CREB is a potential marker associated with drug-induced liver injury: Identification and validation through transcriptome database analysis.

Drug-induced liver injury (DILI) is the main cause of failure in drug development and postapproval withdrawal. Although toxicogenomic techniques provide an unprecedented opportunity for mechanistic assessment and biomarker discovery, they are not suitable for the screening of large numbers of exploratory compounds in early drug discovery. Using a comprehensive analysis of toxicogenomics (TGx) data, we aimed to find DILI-relevant transcription factors (TFs) that could be incorporated into a reporter gene assay system. Gene set enrichment analysis (GSEA) of the Open TG-GATEs dataset highlighted 4 DILI-relevant TFs, including CREB, NRF2, ELK-1, and E2F. Using ten drugs with already assigned idiosyncratic toxicity (IDT) risks, reporter gene assays were conducted in HepG2 cells in the presence of the S9 mix. There were weak correlations between NRF2 activity and IDT risk, whereas strong correlations were observed between CREB activity and IDT risk. In addition, CREB activation associated with 3 Withdrawn/Black box Warning drugs was reversed by pretreatment with a PKA inhibitor. Collectively, we suggest that CREB might be a sensitive biomarker for DILI prediction, and its response to stress induced by high-risk drugs might be primarily regulated by the PKA/CREB signaling pathway.

Advocation and advancements of EPR effect theory in drug delivery science: A commentary.

To honor the contributions of Professor Hiroshi Maeda to the progress of targeted drug delivery research, a brief review of enhanced permeability and retention (EPR) effect theory proposed by him as the physiology-based principal mechanism of intra-tumoral accumulation of large molecules and small particles is presented. Under historical and practical backgrounds in developments of various drug delivery systems including macromolecular conjugates, the concept of EPR effect was advocated in mid1980s and has cultivated new cancer chemotherapeutic modalities until recently. Namely, nanoplatforms such as polymer conjugates, liposomes, polymeric micelles, and nanoparticles have been studied as a promising fusion area for nanotechnology and medicine. Modulation of EPR effect by chemical and/or mechanical approaches to achieve tumor vascular and tissue modification would further lead to sophistication of cancer chemotherapy employing nanomedicines.

Evaluation of transgene expression characteristics and DNA vaccination against melanoma metastasis of an intravenously injected ternary complex with biodegradable dendrigraft poly-L-lysine in mice.

We developed a biocompatible splenic vector for a DNA vaccine against melanoma. The splenic vector is a ternary complex composed of plasmid DNA (pDNA), biodegradable dendrigraft poly-L-lysine (DGL), and γ-polyglutamic acid (γ-PGA), the selective uptake of which by the spleen has already been demonstrated. The ternary complex containing pDNA encoding luciferase (pCMV-Luc) exhibited stronger luciferase activity for RAW264.7 mouse macrophage-like cells than naked pCMV-Luc. Although the ternary complex exhibited strong luciferase activity in the spleen after its tail vein injection, luciferase activity in the liver and spleen was significantly decreased by a pretreatment with clodronate liposomes, which depleted macrophages in the liver and spleen. These results indicate that the ternary complex is mainly transfected in macrophages and is a suitable formulation for DNA vaccination. We applied the ternary complex to a pUb-M melanoma DNA vaccine. The ternary complex containing pUb-M suppressed the growth of melanoma and lung metastasis by B16-F10 mouse melanoma cells. We also examined the acute and liver toxicities of the pUb-M ternary complex at an excess pDNA dose in mice. All mice survived the injection of the excess amount of the ternary complex. Liver toxicity was negligible in mice injected with the excess amount of the ternary complex. In conclusion, we herein confirmed that the ternary complex was mainly transfected into macrophages in the spleen after its tail vein injection. We also showed the prevention of melanoma metastasis by the DNA vaccine and the safety of the ternary complex.

Minocycline Hydrochloride Controlled-release Microsphere Preparation Process Optimization Based on the Robust Design Method.

OBJECTIVES: The objective of the present study is to establish a robust preparation method that could steadily produce minocycline hydrochloride (MCH) microspheres regardless of used polymer types. MATERIALS AND METHODS: Taguchi's Robust Experimental Design methodology was employed to optimize the process parameters for MCH-loaded poly(D,L-lactide-co-glycolide) (PLGA) microspheres. In the experimental design, seven controllable factors, i.e., preparation method, pH of the aqueous phase, volume of the aqueous phase, volume of dichloromethane, rotation speed, temperature, and amount of polyvinyl alcohol, were considered for the optimization of process parameters. PLGA types with different lactide/glycolide ratios were considered the uncontrollable (noise) factor. Based on the L18 orthogonal array, 18 experimental runs were conducted for each type of PLGA. The encapsulation efficiency (EE) and in vitro release rate were evaluated for all the prepared formulations. RESULTS: Regardless of the PLGA type with different lactic/glycolic acid ratios, microspheres prepared via the solid-in-oil-in-water (S/O/W) method, showed a much higher EE and faster drug release than the microspheres prepared via the co-solvent method. Preparation methods, pH of the aqueous phase, and volume of the aqueous phase were the most influencing parameters on the EE. The confirmation experiment results indicated that the signal-to-noise ratio increased by 5.76 db from that of an initial condition. The release of minocycline was fastest with the PLGA (50:50) microspheres, followed by PLGA (75:25) and PLGA (85:15). CONCLUSION: Although the interaction between the selected factors in the evaluation was ignored, the orthogonal array design of the experiment based on Taguchi's robust experimental design methodology was sufficient to optimize the process parameters for the PLGA microspheres of MCH. The S/O/W was the main factor affecting the EE. Microspheres prepared via the S/O/W method exhibited a higher EE and faster drug release than the microspheres prepared via co-solvent method. The pH and volume of the aqueous phase were also effective parameters on the EE. A robust experimental design has been successfully applied to the optimization of the process parameters for microsphere preparation.

Role of pharmacokinetic consideration for the development of drug delivery systems: A historical overview.

Drug delivery system is defined as a system or technology to achieve optimum therapeutic effects of drugs through precise control of their movements in the body. In order to optimize function of drug delivery systems aiming at targeting, their whole-body distribution profiles should be systematically evaluated and analyzed, where pharmacokinetic analysis based on the clearance concepts plays important role. Organ perfusion experiments combined with statistical moment analysis further supply detailed information on drug disposition at organ and cellular levels. Based on general relationship between physicochemical properties and distribution profile, macromolecular prodrugs or polymer conjugates of proteins are rationally designed and further introduction of ligand structure brings cell-specific delivery for them. These approaches are also applicable for particulate carriers such as liposomes and offer various opportunities for biological drugs such as nucleic acid drugs for their delivery. Mechanistic approach for dermal absorption analysis based on physiological skin model offers another opportunity in rational design of drug delivery. Potential of drug delivery technology in future medicines such as cell therapy and nanomaterial platform application is further discussed in relation to pharmacokinetic consideration.

Development of a DNA Vaccine for Melanoma Metastasis by Inhalation Based on an Analysis of Transgene Expression Characteristics of Naked pDNA and a Ternary Complex in Mouse Lung Tissues.

The present study investigated a pulmonary delivery system of plasmid DNA (pDNA) and its application to melanoma DNA vaccines. pCMV-Luc, pEGFP-C1, and pZsGreen were used as a model pDNA to evaluate transfection efficacy after inhalation in mice. Naked pDNA and a ternary complex, consisting of pDNA, dendrigraft poly-l-lysine (DGL), and γ-polyglutamic acid (γ-PGA), both showed strong gene expression in the lungs after inhalation. The transgene expression was detected in alveolar macrophage-rich sites by observation using multi-color deep imaging. On the basis of these results, we used pUb-M, which expresses melanoma-related antigens (ubiquitinated murine melanoma gp100 and tyrosinase-related protein 2 (TRP2) peptide epitopes), as DNA vaccine for melanoma. The inhalation of naked pUb-M and its ternary complex significantly inhibited the metastasis of B16-F10 cells, a melanoma cell line, in mice. The levels of the inflammatory cytokines, such as TNF-α, IFN-γ, and IL-6, which enhance Th1 responses, were higher with the pUb-M ternary complex than with naked pUb-M and pEGFP-C1 ternary complex as control. In conclusion, we clarified that the inhalation of naked pDNA as well as its ternary complex are a useful technique for cancer vaccination.

The Application of the in-Situ Hyperthermia Emission from Acoustic Nanodroplets for Theranostic Dual-Imaging and Antitumor Modalities.

In this study, we have developed a theranostic nanocarrier that can emit heat upon the exposure to ultrasound (US) irradiation as well as the generation of a contrast signal that can be detected with ultrasonography. The prepared acoustic nanodroplets (NDs) made with liquid perfluporopentane (PFPn) had an average size of 197.7 ± 3.6 nm in diameter and were stable in vitro for 60 min. US irradiation at 2 W.cm-2 induced phase change of NDs into bubbles in vitro. On the other hand, the intra-tumor injection of NDs in combination with US irradiation induced thermal emission in situ in B16BL6 melanoma tumor implanted into mice and the emission areas have mostly covered the tumor site. Also, the combination between NDs and US irradiation has inhibited the tumor growth. Under this condition, the heat shock protein (HSP70) in tumor was significantly upregulated after 6 h of the treatment of NDs with US. Thus, we have developed a therapeutic system with multiple theranostic modalities composed of acoustic NDs and US irradiation applicable to the tumor treatment on the external surface of the body.

Tissue suction-mediated gene transfer to the beating heart in mice.

We previously developed an in vivo site-specific transfection method using a suction device in mice; namely, a tissue suction-mediated transfection method (tissue suction method). The aim of this study was to apply the tissue suction method for cardiac gene transfer. Naked plasmid DNA (pDNA) was intravenously injected in mice, followed by direct suction on the beating heart by using a suction device made of polydimethylsiloxane. We first examined the effects of suction conditions on transgene expression and toxicity. Subsequently, we analyzed transgene-expressing cells and the transfected region of the heart. We found that heart suction induced transgene expression, and that -75 kPa and -90 kPa of suction achieved high transgene expression. In addition, the inner diameter of the suction device was correlated with transgene expression, but the pressure hold time did not change transgene expression. Although the tissue suction method at -75 kPa induced a transient increase in the serum cardiac toxicity markers at 6 h after transfection, these markers returned to normal at 24 h. The cardiac damage was also analyzed through the measurement of hypertrophic gene expression, but no significant differences were found. In addition, the cardiac function monitored by echocardiography remained normal at 11 days after transfection. Immunohistochemical analysis revealed that CD31-positive endothelial cells co-expressed the ZsGreen1-N1 reporter gene. In conclusion, the tissue suction method can achieve an efficient and safe gene transfer to the beating heart in mice.

Effects of Tissue Pressure on Transgene Expression Characteristics via Renal Local Administration Routes from Ureter or Renal Artery in the Rat Kidney.

We previously developed a renal pressure-mediated transfection method (renal pressure method) as a kidney-specific in vivo gene delivery system. However, additional information on selecting other injection routes and applicable animals remains unclear. In this study, we selected renal arterial and ureteral injections as local administration routes and evaluated the characteristics of gene delivery such as efficacy, safety, and distribution in pressured kidney of rat. Immediately after the naked pDNA injection, via renal artery or ureter, the left kidney of the rat was pressured using a pressure controlling device. Transfection efficiency of the pressured kidney was about 100-fold higher than that of the injection only group in both administration routes. The optimal pressure intensity in the rat kidney was 1.2 N/cm2 for renal arterial injection and 0.9 N/cm2 for ureteral injection. We found that transgene expression site differs according to administration route: cortical fibroblasts and renal tubule in renal arterial injection and cortical and medullary tubule and medullary collecting duct in ureteral injection. This is the first report to demonstrate that the renal pressure method can also be effective, after renal arterial and ureteral injections, in rat kidney.

Evaluation of the Theranostic Potential of Perfluorohexane-Based Acoustic Nanodroplets.

In this study, we have prepared perfluorohexane (PFH)-based acoustic nanodroplets (PFH-NDs) and evaluated their theranostic characteristics. Nile Red (NR) was incorporated into PFH-NDs as a model of hydrophobic drugs (NR-PFH-NDs). The mean particle diameters of PFH-NDs and NR-PFH-NDs were 205 ± 1.8 nm and 346.3 ± 6 nm, respectively. There was no significant PFH leakage from PFH-NDs during 90 min incubation at 37°C in the presence of 10% rat serum. The in vitro ultrasonography showed that the phase transition of PFH-NDs from liquid droplets to gassed bubbles could be induced by therapeutic low-intensity ultrasound with a frequency of 1 MHz and an intensity of 5 W/cm2. Irradiation of ultrasound in combination with NR-PFH-NDs enhanced uptake of NR in murine adenocarcinoma cells (C26). After intravenous injection of PFH-NDs to mice, PFH gradually disappeared from blood circulation with an elimination half-life of 43.3 min. Intravenous injection of PFH-NDs also resulted in significant contrast enhancement in the mouse carotid artery upon therapeutic low-intensity ultrasound irradiation. These results suggest the potential of PFH-NDs as a novel contrast agent for further theranostic applications.

Development of mKO2 fusion proteins for real-time imaging and mechanistic investigation of the degradation kinetics of human IκBα in living cells.

In resting cells, the nuclear factor kappa B (NF-κB) family of transcription factors is stabilized by complexation with the cytoplasmic inhibitor of kappa B alpha (IκBα). Extracellular stimuli, such as tumor necrosis factor alpha (TNFα) or bacterial lipopolysaccharide activate NF-κB through IκBα phosphorylation and ubiquitin-proteasomal degradation. Herein, we developed a novel biosensor, by fusing the monomeric fluorescent protein Kusabira-Orange 2 to IκBα (mKO2-IκBα), to study the dynamics and structure-activity relationship of IκBα degradation. Site-specific deletion studies on the IκBα sequence revealed that the C-terminal PEST domain is required in signal-induced proteasomal degradation of IκBα and functions independently from ankyrin repeats. Using deletion mutants, we show that IκBα ankyrin repeats do not affect IκBα degradability but affect its degradation rate. We demonstrate, by both real-time confocal microscopy and western blot analysis, that the half-life of mKO2-IκBα in response to TNFα is approximately 35 min, which is similar to the half-life of endogenous IκBα. Using this biosensor we also show that selective proteasome inhibitors, such as lactacystin and MG132, inhibit degradation and affect the kinetics of IκBα in a dose-dependent manner. The techniques described here can have a range of possible applications, such as facilitating studies associated with IκBα dynamics and biochemical characteristics, as well as the screening of potential proteasome inhibitors.

Sialyl LewisX mimic-decorated liposomes for anti-angiogenic everolimus delivery to E-selectin expressing endothelial cells.

In this study, we developed novel E-selectin-targeting liposomes, i.e., 3'-(1-carboxy)ethyl sialyl LewisX (3'-CE sLeX) mimic liposomes, for targeted delivery of everolimus (EVE) in anti-angiogenic therapy. We investigated the uptake and efficacy of these E-selectin targeting liposomes in inflammatory cytokine-treated human umbilical vein endothelial cells (HUVECs). The uptake of EVE in 3'-CE sLeX mimic liposomes increased steadily and almost caught up with the uptake of plain EVE at 3 h, which was higher than that in PEGylated liposomes (PEG-liposomes). Inhibition of uptake by anti-E-selectin antibody suggested involvement of E-selectin-mediated endocytotic processes. Migration in cells treated with EVE/3'-CE sLeX mimic liposomes was suppressed by more than half when compared to the control. This treatment was also seen to significantly inhibit the formation of capillary tubes and networks. In addition, Thr389 phosphorylation of pS6 kinase, as a marker of mTOR activity, was remarkably suppressed to less than endogenous levels by EVE/3'-CE sLeX mimic liposomes. In conclusion, the present study demonstrated that EVE/3'-CE sLeX mimic liposomes were intracellularly taken up by E-selectin and prompted anti-angiogenic effects of EVE involved in the mTOR signaling pathway. However, moderate retention of EVE in the liposomes might limit the targeting ability of 3'-CE sLeX mimic liposomes.

Binding and structure-kinetic relationship analysis of selective TLR4-targeted immunosuppressive self-assembling heparin nanoparticles.

Self-assembling aliphatic heparin derivatives were shown to inhibit the immune system by antagonizing Toll-like receptor 4/myeloid differentiation protein 2 (TLR4/MD2). In the present study, glycol split heparin-d-erythro-sphingosine conjugates (NAHNP) and its regioselectively desulfated derivatives with shortened aliphatic chains were investigated regarding their biophysical properties in the interaction with TLR4/MD2. Two-dimensional nuclear Overhauser effect spectroscopy studies showed that upon glycol splitting, the heparin backbone gains extra adaptability that facilitates binding to proteins. However, unlike native heparin or glycol split non-anticoagulant heparin (NAH), hydrophobic derivatization of NAH forces sulfated iduronic acid residues to change configuration from a 2S0 skew-boat to a 1C4 chair form. Whereas neither heparin nor NAH had any appreciable effect, NAHNP significantly inhibited lipopolysaccharide-induced activation of the NF-κB transcription factor. We showed that NAHNP binds to TLR4/MD2 with an affinity of 62.3 nM. In line with computational studies, biosensor-based structure-kinetic relationship studies demonstrated that 6-O-sulfo groups of d-glucosamine residue were essential in binding to arginines of both TLR4 and MD2 domains of the receptor complex. The desulfation of 6-O-sulfo groups decreases the association kinetics from 4.2 × 104 M-1 s-1 to 3.8 × 103 M-1 s-1, which results in a decreased affinity of 800 nM. Two aliphatic chains of NAHNP bound to the MD2 pocket similarly to lipopolysaccharide. A decrease in chain length resulted in a loss of inhibitory activity on NF-κB transcription and binding affinity to TLR4/MD2. In conclusion, the present study characterizes the immunosuppressive effect of aliphatic heparin derivatives and provides a promising strategy to develop selective immunosuppressants for acute and chronic inflammatory disorders.

Urokinase injection-triggered clearance enhancement of a 4-arm PEG-conjugated 64Cu-bombesin analog tetramer: A novel approach for the improvement of PET imaging contrast.

Radiolabeled antibodies, polyethylene glycol-conjugated (PEGylated) peptides, liposomes, and other materials were investigated as positron-emission tomography (PET) probes. These substances accumulate in tumors but often remain too long in circulation. We investigated the combination of intravenous urokinase injection and its substrate linker as a triggered radioisotope clearance enhancement system to improve imaging contrast. To this end, we synthesized a four-arm PEGylated 64Cu-bombesin analog tetramer with a urokinase substrate linker. In mouse blood, it was almost perfectly cleaved and degraded into smaller radioactive fragments in vitro with urokinase (≥20,000 IU/mL). In mouse blood circulation, ∼50-65% of the probe was rapidly degraded after the urokinase injection and the radioactive fragments were eliminated mainly from the kidney. In contrast, tumor radioactivity levels did not change, and therefore, the tumors were clearly visualized. The tumor/blood ratio, an indicator of imaging contrast, increased 2.5 times, while elimination of the radioisotope from the blood was enhanced. This approach has the potential to improve imaging contrast using various PET probes. It could also shorten the time required to obtain sufficient contrast and decrease patient radiation exposure.

High-density lipoprotein mutant eye drops for the treatment of posterior eye diseases.

Age-related macular degeneration (AMD), in which choroidal neovascularization (CNV) affects the center of the retina (macula), leads to the irreversible visual loss. The intravitreal injection of anti-angiogenesis antibodies improved the prognosis of AMD, but relatively less invasive therapies should be explored. In the present study, we show that a high-density lipoprotein (HDL) mutant is a therapeutically active drug carrier capable of treating a posterior eye disease in mice via instillation. Various HDL mutants were prepared with apoA-I proteins fused with different cell-penetrating peptides (CPPs) and phospholipids with different alkyl chain lengths; their sizes were further controlled in the range of 10-25nm. They were screened based on the efficiency of fluorescent dye delivery to the inner retinal layer in mice. The best mutant was found to have penetratin (PEN) as a CPP, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and a size of 15nm. In preclinical studies on a laser-induced CNV murine model, 1week of instillation of the best mutant carrying the anti-angiogenesis drug pazopanib had dramatic therapeutic effects in reducing the CNV size. Importantly, the HDL mutant by itself contributed to the therapeutic effects. Future clinical trials for treating AMD with instillation of the HDL mutant are expected.

An Evolutionary Search Algorithm for Covariate Models in Population Pharmacokinetic Analysis.

Building a covariate model is a crucial task in population pharmacokinetics. This study develops a novel method for automated covariate modeling based on gene expression programming (GEP), which not only enables covariate selection, but also the construction of nonpolynomial relationships between pharmacokinetic parameters and covariates. To apply GEP to the extended nonlinear least squares analysis, the parameter consolidation and initial parameter value estimation algorithms were further developed and implemented. The entire program was coded in Java. The performance of the developed covariate model was evaluated for the population pharmacokinetic data of tobramycin. In comparison with the established covariate model, goodness-of-fit of the measured data was greatly improved by using only 2 additional adjustable parameters. Ten test runs yielded the same solution. In conclusion, the systematic exploration method is a potentially powerful tool for prescreening covariate models in population pharmacokinetic analysis.

Anti-inflammatory Effect of Self-assembling Glycol-Split Glycosaminoglycan-Stearylamine Conjugates in Lipopolysaccharide-Stimulated Macrophages.

Glycosaminoglycans (GAGs) play important roles in various biological processes such as cell adhesion and signal transduction, as well as promote anti-inflammatory activity. We previously revealed that glycol-split heparin (HP)-aliphatic amine conjugates form self-assembled nanoparticles and suppress the production of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1ß in lipopolysaccharide (LPS)-stimulated macrophages much more strongly than native HP (J. CONTROL: Release, 194, 2014, Babazada et al.). Considering that HP is not the only GAG to have anti-inflammatory activity, the present study was initiated to examine whether conjugation of GAGs with aliphatic amines is generally effective in their activity augmentation against LPS-stimulated macrophages. We newly synthesized the stearylamine conjugates of chondroitin sulfate (CS), hyaluronic acid (HA), and low-molecular-weight heparin (LH), and investigated the effect of the position and degree of sulfation and molecular weight of GAGs on their anti-inflammatory activity. All of the conjugates formed self-assembled nanoparticles in aqueous solution. The IC50 value for suppression of TNF-α production from the macrophages was the smallest with the derivative of LH, followed by HP, CS, and HA. The degree of sulfation appeared to be important in determining their anti-inflammatory activity, which would correspond to previous results using the derivatives of site-selectively desulfated HP. Comparison of HP and LH derivatives revealed that fractionated smaller heparin has greater anti-inflammatory activity.

Quantitative prediction of ionization effect on human skin permeability.

Although skin permeability of an active ingredient can be severely affected by its ionization in a dose solution, most of the existing prediction models cannot predict such impacts. To provide reliable predictors, we curated a novel large dataset of in vitro human skin permeability coefficients for 322 entries comprising chemically diverse permeants whose ionization fractions can be calculated. Subsequently, we generated thousands of computational descriptors, including LogD (octanol-water distribution coefficient at a specific pH), and analyzed the dataset using nonlinear support vector regression (SVR) and Gaussian process regression (GPR) combined with greedy descriptor selection. The SVR model was slightly superior to the GPR model, with externally validated squared correlation coefficient, root mean square error, and mean absolute error values of 0.94, 0.29, and 0.21, respectively. These models indicate that Log D is effective for a comprehensive prediction of ionization effects on skin permeability. In addition, the proposed models satisfied the statistical criteria endorsed in recent model validation studies. These models can evaluate virtually generated compounds at any pH; therefore, they can be used for high-throughput evaluations of numerous active ingredients and optimization of their skin permeability with respect to permeant ionization.