EMSA), simulation docking and surface plasmon resonance (SPR). Further, we showed that eltrombopag inhibited in vitro cell proliferation of multiple cancer cell lines and macrophages, and the in vivo anti-tumor activity was also demonstrated in a 4T1 tumor-bearing mouse model. The in vivo data showed that eltrombopag was efficient in reducing microvessels in tumor tissues. We then confirmed the HuR-dependent anti-angiogenesis effect of eltrombopag in 4T1 cells and RAW264.7 macrophages with qRT-PCR, HuR-overexpression and HuR-silencing assays, RNA stability assays, RNA immunoprecipitation and luciferase assays. Finally, we analyzed the in vitro anti-angiogenesis effect of eltrombopag on human umbilical vein endothelial cells (HUVECs) mediated by macrophages with cell scratch assay and in vitro Matrigel angiogenesis assay. With these data, we revealed the HuR-dependent anti-angiogenesis effect of eltrombopag in breast tumor, suggesting that the existing drug eltrombopag may be used as an anti-cancer drug.     

Cancer-specific calcium nanoregulator suppressing the generation and circulation of circulating tumor cell clusters for enhanced anti-metastasis combinational chemotherapy

Tumor metastasis is responsible for chemotherapeutic failure and cancer-related death. Moreover, circulating tumor cell (CTC) clusters play a pivotal role in tumor metastasis. Herein, we develop cancer-specific calcium nanoregulators to suppress the generation and circulation of CTC clusters by cancer membrane-coated digoxin (DIG) and doxorubicin (DOX) co-encapsulated PLGA nanoparticles (CPDDs). CPDDs could precisely target the homologous primary tumor cells and CTC clusters in blood and lymphatic circulation. Intriguingly, CPDDs induce the accumulation of intracellular Ca²⁺ by inhibiting Na⁺/K⁺-ATPase, which help restrain cell–cell junctions to disaggregate CTC clusters. Meanwhile, CPDDs suppress the epithelial–mesenchymal transition (EMT) process, resulting in inhibiting tumor cells escape from the primary site. Moreover, the combination of DOX and DIG at a mass ratio of 5:1 synergistically induces the apoptosis of tumor cells. In vitro and in vivo results demonstrate that CPDDs not only effectively inhibit the generation and circulation of CTC clusters, but also precisely target and eliminate primary tumors. Our findings present a novel approach for anti-metastasis combinational chemotherapy.