Human Plasma Proteins Research Articles

Molecular interaction of fluorescent carbon dots from mature vinegar with human hemoglobin: Insights from spectroscopy, thermodynamics and AFM

Foodborne nanoparticles have attracted considerable interest due to their distinctive fluorescence and physicochemical properties. The discovery of vinegar carbon dots (VCDs) has drawn our attention to study their effect on human plasma protein. Herein, spectral, constructional, morphological, and enzymatic activity assessments were carried out to investigate the interaction of VCDs with human hemoglobin (HHb). The intrinsic fluorescence of HHb was quenched significantly by the VCDs through a static quenching process. Furthermore, binding constants and important thermodynamic parameters were calculated, the negative enthalpy and entropy changes were accompanied by a negative Gibbs energy, which proposed the binding between VCDs with HHb was spontaneous. Moreover, negative enthalpy and entropy change corroborated the involvement of van der Waals force and hydrogen bonds in the binding process. Results from FTIR, atomic force microscopy and circular dichroism revealed change of HHB after binding with VCDs although their essential morphological features were unaffected. The esterase activity of HHb decreased after VCDs treatment in a dose-dependent manner, which further confirmed the effect of VCDs on HHb. The results offered detailed information about the interaction between VCDs and HHb.

The GWAS-MAP platform for aggregation of results of genome-wide association studies and the GWAS-MAP|homo database of 70 billion genetic associations of human traits.

Hundreds of genome-wide association studies (GWAS) of human traits are performed each year. The results of GWAS are often published in the form of summary statistics. Information from summary statistics can be used for multiple purposes – from fundamental research in biology and genetics to the search for potential biomarkers and therapeutic targets. While the amount of GWAS summary statistics collected by the scientific community is rapidly increasing, the use of this data is limited by the lack of generally accepted standards. In particular, the researchers who would like to use GWAS summary statistics in their studies have to become aware that the data are scattered across multiple websites, are presented in a variety of formats, and, often, were not quality controlled. Moreover, each available summary statistics analysis tools will ask for data to be presented in their own internal format. To address these issues, we developed GWAS-MAP, a high-throughput platform for aggregating, storing, analyzing, visualizing and providing access to a database of big data that result from region- and genome-wide association studies. The database currently contains information on more than 70 billion associations between genetic variants and human diseases, quantitative traits, and “omics” traits. The GWAS-MAP platform and database can be used for studying the etiology of human diseases, building predictive risk models and finding potential biomarkers and therapeutic interventions. In order to demonstrate a typical application of the platform as an approach for extracting new biological knowledge and establishing mechanistic hypotheses, we analyzed varicose veins, a disease affecting on average every third adult in Russia. The results of analysis confirmed known epidemiologic associations for this disease and led us to propose a hypothesis that increased levels of MICB and CD209 proteins in human plasma may increase susceptibility to varicose veins.

Stabilization of a potential anticancer thiosemicarbazone derivative in Sudlow site I of human serum albumin: In vitro spectroscopy coupled with molecular dynamics simulation

Human Serum Albumin (HSA) is the most important protein in human blood plasma and can acts as a major transporting agent for various drug molecules with flexible binding interaction. To elucidate the interaction of a newly designed potential anticancer thiosemicarbazone based luminophore (E)-1-(4-(diethylamino)-2-hydroxybenzylidene)-4,4-dimethyl-thiosemicarbazide (DAHTS) with HSA under physiological condition, in vitro optical spectroscopic experiments viz UV–Vis absorption, steady state fluorescence, fluroscence anisotropy, time resolved fluorscence (TRF) and cicular dichroism (CD) spectroscopy have been scrutinised. The experimental findings have been corroborated with in silico molecular docking analysis and Molecular Dynamics (MD) simulation. The spectroscopic results demonstrated that the conventionally anion-favouring Sudlow site I of HSA copiously adapt neutral DAHTS molecule with moderate binding affinity. The mean fluorescence lifetime of the sole tryptophan (Trp-214) present in the macromolecule experiences an appreciable diminution with an increase in concentration of the synthesized molecule. DAHTS localize itself close to Trp-214 within subdomain IIA (Sudlow site I) and surrounded by multiple hydrophobic amino acid residues (Val-235, Val-231, Ala-229, Phe-228, Val-325, Phe-326, Leu-327, Met-329, Phe-330, Leu-331, Tyr-332, Leu-346, Leu-347, Val-482, Leu-349, Ala-350, Ala-210, Trp-214, Ala- 213 and Val-216) in HSA. The distinct fluorescence lifetime, diverse pathways and changing rate of population indicates that the rotamerisation of Trp-214 residue is controlled by the guest molecule. Sudlow site I of HSA behaves flexibly and induces an allosteric modulation in the macromolecule resulting a minor deformation in the protein secondary structure as observed in CD (observed 11% change of α-helix content) as well as in MD simulation. The integrated multi-spectroscopic research described herein provides several important information about the binding interaction of a thiosemicarbazone Schiff base with HSA, which can be very significant for thiosemicarbazone based drug designing for academia as well as industry.

FRACPRED‐2D‐PRM: A Fraction Prediction Algorithm‐Assisted 2D Liquid Chromatography‐Based Parallel Reaction Monitoring‐Mass Spectrometry Approach for Measuring Low‐Abundance Proteins in Human Plasma

Multidimensional fractionation-based enrichment methods improve the sensitivity of proteomic analysis for low-abundance proteins. However, a major limitation of conventional multidimensional proteomics is the extensive labor and instrument time required for analyzing many fractions obtained from the first dimension separation. Here, a fraction prediction algorithm-assisted 2D LC-based parallel reaction monitoring-mass spectrometry (FRACPRED-2D-PRM) approach for measuring low-abundance proteins in human plasma is presented. Plasma digests are separated by the first dimension high-pH RP-LC with data-dependent acquisition (DDA). The FRACPRED algorithm is then usedto predict the retention times of undetectable target peptides according to those of other abundant plasma peptides during the first dimension separation. Fractions predicted to contain target peptides are analyzed by the second dimension low-pH nano RP-LC PRM. The accuracy and robustness of fraction prediction with the FRACPRED algorithm are demonstrated by measuring two low-abundance proteins, aldolase B and carboxylesterase 1, in human plasma. The FRACPRED-2D-PRM proteomics approach demonstrates markedly improved efficiency and sensitivity over conventional 2D-LC proteomics assays. It is expected that this approach will be widely used in the study of low-abundance proteins in plasma and other complex biological samples.

Sample Displacement Batch Chromatography of Proteins.

In downstream processing, large-scale chromatography plays an important role. For its development, screening experiments followed by pilot-plant chromatography are mandatory steps. Here we describe fast, simple, and inexpensive methods for establishing a preparative chromatography for the separation of complex protein mixtures, based on sample displacement batch chromatography. The methods are demonstrated by anion-exchange chromatography of a human plasma protein fraction (Cohn IV-4), including the screening step and upscaling of the chromatography by a factor of one hundred. The results of the screening experiments and the preparative chromatography are monitored by SDS-PAGE electrophoresis. In summary, we provide a protocol, which should be easily adaptable for the chromatographic large-scale purification of other proteins, in the laboratory as well as in the manufacturing of biopharmaceuticals. These protocols cover the initial piloting steps for establishing a large-scale sample batch chromatography. The results from the piloting steps may also be applied for packed columns for performing simulated-moving-bed (SMB) chromatography rather than batch chromatography.

Applicability of capillary electrophoresis-frontal analysis for displacement studies: Effect of several drugs on l-tryptophan and lidocaine binding to human serum albumin.

The effective concentration of a drug in the blood, i.e. the concentration of a free drug in the blood, is influenced by the strength of drug binding onto plasma proteins. Besides its efficacy, these interactions subsequently influence the liberation, absorption, distribution, metabolism, excretion, and toxicological properties of the drug. It is important to not only determine the binding strength and stoichiometry, but also the binding site of a drug on the plasma protein molecule, because the co-administration of drugs with the same binding site can affect the above-mentioned concentration and as a result the pharmacological behavior of the drugs and lead to side effects caused by the change in free drug concentration, its toxicity. In this study, the binding characteristics of six drugs with human serum albumin, the most abundant protein in human plasma, were determined by capillary electrophoresis-frontal analysis, and the obtained values of binding parameters were compared with the literature data. The effect of several drugs and site markers on the binding of l-tryptophan and lidocaine to human serum albumin was investigated in subsequent displacement studies which thus demonstrated the usability of capillary electrophoresis as an automated high-throughput screening method for drug-protein binding studies.

Crystal structure of pharmaceutical-grade human serum albumin

Human serum albumin (HSA) is the most abundant protein in human plasma and plays versatile biological role. HSA has been widely used to treat several diseases and develop biocompatible biomaterials for biomedical applications. However, pharmaceutical-grade HSA (p-HSA) showed the altered oxidative and ligand-binding properties compare to native HSA. To investigate the influences of the manufacturing process on the molecular state of HSA, we determined the first crystal structure of p-HSA using the commercial HSA solution without any defatting step and further purification and carried out mass spectrometry to identify bound ligands. The crystal structure of p-HSA revealed that medium- and long-chain fatty acids and tryptophan are bound to p-HSA and one free cysteine is oxidized to cysteine-sulfenic acid. The mass spectra of p-HSA also confirmed the existence of fatty acids and tryptophan in p-HSA. Our results enhance understanding of the molecular state of p-HSA and can be utilized to produce p-HSA solutions and HSA-based biomaterials that has a higher biorelevance.

Engineered polymer nanoparticles incorporating l-amino acid groups as affinity reagents for fibrinogen

Synthetic polymer hydrogel nanoparticles (NPs) were developed to function as abiotic affinity reagents for fibrinogen. These NPs were made using both temperature-sensitive N-isopropyl acrylamide (NIPAm) and l-amino acid monomers. Five kinds of l-amino acids were acryloylated to obtain functional monomers: l-phenylalanine (Phe) and l-leucine (Leu) with hydrophobic side chains, l-glutamic acid (Glu) with negative charges, and l-lysine (Lys) and l-arginine (Arg) with positive charges. After incubating the NPs with fibrinogen, γ-globulin, and human serum albumin (HSA) respectively, the NPs that incorporated N-acryloyl-Arg monomers (AArg@NPs) showed the strongest and most specific binding affinity to fibrinogen, when compared with γ-globulin and HSA. Additionally, the fibrinogen-AArg binding model had the best docking scores, and this may be due to the interaction of positively charged AArg@NPs and the negatively charged fibrinogen D domain and the hydrophobic interaction between them. The specific adsorption of AArg@NPs to fibrinogen was also confirmed by the immunoprecipitation assay, as the AArg@NPs selectively trapped the fibrinogen from a human plasma protein mixture. AArg@NPs had a strong selectivity for, and specificity to, fibrinogen and may be developed as a potential human fibrinogen-specific affinity reagent.

Data mining of human plasma proteins generates a multitude of highly predictive aging clocks that reflect different aspects of aging.

ABSTRACTWe previously identified 529 proteins that had been reported by multiple different studies to change their expression level with age in human plasma. In the present study, we measured the q‐value and age coefficient of these proteins in a plasma proteomic dataset derived from 4263 individuals. A bioinformatics enrichment analysis of proteins that significantly trend toward increased expression with age strongly implicated diverse inflammatory processes. A literature search revealed that at least 64 of these 529 proteins are capable of regulating life span in an animal model. Nine of these proteins (AKT2, GDF11, GDF15, GHR, NAMPT, PAPPA, PLAU, PTEN, and SHC1) significantly extend life span when manipulated in mice or fish. By performing machine‐learning modeling in a plasma proteomic dataset derived from 3301 individuals, we discover an ultra‐predictive aging clock comprised of 491 protein entries. The Pearson correlation for this clock was 0.98 in the learning set and 0.96 in the test set while the median absolute error was 1.84 years in the learning set and 2.44 years in the test set. Using this clock, we demonstrate that aerobic‐exercised trained individuals have a younger predicted age than physically sedentary subjects. By testing clocks associated with 1565 different Reactome pathways, we also show that proteins associated with signal transduction or the immune system are especially capable of predicting human age. We additionally generate a multitude of age predictors that reflect different aspects of aging. For example, a clock comprised of proteins that regulate life span in animal models accurately predicts age.

Investigation of the influence of lead compounds with particles of different dispersity on human blood plasma proteins as an express method for evaluation of their safety

Investigation of the influence of lead compounds with particles of different dispersity on human blood plasma proteins as an express method for evaluation of their safety

A Novel Promazine Derivative Shows High in vitro and in vivo Antimicrobial Activity Against Staphylococcus aureus.

The emergence of multidrug-resistant bacteria constitutes a significant public health issue worldwide. Consequently, there is an urgent clinical need for novel treatment solutions. It has been shown in vitro that phenothiazines can act as adjuvants to antibiotics whereby the minimum inhibitory concentration (MIC) of the antibiotic is decreased. However, phenothiazines do not perform well in vivo, most likely because they can permeate the blood-brain (BBB) barrier and cause severe side-effects to the central nervous system. Therefore, the aim of this study was to synthesize a promazine derivate that would not cross the BBB but retain its properties as antimicrobial helper compound. Surprisingly, in vitro studies showed that the novel compound, JBC 1847 exhibited highly increased antimicrobial activity against eight Gram-positive pathogens (MIC, 0.5–2 mg/L), whereas a disc diffusion assay indicated that the properties as an adjuvant were lost. JBC 1847 showed significant (P < 0.0001) activity against a Staphylococcus aureus strain compared with the vehicle, in an in vivo wound infection model. However, both in vitro and in silico analyses showed that JBC 1847 possesses strong affinity for human plasma proteins and an Ames test showed that generally, it is a non-mutagenic compound. Finally, in silico predictions suggested that the compound was not prone to pass the BBB and had a suitable permeability to the skin. In conclusion, JBC 1847 is therefore suggested to hold potential as a novel topical agent for the clinical treatment of S. aureus skin and soft tissue infections, but pharmacokinetics and pharmacodynamics need to be further investigated.

Protein adsorption and bioactivity of functionalized electrospun membranes for bone regeneration

ObjectivesTo evaluate the adsorption of bone related proteins and bioactivity of experimental functionalized (carboxylated or aminated) polymeric membranes for bone regeneration. MethodsPolymethylmethacrylate-based membranes functionalized with carboxyl or amino radicals were tested. Membranes were zinc loaded and the adsorption isotherms of zinc were studied. Human plasma proteins, bovine serum albumin, fibrinogen and fibronectin adsorption were measured with a spectrophotometer applying an acid determination protocol. Biomimetic calcium phosphate precipitation on polymeric membranes was also assessed after simulated body fluid immersion. Scanning electron microscopy and elemental analysis by means of an energy dispersive system were used for mineral deposits identification. A commercially available polytetrafluoroethylene membrane was used as control. ResultsBoth experimental membranes produced higher protein adsorption than the commercial control that does not adsorb proteins. Carboxylated membranes adsorbed significantly more albumin than the aminated ones, the opposite occurred with fibrinogen. With plasma and fibronectin proteins both type of membranes performed similarly. Only carboxylated membranes were bioactive and precipitated calcium and phosphate on their surfaces. ConclusionsThe polymethylmethacrylate zinc-loaded membranes functionalized with carboxyl groups performed as high adsorbable membranes for bone regeneration related proteins. They also served as templates for mineralization of hydroxyapatite. Clinical significanceProtein adsorption is the initial reaction after the implantation of a biomaterial into the body and will influence subsequent cell function. The adsorption of bone related proteins together with the observed biomimetic calcium deposition on the experimental carboxylated membranes could be associated with their ability to promote bone regeneration.

Transthyretin Amyloidogenesis Inhibitors: From Discovery to Current Developments.

Transthyretin (TTR) is a homotetrameric protein in human plasma. The dissociation of the TTR tetramer and misfolding of the TTR monomer result in the formation of amyloid fibrils. Hereditary TTR amyloidosis is characterized by the extracellular deposition of amyloid fibrils containing TTR variants. The development of small molecules that kinetically stabilize the TTR tetramer is one of the effective strategies for the treatment of hereditary TTR amyloidosis. So far, several stabilizers have been discovered. Tafamidis is the only approved stabilizer for treatment of hereditary TTR amyloidosis, although two nucleic acid medicines that inhibit TTR synthesis, inotersen and patisiran, were recently approved for treatment of this disorder. In this Perspective, we seek to describe the representative kinetic stabilizers from discovery to development, interweaving the crystallographic study of the complex structures.

Plasma proteins facilitates placental transfer of polystyrene particles

BackgroundNanoparticles, which are exposed to biological fluids are rapidly interacting with proteins and other biomolecules forming a corona. In addition to dimension, charge and material the distinct protein corona influences the interplay of nanoparticles with tissue barriers. In this study we were focused on the impact of in situ formed human plasma protein corona on the transfer of 80 nm polystyrene nanoparticles (PS-particles) across the human placenta. To study materno-to fetal PS transfer we used the human ex vivo placental perfusion approach, which represents an intact and physiological tissue barrier. To analyze the protein corona of PS particles we performed shotgun proteomics of isolated nanoparticles before and after tissue exposure.ResultsHuman plasma incubated with PS-particles of 80 nm and subsequent formed protein corona enhanced the transfer across the human placenta compared to PS-corona formed by bovine serum albumin and dextran which served as a control. Quantitative and qualitative changes of plasma proteins determined the changes in PS transfer across the barrier. Based on the analysis of the PS-proteome two candidate proteins, namely human albumin and immunoglobulin G were tested if these proteins may account for the enhanced PS-transfer across the placenta. Interestingly, the protein corona formed by human albumin significantly induced the transfer of PS-particles across the tissue compared to the formed IgG-corona.ConclusionIn total we demonstrate the PS corona dynamically and significantly evolves upon crossing the human placenta. Thus, the initial composition of PS particles in the maternal circulation is not predictive for their transfer characteristics and performance once beyond the barrier of the placenta. The precise mechanism of these effects remains to be elucidated but highlights the importance of using well designed biological models when testing nanoparticles for biomedical applications.

Elucidation of molecular interaction of bioactive flavonoid luteolin with human serum albumin and its glycated analogue using multi-spectroscopic and computational studies

Human serum albumin (HSA), an abundant protein in human plasma, which is associated with the transportation of numerous drugs, fatty acids to their targets and regulates the blood pH levels, is exposed to non-enzymatic glycation by reducing sugars. The existence of elevated levels of glucose during diabetes mellitus mediates the glycation of HSA which leads to structural and functional modification of the protein. The aim of our present study is to determine the effect of non-enzymatic glycation on the binding of a bioactive flavonoid luteolin to HSA using multi-spectroscopic and computational studies. The intrinsic fluorescence exhibited by the proteins (HSA and gHSA) were quenched by luteolin through static quenching mechanism. The binding constant for the interaction of HSA with luteolin was found higher as compared to that of gHSA-Luteolin at the experimental temperatures (290, 300 and 310 K). The ∆G values for the complexes formed between HSA/gHSA and luteolin were observed to be negative, indicating the spontaneity of the binding processes. Both enthalpy and entropy factors contribute to the binding of luteolin to HSA, whereas, enthalpy factors played a major role in the binding of luteolin to gHSA. A reduction in the α-helical content of HSA-Luteolin complex was observed, but no significant change was found for gHSA-Luteolin complex. Site probe displacement and related binding studies indicated that luteolin binds to both Subdomain IIA and IIIA of HSA and gHSA but with different affinities. Further molecular docking and MD simulation studies confirmed that luteolin preferably binds to the Subdomain IIA of HSA and IIIA of gHSA. These information, in turn, would give a better understanding of the functional changes occurred due to structural modification of HSA induced by glycation and may have some useful impact on the field of pharmaceutical sciences.

Dickkopf-1 Overexpression in vitro Nominates Candidate Blood Biomarkers Relating to Alzheimer's Disease Pathology.

Background:Previous studies suggest that Dickkopf-1 (DKK1), an inhibitor of Wnt signaling, plays a role in amyloid-induced toxicity and hence Alzheimer’s disease (AD). However, the effect of DKK1 expression on protein expression, and whether such proteins are altered in disease, is unknown.Objective:We aim to test whether DKK1 induced protein signature obtained in vitro were associated with markers of AD pathology as used in the amyloid/tau/neurodegeneration (ATN) framework as well as with clinical outcomes.Methods:We first overexpressed DKK1 in HEK293A cells and quantified 1,128 proteins in cell lysates using aptamer capture arrays (SomaScan) to obtain a protein signature induced by DKK1. We then used the same assay to measure the DKK1-signature proteins in human plasma in two large cohorts, EMIF (n = 785) and ANM (n = 677).Results:We identified a 100-protein signature induced by DKK1 in vitro. Subsets of proteins, along with age and apolipoprotein E ɛ4 genotype distinguished amyloid pathology (A + T–N–, A+T+N–, A+T–N+, and A+T+N+) from no AD pathology (A–T–N–) with an area under the curve of 0.72, 0.81, 0.88, and 0.85, respectively. Furthermore, we found that some signature proteins (e.g., Complement C3 and albumin) were associated with cognitive score and AD diagnosis in both cohorts.Conclusions:Our results add further evidence for a role of DKK regulation of Wnt signaling in AD and suggest that DKK1 induced signature proteins obtained in vitro could reflect theATNframework as well as predict disease severity and progression in vivo.

Recombinant Spider Silk Forms Tough and Elastic Nanomembranes that are Protein‐Permeable and Support Cell Attachment and Growth

AbstractBiologically compatible membranes are of high interest for several biological and medical applications. Tissue engineering, for example, would greatly benefit from ultrathin, yet easy‐to‐handle, biodegradable membranes that are permeable to proteins and support cell growth. In this work, nanomembranes are formed by self‐assembly of a recombinant spider silk protein into a nanofibrillar network at the interface of a standing aqueous solution. The membranes are cm‐sized, free‐standing, bioactive and as thin as 250 nm. Despite their nanoscale thickness, the membranes feature an ultimate engineering strain of over 220% and a toughness of 5.2 MPa. Moreover, they are permeable to human blood plasma proteins and promote cell adherence and proliferation. Human keratinocytes seeded on either side of the membrane form a confluent monolayer within three days. The significance of these results lays in the unique combination of nanoscale thickness, elasticity, toughness, biodegradability, protein permeability and support for cell growth, as this may enable new applications in tissue engineering including bi‐layered in vitro tissue models and support for clinical transplantation of coherent cell layers.

Abstract 578: Pharmacokinetic proprieties of a locked nucleic acid oligonucleotide targeting miR-221

Abstract MiR-221 is overexpressed in several malignancies where it promotes tumor growth and survival by interfering with gene transcripts, including p27Kip1, PUMA, PTEN, and p57Kip2. We previously demonstrated that a novel 13-mer miR-221 inhibitor (LNA-i-miR-221) exerts antitumor activity against human cancer with a pilot favorable pharmacokinetics and safety profile in mice and non-naive monkeys. Next we performed and here report a non-GLP/GLP dose-finding investigation of LNA-i-miR-221 in Sprague Dawley rats. The safety of the intravenous dose (125 mg/kg/day) for four consecutive days, two treatment cycles, was investigated by a first non-GLP study. Toxicokinetics profile of LNA-i-miR-221 was next explored in a GLP study at three different doses (5, 12.5 and 125 mg/kg/day). Slight changes in blood parameters and histological findings in kidney were observed at the highest dose. These effects were reversible and consistent with in vivo ASO class effect. Based on these findings the NOAEL was established at 5 mg/kg/day. The plasma exposure of LNA-i-miR-221, based on C0 and AUC, suggested no differential sex effect. Slight accumulation occurred between cycles one and two but was not observed after four consecutive administrations. These findings demonstrate a safety profile of LNA-i-miR-221 in Sprague Dawley rats and provide a reference translational framework and path for the development of other LNA miR inhibitors in Phase I clinical study. In addition to understand the pharmacokinetics properties of this new agent, including unbound/total clearance, we investigated the LNA-i-miR-221 protein binding in three different species including rat (Sprague Dawley), monkey (Macaca fascicularis) and human. To this aim, we generated a suitable ultrafiltration method to study the binding of LNA-i-miR-221 to plasma proteins. We identified that the fraction of LNA-i-miR-221 (at concentration of 1 and 10 µM) bound to rat, monkey and human plasma proteins is high and ranges from 98.2 to 99.05%. This high protein binding of LNA-i-miR-221 to plasma proteins in all the species tested translates into a pharmacokinetic advantage by preventing rapid renal clearance. The integration of these results into multiple allometric interspecies scaling methods have been then used to draw inferences about LNA-i-miR-221 pharmacokinetics in humans, therefore providing a framework for definition of safe starting and escalation doses toward a first-in-human clinical trial of LNA-i-miR-221. This work was supported by the Italian Association for Cancer Research (AIRC), PI: PT. “Special Program Molecular Clinical Oncology - 5 per mille" n. 9980, 2010/15 and its Extension Program 2016/17 n.9980. Citation Format: Maria Teresa Di Martino. Pharmacokinetic proprieties of a locked nucleic acid oligonucleotide targeting miR-221 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 578.

Production of Recombinant Polypeptides Binding α2-Macroglobulin and Analysis of Their Ability to Bind Human Serum α2-Macroglobulin.

Many streptococcal strains bind to two main human blood plasma proteins: IgG and human serum albumin (HSA). Protein G expressed in group C and G streptococci has specific binding regions for these proteins. Protein G in group G streptococcal strains also contains a region binding another human plasma protein, α2-macroglobulin (α2-М), upstream to the HSA-binding domain. Two recombinant polypeptides GM and GM1 capable of binding to α2-М were obtained using the G4223 strain of a group G Streptococcus, protein G molecule of which interacts with three human blood serum proteins (IgG, HSA, and α2-М). However, polypeptide GM containing three IgG-binding and three HSA-bindings domains and the region binding α2-М has higher molecular mass and higher affinity to α2-М than polypeptide GM1 that includes only the α2-М binding region.

Plasma protein binding, metabolism, reaction phenotyping and toxicokinetic studies of fenarimol after oral and intravenous administration in rats

Fenarimol (FNL), an organic chlorinated fungicide, is widely used in agriculture for protection from fungal spores and fungi. Despite being an endocrine disruptor, no toxicokinetic data is reported for this fungicide. In the present work, we determined the plasma protein binding, metabolic pathways and toxicokinetics of FNL in rats. In vitro binding of FNL to rat and human plasma proteins was ∼90%, suggesting that FNL is a highly protein bound fungicide. The predicted in vivo hepatic clearance of FNL in rats and humans was estimated to be 36.71 and 14.39 mL/min/kg, respectively, indicating it to be an intermediate clearance compound. Reaction phenotyping assay showed that CYP3A4 mainly contributed to the overall metabolism of FNL. The oral toxicokinetic study of FNL in rats at no observed adverse effect level dose (1 mg/kg) showed maximum plasma concentration (C max) of 33.97 ± 4.45 ng/mL at 1 h (T max). The AUC0–∞ obtained was 180.18 ± 17.76 h*ng/mL, whereas, the t 1/2 was ∼4.74 h. Following intravenous administration, FNL displayed a clearance of 42.48 mL/min/kg which was close to the predicted in vivo hepatic clearance. The absolute oral bioavailability of FNL at 1 mg/kg dose in rats was 45.25%. FNL at 10 mg/kg oral dose exhibited non-linear toxicokinetics with greater than dose-proportional increase in the systemic exposure (AUC0–∞ 8270.53 ± 1798.59 h*ng/mL).