Mogrol stimulates G-protein-coupled bile acid receptor 1 (GPBAR1/TGR5) and insulin secretion from pancreatic ß-cells and alleviates hyperglycemia in mice.

Target identification is a crucial step in elucidating the mechanisms by which functional food components exert their functions. Here, we identified the G-protein-coupled bile acid receptor 1 (GPBAR1, also known as TGR5) as a target of the triterpenoid mogrol, a class of aglycone mogroside derivative from Siraitia grosvenorii. Mogrol, but not mogrosides, activated cAMP-response element-mediated transcription in a TGR5-dependent manner. Additionally, mogrol selectively activated TGR5 but not the other bile acid-responsive receptors (i.e., farnesoid X receptor, vitamin D receptor, or muscarinic acetylcholine receptor M3). Several amino acids in TGR5 (L71A2.60, W75AECL1, Q77AECL1, R80AECL1, Y89A3.29, F161AECL2, L166A5.39, Y240A6.51, S247A6.58, Y251A6.62, L262A7.35, and L266A7.39) were found to be important for mogrol-induced activation. Mogrol activated insulin secretion under low-glucose conditions in INS-1 pancreatic ß-cells, which can be inhibited by a TGR5 inhibitor. Similar effects of mogrol on insulin secretion were observed in the isolated mouse islets. Mogrol administration partially but significantly alleviated hyperglycemia in KKAy diabetic mice by increasing the insulin levels without affecting the ß-cell mass or pancreatic insulin content. These results suggest that mogrol stimulates insulin secretion and alleviates hyperglycemia by acting as a TGR5 agonist.

Non-electrostatic interactions associated with aggregate formation between polyallylamine and Escherichia coli.

Bacterial aggregation by mixing with polymers is applied as pretreatment to identify pathogens in patients with infectious diseases. However, the detailed interaction between polymers and bacteria has yet to be fully understood. Here, we investigate the interaction between polyallylamine and Escherichia coli by isothermal titration calorimetry. Aggregation was observed at pH 10 and the binding was driven by favorable enthalpic gain such as the electrostatic interaction. Neither aggregation nor the apparent heat of binding was observed at pH 4.0, despite the strong positive charge of polyallylamine. These results suggest that intermolecular repulsive forces of the abundant positive charge of polyallylamine cause an increased loss of conformational entropy by binding. Non-electrostatic interaction plays a critical role for aggregation.

Structural and interaction analysis of human lipocalin-type prostaglandin D synthase with the poorly water-soluble drug NBQX.

Lipocalin-type prostaglandin D synthase (L-PGDS) is a secretory lipid-transporter protein that was shown to bind a wide variety of hydrophobic ligands in vitro. Exploiting this function, we previously examined the feasibility of using L-PGDS as a novel delivery vehicle for poorly water-soluble drugs. However, the mechanism by which human L-PGDS binds to poorly water-soluble drugs is unclear. In this study, we determined the solution structure of human L-PGDS and investigated the mechanism of L-PGDS binding to 6-nitro-7-sulfamoyl-benzo[f]quinoxalin-2,3-dione (NBQX), an α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor antagonist. NMR experiments showed that human L-PGDS has an eight-stranded antiparallel ß-barrel structure that forms a central cavity, a short 310 -helix and two α-helices. Titration with NBQX was monitored using 1 H-15 N HSQC spectroscopy. At higher NBQX concentrations, some cross-peaks of the protein exhibited fast-exchanging shifts with a curvature, indicating at least two binding sites. These residues were located in the upper portion of the cavity. Singular value decomposition analysis revealed that human L-PGDS has two NBQX binding sites. Large chemical shift changes were observed in the H2-helix and A-, B-, C-, D-, H- and I-strands and H2-helix upon NBQX binding. Calorimetric experiments revealed that human L-PGDS binds two NBQX molecules with dissociation constants of 46.7 µm for primary binding and 185.0 µm for secondary binding. Molecular docking simulations indicated that these NBQX binding sites are located within the ß-barrel. These results provide new insights into the interaction between poorly water-soluble drugs and human L-PGDS as a drug carrier.

Thermodynamic stability of human lipocalin-type prostaglandin D synthase under various pH conditions.

Lipocalin-type prostaglandin D synthase (L-PGDS) binds various hydrophobic small molecules. Since we aim to use human L-PGDS as a carrier in a drug delivery system (DDS) for poorly water-soluble drugs, quality control of the protein is indispensable. In this study, we investigated the thermodynamic stability of human L-PGDS under various pH conditions. Differential scanning calorimetry revealed that the thermal unfolding of L-PGDS was an almost-reversible two-state transition between the native and unfolded states over the pH range from 2.5 to 7.4. The linear relationship of ΔH(Tm) to Tm in this pH range gave a heat capacity change (ΔCp) of 4.76 kJ/(K·mol), which was small compared to those commonly found in globular proteins. The temperature-dependent free energy of unfolding, ΔG(T), specified by Tm, ΔH(Tm) and ΔCp, showed a pH dependence with the highest value at pH 7.4 closest to the isoelectric point of 8.3. The small value of Cp resulted in a large value of ΔG(T), which contributed to the stability of the protein. Taken together, these results demonstrated that human L-PGDS is sufficiently thermostable for storage and practical use and can be useful as a delivery vehicle of protein-based DDS.

Transport Form and Pathway from the Intestine to the Peripheral Tissues and the Intestinal Absorption and Metabolism Properties of Oleamide.

This study aimed to obtain information on the transport form and pathway from the intestine to the peripheral tissues and on the intestinal absorption and metabolism properties of oleamide (cis-9-octadecenamide). Oleamide was primarily transported via the portal vein. Density gradient centrifugation indicated that plasma oleamide was enriched in the fractions containing albumin in the portal and peripheral blood. Oleamide formed a complex with albumin in an endothermic reaction (apparent Kd = 4.4 µM). The CD36 inhibitor inhibited the oleamide uptake into the intestinal epithelial Caco-2 cells, and oleamide decreased the cell surface CD36 level. The fatty acid amide hydrolase (FAAH) inhibitor increased the transepithelial transport of oleamide across Caco-2 cells and the plasma oleamide concentration in mice intragastrically administered with oleamide. These results indicate that oleamide is transported primarily via the portal vein as a complex with albumin. Furthermore, we suggest that oleamide is taken up via CD36 in the small intestine and degraded intracellularly by FAAH.

The Role of miR-217-5p in the Puromycin Aminonucleoside-Induced Morphological Change of Podocytes.

Podocytes, alternatively called glomerular epithelial cells, are terminally differentiated cells that wrap around glomerular capillaries and function as a part of the glomerular filtration barrier in the kidney. Therefore, podocyte injury with morphological alteration and detachment from glomerular capillaries leads to severe proteinuria and subsequent renal failure through glomerulosclerosis. Previous RNA sequencing analysis of primary rat podocytes exposed to puromycin aminonucleoside (PAN), a well-known experimental model of injured podocytes, identified several transcripts as being aberrantly expressed. However, how the expression of these transcripts is regulated remains unclear. MicroRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally inhibit the expression of their target transcripts. In this study, using small RNA sequencing analysis, miR-217-5p was identified as the most upregulated transcript in PAN-treated rat podocytes. MiR-217-5p overexpression in E11 podocyte cells led to shrunken cells with abnormal actin cytoskeletons. Consistent with these changes in cell morphology, gene ontology (GO) enrichment analysis showed that interactive GO terms related to cell morphogenesis were enriched with the predicted targets of miR-217-5p. Of the predicted targets highly downregulated by PAN, Myosin 1d (Myo1d) is a nonmuscle myosin predicted to be involved in actin filament organization and thought to play a role in podocyte morphogenesis and injury. We demonstrated that miR-217-5p targets Myo1d by luciferase assays, qRT-PCR, and Western blotting. Furthermore, we showed that miR-217-5p was present in urine from PAN- but not saline-administrated rats. Taken together, our data suggest that miR-217-5p may serve as a therapeutic target and a biomarker for podocyte injury.

Structure-based prediction of the IgE epitopes of the major dog allergen Can f 1.

Allergy to dogs has become increasingly prominent worldwide. Seven dog allergens have been identified, including Canis familiaris allergen 1-7 (Can f 1-7). Although Can f 1 is a major dog allergen sensitized to 50-75% of dog-allergic subjects, its IgE epitopes have not been identified. The structural analysis of an allergen is important to identify conformational epitopes. In this study, we generated a recombinant Can f 1 protein and determined its crystal structure using X-ray crystallography. Can f 1 had a typical lipocalin fold, which is composed of an eight-stranded ß-barrel and α-helix, and has high similarity to Can f 2, Can f 4, and Can f 6 in overall structure. However, the localizations of surface charges on these proteins were quite different. Based on sequence alignment and tertiary structure, we predicted five critical residues (His86, Glu98, Arg111, Glu138, and Arg152) for the IgE epitopes. The relevance of these residues to IgE reactivity was assessed by generating Can f 1 mutants with these residues substituted for alanine. Although the effects of the mutation on IgE binding depended on the sera of dog-allergic patients, H86A and R152A mutants showed reduced IgE reactivity compared with wild-type Can f 1. These results suggest that Can f 1 residues His86 and Arg152 are candidates for the IgE conformational epitope.

Zinc mediates the interaction between ceruloplasmin and apo-transferrin for the efficient transfer of Fe(III) ions.

Fe(II) exported from cells is oxidized to Fe(III), possibly by a multicopper ferroxidase (MCF) such as ceruloplasmin (CP), to efficiently bind with the plasma iron transport protein transferrin (TF). As unbound Fe(III) is highly insoluble and reactive, its release into the blood during the transfer from MCF to TF must be prevented. A likely mechanism for preventing the release of unbound Fe(III) is via direct interaction between MCF and TF; however, the occurrence of this phenomenon remains controversial. This study aimed to reveal the interaction between these proteins, possibly mediated by zinc. Using spectrophotometry, isothermal titration calorimetry, and surface plasmon resonance methods, we found that Zn(II)-bound CP bound to iron-free TF (apo-TF) with a Kd of 4.2 µM and a stoichiometry CP:TF of ∼2:1. Computational modeling of the complex between CP and apo-TF predicted that each of the three Zn(II) ions that bind to CP further binds to an acidic amino acid residue of apo-TF to play a role as a cross-linker connecting both proteins. Domain 4 of one CP molecule and domain 6 of the other CP molecule fit tightly into the clefts in the N- and C-lobes of apo-TF, respectively. Upon the binding of two Fe(III) ions to apo-TF, the resulting diferric TF [Fe(III)2TF] dissociated from CP by conformational changes in TF. In human blood plasma, zinc deficiency reduced the production of Fe(III)2TF and concomitantly increased the production of non-TF-bound iron. Our findings suggest that zinc may be involved in the transfer of iron between CP and TF.

Different hydration states and passive tumor targeting ability of polyethylene glycol-modified dendrimers with high and low PEG density.

It has been reported that the amount of intermediate water, defined as water molecules loosely bound to a material, is a useful index of the material's bio-inert properties. Polyethylene glycol (PEG) is a well-known biocompatible polymer with a large amount of intermediate water. Many researchers have showed that PEGylated nanoparticles are passively accumulated in tumor tissues owing to their enhanced permeability and retention (EPR) effects. Dendrimers are regularly branched polymers with highly controllable size and structure, which can be exploited as potent drug carriers. In this study, we investigated the tripartite relationship among the PEG density, the hydration state, and the passive tumor targeting property, using PEGylated dendrimers. The fully PEGylated dendrimer, PEG64-den, showed similar hydration behavior to PEG and a passive tumor targeting property. In contrast, the hydration state of the partly PEGylated dendrimer, PEG5-den, was different from that of PEG64-den, and the passive tumor targeting property was not observed. This is the first report to show the hydration state of a drug carrier as well as discuss a relationship between the hydration state and biodistribution.

Allosteric regulation accompanied by oligomeric state changes of Trypanosoma brucei GMP reductase through cystathionine-ß-synthase domain.

Guanosine 5'-monophosphate reductase (GMPR) is involved in the purine salvage pathway and is conserved throughout evolution. Nonetheless, the GMPR of Trypanosoma brucei (TbGMPR) includes a unique structure known as the cystathionine-ß-synthase (CBS) domain, though the role of this domain is not fully understood. Here, we show that guanine and adenine nucleotides exert positive and negative effects, respectively, on TbGMPR activity by binding allosterically to the CBS domain. The present structural analyses revealed that TbGMPR forms an octamer that shows a transition between relaxed and twisted conformations in the absence and presence of guanine nucleotides, respectively, whereas the TbGMPR octamer dissociates into two tetramers when ATP is available instead of guanine nucleotides. These findings demonstrate that the CBS domain plays a key role in the allosteric regulation of TbGMPR by facilitating the transition of its oligomeric state depending on ligand nucleotide availability.

Datasets of microarray analysis to identify Gpr137b-dependent interleukin-4-responsive genes in the mouse macrophage cell line RAW264.

Macrophages are classified mainly into two subtypes, M1 and M2, which exhibit distinct phenotypes, based on their microenvironment. We have recently demonstrated that Gpr137b is abundantly expressed in RAW264 macrophages, "Gpr137b is an orphan G-protein-coupled receptor associated with M2 macrophage polarization" (Islam et al., in press) [1]. Although recent studies have suggested that G-protein-coupled receptors (GPCRs) are associated with M1/M2 macrophage polarization ("G-protein-coupled bile acid receptor 1 (GPBAR1, TGR5) agonists reduce the production of proinflammatory cytokines and stabilize the alternative macrophage phenotype" (Hogenauer et al., 2014) [2], "Leukotriene B4 promotes neovascularization and macrophage recruitment in murine wet-type AMD models" (Sasaki et al., 2018) [3]), available information about GPCR-mediated macrophage polarization is still limited. This prompted us to generate Gpr137b-knockout (KO) RAW264 clones using the CRISPR/Cas9 genome editing system to elucidate the function of Gpr137b in interleukin (IL)-4-induced M2 macrophage polarization (Islam et al., in press) [1]. Here we present the datasets of a microarray analysis to identify Gpr137b-dependent IL-4-responsive genes in RAW264 cells. The raw microarray data are available in the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE117578, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE117578.

Crystal structure of the dog allergen Can f 6 and structure-based implications of its cross-reactivity with the cat allergen Fel d 4.

Several dog allergens cause allergic reactions in humans worldwide. Seven distinct dog allergens, designated Canis familiaris allergen 1 to 7 (Can f 1-Can f 7), have been identified thus far. Can f 6 shows high sequence similarity and cross-reactivity with Fel d 4 and Equ c 1, major cat and horse allergens, respectively. This study was conducted on the allergenic epitopes of Can f 6 based on its structural characterization. We demonstrated that sera from 18 out of 38 (47%) dog-sensitized patients reacted to recombinant Can f 6 protein (rCan f 6). We then determined the crystal structure of rCan f 6 by X-ray crystallography, which exhibited a conserved tertiary structural architecture found in lipocalin family proteins. Based on the tertiary structure and sequence similarities with Fel d 4 and Equ c 1, we predicted three IgE-recognizing sites that are possibly involved in cross-reactivity. Substituting three successive amino acids in these sites to triple alanine decreased IgE reactivity to the allergen. However, the degree of reduction in IgE reactivity largely depended on the site mutated and the serum used, suggesting that Can f 6 is a polyvalent allergen containing multiple epitopes and Can f 6-reactive sera contain varied amounts of IgE recognising individual Can f 6 epitopes including those predicted in this study. We also demonstrated that the predicted epitopes are partly involved in IgE cross-reactivity to Fel d 4. Interestingly, the effect of the mutation depended on whether the protein was structured or denatured, indicating that the bona fide tertiary structure of Can f 6 is essential in determining its IgE epitopes.

Gpr137b is an orphan G-protein-coupled receptor associated with M2 macrophage polarization.

Macrophages are classified mainly into two subtypes, M1 and M2, which exhibit distinct phenotypes, based on their microenvironment. Although recent studies have suggested that G-protein-coupled receptors (GPCRs) are associated with M1/M2 macrophage polarization, available information on GPCR-mediated macrophage polarization is still limited. In the present study, we identified Gpr137b as an orphan GPCR abundantly expressed in RAW264, a mouse macrophage cell line, and illuminated its role in M2 macrophage polarization. We generated Gpr137b-knockout (Gpr137b-KO) clones of RAW264 cells using the CRISPR/Cas9 genome editing system. Two independent Gpr137b-KO clones were isolated, which were demonstrated to have frameshifting 188-nucleotide deletions at a region containing the ATG start codon of Gpr137b. Consistently, qRT-PCR analysis revealed that the deleted region is not transcribed. We then treated the Gpr137b-KO and wildtype RAW264 cells with interleukin-4 (IL-4) to induce M2 macrophage polarization. Microarray analysis revealed that the IL-4-induced gene expression of representative M2 macrophage markers was significantly reduced in the Gpr137b-KO cells, and this was validated by qRT-PCR analysis. By contrast, M1 macrophage marker gene expression induced by lipopolysaccharide was unaffected by Gpr137b-KO. Collectively, the current study shows that Gpr137b is a possible regulator of M2 macrophage polarization.

A novel splicing variant of small nucleolar RNA host gene 4 is a podocyte-selective non-coding RNA upregulated in response to puromycin aminonucleoside-induced podocyte injury.

Podocytes are terminally differentiated cells that function as the glomerular filtration barrier in the kidney, and podocyte injury leads to serious proteinuria and podocyte leakage into urine. Recent studies have demonstrated that the number of urinary podocytes is correlated with the progression of glomerular diseases. Therefore, urinary podocytes may serve as an indicator of podocyte injury. In this study, to explore podocyte injury-related genes, we performed comprehensive transcriptome analysis of primary rat podocytes cultured in the presence or absence of puromycin aminonucleoside (PAN), an agent commonly used to induce podocyte injury. RNA-seq revealed that a transcript containing the intronic sequence of small nucleolar RNA host gene 4 (Snhg4) was expressed in podocytes and upregulated by PAN. RT-qPCR analysis demonstrated that this transcript, but not Snhg4, was selectively expressed in podocytes. Therefore, we designated the novel transcript Snhg4-pod. 5'- and 3'-RACE experiments revealed that Snhg4-pod is a novel splice variant of Snhg4 lacking a poly(A) tail. PAN induced Snhg4-pod expression in podocytes in a dose-dependent manner along with their mitochondria-mediated apoptotic cell death. Further, Snhg4-pod was detected in urinary sediments from PAN-induced nephrotic rats. Our findings suggest that Snhg4-pod may serve as a novel marker for the diagnosis of glomerular injury.

Proteinase K treatment improves RNA recovery from thyroid cells fixed with liquid-based cytology solution.

OBJECTIVE: Fine-needle aspiration biopsy (FNAB), an important diagnostic tool given its simplicity, safety, and cost-effectiveness, is fast becoming a popular procedure in the diagnosis of thyroid diseases. Generally, cells isolated from biopsies are transferred directly to microscope slides to prepare smears for cytopathological examination; however, the technical difficulties of this procedure often cause poor reproducibility, which limits the accuracy of diagnostic results. Liquid-based cytology (LBC), in which isolated cells are collected in a fixative solution, is advantageous in that it facilitates the preparation of homogenous cytological specimens. However, LBC has not been applied to molecular diagnoses, such as RNA expression-based diagnosis, mainly because of difficulties in cell recovery and RNA isolation. This study was aimed to improve RNA extraction from papillary cancer-derived K1 cells and thyroid FNAB specimens suspended in LBC solutions. RESULTS: K1 cells suspended in CytoRich-Red and CytoRich-Blue, fixatives for LBC, were efficiently recovered by trapping to glass-fiber filters. Importantly, subsequent Proteinase K treatment was essential for efficient RNA extraction from the fixed cells. This finding was also applicable to RNA extraction from CytoRich-Red-fixed thyroid FNAB specimens processed in the same way. Consistently, U6 small nuclear RNA was detected in these RNA samples by reverse transcription-polymerase chain reaction.

MiR-141-3p is upregulated in esophageal squamous cell carcinoma and targets pleckstrin homology domain leucine-rich repeat protein phosphatase-2, a negative regulator of the PI3K/AKT pathway.

The phosphatidylinositol-3-kinase (PI3K)/AKT pathway is frequently activated in various human cancers and plays essential roles in their development and progression. Accumulating evidence suggests that dysregulated expression of microRNAs (miRNAs) is closely associated with cancer progression and metastasis. Here, we focused on miRNAs that could regulate genes related to the PI3K/AKT pathway in esophageal squamous cell carcinoma (ESCC). To identify upregulated miRNAs and their possible target genes in ESCC, we performed microarray-based integrative analyses of miRNA and mRNA expression levels in three human ESCC cell lines and a normal esophageal epithelial cell line. The miRNA microarray analysis revealed that miR-31-5p, miR-141-3p, miR-200b-3p, miR-200c-3p, and miR-205-5p were expressed at higher levels in the ESCC cell lines than the normal esophageal epithelial cell line. Bioinformatical analyses of mRNA microarray data identified several AKT/PI3K pathway-related genes as candidate targets of these miRNAs, which include tumor suppressors such as DNA-damage-inducible transcript 4 and pleckstrin homology domain leucine-rich repeat protein phosphatase-2 (PHLPP2). To validate the targets of relevant miRNAs experimentally, synthetic mimics of the miRNAs were transfected into the esophageal epithelial cell line. Here, we report that miR-141-3p suppress the expression of PHLPP2, a negative regulators of the AKT/PI3K pathway, as a target in ESCC.

Solubility-Improved 10-O-Substituted SN-38 Derivatives with Antitumor Activity.

With the objective of improving the poor water solubility of the potent antitumor compound SN-38, 10-O-substituted SN-38 derivatives were developed by the introduction of fluoroalkyl, fluorobenzoyl, or bromobenzoyl groups. The 10-O-fluoropropyl-substituted compound 2 {(S)-4,11-diethyl-9-(3-fluoropropoxy)-4-hydroxy-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione} was found to be 17-fold more soluble than SN-38 in phosphate-buffered saline, and it exhibited a level of biological activity ≈50 % that of SN-38 in a cytotoxicity assay using the prostate cancer cell line PC-3. Five other derivatives did not show solubility improvements to the same extent, but their activities in cytotoxicity assays were nearly the same as that of SN-38. In vivo studies of 2 with PC-3 tumor-bearing mice revealed that it has higher antitumor activity than SN-38, even at lower dosage. These results will promote the medicinal chemistry application of 10-O-modifications of SN-38 and help reestablish the potential this drug. Furthermore, the inclusion of fluoro and bromo substituents means that the synthetic strategy developed here may be used to obtain 18 F- or 76 Br-labeled SN-38 derivatives for in vivo positron emission tomography studies.

High titers of both rheumatoid factor and anti-CCP antibodies at baseline in patients with rheumatoid arthritis are associated with increased circulating baseline TNF level, low drug levels, and reduced clinical responses: a post hoc analysis of the RISING study.

BACKGROUND: Although both rheumatoid factor (RF) and anticyclic citrullinated peptide antibodies (anti-CCP) are useful for diagnosing rheumatoid arthritis (RA), the impact of these autoantibodies on the efficacy of tumor necrosis factor (TNF) inhibitors has been controversial. The aim of this post hoc analysis of a randomized double-blind study (the RISING study) was to investigate the influences of RF and anti-CCP on the clinical response to infliximab in patients with RA. METHODS: Methotrexate-refractory patients with RA received 3 mg/kg of infliximab from weeks 0 to 6 and then 3, 6, or 10 mg/kg every 8 weeks from weeks 14 to 46. In this post hoc analysis, patients were stratified into three classes on the basis of baseline RF/anti-CCP titers: "low/low-C" (RF < 55 IU/ml, anti-CCP < 42 U/ml), "high/high-C" (RF ≥ 160 IU/ml, anti-CCP ≥ 100 U/ml), and "middle-C" (neither low/low-C nor high/high-C). Baseline plasma TNF level, serum infliximab level, and disease activity were compared between the three classes. RESULTS: Baseline RF and anti-CCP titers showed significant correlations with baseline TNF and infliximab levels in weeks 2-14. Comparison of the three classes showed that baseline TNF level was lowest in the low/low-C group and highest in the high/high-C group (median 0.73 versus 1.15 pg/ml), that infliximab levels at week 14 were highest in the low/low-C group and lowest in the high/high-C group (median 1.0 versus 0.1 µg/ml), and that Disease Activity Score in 28 joints based on C-reactive protein at week 14 was lowest in the low/low-C group and highest in the high/high-C group (median 3.17 versus 3.82). A similar correlation was observed at week 54 in the 3 mg/kg dosing group, but not in the 6 or 10 mg/kg group. Significant decreases in both RF and anti-CCP were observed during infliximab treatment. CONCLUSIONS: RF/anti-CCP titers correlated with TNF level. This might explain the association of RF/anti-CCP with infliximab level and clinical response in patients with RA. Baseline RF/anti-CCP titers may serve as indices that aid infliximab treatment. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00691028 . Retrospectively registered on 3 June 2008.

Comprehensive Evaluation of the Binding of Lipocalin-Type Prostaglandin D Synthase to Poorly Water-Soluble Drugs.

Low water solubility of candidate drug compounds is a major problem in pharmaceutical research and development. We developed a novel drug delivery system (DDS) for poorly water-soluble drugs using lipocalin-type prostaglandin D synthase (L-PGDS), which belongs to the lipocalin superfamily and binds a large variety of hydrophobic molecules. In this study, we comprehensively evaluated the capability of L-PGDS to bind and solubilize various poorly water-soluble drugs using structure-based docking. Docking simulations of 2892 commercially available approved drugs indicated that L-PGDS shows higher binding affinities for various drugs compared with 2-hydroxypropyl-ß-cyclodextrin. Five drugs selected from the top 100 with the highest binding affinities for L-PGDS exhibited very low solubility in PBS (pH 7.4). However, in the presence of 1 mM L-PGDS, the apparent solubility of all drugs improved markedly, from 19.5- to 166-fold. Calorimetric experiments on two drugs, telmisartan and imatinib, revealed that L-PGDS forms a 1:2 complex with each drug, with dissociation constants of 0.4-40.0 µM. Kinetic simulations of drug dissolution with L-PGDS indicated that the difference in free energy change (ΔΔG) between the insoluble state and the L-PGDS-bound state are within the range from -10 to +5 kJ mol-1. The ΔΔG value is a critical factor in evaluating whether a poorly water-soluble drug can be solubilized by L-PGDS. Collectively, these results demonstrate that in silico docking is a promising approach for identifying drug molecules suitable for the L-PGDS-based DDS.

MEK and PI3K catalytic activity as predictor of the response to molecularly targeted agents in triple-negative breast cancer.

Hyper-activation of the MAPK and PI3K-AKT pathways is linked to tumour progression in triple-negative breast cancer (TNBC). However, clinically effective predictive markers for drugs targeted against protein kinases involved in these pathways have not been identified. We investigated the ability of MEK and PI3K catalytic activity to predict sensitivity to trametinib and wortmannin in TNBC. MEK and PI3K activities correlated strongly with each other only in cell lines showing wortmannin-specific sensitivity, as shown by a linear regression curve (R = 0.951). Accordingly, we created a new parameter that distinguishes trametinib and wortmannin sensitivity in vitro and in vivo. Our findings suggest that the catalytic activities of MEK and PI3K might predict the response of TNBC to trametinib and wortmannin.