An electrochemical in-vitro tool for study of in-vivo relevant biochemical oxidation/reduction of sulfide ion by human whole blood: Evidence for the biological detoxification of hydrogen sulfide

2017 ◽  
Vol 790 ◽  
pp. 20-26 ◽  
Author(s):  
Khairunnisa Amreen ◽  
Annamalai Senthil Kumar
Keyword(s):  
Hydrogen Sulfide ◽  
Whole Blood ◽  
Human Whole Blood ◽  
Sulfide Ion ◽  
Biochemical Oxidation ◽  
Oxidation Reduction ◽  
Biological Detoxification

scholarly journals AB0095 PRECLINICAL CHARACTERIZATION OF CJ-15314, A HIGHLY SELECTIVE JAK1 INHIBITOR, FOR THE TREATMENT OF AUTOIMMUNE DISEASES

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1347.2-1347
Author(s):  
S. Y. Ki ◽  
H. Shin ◽  
Y. Lee ◽  
H. R. Bak ◽  
H. Yu ◽  
...  
Keyword(s):  
Autoimmune Disease ◽  
Autoimmune Diseases ◽  
Whole Blood ◽  
Inflammatory Diseases ◽  
Selective Inhibition ◽  
Janus Kinase ◽  
Dependent Manner ◽  
Human Whole Blood

Background:Janus kinases (JAK1, JAK2, JAK3, and TYK2) play critical roles in mediating various cytokine signaling, and has been developed as a target for autoimmune diseases such as RA. Tofacitinib, oral Pan-JAK inhibitor, demonstrated efficacy in RA patients, but its widespread use is limited by safety issues. Baricitinib, JAK1/2 inhibitor, is also known to interfere with the hematopoiesis system, such as anemia and thrombocytopenia associated with suppression of JAK2 signals. Therefore, it is necessary to develop a new potent compound that selectively inhibits JAK1 over JAK2, 3Objectives:To identify the pharmacological characteristic based on efficacy of CJ-15314 as potent and selective JAK1 inhibitor for treatment of autoimmune disease.Methods:In vitro, cell-based, kinase panel, Kd value and human whole blood assay were performed to determine the inhibition potency and selectivity for JAK subfamily kinases. In vivo therapeutic potential was evaluated by RA model including rat Adjuvant-Induced Arthritis (AIA) and collagen-induced arthritic (CIA). To confirm the possibility of further expansion into the autoimmune disease, BioMAP® Diversity PLUS® Panel was performed by discoverX.Results:In vitro assay, CJ-15314 inhibited JAK kinase family in a concentration-dependent manner with IC50 values of 3.8 nM against JAK1, Selectivity for JAK1 over JAK2, 3 was approximately 18, 83 fold greater for CJ-15314. In 1mM ATP condition, CJ-15314 has been confirmed to have the highest JAK1 selectivity over competing drugs, under 1 mM ATP condition that reflects the physiological environment in the body. Similarly, Kd values has also confirmed the selectivity of JAK1, which is 10 fold higher than JAK2, 3. Accordingly, in human whole blood assays, CJ-15314 is 11 fold more potent against IL-6 induced pSTAT1 inhibition through JAK1 (IC50 value: 70 nM) than GM-CSF-induced pSTAT5 inhibition (JAK2) whereas baricitinib and filgotinib exhibited only 2 fold and 7 fold respectively.In vivo efficacy model, CJ-15314 inhibited disease severity scores in a dose dependent manner. In the rat AIA model, CJ-15314 at 30 mg/kg dose showed 95.3% decrease in arthritis activity score, 51.2% in figotinib at 30 mg/kg, 97.7% showed baricitinib at 10 mg/kg. CJ-15314 showed superior anti-arthritic efficacy than filgotinib. CJ-15314 also minimally affected anemia-related parameters but not bricitinib end of the 2-week treatment. In the rat CIA model, like 10 mg/kg of bricitinib, 30 mg/kg of CJ-15314 also has a similar effect, with a significant reduction in histopathological scores.In biomap diversity panel, CJ-15314 inhibited the expression of genes such as MCP-1, VCAM-1, IP-10, IL-8, IL-1, sTNF-α and HLA-DR confirming the possibility of expansion into other diseases beyond arthritis.Conclusion:CJ-15314 is a highly selective JAK1 inhibitor, demonstrates robust efficacy in RA animal model and is good candidate for further development for inflammatory diseases.* CJ-15314 is currently conducting a phase I trial in south Korea.References:[1]Clark JD et al. Discovery and development of Janus kinase (JAK) inhibitors for inflammatory diseases. J Med Chem. 2014; 57(12):5023-38.[2]Burmester GR et al. Emerging cell and cytokine targets in rheumatoid arthritis. Nat Rev Rheumatol. 2014; 10(2):77-88[3]Jean-Baptiste Telliez et al. Discovery of a JAK3-selective inhibitor: functional differentiation of JAK3-selective inhibition over pan-JAK or JAK1-selective inhibition. ACS Chem. Biol., 2016; 11 (12):3442-3451Disclosure of Interests:so young Ki Employee of: CJ healthcare, hyunwoo shin Employee of: CJ healthcare, yelim lee Employee of: CJ healthcare, Hyoung rok Bak Employee of: CJ healthcare, hana yu Employee of: CJ healthcare, Seung Chan Kim Employee of: CJ healthcare, juhyun lee Employee of: CJ healthcare, donghyun kim Employee of: CJ healthcare, Dong-hyun Ko Employee of: CJ Healthcare, dongkyu kim Employee of: CJ healthcare

Acidosis in Human Whole Blood Does Not Necessarily Impair the Coagulation and the Effect of RFVIIa.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3930-3930
Author(s):  
Dorthe Viuff ◽  
Marianne Kjalke ◽  
Vivian Lind ◽  
Egon Persson ◽  
Mirella Ezban
Keyword(s):  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Whole Blood ◽  
Annexin A5 ◽  
In Vivo Models ◽  
Human Whole Blood ◽  
Major Interest ◽  
R Value

Abstract Introduction: Acidosis is associated with high mortality in trauma patients. Therefore there is a major interest in generating acidosis models in vitro and in vivo to determine the effect of acidosis on coagulation and to develop treatments. The aim of this study was to examine the effect of acidosis induction in human whole blood using HCl versus Hepes and to analyze the subsequent effect of rFVIIa (NovoSeven®). Materials and Methods: Native human whole blood was obtained from healthy volunteers (n=6) and pH was adjusted to 6.8 using 1 M HCl or 1 M Hepes (pH 6.8). Coagulation was triggered with kaolin or tissue factor (TF, Innovin, final dilution 1:42500) and measured by thrombelastography (TEG, Haemoscope®). Furthermore, the effect of rFVIIa (25nM ∼ 90 mcg/kg) was measured. The TEG parameters R (sec), angle (deg) and maximum amplitude (MA, mm) were recorded and presented as mean±SD. A shorter R and greater angle and MA values are indicative of a more robust clot formation. Statistical analysis was performed by a two-way ANOVA-model. Platelet function was analyzed by platelet aggregation using Multiplate (Dynabyte Medical). Exposure of P-selectin, negatively charged phospholipids (annexin A5 binding) and induction of the active conformation of the fibrinogen receptor GPIIb/IIIa (PAC-1 binding) on platelets after TRAP-stimulation of whole blood was analyzed using a FACS Canto flow cytometer (BD). Results: TEG, platelet aggregation and flow cytometry indicated that lowering the pH to 6.8 by HCl affected the blood significantly different than when pH was lowered by addition of Hepes. HCl-treated blood triggered with either kaolin or TF showed a significantly decreased R value (378±45 or 661±130 vs 539±98 or 888±353 in untreated controls), significantly decreased MA (52±6 or 51±9 vs 66±8 or 62±13) and decreased angle (50±7 or 36±10 vs control 57±10 or 44±19, not significant). Hepes-treated blood triggered with kaolin showed no difference in R (458±52), angle (64±4) and MA (58±9) compared to untreated controls, whereas blood triggered with TF showed significantly shortened R-value (461±91) and enhanced angle (63±5) compared to untreated controls. Hepes treatment had no effect on MA (64±12). rFVIIa significantly shortened R irrespective of the acidosis inducer or clot trigger(HCl/kaolin 283±34, HCl/TF 307±52; Hepes/kaolin 363±32, Hepes/TF 313±46). Although the other TEG parameters were also improved, the effect was only significant when blood was treated with HCl and clotting initiated with TF (angle 48±11, MA 56±10). HCl-induced acidosis abolished platelet aggregation, whereas Hepes-induced acidosis did not alter platelet aggregation compared to normal blood. Flow cytometry showed that platelets from HCl-treated blood were pre-activated as evidence by expression of P-selectin on 70% of the platelets, annexin A5 binding to 14% of the platelets and PAC-1 binding to 62% of the platelets before stimulation. TRAP-stimulation increased P-selectin expression, and PAC1 and Annexin A5 binding to platelets in HCl-treated blood. In contrast, Hepes-treatment did not pre-activate the platelets and the increase in P-selectin expression, and annexin A5 and PAC-1 binding after TRAP-stimulation was as seen for control blood. Conclusion: The method used to lower pH in human blood strongly influences the functionality of the platelets and coagulation factors independent of the final pH. It is therefore important in experimental in vitro and in vivo models to be aware of these dramatically different effects in order to draw correct conclusions.

The Effect of Soluble P-Selectin in Coagulation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4106-4106
Author(s):  
Ingrid Korakas ◽  
Darlene Guillen ◽  
Erika Martin ◽  
Mikael Tranholm ◽  
Thomas Barnett ◽  
...  
Keyword(s):  
Whole Blood ◽  
Thrombus Formation ◽  
Membrane Surface ◽  
Control Group ◽  
Human Igg ◽  
Human Whole Blood ◽  
Significant Difference ◽  
Selectin Family

Abstract P-selectin, a member of the selectin family, is a vascular cell adhesion molecule. It is expressed and stored in alpha granules of platelets and in Weibel-Palade bodies of endothelial cells, and upon activation, P-selectin is translocated/transferred to the membrane surface. A key function of the P-selectin is to mediate leukocyte, lymphocyte and platelet interactions in inflammation and possibly in the thrombus formation. A soluble variant, S-Psel, comprising the extracellular domain of P-selectin, has been identified in healthy individuals, but is markedly elevated in patient with vascular disorders. Recent work on S-Psel suggested that S-Psel may play a role in hemostasis/coagulation through the generation of procoagulant TF-bearing microparticles (MP), and therefore, has potential in treating patients with the bleeding disorders, like hemophilia. The aim of this study is to verify studies reporting that S-Psel exhibits in vitro and in vivo pro-coagulant activity. S-Psel and S-Psel-Fc (IgG) fusion were purchased commercially or prepared at Novo Nordisk Research US (NNRUS) and their biological activity was verified by P-selectin/Pselectin glycoprotein ligand-1(PSGL-1) interaction in vitro. The clotting times of human whole blood and plasma treated with S-Psel or S-Psel-Fc, or with an irrelevant human IgG control protein, were measured by thromboelastography and aggregometry respectively. After up to 8 hours of incubation with S-Psel and S-Psel-Fc at a concentration of 15ug/ml, we found no significant difference between samples treated with S-Psel, S-Psel-Fc and the IgG controls. The ability of S-Psel to generate TF-bearing microparticles in human whole blood was examined in a FXa substrate cleavage assay; however, no significant difference in cleavage was observed. Finally, we evaluated S-Psel in vivo. Hemophilia A mice were injected with recombinant mouse S-Psel-IgG or S-Psel-Fc (IgG) at the concentration of 1.2 mg/Kg body weight and human IgG was used as control. As suggested from published results, the effect of S-Psel was determined 6 h after the treatment. Contrary to previous reports, the results revealed no significant difference in bleed time and blood loss between the experimental and control group. In conclusion, we were unable to demonstrate the procoagulant activity of S-Psel in our laboratory either in vitro or in vivo.

scholarly journals OBSERVATIONS ON THE PHAGOCYTOSIS OF THE PNEUMOCOCCUS BY HUMAN WHOLE BLOOD

1930 ◽  
Vol 51 (5) ◽  
pp. 685-702 ◽  
Author(s):  
Hugh K. Ward
Keyword(s):  
Human Blood ◽  
Whole Blood ◽  
Inhibition Zone ◽  
Type I ◽  
Soluble Substance ◽  
Human Whole Blood ◽  
Type Iii ◽  
Serum Therapy

1. In vitro phagocytic experiments with human blood, antipneumococcus serum, pneumococcus specific soluble substance, and living virulent pneumococci show that there is a definite phagocytic inhibition zone when strong antiserum is used. 2. If the antiserum is further diluted, there is a zone where phagocytosis is effective. If the serum is diluted still more, phagocytosis gradually falls off, as the very dilute antiserum fails to neutralize the specific carbohydrate, which has a specific antiphagocytic action. 3. The inhibition zone is apparently caused by the specific precipitate (formed by the antiserum and the specific carbohydrate) interfering, perhaps mechanically, with the ingestion of the pneumococci by the leucocytes. 4. The inhibition zone is better marked with Type III than with Type I pneumococcus. 5. As the concentration of antiserum in the zone of effective phagocytosis in vitro does not correspond with the concentration of antiserum generally used in vivo in the serum therapy of pneumonia, this question is discussed.

Endogenous hydrogen sulfide sulfhydrates IKKβ at cysteine 179 to control pulmonary artery endothelial cell inflammation

2019 ◽  
Vol 133 (20) ◽  
pp. 2045-2059 ◽  
Author(s):  
Da Zhang ◽  
Xiuli Wang ◽  
Siyao Chen ◽  
Selena Chen ◽  
Wen Yu ◽  
...  
Keyword(s):  
Hydrogen Sulfide ◽  
Endothelial Cell ◽  
Pulmonary Artery ◽  
Cell Model ◽  
Site Directed Mutagenesis ◽  
Critical Event ◽  
Hypertensive Rats ◽  
Pulmonary Artery Endothelial Cell

Abstract Background: Pulmonary artery endothelial cell (PAEC) inflammation is a critical event in the development of pulmonary arterial hypertension (PAH). However, the pathogenesis of PAEC inflammation remains unclear. Methods: Purified recombinant human inhibitor of κB kinase subunit β (IKKβ) protein, human PAECs and monocrotaline-induced pulmonary hypertensive rats were employed in the study. Site-directed mutagenesis, gene knockdown or overexpression were conducted to manipulate the expression or activity of a target protein. Results: We showed that hydrogen sulfide (H2S) inhibited IKKβ activation in the cell model of human PAEC inflammation induced by monocrotaline pyrrole-stimulation or knockdown of cystathionine γ-lyase (CSE), an H2S generating enzyme. Mechanistically, H2S was proved to inhibit IKKβ activity directly via sulfhydrating IKKβ at cysteinyl residue 179 (C179) in purified recombinant IKKβ protein in vitro, whereas thiol reductant dithiothreitol (DTT) reversed H2S-induced IKKβ inactivation. Furthermore, to demonstrate the significance of IKKβ sulfhydration by H2S in the development of PAEC inflammation, we mutated C179 to serine (C179S) in IKKβ. In purified IKKβ protein, C179S mutation of IKKβ abolished H2S-induced IKKβ sulfhydration and the subsequent IKKβ inactivation. In human PAECs, C179S mutation of IKKβ blocked H2S-inhibited IKKβ activation and PAEC inflammatory response. In pulmonary hypertensive rats, C179S mutation of IKKβ abolished the inhibitory effect of H2S on IKKβ activation and pulmonary vascular inflammation and remodeling. Conclusion: Collectively, our in vivo and in vitro findings demonstrated, for the first time, that endogenous H2S directly inactivated IKKβ via sulfhydrating IKKβ at Cys179 to inhibit nuclear factor-κB (NF-κB) pathway activation and thereby control PAEC inflammation in PAH.

scholarly journals In-vitro and in-vivo metabolism of different aspirin formulations studied by a validated liquid chromatography tandem mass spectrometry method

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Michele Dei Cas ◽  
Jessica Rizzo ◽  
Mariangela Scavone ◽  
Eti Femia ◽  
Gian Marco Podda ◽  
...  
Keyword(s):  
Oral Administration ◽  
Whole Blood ◽  
Serum Levels ◽  
Reversed Phase ◽  
Weekly Treatment ◽  
Low Dose Aspirin ◽  
Liquid Chromatography Tandem Mass ◽  
Enteric Coated

AbstractLow-dose aspirin (ASA) is used to prevent cardiovascular events. The most commonly used formulation is enteric-coated ASA (EC-ASA) that may be absorbed more slowly and less efficiently in some patients. To uncover these “non-responders” patients, the availability of proper analytical methods is pivotal in order to study the pharmacodynamics, the pharmacokinetics and the metabolic fate of ASA. We validated a high-throughput, isocratic reversed-phase, negative MRM, LC–MS/MS method useful for measuring circulating ASA and salicylic acid (SA) in blood and plasma. ASA-d4 and SA-d4 were used as internal standards. The method was applied to evaluate: (a) the "in vitro" ASA degradation by esterases in whole blood and plasma, as a function of time and concentration; (b) the "in vivo" kinetics of ASA and SA after 7 days of oral administration of EC-ASA or plain-ASA (100 mg) in healthy volunteers (three men and three women, 37–63 years). Parameters of esterases activity were Vmax 6.5 ± 1.9 and Km 147.5 ± 64.4 in plasma, and Vmax 108.1 ± 20.8 and Km 803.2 ± 170.7 in whole blood. After oral administration of the two formulations, tmax varied between 3 and 6 h for EC-ASA and between 0.5 and 1.0 h for plain-ASA. Higher between-subjects variability was seen after EC-ASA, and one subject had a delayed absorption over eight hours. Plasma AUC was 725.5 (89.8–1222) for EC-ASA, and 823.1(624–1196) ng h/mL (median, 25–75% CI) for plain ASA. After the weekly treatment, serum levels of TxB2 were very low (< 10 ng/mL at 24 h from the drug intake) in all the studied subjects, regardless of the formulation or the tmax. This method proved to be suitable for studies on aspirin responsiveness.

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