Bioanalytical Assessment Research Articles

DDDR-14. IMPROVED ANTI-GLIOBLASTOMA EFFICACY BY BRG399, A NOVEL ORAL MICROTUBULE BINDING AGENT

Abstract Glioblastoma (GBM) is a highly malignant form of brain tumor with a poor prognosis despite advances in treatments. Therapeutic resistance/relapse remains a significant challenge necessitating the development of novel approaches. BRG399 is a brain permeant, non P-gp substrate, microtubule-targeting agent that displays anti-cancer activity in vitro and in vivo. BRG399 was well tolerated in rat and dog toxicology studies. Anti-cancer activity (IC50) of BRG399 in in vitro models of GBM was assessed to have potency ranging from 42nm to 89nM in the 3 cell lines tested. Here, in vivo tumor activity of BRG399 was assessed in Sprague Dawley rats implanted with 3x105 C6 rat glioma cells into the right entorhinal cortex/subiculum region. 14 days post successful implantation tumors were assessed by MRI, rats were randomized to receive vehicle (2% Labrasol) or BRG399 (5, 10, 20 mg/kg groups) twice daily by oral gavage for 16 days, and MRIs were performed on survivors on days 31 and 45 post implantation. BRG399 administration resulted in a marked improvement in survival (from 7.7 ± 2.6 days to 25.5 ± 15.6 days, log-rank test, p:0.007) compared to controls. Six animals receiving BRG399 remained alive beyond day 60 of post-treatment initiation. To assess biodistribution of BRG399 in the brain, rats were implanted with C6 cells and tumor establishment assessed 12 days later by MRI. Animals were treated with vehicle or BRG399 (10, 20, 25 mg/kg) and plasma and brains were collected for bioanalytical assessment. Tissue single cell biodistribution was performed using TIMSTOF FLEX spatial OMICS. These results demonstrate a therapeutic effect of BRG399 on intracranial glioma-bearing rats with survival of the BRG399 treatment groups of strong statistical significance over the control group. Analysis of drug levels in plasma and brain will serve as guidance for dose and scheduling decisions for further therapeutic development.

A guidance for the enrichment of micropollutants from wastewater by solid-phase extraction before bioanalytical assessment

BackgroundWastewater can contain a complex mixture of organic micropollutants, with both chemical analysis and effect-based methods needed to identify relevant micropollutants and detect mixture effects. Solid-phase extraction (SPE) is commonly used to enrich micropollutants prior to analysis. While the recovery and stability of individual micropollutants by SPE has been well studied, few studies have optimized SPE for effect-based methods. The aim of the current study was to develop and evaluate two standard operating procedures (SOPs) for the enrichment of micropollutants in preparation for chemical analysis and bioanalysis, one covering a broad range of chemicals and the other selective for estrogenic chemicals.ResultsPristine surface water spiked with > 600 micropollutants was used to develop a generic extraction method for micropollutants with a wide range of physiochemical properties, while water spiked with estrogenic chemicals was used to identify a selective extraction method. Three different SPE sorbents were tested, with recoveries of individual chemicals and effect in assays indicative of mutagenicity, estrogenic activity, and fish embryo toxicity assessed. The sorbent HRX at pH 7 was selected for the generic extraction method as it showed the best recovery of both individual chemicals and effect in the bioassays. The sorbent HLB at pH 3 showed optimal recovery of estrogenic chemicals and estrogenic activity. The two optimal SPE methods were applied to spiked and unspiked wastewater effluents, with the concentrations of detected chemicals and observed effects similar to those of previous studies. The long-term storage of both extracts and SPE cartridges for estrogens and estrogenic activity after extraction with the HRX and HLB methods were evaluated, with estrogenic effectiveness close to 100% after 112 days when HLB was used.ConclusionsHRX is recommended for generic extraction, while HLB is optimal for the selective extraction of estrogenic micropollutants. However, if a laboratory only wants to use a single SPE sorbent, HLB can be used for both generic and selective extraction as it yielded similar chemical and effect recovery as HRX for a wide range of micropollutants. This paper is supplemented by the final SOP that includes a variant for generic extraction and one for the extraction of estrogenic chemicals.

Separating the Living from the Dead: An Electrophoretic Approach.

Cell viability studies are essential in numerous applications, including drug development, clinical analysis, bioanalytical assessments, food safety, and environmental monitoring. Microfluidic electrokinetic (EK) devices have been proven to be effective platforms to discriminate microorganisms by their viability status. Two decades ago, live and dead Escherichia coli (E. coli) cells were trapped at distinct locations in an insulator-based EK (iEK) device with cylindrical insulating posts. At that time, the discrimination between live and dead cells was attributed to dielectrophoretic effects. This study presents the continuous separation between the live and dead E. coli cells, which was achieved primarily by combining linear and nonlinear electrophoretic effects in an iEK device. First, live and dead E. coli cells were characterized in terms of their electrophoretic migration, and then the properties of both live and dead E. coli cells were input into a mathematical model built using COMSOL Multiphysics software to identify appropriate voltages for performing an iEK separation in a T-cross iEK channel. Subsequently, live and dead cells were successfully separated experimentally in the form of an electropherogram, achieving a separation resolution of 1.87. This study demonstrated that linear and nonlinear electrophoresis phenomena are responsible for the discrimination between live and dead cells under DC electric fields in iEK devices. Continuous electrophoretic assessments, such as the one presented here, can be used to discriminate between distinct types of microorganisms including live and dead cell assessments.

Exploring the Impacts of Angelica purpurascens Extracts on Anticholinergic, Antidiabetic, Antibacterial Potential, and Antioxidant Capacity

In Kars-Sarıkamış-Soğanlı, Angelica purpurascens (A. purpurascens) emerges as a promising natural antioxidant source. Extracts from its leaves, branches, and flowers underwent thorough bioanalytical assessments. The leaf extract exhibited the highest concentrations of herbal flavonoids (45.22 µg QE/mg extract) and total phenolics (28.96 µg GAE/mg extract). Branch extracts demonstrated significant enzymatic activity against AChE and BChE with IC50 values of 37.26 mg/mL and 9.08 mg/mL respectively. The flower extract displayed notable antibacterial properties. This study sheds light on the therapeutic potential of A. purpurascens ethanol extracts, suggesting benefits for Alzheimer’s, cell damage-induced diseases, and diabetes mellitus. It pioneers new enzymatic and antioxidant insights and contributes to understanding this abundant Türkiye species. A. purpurascens holds promise for pharmaceutical exploration, offering potential solutions for challenging health conditions

Validation of Anti-Adeno Associated Virus Serotype rh10 (AAVrh.10) Total and Neutralizing Antibody Immunogenicity Assays

Immunogenicity assessment of Adeno-Associated Virus (AAV) vectors is a critical part of gene therapy drug development. Whether the assays are used for inclusion/exclusion criteria or to monitor the safety and efficacy of the gene therapy, they are critical bioanalytical assessments. While total anti-AAV assays are perceived as easier to develop and implement than neutralizing anti-AAV assays, the gene therapy field is still nascent, and it is not yet clear which of the assays should be implemented at what stage of drug development. Recently AAVrh.10 has gained interest for use in gene therapies targeting cardiac, neurological, and other diseases due to its enhanced transduction efficiency. There is limited information on anti-AAVrh.10 antibodies and their clinical impact; thus, the information presented herein documents the validation of both a total antibody assay (TAb) and a neutralizing antibody (NAb) assay for anti-AAVrh.10 antibodies. In this manuscript, the validation was performed in accordance with the 2019 FDA immunogenicity guidance with additional evaluations to comply with CLIA where applicable. The AAVrh.10 TAb and NAb assays were compared in terms of sensitivity, drug tolerance, and precision, along with a concordance analysis using the same individual serum samples. This comparison gave insight into the utility of each format as a screening assay for inclusion into clinical studies.

Bioanalytical and chemical characterization of organic micropollutant mixtures in long-term exposed passive samplers from the Joint Danube Survey 4: Setting a baseline for water quality monitoring

Monitoring methodologies reflecting the long-term quality and contamination of surface waters are needed to obtain a representative picture of pollution and identify risk drivers. This study sets a baseline for characterizing chemical pollution in the Danube River using an innovative approach, combining continuous three-months use of passive sampling technology with comprehensive chemical (747 chemicals) and bioanalytical (seven in vitro bioassays) assessment during the Joint Danube Survey (JDS4). This is one of the world's largest investigative surface-water monitoring efforts in the longest river in the European Union, which water after riverbank filtration is broadly used for drinking water production. Two types of passive samplers, silicone rubber (SR) sheets for hydrophobic compounds and AttractSPETM HLB disks for hydrophilic compounds, were deployed at nine sites for approximately 100 days. The Danube River pollution was dominated by industrial compounds in SR samplers and by industrial compounds together with pharmaceuticals and personal care products in HLB samplers. Comparison of the Estimated Environmental Concentrations with Predicted No-Effect Concentrations revealed that at the studied sites, at least one (SR) and 4–7 (HLB) compound(s) exceeded the risk quotient of 1. We also detected AhR-mediated activity, oxidative stress response, peroxisome proliferator-activated receptor gamma-mediated activity, estrogenic, androgenic, and anti-androgenic activities using in vitro bioassays. A significant portion of the AhR-mediated and estrogenic activities could be explained by detected analytes at several sites, while for the other bioassays and other sites, much of the activity remained unexplained. The effect-based trigger values for estrogenic and anti-androgenic activities were exceeded at some sites. The identified drivers of mixture in vitro effects deserve further attention in ecotoxicological and environmental pollution research. This novel approach using long-term passive sampling provides a representative benchmark of pollution and effect potentials of chemical mixtures for future water quality monitoring of the Danube River and other large water bodies.

In vitro bioanalytical assessment of the occurrence and removal of bioactive chemicals in municipal wastewater treatment plants in Korea

Effluents of wastewater treatment plants (WWTPs) contain various organic micropollutants, some of which can exert negative effects on the quality of receiving waters or drinking water sources. This study monitored two full-scale WWTPs in Korea for the occurrence and removal of bioactive chemicals for a one-year period using a battery of in vitro bioassays as a complementary approach to chemical analysis. Bioassays covering different endpoints were employed, such as hormone receptor activation (AR and ERα), xenobiotic metabolism (PAH and PXR), oxidative stress response (Nrf2), and cytotoxicity. The WWTP influents showed AR, ERα, and PAH activities at ng/L - μg/L and PXR and Nrf2 activities at μg/L - mg/L as bioanalytical equivalent concentrations of a reference compound for each bioassay. These bioactivities decreased along with the WWTP treatment train, with significant removals achieved by the secondary biological treatment processes. Cytotoxicity was observed only for some municipal wastewater (M-WWTP) influents but was below the limit of quantification for most cases. The influent and effluent bioactivities observed in this study were mostly comparable to those reported in other WWTPs in the literature. Comparison of the bioactivities with the effect-based trigger (EBT) values indicates that the impact of WWTP effluents on receiving water quality was low for most endpoints. For Nrf2, however, further investigation is required to evaluate the observed high bioactivities compared with the current EBT. The observed ERα activity could partly be explained by the presence of some steroid estrogens. Overall, our results contribute to an important database for the concentrations and removal efficiencies of bioactive chemicals in WWTPs and demonstrate bioassays as a useful tool for urban water quality monitoring.

Selective reversed-phase high-performance liquid chromatography method for the determination of intact SARS-CoV-2 spike protein

Protein-based vaccines are playing an increasingly important role in the COVID-19 pandemic. As late-stage clinical data are finalized and released, the number of protein-based vaccines expected to enter the market will increase significantly. Most protein-based COVID-19 vaccines are based on the SARS-CoV-2 spike protein (S-protein), which plays a major role in viral attachment to human cells and infection. As a result, in order to develop and manufacture quality vaccines consistently, it is imperative to have access to selective and efficient methods for the bioanalytical assessment of S-protein. In this study, samples of recombinant S-protein (hexS-protein) and commercial S-protein were used to develop a selective reversed-phase HPLC (RP-HPLC) method that enabled elution of the intact S-protein monomer as a single peak on a wide pore, C8-bonded chromatographic column. The S-protein subunits, S1 and S2 subunits, were clearly separated from intact S-protein and identified. The results of this study set the foundation for reversed-phase HPLC method development and analysis for selective and efficient separation of S-protein monomer from its subunits.

Evaluation of Cilnidipine Loaded Selfmicroemulsifying Drug Delivery System (Smedds) by Quantification of Comparative Pharmacokinetic Parameters Using Validated Lc-Esi-Ms/Ms Bioanalytical Method and Pharmacodynamic Assessment

Evaluation of Cilnidipine Loaded Selfmicroemulsifying Drug Delivery System (Smedds) by Quantification of Comparative Pharmacokinetic Parameters Using Validated Lc-Esi-Ms/Ms Bioanalytical Method and Pharmacodynamic Assessment

Instrumental and bioanalytical assessment of pharmaceuticals and hormone-like compounds in a major drinking water source-wastewater receiving Zayandeh Rood river, Iran.

Zayandeh Rood river is the most important river in central Iran supplying water for a variety of uses including drinking water for approximately three million inhabitants. The study aimed to investigate the quality of water concerning the presence of pharmaceutical active compounds (PhACs) and hormonelike compounds, which have been only poorly studied in this region. Sampling was performed at seven sites along the river (from headwater sites to downstream drinking water source, corresponding drinking water, and treated wastewater) affected by wastewater effluents, specific drought conditions, and high river-water demand. The targeted and nontargeted chemical analyses and in vitro bioassays were used to evaluate the presence of PhACs and hormonelike compounds in river water. In the samples, 57 PhACs and estrogens were detected with LC-MS/MS with the most common and abundant compounds valsartan, carbamazepine, and caffeine present in the highest concentrations in the treated wastewater in the concentrations of 8.4, 19, and 140 μg/L, respectively. A battery of in vitro bioassays detected high estrogenicity, androgenicity, and AhR-mediated activity (viz., in treated wastewater) in the concentrations 24.2 ng/L, 62.2 ng/L, and 0.98 ng/L of 17β-estradiol, dihydrotestosterone and 2,3,7,8-TCDD equivalents, respectively. In surface water samples, estrogenicity was detected in the range of <0.42 (LOD) to 1.92 ng/L of 17β-estradiol equivalents, and the drinking water source contained 0.74 ng/L of 17β-estradiol equivalents. About 19% of the estrogenicity could be explained by target chemical analyses, and the remaining estrogenicity can be at least partially attributed to the potentiation effect of detected surfactant residues. Drinking water contained several PhACs and estrogens, but the overall assessment suggested minor human health risk according to the relevant effect-based trigger values. To our knowledge, this study provides some of the first comprehensive information on the levels of PhACs and hormones in Iranian waters.

In vitro bioanalytical assessment of toxicity of wetland samples from Spanish Mediterranean coastline

BackgroundFresh water bodies represent less than 1% of overall amount of water on earth and ensuring their quality and sustainability is pivotal. Although several campaigns have been performed to monitor the occurrence of micropollutants by means of chemical analysis, this might not cover the whole set of chemicals present in the sample nor the potential toxic effects of mixtures of natural and anthropogenic chemicals. In this sense, by selecting relevant toxicity endpoints when performing in vitro bioanalysis, effect-based methodologies can be of help to perform a comprehensive assessment of water quality and reveal biological activities relevant to adverse health effects. However, no prior bioanalytical study was performed in wetland water samples from the Spanish Mediterranean coastline.MethodsEleven samples from relevant water bodies from the Spanish Mediterranean coastline were collected to monitor water quality on 8 toxicity endpoints. Aryl hydrocarbon receptor (AhR), androgenicity (AR+ and AR−), estrogenicity (ER+ and ER−), oxidative stress response (Nrf2) and vitamin D receptor (VDR+ and VDR−) reporter gene assays were evaluated.ResultsAhR was the reporter gene assay showing a more frequent response over the set of samples (activated by 9 out of 11 samples), with TCDD-eq in the range 7.7–22.2 pM. For AR, ER and VDR assays sporadic activations were observed. Moreover, no activity was observed on the Nrf2 reporter gene assay. Wastewater and street runaway streams from Valencia could be responsible for enhanced activities in one of the water inputs in the Natural Park ‘L’Albufera’.ConclusionsWater quality of relevant wetlands from the Spanish Mediterranean coastline has been evaluated. The utilization of a panel of 5 different bioassays to cover for different toxicity endpoints has demonstrated to be a good tool to assess water quality.

Chemical and in vitro bioanalytical assessment of drinking water quality in Manhiça, Mozambique

The chemical quality of drinking water is widely unknown in low-income countries. We conducted an exploratory study in Manhiça district (Mozambique) to evaluate drinking water quality using chemical analyses and cell-based assays. We measured nitrate, fluoride, metals, pesticides, disinfection by-products, and industrial organochlorinated chemicals, and conducted the bioassays Ames test for mutagenicity, micronuclei assay (MN-FACS), ER-CALUX, and antiAR-CALUX in 20 water samples from protected and unprotected sources. Nitrate was present in all samples (median 7.5 mg/L). Manganese, cobalt, chromium, aluminium, and barium were present in 90-100% of the samples, with median values of 32, 0.6, 2.0, 61, 250 μg/l, respectively. Manganese was above 50 μg/l (EU guideline) in eight samples. Arsenic, lead, nickel, iron, and selenium median values were below the quantification limit. Antimony, cadmium, copper, mercury, zinc and silver were not present. Trihalomethanes, haloacetic acids, haloacetonitriles and haloketones were present in 5-28% samples at levels ≤4.6 μg/l. DDT, dieldrin, diuron, and pirimiphos-methyl were quantified in 2, 3, 3, and 1 sample, respectively (range 12-60 ng/L). Fluoride was present in one sample (0.11 mg/l). Trichloroethene and tetrachloroethene were not present. Samples were negative in the in vitro assays. Results suggest low exposure to chemicals, mutagenicity, genotoxicity and endocrine disruption through drinking water in Manhiça population. High concentration of manganese in some samples warrants confirmatory studies, given the potential link to impaired neurodevelopment.

Advancements in effect-based surface water quality assessment

Legally-prescribed chemical monitoring is unfit for determining the pollution status of surface waters, and there is a need for improved assessment methods that consider the aggregated risk of all bioavailable micropollutants present in the aquatic environment. Therefore, the present study aimed to advance effect-based water quality assessment by implementing methodological improvements and to gain insight into contamination source-specific bioanalytical responses. Passive sampling of non-polar and polar organic compounds and metals was applied at 14 surface water locations that were characterized by two major anthropogenic contamination sources, agriculture and wastewater treatment plant (WWTP) effluent, as well as reference locations with a low expected impact from micropollutants. Departing from the experience gained in previous studies, a battery of 20 in vivo and in vitro bioassays was composed and subsequently exposed to the passive sampler extracts. Next, the bioanalytical responses were divided by their respective effect-based trigger values to obtain effect-based risk quotients, which were summed per location. These cumulative ecotoxicological risks were lowest for reference locations (4.3–10.9), followed by agriculture locations (11.3–27.2) and the highest for WWTP locations (12.8–47.7), and were mainly driven by polar organic contaminants. The bioanalytical assessment of the joint risks of metals and (non-)polar organic compounds resulted in the successful identification of pollution source-specific ecotoxicological risk profiles: none of the bioassays were significantly associated with reference locations nor with multiple location types, while horticulture locations were significantly characterized by anti-AR and anti-PR activity and cytotoxicity, and WWTP sites by ERα activity and toxicity in the in vivo bioassays. It is concluded that the presently employed advanced effect-based methods can readily be applied in surface water quality assessment and that the integration of chemical- and effect-based monitoring approaches will foster future-proof water quality assessment strategies on the road to a non-toxic environment.

Bioanalytical Assessment of Plasma Concentrations of Angiotensin-Converting Enzyme II Inhibitors and Angiotensin Receptor Blockers: A Pilot Study Among Patients Hospitalized With Acute Heart Failure.

Although angiotensin-converting enzyme II inhibitors (ACEIs) and angiotensin II receptor blockers (ARBs) improve chronic heart failure (HF) outcomes, their potential harms and benefits in acute HF (AHF) is less clear. We explored the relationship between ACEI or ARB plasma concentrations among patients with AHF with in-hospital change in estimated glomerular filtration rate (eGFR). From August 2016-June 2017, patients with AHF prescribed an outpatient ACEI or ARB were enrolled before AHF treatment. All patients were given twice their home dose of diuretic intravenously and received clinical care at the discretion of the medical team. Of 61 patients in the parent study, saved plasma from 34 who were prescribed an outpatient ACEI or ARB was included in this substudy. Liquid chromatography-tandem mass spectrometry was performed to assess ACEI or ARB plasma concentrations before AHF treatment. Change in eGFR was computed using the Chronic Kidney Disease Epidemiology Collaboration equation, which adjusts for age, sex, and race; diuretic dose and enrollment eGFR were used to adjust for HF severity. Multiple linear regression adjusting for enrollment eGFR and diuretic dose was performed to examine the relationship between drug concentration (undetectable/low vs. in/above-range) and in-hospital change in eGFR. Of 34 patients with AHF, median age was 63 years (interquartile range, 58-78 years), 19 (55.9%) were women, median eGFR at enrollment was 55.6 mL/min (interquartile range, 35.2-75.3 mL/min), and for 11 (32.4%), no ACEI or ARB was detectable in plasma. Medication concentrations in- or above-reference range were associated with in-hospital decrease in eGFR of 8.3 mL/min (95% confidence interval, 15.3-1.3 mL/min decrease), after adjusting for enrollment eGFR and diuretic treatment. Bioanalytical assessment of medication levels may be useful to guide in-hospital ACEI and ARB therapy for patients with AHF.

Receptor-mediated potencies of polycyclic aromatic hydrocarbons in urban sediments: comparisons of toxic equivalency risk assessment

The Klip River, flowing through South Africa’s most populated urban area—Soweto and Lenasia—is subject to various pollution and anthropogenic influences, including great concentrations of polycyclic aromatic hydrocarbons. The aims were to determine the aryl-hydrocarbon receptor-mediated potencies of the 16 priority polycyclic aromatic hydrocarbons in sediments of the Klip River, using chemical- and bio-analytical assessments of hazard, and to compare these results with international sediment quality guidelines. Sediment samples were collected from nine sites during the dry seasons of 2013 and 2014. Two sets of toxic equivalents were calculated from analytically obtained polycyclic aromatic hydrocarbon concentrations using: (1) 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalency factors and (2) relative potency factors for fish. The fraction of the sediment extracts containing polycyclic aromatic hydrocarbons was assayed with the H4IIE-luc reporter gene bio-assay, and the aryl-hydrocarbon receptor potency expressed as bio-assay equivalents. The bio-assay equivalents and tetrachlorodibenzo-p-dioxin equivalency factors were compared to Canadian sediment quality guidelines and of the three approaches, the bio-assay equivalents and the relative potency factors for fish proved the most protective. Results of this study are proof of the utility of combining biological analysis with instrumental analysis when predicting hazard. Even though there were instances where the bio-assay equivalents were orders of magnitude greater than the tetrachlorodibenzo-p-dioxin equivalency factors, the results still showed similar trends. It was concluded that hazard from aryl-hydrocarbon receptor-mediated potency to adversely affect aquatic organisms in the Klip River was relatively great, which indicated the need for further investigation into possible mitigations.

Multiplexed immunoassay approach to characterize antidrug antibody like specific reactivity.

Aim: Characterization of antidrug antibody (ADA)-like reactivity has emerged as critical element of bioanalytical design and assessment of compound immunogenicity risk. Materials& methods: Multiplex immunoassay was applied to detect and characterize ADA like reactivity using Photonic Ring Immunoassay platform (Genalyte). Specific binding to human IgE or human recombinant IL21-receptor-Fc fusion using exogenous reagents as surrogates for drug-specific reactivity was investigated. Results: Multiplexed assay format allowed identification of spiked antihuman IgE reactivity as murine IgG1 and endogenous antihuman recombinant IL21-receptor-Fc reactivity in rheumatoid arthritis sera as antihuman Fc-specific binding. Conclusion:The ability of a multiplex immunoassay platform to identify isotype and domain specificity of antidrug immunoglobulins was shown to be effective and should be considered when screening and characterizing pre- and post-dose ADA reactivity.

40EMF Outpatient Lisinopril and Losartan Blood Levels Are Associated With In-Hospital Acute Kidney Injury Among Patients With Acute Heart Failure

We report a novel tandem liquid chromatography mass spectrometry (LC/MS/MS) method for the simultaneous bioanalytical assessment of blood levels for multiple commonly prescribed medications, including angiotensin-converting enzyme inhibitors (ACEI) or angiotensin II receptor blockers (ARB). Despite morbidity and mortality benefits of ACEI/ARBs, these medications may cause a transient rise in creatinine and occasionally contribute to hypotension, which in turn may result in acute kidney injury (AKI). Previous studies of AKI in the setting of ACEI/ARBs have not assessed medication levels. We hypothesized ACEI/ARB plasma levels in/above reference range for patients with AHF treated by intravenous (IV) diuretics would be associated with increased risk of in-hospital AKI compared to undetectable/low ACEI/ARB plasma levels. From June 2016 to June 2017, adults with AHF were approached for consent. Exclusions were: 1) no outpatient ACEI/ARB, 2) systolic blood pressure <90 mm Hg, 3) IV diuretic before enrollment, 4) allergy to furosemide or bumetanide, or 5) any dialysis. Patients were given an initial IV dose of diuretic (2x their daily dose) and received standard clinical care during hospitalization. Medication plasma levels at ED presentation, before AHF therapy, were assessed using LC/MS/MS. Standardized plasma concentrations for each medication were computed using the median published reference range. AKI was defined as a rise in creatinine at 48 hours (or at hospital discharge, if unavailable) of ≥0.3 mg/dL or ≥1.5x baseline. Mean age for the 37 patients was 65.9 years (sd 14.6), 20 (54.0%) were female, 11 (29.7%) were African American, 10 (32.3%) had an ejection fraction <40%, and 11 (29.8%) did not have detectible ACEI/ARB levels. Of the 5 (13.5%) patients who developed in-hospital AKI, 4 had medications in/above reference range. ACEI/ARB levels were higher for patients with AKI (Figure), and levels in/above reference range were associated with a rise in creatinine (beta 0.22, 95% confidence interval 0.02-0.42, P=0.03, adjusted for age, sex, and race). Patients with AHF may be more likely to develop AKI in the hospital if they have normal/high ACEI/ARB blood levels at ED presentation. Bioanalytical assessment of ACEI/ARB levels may prove useful for guiding in-hospital medical therapy for AHF.

Analytical and bioanalytical assessments of organic micropollutants in the Bosna River using a combination of passive sampling, bioassays and multi-residue analysis

Complex mixtures of contaminants from multiple sources, including agriculture, industry or wastewater enter aquatic environments and might pose hazards or risks to humans or wildlife. Targeted analyses of a few priority substances provide limited information about water quality. In this study, a combined chemical and effect screening of water quality in the River Bosna, in Bosnia and Herzegovina was carried out, with focus on occurrence and effects of contaminants of emerging concern. Chemicals in water were sampled at 10 sites along the Bosna River by use of passive sampling. The combination of semipermeable membrane devices (SPMDs) and polar organic chemical integrative samplers (POCIS) enabled sampling of a broad range of contaminants from hydrophobic (PAHs, PCBs, OCPs) to hydrophilic compounds (pesticides, pharmaceuticals and hormones), which were determined by use of GC–MS and LC-MS (MS). In vitro, cell-based bioassays were applied to assess (anti)androgenic, estrogenic and dioxin-like potencies of extracts of the samplers. Of a total of 168 targeted compounds, 107 were detected at least once. Cumulative pollutant concentrations decreased downstream from the city of Sarajevo, which was identified as the major source of organic pollutants in the area. Responses in all bioassays were observed for samples from all sites. In general, estrogenicity could be well explained by analysis of target estrogens, while the drivers of the other observed effects remained largely unknown. Profiling of hazard quotients identified two sites downstream of Sarajevo as hotspots of biological potency. Risk assessment of detected compounds revealed, that 7 compounds (diazinon, diclofenac, 17β-estradiol, estrone, benzo[k]fluoranthene, fluoranthene and benzo[k]fluoranthene) might pose risks to aquatic biota in the Bosna River. The study brings unique results of a complex water quality assessment in a region with an insufficient water treatment infrastructure.

Antibody-drug conjugates: integrated bioanalytical and biodisposition assessments in lead optimization and selection

Therapies based on monoclonal antibodies (mAbs) have delivered an impressive success in the clinics due to their exquisite specificity, potential for agonistic or antagonistic responses, tunable effector function, and optimal pharmacokinetic properties. Building on these inherent antibody properties, the design and development of antibody-drug conjugates (ADCs) with improved or gained therapeutic activity and safety has been successfully demonstrated in oncological applications. There is enormous potential for this new type of hybrid biologics but there are also significant engineering, manufacturing and bioanalytical challenges. In this manuscript, we highlight the range and diversity of assays that are critical to characterize the individual components of ADCs-linker, carrier, and payload. We discuss a series of in vitro and in vivo preclinical experimental approaches we implemented to characterize two anti-inflammatory steroid bearing ADCs, and an ADC bearing a modified glucagon-like peptide 1 receptor/glucagon receptor co-agonist peptide.

Outpatient Lisinopril and Losartan Blood Levels are Associated With In-Hospital Acute Kidney Injury Among Patients with Acute Heart Failure

IntroductionWe report on a novel tandem liquid chromatography mass spectrometry (LC/MS/MS) method for the simultaneous bioanalytical assessment of blood levels for multiple commonly prescribed medications, including angiotensin-converting enzyme inhibitors (ACEI) and angiotensin II receptor blockers (ARB). Despite morbidity and mortality benefits of ACEI/ARBs, these medications may cause transient rise in creatinine and occasionally contribute to hypotension, which in turn may result in acute kidney injury (AKI). Previous studies of AKI in the setting of ACEI/ARBs have not assessed medication levels.HypothesisWe hypothesized ACEI/ARB plasma levels in/above reference range for patients with AHF treated by intravenous (IV) diuretics would be associated with increased risk of in-hospital AKI compared to undetectable/low ACEI/ARB plasma levels.MethodsFrom June 2016 to June 2017, adults with AHF were approached for consent. Patients were excluded for: 1) no outpatient ACEI/ARB, 2) systolic blood pressure <90 mm Hg, 3) IV diuretic before enrollment, 4) allergy to furosemide or bumetanide, or 5) any dialysis. Patients were given an initial IV dose of diuretic (2x their daily dose) and received standard clinical care during hospitalization. LC/MS/MS assessed medication plasma levels at ED presentation, before AHF therapy. Standardized plasma concentrations for each medication were computed using the median published reference range. AKI was defined as a rise in creatinine at 48 hours (or at hospital discharge, if unavailable) of ≥0.3 mg/dL or ≥1.5x baseline.ResultsMean age for the 37 patients was 65.9 years (sd 14.6), 20 (54.0%) were female, 11 (29.7%) were African American, 10 (32.3%) had an ejection fraction <40%, and 11 (29.8%) did not have detectible ACEI/ARB levels. Of the 5 (13.5%) patients who developed in-hospital AKI, 4 had medications in/above reference range. ACEI/ARB levels were higher for patients with AKI (Figure), and ACEI/ARB levels in/above reference range at the time of ED presentation were associated with an in-hospital rise in creatinine (beta 0.22, 95% confidence interval 0.02-0.42, P=0.03, adjusted for age, sex, and race).ConclusionsPatients with AHF may be more likely to develop AKI in the hospital if they have ACEI/ARB blood levels in/above reference range at ED presentation. Bioanalytical assessment of ACEI/ARB levels may prove useful for guiding in-hospital medical therapy for AHF. We report on a novel tandem liquid chromatography mass spectrometry (LC/MS/MS) method for the simultaneous bioanalytical assessment of blood levels for multiple commonly prescribed medications, including angiotensin-converting enzyme inhibitors (ACEI) and angiotensin II receptor blockers (ARB). Despite morbidity and mortality benefits of ACEI/ARBs, these medications may cause transient rise in creatinine and occasionally contribute to hypotension, which in turn may result in acute kidney injury (AKI). Previous studies of AKI in the setting of ACEI/ARBs have not assessed medication levels. We hypothesized ACEI/ARB plasma levels in/above reference range for patients with AHF treated by intravenous (IV) diuretics would be associated with increased risk of in-hospital AKI compared to undetectable/low ACEI/ARB plasma levels. From June 2016 to June 2017, adults with AHF were approached for consent. Patients were excluded for: 1) no outpatient ACEI/ARB, 2) systolic blood pressure <90 mm Hg, 3) IV diuretic before enrollment, 4) allergy to furosemide or bumetanide, or 5) any dialysis. Patients were given an initial IV dose of diuretic (2x their daily dose) and received standard clinical care during hospitalization. LC/MS/MS assessed medication plasma levels at ED presentation, before AHF therapy. Standardized plasma concentrations for each medication were computed using the median published reference range. AKI was defined as a rise in creatinine at 48 hours (or at hospital discharge, if unavailable) of ≥0.3 mg/dL or ≥1.5x baseline. Mean age for the 37 patients was 65.9 years (sd 14.6), 20 (54.0%) were female, 11 (29.7%) were African American, 10 (32.3%) had an ejection fraction <40%, and 11 (29.8%) did not have detectible ACEI/ARB levels. Of the 5 (13.5%) patients who developed in-hospital AKI, 4 had medications in/above reference range. ACEI/ARB levels were higher for patients with AKI (Figure), and ACEI/ARB levels in/above reference range at the time of ED presentation were associated with an in-hospital rise in creatinine (beta 0.22, 95% confidence interval 0.02-0.42, P=0.03, adjusted for age, sex, and race). Patients with AHF may be more likely to develop AKI in the hospital if they have ACEI/ARB blood levels in/above reference range at ED presentation. Bioanalytical assessment of ACEI/ARB levels may prove useful for guiding in-hospital medical therapy for AHF.