Production of monoclonal antibodies for sandwich immunoassay detection of Pacific ciguatoxins

Ciguatoxins are the major causative toxins of ciguatera seafood poisoning. Limited availability of ciguatoxins has hampered the development of a reliable and specific immunoassay for detecting these toxins in contaminated fish. Monoclonal antibodies (mAbs) specific against both ends of Pacific ciguatoxins CTX3C and 51-hydroxyCTX3C were prepared by immunization of mice with the protein conjugates of rationally designed synthetic haptens in place of the natural toxin. Haptenic groups that possess a surface area larger than 400 Å² were required to produce mAbs that can bind strongly to CTX3C or 51-hydroxyCTX3C. A direct sandwich enzyme-linked immunosorbent assay (ELISA) using these mAbs was established to detect CTX3C and 51-hydroxyCTX3C at the ppb level with no cross-reactivity against the other marine toxins, including brevetoxin A, brevetoxin B, okadaic acid, or maitotoxin.     

Cytokine expression in trichloroethylene-induced hypersensitivity dermatitis: An in vivo and in vitro study

The purpose of this study was to address the association between cytokine expression and the hypersensitivity dermatitis induced by trichloroethylene (TCE) exposure. 28 TCE-induced hypersensitivity dermatitis patients, 22 TCE exposed workers and 22 non-exposed controls were enrolled in the study. The serum levels of interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor (TNF)-α were analyzed using a magnetic colorbead-based multiplex assay. The patients showed significantly higher levels of serum IL-1β (p = 0.033 and p = 0.015), IL-6 (p < 0.001), IL-8 (p < 0.001 and p = 0.002) and TNF-α (p = 0.009 and p = 0.005) than the TCE exposed workers and non-exposed controls. There was a significantly positive correlation among these cytokine concentrations, but no significant correlation was found between these cytokine concentrations and the disease duration in patient group. We further compared the effects of trichloroethanol (TCOH) and trichloroacetic acid (TCA), two major metabolites of TCE, on cytokine expression in keratinocyte cell line (HaCaT). IL-1α, IL-6, IL-8 and TNF-α concentrations were tested using enzyme-linked immunosorbent assay (ELISA) after HaCaT cells were treated with different concentrations of TCOH or TCA for 24 h. We found that TCOH, but not TCA, increased the levels of IL-1α and IL-6 in a dose-dependent manner. We also found that TCOH activated the nuclear factor kappa B (NF-κB) pathway. Bay 11-7082 (NF-κB inhibitor) significantly attenuated the TCOH-induced production of IL-6 in HaCaT cells, but IL-1α production was not affected. In conclusions, it is suggested that IL-1β, IL-6, IL-8 and TNF-α were associated with TCE-induced hypersensitivity dermatitis. TCOH induced IL-6 expression through activation of the NF-κB pathway in HaCaT cells and may play an integral role in TCE-induced skin hypersensitivity.     

Evaluation of the Architect tacrolimus assay in kidney,liver, and heart transplant recipients

The narrow therapeutic range of tacrolimus requires therapeutic drug monitoring to prevent transplant rejection and to minimize nephrotoxicity. The aim of this study was to evaluate the analytical performance of the tacrolimus chemiluminescent microparticle immunoassay (CMIA) in everyday practice comparatively with other methods. CMIA imprecision and accuracy were tested using low, medium, and high concentrations in control samples. The limits of quantification (LOQ) of CMIA and antibody-conjugated magnetic immunoassay (ACMIA) were evaluated using negative whole-blood samples containing 0.4–5.7 ng/ml of tacrolimus from a stock solution. CMIA was compared with ACMIA, enzyme multiplied immunoassay (EMIT), and liquid chromatography–tandem mass spectrometry (LC–MS/MS), using 176 samples from recipients (135 men and 41 women) of heart (n = 19), kidney (n = 107), or liver (n = 50) transplants. CMIA total precision was 5.7%, 3.7% and 3.6% with the low-, medium-, and high-concentration controls, respectively; corresponding values for accuracy were 98%, 104%, and 104%. LOQ was 0.5 (95%CI, 0.22–1.38) with CMIA and 2.5 ng/ml with ACMIA. Linear regression results were as follows: CMIA = 1.2LC–MS/MS + 0.14 (r = 0.98); CMIA = 0.93EMIT + 0.36 (r = 0.975); CMIA = 1.15ACMIA − 0.25 (r = 0.988); and, for tacrolimus concentrations in the 1–15 ng/ml range, of special interest as many transplant recipients are given low-dose tacrolimus, CMIA = 1.05LC–MS/MS + 0.38 (r = 0.94). Two patients had falsely elevated tacrolimus concentrations due to interference in the ACMIA assay; one was a renal transplant recipient who stopped her treatment and had tacrolimus concentrations of 12.5 ng/ml by ACMIA and <0.5 ng/ml by CMIA; the other was an HIV-positive renal transplant recipient whose tacrolimus concentrations by ACMIA were 1.8–43.7-fold those by CMIA. Such interferences with ACMIA, which may be related to endogenous antibodies in the plasma, are likely to negatively impact patient care. In conclusion, the tacrolimus CMIA assay is suitable for routine laboratory use and does not suffer from the interferences seen with ACMIA in some patients.     

Qualitative and quantitative studies on human B7.1-Fc fusion protein and the application in pharmacokinetic study in rhesus monkeys

A sensitive, accurate, and precise enzyme immunoassay (EIA) for the quantification of intact human B7.1-Fc in rhesus monkey serum was validated, and the characteristics of B7.1 and Fc moiety of fusion protein were identified by surface plasmon resonance (SPR) and flow-cytometric method, respectively. B7.1-Fc bound to CD28 and CTLA-4 with K_d values of 45.1 and 9.58 nM, respectively, which were very closed to the previous reports and the function of Fc moiety of fusion protein was also confirmed by Fc receptor binding assay and IL-8 releasing assay. To monitor the intact protein, the EIA method employed a sandwich scheme in which a multiclonal anti-human IgG (Fc specific) antibody and a monoclonal anti-human B7.1 antibody were served as capture and detection antibody, respectively. This EIA has a range of reliable response of 0.5–32 ng/ml. The LLOQ was established at 0.5 ng/ml. The intra-assay precision and accuracy were 6.1–8.8% and (3.0–9.0)%, respectively with the inter-assay precision and accuracy were 5.7–11.5% and (10.7–9.1)%, respectively. Stability was established under certain conditions and no significant differences were found. This validated EIA assay was then successfully employed in the assessment of pharmacokinetic behavior of B7.1-Fc in rhesus monkeys after intravenous infusion, and a non-linear characteristics was established across the investigated dosage range (32–320 μg/kg).     

Use of Monoclonal Antibodies in the Sensitive Detection and Neutralization of Botulinum Neurotoxin Serotype B

Botulinum neurotoxins (BoNT) are some of nature’s most potent toxins. Due to potential food contamination, and bioterrorism concerns, the development of detection reagents, therapeutics and countermeasures are of urgent interest. Recently, we have developed a sensitive electrochemiluminescent (ECL) immunoassay for BoNT/B, using monoclonal antibodies (mAbs) MCS6-27 and anti-BoNT/B rabbit polyclonal antibodies as the capture and detector. The ECL assay detected as little as 1 pg/mL BoNT/B in the buffer matrix, surpassing the detection sensitivities of the gold standard mouse bioassays. The ECL assay also allowed detection of BoNT/B in sera matrices of up to 100% sera with negligible matrix effects. This highly-sensitive assay allowed the determination of the biological half-lives of BoNT/B holotoxin in vivo. We further tested the toxin neutralization potential of our monoclonal antibodies using the mouse systemic and oral intoxication models. A combination of mAbs protected mice in both pre- and post-exposure models to lethal doses of BoNT/B. MAbs were capable of increasing survival of animals when administered even 10 h post-intoxication in an oral model, suggesting a likely time for BoNT/B complexes to reach the blood stream. More sensitive detection assays and treatments against BoNT intoxication will greatly enhance efforts to combat botulism.     

miR-3940-5p associated with genetic damage in workers exposed to hexavalent chromium

To understand the regulation of genetic damage by epigenetics at the early stage of carcinogenesis after hexavalent chromium (Cr(VI)) and assessed genetic damage to explore their association with DNA repair genes mediated by differently expressed miRNA. Genetic damages were evaluated using cytokinesis-block micronucleus assay (CBMN) and serum 8-hydroxyguanine (8-OHdG) ELISA assay. Blood Cr level showed significant association with plasma miR-3940-5p level (r = −0.33, P = 0.001) and non-linear relationship with micronuclei frequency in CBMN and serum 8-OHdG level (β_std = 0.29, P = 0.039; β_std = 0.35, P = 0.001), with micronuclei frequency not increasing apparently under high Cr exposure. In contrast, no significant association was found between plasma miR-3940-5p level and the two genetic indicators. However, plasma miR-3940-5p level was linked to micronuclei frequency under high blood Cr level (β_std = 0.18, P = 0.015). To explore the effect of miR-3940-5p on genetic damage under high Cr exposure, the protein expression levels of miR-3940-5p-mediated DNA repair genes in leukocytes were quantified using enzyme-linked immunosorbent assay for subjects with high blood Cr level. The results showed that XRCC2 and BRCC3 protein levels were statistically associated with miR-3940-5p level respectively (β_std = −0.31, P = 0.010; β_std = −0.24, P = 0.037). Meanwhile, a weak but statistically negative association between XRCC2 level and micronuclei frequency was found (β_std = −0.15, P = 0.027). These data suggests that high Cr(VI) does not always aggravate genetic damage after reaching a high Cr(VI) exposure in real situation, which may be due to the regulation of miRNA on DNA repair genes responsive to high Cr(VI) exposure.     

Quantification of oltipraz using liquid chromatography-tandem mass spectrometry and its application to a pharmacokinetic study in rat plasma

An assay method for the determination of oltipraz, a candidate drug for the treatment of liver fibrosis and liver cirrhosis, was developed in rat plasma using a fast-flow protein precipitation (FF-PPT) method coupled with LC-MS/MS for quantification to reduce the labor and to improve the speed of analysis. The applicability of the assay to pharmacokinetic studies was also evaluated. Oltipraz and ethyl-oltipraz, an internal standard (IS), were analyzed by multiple reaction monitoring (MRM) at m/z transitions of 227 → 193 and 241 → 174, respectively. A lower limit of quantification (LLOQ) of 20 ng/mL was observed, with a linear dynamic range from 20 to 4000 ng/mL (R > 0.997). The accuracy, precision, dilution, recovery, and stability of the assay were deemed acceptable according to FDA guidelines. Oltipraz concentrations were measured successfully in plasma samples up to 12 h post-dose in rats that had received an oral dose of 60 mg/kg. The findings indicate that the assay method is rapid and sensitive to oltipraz, showing applicability for pharmacokinetics (PK) studies of oltipraz in other small animals, including rats.     

LOX-1 is involved in IL-1β production and extracellular matrix breakdown in dental peri-implantitis

PurposeTo explore whether lectin-type oxidized LDL receptor 1 (LOX-1), interleukin 1 beta (IL-1β), matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9) are involved in the nosogenesis of human dental peri-implantitis and determine the role of LOX-1 in IL-1β, MMP2 and MMP9 production in response to Porphyromonas gingivalis.MethodsPeri-implant crevicular fluid (PICF) was collected from ten patients with healthy implants and ten patients with peri-implantitis. The LOX-1 protein in PICF was detected by Western-blot, and the expression of LOX-1 in superficial gingiva of peri-implantitis patients was detected by immunofluorescence staining. The IL-1β, MMP2 and MMP9 proteins in PICF were detected by enzyme-linked immunosorbent assay (ELISA). THP-1 macrophages were pretreated with neutralizing antibody (LOX-1) and inhibitors (LOX-1 and c-Jun N-terminal kinase, JNK) to evaluate the role of LOX-1 and JNK in IL-1β production, as well as the role of LOX-1 in MMP2 and MMP9 production in response to P. gingivalis by quantitative polymerase chain reaction (RT-PCR) and Western-blot.ResultsLOX-1, IL-1β, MMP2 and MMP9 increased in PICF of peri-implantitis patients and in THP-1 macrophages on P. gingivalis stimulation. IL-1β, MMP2 and MMP9 production in response to P. gingivalis in THP-1 macrophages was dependent on LOX-1. JNK was responsible for LOX-1 induced IL-1β production as a result of P. gingivalis infection.ConclusionLOX-1 is involved in IL-1β production and extracellular matrix breakdown is a novel inflammatory pathway trigger and potential drug target in human dental peri-implantitis.     

Protective effects of memantine and epicatechin on catechol-induced toxicity on Müller cells in vitro

This study evaluates the toxic effects of catechol (a component from cigarette smoke) on Müller cells (MIO-M1) in vitro, and investigates the inhibitors memantine and epicatechin to determine if they can reverse the catechol toxic effects. MIO-M1 cells were exposed to varying concentrations of catechol with or without memantine or epicatechin. Cell viability (CV) was measured by a trypan blue dye-exclusion assay. Caspase-3/7 activity was measured by fluorochrome assay. The production of reactive oxygen/nitrogen species (ROS/RNS) was measured with 2′,7′-dichlorodihydrofluorescein diacetate dye assay. Mitochondrial membrane potential (ΔΨm) was measured using JC-1 assay. Intracellular ATP content was determined by the ATPLite kit. MIO-M1 cells showed significant decrease in cell viability, increased caspase-3/7 activity, elevated ROS/RNS levels, decreased ΔΨm value, and decreased intracellular ATP content after exposure to catechol 150, 300, and 600 μM compared with control. Pre-treatment with memantine 10 μM or epicatechin 15 μM reversed loss of cell viability in catechol 150 μM-treated cultures (22.3%, p < 0.01 and 17.8%, p < 0.05), respectively. Similarly, pre-treatment with memantine 10 μM and epicatechin 15 μM prior to catechol resulted in decreased caspase-3/7 activities (77% and 64.2%, p < 0.001), decreased ROS/RNS levels (82.3% and 79%, p < 0.001), increased ΔΨm value (76.4% and 72.2%, p < 0.001), and increased ATP levels (46.6% and 40.4%, p < 0.001) compared to 150 μM catechol-treated cultures. Catechol, a component of smoking, can diminish cell viability and mitochondrial function in MIO-M1 cells in vitro. However, memantine and epicatechin can partially reverse the cytotoxic effect of catechol. Their administration may reduce or prevent Müller cells degeneration in AMD or other retinal degenerative disorders.     

血清凝集素样氧化低密度脂蛋白受体-1水平与急性冠脉综合征关系的研究

目的:探讨血清中凝集素样氧化低密度脂蛋白受体-1(LOX-1)水平与急性冠脉综合征(ACS)的关系。方法:经冠状动脉造影(CAG)确诊的冠心病患者121例,其中ACS组75例,稳定型心绞痛组(SAP)46例;另设经CAG证实冠脉无狭窄者50例为对照组。采用酶联免疫吸附法(ELISA)测定3组入选者入院即刻,ACS组与SAP组经皮冠状动脉介入治疗(PCI)术后即刻、对照组CAG术后即刻,ACS组与SAP组PCI术后1周血清sLOX-1浓度。结果:入院即刻及术后即刻,ACS组外周血清sLOX-1水平高于SAP组和对照组,SAP组高于对照组(P<0.05或P<0.01);PCI术后1周,ACS组与SAP组之间差异无统计学意义(P>0.05)。各组内比较,3组术后即刻血清sLOX-1水平与入院即刻相比,差异均无统计学意义(P>0.05);PCI术后1周血清sLOX-1水平较入院即刻有所降低,但差异无统计学意义(P>0.05)。结论:ACS患者外周血清LOX-1浓度明显升高,其水平与冠状动脉内粥样硬化斑块的稳定性密切相关,其可作为动脉粥样硬化斑块不稳定的血清学指标。     

Noninvasive measurement of smoking-associated N-ethyladenine and N-ethylguanine in human salivary DNA by stable isotope dilution nanoflow liquid chromatography–nanospray ionization tandem mass spectrometry

Evidence showed that ethylating agents are contained in cigarette smoke, which damage DNA producing ethylated DNA adducts, including N³-ethyladenine (3-EtAde) and N⁷-ethylguanine (7-EtGua). These two ethylpurines can be depurinated spontaneously and be repaired by enzymes and they have been detected in human urine. In this study, a highly specific and sensitive assay based on stable isotope dilution nanoflow liquid chromatography nanospray ionization tandem mass spectrometry (nanoLC–NSI/MS/MS) was used to measure 3-EtAde and 7-EtGua in human salivary DNA. These ethylpurines were released from DNA by neutral thermal hydrolysis and then enriched by a solid-phase extraction column before nanoLC–NSI/MS/MS analysis. The detection limits (S/N ≥ 3) of 3-EtA and 7-EtG were 15 fg (92 amol) and 10 fg (56 amol), respectively, injected on-column. The lower quantification limits of 3-EtAde and 7-EtGua were both 100 fg, i.e. 620 and 560 amol, respectively, corresponding to 9.4 and 8.6 adducts in 10⁹ normal nucleotides, respectively, starting with as little as 20 μg of DNA isolated from an average of 3 mL of saliva. The mean (±SD) levels of 3-EtAde in 15 smokers and 15 nonsmokers were 12.6 ± 7.0 and 9.7 ± 5.3 in 10⁸ normal nucleotides, respectively, while those of 7-EtGua were 14.1 ± 8.2 and 3.8 ± 2.8 in 10⁸ normal nucleotides in smokers and nonsmokers, respectively. Levels of 7-EtGua, but not 3-EtAde, were statistically significantly higher in smokers than in nonsmokers (p < 0.0001). Furthermore, salivary 7-EtGua levels are significantly correlated with the number of cigarettes smoked per day as well as with the smoking index. This highly specific and sensitive stable isotope dilution nanoLC–NSI/MS/MS assay might be feasible in measuring 7-EtGua in human salivary DNA as a noninvasive biomarker for DNA damage induced by cigarette smoking.     

Forced degradation of fentanyl: Identification and analysis of impurities and degradants

Fentanyl, N-(1-phenethylpiperidin-4-yl)-N-phenylpropionamide is a rapid-acting, powerful opioid analgesic used extensively for anesthesia and chronic pain management. A forced degradation study of fentanyl active pharmaceutical ingredient (API) was performed using light, acid, base, heat and oxidation. Under acidic conditions, fentanyl was shown to degrade to N-phenyl-1-(2-phenylethyl)-piperidin-4-amine (PPA¹). Fentanyl was stable to light exposure and base treatment with no degradation observed. Oxidation with hydrogen peroxide produced fentanyl N-oxide by rapidly oxidizing the nitrogen on the piperidine ring. Five degradants were formed during thermal degradation of fentanyl. The two known degradants included propionanilide (PRP²) and norfentanyl (NRF³). The three unknown degradants were first identified by mass using LC/MS, and postulated compounds were synthesized and confirmed by LC/MS and ¹H NMR. These degradants were identified as 1-phenethylpyridinium salt (1-PEP⁴), 1-phenethyl-1H-pyridin-2-one (1-PPO⁵), and 1-styryl-1H-pyridin-2-one (1-SPO⁶). In addition to the seven degradants, three known process impurities, acetyl fentanyl, pyruvyl fentanyl and butyryl fentanyl were also detected by reverse-phase high performance liquid chromatography (HPLC) with UV detection. All degradants and impurities were identified and confirmed using authentic materials. Method validation was performed for the assay of fentanyl and its related compounds in accordance to ICH guideline Q2(R1), and the method was demonstrated to be specific, linear (r > 0.999 for fentanyl assay and r > 0.996 for related compounds), accurate (recovery > 99.6% for fentanyl assay and recovery > 91.0 for related compounds), precise (%RSD < 0.8% for fentanyl assay and <4.8% for related compounds), sensitive (limit of detection = 0.08 μg/mL or 0.016% of nominal concentration), robust and suitable for its intended use. The chemical structures for the degradants and impurities were submitted to three in silico toxicity programs to identify any structural alerts.     

Simultaneous detection and quantification of 3-nitrotyrosine and 3-bromotyrosine in human urine by stable isotope dilution liquid chromatography tandem mass spectrometry

Nitration and bromination of proteins, giving rise to the respective 3-nitrotyrosine (3NT) and 3-bromotyrosine (3BT), are implicated in asthma, allergic inflammatory disorders, and cancer. We have developed an isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS) assay for simultaneous analysis of protein-bound 3NT and 3BT in human urine. The detection limits (S/N = 3) were 10 pg (44 fmol) for 3NT and 5.0 pg (19 fmol) for 3BT injected on-column. The average levels of protein-bound 3NT and 3BT in 23 healthy individuals were 9.7 ± 11.0 (mean ± S.D.) in 10⁵ tyrosine and 4.4 ± 3.9 (mean ± S.D.) in 10³ tyrosine, respectively, using this highly sensitive LC/MS/MS under the selective reaction monitoring mode. Furthermore, the levels of urinary 3NT and 3BT show a statistically significant correlation (R² = 0.55, p = 0.0065, n = 23). The high specificity and accuracy of this LC/MS/MS method render it a valuable tool in measurement of 3NT and 3BrT in the human urinary protein as promising noninvasive biomarkers for protein tyrosine nitration and bromination in vivo.     

Inhibitory effects of melittin on the production of lipopolysaccharide-induced matrix metalloproteinase 3 in human osteoarthritic chondrocytes

In continuation of our previous study which explored the effect of bee venom (BV) on the global gene expression profiles in lipopolysaccharide (LPS)-treated human chondrosarcoma cells, we investigated herein the effect of melittin, a major component of BV, on the productions of matrix metalloproteinases (MMPs) 1, 3, and 13 in primary cultured human arthritic chondrocytes. Increased generations of MMPs 1, 3, and 13 were observed by MMPs stimulating agents LPS, tumor necrosis factor α (TNF-α), and interleukin 1β (IL-1β). The generations of LPS (1 μg/ml)-induced MMPs 1 and 13 were not decreased by melittin, whereas that of LPS-stimulated MMP 3 was significantly inhibited by melittin. IL-1β (10 ng/ml) and TNF-α (10 ng/ml)-induced MMPs 1, 3 and 13, however, were not decreased by melittin. Immunoblot analysis revealed that melittin exerted no effect on the LPS-stimulated expression levels of MMPs 1 and 13 but attenuated the LPS-induced MMP 3, which is consistent with the enzyme-linked immunosorbent assay (ELISA) data. Taken together, these findings suggest melittin may exert its anti-arthritic effect, at least in part, by inhibiting LPS-stimulated MMP 3 production in human osteoarthritic chondrocytes.     

Modulation of DNA repair capacity and mRNA expression levels of XRCC1, hOGG1 and XPC genes in styrene-exposed workers

Decreased levels of single-strand breaks in DNA (SSBs), reflecting DNA damage, have previously been observed with increased styrene exposure in contrast to a dose-dependent increase in the base-excision repair capacity. To clarify further the above aspects, we have investigated the associations between SSBs, micronuclei, DNA repair capacity and mRNA expression in XRCC1, hOGG1 and XPC genes on 71 styrene-exposed and 51 control individuals. Styrene concentrations at workplace and in blood characterized occupational exposure. The workers were divided into low (below 50 mg/m³) and high (above 50 mg/m³) styrene exposure groups. DNA damage and DNA repair capacity were analyzed in peripheral blood lymphocytes by Comet assay. The mRNA expression levels were determined by qPCR. A significant negative correlation was observed between SSBs and styrene concentration at workplace (R = − 0.38, p = 0.001); SSBs were also significantly higher in men (p = 0.001). The capacity to repair irradiation-induced DNA damage was the highest in the low exposure group (1.34 ± 1.00 SSB/10⁹ Da), followed by high exposure group (0.72 ± 0.81 SSB/10⁹ Da) and controls (0.65 ± 0.82 SSB/10⁹ Da). The mRNA expression levels of XRCC1, hOGG1 and XPC negatively correlated with styrene concentrations in blood and at workplace (p < 0.001) and positively with SSBs (p < 0.001). Micronuclei were not affected by styrene exposure, but were higher in older persons and in women (p < 0.001). In this study, we did not confirm previous findings on an increased DNA repair response to styrene-induced genotoxicity. However, negative correlations of SSBs and mRNA expression levels of XRCC1, hOGG1 and XPC with styrene exposure warrant further highly-targeted study.     

Detection of low-affinity anti-drug antibodies and improved drug tolerance in immunogenicity testing by Octet biolayer interferometry

We assessed the utility of the FortéBio Octet^® system for detection of anti-drug antibodies (ADAs) against an investigational therapeutic human IgG1 monoclonal antibody (mAb), CNTO X. To understand the relative merits of this technology, key performance requirements were compared with two popularly accepted ADA detection methods, a step-wise bridging ELISA and a Meso Scale Discovery (MSD) homogeneous (single step binding) bridging ECLIA. When used to detect 13 monoclonal ADAs of varying affinities and one polyclonal ADA, all three methods demonstrated their greatest apparent sensitivity to the polyclonal sample (1, 6, and 130 ng/mL, respectively for ECLIA, ELISA, and Octet). Sensitivity to monoclonal ADAs tended to vary in accordance with their affinities, however, the sensitivity of the Octet method varied much less between ADAs. As a result, the above ranking became reversed such that Octet was the most and ELISA least sensitive for detection of low-affinity ADAs. With regard to drug tolerance, the presence of CNTO X could lead to false-negative assay results, although each method was affected to a different degree, with the Octet method tolerating up to 10 times more drug than the ECLIA method, which in turn tolerated up to 10 times more than the ELISA. Finally, the ECLIA and Octet methods were applied to the bioanalysis of cynomolgus monkey sera from a pre-clinical multiple dose study of CNTO X. Octet indicated 3 positive animals developed ADA as early as day 15 of the dosing phase while drug was present at nearly 1 mg/mL. ECLIA detected only one of these, and only in a day 57 recovery sample after drug had cleared from circulation. We conclude that the Octet is a promising platform for detection of lower affinity ADAs and is particularly suitable for ADA detection when drug persists at levels that negatively impact bridging immunoassays.     

Simultaneous determination of rupatadine and its metabolite desloratadine in human plasma by a sensitive LC–MS/MS method: Application to the pharmacokinetic study in healthy Chinese volunteers

A sensitive liquid chromatography/tandem mass spectrometry (LC–MS/MS) method was developed for simultaneous determination of rupatadine and its metabolite desloratadine in human plasma. After the addition of diphenhydramine, the internal standard (IS), plasma samples were extracted with a mixture of methyl tert-butyl ether and n-hexane (1:1, v/v). The analysis was performed on a Ultimate™ AQ-C18 (4.6 mm × 100 mm, 5 μm) column using a mobile phase consisting of a 80/20 mixture of methanol/water containing 0.0005% formic acid pumped at 0.3 ml min⁻¹. The analytes and the IS were detected in positive ionization mode and monitoring their precursor → product ion combinations of m/z 416 → 309, 311 → 259, and 256 → 167, respectively, in multiple reaction monitoring mode. The linear ranges of the assay were 0.1–50 and 0.1–20 ng ml⁻¹ for rupatadine and desloratadine, respectively. The lower limits of reliable quantification for both rupatadine and desloratadine were 0.1 ng ml⁻¹, which offered high sensitivity and selectivity. The within- and between-run precision was less than 7.2%. The accuracy ranged from −9.2% to +6.4% and −7.2% to +7.2% for rupatadine and desloratadine in quality control samples at three levels, respectively. The method has been successfully applied to a pharmacokinetic study of rupatadine and its major metabolite after oral administration of 10, 20 and 40 mg rupatadine tablets to healthy Chinese volunteers.     

Development and validation of a LC–MS/MS method for determination of bivalirudin in human plasma: Application to a clinical pharmacokinetic study

A simple, sensitive and rapid LC–MS/MS method has been developed and validated for the identification and quantification of bivalirudin in human plasma using nafarelin as the internal standard. Following protein precipitation with methanol, the analytes were separated on a C₁₈ column interfaced with a triple-quadrupole tandem mass spectrometer using positive electrospray ionization. Quantification of bivalirudin was conducted by multiple reaction monitoring (MRM) of the transitions of m/z 1091.4 → (356.4 + 227.4) for bivalirudin and m/z 662.4 → 328.5 for IS. The lower limit of quantification was 1.25 ng/ml, and the assay exhibited a linear range of 1.25–500 ng/ml. The developed assay method was successfully applied to a pharmacokinetic (PK) study in healthy volunteers after intravenous administration of bivalirudin.     

Protective effects of a polysaccharide from Hizikia fusiformis against ethanol toxicity in rats

Hizikia fusiformis is an edible brown alga that is widely consumed in Korea, Japan, and China and possesses a number of potentially beneficial compounds, including antioxidants and anticoagulants. No reports have investigated potential H. fusiformis protectants against ethanol-induced peptic injury. We extracted a polysaccharide from H. fusiformis (Hf–PS-1) that exhibited protective effects against ethanol-induced peptic injury and related mechanisms in rats. Experimental animals were divided into three groups: control, ethanol-only, and ethanol + Hf–PS-1. The ethanol-only group exhibited decreased levels of total glutathione (GSH) and increased levels of jun N-terminal kinase (JNK) phosphorylation relative to the control group, whereas levels were significantly increased and decreased, respectively, in the ethanol + Hf–PS-1 group. The ethanol-only group also exhibited increased levels of extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation relative to the control group; these levels were not significantly different in the ethanol + Hf–PS-1 group. Hf–PS-1 appeared to reduce ethanol-induced gastric injury. Therefore, we suggest that Hf–PS-1 could protect against ethanol-induced peptic ulcers primarily through a mechanism associated with the inhibition of JNK activation.     

In vivo CYP2E1 phenotyping as a new potential biomarker of occupational and experimental exposure to benzene

Assessing CYP2E1 phenotype in vivo may be important to predict individual susceptibility to those chemicals, including benzene, which are metabolically activated by this isoenzyme. Chlorzoxazone (CHZ), a specific CYP2E1 substrate, is readily hydroxylated to 6-OH-chlorzoxazone (6-OH-CHZ) by liver CYP2E1 and the metabolic ratio 6-OH-CHZ/CHZ in serum (MR) is a specific and sensitive biomarker of CYP2E1 activity in vivo in humans. We used this MR as a potential biomarker of effect in benzene-treated rats and, also, in humans occupationally exposed to low levels of benzene. Male Sprague-Dawley rats (375–400 g b.w.) were treated i.p. for 3 days with either a 0.5 ml solution of benzene (5 mmol/kg b.w.) in corn oil, or 0.5 ml corn oil alone. Twenty-four hours after the last injection, a polyethylene glycol (PEG) solution of CHZ (20 mg/kg b.w.) was injected i.p. in both treated and control animals. After 2, 5, 10, 15, 20, 30, 45, 60, 90, 120, 180, and 240 min from injection, 0.2 ml blood was taken from the tip tail and stored at −20 °C until analysis. A modified reverse phase HPLC method using a 5 μm Ultrasphere C18 column equipped with a direct-connection ODS guard column, was used to measure CHZ and its metabolite 6-OH-CHZ in serum. No statistically significant difference in the MR was observed, at any sampling time, between benzene-treated and control rats. The concentration-versus-time area under the curve (AUC), however, was lower (p < 0.05, Mann–Whitney test), whereas the systemic clearance was higher (p < 0.05) in treated than in control rats. Eleven petrochemical workers occupationally exposed to low levels of airborne benzene (mean ± SD, 25.0 ± 24.4 μg/m³) and 13 non-exposed controls from the same factory (mean ± SD, 6.7 ± 4.0 μg/m³) signed an informed consent form and were administered 500 mg CHZ p.o. Two hours later a venous blood sample was taken for CHZ and 6-OH-CHZ measurements. Despite exposed subjects showed significantly higher levels of t,t-MA and S-PMA, two biomarkers of exposure to benzene, than non-exposed workers, no difference in the MR mean values ± SD was found between exposed (0.59 ± 0.29) and non-exposed (0.57 ± 0.23) subjects. So, benzene was found to modify CHZ disposition, but not CYP2E1 phenotype in benzene-treated rats, nor in workers exposed to benzene, probably due to the levels of exposure being too low.