Radiosynthesis and Preclinical Evaluation of an α2A-Adrenoceptor Tracer Candidate, 6-[18F]Fluoro-marsanidine.

PURPOSE: The α2-adrenoceptors mediate many effects of norepinephrine and epinephrine, and participate in the regulation of neuronal, endocrine, cardiovascular, vegetative, and metabolic functions. Of the three receptor subtypes, only α2A and α2C are found in the brain in significant amounts. Subtype-selective positron emission tomography (PET) imaging of α2-adrenoceptors has been limited to the α2C subtype. Here, we report the synthesis of 6-[18F]fluoro-marsanidine, a subtype-selective PET tracer candidate for α2A-adrenoceptors, and its preclinical evaluation in rats and mice. PROCEDURES: 6-[18F]Fluoro-marsanidine was synthesized using electrophilic F-18 fluorination with [18F]Selectfluor bis(triflate). The tracer was evaluated in Sprague Dawley rats and in α2A-knockout (KO) and wild-type (WT) mice for subtype selectivity. In vivo PET imaging and ex vivo brain autoradiography were performed to determine the tracer distribution in the brain. The specificity of the tracer for the target was determined by pretreatment with the subtype-non-selective α2-agonist medetomidine. The peripheral biodistribution and extent of metabolism of 6-[18F]fluoro-marsanidine were also analyzed. RESULTS: 6-[18F]Fluoro-marsanidine was synthesized with [18F]Selectfluor bis(triflate) in a radiochemical yield of 6.4 ± 1.7 %. The molar activity was 3.1 to 26.6 GBq/µmol, and the radiochemical purity was > 99 %. In vivo studies in mice revealed lower uptake in the brains of α2A-KO mice compared to WT mice. The results for selectivity were confirmed by ex vivo brain autoradiography. Blocking studies revealed reduced uptake in α2A-adrenoceptor-rich brain regions in pretreated animals, demonstrating the specificity of the tracer. Metabolite analyses revealed very rapid metabolism of 6-[18F]fluoro-marsanidine with blood-brain barrier-permeable metabolites in both rats and mice. CONCLUSION: 6-[18F]Fluoro-marsanidine was synthesized and evaluated as a PET tracer candidate for brain α2A-adrenoceptors. However, rapid metabolism, extensive presence of labeled metabolites in the brain, and high non-specific uptake in mouse and rat brain make 6-[18F]fluoro-marsanidine unsuitable for α2A-adrenoceptor targeting in rodents in vivo.

Nitrogen doped carbon nanofibers loaded with hierarchical vanadium tetrasulfide for the voltammetric detection of the non-steroidal anti-prostate cancer drug nilutamide.

An electrochemical sensor is described for the determination of nilutamide (NLM) in biological fluids. A flexible mat of nitrogen-doped carbon nanofibers (NCNFs) was prepared by electrospinning. This was followed by carbonization and the growth of one-dimensional vanadium tetrasulfide (VS4) nanorods in the circumference of the NCNFs by a solvothermal route. The material was used to modify a glassy carbon electrode (GCE). Cyclic voltammetry and amperometry revealed an excellent electrocatalytic activity of the VS4/NCNF mat towards NLM at a working potential of - 0.57 V (vs Ag/AgCl). The modified GCE has a wide linear range (0.001-760 µM), a low limit of detection (90 pM), good stability, and fast response. It was used to detect NLM in spiked serum and urine samples. Graphical abstract Schematic presentation of a glassy carbon electrode (GCE) composed of free-standing nitrogen-doped carbon nanofibers decorated with vanadium tetrasulfide (Patronite) nanorods (VS4/NCNF). It was fabricated by electrospinning followed by stabilization and carbonization. The GCE responds to nilutamide (NLM) with excellent selectivity and nanomolar sensitivity.

Population Pharmacokinetic Modeling of JNJ-53718678, a Novel Fusion Inhibitor for the Treatment of Respiratory Syncytial Virus: Results from a Phase I, Double-Blind, Randomized, Placebo-Controlled First-in-Human Study in Healthy Adult Subjects.

BACKGROUND: JNJ-53718678 is a potent small-molecule inhibitor of the F-glycoprotein-mediated complex membrane fusion process of the respiratory syncytial virus. Here, we report the pharmacokinetics, the population pharmacokinetic modeling, and the safety and tolerability of JNJ-53718678 from the first-in-human, double-blind, randomized, placebo-controlled phase I study. METHODS: Healthy subjects were randomized (6:3) into five single-dose groups (25-1000 mg) or three multiple-dose groups [250 mg every 24 h (q24h), 500 mg q24h, 250 mg every 12 h; fed conditions for 8 days] to receive JNJ-53718678 or placebo. Blood and urine samples were collected at several timepoints up to 72 h after intake of JNJ-53718678 and analyzed using validated liquid chromatography-mass spectrometry methods. A population pharmacokinetic model was developed and validated. RESULTS: Peak plasma concentrations of JNJ-53718678 increased with increasing single (159 ± 54.9 to 6702 ± 1733 ng/mL) and multiple (on day 8, 1528 ± 256 to 2655 ± 591 ng/mL) doses. Steady-state conditions were reached on day 2 of the 8-day dosing regimen. Less than 4% of JNJ-53718678 was excreted in urine across all dose groups. Mean exposure of JNJ-53718678 was 7% lower in the fed state compared with the fasted state at the same dose. A two-compartment model with first-order absorption with parallel linear and non-linear elimination best described the pharmacokinetics of JNJ-53718678. No covariate effects were observed. CONCLUSIONS: A population pharmacokinetic model that describes the concentration data well with good precision of all parameter estimates was developed and validated. JNJ-53718678 was well tolerated at all single and multiple doses studied.

A highly sensitive and selective electrochemical determination of non-steroidal prostate anti-cancer drug nilutamide based on f-MWCNT in tablet and human blood serum sample.

A novel electrochemical sensor based on the functionalized multiwalled carbon nanotube (f-MWCNT) was successfully developed for the sensitive and selective determination of non-steroidal prostate anti-cancer drug nilutamide in tablet and blood serum samples. The f-MWCNT was prepared by the simple reflux method and characterized by the scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), Raman spectroscopy, X-ray powder diffraction (XRD) and fourier transform infrared spectroscopy (FT-IR). Interestingly, the f-MWCNT was exhibited a superior electrocatalytic activity towards the anti-cancer drug nilutamide when compared with pristine MWCNT and unmodified electrodes. Besides, the electrochemical sensor was revealed an excellent current response for the determination of nilutamide with wide linear ranges (0.01-21µM and 28-535µM), high sensitivity (11.023 and 1.412µA µM-1cm2) and very low detection limit (LOD) 0.2nM. The developed electrochemical sensor was showed an excellent selectivity even in the presence of electrochemically active biological substances and nitro aromatic compounds. Moreover, it manifested a good reproducibility and stability. In addition, the f-MWCNT modified glassy carbon electrode (GCE) sensor was successfully applied for the detection of nilutamide in tablet and blood serum sample.

Diet low in advanced glycation end products increases insulin sensitivity in healthy overweight individuals: a double-blind, randomized, crossover trial.

BACKGROUND: The consumption of advanced glycation end products (AGEs) has increased because of modern food processing and has been linked to the development of type 2 diabetes in rodents. OBJECTIVE: We determined whether changing dietary AGE intake could modulate insulin sensitivity and secretion in healthy, overweight individuals. DESIGN: We performed a double-blind, randomized, crossover trial of diets in 20 participants [6 women and 14 men; mean ± SD body mass index (in kg/m(2)): 29.8 ± 3.7]. Isoenergetic- and macronutrient-matched diets that were high or low in AGE content were alternately consumed for 2 wk and separated by a 4-wk washout period. At the beginning and end of each dietary period, a hyperinsulinemic-euglycemic clamp and an intravenous glucose tolerance test were performed. Dietary, plasma and urinary AGEs N(€)-(carboxymethyl)lysine (CML), N(€)-(carboxyethyl)lysin (CEL), and methylglyoxal-derived hydroimadazolidine (MG-H1) were measured with the use of mass spectrometry. RESULTS: Participants consumed less CML, CEL, and MG-H1 during the low-AGE dietary period than during the high-AGE period (all P < 0.05), which was confirmed by changes in urinary AGE excretion. There was an overall difference in insulin sensitivity of -2.1 mg · kg(-1) · min(-1) between diets (P = 0.001). Insulin sensitivity increased by 1.3 mg · kg(-1) · min(-1) after the low-AGE diet (P = 0.004), whereas it showed a tendency to decrease by 0.8 mg · kg(-1) · min(-1) after the high-AGE diet (P = 0.086). There was no difference in body weight or insulin secretion between diets (P = NS). CONCLUSIONS: A diet that is low in AGEs may reduce the risk of type 2 diabetes by increasing insulin sensitivity. Hence, a restriction in dietary AGE content may be an effective strategy to decrease diabetes and cardiovascular disease risks in overweight individuals. This trial was registered at clinicaltrials.gov as NCT00422253.

Development and validation of a UHPLC-MS/MS bioanalytical method to quantify in plasma the analgesic candidate PT-31 following a preliminary pharmacokinetic study in rats.

A selective and sensitive UHPLC-MS/MS bioanalytical method to determine PT-31, an analgesic drug candidate, in rat plasma was developed and validated. Analyses were performed using a UHPLC-MS/MS system equipped with an electrospray ionization interface operating in the positive ionization mode using a C18 reversed-phase column with a mobile phase of water:acetonitrile (68:31, v/v) containing 0.1% acetic acid eluting in a gradient mode with a flow rate of 0.3 mL/min. Plasma samples were deproteinized with cold acetonitrile containing 0.01% TFA (1:2, v/v) and 50 µL of the supernatant were injected into the system. PT-31 and phenytoin (internal standard) retention times were roughly 1.0 and 1.5 min, respectively. Linear standard curves were plotted for the 0.01-10 µg/mL concentration range, with a coefficient of determination > 0.99. The method's precision was over 88%. Maximum intra- and inter-day relative standard deviations were 14.6% and 11.6%, respectively. Interfering substances were not detected in the chromatogram, indicating that the method was specific. PT-31 stability was assessed under different temperature and storage settings. The method was used to characterize PT-31 plasma pharmacokinetics following administration of 5 mg/kg i.v. to Wistar rats. Therefore, the method described is sensitive, linear, precise and specific enough to determine PT-31 in preclinical pharmacokinetic investigations. Copyright © 2015 John Wiley & Sons, Ltd.

The safety, tolerability, pharmacokinetics, and pharmacodynamics of single oral doses of RO5068760, an MEK inhibitor, in healthy volunteers: assessment of target suppression.

RO5068760, a substituted hydantoin, represents a new class of potent, highly selective, non-adenosine triphosphate (ATP)-competitive MEK1/2 inhibitors. The study aimed to determine the safety/tolerability, pharmacokinetics, and pharmacodynamics of single ascending doses of RO5068760 in human healthy volunteers. All participants received a single dose followed by 48 hours of pharmacokinetics, pharmacodynamics, and safety/tolerability assessments. The pharmacodynamics were measured by changes in ERK phosphorylation (pERK) in peripheral blood mononuclear cells, ex vivo stimulated by phorbol 12-myristate 13-acetate (PMA). Forty-eight participants received 6 doses (50, 100, 200, 400, 600, 800 mg). RO5068760 was well tolerated up to 800 mg. There were no clinically significant safety findings, including laboratory, electrocardiogram, ophthalmological assessment, and fecal occult blood tests. Of the total 13 adverse events (n = 12), 11 were mild, 2 were moderate, and none were severe, and only 5 were considered by the investigator as possibly related to treatment. RO5068760 was absorbed with a t(max), of 2 hours. Disposition appeared to be biphasic with a terminal elimination t(1/2) of 5 to 9 hours. The variability was moderate to high, ranging from 38% to 62% for C(max) and 41% to 69% AUC. Within the dose range tested, pERK inhibition was relatively modest with a mean maximal pERK suppression of 55%.

Preclinical in vivo evaluation of efficacy, pharmacokinetics, and pharmacodynamics of a novel MEK1/2 kinase inhibitor RO5068760 in multiple tumor models.

Targeting the Ras/Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway represents a promising anticancer strategy. Recently, we have reported a novel class of potent and selective non-ATP-competitive MEK1/2 inhibitors with a unique structure and mechanism of action. RO5068760 is a representative of this class showing significant efficacy in a broad spectrum of tumors with aberrant mitogen-activated protein kinase pathway activation. To understand the relationship between systemic exposures and target (MEK1/2) inhibition as well as tumor growth inhibition, the current study presents a detailed in vivo characterization of efficacy, pharmacokinetics, and pharmacodynamics of RO5068760 in multiple xenograft tumor models. For inhibition of MEK1/2 as measured by the phosphorylated ERK levels, the estimated EC(50)s in plasma were 1.36 micromol/L (880 ng/mL) and 3.35 micromol/L (2168 ng/mL) in LOX melanoma and HT-29 colorectal cancer models, respectively. A similar EC(50) (1.41 micromol/L or 915 ng/mL) was observed in monkey peripheral blood lymphocytes. To achieve tumor growth inhibition (>or=90%), an average plasma drug concentration of 0.65 or 5.23 micromol/L was required in B-RafV600E or K-Ras mutant tumor models, respectively, which were remarkably similar to the IC(90) values (0.64 or 4.1 micromol/L) determined in vitro for cellular growth inhibition. With equivalent in vivo systemic exposures, RO5068760 showed superior efficacy in tumors harboring B-RafV600E mutation. The plasma concentration time profiles indicate that constant p-ERK suppression (>50%) may not be required for optimal efficacy, especially in highly responsive tumors. This study may facilitate future clinical trial design in using biochemical markers for early proof of mechanism and in selecting the right patients and optimal dose regimen.

Guadinomines, Type III secretion system inhibitors, produced by Streptomyces sp. K01-0509. I: taxonomy, fermentation, isolation and biological properties.

Enteropathogenic Escherichia coli (EPEC) expressing the Type III secretion system (TTSS) induced hemolysis of sheep blood cells. Using this assay, six structurally related compounds designated as guadinomines were isolated as inhibitors of TTSS-induced hemolysis by ion exchange column chromatography and HPLC from the culture broth of Streptomyces sp. K01-0509. Guadinomines A and B showed potent inhibition with IC50 values of 0.02 and 0.007 microg/ml, respectively, guadinomine D showed moderate activity (IC50: 8.5 microg/ml), while guadinomines C1 and C2 and guadinomic acid had no activity.

Clearance of imidapril, an Angiotensin-converting enzyme inhibitor, during hemodialysis in hypertensive renal failure patients: comparison with quinapril and enalapril.

The dialyzability of imidaprilat, an active metabolite of the angiotensin-converting enzyme (ACE) inhibitor imidapril, was determined and compared with those of enalaprilat and quinaprilat in hypertensive patients on chronic hemodialysis. Imidapril (5 mg/d, n = 6), enalapril (2.5 mg/d, n = 6), or quinapril (2.5 mg/d, n = 6) was given for at least 8 weeks prior to the trial. During dialysis, enalaprilat, but not imidaprilat or quinaprilat, concentrations in both sides decreased significantly. Compared to enalaprilat, the dialyzabilities of imidaprilat and quinaprilat were significantly lower (dialyzer clearance [mL/min/m(2)]: enalaprilat, 41.8 +/- 7.4; imidaprilat, 19.0 +/- 7.8; quinaprilat, 8.9 +/- 1.3). The dialyzabilities of the 3 drugs were negatively correlated with their respective protein-binding rates. During hemodialysis, blood pressure did not change significantly in any group. These results suggest that imidapril provides good blood pressure control without a large fluctuation of drug concentration in hypertensive patients undergoing chronic hemodialysis.

Associations between CYP11B2 gene polymorphisms and the response to angiotensin-converting enzyme inhibitors.

OBJECTIVE: Our objective was to investigate the association between the -344C/T or A6547G polymorphism of the aldosterone synthase gene and the blood pressure response to angiotensin-converting enzyme inhibitors in a hypertensive cohort. METHODS: After a 2-week single-blind placebo run-in period, either benazepril or imidapril was administered for 6 weeks to 509 patients with mild to moderate essential hypertension. Polymerase chain reaction combined with restriction enzyme digestion was used to detect the 2 polymorphisms. The achieved changes in systolic and diastolic blood pressure were analyzed for their association with genotypes at the aldosterone synthase gene loci. RESULTS: Regarding the -344C/T polymorphism, we observed the CC genotype in 53 patients (10.4%), the CT genotype in 204 (40.1%), and the TT genotype in 252 (49.5%). After 6 weeks of treatment, the reductions in diastolic blood pressure were significantly greater in patients carrying the TT or CT genotype compared with those carrying the CC genotype (9.1+/-7.0 mm Hg or 8.9+/-7.0 mm Hg versus 5.1+/-7.3 mm Hg, respectively; P=.001, ANOVA). Regarding the A6547G polymorphism, we observed the AA genotype in 19 patients (3.7%), the AG genotype in 184 (36.2%), and the GG genotype in 306 (60.1%). There were no significant differences in the blood pressure reductions after treatment among the 3 genotype groups, and there was no interaction between it and the -344C/T polymorphism. Stepwise multiple regression analysis showed that the significant predictors of diastolic blood pressure reduction at 6 weeks were baseline diastolic blood pressure (P<.001), -344C/T genotype (P=.007), and sex (P=.033). CONCLUSIONS: The -344C/T variant, but not the A6547G variant, of the aldosterone synthase gene may be a determinant of the blood pressure response to angiotensin-converting enzyme inhibitors in hypertensive patients.

The metabolic profile of azimilide in man: in vivo and in vitro evaluations.

The metabolic fate of azimilide in man is unusual as it undergoes a cleavage in vivo resulting in the formation of two classes of structurally distinct metabolites. During a metabolite profiling study conducted in human volunteers to assess the contribution of all pathways to the clearance of (14)C-azimilide, greater than 82% of radioactivity was recovered in urine (49%-58%) and feces (33%). Urine, feces, and plasma were profiled for metabolites. A cleaved metabolite, 4-chloro-2-phenyl furoic acid was present at high concentration in plasma (metabolite/parent AUC ratio approx. 4), while other plasma metabolites, azimilide N-oxide (metabolite/parent AUC ratio 0.001), and a cleaved hydantoin metabolite (metabolite/parent AUC ratio = 0.3) were present at lower concentrations than azimilide. In urine, the cleaved metabolites were the major metabolites, (> 35% of the dose) along with phenols (as conjugates, 7%-8%), azimilide N-oxide (4%-10%), a butanoic acid metabolite (2%-3%), and desmethyl azimilide (2%). A limited investigation of fecal metabolites indicated that azimilide (3%-5%), desmethyl azimilide (1%-3%), and the butanoic acid metabolite (< 1%) were present. Contributing pathways for metabolism of azimilide, identified through in vitro and in-vivo studies, were CYPs 1A1 (est. 28%), 3A4/5 (est. 20%), 2D6 (< 1%), FMO (est. 14%), and cleavage (35%). Enzyme(s) involved in the cleavage of azimilide were not identified.

Influence of coadministration on the pharmacokinetics of azimilide dihydrochloride and digoxin.

The influence of coadministration on digoxin and azimilide pharmacokinetics/pharmacodynamics was assessed in a randomized, 3-way crossover study in 18 healthy men. Serial blood and urine samples were obtained for azimilide and digoxin quantitation. Treatment effects on pharmacokinetics were assessed using analysis of variance. The relationship between azimilide blood concentrations and QT(c) prolongation was characterized by an E(max) model. Effects of coadministration on pharmacodynamics were assessed using a mechanistic-based inhibition model. Azimilide pharmacokinetics was unaffected by digoxin, except for a 36% increase in CL(r) (P = .0325), with no change in CL(o). Digoxin pharmacokinetics was unaffected by azimilide, except for a 21% increase in C(max) (P = .0176) and a 10% increase in AUC(tau) (P = .0121). Digoxin coadministration increased the apparent EC(50) with no effect on E(max), consistent with competitive inhibition (K(i) = 0.899 ng/mL). The pharmacokinetic and pharmacodynamic changes observed upon coadministration were small and are not expected to be clinically important.

LC-MS determination and bioavailability study of imidapril hydrochloride after the oral administration of imidapril tablets in human volunteers.

The purpose of the present study was to develop a standard protocol for imidapril hydrochloride bioequivalence testing. For this reason, a specific LC-MS method was developed and validated for the determination of imidapril in human plasma. A solid-phase extraction cartridge, Sep-pak C18, was used to extract imidapril and ramipril (an internal standard) from deproteinized plasma. The compounds were separated using a XTerra MS C18 column (3.5 microm, 2.1 x 150 mm) and acetonitrile-0.1% formic acid (67:33, v/v) adjusted to pH 2.4 by 2 mmol/L ammonium formic acid, as mobile phase at 0.3 mL/min. Imidapril was detected as m/z 406 at a retention time of ca. 2.3 min, and ramipril as m/z 417 at ca. 3.6 min. The described method showed acceptable specificity, linearity from 0.5 to 100 ng/mL, precision (expressed as a relative standard deviation of less than 15%), accuracy, and stability. The plasma concentration-versus-time curves of eight healthy male volunteers administered a single dose of imidapril (10 mg), gave an AUC12hr of imidapril of 121.48 +/- 35.81 ng mL(-1) h, and Cmax and Tmax values of 32.59 +/- 9.76 ng/mL and 1.75 +/- 0.27 h. The developed method should be useful for the determination of imidapril in plasma with sufficient sensitivity and specificity in bioequivalence study.

Influence of ketoconazole on azimilide pharmacokinetics in healthy subjects.

AIM: To assess the influence of ketoconazole on azimilide pharmacokinetics. METHODS: A two-period randomized crossover study was conducted in healthy male and female subjects (19-45 years). Placebo or 200 mg ketoconazole were administered orally every 24 h for 29 days. On day 8, a single oral dose of 125 mg azimilide dihydrochloride was coadministered following an overnight fast. Blood samples were obtained prior to and for 22 days following azimilide dihydrochloride administration. The plasma protein binding of azimilide was also assessed at 6 h after dosing. RESULTS: Following ketoconazole administration, a 16% increase in azimilide AUC (90% confidence interval (CI) 112%, 120%), a 12% increase in C(max) (95% CI 107%, 116%), a 13% increase in t(1/2,z) (95% CI 107%, 120%) and a 14% decrease in CL(o) (95% CI 82%, 90%) were observed. CONCLUSIONS: The changes in azimilide pharmacokinetics following ketoconazole treatment are not clinically important since the 90% CI for the AUC fell within the prespecified range of 80-125%. Thus, no clinically important drug interactions are expected when azimilide dihydrochloride is coadministered with CYP3A4 inhibitors.