Butyrylated starch protects mice from DSS-induced colitis: combined effects of butyrate release and prebiotic supply.

Butyrate has recently emerged as a promising substance for the therapy of colitis. To overcome the shortcomings implicated in the existing delivery systems of butyrate, we utilized butyrylated starch to specifically deliver butyrate to the colon. Herein, we describe the stable loading of butyrate via chemical bonds with a heterogeneous distribution throughout the particle. Butyrylated starch supply increased butyrate as well as total short-chain fatty acid contents at the end of the intervention period. Moreover, butyrylated starch showed multiple effects on the suppression of DSS-induced colitis. From the observation of the gut-liver axis, reduced hepatic inflammation and hepatocyte damage further confirmed alleviated colonic inflammation. Given that butyrylated starch has the combined effects of specific release of butyrate in the colon and extra supply of fermentable substrates for gut microbiota, this work provides an effective strategy for the assistant therapy of colitis.

Translational pharmacology of an inhaled small molecule αvß6 integrin inhibitor for idiopathic pulmonary fibrosis.

The αvß6 integrin plays a key role in the activation of transforming growth factor-ß (TGFß), a pro-fibrotic mediator that is pivotal to the development of idiopathic pulmonary fibrosis (IPF). We identified a selective small molecule αvß6 RGD-mimetic, GSK3008348, and profiled it in a range of disease relevant pre-clinical systems. To understand the relationship between target engagement and inhibition of fibrosis, we measured pharmacodynamic and disease-related end points. Here, we report, GSK3008348 binds to αvß6 with high affinity in human IPF lung and reduces downstream pro-fibrotic TGFß signaling to normal levels. In human lung epithelial cells, GSK3008348 induces rapid internalization and lysosomal degradation of the αvß6 integrin. In the murine bleomycin-induced lung fibrosis model, GSK3008348 engages αvß6, induces prolonged inhibition of TGFß signaling and reduces lung collagen deposition and serum C3M, a marker of IPF disease progression. These studies highlight the potential of inhaled GSK3008348 as an anti-fibrotic therapy.

Modelled plasma concentrations of pemafibrate with co-administered typical cytochrome P450 inhibitors clopidogrel, fluconazole or clarithromycin predicted by physiologically based pharmacokinetic modelling in virtual populations.

Oral antidyslipidaemic drug pemafibrate is cleared from human plasma via hepatic uptake by organic anion transporting polypeptide (OATP) 1B1 and oxidation by cytochromes P450 (P450) 2C8, 2C9 and 3A4. The pharmacokinetic profiles of pemafibrate with virtual administrations of P450 inhibitors and/or disease interactions were generated using a physiologically based pharmacokinetic (PBPK) model previously established for co-administration of pemafibrate with OATP1B1 inhibitors. This PBPK model was validated in the current study using reported maximum pemafibrate plasma concentrations and areas under the curve from interaction studies in healthy subjects co-administered with clopidogrel (P450 2C8 inhibitor), fluconazole (P450 2C9/3A4 inhibitor) or clarithromycin (P450 3A4 inhibitor). Virtual co-administrations of pemafibrate with clopidogrel, fluconazole or clarithromycin increased the predicted plasma exposures of pemafibrate 1.4-1.7-fold, 1.2-1.4-fold and 2.9-11-fold, respectively, in subjects with or without moderate or severe renal impairment or Child-Pugh A or B liver cirrhosis. Some of the exposure-enhancing effects of clarithromycin may originate from its inhibitory potential toward OATP1B1, because the estimated effects of itraconazole (a P450 3A4 inhibitor) were only minor. Simulations using the current PBPK model in groups of virtual subjects with or without renal or hepatic impairment revealed modified pharmacokinetic profiles for pemafibrate following co-administration of typical P450 inhibitors.

A positron emission tomography imaging study to confirm target engagement in the lungs of patients with idiopathic pulmonary fibrosis following a single dose of a novel inhaled αvß6 integrin inhibitor.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disease with poor prognosis and a significant unmet medical need. This study evaluated the safety, pharmacokinetics (PK) and target engagement in the lungs, of GSK3008348, a novel inhaled alpha-v beta-6 (αvß6) integrin inhibitor, in participants with IPF. METHODS: This was a phase 1b, randomised, double-blind (sponsor unblind) study, conducted in the UK (two clinical sites, one imaging unit) between June 2017 and July 2018 (NCT03069989). Participants with a definite or probable diagnosis of IPF received a single nebulised dose of 1000 mcg GSK3008348 or placebo (ratio 5:2) in two dosing periods. In period 1, safety and PK assessments were performed up to 24 h post-dose; in period 2, after a 7-day to 28-day washout, participants underwent a total of three positron emission tomography (PET) scans: baseline, Day 1 (~ 30 min post-dosing) and Day 2 (~ 24 h post-dosing), using a radiolabelled αvß6-specific ligand, [18F]FB-A20FMDV2. The primary endpoint was whole lung volume of distribution (VT), not corrected for air volume, at ~ 30 min post-dose compared with pre-dose. The study success criterion, determined using Bayesian analysis, was a posterior probability (true % reduction in VT > 0%) of ≥80%. RESULTS: Eight participants with IPF were enrolled and seven completed the study. Adjusted posterior median reduction in uncorrected VT at ~ 30 min after GSK3008348 inhalation was 20% (95% CrI: - 9 to 42%). The posterior probability that the true % reduction in VT > 0% was 93%. GSK3008348 was well tolerated with no reports of serious adverse events or clinically significant abnormalities that were attributable to study treatment. PK was successfully characterised showing rapid absorption followed by a multiphasic elimination. CONCLUSIONS: This study demonstrated engagement of the αvß6 integrin target in the lung following nebulised dosing with GSK3008348 to participants with IPF. To the best of our knowledge this is the first time a target-specific PET radioligand has been used to assess target engagement in the lung, not least for an inhaled drug. TRIAL REGISTRATION: clinicaltrials.gov: NCT03069989; date of registration: 3 March 2017.

Pemafibrate, a New Selective PPARα Modulator: Drug Concept and Its Clinical Applications for Dyslipidemia and Metabolic Diseases.

PURPOSE OF REVIEW: Reduction of serum low-density lipoprotein cholesterol (LDL-C) levels by statins, ezetimibe and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors has been shown to significantly reduce cardiovascular events risk. However, fasting and postprandial hypertriglyceridemia as well as reduced high-density lipoprotein cholesterol (HDL-C) remain as residual risk factors of atherosclerotic cardiovascular diseases (ASCVD). To treat patients with hypertriglyceridemia and/or low HDL-C, drugs such as fibrates, nicotinic acids, and n-3 polyunsaturated fatty acids have been used. However, fibrates were demonstrated to cause side effects such as liver dysfunction and increase in creatinine levels, and thus large-scale clinical trials of fibrates have shown negative results for prevention of ASCVD. The failure could be attributed to their low selectivity and potency for binding to peroxisome proliferator-activated receptor (PPAR) α. To resolve these issues, the concept of selective PPARα modulator (SPPARMα) with a superior balance of efficacy and safety has been proposed and pemafibrate (K-877) has been developed. RECENT FINDINGS: Pemafibrate, one of SPPARMsα, was synthesized by Kowa Company, Ltd. for better efficiency and safety. Clinical trials in Japan have established the superiority of pemafibrate on effects on serum triglycerides (TG) reduction and HDL-C elevation as well safety. Although available fibrates showed worsening of liver and kidney function test values, pemafibrate indicated improved liver function test values and was less likely to increase serum creatinine or decrease estimated glomerular filtration rate (eGFR). Very few drug-drug interactions were observed even when used concomitantly with statins. Furthermore, pemafibrate is metabolized in the liver and excreted into the bile, while many of available fibrates are mainly excreted from the kidney. Therefore, pemafibrate can be used safely even in patients with impaired renal function since there is no significant increase in its blood concentration. A large-scale trial of pemafibrate, PROMINENT, for dyslipidemic patients with type 2 diabetes is ongoing. Pemafibrate is one of novel SPPARMsα and has superior benefit-risk balance compared to conventional fibrates and can be applicable for patients for whom the usage of existing fibrates is difficult such as those who are taking statins or patients with renal dysfunction. In the current review, all the recent data on pemafibrate will be summarized.

Pharmacokinetics and metabolism of pemafibrate, a novel selective peroxisome proliferator-activated receptor-alpha modulator, in rats and monkeys.

The metabolic profiles and pharmacokinetics of pemafibrate, a novel selective peroxisome proliferator activated receptor-alpha modulator currently launched as an antidyslipidemic drug, were investigated in vitro using hepatocytes from rats, monkeys and humans and in vivo in rats and monkeys. Hepatocytes from rats, monkeys and humans all biotransformed pemafibrate to its demethylated form (M1). The bioavailabilities of pemafibrate in Sprague-Dawley rats and cynomolgus monkeys were 15% and 87%, respectively, after a single oral administration of pemafibrate (1 mg/kg). In rat plasma, unmetabolized pemafibrate was the major form, accounting for 29% of the area under the curve (AUC) of total radioactivity. In monkey plasma, in contrast, the major circulating metabolites were M2/3 (dearylated/dicarboxylic acid forms, 15%), M4 (N-dealkylated form, 21%) and M5 (benzylic oxidative form, 9%), but pemafibrate was the notable minor form (3%). These results, in combination with the reported findings in humans, suggest that the metabolite profile of pemafibrate in plasma was different for rats and monkeys, and that monkeys could be a suitable animal model for further pharmacokinetic studies of pemafibrate in humans.

The safety, tolerability, and pharmacokinetic profile of GSK2838232, a novel 2nd generation HIV maturation inhibitor, as assessed in healthy subjects.

This work aimed to assess the safety, tolerability, pharmacokinetics (PK), and relative bioavailability of GSK2838232, an investigational HIV maturation inhibitor. GSK2838232 was administered over four dose-escalation studies in healthy subjects which assessed single oral doses (5-250 mg) and repeat doses (up to 200 mg once or twice daily) ±100 mg ritonavir (RTV) once daily. GSK2838232 administration (up to 250 mg) to 124 subjects across four studies resulted in few mild adverse events (AEs) with similar frequencies to placebo. There were no clearly identified drug-related AEs. GSK2838232 tested fasted was quickly absorbed with a tmax of 2-3 hours. With food, the absorption was delayed and more variable, with ~60% increase in AUC and Cmax. Overall, following single doses GSK2838232 AUC and Cmax generally exhibited proportional PK from 50 to 100 mg dose without RTV and from 50 to 250 mg with RTV and following repeated doses of 20-200 mg with RTV. In relative bioavailability studies, a micronized formulation was found to be suitable for development. At steady state, RTV increased GSK2838232 AUC and Cmax by 10- and 3-fold, respectively. Half-life was prolonged from ~17 hours nonboosted to ~34 hours with RTV. This boosting effect was also seen in repeat-dose GSK2838232 studies, which achieved the targeted plasma exposure with GSK2838232 as a once-daily regimen of up to 200 mg with RTV. The results of these studies demonstrated a favorable safety and PK profile for GSK2838232 and support its investigation for the treatment of HIV infection.

Safety, tolerability and pharmacokinetics of GSK3008348, a novel integrin αvß6 inhibitor, in healthy participants.

PURPOSE: Inhaled drug delivery is an attractive route by which to deliver drugs to lungs of patients with idiopathic pulmonary fibrosis (IPF). GSK3008348 is a potent and selective small molecule being developed as the first inhaled inhibitor of the αvß6 integrin for the treatment of IPF. The phase 1 first-time-in-human clinical trial (NCT02612051) presented here was designed to investigate the safety, tolerability and pharmacokinetic (PK) profile of single doses of GSK3008348 in healthy participants. METHODS: Single ascending doses of GSK3008348 were administered to three cohorts of eight healthy participants in a randomised, double-blind, placebo-controlled, 4-period crossover design. Safety, tolerability and PK were assessed after single doses of 1-3000 mcg given by nebulisation. RESULTS: A total of 29 participants were enrolled and received at least one dose of study treatment. There were no serious adverse events (AE) reported in any participant. No trends or clinically important differences were noted in the incidence or intensity of AEs or other safety assessments. Maximum plasma concentrations of GSK3008348 were generally attained within approximately 30 min after start of nebulisation, with geometric mean terminal elimination half-lives ranging from 7.95 to 10.2 h. Exposures, as measured by area under the plasma concentration-time curve (AUC), were dose proportional across all doses where estimates were possible (100-3000 mcg). Dose normalised geometric mean Cmax increased with dose up to 3000 mcg. This supra proportionality was relatively modest, with a less than 3-fold increase over the range from 30 to 3000 mcg. The reason(s) for this observation are currently not known but may be due to slower absorption at the lowest doses. All exposures were within the exposure margins set by the non-clinical toxicity studies and so this is not expected to have any impact on safety. CONCLUSIONS: In summary, GSK3008348 was well tolerated at single doses up to 3000 mcg in healthy participants, and its PK profile was dose proportional at potentially clinically relevant doses (300-3000 mcg). These findings support further development of GSK3008348 as a novel inhaled treatment option for IPF.

Natural Korean Medicine Dang-Gui: Biosynthesis, Effective Extraction and Formulations of Major Active Pyranocoumarins, Their Molecular Action Mechanism in Cancer, and Other Biological Activities.

Angelica gigas Nakai (AGN) is a crucial oriental medicinal herb that grows especially in Korea and the Far-East countries. It contains chemically active compounds like pyranocoumarins, polyacetylenes and essential oils, which might be useful for treatment of several chronic diseases. It has been used for centuries as a traditional medicine in Southeast Asia, but in Western countries is used as a functional food and a major ingredient of several herbal products. The genus Angelica is also known as 'female ginseng' due to its critical therapeutic role in female afflictions, such as gynecological problems. However, it is well-documented that the AGN pyranocoumarins may play vital beneficial roles against cancer, neurodisorders, inflammation, osteoporosis, amnesia, allergies, depression, fungi, diabetes, ischemia, dermatitis, reactive oxygen species (ROS) and androgen. Though numerous studies revealed the role of AGN pyranocoumarins as therapeutic agents, none of the reviews have published their molecular mechanism of action. To the best of our knowledge, this would be the first review that aims to appraise the biosynthesis of AGN's major active pyranocoumarins, discuss effective extraction and formulation methods, and detail the molecular action mechanism of decursin (D), decursinol angelate (DA) and decursinol (DOH) in chronic diseases, which would further help extension of research in this area.

Prostate Cancer Xenograft Inhibitory Activity and Pharmacokinetics of Decursinol, a Metabolite of Angelica gigas Pyranocoumarins, in Mouse Models.

We have previously shown that the ethanol extract of dried Angelica gigas Nakai (AGN) root exerts anticancer activity against androgen receptor (AR)-negative human DU145 and PC-3 prostate cancer xenografts and primary carcinogenesis in the transgenic adenocarcinoma of mouse prostate (TRAMP) model. The major pyranocoumarin isomers decursin (D) and decursinol angelate (DA), when provided at equi-molar intake to that provided by AGN extract, accounted for the inhibitory efficacy against precancerous epithelial lesions in TRAMP mice. Since we and others have shown in rodents and humans that D and DA rapidly and extensively convert to decursinol, here we tested whether decursinol might be an in vivo active compound for suppressing xenograft growth of human prostate cancer cells expressing AR. In SCID-NSG mice carrying subcutaneously inoculated human LNCaP/AR-Luc cells overexpressing the wild type AR, we compared the efficacy of 4.5[Formula: see text]mg decursinol per mouse with equi-molar dose of 6[Formula: see text]mg D/DA per mouse. The result showed that decursinol decreased xenograft tumor growth by 75% and the lung metastasis, whereas D/DA exerted a much less effect. Measurement of plasma decursinol concentration, at 3[Formula: see text]h after the last dose of respective dosing regimen, showed higher circulating level in the decursinol-treated NSG mice than in the D/DA-treated mice. In a subsequent single-dose pharmacokinetic experiment, decursinol dosing led to 3.7-fold area under curve (AUC) of plasma decursinol over that achieved by equi-molar D/DA dosing. PK advantage notwithstanding, decursinol represents an active compound to exert in vivo prostate cancer growth and metastasis inhibitory activity in the preclinical model.

Nanocomposites based on Soluplus and Angelica gigas Nakai extract fabricated by an electrohydrodynamic method for oral administration.

Nanocomposites (NCs) based on Soluplus (SP) were fabricated by an electrohydrodynamic (EHD) method for the oral delivery of Angelica gigas Nakai (AGN). Nano-sized particles were obtained after dispersing the resultant, produced by the EHD technique, in the aqueous environment. AGN/SP2 (AGN:SP=1:2, w/w) NC dispersion in aqueous media exhibited a 130nm mean diameter, narrow size distribution, and robust stability in the tested concentration range of the ethanol extract of AGN (AGN EtOH ext) and at pH 1.2 and 6.8. Amorphization of the components of AGN and their interactions with SP in the AGN/SP2 NC formulation were demonstrated by X-ray diffractometry (XRD) analysis. The released amounts of decursin (D) and decursinol angelate (DA), major components of AGN, from NCs were improved compared with those from the AGN EtOH ext group at both pH 1.2 and 6.8. As D and DA can be metabolized into decursinol (DOH) in the liver after oral administration, the DOH concentrations in plasma were quantitatively determined to evaluate the oral absorption of AGN. In a pharmacokinetic study in rats, higher oral absorption and the maximum concentration in plasma (Cmax) were presented in the AGN/SP2 NC group compared with the AGN EtOH ext and AGN NC groups. These findings indicate the successful application of developed SP-based NCs for the oral delivery of AGN.

Safety, pharmacokinetics and pharmacodynamics of GLPG0974, a potent and selective FFA2 antagonist, in healthy male subjects.

AIMS: Free fatty acids (FFA) can act as direct signalling molecules through activation of several membrane-bound G-protein coupled receptors. The FFA2 receptor (known as GPR43) is activated by short chain fatty acids (SCFA) such as acetate and has been shown to play a major role in SCFA-induced neutrophil activation and migration and to contribute in the development and control of inflammation. GLPG0974 is a potent and selective antagonist of the human FFA2. The main objectives of the two phase 1 trials were to characterize the safety, tolerability, pharmacokinetics and pharmacodynamics of GLPG0974. METHODS: Two consecutive randomized, double-blind, placebo-controlled, single centre trials in healthy subjects were performed. In the first, GLPG0974 was administered as single doses up to 250 mg and in the second, multiple daily doses up to 400 mg for 14 days were evaluated. Non-compartmental analysis was used to determine GLPG0974 pharmacokinetics while target engagement was investigated through the inhibition of neutrophils in acetate-simulated whole blood samples using surface expression of CD11b activated epitope as a marker of neutrophil activation. RESULTS: The investigation of safety/tolerability and pharmacokinetics in the early development phase showed that GLPG0974 was safe and well tolerated up to a daily dose of 400 mg. GLPG0974 showed good and dose proportional exposure up to 400 mg daily as well as a substantial and sustained inhibition of acetate-stimulated neutrophil activation. CONCLUSION: Based on these results, a proof-of-concept study was initiated to evaluate the safety, tolerability and efficacy of GLPG0974 in patients with mild to moderate ulcerative colitis.

Protective effects of butyrate-based compounds on a mouse model for spinal muscular atrophy.

Proximal spinal muscular atrophy (SMA) is a childhood-onset degenerative disease resulting from the selective loss of motor neurons in the spinal cord. SMA is caused by the loss of SMN1 (survival motor neuron 1) but retention of SMN2. The number of copies of SMN2 modifies disease severity in SMA patients as well as in mouse models, making SMN2 a target for therapeutics development. Sodium butyrate (BA) and its analog (4PBA) have been shown to increase SMN2 expression in SMA cultured cells. In this study, we examined the effects of BA, 4PBA as well as two BA prodrugs-glyceryl tributyrate (BA3G) and VX563-on the phenotype of SMNΔ7 SMA mice. Treatment with 4PBA, BA3G and VX563 but not BA beginning at PND04 significantly improved the lifespan and delayed disease end stage, with administration of VX563 also improving the growth rate of these mice. 4PBA and VX563 improved the motor phenotype of SMNΔ7 SMA mice and prevented spinal motor neuron loss. Interestingly, neither 4PBA nor VX563 had an effect on SMN expression in the spinal cords of treated SMNΔ7 SMA mice; however, they inhibited histone deacetylase (HDAC) activity and restored the normal phosphorylation states of Akt and glycogen synthase kinase 3ß, both of which are altered by SMN deficiency in vivo. These observations show that BA-based compounds with favorable pharmacokinetics ameliorate SMA pathology possibly by modulating HDAC and Akt signaling.

Single oral dose pharmacokinetics of decursin and decursinol angelate in healthy adult men and women.

UNLABELLED: The ethanol extract of Angelica gigas Nakai (AGN) root has promising anti-cancer and other bioactivities in rodent models. It is currently believed that the pyranocoumarin isomers decursin (D) and decursinol angelate (DA) contribute to these activities. We and others have documented that D and DA were rapidly converted to decursinol (DOH) in rodents. However, our in vitro metabolism studies suggested that D and DA might be metabolized differently in humans. To test this hypothesis and address a key question for human translatability of animal model studies of D and DA or AGN extract, we conducted a single oral dose human pharmacokinetic study of D and DA delivered through an AGN-based dietary supplement Cogni.Q (purchased from Quality of Life Labs, Purchase, NY) in twenty healthy subjects, i.e., 10 men and 10 women, each consuming 119 mg D and 77 mg DA from 4 vegicaps. Analyses of plasma samples using UHPLC-MS/MS showed mean time to peak concentration (Tmax) of 2.1, 2.4 and 3.3 h and mean peak concentration (Cmax) of 5.3, 48.1 and 2,480 nmol/L for D, DA and DOH, respectively. The terminal elimination half-life (t1/2) for D and DA was similar (17.4 and 19.3 h) and each was much longer than that of DOH (7.4 h). The mean area under the curve (AUC0-48h) for D, DA and DOH was estimated as 37, 335 and 27,579 h∙nmol/L, respectively. Gender-wise, men absorbed the parent compounds faster and took shorter time to reach DOH peak concentration. The human data supported an extensive conversion of D and DA to DOH, even though they metabolized DA slightly slower than rodents. Therefore, the data generated in rodent models concerning anti-cancer efficacy, safety, tissue distribution and pharmacodynamic biomarkers will likely be relevant for human translation. TRIAL REGISTRATION: ClinicalTrials.gov NCT02114957.

[Simultaneous determination of clevidipine butyrate and its metabolite clevidipine acid in dog blood by liquid chromatography-tandem mass spectrometry].

A rapid, sensitive and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of clevidipine butyrate and its primary metabolite clevidipine acid in dog blood. After one-step protein precipitation with methanol, the chromatographic separation was carried out on an Ecosil C18 column (150 mm x 4.6 mm, 5 µm) with a gradient mobile phase consisting of methanol and 5 mmol · L(-1) ammonium formate. A chromatographic total run time of 13.0 min was achieved. The quantitation analysis was performed using multiple reaction monitoring (MRM) at the specific ion transitions of m/z 454.1 [M-H]- --> m/z 234.1 for clevidipine butyrate, m/z 354.0 [M-H]- --> m/z 208.0 for clevidipine acid and m/z 256.1 [M-H]- --> m/z 227.1 for elofesalamide (internal standard, IS) in the negative ion mode with electrospray ionization (ESI) source. The linear calibration curves for clevidipine butyrate and clevidipine acid were obtained in the concentration ranges of 0.5-100 ng · mL and 1-200 ng · mL(-1), separately. The lower limit of quantification of clevidipine butyrate and clevidipine acid were 0.5 ng · mL(-1) and 1 ng · mL(-1). The intra and inter-assay precisions were all below 12.9%, the accuracies were all in standard ranges. Stability testing indicated that clevidipine butyrate and clevidipine acid in dog blood with the addition of denaturant methanol was stable under various processing and/or handling conditions. The validated method has been successfully applied to a pharmacokinetic study of clevidipine butyrate injection to 8 healthy Beagle dogs following intravenous infusion at a flow rate of 5 mg · h(-1) for 0.5 h.

Discovery and optimization of an azetidine chemical series as a free fatty acid receptor 2 (FFA2) antagonist: from hit to clinic.

FFA2, also called GPR43, is a G-protein coupled receptor for short chain fatty acids which is involved in the mediation of inflammatory responses. A class of azetidines was developed as potent FFA2 antagonists. Multiparametric optimization of early hits with moderate potency and suboptimal ADME properties led to the identification of several compounds with nanomolar potency on the receptor combined with excellent pharmacokinetic (PK) parameters. The most advanced compound, 4-[[(R)-1-(benzo[b]thiophene-3-carbonyl)-2-methyl-azetidine-2-carbonyl]-(3-chloro-benzyl)-amino]-butyric acid 99 (GLPG0974), is able to inhibit acetate-induced neutrophil migration strongly in vitro and demonstrated ability to inhibit a neutrophil-based pharmacodynamic (PD) marker, CD11b activation-specific epitope [AE], in a human whole blood assay. All together, these data supported the progression of 99 toward next phases, becoming the first FFA2 antagonist to reach the clinic.

Tailor-made fluorescent trilobolide to study its biological relevance.

Trilobolide (Tb) is a potent natural counterpart of thapsigargin, which has shown promising results in cancer clinical trials. Here, we report a rational approach to study intracellular localization and biological activity of this sesquiterpene lactone. We conjugated Tb with a green-emitting Bodipy dye attached by alternative linkers of different lengths. The live-cell imaging of the prepared bioconjugates brought clear evidence that Tb-Bodipy localized in the endoplasmic reticulum (ER) of various cancer cell lines. The localization signal was compared with ER-specific dyes. Cytotoxicity of Tb conjugates and impact on the mitochondrial physiology and nitric oxide release were also studied. The nitric oxide production and cytokine secretion in rat peritoneal cells indicate immunobiological potential of these lactone bioconjugates. In summary, our Tb-Bodipy conjugates could help us to reveal the molecular mechanism of trilobolide for its further potential use in biomedical applications.

Characterization of butyrate transport across the luminal membranes of equine large intestine.

The diet of the horse, pasture forage (grass), is fermented by the equine colonic microbiota to short-chain fatty acids, notably acetate, propionate and butyrate. Short-chain fatty acids provide a major source of energy for the horse and contribute to many vital physiological processes. We aimed to determine both the mechanism of butyrate uptake across the luminal membrane of equine colon and the nature of the protein involved. To this end, we isolated equine colonic luminal membrane vesicles. The abundance and activity of cysteine-sensitive alkaline phosphatase and villin, intestinal luminal membrane markers, were significantly enriched in membrane vesicles compared with the original homogenates. In contrast, the abundance of GLUT2 protein and the activity of Na(+)-K(+)-ATPase, known markers of the intestinal basolateral membrane, were hardly detectable. We demonstrated, by immunohistochemistry, that monocarboxylate transporter 1 (MCT1) protein is expressed on the luminal membrane of equine colonocytes. We showed that butyrate transport into luminal membrane vesicles is energized by a pH gradient (out < in) and is not Na(+) dependent. Moreover, butyrate uptake is time and concentration dependent, with a Michaelis-Menten constant of 5.6 ± 0.45 mm and maximal velocity of 614 ± 55 pmol s(-1) (mg protein)(-1). Butyrate transport is significantly inhibited by p-chloromercuribenzoate, phloretin and α-cyano-4-hydroxycinnamic acid, all potent inhibitors of MCT1. Moreover, acetate and propionate, as well as the monocarboxylates pyruvate and lactate, also inhibit butyrate uptake. Data presented here support the conclusion that transport of butyrate across the equine colonic luminal membrane is predominantly accomplished by MCT1.

Development of a pharmacokinetic/pharmacodynamic/disease progression model in NC/Nga mice for development of novel anti-atopic dermatitis drugs.

1. JHL45, a novel immune modulator against atopic dermatitis (AD), was synthesized from decursin isolated from Angelica gigas. The goal is to evaluate the lead compound using quantitative modeling approaches to novel anti-AD drug development. 2. We tested the anti-inflammatory effect of JHL45 by in vitro screening, characterized its in vitro pharmacokinetic (PK) properties. The dose-dependent efficacy of JHL45 was developed using a pharmacokinetics/pharmacodynamics/disease progression (PK/PD/DIS) model in NC/Nga mice. 3. JHL45 has drug-like properties and pharmacological effects when administered orally to treat atopic dermatitis. The developed PK/PD/DIS model described well the rapid metabolism of JHL45, double-peak phenomenon in the PK of decursinol and inhibition of IgE generation by compounds in NC/Nga mice. Also, a quantitative model was developed and used to elucidate the complex interactions between serum IgE concentration and atopic dermatitis symptoms. 4. Our findings indicate that JHL45 has good physicochemical properties and powerful pharmacological effects when administered orally for treatment of AD in rodents.

Al18F-NODA-butyric acid: biological evaluation of a new PET renal radiotracer.

INTRODUCTION: Renal scintigraphy is an important imaging modality for the diagnosis and management of a variety of renal diseases including obstruction and renovascular hypertension as well as the evaluation of absolute and relative kidney function. The goal of this work was to evaluate Al(18)F-NODA-butyric acid (Al(18)F-1) as a potential PET tracer to image the kidneys and monitor renal function by comparing its pharmacokinetic properties with those of (131)I-o-iodohippurate ((131)I-OIH), the radioactive standard for the measurement of effective renal plasma flow. METHODS: Al(18)F-1 was prepared in aqueous conditions using a one-pot Al(18)F-radiofluorination method and its radiochemical purity was determined by HPLC. Biodistribution studies, using (131)I-OIH as an internal control, were performed in normal rats and in rats with renal pedicle ligation. In vitro stability and metabolism of Al(18)F-1 were analyzed by HPLC. Dynamic microPET/CT studies were conducted in normal rats. RESULTS: Al(18)F-1 showed excellent stability in vitro and in vivo. Biodistribution studies in normal rats and in rats with simulated renal failure confirmed that Al(18)F-1 was exclusively cleared through the renal-urinary pathway and that the hepatic/gastrointestinal activity was less for Al(18)F-1 than for (131)I-OIH both at 10 and 60 min. Dynamic PET showed a rapid transit of Al(18)F-1 through the kidneys into the bladder. CONCLUSION: These results suggest that the easily labeled Al(18)F-based compounds provide a highly promising approach for the development of a PET renal radiotracer that combines superior imaging qualities with a reliable measure of effective renal plasma flow.