Simultaneous determination of naltrexone and bupropion in their co-formulated tablet utilizing green chromatographic approach with application to human urine

A rapid, simple and accurate micellar HPLC-method was adopted and validated for concurrent quantification of naltrexone hydrochloride (NTX) and bupropion hydrochloride (BUP). The proposed method was conducted on RP-18 LiChrosorb® column (150?mm?×?4.6?mm i.d. 5-µm particle size) at 25?°C, as a stationary phase and a mixture of 0.175?M sodium dodecyl sulphate (SDS), 0.3% triethanolamine (TEA) and 12% n-propanol in 0.02?M ortho (o)-phosphoric acid of pH 3.5 as a developing system. It was pumped at a flow rate of 1.2?mL/min, with ultraviolet detection at 210?nm. The linearity ranges were 0.5–15.0?µg/mL and 1.2–18.0?µg/mL, with detection limits of 0.10 and 0.31?µg/mL and quantification limits of 0.30 and 0.93?µg/mL for NTX and BUP, respectively. The studied drugs were successfully quantified by applying the proposed method in their co-formulated tablet. The cited method was also applied for in-vitro quantification of BUP in spiked human urine without prior extraction.     

Interpretation of urinary concentrations of pseudoephedrine and its metabolite cathine in relation to doping control

Until the end of 2003 a urinary concentration of pseudoephedrine exceeding 25 µg/mL was regarded as a doping violation by the World Anti‐Doping Agency. Since its removal from the prohibited list in 2004 the number of urine samples in which pseudoephedrine was detected in our laboratory increased substantially. Analysis of 116 in‐competition samples containing pseudoephedrine in 2007 and 2008, revealed that 66% of these samples had a concentration of pseudoephedrine above 25 µg/mL. This corresponded to 1.4% of all tested in competition samples in that period. In the period 2001–2003 only 0.18% of all analysed in competition samples contained more than 25 µg/mL. Statistical comparison of the two periods showed that after the removal of pseudoephedrine from the list its use increased significantly. Of the individual sports compared between the two periods, only cycling is shown to yield a significant increase. Analysis of excretion urine samples after administration of a therapeutic daily dose (240 mg pseudoephedrine) in one administration showed that the threshold of 25 µg/mL can be exceeded. The same samples were also analysed for cathine, which has currently a threshold of 5 µg/mL on the prohibited list. The maximum urinary concentration of cathine also exceeded the threshold for some volunteers. Comparison of the measured cathine and pseudoephedrine concentrations only indicated a poor correlation between them. Hence, cathine is not a good indicator to control pseudopehedrine intake. To control the (ab)use of ephedrines in sports it is recommended that WADA reintroduce a threshold for pseudoephedrine. Copyright © 2009 John Wiley & Sons, Ltd.     

RP-HPLC法测定法罗培南血药浓度和尿药浓度

目的：建立测定法罗培南血药浓度和尿药浓度的RP-HPLC法。方法：采用Diamond C18色谱柱（250mm×4．6mm,5μm）,血样与尿样测定流动相分别为0．02mol／L磷酸二氢钾缓冲液（pH=2．95）：乙腈=75：25和81：19,流速均为1．0mL／min,检测波长均为318nm。结果：血浆样品的线性范围为0．03125～2．5μg／mL（r=0．9999）、2．5～120μg／mL（r=0．9997）,最低检测浓度为31．25ng／mL,绝对回收率为68．9％～77．8％;尿液样品的线性范围为0．5～20μg／mL（r=0．9999）、20～1600μg／mL（r=0．9997）,最低检测浓度为0．5μg／mL,绝对回收率为96．5％～106．0％。日内及日间精密度均〈11．0％。结论：本方法简单、快速,灵敏度和准确度较高,能满足法罗培南钠注射液临床药动学研究的需要。     

A novel chemiluminescence quenching method for determination of sulfonamides in pharmaceutical and biological fluid based on luminol-Ag(III) complex reaction in alkaline solution

A novel chemiluminescence (CL) quenching method for the determination of sulfonamides is proposed. The CL reaction between Ag(III) complex [Ag(HIO₆)₂]^5? and luminol in alkaline solution was investigated. The quenching effect of sulfonamides on CL emission of [Ag(HIO₆)₂]^5?–luminol system was found. Quenching degree of CL emission was proportional to sulfonamide concentration. The effects of the reaction conditions on CL emission and quenching were examined. Under optimal conditions, the detection limits (s/n = 3) were 7.2, 17 and 8.3 ng/mL for sulfadiazine, sulfameter, and sulfadimethoxine, respectively. The recoveries of the three drugs were in the range of 91.3–110% with RSDs of 1.9–2.7% for urine samples, and 106–112% with RSDs of 1.6–2.8% for serum samples. The proposed method was used for the determination of sulfadiazine at clinically relevant concentrations in real urine and serum samples with satisfactory results. Copyright © 2011 John Wiley & Sons, Ltd.     

The influence of exercise and dehydration on the urine concentrations of salbutamol after inhaled administration of 1600 µg salbutamol as a single dose in relation to doping analysis

The present study investigated the influence of exercise and dehydration on the urine concentrations of salbutamol after inhalation of that maximal permitted (1600 µg) on the 2015 World Anti‐Doping Agency (WADA) prohibited list. Thirteen healthy males participated in the study. Urine concentrations of salbutamol were measured during three conditions: exercise (EX), exercise+dehydration (EXD), and rest (R). Exercise consisted of 75 min cycling at 60% of VO_2max and a 20‐km time‐trial. Fluid intake was 2300, 270, and 1100 mL during EX, EXD, and R, respectively. Urine samples of salbutamol were collected 0–24 h after drug administration. Adjustment of urine concentrations of salbutamol to a specific gravity (USG) of 1.020 g/mL was compared with no adjustment. The 2015 WADA decision limit (1200 ng/mL) for salbutamol was exceeded in 23, 31, and 10% of the urine samples during EX, EXD, and R, respectively, when unadjusted for USG. When adjusted for USG, the corresponding percentages fell to 21, 15, and 8%. During EXD, mean urine concentrations of salbutamol exceeded (1325±599 ng/mL) the decision limit 4 h after administration when unadjusted for USG. Serum salbutamol C_max was lower (P<0.01) for R(3.0±0.7 ng/mL) than EX(3.8±0.8 ng/mL) and EXD(3.6±0.8 ng/mL). AUC was lower for R (14.1±2.8 ng/mL·?h) than EX (16.9±2.9 ng/mL·?h)(P<0.01) and EXD (16.1±3.2 ng/mL·?h)(P<0.05). In conclusion, exercise and dehydration affect urine concentrations of salbutamol and increase the risk of Adverse Analytical Findings in samples collected after inhalation of that maximal permitted (1600 µg) for salbutamol. This should be taken into account when evaluating doping cases of salbutamol. Copyright © 2015 John Wiley & Sons, Ltd.     

Enantioselective disposition of (R)‐salmeterol and (S)‐salmeterol in urine following inhaled dosing and application to doping control

Salmeterol (USAN, INN, BAN) is a long‐acting beta2‐adrenoceptor agonist (LABA) widely used in the treatment of airways disease. Although salmeterol is permitted via inhalation by athletes and supratherapeutic dosing may enhance performance, no urine threshold has been established by the World Anti‐Doping Agency (WADA). Salmeterol is a chiral compound consisting of (R)‐ and (S)‐enantiomers, normally administered as racemic (rac‐ ) mixture via inhalation. Levels of rac‐ salmeterol in urine are often below detectable levels and there is surprisingly little information regarding the enantioselectivity of salmeterol pharmacokinetics. In this study, subjects inhaled either 50 (n = 6) or 200 µg (n = 4; generally regarded as maximum therapeutic dose) of salmeterol and urine was then collected for 24 h and analyzed by enantioselective ultra performance liquid chromatography‐tandem mass spectrometry (UPLC‐MS/MS). Maximum rac‐ salmeterol urine concentrations were obtained at 2 h for both doses with medians of 0.084 ng/mL after the 50 µg dose and 2.1 ng/mL after the 200 µg dose, with an individual maximum of 5.7 ng/mL. Levels were detectable at 24 h for both doses. Salmeterol displayed enantioselective pharmacokinetics, with a mean ± SD log (S):(R) = 0.055 ± 0.025 (P < 0.0001) equivalent to (S):(R) of 1.13. In conclusion, rac‐ salmeterol by inhalation exhibits modest enantioselectivity in urine following single dose administration and can be detected following a single 50 µg dose for up to 24 h after inhalation. The present findings are of relevance if a urine threshold limit is to be introduced for salmeterol on the list of prohibited substances. The application of an enantiomer ratio analysis may offer improved discriminatory detection capability for doping control analysis applications. Copyright © 2016 John Wiley & Sons, Ltd.     

Antioxidant,antibacterial, and anti-inflammatory activities of standardized brazilin-rich Caesalpinia sappan extract

Abstract

Context: Brazilin is a major active principle of Caesalpinia sappan L. (Leguminosae or Fabaceae). For industry aspects, brazilin-rich extract (BRE) has been prepared and standardized to contain 39% w/w brazilin. BRE may have more advantages than brazilin in term of a lower-cost production process.

Objectives: To investigate the antioxidant, antibacterial, and anti-inflammatory activities of BRE.

Material and methods: BRE was prepared by a simple one-step purification of the crude ethanol extract of C. sappan heartwood (CSE) using a Diaion® HP-20 column. The antioxidant activities were determined using three methods, including DPPH radical scavenging, reducing power, and β-carotene bleaching assays, at concentration ranges of 1–10, 10–100, and 10–100?µg/mL, respectively. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of BRE (15.6–1000?µg/mL) against Gram-positive and Gram-negative bacteria were determined by the broth microdilution method. Anti-inflammatory activity of BRE (0.1–5?µg/mL) was evaluated as anti-denaturation activity using bovine serum albumin as a substrate.

Results and discussion: On the basis of β-carotene bleaching assay, BRE showed antioxidant activity with an EC₅₀ value of 60.5?µg/mL, which was almost equal to that of pure brazilin (52.1?µg/mL). Gram-positive bacteria were more sensitive to all tested samples than Gram-negative bacteria. BRE possessed higher antibacterial activities than CSE, but lower than brazilin. MIC/MBC values of 62.5–125/125 and 250–500/250–500?µg/mL were obtained for BRE against Gram-positive and Gram-negative bacteria, respectively. A low concentration (0.1?µg/mL) of brazilin, BRE, and CSE showed anti-inflammatory activity by inhibiting protein denaturation up to 46.8, 54.1, and 61.9%, respectively.     

Inhibition of herpes simplex virus 1 (HSV-1) and poliovirus (PV-1) by bacteriocins from lactococcus lactis subsp. lactis and enterococcus durans strains isolated from goat milk

Bacteriocins have unusual inhibitory activity, including antiviral properties, and this can be exploited to give alternative applications. Semi–purified bacteriocins of six lactic acid bacteria (LAB) strains isolated from goat milk (two Lactococcus lactis: GLc03 and GLc05, and four Enterococcus durans: GEn09, GEn12, GEn14 and GEn17) were tested for cytotoxicity in Vero cells (50% Cytotoxicity Concentration: CC₅₀), and for their antiviral activities against herpes simplex virus 1 (HVS-1) and poliovirus (PV-1). Semi-purified bacteriocins presented low cytotoxicity, with CC₅₀ varying from 256.2?µg/mL (GLc05) to 1084.5?µg/mL (GEn14). CC₁₀ was determined for all isolates (GLc03: 36.9?µg/mL; GLc05: 51.2?µg/mL; GEn09: 88.1?µg/mL; GEn12: 99.9?µg/mL; GEn14: 275?µg/mL; and GEn17: 62.2?µg/mL) and considered for antiviral activity assays. Antiviral activity before virus adsorption was recorded against PV-1 for GLc05 (4.9%), GEn09 (3.4%), GEn12 (24.7%) and GEn17 (23.5%), and against HSV-1 for GEn12 (27.9%), GEn14 (58.7%) and GEn17 (39.2%). Antiviral activity after virus adsorption was identified against PV-1 for GLc05 (32.7%), GEn09 (91.0%), GEn12 (93.7%) and GEn17 (57.2%), and against HSV-1 for GEn17 (71.6%). The results obtained indicate the potential of some bacteriocins, particularly those produced by E. durans strains investigated in the present study, in viral inhibition and their application as new antiviral agents.     

Disposition,metabolism and mass balance of delafloxacin in healthy human volunteers following intravenous administration

Abstract

1. The pharmacokinetics and disposition of delafloxacin was investigated following a single intravenous (300?mg, 100?µCi) dose to healthy male subjects.

2. Mean C_max, AUC_0–∞, T_max and t_1/2 values for delafloxacin were 8.98?µg/mL, 21.31?µg?h/mL, 1?h and 2.35?h, respectively, after intravenous dosing.

3. Radioactivity was predominantly excreted via the kidney with 66% of the radioactive dose recovered in the urine. Approximately 29% of the radioactivity was recovered in the faeces, giving an overall mean recovery of 94% administered radioactivity.

4. The predominant circulating components were identified as delafloxacin and a direct glucuronide conjugate of delafloxacin.     

An evaluation of the DRI‐ETG EIA method for the determination of ethyl glucuronide concentrations in clinical and post‐mortem urine

A commercial enzyme immunoassay for the qualitative and semi‐quantitative measurement of ethyl glucuronide (EtG) in urine was evaluated. Post‐mortem (n=800), and clinical urine (n=200) samples were assayed using a Hitachi 902 analyzer. The determined concentrations were compared with those obtained using a previously published liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method for the quantification of EtG and ethyl sulfate. Using a cut‐off of 0.5 µg/ml and LC‐MS/MS limit of reporting of 0.1 µg/ml, there was a sensitivity of 60.8% and a specificity of 100% for clinical samples. For post‐mortem samples, sensitivity and specificity were 82.4% and 97.1%, respectively. When reducing the cut‐off to 0.1 µg/ml, the sensitivity and specificity were 83.3% and 100% for clinical samples whereas for post‐mortem samples the sensitivity and specificity were 90.3 % and 88.3 %, respectively. The best trade‐offs between sensitivity and specificity for LC‐MS/MS limits of reporting of 0.5 and 0.1 µg/ml were achieved when using immunoassay cut‐offs of 0.3 and 0.092 µg/ml, respectively. There was good correlation between quantitative results obtained by both methods but analysis of samples by LC‐MS/MS gave higher concentrations than by enzyme immunoassay (EIA), with a statistically significant proportional bias (P<0.0001, Deming regression) for both sample types. The immunoassay is reliable for the qualitative and semi‐quantitative presumptive detection of ethyl glucuronide in urine. Copyright © 2012 John Wiley & Sons, Ltd.     

Simultaneous determination of a new antituberculosis agent KBF-611 and its de-acetylated metabolite in mouse and rabbit plasma by HPLC

KBF-611 is a new thiosemicarbazone derivative which has demonstrated significant antituberculosis effect. A sensitive and specific HPLC method was established and validated for the determination of KBF-611 and its deacetylated metabolite (KM) in mouse and rabbit plasma. Chromatographic separation was achieved on a Eurospher-100 C8 column using acetonitrile, methanol, phosphate buffer (pH 7) and TEA (25:5:70:0.1, v/v), as mobile phase at a flow rate of 1 mL/min. KBF-611, KM and internal standard (4-acetamido-3-chlorobenzaldehyde thiosemicarbazone) were detected at the wavelength of 323 nm. The calibration curves were linear within the concentration range from 0.02–5 µg/mL and 0.02–1 µg/mL for KBF-611 and KM respectively. The limit of detection and the limit of quantitation were 6 ng/mL and 20 ng/mL respectively for both KBF-611 and KM. The relative standard deviation for intra- and inter-day precision was less than 7.5%. Average recoveries were 70.8% and 75.0% for KBF-611 and KM respectively. The established HPLC method was validated to be a simple, rapid and reliable procedure and successfully applied to study the preclinical pharmacokinetics of KBF-611 and KM in mice and rabbits.     

In vitro activity of cefiderocol,a siderophore cephalosporin,against a recent collection of clinically relevant carbapenem-non-susceptible Gram-negative bacilli,including serine carbapenemase- and metallo-β-lactamase-producing isolates (SIDERO-WT-2014 Study)

Cefiderocol is a siderophore cephalosporin in development for treatment of infections caused by Gram-negative bacilli, including carbapenem-resistant and multidrug-resistant isolates. β-Lactamase carriage and in vitro activity of cefiderocol were determined against 1272 meropenem-non-susceptible isolates of Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii collected as part of the SIDERO-WT-2014 surveillance study. Minimum inhibitory concentration (MIC) values for cefiderocol were ≤4 µg/mL against 97.7% of tested isolates, including 100% of IMP-positive (range, 1-2 µg/mL), OXA-58-positive (MIC₉₀, 1 µg/mL), KPC-positive (MIC₉₀, 2 µg/mL), VIM-positive (MIC₉₀, 2 µg/mL), and OXA-48-like-positive (MIC₉₀, 4 µg/mL) isolates; 99.3% of carbapenemase-negative isolates (MIC₉₀, 1 µg/mL); 97.2% of OXA-23-positive isolates (MIC₉₀, 1 µg/mL); 95.2% of OXA-24-positive isolates (MIC₉₀, 1 µg/mL); 91.7% of GES-positive isolates (MIC₉₀, 4 µg/mL); and 64.3% of NDM-positive isolates (MIC₉₀, 8 µg/mL). A total of 29 isolates (2.3%; 15 OXA-23-producers, 6 OXA-24-producers, 5 NDM-producers, and 3 carbapenemase-negative isolates) exhibited cefiderocol MIC ≥8 µg/mL, confirming there was no clear correlation between carriage of β-lactamases included in the molecular testing algorithm and elevated cefiderocol MICs. Similarly, no correlation was observed between cefiderocol MICs and truncation or loss of porin proteins in meropenem-non-susceptible isolates of E. coli and K. pneumoniae. Cefiderocol MICs were also ≤4 µg/mL against 99.3% of 136 colistin-resistant Enterobacteriaceae collected as part of the SIDERO-WT-2014 study, including isolates carrying mcr-1 (MIC₉₀, 2 µg/mL). Cefiderocol demonstrated potent in vitro activity against a collection of carbapenemase-producing and carbapenemase-negative meropenem-non-susceptible Gram-negative bacilli for which few treatment options are available, including the majority of metallo-β-lactamase producing isolates identified.     

Human exposure to airborne aniline and formation of methemoglobin: a contribution to occupational exposure limits

Aniline is an important starting material in the manufacture of polyurethane-based plastic materials. Aniline-derived methemoglobinemia (Met-Hb) is well described in exposed workers although information on the dose–response association is limited. We used an experimental design to study the association between aniline in air with the formation of Met-Hb in blood and the elimination of aniline in urine. A 6-h exposure of 2 ppm aniline in 19 non-smoking volunteers resulted in a time-dependent increase in Met-Hb in blood and aniline in urine. The maximum Met-Hb level in blood (mean 1.21 ± 0.29 %, range 0.80–2.07 %) and aniline excretion in urine (mean 168.0 ± 51.8 µg/L, range 79.5–418.3 µg/L) were observed at the end of exposure, with both parameters rapidly decreasing after the end of exposure. After 24 h, the mean level of Met-Hb (0.65 ± 0.18 %) returned to the basal level observed prior to the exposure (0.72 ± 0.19 %); whereas, slightly elevated levels of aniline were still present in urine (means 17.0 ± 17.1 vs. 5.7 ± 3.8 µg/L). No differences between males and females as well as between slow and fast acetylators were found. The results obtained after 6-h exposure were also comparable to those observed in four non-smoking volunteers after 8-h exposure. Maximum levels of Met-Hb and aniline in urine were 1.57 % and 305.6 µg/L, respectively. Overall, our results contribute to the risk assessment of aniline and as a result, the protection of workers from aniline-derived adverse health effects at the workplace.     

Urine naloxone concentration at different phases of buprenorphine maintenance treatment

In spite of the benefits of buprenorphine‐naloxone co‐formulation (BNX) in opioid maintenance treatment, the naloxone component has not prevented parenteral use of BNX. Current laboratory methods are not sufficient to differentiate between therapeutic and illicit use of buprenorphine, and little is known about urine naloxone concentrations. Measurement of urine naloxone, together with buprenorphine and norbuprenorphine, might help to determine the naloxone source and administration route. A liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method was developed and validated for this purpose. Naloxone, buprenorphine, and norbuprenorphine total concentrations were measured in urine samples from opioid‐dependent patients before and during stable and unstable phases of maintenance treatment with BNX. The limit of quantification in urine was 1.0 µg/L for naloxone, buprenorphine and norbuprenorphine. Before treatment, all samples contained buprenorphine but the median naloxone concentration was 0 µg/L. During the maintenance treatment with BNX all urine samples were positive for naloxone, buprenorphine and norbuprenorphine. The naloxone concentration at a stable phase of treatment (median 60 µg/L, range 5–200 µg/L) was not different from the naloxone concentration at an unstable phase (70 µg/L, 10–1700 µg/L). Applying an upper limit of 200 µg/L to the sample, the median naloxone/buprenorphine ratio was higher in the high than in the low naloxone concentration group (0.9 vs 0.3, respectively). This study suggests that naloxone in urine can act as an indicator of compliance with BNX. Parenteral use of BNX was associated with a high naloxone/buprenorphine ratio. Negative naloxone with positive buprenorphine suggests the use/abuse of buprenorphine alone. Copyright © 2013 John Wiley & Sons, Ltd.     

Performance characteristics of an ELISA screening assay for urinary synthetic cannabinoids

Synthetic cannabinoids are marketed as legal alternatives to cannabis, as routine urine cannabinoid immunoassays do not detect synthetic cannabinoids. Laboratories are challenged to identify these new designer drugs that are widely available and represent a major public health and safety problem. Immunoassay testing offers rapid separation of presumptive positive and negative specimens, prior to more costly and time‐consuming chromatographic confirmation. The Neogen SPICE ELISA kit targets JWH‐018 N‐pentanoic acid as a marker for urinary synthetic cannabinoids. Assay performance was evaluated by analyzing 2469 authentic urine samples with the Neogen immunoassay and liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). Two immunoassay cut‐off concentrations, 5 and 10 µg/L, classified samples as presumptive positive or negative, followed by qualitative LC‐MS/MS confirmation for 29 synthetic cannabinoids markers with limits of detection of 0.5–10 µg/L to determine the assay's sensitivity, specificity and efficacy. Challenges at ±25% of each cut‐off also were investigated to determine performance around the cut‐off and intra‐ and inter‐plate imprecision. The immunoassay was linear from 1 to 250 µg/L (r² = 0.992) with intra‐ and inter‐plate imprecision of ≤5.3% and <9%, respectively. Sensitivity, specificity, and efficiency results with the 5 µg/L cut‐off were 79.9%, 99.7%, and 97.4% and with the 10 µg/L cut‐off 69.3%, 99.8%, and 96.3%, respectively. Cross‐reactivity was shown for 18 of 73 synthetic cannabinoids markers evaluated. Good sensitivity, specificity, and efficiency, lack of sample preparation requirements, and rapid semi‐automation documented that the Neogen SPICE ELISA kit is a viable method for screening synthetic cannabinoids in urine targeting JWH‐018 N‐pentanoic acid. Copyright © 2014 John Wiley & Sons, Ltd.     

Alkaloid presence and brine shrimp (Artemia salina) bioassay of medicinal species of eastern Nicaragua

We used an alkaloid test and a brine shrimp bioassay to assess the bioactivity of the medicinal plants used by eastern Nicaraguan healers in traditional medicine. Ethnomedicinal uses were obtained from interviews of traditional healers. Aqueous extracts derived from 30 species of angiosperms were assayed for the presence of alkaloids and toxicity. Species tested are distributed in 30 genera and 21 families. Of the 30 species tested for alkaloids with Dragendorff’s reagent, 29 contained alkaloids. Toxicological analysis was conducted using the brine shrimp lethal assay (BSLA). Biological activity using BSLA was recorded as the median lethal concentration (LC₅₀) that kills 50% of the larvae within 24?h of contact with the aqueous plant extracts. The LC₅₀ of the shrimp was less than 2500 µg/mL for 3 (10%) species, 2500-5000 µg/mL for 9 (30%), 5001-7500 µg/mL for 7 (23%), 7501-10000 µg/mL for 3 (10%), and greater than 10000 µg/mL for 8 (27%) of the species. The members of the orders Santales and Rubiales in general contained species with greater toxicity than any other group. Struthanthus cassythoides (Struthanthus cassythoides Millsp.(Loranthaceae)). (LC₅₀ 1574 µg/mL) and Alibertia edulis (Rich.) A. Rich. (Rubiaceae) (LC₅₀ 1741 µg/mL) were the most toxic.     

Comparative analysis of nanosystems’ effects on human endothelial and monocytic cell functions

Abstract

The objective of our work was to investigate the effects of different types of nanoparticles on endothelial (HUVEC) and monocytic cell functions. We prepared and tested 14 different nanosystems comprising liposomes, lipid nanoparticles, polymer, and iron oxide nanoparticles. Some of the tested nanosystems contained targeting, therapeutic, or contrast agent(s). The effect of particles (0–400 µg/mL) on endothelial-monocytic cell interactions in response to TNF-α was investigated using an arterial bifurcation model and dynamic monocyte adhesion assay. Spontaneous HUVEC migration (0–100 µg/mL nanoparticles) and chemotaxis of monocytic cells towards MCP-1 in presence of particles (0–400 µg/mL) were determined using a barrier assay and a modified Boyden chamber assay, respectively. Lipid nanoparticles dose-dependently reduced monocytic cell chemotaxis and adhesion to activated HUVECs. Liposomal nanoparticles had little effect on cell migration, but one formulation induced monocytic cell recruitment by HUVECs under non-uniform shear stress by about 50%. Fucoidan-coated polymer nanoparticles (25–50 µg/mL) inhibited HUVEC migration and monocytic cell chemotaxis, and had a suppressive effect on monocytic cell recruitment under non-uniform shear stress. No significant effects of iron oxide nanoparticles on monocytic cell recruitment were observed except lauric acid and human albumin-coated particles which increased endothelial-monocytic interactions by 60–70%. Some of the iron oxide nanoparticles inhibited HUVEC migration and monocytic cell chemotaxis. These nanoparticle-induced effects are of importance for vascular cell biology and function and must be considered before the potential clinical use of some of the analyzed nanosystems in cardiovascular applications.     

Interaction of N-acetylcysteine and Cysteine in Human Plasma

N-acetyl-l-cysteine (NAC), a well-known antioxidant, has been successfully used as adjuvant therapy for late-stage childhood cerebral adrenoleukodystrophy (c-ALD); however, the mechanisms of NAC action are poorly understood. Previous research indicates that NAC serves as a precursor to l-cysteine (Cys), the rate-limiting substrate in the biosynthesis of glutathione (GSH), a potent, endogenous antioxidant. We hypothesized that NAC acts by liberating protein-bound Cys in plasma in an NAC concentration-dependent manner, which increases unbound Cys available for GSH biosynthesis. Human plasma was incubated for 1 h with varying, clinically relevant concentrations of NAC (0–1000 µg/mL). The effect of this interaction over time was evaluated by incubating plasma for 5–90 min with 100 µg/mL NAC. Unbound and bound Cys and NAC were separated by ultrafiltration, and concentrations were measured using high-performance liquid chromatography–mass spectrometry. Significant increases in unbound Cys were observed with increasing NAC concentrations. Also, Cys plasma protein binding decreased from 85% (10 µg/mL NAC) to approximately 0% (1000 µg/mL). Total endogenous Cys was 66% unbound at 5 min after incubation. These results demonstrate that NAC liberates endogenous, protein-bound Cys in human plasma at clinically relevant NAC concentrations. A greater understanding of NAC actions will aid in the optimization of NAC therapy including its use in c-ALD.     

An approach to enhanced stability: Formulation and characterization of Solanum lycopersicum derived lycopene based topical emulgel

Focus of the study was to design a novel and cost effective extraction technique for the lycopene from Lycopersicum esculentum L. fruit and to develop and characterize a stable emulgel formulation containing lycopene as an active ingredient as well as to design an analytical method to determine lycopene concentration in emulgel. Emulgel formulation was prepared and evaluated for its stability at different storage conditions, 8?°C, 25?°C, 40?°C, 40?°C?+?75% relative humidity (RH) and 50?°C, for 6?months. Results were statistically analyzed using two way ANOVA, Post-Hoc test and paired sample t-test at 5% significance level. Designed extraction technique presented comparable yield, 154.83?mg/Kg of tomato fruit, with all recoveries in the range of 145–156?mg/Kg of tomato. “P-values” calculated for different levels of stability parameters were <0.05, except at 50?°C and time points of 60th day and later. Analytical method designed was having linear range of lycopene 1–10?µg/mL with limit of detection 0.11?µg/mL and limit of quantification 0.34?µg/mL. All inter-day and intra-day recoveries were in the range of 94–105% while in all measurements RSD % was ≤5.36. It can be concluded that the extraction technique was cost effective with comparable results and analytical method was simple, robust, specific and sensitive enough to be used for lycopene concentration determination in emulgel formulation. Furthermore, designed formulation was stable even at high temperature of 40?°C and RH 75%.     

Determining therapeutic trough ranges for linezolid

Linezolid (LZD) is an oxazolidinone approved for the treatment of gram-positive infections. Therapeutic drug monitoring is increasingly used to optimize LZD dosing. The therapeutic target for LZD is to achieve an area under the concentration-time curve over 24 h divided by the MIC (AUC/MIC) > 100. In this study, we determined the trough ranges associated with this therapeutic AUC. Concentration-time profiles for 999 virtual patients were simulated using a previously published pharmacokinetic model for LZD. AUC was estimated for each virtual patient using the trapezoidal method. We determined the trough ranges that achieve the therapeutic target of AUC/MIC > 100 at different MIC values of 1, 2 and 4 μg/mL. Trough samples correlated well with LZD AUC (R² = 0.87). For trough concentration of 2–5 μg/mL, 99% had an AUC_0–24 > 100 µg⋅h⋅ml⁻¹, 23% had an AUC_0–24 > 200 µg⋅h⋅ml⁻¹ and none had an AUC_0–24 > 400 µg⋅h⋅ml⁻¹. For trough concentrations of 5–8 µg/ml, 87% of the patients had an AUC_0–24 > 200 µg⋅h⋅ml⁻¹ and none had an AUC_0–24 > 400 µg⋅h⋅ml⁻¹ To achieve the therapeutic target of an AUC/MIC > 100, it is suggested that trough ranges be set at 2–5 µg/ml if the MIC < 2 and 5–8 µg/ml if the MIC = 2; however, at an MIC of 4 µg/ml, it is difficult to achieve an AUC/MIC > 100 without increasing the risk of LZD toxicity.