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Using venom for predation often leads to the evolution of resistance in prey. Understanding individual variation in venom resistance is key to unlocking basic mechanisms by which antagonistic coevolution can sustain variation in traits under selection. For prey, the opposing challenges of predator avoidance and resource acquisition often lead to correlated levels of risk and reward, which in turn can favor suites of integrated morphological, physiological and behavioral traits. We investigate the relationship between risk-sensitive behaviors, physiological resistance to rattlesnake venom, and stress in a population of California ground squirrels. For the same individuals, we quantified foraging decisions in the presence of snake predators, fecal corticosterone metabolites (a measure of "stress"), and blood serum inhibition of venom enzymatic activity (a measure of venom resistance). Individual responses to snakes were repeatable for three measures of risk-sensitive behavior, indicating that some individuals were consistently risk-averse whereas others were risk tolerant. Venom resistance was lower in squirrels with higher glucocorticoid levels and poorer body condition. Whereas resistance failed to predict proximity to and interactions with snake predators, individuals with higher glucocorticoid levels and in lower body condition waited the longest to feed when near a snake. We compared alternative structural equation models to evaluate alternative hypotheses for the relationships among stress, venom resistance, and behavior. We found support for stress as a shared physiological correlate that independently lowers venom resistance and leads to squirrels that wait longer to feed in the presence of a snake, whereas we did not find evidence that resistance directly facilitates latency to forage. Our findings suggest that stress may help less-resistant squirrels avoid a deadly snakebite, but also reduces feeding opportunities. The combined lethal and non-lethal effects of stressors in predator-prey interactions simultaneously impact multiple key traits in this system, making environmental stress a potential contributor to geographic variation in trait expression of toxic predators and resistant prey.

Objective: The objective of this study was to use LC-MS/MS to compare the pharmacodynamic properties and bioequivalence of two 200-mg formulations of racecadotril: suspension formulation (test) and granule formulation (reference) in healthy Chinese subjects., Materials and Methods: A single-dose, randomized, two-period crossover study was conducted in fasted healthy Chinese subjects, who received a single oral dose of the test or reference formulation, followed by a 7-day washout period and administration of the alternate formulation., Results: The rapid and highly sensitive LC-MS/MS method exhibited a reasonable linearity range (2.324 - 952.000 ng/mL) and high sensitivity (LLOQ of 2.324 ng/mL). The within- and between-run precision, accuracy, and stability results were within the acceptable limits, and no matrix effect was observed. The 90% CI of the ratio of geometric means for AUC 0-t , AUC 0-∞ , and C max were 88.1 - 102.3%, 87.9 - 101.5% and 99.5 - 113%, respectively, which met the regulatory criteria for bioequivalence., Conclusion: The method is suitable for quantification of thiorphan in human plasma. In addition, the results indicated that the test and reference formulations were bioequivalent in terms of both rate and extent of absorption.

Objectives: The mechanism of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in diabetic foot ulcers (DFUs) is unclear. The purpose of this study was to describe changes in MMP-1, MMP-9, and TIMP-1 levels during DFU healing, and to search for any correlation in the changes in MMP levels with wound healing, in order to find possible predictors of healing., Methods: Patients with a DFU were recruited and placed into two groups, according to the degree of wound healing: 'good healers' and 'poor healers'. Levels of MMP-1, MMP-9, and TIMP-1 were analysed by ELISA (enzyme-linked immunosorbent assay)., Results: A total of 22 patients participated in the study. The MMP-1 level was significantly higher at weeks zero (W0) and 12 (W12) in 'good healers' than in 'poor healers' (p=0.045 and 0.008, respectively). In contrast, the MMP-9 level was significantly lower in 'good healers' than in 'poor healers' at W0, W4, and W12 (p=0.001, 0.001 and 0.028, respectively). Receiver operator curve (ROC) analysis of the MMP-9 level, MMP-1/TIMP-1 ratio, and MMP-9/TIMP-1 ratio at W0 provided cut-off levels of 0.38, 0.056, and 9.06, respectively, which were best predictive of a reduction in wound area at W4 ('good healers' versus 'poor healers'; thereby predicting wound healing condition at W12) with a sensitivity of 81.8%, 81.8%, and 90.9%, and a specificity of 64.6%, 55%, and 64.6%, respectively., Conclusion: A 'poor healing scoring system' is therefore proposed that could be determined on patient admission, which has the potential to be used clinically as a predictor of healing, thus allowing an appropriate treatment plan to be developed.

Aims: Liver injury can occur during antiviral therapies with direct-acting antivirals (DAAs), potentially necessitating discontinuation of the therapies, with consequent worsening of the sustained viral response (SVR) rates, in patients with hepatitis C virus (HCV). To clarify the mechanisms involved in serum transaminase level elevation, we performed a retrospective evaluation of the serum concentrations of daclatasvir and asunaprevir, both classified as DAAs, in patients receiving treatment with a combination of the two drugs., Methods: Subjects were 278 Japanese patients with genotype-1b HCV who received daclatasvir plus asunaprevir therapy for more than 4 weeks. Serum concentrations of both the DAAs were measured at 4 weeks after the initiation of therapy., Result: Liver injuries including serum AST and/or ALT level elevation to 150 U/L or over were found in 34 patients (12.2%). Multivariate logistic regression analysis identified serum asunaprevir concentrations as being significantly associated with developing liver injury, with an odds ratio of 1.046 (95% confidence interval 1.011-1.082, p<0.05). Serum asunaprevir concentrations showed correlation with the extent of liver fibrosis, estimated by peripheral platelets counts and serum albumin levels and baseline and FIB4 index and serum Mac-2 binding protein glycosylation isomer (M2BPGi) levels at 4 weeks of the therapy; the concentrations were significantly higher among patients showing 3.0 or more of M2BPGi levels than among those with the levels less than 3.0; on the other hand, no such correlation/difference was found in serum daclatasvir concentrations., Conclusion: High serum concentrations of serum asunaprevir, which were associated with the extent of liver fibrosis, appear to provoke the occurrence of liver injury in patients with genotype-1b HCV receiving combined daclatasvir plus asunaprevir therapy., Competing Interests: SM has received patent royalties from SRL Inc., has received speaking fees or hon-oraria from AbbVie GK, Ajinomoto Pharmaceuticals Co. Ltd., Bristol Myers Squibb Co., Gilead Sciences Inc., MSD K.K., Sumitomo Dainippon Pharma Co., Toray Medical Co. Ltd., has received research grants from A2 Healthcare Co., AbbVie GK, Bristol Myers Squibb Co., Chugai Pharma-ceutical Co. Ltd., EA Pharma Co. Ltd., Mitsubishi Tanabe Pharma Co., MSD K.K., Sumitomo Dainippon Pharma Co., Toray Medical Co. Ltd. YU has received research grants from AbbVie GK. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Serum canine α 1 -proteinase inhibitor (cα 1 -PI) concentrations were evaluated in dogs with pancreatitis (n=24), exocrine pancreatic insufficiency (EPI; n=29), chronic hepatitis (CH; n=11) or proteinuric chronic kidney disease (CKD-P; n=61) to determine whether systemic proteinase/proteinase-inhibitor balance is altered in these conditions. Dogs with CKD-P had significantly lower cα 1 -PI concentrations than dogs with pancreatitis, EPI or CH; 16% of dogs with CKD-P had serum cα 1 -PI concentrations below the reference interval. Serum and urine cα 1 -PI concentrations were inversely correlated in dogs with CKD-P, but not in dogs with CH. This suggests that renal loss of cα 1 -PI contributes to decreased serum concentrations in dogs with CKD-P, while hepatic cα 1 -PI synthesis with CH either is not compromised or is counterbalanced by extrahepatic production., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Experimental studies showed that certain angiotensin-converting enzyme inhibitors and angiotensin AT 1 receptor antagonists can decrease seizure severity in rodents. Additionally, some of these blockers of the renin-angiotensin system have been documented to enhance the anticonvulsant activity of antiepileptic drugs against maximal electroshock-induced seizures. The aim of the current study was to investigate the effect of aliskiren, a direct renin inhibitor and a novel antihypertensive drug, on the protective action of numerous antiepileptic drugs (carbamazepine, valproate, clonazepam, phenobarbital, oxcarbazepine, lamotrigine, topiramate and pregabalin) in the test of maximal electroshock in mice. The examined drugs were administered intraperitoneally. Aliskiren up to a dose of 75mg/kg did not affect the threshold for electroconvulsions, however, aliskiren (75mg/kg) enhanced the anticonvulsant action of clonazepam and valproate. Following aliskiren treatment, a higher brain concentration of valproate was noted, suggesting a pharmacokinetic interaction. In the rota-rod test, the concomitant treatment with aliskiren (50 or 75mg/kg) and clonazepam (22.6mg/kg) impaired motor coordination while clonazepam (22.6mg/kg) alone showed strong tendency towards this impairment. The combination of aliskiren (75mg/kg) with phenobarbital (25.5mg/kg) caused long-term memory deficits in the passive avoidance task. This study shows that there are no negative interactions between aliskiren and the examined antiepileptic drugs as concerns their anticonvulsant activity. Aliskiren even potentiated the anticonvulsant action of clonazepam and valproate against maximal electroshock. The impact of aliskiren alone on seizure activity or on the anticonvulsant and adverse activity of antiepileptic drugs needs further evaluation in other animal models of seizures., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Activation and regulation of the cascade systems of the blood (the complement system, the coagulation/contact activation/kallikrein system, and the fibrinolytic system) occurs via activation of zymogen molecules to specific active proteolytic enzymes. Despite the fact that the generated proteases are all present together in the blood, under physiological conditions, the activity of the generated proteases is controlled by endogenous protease inhibitors. Consequently, there is remarkable little crosstalk between the different systems in the fluid phase. This concept review article aims at identifying and describing conditions where the strict system-related control is circumvented. These include clinical settings where massive amounts of proteolytic enzymes are released from tissues, e.g., during pancreatitis or post-traumatic tissue damage, resulting in consumption of the natural substrates of the specific proteases and the available protease inhibitor. Another example of cascade system dysregulation is disseminated intravascular coagulation, with canonical activation of all cascade systems of the blood, also leading to specific substrate and protease inhibitor elimination. The present review explains basic concepts in protease biochemistry of importance to understand clinical conditions with extensive protease activation.

Background: Proteases produced by many microorganisms, including oomycetes, are crucial for their growth and development. They may also play a critical role in disease manifestation. Epizootic ulcerative syndrome is one of the most destructive fish diseases known. It is caused by the oomycete Aphanomyces invadans and leads to mass mortalities of cultured and wild fish in many countries. The areas of concern are Australia, China, Japan, South and Southeast Asian countries and the USA. Extracellular proteases produced by this oomycete are believed to trigger EUS pathogenesis in fish. To address this activity, we collected the extracellular products (ECP) of A. invadans and identified the secreted proteins using SDS-PAGE and mass spectrometery. A. invadans was cultivated in liquid Glucose-Peptone-Yeats media. The culture media was ultra-filtered through 10 kDa filters and analysed using SDS-PAGE. Three prominent protein bands from the SDS gel were excised and identified by mass spectrometery. Furthermore, we assessed their proteolytic effect on casein and immunoglobulin M (IgM) of rainbow trout (Oncorhynchus mykiss) and giant gourami (Osphronemus goramy). Antiprotease activity of the fish serum was also investigated., Results: BLASTp analysis revealed that the prominent secreted proteins were proteases, mainly of the serine and cysteine types. Proteins containing fascin-like domain and bromodomain were also identified. We could demonstrate that the secreted proteases showed proteolytic activity against the casein and the IgM of both fish species. The anti-protease activity experiment showed that the percent inhibition of the common carp serum was 94.2% while that of rainbow trout and giant gourami serum was 7.7 and 12.9%, respectively., Conclusions: The identified proteases, especially serine proteases, could be the potential virulence factors in A. invadans and, hence, are candidates for further functional and host-pathogen interaction studies. The role of identified structural proteins in A. invadans also needs to be investigated further.

Alarm antiproteases, i.e. secretory leukocyte protease inhibitor ad elafin, are key mediators in innate immune response and integrate innate and adaptive immunity systems. The aim of the study was to assess clinical significance of serum levels of alarm antiproteases, elafin and secretory leukocyte protease inhibitor (SLPI) in patients with systemic sclerosis (SSc). Twenty-eight patients with SSc, 25 patients with rheumatoid arthritis (RA) and 22 healthy controls were recruited. Serum elafin and SLPI levels were examined using enzyme-linked immunosorbent assay (ELISA). The patients with SSc had significantly increased serum levels of SLPI in comparison with the RA patients and the healthy controls (p<0.01), and the RA patients presented significantly higher serum levels of elafin in comparison with the controls (p=0.003). In the SSc subgroup serum SLPI level negatively correlated with diffusing capacity of the lung for carbon monoxide (DLCO) (r=-0.41, p=0.03) and total lung capacity (r=-0.42, p=0.03). Both alarm antiproteases, elafin and SLPI could be potentially implicated in the pathogenesis of SSc and SLPI may be considered a candidate for serum biomarker of lung involvement in SSc., (Copyright © 2017. Published by Elsevier Inc.)

Resistance of snakes and some other animals to snake envenomation has been attributed to soluble factors present in their tissues. Here we report the isolation of a novel metalloprotease inhibitor from Bothrops alternatus snake serum (named BaltMPI) with high purity, using a four-step chromatographic method. BaltMPI has molecular weights of 60.5 and 42.4kDa, as determined by SDS-PAGE and mass spectrometry, respectively, and pI=5.27. The first 60 amino acids from the N-terminal region of BaltMPI, determined by Edman's degradation, showed high homology (97%) with the snake venom metalloprotease inhibitor (SVMPI) BJ46a and other SVMPIs (78-82%). The chromatographic fractions and purified BaltMPI exhibited anti-hemorrhagic activity against Batroxase and BjussuMP-I. BaltMPI was stable over wide ranges of pH (1, 5, 8, and 9) and temperature (-80, -20, 4, 60, and 100°C), and suppressed the fibrinogenolytic, fibrinolytic, and azocaseinolytic activities of Batroxase. BaltMPI specifically inhibited the activity of metalloproteases, without affecting the activity of serine proteases. Together, our results suggest that BaltMPI and other SVMPIs are promising molecules for the treatment of snake envenomation, in particular that caused by Bothrops sp., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Asunaprevir is an inhibitor of the hepatitis C virus (HCV) NS3/4A protease, demonstrating efficacy in clinical studies in patients infected with HCV genotype 1 or 4, with either peginterferon/ribavirin or combinations of direct-acting antivirals. Because of preferential distribution of asunaprevir to the liver via organic anion-transporting polypeptide (OATP)-mediated transport, asunaprevir demonstrates high apparent oral clearance and very low plasma concentrations. Asunaprevir plasma concentrations are markedly increased by single-dose rifampin (an OATP inhibitor) and in subjects with moderate to severe hepatic impairment. In addition, modestly higher plasma concentrations of asunaprevir have been noted in subjects infected with HCV relative to healthy subjects and in Asian subjects relative to whites. At the marketed dose, infrequent hepatic transaminase abnormalities were poorly predicted by plasma concentrations. For a compound with these characteristics, hepatic concentrations may have provided an improved understanding of the in vivo pharmacokinetic and pharmacodynamic data to support decision making during development., (© 2017, The American College of Clinical Pharmacology.)

Aim: Fotagliptin is a novel dipeptidyl peptidase IV inhibitor under clinical development for the treatment of Type II diabetes mellitus. The objective of this study was to develop and validate a specific and sensitive ultra-performance liquid chromatography (UPLC)-MS/MS method for simultaneous determination of fotagliptin and its two major metabolites in human plasma and urine. Methodology & results: After being pretreated using an automatized procedure, the plasma and urine samples were separated and detected using a UPLC-ESI-MS/MS method, which was validated following the international guidelines., Conclusion: A selective and sensitive UPLC-MS/MS method was first developed and validated for quantifying fotagliptin and its metabolite in human plasma and urine. The method was successfully applied to support the clinical study of fotagliptin in Chinese healthy subjects.

The pathological consequences of decreased protein Z (PZ) and/or Z-dependent protease inhibitor (ZPI) levels remain as yet unclear, despite a growing body of evidence which supports their involvement in an increased thrombotic risk. The purpose of the present study was 2-fold: to evaluate plasma concentrations of protein Z and ZPI in patients with essential thrombocythemia (ET) and to determine their significance in thrombotic complications. The median (range) plasma concentrations of PZ in our patients with ET were lower, but not significantly, than in healthy individuals: PZ (1.42 µg/mL, 0.36-3.14 µg/mL vs 1.6 µg/mL, 0.75-2.56 µg/mL, P = .08). On the other hand, the median (range) plasma concentrations of ZPI in the said patients with ET were meaningfully lower than in the reference group: ZPI (3.22 µg/mL, 0.85-6.97 µg/mL vs 4.41 µg/mL, 1.63-7.83 µg/mL, P = .0004). More importantly, the study revealed a statistically significant lower concentration of PZ and ZPI in patients with the presence of the JAK2V617F mutation relative to patients without the mutation, for PZ: 1.38 µg/mL, 0.36-2.6 µg/mL versus 1.63 µg/mL, 0.88-3.14 µg/mL, P = .03, and ZPI 2.89 µg/mL, 0.85-5.91 µg/mL versus 3.61 µg/mL, 1.53-6.97 µg/mL, P = .002. Additionally, significant differences between the concentrations of PZ and ZPI were found in patients with venous thrombotic episodes compared to healthy individuals, for PZ: 1.23 µg/mL, 0.82-1.99 µg/mL versus 1.6 µg/mL, 0.75-2.56 µg/mL, P = .043, and ZPI: 2.42 µg/mL, 0.85-4.21 µg/mL versus 4.41 µg/mL, 1.63-7.83 µg/mL, P < .0001. To recapitulate, our results suggest that the deficiency of PZ may increase tendency to thrombosis in patients with ET., (© The Author(s) 2015.)

Introduction: Quantitation of ADAMTS13 activity, functional inhibitors, and autoantibodies is crucial in diagnosis and management of thrombotic thrombocytopenic purpura. We compared and optimized commercial assay kits and validated a testing panel., Methods: Citrated plasma specimens from healthy volunteers and residual samples submitted for clinical testing were used in the study. Commercially available ADAMTS13 activity assays including ACTIFLUOR(™) ADAMTS13 (Sekisui Diagnostics, Stamford, CT, USA), LIFECODES ATS-13 (Gen-Probe Inc., San Diego, CA, USA), and TECHNOZYM(®) ADAMTS-13 (Technoclone, Vienna, Austria) were evaluated. Functional inhibitor assays were performed using internally developed mixing protocols. Two autoantibody assays were also evaluated: IMUBIND(®) (Sekisui Diagnostics) and TECHNOZYM(®) ADAMTS-13 INH ELISA kits (Technoclone)., Results: A laboratory-developed assay using ACTIFLUOR(™) reagents showed best agreement with the reference method, and full validation showed a reportable range of 5% (LLOQ) to 114% with a reference interval of ≥68%. Both intra- and interassay coefficients of variation were <10%. Inhibitor assays performed with the kits showed 95% overall agreement with the reference method. A modification of the TECHNOZYM(®) autoantibody assay showed 85% overall agreement with the reference method with imprecision approximately 20%., Conclusion: ADAMTS13 activity and inhibitor tests using ACTIFLUOR(™) reagents and modified TECHNOZYM(®) autoantibody ELISA showed superior performance compared to the other kits for clinical use in this study., (© 2016 John Wiley & Sons Ltd.)

Paritaprevir (also known as ABT-450), a potent NS3-4A serine protease inhibitor [identified by AbbVie (North Chicago, IL) and Enanta Pharmaceuticals (Watertown, MA)] of the hepatitis C virus (HCV), has been developed in combination with ombitasvir and dasabuvir in a three-direct-acting antiviral agent (DAA) oral regimen for the treatment of patients infected with HCV genotype 1. This article describes the mass balance, metabolism, and disposition of paritaprevir in humans. After the administration of a single 200-mg oral dose of [(14)C]paritaprevir coadministered with 100 mg of ritonavir to four male healthy volunteers, the mean total percentage of the administered radioactive dose recovered was 96.5%, with recovery in individual subjects ranging from 96.0% to 96.9%. Radioactivity derived from [(14)C]paritaprevir was primarily eliminated in feces (87.8% of the dose). Radioactivity recovered in urine accounted for 8.8% of the dose. The biotransformation of paritaprevir in humans involves: 1) P450-mediated oxidation on the olefinic linker, the phenanthridine group, the methylpyrazinyl group, or combinations thereof; and 2) amide hydrolysis at the acyl cyclopropane-sulfonamide moiety and the pyrazine-2-carboxamide moiety. Paritaprevir was the major component in plasma [90.1% of total radioactivity in plasma, AUC from time 0 to 12 hours (AUC0-12hours) pool]. Five minor metabolites were identified in plasma, including the metabolites M2, M29, M3, M13, and M6; none of the metabolites accounted for greater than 10% of the total radioactivity. Paritaprevir was primarily eliminated through the biliary-fecal route followed by microflora-mediated sulfonamide hydrolysis to M29 as a major component in feces (approximately 60% of dose). In summary, the biotransformation and clearance pathways of paritaprevir were characterized, and the structures of metabolites in circulation and excreta were elucidated., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

γ-Secretase mediates amyloid production in Alzheimer's disease (AD) and oncogenic activity of Notch. γ-Secretase inhibitors (GSIs) are thus of interest for AD and oncology. A peripheral biomarker of Notch activity would aid determination of the therapeutic window and dosing regimen for GSIs, given toxicities associated with chronic Notch inhibition. This study examined the effects of GSI MK-0752 on blood and hair follicle transcriptomes in healthy volunteers. The effects of a structurally diverse GSI on rhesus blood and hair follicles were also compared. Significant dose-related effects of MK-0752 on transcription were observed in hair follicles, but not blood. The GSI biomarker identified in follicles exhibited 100% accuracy in a clinical test cohort, and was regulated in rhesus by a structurally diverse GSI. This study identified a translatable, accessible pharmacodynamic biomarker of GSI target engagement and provides proof of concept of hair follicle RNA as a translatable biomarker source., (© 2016 American Society for Clinical Pharmacology and Therapeutics.)

Background: Elafin inhibits serine proteases, such as human neutrophil elastase and proteinase 3, to prevent excessive damage during inflammation. However, the relationship between elafin and asthma is still unclear. Microarray technology was used to evaluate smoking- and asthma-related biomarkers in a discovery-driven manner. We identified candidate genes, e.g., proteinase inhibitor 3 (PI3), related to asthma and smoking from gene expression microarray data sets and evaluated their potential as biomarkers for asthma., Methods: We used human genome microarray data sets from smoking- and asthma-related gene expression data sets and performed real-time quantitative polymerase chain reaction to measure and validate differences in gene expression. We also recruited adult patients with asthma and age- and sex-matched control patients who were administered a structured questionnaire and evaluated for lung function and plasma elafin levels, which are encoded by the PI3 gene., Results: Six significantly altered candidate genes, PI3, protein kinase C iota, phosphoserine phosphatase, IQ motif-containing GTPase activating protein 1, interleukin 13 receptor α 1, and signal transducing adaptor molecule SH3 domain and ITAM motif 2, were identified from comparisons across the four asthma- and four smoking-related data sets included in this study. An in vitro study of human airway epithelial cells (A549) and a human monocytic cell line (THP-1) demonstrated that PI3 messenger RNA levels were significantly altered by nicotine exposure. Elafin concentration was significantly higher in control patients than in patients with asthma (p < 0.001). The plasma elafin concentration in the highest quartile (≥12.69 ng/mL) was inversely associated with asthma (adjusted odds ratio 0.122 [95% confidence interval, 0.053-0.278]) compared with the lowest quartile (<5.82 ng/mL) after adjusting for age, sex, smoking status, waist-to-hip ratio, percentage predicted forced expiratory volume in 1 second, cockroaches in the home, incense burning, and family history., Conclusion: Our study revealed that high elafin levels identified in smoking- and asthma-related microarray data sets and an epidemiologic study significantly reduced the risk of asthma. Further studies of elafin as a potential therapy for asthma are warranted.

The immunostimulatory effect of phospholipopeptide biosurfactant from Staphylococcus hominis (GenBank Accession No: KJ564272) was assessed with Oreochromis mossambicus. The non-specific (serum lysozyme activity, serum antiprotease activity, serum peroxidase activity and serum bactericidal activity), specific (bacterial agglutination assay) immune responses and disease resistance activity against Aeromonas hydrophila were examined. Fish were intraperitonially injected with water soluble secondary metabolite (biosurfactant) of S. hominis at a dose of 2 mg, 20 mg and 200 mg kg(-1) body weight. Commercial surfactant surfactin (sigma) at 20 mg kg(-1) was used as standard and saline as negative control. All the doses of water soluble biosurfactant tested, significantly enhanced the specific, nonspecific immunity and disease resistance from the day of post administration of phospholipopeptide biosurfactant till the tail of the experimental period. These results clearly indicated that the secondary metabolite isolated from S. hominis stimulates the immunity of finfish thereby could enhance aquaculture production., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

LCZ696 is a first-in-class angiotensin receptor neprilysin inhibitor in development for treatments of hypertension and heart failure indications. In 3 separate studies, pharmacokinetic drug-drug interactions (DDIs) potential was assessed when LCZ696 was coadministered with hydrochlorothiazide (HCTZ), amlodipine, or carvedilol. The studies used a open-label, single-sequence, 3-period, crossover design in healthy subjects. Blood samples were collected to determine the pharmacokinetic parameters of LCZ696 analytes (AHU377, LBQ657, and valsartan), HCTZ, amlodipine, or carvedilol (R[+]- and S[-]-carvedilol) for statistical analysis. When coadministered LCZ696 with HCTZ, the 90% CIs of the geometric mean ratios of AUCtau,ss of HCTZ and that of LBQ657 were within a 0.80-1.25 interval, whereas HCTZ Cmax,ss decreased by 26%, LBQ657 Cmax,ss increased by 19%, and the AUCtau,ss and Cmax,ss of valsartan increased by 14% and 16%, respectively. Pharmacokinetics of amlodipine, R(+)- and S(-)-carvedilol, or LBQ657 were not altered after coadministration of LCZ696 with amlodipine or carvedilol. Coadministration of LCZ696 400 mg once daily (qd) with HCTZ 25 mg qd, amlodipine 10 mg qd, or carvedilol 25 mg twice a day (bid) had no clinically relevant pharmacokinetic drug-drug interactions. LCZ696, HCTZ, amlodipine, and carvedilol were safe and well tolerated when given alone or concomitantly in the investigated studies., (© 2015, The American College of Clinical Pharmacology.)

Background: Plasma exchange (PE) is the first-line treatment for primary acquired thrombotic thrombocytopenic purpura (aTTP) with severe deficiency of ADAMTS13 activity (ADAMTS13:AC). Some patients are poor responders to PE, raising concern over multiple pathogenetic pathways., Study Design and Methods: Based on 52 aTTP patients in our national cohort study, we monitored plasma levels of ADAMTS13, clinical and laboratory findings, and outcomes. In a representative poor responder to PE, we examined an ADAMTS13 inhibitor (ADAMTS13:INH) complex in plasma milieu, by means of a large-pore isoelectric focusing (IEF) analysis., Results: Of 52 aTTP patients, 20 were good responders and 32 were poor responders. In the latter group, plasma ADAMTS13:AC levels never increased to more than 10% of normal during 14 days after PE initiation. Mean (±SD) plasma ADAMTS13:INH titers (Bethesda unit/mL) were 5.7 (±4.5) before PE, but decreased to 1.4 (±0.8) on the fourth PE day and then remarkably increased to 14.8 (±10.0) on the 10th PE day, termed "inhibitor boosting," and then slowly decreased to undetectable level over 1 month. On admission, none of the routinely available clinical and laboratory markers differentiated these two groups. However, elevated pre-PE levels of ADAMTS13:INH were correlated with a poor response. We visualized an ADAMTS13:INH (immunoglobulin G) complex in a patient plasma by an IEF analysis and found proteolytic fragment of ADAMTS13 antigen by a two-dimensional IEF and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis., Conclusion: Findings from this cohort of aTTP patients demonstrated that inhibitor boosting often occurs in aTTP patients in Japan. Poor responders could be predicted by elevated pre-PE ADAMTS13:INH levels on admission, but not by routinely collected clinical or laboratory data., (© 2015 AABB.)

Background: Simeprevir is a N3/4 protease inhibitor approved for the treatment of hepatitis C virus (HCV) infection. HCV prevalence is higher in patients with chronic kidney disease compared with the general population; safe and efficacious therapies in renal impairment are needed., Objectives: To evaluate simeprevir renal excretion in healthy subjects and to compare the simeprevir steady-state pharmacokinetics between subjects with severe renal impairment and healthy subjects., Methods: In the mass balance study, healthy adults received a single 200-mg dose of (14)C-simeprevir; radioactivity in the urine and feces was quantified until concentrations were <2% of the administered dose and seven or more stools were produced. In the pharmacokinetic study, non-HCV-infected adults with severe renal impairment (estimated glomerular filtration rate ≤29 mL/min/1.73 m(2)) and matched healthy subjects (estimated glomerular filtration rate ≥80 mL/min/1.73 m(2)) received 150 mg simeprevir for 7 days. Pharmacokinetic analysis was performed post-dose on Day 7., Results: (14)C-simeprevir recovery from the urine was low (0.009-0.138% of total dose). The minimum plasma concentration, maximum plasma concentration, and area under the plasma concentration-time curve at 24 h were 71, 34, and 62% higher, respectively, in subjects with severe renal impairment compared with healthy subjects. The mean fraction of simeprevir unbound to protein was <0.0001 (all subjects). Most adverse events were grade I or II; one subject with renal impairment who was receiving fenofibrate presented with grade 3 rhabdomyolysis., Conclusions: Simeprevir plasma concentrations were mildly elevated in subjects with severe renal impairment. The results suggest that simeprevir may be administered without dose adjustment in patients with renal impairment.

Background: Bilirubin is a yellow breakdown product of heme catabolism. Increased serum levels of unconjugated bilirubin are conditions commonly seen in premature neonates and adults with acute hemolysis including thrombotic microangiopathy. Previous studies have shown that unconjugated bilirubin lowers plasma ADAMTS13 activity, but the mechanism is not fully understood., Objectives: The study is to determine whether unconjugated bilirubin directly inhibits the cleavage of von Willebrand factor (VWF) and its analogs by ADAMTS13., Methods: Fluorogenic, surface-enhanced laser desorption/ionization time-of-flight mass spectrometric assay, and Western blotting analyses were used to address this question., Results: Unconjugated bilirubin inhibits the cleavage of F485-rVWF73-H, D633-rVWF73-H, and GST-rVWF71-11K by ADAMTS13 in a concentration-dependent manner with a half-maximal inhibitory concentration of ~13, ~70, and ~17 μmol L(-1) , respectively. Unconjugated bilirubin also dose-dependently inhibits the cleavage of multimeric VWF by ADAMTS13 under denaturing conditions. The inhibitory activity of bilirubin on the cleavage of D633-rVWF73-H and multimeric VWF, but not F485-rVWF73-H, was eliminated after incubation with bilirubin oxidase that converts bilirubin to biliverdin. Furthermore, plasma ADAMTS13 activity in patients with hyperbilirubinemia increased after treatment with bilirubin oxidase., Conclusions: Unconjugated bilirubin directly inhibits ADAMTS13's ability to cleave both peptidyl and native VWF substrates in addition to its interference with certain fluorogenic assays. Our findings may help proper interpretation of ADAMTS13 results under pathological conditions. Whether elevated serum unconjugated bilirubin has prothrombotic effect in vivo remains to be determined in our future study., (© 2015 International Society on Thrombosis and Haemostasis.)

Background and Objective: Chronic hepatitis C virus (HCV) infection is a major cause of liver transplantation. Drug-drug interactions (DDIs) with cyclosporine and tacrolimus hindered the use of first-generation protease inhibitors in transplant recipients. The current study investigated DDIs between daclatasvir-a pan-genotypic HCV NS5A inhibitor with clinical efficacy in multiple regimens (including all-oral)-and cyclosporine or tacrolimus in healthy subjects., Methods: Healthy fasted subjects (aged 18-49 years; body mass index 18-32 kg/m(2)) received single oral doses of cyclosporine 400 mg on days 1 and 9, and daclatasvir 60 mg once daily on days 4-11 (group 1, n = 14), or a single oral dose of tacrolimus 5 mg on days 1 and 13, and daclatasvir 60 mg once daily on days 8-19 (group 2, n = 14). Blood samples for pharmacokinetic analysis [by liquid chromatography with tandem mass spectrometry (LC-MS/MS)] were collected on days 1 and 9 for cyclosporine (72 h), on days 1 and 13 for tacrolimus (168 h) and on days 8 and 9 (group 1) or on days 12 and 13 (group 2) for daclatasvir (24 h). Plasma concentrations were determined by validated LC-MS/MS methods., Results: Daclatasvir did not affect the pharmacokinetic parameters of cyclosporine or tacrolimus, and tacrolimus did not affect the pharmacokinetic parameters of daclatasvir. Co-administration of cyclosporine resulted in a 40 % increase in the area under the concentration-time curve of daclatasvir but did not affect its maximum observed concentration., Conclusion: On the basis of these observations in healthy subjects, no clinically relevant DDIs between daclatasvir and cyclosporine or tacrolimus are anticipated in liver transplant recipients infected with HCV; dose adjustments during co-administration are unlikely to be required.

Anemia is a well-known RBV-related event in HCV therapy which is exacerbated by the addition of telaprevir and boceprevir. This retrospective study evaluated and compared ribavirin exposure and parameters able to influence hemoglobin decrease in a large population of patients treated with dual or triple therapy. Patients on triple therapy had higher ribavirin concentrations at week 12 of treatment (3460 ng/mL vs. 1843 ng/mL; P < .0001). An association was also observed between week 12 eGFR and ribavirin concentration only for patients on triple therapy (P = .002). The proportion of patients with a  >20 mL/min/1.73 m(2) decrease in eGFR at week 12 was higher among patients on triple therapy: 32%, 14%, and 5% for boceprevir, telaprevir, and dual therapy, respectively (P = .025 and .026). No correlation was observed between boceprevir and telaprevir concentrations and hemoglobin or eGFR decrease. Exacerbation of anemia in patients on triple therapy is related to higher ribavirin concentrations. We provide an explanation for this increase in plasma RBV concentration. Triple therapy with PEG-IFN, RBV, and telaprevir or boceprevir will remain the only HCV treatment option for many patients. Our data show that the RBV dose can be decreased while maintaining adequate plasma concentrations and reducing anemia., (© 2015, The American College of Clinical Pharmacology.)

Purpose: Intravenous opioid use is a common route of hepatitis C virus (HCV) infection; consequently, the prevalence of HCV is high among patients on methadone or buprenorphine/naloxone. The authors evaluated the pharmacokinetic interaction of boceprevir with methadone or buprenorphine/naloxone in patients on stable maintenance therapy., Methods: This was a two-center, open-label, fixed-sequence study in 21 adult volunteers on stable maintenance therapy. Oral methadone (20-150 mg once daily) or sublingual buprenorphine/naloxone (8/2-24/6 mg once daily) was administered alone or in combination with boceprevir (800 mg every 8 h) on days 2-7. Pharmacokinetic sampling occurred before and up to 24 h after the dose on days 1 and 7., Results: Coadministration of boceprevir reduced the area under the concentration-time curve during a dosing interval τ (AUC τ ) and maximum observed plasma (or serum) concentration (C max) of R-methadone (geometric mean ratios (GMRs) [90 % confidence intervals (CIs)], 0.85 [0.74, 0.96] and 0.90 [0.71, 1.13]) and S-methadone (GMRs [90 % CIs], 0.78 [0.66, 0.93] and 0.83 [0.64, 1.09]). Boceprevir increased the AUC τ and C max of buprenorphine (GMRs [90 % CIs], 1.19 [0.91, 1.58] and 1.18 [0.93, 1.50]) and naloxone (GMRs [90 % CIs], 1.33 [0.90, 1.93] and 1.09 [0.79, 1.51]). Boceprevir exposure upon methadone or buprenorphine/naloxone coadministration was not clinically different from historical controls and there was no evidence of opioid withdrawal or excess., Conclusions: There was no clinically meaningful impact of boceprevir on methadone or buprenorphine pharmacokinetics, suggesting that methadone/buprenorphine dose adjustments are not required upon coadministration with boceprevir. Individual patients may differ in their clinical experience and clinicians should maintain vigilance when coadministering these medications.

Objective: Pigment epithelium-derived factor (PEDF) is a potent inhibitor of angiogenesis and an important target molecule for preventing the progression of atherosclerosis. However, the relationship between PEDF and coronary atherosclerosis has not been fully examined. The aim of the present study is to evaluate the effects of statins on serum PEDF levels and the association between PEDF and coronary atherosclerosis., Patients and Methods: Coronary atherosclerosis in nonculprit lesions in the vessel of patients undergoing a percutaneous coronary intervention was evaluated using virtual histology intravascular ultrasound in 99 patients during percutaneous coronary intervention and after 8 months of statin therapy., Results: Serum PEDF levels at baseline and at the 8-month follow-up did not differ. A significant decrease in the fibro-fatty component (-0.24 mm³/mm, P=0.0003) and increases in the necrotic core (0.13 mm³/mm, P=0.02) and dense calcium components (0.11 mm³/mm, P<0.0001) were observed during the 8-month statin therapy. On univariate regression analyses, serum PEDF levels (r=0.291, P=0.004) and unstable angina pectoris (r=0.203, P=0.04) showed significant positive correlations with the percentage change in necrotic core volume. Multivariate regression analysis showed that serum PEDF level was a significant independent predictor associated with necrotic core progression during statin therapy (β=0.218, P=0.04)., Conclusion: Statin therapy had no effects on serum PEDF levels. Serum PEDF was a useful biomarker for predicting necrotic core progression during statin therapy, and its levels could be elevated as a counter-regulatory response mechanism to protect against necrotic core progression.

With the advent of the Highly Active Antiretroviral Therapy, the morbidity and the mortality associated to HIV have been considerably reduced. However, 35-40 million people bear the infection worldwide. One of the main causes of therapeutic failure is the frequent administration of several antiretrovirals that results in low patient compliance and treatment cessation. In this work, we have developed an innovative Nanoparticle-in-Microparticle Delivery System (NiMDS) comprised of pure drug nanocrystals of the potent protease inhibitor indinavir free base (used as poorly water-soluble model protease inhibitor) produced by nanoprecipitation that were encapsulated within mucoadhesive polymeric microparticles. Pure drug nanoparticles and microparticles were thoroughly characterized by diverse complementary techniques. NiMDSs displayed an encapsulation efficiency of approximately 100% and a drug loading capacity of up to 43% w/w. In addition, mucoadhesiveness assays ex vivo conducted with bovine gut showed that film-coated microparticles were retained for more than 6 h. Finally, pharmacokinetics studies in mongrel dogs showed a dramatic 47- and 95-fold increase of the drug oral bioavailability and half-life, respectively, with respect to the free unprocessed drug. These results support the outstanding performance of this platform to reduce the dose and the frequency of administration of protease inhibitors, a crucial step to overcome the current patient-incompliant therapy., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Introduction: Antiviral drugs for the treatment of hepatitis C virus (HCV) infections have a large number of interactions. The aim of this study was to describe the interactions of telaprevir, boceprevir and sofosbuvir with immunosuppressive drugs in liver transplant recipients., Methods: A retrospective observational study was performed in liver transplant patients with HCV infection who started treatment with telaprevir, boceprevir or sofosbuvir. Dose, regimens and plasma levels of tacrolimus, cyclosporine and sirolimus before and after antiviral treatment initiation were collected. Average variations in dose, dosing interval and immunosuppressive plasma levels after the start of treatment were calculated., Results: Thirty-five patients were included. In patients treated with telaprevir (n = 18), the cyclosporine dose was reduced by an average of 59.1% (SD = 14.6%), yielding an average reduction of 14.6% (18.8%) in plasma levels. The dose of tacrolimus was reduced by 34.3% (31.7%), increasing the dosing interval by a mean of 73.4 (38.2) hours. After this variation, tacrolimus levels were increased by an average of 59.7% (89.6%). In patients treated with boceprevir (n = 4), tacrolimus started with a reduction of 18.1% (9.8%) of the initial dose and an average increase in the dosing interval of 12.0 (16.9) hours, showing a mean reduction in plasma levels of 37.7% (21.8%). Sofosbuvir therapy (n = 13) showed no significant variations in immunosuppressive drug levels., Conclusions: The interaction of telaprevir and boceprevir with immunosuppressive drugs requires a substantial dose reduction at the beginning of treatment and close monitoring of plasma levels., (Copyright © 2014 Elsevier España, S.L.U. and AEEH y AEG. All rights reserved.)

Alpha-2-macroglobulin (α-2-M) is a broad spectrum protease inhibitor which is abundant in the plasma of vertebrates and several invertebrates. The α-2-M was purified from cobia (Rachycentron canadum) plasma by a four-step procedure: poly ethylene glycol fractionation, affinity chromatography, hydrophobic interaction chromatography and ion exchange chromatography on Fast Protein liquid chromatography system in the present study. It migrated as one protein band with a molecular mass of about 360 kDa in the native state, whereas in SDS-PAGE it was about 180 kDa under non-reducing condition. This result revealed that the native protein was a dimer. In addition, it was cleaved into two different fragments of molecular mass about 93 and 87 kDa when reduced by dithiothreitol (DTT). The anti-protease activity of the purified α-2-M was apparently decreased as temperature elevated above 50 °C. The α-2-M exhibited highest protease inhibitory activity at pH 9. The results indicate that the α-2-M is a heat-labile and alkaline protease inhibitor. The purified α-2-M exhibited more than 50% protease inhibitory activity against extracellular products (ECP) of Vibrio alginolytius isolated from diseased cobia. It seems that the protease activities in ECP may be affected by the plasma α-2-M. The protease inhibitory activities of cobia plasma or purified α-2-M were decreased when incubated with 10 mM methylamine for 30 min. The α-2-M cDNA consisted of 4611 bp with an open reading frame of 4374 bp had been cloned from cobia liver. This sequence contained thioester domain (GCGEQ) and thirteen predicted N-linked glycosylation sites. In addition, the amino acid sequence of thioester domain and genes of adjacent regions of cobia α-2-M were further compared with sequences of known fish species in GenBank. The unweighted pair group method using arithmetic average (UPGMA) was employed to construct the phylogenetic trees of α-2-M among different fish species (freshwater fish, sea water fish and primitive fish), and all these fish species were then clustered into three groups. The cobia α-2-M was closer to that of sea water fish than that of freshwater fish compared basing on its similarity of amino acid sequence and phylogenetic analysis of the partial gene., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

This Phase I, randomized, open-label study evaluated the gastric pH-altering effects of omeprazole, a proton pump inhibitor, and the CYP3A enzyme/P-glycoprotein (Pgp)-inhibitory effects of ritonavir, an HIV protease inhibitor, on the pharmacokinetics and safety of the hepatitis C virus (HCV) non-structural protein 5A (NS5A) inhibitor GSK2336805 in healthy male and female subjects. Co-administration of GSK2336805 60 mg with omeprazole decreased GSK2336805 plasma AUC(0-∞) by 10% and Cmax by 18%; no marked effect was observed on t½ . Co-administration of GSK2336805 30 mg with ritonavir increased GSK2336805 plasma AUC(0-∞) by 52%, Cmax by 43%, and t½ by 40%; CL/F was decreased by 34%. All adverse events were minor in intensity. The gastric acid-suppressive effect of omeprazole had minimal impact on the extent and rate of GSK2336805 absorption in vivo; therefore, GSK2336805 may be co-administered with omeprazole without concern about lower GSK2336805 exposures and compromised antiviral efficacy. The modest increases in AUC and Cmax following co-administration of GSK2336805 plus ritonavir suggest that GSK2336805 when given concomitantly with a single CYP3A/Pgp inhibiting drug will not likely require dose adjustment. Final dose recommendation will be based on GSK2336805 efficacy and safety profiles from Phase III trials in HCV-infected patients., (© 2014, The American College of Clinical Pharmacology.)

Modulating deposition of Aβ-containing plaques in the brain may be beneficial in treating Alzheimer's disease. β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors have been shown to reduce Aβ in plasma and CSF in healthy volunteers. In this study safety, pharmacokinetics and pharmacodynamics that is reduction of the plasma biomarkers Aβ40 and Aβ42 , of the BACE1 inhibitor AZD3839 were evaluated. Single oral ascending doses (1-300 mg) of AZD3839 were administered to 54 young healthy volunteers in a randomized, double-blind, placebo-controlled study. The data was analyzed using non-linear mixed effects modeling. AZD3839 reduced Aβ40 and Aβ42 in plasma with estimated potencies (EC50 ) of 46 and 59 nM, respectively, and a maximum effect of approximately 55%. This was in excellent agreement with the concentration-response relationships obtained in mouse and guinea pig. AZD3839 exposure displayed non-linear kinetics, described by a three-compartment model with a saturated binding compartment and an increase in bioavailability with dose. AZD3839 was safe, although, a dose-dependent QTcF prolongation was observed (mean 20 milliseconds at 300 mg). In conclusion, AZD3839 reduced plasma Aβ40 and Aβ42 , demonstrating clinical peripheral proof of mechanism. Pre-clinical models were predictive for the effect of AZD3839 on the human plasma biomarker in a strictly quantitative manner., (© 2014, The American College of Clinical Pharmacology.)

Simeprevir (also known as TMC435 or TMC435350) is a novel hepatitis C protease inhibitor. A validated high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the sensitive and selective quantification of simeprevir in human EDTA plasma is described. During assay development, special attention was given to light instability of the drug in plasma and blood. The method consisted of precipitation of plasma proteins with acetonitrile after which the supernatant was analyzed using electrospray LC-MS/MS. The linearity was confirmed in the concentration range from 2.00 to 2000ng/mL, with 50-fold dilution extending to 100,000ng/mL. The precision of this assay, expressed as CV, ranged between 4.4% and 8.5% over the entire concentration range with assay accuracy between -0.3% and 8.5%. The method was applied successfully in many clinical studies to document the pharmacokinetics of simeprevir in plasma from healthy volunteers and patients., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The discovery of asunaprevir (BMS-650032, 24) is described. This tripeptidic acylsulfonamide inhibitor of the NS3/4A enzyme is currently in phase III clinical trials for the treatment of hepatitis C virus infection. The discovery of 24 was enabled by employing an isolated rabbit heart model to screen for the cardiovascular (CV) liabilities (changes to HR and SNRT) that were responsible for the discontinuation of an earlier lead from this chemical series, BMS-605339 (1), from clinical trials. The structure-activity relationships (SARs) developed with respect to CV effects established that small structural changes to the P2* subsite of the molecule had a significant impact on the CV profile of a given compound. The antiviral activity, preclincial PK profile, and toxicology studies in rat and dog supported clinical development of BMS-650032 (24).

The present study was designed to test the immunostimulatory efficacy of poly-β hydroxybutyrate-hydroxyvalerate (PHB-HV) extracted from Bacillus thuringiensis B.t.A102 on the immune system of Oreochromis mossambicus. Fish were fed with 0%, 1%, 3% or 5% PHB-HV supplemented feed and were bled at regular intervals of 5 days. The specific immune response was measured in terms of antibody response to sheep red blood cells, the nonspecific immune mechanisms were analysed in terms of serum lysozyme activity, total peroxidases activity and antiprotease activity. The overall functional immunity was tested by experimental challenge with live virulent Aeromonas hydrophila. The results revealed that all the doses of PHB-HV supplementation in feed were effective in stimulating both specific and nonspecific immune mechanisms. The bacterial challenge experiment showed that highest dose of 5% PHB-HV supplementation was more effective than 1% and 3% doses. The study concludes that PHB-HV can be used as a potential immunostimulant in finfish aquaculture., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Many HIV-treated patients travel to malaria-infected zones, but very few data are available on potential interactions between antiretroviral and antimalarial drugs., Method: We performed a pharmacokinetic study on the interaction of doxycycline (100 mg/d) on 2 protease inhibitors (PIs), atazanavir and lopinavir, and 2 non-nucleoside reverse transcriptase inhibitors (NNRTIs), efavirenz and nevirapine, given at usual daily doses in HIV-infected migrants native from sub-Saharan Africa included in the VIHVO ANRS-study before travelling to a sub-Saharan country. Antiretroviral trough plasma concentrations were measured at enrollment visit during the month preceding the travel before doxycycline introduction and on the week following the patients' return to France when they had been taking doxycycline for at least 15 days. Impact of doxycycline on antiretroviral concentrations was tested either with antiretroviral drugs separately or within the therapeutic classes (PI or NNRTI) in patients with an HIV RNA level <50 copies/mL at both visits and with good declared adherence. The Two One-Sided Test that was adapted to the Wilcoxon test was used to evidence the lack of interaction. Sixty-five patients receiving regimens containing atazanavir (n = 1), ritonavir-boosted atazanavir (n = 14), ritonavir-boosted lopinavir (n = 23), efavirenz (n = 17), nevirapine (n = 10) were included., Results: Lack of pharmacokinetic interaction was statistically significant when tested by therapeutic class (PI, P = .02; NNRTI, P = .005) and was not demonstrated for each antiretroviral when tested separately., Conclusion: This study is the first to assess the interaction of doxycycline on PI and NNRTI. This lack of pharmacokinetic interaction supports the choice of doxycycline as the antimalarial drug in patients treated with PI or NNRTI.

The present study evaluated the effect of dietary andrographolide (EC 50%) on growth, non-specific immune parameters and disease resistance against Aeromonas hydrophila infection in Indian major carp, Labeo rohita fingerlings. Fishes were fed with formulated diet containing andrographolide as T0 (0.00%), T1 (0.05%), T2 (0.10%), T3 (0.20%), T4 (0.40%) and T5 (0.80%) for 42 days. Fishes were challenged with A. hydrophila 42 days post feeding and relative percentage survival (RPS) was recorded over 14 days post challenge. Blood and serum samples were collected for nonspecific immune parameters on 14, 28 and 42 days of feeding and growth performance was evaluated at the end of experiment. The results revealed that fishes fed with andrographolide showed significant (p < 0.05) increase in NBT levels, myeloperoxidase activity, phagocytic activity, serum lysozyme activity, and serum antiprotease activity when compared to the control group. The weight gain, specific growth rate, feed conversion ratio and protein efficiency ratio of fishes fed with andrographolide were found to be significantly (p < 0.05) differed compared with control. Dietary andrographolide at the level of 0.10% showed significantly (P < 0.05) higher RPS (74.06%) against A. hydrophila infection than control. The results revealed that andrographolide supplemented diet has a stimulatory effect on non-specific immune parameters along with improved growth performance and increased disease resistance against A. hydrophila infection in L. rohita fingerlings., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Epithelial ovarian cancer (EOC) is the fifth leading cause of cancer-related death in females and leading gynecologic cause of cancer-related death. Despite the identification of a number of serum biomarkers, methods to identify early-stage disease and predict prognosis remain scarce. We have evaluated two biologically connected serum biomarkers, serum leukocyte protease inhibitor (SLPI) and progranulin (PGRN)., Methods: Two-hundred frozen plasma samples were acquired from the Mayo Clinic Biospecimen Repository for Ovarian Cancer Research. Samples were obtained from 50 patients with benign conditions, 50 with American Joint Committee on Cancer (AJCC) stage I and II EOC, and 100 with AJCC stage III and IV EOC. Samples were obtained before surgical resection of a mass and were analyzed for absolute levels of SLPI and PGRN using ELISA assays. Receiver-operator characteristic curves were generated for SLPI and PGRN. Median follow-up was 48 months., Results: Absolute levels of SLPI were significantly elevated in patients with EOC compared with benign disease and predicted the presence of EOC (AUC of 0.812; P = 0.04); SLPI remained elevated in the subset of patients with normal CA-125. PGRN levels were not significantly increased in patients with early-stage or late-stage EOC as a whole, but an increase in PGRN levels was associated with decreased overall survival in advanced EOC., Conclusions: SLPI levels are elevated in EOC, and SLPI shows promise as a diagnostic biomarker for patients with both elevated and normal CA-125 levels. An increase in PGRN is associated with decreased overall survival., Impact: SLPI is elevated in EOC and warrants investigation in a screening study in women at risk for EOC.

A sensitive high-performance reverse phase liquid chromatography-positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of telaprevir and its inactive R-diastereomer (VRT-127394) in human plasma. The analytes and the internal standard (telaprevir-d11) were extracted from plasma by liquid-liquid extraction using tert-Butyl methyl ether (TBME). Chromatographic separation was achieved on a reversed-phase Accucore C18 column with a gradient programme consisting of water:ammonia (25%), 100:0.01 (v/v) (mobile phase A) and ACN:MeOH:ammonia (25%), 15:85:0.01 (v/v/v) (mobile phase B). The MS acquisition was performed with selective reaction monitoring mode using the respective [M+H](+) ions, m/z 680.59→322.42 for telaprevir and VRT-127394, and 691.15→110.13 for telaprevir-d11. The assay exhibited a linear dynamic range of 5-5000ng/mL for telaprevir and VRT-127394. Acceptable precision (%RSD<6.5%) and accuracy (94-108%) were obtained for concentrations over the range of the standard curve. A procedure was established to stabilise the plasma to prevent ex vivo interconversion of the isomers., (Copyright © 2013 Elsevier B.V. All rights reserved.)

HCV infection affects over 170 milions people in the world. Up to now standard-of-care therapy consisted in ribavirin plus interferon-α 2a or 2b. Recently, two new Direct Acting Antivirals (DAA), inhibitors of NS3 HCV protease, have been developed and approved for the routine use on HCV genotype 1 infected patients: boceprevir and telaprevir. Protease inhibitors show complex pharmacokinetics (strong metabolism of both drugs by CYP3A and drug interactions), they require a TID dosage and, furthermore, they are present in plasma patients in two different isomeric forms. In this work, we developed and validated an UPLC-tandem mass spectrometry assay method capable of monitoring the telaprevir and boceprevir concentrations in plasma, discriminating each isomer of both drugs. Blank plasma used for Standards and QCs preparation, was obtained from blood of healthy donors. The calibration curves for each isomer of telaprevir and boceprevir were linear in a range from 46.87 ng/mL to 6000 ng/mL and from 23.43 to 3000 ng/mL, respectively (mean r(2)>0.998 for all analytes). QCs at three different concentration were prepared: High, Medium and Low. Each Standard, QC and patient sample was treated with a protein precipitation protocol with a solution containing acidified acetonitrile and Internal Standard (6,7-Dimethyl-2,3-di(2-pyridyl)quinoxaline). An aliquot of supernatant was diluted and then directly analyzed by reverse-phase UPLC-MS/MS. Accuracy (mean 98.47%), intra-day (mean <5.21%) and inter-day (mean <7.57%) precision for telaprevir and boceprevir quality controls fitted all FDA guidelines. All compounds resulted stable in our method conditions and the extraction procedure showed a recovery near to 100%. Finally, we tested this method by monitoring plasma concentrations in 9 HCV+ patients, for each triple combined therapy, with good results. The observed median ratio between S and R isomers for boceprevir and telaprevir were 1.22 (IQR 1.10-1.33) and 1.52 (IQR 1.21-1.67), respectively. The use of this simple assay method could be an important tool for management of HCV-1 DAAs treated patients., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Fecal canine alpha1-proteinase inhibitor (cα1-PI) concentration has been reported to be increased in dogs with protein-losing enteropathy due to the loss of cα1-PI into the gastrointestinal tract. A chronic loss of cα1-PI may theoretically deplete serum cα1-PI, potentially altering the proteinase-to-proteinase inhibitor balance. Protein-losing enteropathy has been reported to occur frequently in certain dog breeds such as Yorkshire Terriers and to be associated with hypocobalaminemia. The objective was to compare serum cα1-PI concentrations in Yorkshire Terriers with and without cobalamin (COB) deficiency. Serum samples from 52 COB-deficient and 69 normocobalaminemic Yorkshire Terriers, which had been submitted to the Gastrointestinal Laboratory (2008-2011; College Station, TX), were included retrospectively. Serum cα1-PI concentrations were measured using an in-house radioimmunoassay and compared between Yorkshire Terriers with and without COB deficiency using a Mann-Whitney U test. A Fisher exact test was used to evaluate whether a decreased serum cα1-PI concentration is associated with COB deficiency in Yorkshire Terriers. Serum cα1-PI concentrations were significantly lower in COB-deficient Yorkshire Terriers (median: 1,016 mg/l, range: 315-3,945 mg/l) than in normocobalaminemic Yorkshire Terriers (median: 1,665 mg/l, range: 900-2,970 mg/l; P < 0.0001). One-fourth (n = 13) of the COB-deficient Yorkshire Terriers had a serum cα1-PI concentration below the lower limit of the reference interval (<732 mg/l), and COB deficiency was associated with decreased serum cα1-PI concentrations (P < 0.0001). In the current study, serum cα1-PI concentrations are significantly lower in COB-deficient Yorkshire Terriers when compared to normocobalaminemic Yorkshire Terriers. Further studies are needed to determine the functional and potential prognostic implications of serum cα1-PI concentrations in dogs with gastrointestinal disease.

New oral anticoagulants are directed towards a single target, essentially factor Xa (FXa) or factor IIa. They do not require routine coagulation monitoring. However, in special clinical settings (emergency surgery, bleeding, thrombosis, control of the patient's compliance, suspected overdose, potential drug interference, and so on), measurement of plasma levels is needed. Several available anti-FXa assays are used for monitoring anticoagulant activity of heparins and fondaparinux. They must be modified and standardized for the measurement of direct FXa inhibitors (rivaroxaban, apixaban, edoxaban, betrixaban and others). The use of calibrators (lyophilized plasma with a known concentration of drug) allows an expression of the results in ng per ml of plasma. Two categories of assays - endogenous and exogenous assays are available. Endogenous assays are useful in pharmaceutical research, while exogenous assays are used in clinical laboratories. The preferred anti-FXa assay is a specific method in contrast to prothrombin time and activated partial thromboplastin time, but it is not available everywhere at any time. A specific measurement of direct FXa inhibitors is feasible with the use of a new test developed by the authors' group. The physicians must be aware of the possibility to measure the plasma concentration of FXa inhibitors in patients at high risk of bleeding and in several other special clinical situations.

Asunaprevir (BMS-650032) is a potent hepatitis C virus (HCV) non-structural protein protease inhibitor currently in Phase III clinical trials for the treatment of HCV infection. A rugged and accurate LC-MS/MS method was developed and validated for the quantitation of asunaprevir in rat, dog, monkey, rabbit and mouse plasma. A systematic method screening and optimization strategy was applied to achieve optimized mass spectrometry, chromatography, and sample extraction conditions. The validated method utilized stable-isotope labeled D9-asunaprevir as the internal standard. The samples were extracted by liquid-liquid extraction using 10% ethyl acetate in hexane. Chromatographic separation was achieved with gradient elution on a Waters Atlantis dC18 analytical column. Analyte and its internal standard were detected by positive ion electrospray tandem mass spectrometry. The standard curve, which ranged from 5.00 to 2000ng/mL for asunaprevir, was fitted to a 1/x(2) weighted linear regression model. The intra-assay precision was within ±3.6% CV, inter-assay precision was within ±4.0% CV, and the assay accuracy was within ±8.1% of the nominal values in all the species. The method was successfully applied to support multiple pre-clinical toxicokinetic studies in different species., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Viperidae snakes containing various venomous proteins also have several anti-toxic proteins in their sera. However, the physiological function of serum protein has been elucidated incompletely. Small serum protein (SSP)-1 is a major component of the SSPs isolated from the serum of a Japanese viper, the habu snake (Trimeresurus flavoviridis). It exists in the blood as a binary complex with habu serum factor (HSF), a snake venom metalloproteinase inhibitor. Affinity chromatography of the venom on an SSP-1-immobilized column identified HV1, an apoptosis-inducing metalloproteinase, as the target protein of SSP-1. Biacore measurements revealed that SSP-1 was bound to HV1 with a dissociation constant of 8.2 × 10⁻⁸ M. However, SSP-1 did not inhibit the peptidase activity of HV1. Although HSF alone showed no inhibitory activity or binding affinity to HV1, the SSP-1-HSF binary complex bound to HV1 formed a ternary complex that non-competitively inhibited the peptidase activity of HV1 with a inhibition constant of 5.1 ± 1.3 × 10⁻⁹ M. The SSP-1-HSF complex also effectively suppressed the apoptosis of vascular endothelial cells and caspase 3 activation induced by HV1. Thus, SSP-1 is a unique protein that non-covalently attaches to HV1 and changes its susceptibility to HSF.

Fasciola hepatica infection results in increased production of reactive oxygen species (ROS) and changes the activity/level of antioxidants in the host organism, which leads to oxidative stress formation and oxidative modifications of lipids and proteins. Taking this into account, the aim of this study was to assess the antioxidant potential and the activity of proteases and their inhibitors in the serum of rats infected with F. hepatica. Wistar rats were infected per os with 30 metacercariae of F. hepatica. The total antioxidant status (TAS) and the activity of cathepsin G and elastase and their inhibitors (alpha1-antitrypsin and alpha2-macroglobulin) were determined at 4, 7, and 10 weeks post infection (wpi). It was confirmed that F. hepatica infection leads to a decrease in the antioxidant capacity of serum, which was manifested as a reduction in total antioxidant status by about 24, 39, and 27%, respectively, at 4, 7, and 10 wpi. At the same time, the activity of proteases increased significantly: cathepsin G by about 25, 37, and 30%, and elastase by about 18, 16, and 9% during the course of F. hepatica infection, compared with the control group. However, the activity of alpha1-antitrypsin was significantly reduced, by 36, 55, and 25%, while alpha2-macroglobulin activity was reduced by about 14, 17, and 8% during the same period of fasciolosis. These results indicate that the shift in protease/antiprotease balance towards protease action observed during the course of fasciolosis may result in a decrease in host antioxidant capacity.

Unlabelled: The hepatitis C virus protease inhibitor boceprevir is a strong inhibitor of cytochrome P450 3A4 and 3A5 (CYP3A4/5). Cyclosporine and tacrolimus are calcineurin inhibitor immunosuppressants used to prevent organ rejection after liver transplantation; both are substrates of CYP3A4. This two-part pharmacokinetic interaction study evaluated boceprevir with cyclosporine (part 1) and tacrolimus (part 2). In part 1, 10 subjects received single-dose cyclosporine (100 mg) on day 1, single-dose boceprevir (800 mg) on day 3, and concomitant cyclosporine/boceprevir on day 4. After washout, subjects received boceprevir (800 mg three times a day) for 7 days plus single-dose cyclosporine (100 mg) on day 6. In part 2A, 12 subjects received single-dose tacrolimus (0.5 mg). After washout, they received boceprevir (800 mg three times a day) for 11 days plus single-dose tacrolimus (0.5 mg) on day 6. In part 2B, 10 subjects received single-dose boceprevir (800 mg) and 24 hours later received boceprevir (800 mg) plus tacrolimus (0.5 mg). Coadministration of boceprevir with cyclosporine/tacrolimus was well tolerated. Concomitant boceprevir increased the area under the concentration-time curve from time 0 to infinity after single dosing (AUC(inf) ) and maximum observed plasma (or blood) concentration (C(max) ) of cyclosporine with geometric mean ratios (GMRs) (90% confidence interval [CI]) of 2.7 (2.4-3.1) and 2.0 (1.7-2.4), respectively. Concomitant boceprevir increased the AUC(inf) and C(max) of tacrolimus with GMRs (90% CI) of 17 (14-21) and 9.9 (8.0-12), respectively. Neither cyclosporine nor tacrolimus coadministration had a meaningful effect on boceprevir pharmacokinetics., Conclusion: Dose adjustments of cyclosporine should be anticipated when administered with boceprevir, guided by close monitoring of cyclosporine blood concentrations and frequent assessments of renal function and cyclosporine-related side effects. Administration of boceprevir plus tacrolimus requires significant dose reduction and prolongation of the dosing interval for tacrolimus, with close monitoring of tacrolimus blood concentrations and frequent assessments of renal function and tacrolimus-related side effects., (Copyright © 2012 American Association for the Study of Liver Diseases.)

Background: Telaprevir is a hepatitis C virus protease inhibitor that is both a substrate and an inhibitor of CYP3A., Study Design: The effect of steady-state telaprevir (administered 750 mg every 8 hours) on the steady-state pharmacokinetics of ethinyl estradiol (EE) and norethindrone (NE) was evaluated in 24 healthy women receiving oral contraceptives (OC) containing 0.5 mg NE and 0.035 mg EE for at least 3 months at the time of screening. This was a phase 1, open-label, single-center, nonrandomized study that included a cycle 1 (OC only for 21 days, followed by no OC for 7 days), cycle 2 (OC plus telaprevir for 21 days, followed by telaprevir alone for 7 days), and a follow-up period., Results: When administration with or without telaprevir was compared, the least-squares mean ratios (90% confidence limits) for EE were 0.74 (0.68; 0.80) for C(max), 0.67 (0.63; 0.71) for C(min), and 0.72 (0.69; 0.75) for AUC; neither NE nor telaprevir exposure was affected., Conclusions: The efficacy of the OC may be compromised by the 26% to 33% reduction in EE exposure. Therefore, alternative methods of nonhormonal contraception should be used when hormonal contraceptives are coadministered with telaprevir and for up to 2 weeks following cessation of telaprevir.

In this open-label study, 24 healthy volunteers received a single intravenous (IV) dose of 0.5 mg of midazolam on day 1 and a single oral dose each of 2 mg of midazolam and 0.5 mg of digoxin on day 3. Telaprevir 750 mg every 8 hours was administered from day 8 through day 23, along with a single IV dose of 0.5 mg of midazolam on day 17 and single oral doses of 2 mg of midazolam and 0.5 mg of digoxin on day 19. Midazolam, 1'-hydroxymidazolam, digoxin, and telaprevir concentrations in plasma and digoxin concentrations in urine were measured and pharmacokinetic parameters calculated. On comparing administration with versus without telaprevir, the geometric least squares mean ratios (with 90% confidence limits) for IV midazolam were 1.02 (0.80, 1.31) for maximum observed concentrations (C(max)) and 3.40 (3.04, 3.79) for area under the curve from 0 to 24 hours (AUC(0-24h)); for oral midazolam 2.86 (2.52, 3.25) for C(max) and 8.96 (7.75, 10.35) for AUC(0-24h); and for oral digoxin 1.50 (1.36, 1.65) for C(max) and 1.85 (1.70, 2.00) for area under the curve from 0 to infinity (AUC(0-∞)). Coadministration of telaprevir with oral midazolam resulted in approximately 3-fold decrease in the mean AUC(0-∞) of 1'-hydroxymidazolam. The renal clearance of digoxin was similar with or without telaprevir. Results show that telaprevir is an inhibitor of CYP3A and P-glycoprotein.

Background and Objective: Boceprevir is a novel inhibitor of the hepatitis C virus NS3 protease and was recently approved for the treatment of patients with chronic hepatitis C infection. The objective of this study was to evaluate the impact of impaired hepatic or renal function on boceprevir pharmacokinetics and safety/tolerability., Methods: We conducted two open-label, single-dose, parallel-group studies comparing the safety and pharmacokinetics of boceprevir in patients with varying degrees of hepatic impairment compared with healthy controls in one study and patients with end-stage renal disease (ESRD) on haemodialysis with healthy controls in the other. Patients with hepatic impairment (mild [n = 6], moderate [n = 6], severe [n = 6] and healthy controls [n = 6]) received a single dose of boceprevir (400 mg) on day 1, and whole blood was collected at selected timepoints up to 72 hours postdose to measure plasma drug concentrations. Patients with ESRD and healthy subjects received a single dose of boceprevir 800 mg orally on days 1 and 4, with samples for pharmacokinetic analyses collected at selected timepoints up to 48 hours postdose on both days. In addition, 4 hours after dosing on day 4, patients with ESRD underwent haemodialysis with arterial and venous blood samples collected up to 8 hours postdose., Results: In the hepatic impairment study, there was a trend toward increased mean maximum (peak) plasma concentration (C(max)) and area under the plasma concentration-time curve (AUC) of boceprevir with increasing severity of liver impairment. Point estimates for the geometric mean ratio for C(max) ranged from 100% in patients with mild hepatic impairment to 161% in patients with severe hepatic impairment, with the geometric mean ratio for AUC ranging from 91% in patients with mild hepatic impairment to 149% for patients with severe hepatic impairment, relative to healthy subjects. The mean elimination half-life (t(1/2;)) and median time to C(max) (t(max)) values of boceprevir were similar in healthy subjects and patients with hepatic impairment. In the renal impairment study, mean boceprevir C(max) and AUC values were comparable in patients with ESRD and in healthy subjects, with point estimates for the geometric mean ratio of 81% and 90%, respectively, compared with healthy subjects. Mean t(1/2;), median t(max) and mean apparent oral total clearance (CL/F) values were similar in healthy subjects and patients with ESRD. Boceprevir exposure was also similar in patients with ESRD on day 1 (no dialysis) and day 4 (dialysis beginning 4 hours postdose), with point estimates for the geometric mean ratio of C(max) and AUC to the last measurable sampling time (AUC(last)) on day 1 compared with day 4 of 88% and 98%, respectively. Treatment-emergent adverse events were reported in one patient with severe hepatic impairment (mild vomiting) and two patients with ESRD (moderate ventricular extrasystoles, flatulence and catheter thrombosis)., Conclusion: In the present studies, the pharmacokinetic properties of boceprevir were not altered to a clinically meaningful extent in patients with impaired liver or renal function. These data indicate that boceprevir dose adjustment is not warranted in patients with impaired hepatic function or ESRD, including those receiving dialysis. Boceprevir is not removed by haemodialysis.