Affinity For Albumin Research Articles

Protein-Bound Uremic Toxins and Immunity.

Protein-bound uremic toxins (PBUTs) are bioactive microbiota metabolites originated exclusively from protein fermentation of the bacterial community resident within the gut microbiota, whose composition and function is profoundly different in the chronic kidney disease (CKD) population. PBUTs accumulate in the later stages of CKD because they cannot be efficiently removed by conventional hemodialysis due to their high binding affinity for albumin, worsening their toxic effects, especially at the cardiovascular level. The accumulation of uremic toxins, along with oxidative stress products and pro-inflammatory cytokines, characterizes the uremic status of CKD patients which is increasingly associated to a state of immune dysfunction including both immune activation and immunodepression. Furthermore, the links between immune activation and cardiovascular disease (CVD), and between immunodepression and infection diseases, which are the two major complications of CKD, are becoming more and more evident. This review summarizes and discusses the current state of knowledge on the role of the main PBUTs, namely indoxyl sulfate and p-cresyl sulfate, as regulators of immune response in CKD, in order to understand whether a microbiota modulation may be useful in the management of its main complications, CVD, and infections. Summarizing the direct effects of PBUT on immune system we may conclude that PCS seemed to be associated to an immune deficiency status of CKD mainly related to the adaptative immune response, while IS seemed to reflect the activation of both innate and adaptative immune systems likely responsible of the CKD-associated inflammation. However, the exact role of IS and PCS on immunity modulation in physiological and pathological state still needs in-depth investigation, particularly in vivo studies.

Vascular <i>in vivo</i> Imaging After Intra-Striatal Injection of DB53 Reveals Two Distinct Kinetics of Glymphatic Efflux

Glymphatic fluid transport eliminates metabolic waste from the brain including amyloid-s and Tau, yet the pathways and mechanisms of clearance remain unclear. Here we develop a new method to evaluate glymphatic clearance in real-time. Direct Blue 53 (DB53), a stable fluorescent compound with low toxicity, is delivered in striatum and imaged in the vascular compartment where it is retained due to its high affinity for albumin. DB53 signal was detectable in femoral veins as early as 20 min post-injection in young mice while aged mice exhibited a sharp reduction in the kinetics of DB53 clearance. Pharmacokinetic modeling revealed that DB53 efflux is consistent with the existence of two efflux paths, with fast (T1/2=32 min) kinetics and slow kinetics (T1/2=170 min), demonstrating that this real-time, in vivo methodology can provide novel insights into the kinetics of brain fluid clearance.

Albumin-binding domain extends half-life of glucagon-like peptide-1

Glucagon-like peptide-1 (GLP-1) is considered to be a promising peptide for the treatment of type 2 diabetes mellitus (T2DM). However, the extremely short half-life of GLP-1 limits its clinical application. Albumin-binding domain (ABD) with high affinity for human serum albumin (HSA) has been used widely for half-life extension of therapeutic peptides and proteins. In the present study, novel GLP-1 receptor agonists were designed by genetic fusion of GLP-1 to three kinds of ABDs with different affinities for HSA: GA3, ABD035 and ABDCon. The bioactivities and half-lives of ABD-fusion GLP-1 proteins with different types and lengths of linkers were investigated in vitro and in vivo. The results demonstrated that ABD-fusion GLP-1 proteins could bind to HSA with high affinity. The blood glucose-lowering effect of GLP-1 was significantly improved and sustained by fusion to ABD. Meanwhile, the fusion proteins significantly inhibited food intake, which was beneficial for T2DM and obesity treatment. The half-life of GLP-1 was substantially extended by virtue of ABD. The in vivo results also showed that a longer linker inserted between GLP-1 and ABD resulted in a higher blood glucose-lowering effect. The fusion proteins generated by fusion of GLP-1 to GA3, ABD035 and ABDCon exhibited similar bioactivities and pharmacokinetics in vivo. These findings demonstrate that ABD-fusion GLP-1 proteins retain the bioactivities of natural GLP-1 and can be further developed for T2DM treatment and weight loss. It also indicates that the ABD-fusion strategy can be generally applicable to any peptide or protein, to improve pharmacodynamic and pharmacokinetic properties.

Synthesis, Characterization and Biological Studies of Organoselenium trans-Palladium(II) Complexes.

Over the years, transition metal complexes have exhibited significant antimicrobial and antitumor activity. It all started with cisplatin discovery, but due to the large number of side effects it shows, there is a growing need to find a new metal-based compound with higher selectivity and activity on more tumors. Two novel trans-palladium(II) complexes with organoselenium compounds as ligands, [Pd(L1)2Cl2] (L1 = 5-(phenylselanylmethyl)-dihydrofuran-2(3H)-one) and [Pd(L2)2Cl2] (L2 = 2- methyl-5-(phenylselanylmethyl)- tetrahydrofuran) were synthesized, in the text referred to as Pd-Se1 and Pd-Se2. Also, a structurally similar trans-palladium(II) complex, [Pd(L3)2Cl2] (L3= 2,2- dimethyl-3-(phenylselanylmethyl)-tetrahydro-2H-pyran) was synthesized according to an already published work and is referred to as Pd-Se3. The interaction of synthesized complexes with DNA and bovine serum albumin was observed. Also, antimicrobial activity and in vitro testing, cell viability, and cytotoxic effects of synthesized ligands and complexes on human epithelial colorectal cancer cell line HCT-116 were studied. Molecular docking simulations were performed to understand better the binding modes of the complexes reported in this paper with DNA and BSA, as well as to comprehend their antimicrobial activity. The interactions of the synthesized complexes with DNA and bovine serum albumin were done using UV-Vis and emission spectral studies as well as docking studies. Antimicrobial activity was tested by determining the minimum inhibitory concentrations (MIC) and minimum microbicidal concentration (MMC) using the resazurin microdilution plate method. Cytotoxic activity on cancer cells was studied by MTT test. The Pd(II) complexes showed a significant binding affinity for calf thymus DNA and bovine serum albumin by UV-Vis and emission spectral studies. The intensity of antimicrobial activity varied with the complexes Pd-Se1 and Pd-Se3, showing significantly higher activity than the corresponding ligand. The most significant activity was shown on Pseudomonas aeruginosa. Under standardized laboratory conditions for in vitro testing, cell viability and cytotoxic effects of synthesized ligands and complexes were studied on human epithelial colorectal cancer cell line HCT-116, where Pd-Se2 showed some significant cytotoxic effects. The newly synthesized complexes have the potential to be further investigated as metallodrugs.

Structure-activity analysis of truncated albumin-binding domains suggests new lead constructs for potential therapeutic delivery

Rapid clearance by renal filtration is a major impediment to the translation of small bioactive biologics into drugs. To extend serum t1/2, a commonly used approach is to attach drug leads to the G-related albumin-binding domain (ABD) to bind albumin and evade clearance. Despite the success of this approach in extending half-lives of a wide range of biologics, it is unclear whether the existing constructs are optimized for binding and size; any improvements along these lines could lead to improved drugs. Characterization of the biophysics of binding of an ABD to albumin in solution could shed light on this question. Here, we examine the binding of an ABD to human serum albumin using isothermal titration calorimetry and assess the structural integrity of the ABD using CD, NMR, and molecular dynamics. A structure-activity analysis of truncations of the ABD suggests that downsized variants could replace the full-length domain. Reducing size could have the benefit of reducing potential immunogenicity problems. We further showed that one of these variants could be used to design a bifunctional molecule with affinity for albumin and a serum protein involved in cholesterol metabolism, PCSK9, demonstrating the potential utility of these fragments in the design of cholesterol-lowering drugs. Future work could extend these in vitro binding studies to other ABD variants to develop therapeutics. Our study presents new understanding of the solution structural and binding properties of ABDs, which has implications for the design of next-generation long-lasting therapeutics.

Synthesis, characterization, theoretical, molecular docking and in vitro biological activity studies of Ru(II) (η6 -p-cymene) complexes with novel aniline substituted aroyl selenoureas

A sequence of aroyl selenourea ligands (L1–L3) substituted by aniline and their Ru(II) (η6 -p-cymene) complexes (1-3), [Ru(II) (η6 -p-cymene) L] (L = monodentate aroyl selenourea ligand) have been synthesized and characterized the composition of the ligands and their metal complexes. The molecular structures of ligand L1 and complex 3 were also confirmed by single XRD crystal method. The single-crystal XRD study showed that aroyl selenourea ligand coordinates with Ru via Se novel neutral monodentate atom. In vitro DNA interaction studies were investigated by Fluorescence and UV-Visible spectroscopic methods which showed that the intercalative mode of binding is in the order of 1 > 2 > 3 with Ru(II) (η6 -p-cymene) complexes. Spectroscopic methods have been used for measuring the binding affinity of bovine serum albumin to complex. Moreover, the cytotoxic study of complexes (1–3) were evaluated against HeLa S3, A549, and IMR90 cells, resulting in complexes 1 and 2 showed promising cytotoxic activity against HeLa S3 cell with IC50 values of 24 and 26 µM, respectively. Also, the morphological changes of HeLa S3 and A549 cells were confirmed by fluorescence microscope in the presence of complexes 1 and 2 using AO (acridine orange, 200 µM) and EB (ethidium bromide, 100 µM). In addition, the docking results strongly support the protein binding studies of the complexes. Communicated by Ramaswamy H. Sarma

Synthetic carboline compounds targeting protein: biophysical and biological perspective

Pictet–Spengler cyclization method has been adopted for the synthesis of three carboline derived compounds: two compounds with tetrahydro gama- and beta-having CF3 group and amino alkyl chain at delta and alpha position, respectively, and another with guanidine alkyl chain at alpha-position. Structure–activity relationship of the analogues with human serum albumin was studied by fluorescence and Fourier-transform infrared spectroscopy followed by molecular docking. The data showed maximum affinity of human serum albumin with comp7 (S0-820) followed by comp3 (S0-1040) and least with comp1 (S0-728). The compounds were tested for cytotoxic potencies. Comp3, showed maximum cytotoxicity with GI50 6.2 µM, against HCT-116, followed by comp7, and poor cytotoxicity with comp1. Comp3 and 7 induced oxidative stress mediated autophagy led programmed cell death in HCT-116. Furthermore, the compounds effectively inhibit DNA topoisomerase I activity and showed anti-inflammatory actions. In vivo studies regarding therapeutic protective action of Comp3, as a representative carboline analogue, against colon toxicant, 1,2-dimethylhydrazine dihydrochloride (DMH), showed the efficacy of the compound against organ toxicity. The existing studies on biological evaluation showed that these synthetic compounds may have a major role as anticancer agents having myriad of proven therapeutic applications. Communicated by Ramaswamy H. Sarma

Enhancing the Stability of Haemonchus contortus Glyceraldehyde-3-Phosphate Dehydrogenase and Binding of Host Albumin to the Parasite Enzyme.

Haemonchus contortus is an economically important parasite of domestic animals. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) excreted in the ES product of H. contortus can be a promising vaccine candidate for controlling the parasite infection. Unfortunately, the parasite enzyme breaks down rapidly. The current study focusses on stabilizing the recombinant GAPDH (rGAPDH) of H. contortus. The rGAPDH was purified and stored in two different buffers (sodium phosphate + EDTA and bicarbonate-sodium chloride) to check the stability. The affinity of the parasite enzyme towards host serum (Goat) components was evaluated by affinity chromatography. The interacting component was identified by mass spectrometry. Here, we report that the enzyme can be stabilized for at least 3months if stored in bicarbonate-sodium chloride. This should facilitate testing of the enzyme in challenge trials. Additionally, we show that the parasite enzyme has affinity for host albumin; this interaction may have significance in host-parasite relationship. The present study reports a combination of sodium bicarbonate (0.1M) with 0.5M sodium chloride as a suitable buffer to enhance the stability of H. contortus GAPDH.

A novel thrombin-based triagonist with diabetes-protective and weight-lowering potential

AimsTo investigate the diabetes-protective effect and weight-lowering potential of a novel long-acting triagonist at three metabolically related hormone receptors including glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), glucagon receptors. Main methodsTriagonist were designed in an iterative manner from native GLP-1, GIP and Glucogan. Main peptide chain (termed TG peptides) and subsequently modified LTG peptides were synthesized via solid phase synthesis. In vitro receptor activity assay was performed to screen the TG peptide with most balanced potency on all three receptors. The in vitro biological activities of modified TG peptides were further investigated by albumin-binding measurement and proteolytic cleavage test. Subsequently, oral glucose tolerance test (OGTT), pharmacokinetic test and chronic study were subjected to the acute and long-term efficacy evaluation of selected fusion peptide, LTG-6. Key findingsTG-8 exhibited equally aligned constituent efficacy and supraphysiological potency on corresponding receptor without cross-reactivity. Modified TG-8, termed LTG-6, exerted the great binding affinity for human serum albumin and the enhanced rational controlled-release of TG-8 in vitro. Further OGTT in different gene knockout mice and diabetic mice demonstrated the promising hypoglycemic and insulinotropic abilities of LTG-6. After long-term treatment for 8 weeks, LTG-6 was proved superior to co-agonists to decrease the body weight and %HbA1c, improve reverse dyslipidemia and glycemic control in the DIO models. SignificanceLTG-6, as a newly designed long-acting triagonist, holds potential to correct the obesity related metabolic disorders.

Synthesis, characterization, in silico and in vitro biological activity studies of Ru(II) (η6-p-cymene) complexes with novel N-dibenzosuberene substituted aroyl selenourea exhibiting Se type coordination

A series of N-dibenzosuberene substituted aroyl selenourea ligands L1–L3 and their Ru(II) (η6-p-cymene) complexes 1–3, [Ru(II) (η6-p-cymene) L] (L = monodentate aroyl selenourea ligand) were synthesized and characterized. The molecular structures of the ligand L3 and complex 3 were confirmed by single-crystal XRD method. The single-crystal XRD study results revealed that aroyl selenourea ligand coordinates with ruthenium via Se neutral monodentate atom. In vitro DNA interaction studies were investigated by UV–Visible and fluorescence spectroscopic methods which showed that the intercalative mode of binding is in the order of 3 > 2 > 1 with the Ru(II) (η6-p-cymene) complexes. The binding affinity of the bovine serum albumin with complexes was calculated using spectroscopic methods. Quantum chemical computations were made using DFT (density functional theory), BL3YP; LANL2DZ basis set in order to determine the frontier molecular orbital parameters and MESP for the newly synthesized complexes. The complexes 1–3 have shown intensive cytotoxicity against the cancer lines HepG-2 and A549 under in vitro conditions. Complex 3 (IC50 = 62 μM) has shown significant cytotoxic activity against HepG-2 compared to cisplatin standard drug. The complexes also examined for their antimicrobial activity. The complex 2 exhibited good activity against B. subtilis (MIC: 13.60 μg/mL), E. coli (MIC: 8.01 μg/mL) and A. flavus (MIC = 15.60 μg/mL), respectively, compared to reference drugs Streptomycin and Ketoconazole.

MON-483 Familial Dysalbuminemic Hyperthyroxinemia with False Hypercortisolemia

Background: Familial dysalbuminemic hyperthyroxinemia (FDH) is an autosomal dominant disease characterized by hyperthyroxinemia without symptoms of thyrotoxicosis, due to a high affinity of mutant albumin for thyroxine. No report has described cortisol-albumin binding in FDH patient, and here we present a case of FDH presenting with false hypercortisolemia.Clinical Case: A 46-year man, previously diagnosed with FDH by genetic test (1), was pointed out hypercortisolemia with normal ACTH level (ACTH 64.7 pg/mL, Cortisol 34.7 μg/dL) during the investigation for Parkinson’s syndrome and referred to our department for further examination. Cortisol was 7.1 μg/dL at midnight, 14.9 μg/dL after low dose dexamethasone overnight test, and DDAVP test was positive. ACTH and cortisol responded to CRH load, although basal and peak cortisol levels were high (35.4 and 53.9 μg/dL, respectively). High dose dexamethasone overnight test showed suppressed ACTH and cortisol, and MRI showed no obvious pituitary adenoma.In spite of significant high cortisol level, no Cushing sign’s or metabolic abnormalities were observed and urinary free cortisol was within the normal range (30.7 μg/day), suggesting the presence of factors affecting the laboratory testing. We removed albumin from serum with immunoprecipitation using anti-albumin antibody and measured cortisol with LC-MS/MS. The decrease of cortisol was 4% in control serum but 38% in the patient serum after removing albumin, suggesting the binding rate of cortisol to mutant albumin in the patient was increased, leading to false hypercortisolemia.Conclusion: This is the first case demonstrating the false hypercortisolemia in a FDH patient. Clinicians should consider the possibility of the abnormal cortisol binding to albumin in differential diagnosis of hypercortisolemia with normal ACTH level.Reference: (1) Norio Wada, et al: A Novel Missense Mutation in Codon 218 of the Albumin Gene in a Distinct Phenotype of Familial Dysalbuminemic Hyperthyroxinemia in a Japanese Kindred. Journal of Clinical Endocrinology and Metabolism 1997;82;3246–3250

Novel mono-PEGylated dimeric GLP-1 conjugate with enhanced receptor activation and prolonged anti-diabetes efficacies

AimsTo design and evaluate novel mono-PEGylated dimeric GLP-1 conjugate with enhanced GLP-1 receptor activation and prolonged anti-diabetes efficacies. Main methodsAll these novel GLP-1 conjugates were produced by using solid-phase synthesis method and further specific cysteine–maleimide modification. In vitro GLP-1R activation assay was performed in CHO cells stably expressing human GLP-1 receptor. The binding affinity for human serum albumin (HSA) in vitro was also conducted using surface plasmon resonance measurement. Subsequently, selected GLP-1 conjugate was subjected to evaluate the acute and chronic efficacies in vivo. Key findingsFour novel glucagon-like peptide-1 (GLP-1) conjugates, termed DIG-1 to DIG-4, were designed and prepared with high purity. Moreover, DIG-1(PEG-5 kDa) and DIG-2 (PEG-10 kDa) exerted ~3-fold and ~2-fold higher potencies of GLP-1R activation than native GLP-1, respectively, and both obviously higher than the DIG-3 (PEG-10 kDa) and DIG-4 (PEG-30 kDa). Then DIG-2 exhibited better in vivo glucose-stabilizing and insulinotropic efficacies than DIG-1 by using multiple oral glucose tests (OGTTs) in SD rats. Furthermore, prolonged glucose-lowering ability of DIG-2 exhibited in hypoglycemic duration test and multiple OGTTs in diabetic db/db mice. Pharmacokinetic data of DIG-2 in cynomolgus monkeys revealed a half-life of ~97.2 h and ~120.4 h after a single subcutaneous (s.c.) administration at doses of 100 and 150 nmol/kg, respectively. Chronic treatment of DIG-2 in db/db mice for consecutive 8-week significantly ameliorate the diabetic symptoms including deteriorative % hemoglobin A1C (HbA1C), glucose tolerance and pancreatic function. SignificanceDIG-2, as a novel mono-PEGylated dimeric GLP-1 conjugate, holds enhanced receptor activation and prolonged anti-diabetes efficacies.

Serum albumin-binding VH Hs with variable pH sensitivities enable tailored half-life extension of biologics.

Prolonged serum half-life is required for the efficacy of most protein therapeutics. One strategy for half-life extension is to exploit the long circulating half-life of serum albumin by incorporating a binding moiety that recognizes albumin. Here, we describe camelid single-domain antibodies (VH Hs) that bind the serum albumins of multiple species with moderate to high affinity at both neutral and endosomal pH and significantly extend the serum half-lives of multiple proteins in rats from minutes to days. We serendipitously identified an additional VH H (M75) that is naturally pH-sensitive: at endosomal pH, binding affinity for human serum albumin (HSA) was dramatically weakened and binding to rat serum albumin (RSA) was undetectable. Domain mapping revealed that M75 bound to HSA domain 1 and 2. Moreover, alanine scanning of HSA His residues suggested a critical role for His247, located in HSA domain 2, in M75 binding and its pH dependence. Isothermal titration calorimetry experiments were suggestive of proton-linked binding of M75 to HSA, with differing binding enthalpies observed for full-length HSA and an HSA domain 1-domain 2 fusion protein in which surface-exposed His residues were substituted with Ala. M75 conferred moderate half-life extension in rats, from minutes to hours, likely due to rapid dissociation from RSA during FcRn-mediated endosomal recycling in tandem with albumin conformational changes induced by M75 binding that prevented interaction with FcRn. Humanized VH Hs maintained in vivo half-life extension capabilities. These VH Hs represent a new set of tools for extending protein therapeutic half-life and one (M75)demonstrates a unique pH-sensitive binding interaction that can be exploited to achieve modest in vivo half-life.

Preclinical Evaluation of a High-Affinity Sarcophagine-Containing PSMA Ligand for 64Cu/67Cu-Based Theranostics in Prostate Cancer.

The application of small molecules targeting prostate-specific membrane antigen (PSMA) has emerged as a highly promising clinical strategy for visualization and treatment of prostate cancer. Ligands that integrate the ability to both quantify the distribution of radioactivity and treat disease through the use of a matched pair of radionuclides have particular value in clinical and regulatory settings. In this study, we describe the development and preclinical evaluation of RPS-085, a ligand that binds PSMA and serum albumin and exploits the 64/67Cu radionuclide pair for prostate cancer theranostics. RPS-085 was synthesized by conjugation of a PSMA-targeting moiety, an Nε-(2-(4-iodophenyl)acetyl)lysine albumin binding group, and a bifunctionalized MeCOSar chelator. The IC50 of the metal-free RPS-085 was determined in a competitive binding assay. The affinity for human serum albumin of the radiolabeled compound was determined by high-performance affinity chromatography. Radiolabeling was performed in NH4OAc buffer at 25 °C. The stability of the radiolabeled compounds was assessed in vitro and in vivo. The biodistribution of [64/67Cu]Cu-RPS-085 was determined following intravenous administration to male BALB/c mice bearing LNCaP tumor xenografts. The radiochemical yields of [64/67Cu]Cu-RPS-085 were nearly quantitative after 20 min. The metal-free complex is a potent inhibitor of PSMA (IC50 = 29 ± 2 nM), and the radiolabeled compound has moderate affinity for human serum albumin (Kd = 9.9 ± 1.7 μM). Accumulation of the tracer in mice was primarily evident in tumor and kidneys. Activity in all other tissues, including blood, was negligible, and the radiolabeled compounds demonstrated high stability in vitro and in vivo. Tumor activity reached a maximum at 4 h post injection (p.i.) and cleared gradually over a period of 96 h. By contrast, activity in the kidney cleared rapidly from 4 to 24 h p.i. As a consequence, by 24 h p.i., the tumor-to-kidney ratio exceeds 2, and the predicted dose to tumors is significantly greater than the dose to kidneys. [64Cu]Cu-RPS-085 combines rapid tissue distribution and clearance with prolonged retention in LNCaP tumor xenografts. The pharmacokinetics should enable radioligand therapy using [67Cu]Cu-RPS-085. By virtue of its rapid kidney clearance, the therapeutic index of [67Cu]Cu-RPS-085 likely compares favorably to its parent structure, [177Lu]Lu-RPS-063, a highly avid PSMA-targeting compound. On this basis, [64/67Cu]Cu-RPS-085 show great promise as PSMA-targeting theranostic ligands for prostate cancer imaging and therapy.

Understanding the Thermodynamic Mechanisms Leading to the Binding of Albumin to Lipid Nanocapsules.

Lipid nanocapsules (LNCs) are drug delivery platforms designed for different administration routes including intravenous delivery. Nanocarrier binding with plasma proteins such as albumin is an important factor that influences the pharmacokinetics of the drug and the drug delivery system. The aim of this paper was to characterize LNCs with different surface compositions and hydrophobicities to study their interactions with albumin: binary LNCs [oil-glyceryl trioctanoate (TG) and PEGylated surfactant macrogol 15-hydroxystearate (MHS)] and ternary LNCs (TG, MHS, and Span 80). Span was found to stabilize and decrease the LNC size. The formation of a stable LNC/albumin complex in the ground state was demonstrated. Thermodynamic parameters indicated that complex formation was exothermic and spontaneous, and the interactions involved van der Waals forces and hydrogen bond formation. Ternary LNCs showed higher affinity for albumin than did binary LNCs (affinity constant 10-fold higher). This study is the first report on the thermodynamic mechanisms that lead to the formation of a complex between albumin and organic nanoparticles with different surface architectures.

Mechanisms of palmitic acid-conjugated antisense oligonucleotide distribution in mice

Conjugation of antisense oligonucleotide (ASO) with a variety of distinct lipophilic moieties like fatty acids and cholesterol increases ASO accumulation and activity in multiple tissues. While lipid conjugation increases tissue exposure in mice and reduces excretion of ASO in urine, histological review of skeletal and cardiac muscle indicates that the increased tissue accumulation of lipid conjugated ASO is isolated to the interstitium. Administration of palmitic acid-conjugated ASO (Palm-ASO) in mice results in a rapid and substantial accumulation in the interstitium of muscle tissue followed by relatively rapid clearance and only slight increases in intracellular accumulation in myocytes. We propose a model whereby increased affinity for lipid particles, albumin, and other plasma proteins by lipid-conjugation facilitates ASO transport across endothelial barriers into tissue interstitium. However, this increased affinity for lipid particles and plasma proteins also facilitates the transport of ASO from the interstitium to the lymph and back into circulation. The cumulative effect is only a slight (∼2-fold) increase in tissue accumulation and similar increase in ASO activity. To support this proposal, we demonstrate that the activity of lipid conjugated ASO was reduced in two mouse models with defects in endothelial transport of macromolecules: caveolin-1 knockout (Cav1−/−) and FcRn knockout (FcRn−/−).

Efficacy of Divinylbenzenic Resin in Removing Indoxyl Sulfate and P-Cresol Sulfate in Hemodialysis Patients: Results From an In Vitro Study and An In Vivo Pilot Trial (xuanro4-Nature 3.2).

High serum levels of microbiota-derived uremic toxins, indoxyl sulfate (IS) and p-cresyl sulfate (PCS), are associated with chronic kidney disease (CKD) progression and cardiovascular complications. IS and PCS cannot be efficiently removed by conventional hemodialysis (HD), due to their high binding affinity for albumin. This study evaluates the efficacy of a divinylbenzene-polyvinylpyrrolidone (DVB-PVP) cartridge and a synbiotic to reduce uremic toxins in HD patients. First, the in vitro efficacy of DVB-PVP in adsorbing IS and PCS was evaluated. Second, a randomized, placebo-controlled pilot study in HD patients was carried out to establish whether the administration of a synbiotic, either individually and in association with DVB-PVP-HD, could reduce the production of uremic toxins. In vitro data showed that DVB-PVP resin removed a mean of 56% PCS and around 54% IS, after 6 h of perfusion. While, in the in vivo study, the DVB-PVP cartridge showed its adsorbing efficacy only for IS plasma levels. The combination of synbiotic treatment with DVB-PVP HD decreased IS and PCS both at pre- and post-dialysis levels. In conclusion, this study provides the first line of evidence on the synergistic action of gut microbiota modulation and an innovative absorption-based approach in HD patients, aimed at reducing plasma levels of IS and PCS.

Binding Constants of Substituted Benzoic Acids with Bovine Serum Albumin.

Experimental data on the affinity of various substances to albumin are essential for the development of empirical models to predict plasma binding of drug candidates. Binding of 24 substituted benzoic acid anions to bovine serum albumin was studied using spectrofluorimetric titration. The equilibrium constants of binding at 298 K were determined according to 1:1 complex formation model. The relationships between the ligand structure and albumin affinity are analyzed. The binding constant values for m- and p-monosubstituted acids show a good correlation with the Hammett constants of substituents. Two- and three-parameter quantitative structure–activity relationship (QSAR) models with theoretical molecular descriptors are able to satisfactorily describe the obtained values for the whole set of acids. It is shown that the electron-density distribution in the aromatic ring exerts crucial influence on the albumin affinity.

Interaction of endothelial cells with plasma-polymer modified surfaces

Endothelialisation of implantable vascular grafts and stents is directed by the adsorbed protein layer. Chemical and mechanical cues sensed at the biomaterial surface by endothelial cells determine their attachment, survival and proliferation. Given the multiplicity of possible interactions we describe the novel application of live cell imaging, factorial analysis and single molecule imaging to investigate higher order interactions between surface chemistry and adsorbed proteins influencing endothelial cell (EC) adhesion dynamics. EC fates were tracked by time-lapse imaging of cell contact area on plasma polymer modified surfaces. The combinatorial effect of plasma polymer chemistry and adsorbed proteins was characterised by factorial experimental design and analysis. Single molecule imaging and counting was used for the first time to quantify binding of fluorescently labelled albumin to plasma polymerized surfaces: Modified glass surfaces with thin plasma polymer coatings rich in primary amine groups had a high affinity for albumin (~2, 650 molecules/μm2) while plasma polymer surfaces functionalised with hydroxyl groups had very low levels of albumin binding (22–30 molecules/μm2). Plasma polymer coatings rich in primary amine groups also promoted endothelial cell adhesion and was superior to tissue culture plastic. An interaction between albumin, heparin and fibronectin promoted adhesion to amine plasma polymer coated glass, while hydroxyl plasma polymer coated glass prevented EC attachment. Tracking cell-specific interactions with the adsorbed protein layer by time-lapse microscopy is more predictive of in vivo cellular responses to biomaterials compared to studies that only measure protein adsorption.

Antioxidant, Antidiabetic and Anticancer Activities of L-Phenylalanine and L-Tyrosine Ester Surfactants: In Vitro and In Silico Studies of their Interactions with Macromolecules as Plausible Mode of Action for their Biological Properties

Background: Aromatic amino acid-based surfactants have been found to have interesting biological properties such as antibacterial and hemolytic activities. Recently, we have reported the antibacterial activity of a range of ester hydrochloride surfactants derived from L-Phenylalanine and LTyrosine. This study aims at assessing the antioxidant, α-glycosidase inhibitory and cytotoxic activities of a series of L-Phenylalanine and L-Tyrosine ester hydrochlorides. Molecular docking and BSA binding studies were also carried out in order to investigate their potential therapeutic targets. Methods: L-Phenylalanine and L-Tyrosine surfactants were tested as potential lipophilic antioxidants using the DPPH and ABTS assays. These surfactants were also tested for their α-glycosidase inhibitory activity using 4-nitrophenyl α -D-glucopyranoside (pNPG) as substrate. Their cytotoxicity effects were screened using HeLa and KB cell lines. Glide version 5.7 as implemented in Schrödinger suite 2013-1, was used for performing docking studies of L-Phenylalanine and L-Tyrosine dodecyl esters. The interaction of the ester hydrochlorides of L-Phenylalanine and L-Tyrosine with bovine serum albumin (BSA) was investigated using fluorometric titration. Results: The presence of the phenolic moiety in L-Tyrosine-based surfactants was found to enhance the antioxidant and α-glucosidase inhibitory activities compared to the L-Phenylalanine derivatives. The α- glucosidase and anticancer activities of the phenylalanine surfactants were found to increase with chain length up to C12 above which the activities exhibited a downward trend. In the case of the tyrosine series, an increase in chain length from C8 to C14 was found to decrease the α-glucosidase inhibitory activity and increase the anticancer activity of the surfactants. Binding studies with bovine serum albumin showed that the tyrosine surfactants displayed greater affinity for the serum albumin, owing to the presence of the phenolic group which altered the orientation of the surfactant molecule within the hydrophobic core of BSA. Conclusion: L-Tyrosine esters having a phenolic moiety were found to possess enhanced biological activity in terms of both the antioxidant and antidiabetic activities as well as also bind more strongly to Bovine serum albumin. Molecular docking studies of the phenylalanine and tyrosine surfactants of similar chain length with target proteins showed direct correlation with their anticancer and antidiabetic activity. Therefore, the findings show that these aromatic based surfactants derived from L-Tyrosine can act as promising antioxidant, antidiabetic and anticancer agents, and they can also be efficiently transported and eliminated in the body, making them useful candidates for drug designs.