Incubation Of Plasma Research Articles

A Lipid Peroxidation Product, 4-Hydroxy-2-Nonenal, Triggers Intravascular Coagulation and Inflammation through Generation of Tissue Factor-Positive Microvesicles

Tissue factor (TF) is a cellular receptor for the clotting protease factor VIIa (FVIIa), and TF-FVIIa complex initiates activation of the coagulation cascade. TF-FVIIa also influences non-hemostatic functions via direct or indirect cell signaling. Constitutive expression of TF is primarily restricted to the surface of perivascular cells, sequestered from circulating blood clotting factors, to prevent aberrant intravascular clotting. It has been suggested that TF is also present in the blood associated with microvesicles (MVs). These circulating TF, however, do not induce any thrombotic complications as they are either cleared from the system before they could accumulate to a pathological level or lack procoagulant activity. However, various pathological conditions such as sepsis, diabetes, bacterial infection, atherosclerosis, and cancer can increase the levels of circulating TF+ MVs, which can then trigger intravascular coagulation and inflammation. We hypothesized that oxidative stress, a pathological condition, central to most of the diseases mentioned above, plays a role in the generation of TF+ MVs in vivo, which could contribute to intravascular coagulation and inflammation. We recently showed that 4-hydroxy-2-nonenal (HNE), the most abundant and stable unsaturated aldehyde produced by the oxidation of ω-6 polyunsaturated fatty acids, enhances TF activity in monocytic cells and macrophages. However, the role of HNE in activation of intravascular coagulation is unknown. To investigate this, C57Bl/6J wild-type mice were administered with HNE intraperitoneally (10 mg/kg body weight) and generation of TF+ MVs, activation of intravascular coagulation, and inflammation was assessed. HNE administration markedly increased TF activity associated with MVs. Nanoparticle tracking analysis (NTA) showed a 3-fold increase in the number of MVs in the plasma of HNE-treated mice. Measurement of thrombin-antithrombin (TAT) complexes revealed a significant increase in TAT levels in HNE treated mice. Pre-treatment of the mice with anti-murine TF antibody attenuated the HNE-induced increase in TAT levels. Measurement of plasma clotting times showed a significant reduction in HNE treated mice (118.3 ± 10.42 sec) compared to saline treated control mice (> 300 sec). Incubation of plasma with anti-TF antibody reversed the reduction observed in the plasma clotting time of HNE treated mice. Assessment of clotting in the saphenous vein injury bleeding model showed an increased number of clots, and reduced blood loss in HNE-administered mice compared to saline treated mice. Blockade of TF with 1H1 anti-TF antibody negated the HNE-mediated reduction in the clotting time. Next, we investigated whether HNE administration also induces a proinflammatory response in mice. Blood cells analysis showed approximately a 4-fold increase in circulating neutrophils in HNE-treated mice at 6 h following HNE treatment. Immunohistochemistry studies showed a robust infiltration of neutrophils into the lungs. Cytokine analysis of plasma showed a significant increase in IL-6 and CXCL1 levels in HNE-treated mice. Blockade of TF using 1H1 anti-TF antibody significantly reduced the HNE-mediated neutrophil infiltration into the lungs and completely abrogated the increase in plasma IL-6 levels. In additional studies, we attempted to identify the source of TF+ MVs. It is unlikely that they are originated from monocytes or endothelial cells as HNE did not induce TF expression in these cell types. LPS, used as a positive control, showed significant induction of TF on both PBMCs and the endothelium Immunohistochemistry revealed that HNE disrupted endothelial barrier integrity of vessel wall, raising a possibility that TF+ MVs could have come from perivascular cells. In summary, our data indicate that oxidative stress could induce the release of TF+ MVs, and this would contribute to intravascular coagulation and inflammation. Our studies also suggest that TF-positive microvesicles can be a useful therapeutic target in the treatment of thromboinflammatory diseases. Disclosures No relevant conflicts of interest to declare.

Evaluation of a Blood Coagulation Factor IX Variant That Functions Independently of Factor VIII As an Alternative Treatment for Hemophilia A

The serine protease factor IXa (FIXa) serves an important role in coagulation by catalyzing the proteolytic activation of factor X (FX) together with its cofactor VIIIa (FVIIIa). Being a critical protease in coagulation, the FIXa structure has evolved to be subjected to strict regulatory mechanisms. While FIXa displays considerable structural homology with other coagulation serine proteases, its active site is uniquely controlled by the 99-loop that blocks access to the active site pocket. Cofactor-mediated interaction of FIXa with its substrate FX induces a conformational change that allows for active site engagement and substrate catalysis. Previously, the molecular constraints of the 99-loop were lifted due to specific modifications in both the 99-loop (K265A), the S1 active site subpocket (V181I, I383V), and the L6F substitution, thereby generating FIX-FIAV [Quade-Lyssy et al. J. Thromb. Haemost. 2014]. As a result, this variant is capable of functioning independently of factor VIII (FVIII). Moreover, FIX-FIAV was demonstrated to ameliorate the hemophilia A phenotype both in vitro and in vivo. To further evaluate its therapeutic potential, FIX-FIAV was stably expressed in HEK293 cells and purified by ion-exchange and hydrophobic interaction chromatography. Evaluation of the kinetics of tissue factor-factor VIIa (TF-FVIIa) activation of FIX-FIAV revealed kinetic parameters similar to those of human wild-type FIX(-WT). Analysis of FIX activation intermediates that are formed upon proteolysis by TF-FVIIa or factor XIa demonstrated prolonged formation of FIX-FIAVα, while no FIXa-WTα could be observed. This is consistent with delayed cleavage at position 180, likely resulting from the V181I substitution in FIX-FIAV. Given that the activation mechanism of FIX-FIAV is unperturbed, we next assessed the specific FVIII clotting activity and demonstrated that FIX-FIAV exhibited significant FVIII-like clotting activity (56 ± 4 U/mg) as opposed to FIX-WT (<13 U/mg). These values correlate with up to 28% of FVIII-independent activity for FIX-FIAV at FIX plasma levels (5 ug/mL), confirming that FIX-FIAV has the potential to enhance thrombin generation in FVIII deficiency. To validate this, tissue factor-initiated (0.5 or 1.0 pM) thrombin generation was assessed in FVIII-immunodepleted plasma, leading to a severely reduced thrombin peak (88% or 81% reduction, respectively) relative to conditions with 100% FVIII. Addition of FIX-FIAV (5 ug/mL) partially restored thrombin generation, demonstrated by an up to ~30% increase in both thrombin peak and endogenous thrombin potential. Evaluation of the FVIII-independent activity of FIX-FIAV in severe hemophilia A patient plasma with or without an inhibitor resulted in an up to 18% or 32% FVIII-like activity, respectively, demonstrating efficacy of FIX-FIAV in the presence of FVIII inhibitors. Although unlikely, it remains to be determined whether specific FVIII-inhibitors may impact FIX-FIAV function. Adding 100% FVIII or low- to mid-range therapeutic concentrations of the bispecific antibody emicizumab to FVIII-deficient plasma incubations with FIX-FIAV resulted in a synergistic enhancement of thrombin generation, demonstrated by a 9-fold increase in thrombin peak. This is consistent with the previously demonstrated hyperactivity of FIX-FIAV in a cofactor-dependent system. In contrast, no synergistic effect on thrombin generation was observed when combining FIX-FIAV with physiologically relevant concentrations of FEIBA or NovoSeven. Summarizing, FIX-FIAV is characterized by a preserved mechanism of activation in addition to being capable of sustaining therapeutic levels of coagulation activity in FVIII deficiency. This provides support for the use of FIX-FIAV as an alternative treatment for hemophilia A. Disclosures Strijbis: uniQure Biopharma B.V.: Research Funding. Konstantinova:uniQure Biopharma B.V.: Employment. Liu:uniQure Biopharma B.V.: Employment. van Deventer:uniQure Biopharma B.V.: Employment. Bos:uniQure Biopharma B.V.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Changes in Organic Anion Transporting Polypeptide Uptake in HEK293 Overexpressing Cells in the Presence and Absence of Human Plasma.

Generating accurate in vitro data is crucial for in vitro to in vivo extrapolation and pharmacokinetic predictions. The use of human embryonic kidney (HEK) 293 cells overexpressing organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 in protein-free buffer and 100% human plasma incubations was explored for the uptake of four OATP substrates: pravastatin, rosuvastatin, repaglinide, and pitavastatin. Differences were observed for each parameter [unbound Michaelis constant (K m,u), V max, intrinsic clearance (CLint), and unbound passive diffusion Pdif,u] obtained from the buffer and plasma incubations in both cells, and the fold differences were greatest for the highly protein bound compounds. The fold change in K m,u values ranged from 1.91 to 619, and the fold change in V max values ranged from 1.22 to 97.4. As a result, in both cells, the CLint values generated in the plasma incubations were higher by 0.762- to 31.7-fold than the values generated in the protein-free buffer. The passive diffusion was also higher in the plasma incubations for all four compounds, with a fold difference range of 1.73-23.4. These shifts in the presence and absence of human plasma suggest that plasma proteins may play a role in both the active uptake and passive diffusion processes. The results also support the idea of a transporter-induced protein-binding shift, where high protein binding may not limit the uptake of compounds that have high affinity for transporters. The addition of plasma to incubations leading to higher CLint values for transporter substrates helps mitigate the underprediction commonly noted with in vitro to in vivo extrapolation. SIGNIFICANCE STATEMENT: The current investigation brings a new perspective on how to mitigate the underprediction commonly noted with in vitro to in vivo extrapolation for OATP substrates by using HEK293 cells overexpressing OATP1B1 and OATP1B3. It also supports the idea of a transporter-induced protein-binding shift, where high protein binding may not limit the uptake of compounds that have high affinity for transporters.

Noncovalent Targeting of Nanocarriers to Immune Cells with Polyphosphoester-Based Surfactants in Human Blood Plasma.

Dendritic cells (DCs) are part of the immune system and can internalize pathogens by carbohydrate receptors. The uptake induces maturation and migration of the DCs resulting in an adaptive immune response by presenting antigens to T‐cells. Thus, targeted delivery to DCs is a powerful tool for immunotherapy. However, in blood, specific targeting is challenging as blood proteins adsorb to the nanocarriers and mask the targeting molecules. Additionally, covalent coupling of targeting groups to nanocarriers requires new chemistry for each nanocarrier, while a general strategy is missing. A general protocol by noncovalent adsorption of mannosylated polyphosphoesters (PPEs) on the nanocarriers' surface resulting in specific uptake into DCs combined with low protein adsorption of PPEs is presented. PPEs with hydrophobic anchors and multiple mannose units are reported and adsorbed to different model nanocarriers. Their protein corona remain similar to pure stealth nanocarriers and prove only low uptake into nontargeted cells (monocytes). Due to the “stealth” properties of PPEs, a high specific uptake into DCs is achieved after incubation in human blood plasma, proving an efficient combination of “stealth” and targeting after simple adsorption of the PPEs. This strategy can transform any nanocarrier into DC‐targeting by noncovalent adsorption of PPEs and will aid in developing novel immunotherapies.

Glycoprotein Ib clustering in platelets can be inhibited by α-linolenic acid as revealed by cryo-electron tomography.

Platelet adhesion to the sub-endothelial matrix and damaged endothelium occurs through a multi-step process mediated in the initial phase by glycoprotein Ib binding to von Willebrand factor (vWF), which leads to the subsequent formation of a platelet plug. The plant-derived ω-3 fatty acid α-linolenic acid is an abundant alternative to fish-derived n-3 fatty acids and has anti-inflammatory and antithrombotic properties. In this study, we investigated the impact of α-linolenic acid on human platelet binding to vWF under high-shear flow conditions (mimicking blood flow in stenosed arteries). Pre-incubation of fresh human blood from healthy donors with α-linolenic acid at dietary relevant concentrations reduced platelet binding and rolling on vWF-coated microchannels at a shear rate of 100 dyn/cm2. Depletion of membrane cholesterol by incubation of platelet-rich plasma with methyl-β cyclodextrin abrogated platelet rolling on vWF. Analysis of glycoprotein Ib by applying cryo-electron tomography to intact platelets revealed local clusters of glycoprotein Ib complexes upon exposure to shear force: the formation of these complexes could be prevented by treatment with α-linolenic acid. This study provides novel findings on the rapid local rearrangement of glycoprotein Ib complexes in response to high-shear flow and highlights the mechanism of in vitro inhibition of platelet binding to and rolling on vWF by α-linolenic acid.

1-Substituted sialorphin analogues\u2014synthesis, molecular modelling and in vitro effect on enkephalins degradation by NEP

Rat sialorphin (Gln-His-Asn-Pro-Arg) is a natural blocker of neprilysin (NEP) that belongs to the family of endogenous opioid peptide-degrading enzymes. Studies have confirmed the efficiency of sialorphin in blocking the activity of NEP, both in vitro and in vivo. It has been demonstrated that this inhibitor has a strong analgesic, anti-inflammatory, immunological and metabolic effect either directly or indirectly by affecting the level of Met/Leu-enkephalins. In this work, sialorphin and their 12 analogues were synthesised using the solid-phase method. The effect of the peptides on the degradation of Met-enkephalin by NEP and metabolic degradation in human plasma was investigated in vitro. We show that the change in the N-terminal amino acid configuration from l to d in almost all peptides, except d-Arg-His-Asn-Pro-Arg (peptide XI), led to the abolition of their inhibitory activity. With molecular modelling technique we explained the structural properties of the l and d-arginine located on the N-terminal part of the peptide. The detailed analysis of the protein binding pocket allowed us to explain why d-arginine is so unique among all d residues. Peptide XI showed the highest stability among the tested peptides in human plasma. For instance sialorphin after a 2-hour incubation in human plasma was almost completely decomposed, while the level of peptide XI dropped to 45% after 48 h under these conditions.

Prevention of Dominant IgG Adsorption on Nanocarriers in IgG-Enriched Blood Plasma by Clusterin Precoating.

Nanocarriers for medical applications must work reliably within organisms, independent of the individual differences in the blood proteome. Variation in the blood proteome, such as immunoglobulin levels, is a result of environmental, nutrition, and constitution conditions. This variation, however, should not influence the behavior of nanocarriers in biological media. The composition of the protein corona is investigated to understand the influence varying immunoglobulin levels in the blood plasma have on the interactions with nanocarriers. Specifically, the composition of the nanocarriers' coronas is analyzed after incubation in plasma with normal or elevated immunoglobulin G (IgG) levels, and cellular uptake is monitored in cell lines containing different immunoglobulin receptors. Here, it is reported that upon doubling the IgG concentration in plasma, the IgG fraction in the protein corona increases by a factor of 40 independent of the nanocarrier material. This results in a significant increase in uptake in cells exhibiting IgG binding receptors. Furthermore, precoating nanocarriers with clusterin successfully prevents dominant IgG‐adsorption and additionally reduces cellular internalization, after incubation with IgG‐enriched plasma. Therefore, precoating nanocarriers may be utilized as a powerful method to reduce the influence of individual variations in blood composition on the protein corona.

Red blood cells are damaged by intraoperative blood salvage via Ca2+-dependent and -independent mechanisms

AimsIntraoperative blood salvage (IBS) is associated with shortened lifespan of red blood cells (RBCs). This study aims to examine how salvaged RBCs are compromised during IBS. Main methodsThirty patients who underwent vertebra surgery with IBS were included in the study. To examine possible mechanisms of IBS-induced injury, both fresh and salvaged RBCs from each patient were mixed with plasma, the Ca2+ ionophore ionomycin or mannitol-adenine-phosphate (MAP) solution (n = 10 patients per condition). Binding of Fluo-3 and/or Annexin V by RBCs was measured. Key findingsThe percentage of Fluo-3-binding RBCs in salvaged samples was 2.83 ± 0.76%, which increased to 15.34 ± 5.99% after 48-h incubation in plasma. These percentages were significantly higher than those observed with fresh RBCs (P < 0.01). Ionomycin dose-dependently increased the percentage of Fluo-3-binding RBCs in salvaged samples, while MAP solution decreased it. Incubating salvaged RBCs in plasma for 48 h increased the percentage of Fluo-3-positive spherocytes from 0.8 ± 0.6% to 11.35 ± 3.96%, and this increase was blocked by MAP solution. Ionomycin increased the percentage of RBCs binding both Fluo-3 and Annexin V, while MAP decreased this percentage. The percentage of Annexin V-binding RBCs was also higher in salvaged samples than in fresh samples, but this percentage was unaffected by either ionomycin or MAP solution. SignificanceOur results suggest that IBS induces a postponed RBC damage by inducing spherocyte formation, which likely reflects Ca2+ entry induced by energy exhaustion, as well as by exposing phosphatidylserine on the RBC surface, which likely occurs via Ca2+ entry and via Ca2+-independent pathways.

Resveratrol improves endothelial cell markers impaired by plasma incubation from women who subsequently develop preeclampsia.

In this study, we demonstrated that plasma collected from women who subsequently developed preeclampsia caused increased heme oxygenase-1 (HO-1) production and decreased levels of nitric oxide (NO) markers in endothelial cells (HUVECs). Conversely, no changes in HO-1 or NO markers were found when HUVECs were treated with plasma from women who remained healthy throughout pregnancy. These alterations in HO-1 and NO markers were prevented by cotreatment with the polyphenol resveratrol, which also improved GSH levels. In addition, we evaluated changes induced by plasma incubation in the expression of genes and their related pathways associated with antioxidant defenses, such as Nrf2, ARE activity, and GSR. Collectively, our findings suggest that even before the appearance of clinical symptoms of preeclampsia, plasma from affected women is able to induce modifications in endothelial cells with respect to HO-1 production and NO markers. We believe that this in vitro strategy may offer an attractive alternative to the exploitation of candidate markers or screening molecules, such as resveratrol, for the prevention and management of preeclampsia.

Development of radiolabeled bis(zinc(II)-dipicolylamine) complexes for cell death imaging.

Although it has been traditionally surmised that phosphatidylserine (PS) externalization is a hallmark of apoptosis, most other non-apoptotic modes of cell death, such as necrosis, are also associated with PS externalization. Bis(zinc-dipicolylamine) (ZnDPA) complexes have been reported to exhibit affinity for PS. The present study aimed to develop novel radiolabeled ZnDPA derivatives for cell death imaging in tumor after treatment with anticancer drugs. [125I]IB-EG2-ZnDPA and [99mTc]Tc-MAG3-EG2-ZnDPA were designed and prepared. The stabilities of these radiotracers were determined in 0.1M phosphate buffer (pH 7.4) or murine plasma at 37°C, and their 1-octanol/water partition coefficients (logP) were measured. The uptake of radioactivity in cancer cells, which were preincubated in a normal medium or in a medium containing 5-FU, was measured after incubation with radiotracers. Accumulation of [99mTc]Tc-MAG3-EG2-ZnDPA in the tumor was evaluated in tumor-bearing mice treated with or without 5-FU, and then TUNEL staining was performed to detect dead cells in the tumor tissue sections. The radiochemical purities of [125I]IB-EG2-ZnDPA and [99mTc]Tc-MAG3-EG2-ZnDPA exceeded 95%. Although [125I]IB-EG2-ZnDPA gradually decomposing with time, more than 90% of [99mTc]Tc-MAG3-EG2-ZnDPA remained in its intact form in phosphate buffer through 6h of incubation. Neither [125I]IB-EG2-ZnDPA nor [99mTc]Tc-MAG3-EG2-ZnDPA decomposed so much after 6-h incubation in murine plasma. [125I]IB-EG2-ZnDPA could not specifically recognize PS on the cell surface because of its high lipophilicity. Conversely, [99mTc]Tc-MAG3-EG2-ZnDPA accumulated in cancer cells after treatment with an anticancer drug both in vitro and in vivo, and its accumulation was correlated with the number of TUNEL-positive cells. However, the biodistribution of [99mTc]Tc-MAG3-EG2-ZnDPA was not suitable for imaging because of its low accumulation in tumor and high uptake in abdomen organs. [99mTc]Tc-MAG3-EG2-ZnDPA could be useful for the early detection of treatment effects after chemotherapy. Since the signal-to-noise ratio is not enough for single photon emission computed tomography imaging, further modification is needed to improve its biodistribution and affinity for PS.

The Presence of a Transporter-Induced Protein Binding Shift: A New Explanation for Protein-Facilitated Uptake and Improvement for In Vitro-In Vivo Extrapolation.

Accurately predicting hepatic clearance is an integral part of the drug-development process, and yet current in vitro to in vivo (IVIVE) extrapolation methods yield poor predictions, particularly for highly protein-bound transporter substrates. Explanations for error include inaccuracies in protein-binding measurements and the lack of recognition of protein-facilitated uptake, where both unbound and bound drug may be cleared, violating the principles of the widely accepted free drug theory. A new explanation for protein-facilitated uptake is proposed here, called a transporter-induced protein binding shift High-affinity binding to cell-membrane proteins may change the equilibrium of the nonspecific binding between drugs and plasma proteins, leading to greater cellular uptake and clearance than currently predicted. The uptake of two lower protein-binding organic anion transporting polypeptide substrates (pravastatin and rosuvastatin) and two higher binding substrates (atorvastatin and pitavastatin) were measured in rat hepatocytes in incubations with protein-free buffer versus 100% plasma. Decreased unbound K m values and increased intrinsic clearance values were seen in the plasma incubations for the highly bound compounds, supporting the new hypothesis and mitigating the IVIVE underprediction previously seen for highly bound transporter substrates.

Inhibition or Stimulation of Ochratoxin A Synthesis on Inoculated Barley Triggered by Diffuse Coplanar Surface Barrier Discharge Plasma.

Ochratoxin A (OTA) is one of the most abundant food-contaminating mycotoxins. Besides their high toxicity, mycotoxins are highly stable to physical, chemical or biological detoxification. Therefore, the treatment with cold atmospheric plasma could be one approach to reduce the amount of mycotoxins in different products. The aim of this study was to determine the influence of cold atmospheric plasma on the inactivation of Aspergillus niger and Penicillium verrucosum inoculated on barley and their production of OTA. Inoculated barley was treated with plasma generated by dry air, CO2 or CO2 + O2 for 1 or 3 min and stored for up to two weeks at 9, 25, or 37°C. Three minutes of air plasma treatment effectively significantly reduced the total mold count of both microorganisms by 2.5–3 log cycles. The production of OTA from A. niger was only low, therefore the treatment effect was indistinguishable. The treatment of P. verrucosum on barley after an incubation of five days using a CO2 + O2 plasma resulted in a reduction of the OTA content from 49.0 (untreated) to 27.5 (1 min) and 23.8 ng/g (3 min), respectively. In contrast, CO2 plasma caused an increase of the OTA amount from 49.0 (untreated) to 55.8 (1 min) and 72.9 ng/g (3 min). Finally, the use of air plasma resulted likewise in a decrease of the OTA concentration from 56.9 (untreated) to 25.7 (1 min) and 20.2 ng/g (3 min), respectively. Reducing the incubation time before the treatment to 24 h caused in contrast an increase of the OTA content from 3.1 (untreated) to 29.1 (1 min) and 20.7 ng/g (3 min). Due to the high standard deviation, these changes were not significant, but the tendencies were clearly visible, showing the strong impact of the plasma gas on the OTA production. The results show, that even if the total mold count was reduced, under certain conditions the OTA amount was yet enhanced, probably due to a stress reaction of the mold. Concluding, the plasma gas and incubation conditions have to be considered to allow a successful inactivation of molds and in particular their toxic metabolites.

Metabolism and disposition of pyrotinib in healthy male volunteers: covalent binding with human plasma protein.

Pyrotinib is a novel irreversible EGFR/HER2 dual tyrosine kinase inhibitor that is used to treat HER2-positive breast cancer. In this study we investigated the metabolism and disposition of pyrotinib in six healthy Chinese men after a single oral dose of 402 mg of [14C]pyrotinib. At 240 h postdose, the mean cumulative excretion of the dose radioactivity was 92.6%, including 1.7% in urine and 90.9% in feces. In feces, oxidative metabolites were detected as major drug-related materials and the primary metabolic pathways were O-depicoline (M1), oxidation of pyrrolidine (M5), and oxidation of pyridine (M6-1, M6-2, M6-3, and M6-4). In plasma, the major circulating entities identified were pyrotinib, SHR150980 (M1), SHR151468 (M2), and SHR151136 (M5), accounting for 10.9%, 1.9%, 1.0%, and 3.0%, respectively, of the total plasma radioactivity based on the AUC0-∞ ratios. Approximately 58.3% of the total plasma radioactivity AUC0-∞ was attributed to covalently bound materials. After incubation of human plasma with [14C]pyrotinib at 37 °C for 2, 5, 8, and 24 h, the recovery of radioactivity by extraction was 97.4%, 91.8%, 69.6%, and 46.7%, respectively, revealing covalent binding occurred independently of enzymes. A group of pyrotinib adducts, including pyrotinib-lysine and pyrotinib adducts of the peptides Gly-Lys, Lys-Ala, Gly-Lys-Ala, and Lys-Ala-Ser, was identified after HCl hydrolysis of the incubated plasma. Therefore, the amino acid residue Lys190 of human serum albumin was proposed to covalently bind to pyrotinib via Michael addition. Finally, the covalently bound pyrotinib could dissociate from the human plasma protein and be metabolized by oxidation and excreted via feces.

Improving Plasma Stability and Bioavailability In Vivo of Gemcitabine Via Nanoparticles of mPEG-PLG-GEM Complexed with Calcium Phosphate.

Despite being widely used for the treatment of several solid tumors, Gemcitabine (GEM) exhibits several suboptimal pharmacokinetic properties. Therefore, the design of nanoparticle delivery systems is a promising strategy to enhance GEM pharmacokinetic properties. In this work, the polymeric material methoxy poly(ethylene glycol)-block-poly(L-glutamic acid)-graft-gemcitabine (mPEG-b-PLG-g-GEM) was synthesized through the covalent conjugation of GEM with the carboxylic group of methoxy poly(ethylene glycol)-block-poly (L-glutamic acid) (mPEG-b-PLG) (mPEG113, Mn = 5000). mPEG-PLG-GEM/CaP nanoparticles were prepared through the simple mixing of calcium and phosphate/mPEG-PLG-GEM solutions. mPEG-PLG-GEM was embedded in the calcium phophate (CaP) backbone via electrostatic interactions. After incubation in plasma at 37°C for 24h, gemcitabine was degraded by 24.6% for the mPEG-PLG-GEM, 14.7% for the mPEG-PLG-GEM/CaP nanoparticles, and 90% for the free gemcitabine solution. It was observed that mPEG-PLG-GEM and mPEG-PLG-GEM/CaP improved the area-under-curve (AUC) values by 5.26-fold and 6.33-fold compared to free drug, respectively. The amide bond linked gemcitabine polymers was able to protect GEM from cytidine deaminase degradation in vivo, and the skeleton formed by the calcium phosphate enhanced the stability and prolonged the half-life of GEM. Importantly, mPEG-PLG-GEM/CaP nanoparticles elevated the GEM plasma concentration in an animal model.

LC-MS/MS Identification and Structural Characterization of Main Biodegradation Products of Nitroproston - A Novel Prostaglandin-based Pharmaceutical Compound.

Nitroproston is a novel prostaglandin-based compound modified by NOdonating groups with potential application in obstructive respiratory diseases such as asthma and obstructive bronchitis. Nitroproston has been extensively studied using various pharmacological models. Its biological stability is still uncertain. The aim of the present study was to evaluate Nitroproston stability in vitro, as well as to identify and characterize its major biodegradation products. The principal biodegradation products of Nitroproston were identified in vitro using liquid chromatography/ion trap - time-of-flight mass-spectrometry. The postulated structure of metabolites was confirmed using authentic reference standards. Rat, rabbit and human plasma and human whole blood samples were used for comparative in vitro degradation study. Nitroproston and its biodegradation products in biological samples were measured by liquid chromatography/triple -stage quadrupole mass spectrometry. Nitroproston is rapidly hydrolyzed in rat plasma to generate glycerol-1,3-dinitrate and prostaglandin E2. The latter can undergo conversion to cyclopentenone prostaglandins A2 and B2. Thereby less than 5% of the parent compound was observed in rat plasma at the first moment of incubation. A similar pattern was observed for rabbit plasma where half-life (T1/2) of Nitroproston was about 2.0 minutes. Nitroproston biodegradation rate for human plasma was the slowest (T1/2 = 2.1 h) among tested species, occurred more rapidly in whole blood (T1/2 = 14.8 min). It was found that Nitroproston is rapidly hydrolyzed in rodent compared to human plasma incubations. Whereas Nitroproston is relatively stable in human plasma an enhanced hydrolytic activity was observed in whole human blood incubations. Extensive metabolism of Nitroproston in human whole blood was mainly associated with red blood cells. The observed interspecies variability highlights the need of suitable animal model selection for Nitroproston follow-up PK/PD studies.

Establishment of a quantitative bioanalytical method for an acetylcholinesterase inhibitor Ethyl 3-(2-(4-fluorophenyl) amino)-4-phenylthiazol-5-yl)-3-oxopropanoate including its physicochemical characterization and in vitro metabolite profiling using Liquid Chromatography-Mass Spectrometry

Ethyl 3-(2-(4-fluorophenyl)amino)-4-phenylthiazo)-5-yl)-3-oxopropanoate is a novel molecule with potent acetylcholinesterase inhibition property. In this research, we have developed a rapid and selective RP-HPLC bioanalytical method for quantitative measurement of the molecule. The method has been validated following the USFDA bioanalytical method validation guideline. In addition, as a part of drug development, in vitro metabolite identification has also been performed. A Kromasil C18 column was used in eluting the molecule chromatographically. The optimized mobile phase was composed of a mixture of acetonitrile and 10 mM ammonium formate buffer (pH 4.6) in 70:30 ratio (v/v). The response of the molecule was found to be linear over a calibration range of 0.2 μg/mL to 12 μg/mL. The inter-day and intra-day accuracy of the method ranged from 89.95% to 101.90% and 99.84% to 104.08%, respectively. On the other hand, the precision (%CV) value for inter-day was in between 3.50% to 6.91% and for intra-day, it was 2.11% to 8.03%. The mean recovery of the molecule at three different quality control levels was more than 85%. The analyte was stable under different stability conditions including 12 h autosampler, 8 h bench top, 15 days long term at −20 °C and three freeze-thaw cycles. The method was applied to determine stability of the molecule in human plasma. The molecule was found to be stable with more than 90% remaining even after 120 min of incubation in plasma. Two metabolites each in rat liver microsome and human liver microsome has been identified.

A new method for the determination of total and released docetaxel from docetaxel-entrapped core-crosslinked polymeric micelles (CriPec®) by LC–MS/MS and its clinical application in plasma and tissues in patients with various tumours

A sensitive, high-performance liquid chromatographic method was developed and validated, for determination of docetaxel from docetaxel-entrapped core-crosslinked polymeric micelles (CriPec®) in human potassium EDTA plasma and released docetaxel to support the clinical development of Cripec® docetaxel. CriPec® docetaxel is a novel formulation of docetaxel – covalently conjugated via a linker agent in a nanoparticle. The analytical characterization of CriPec® docetaxel comprises determination of both released and total docetaxel, the first being the already deconjugated docetaxel, whereas total is representative of all docetaxel (deconjugated as well as CriPec® nanoparticle conjugated material). Total docetaxel was determined by incubation of human plasma with 0.5 M ammonium acetate buffer pH 7.4 for 3-days at 37 °C. Hereafter, a liquid-liquid extraction with 1-chlorobutane was performed using paclitaxel as internal standard. Released docetaxel from CriPec® docetaxel nanoparticles was determined in human plasma stabilized with 5 M ammonium acetate, pH 5.0. Hereafter, a liquid-liquid extraction with 1-chlorobutane was performed using docetaxel-d5 in acetonitrile as internal standard. Released docetaxel and its internal standard were eluted. The validated ranges for total docetaxel were 2,000–100,000 ng/mL for the high concentrations and 2–500 ng/mL for the low concentrations and 0.250–100 ng/mL for released docetaxel.In conclusion the newly developed assay met the required standards for validation and was applied successfully to support pharmacokinetic analysis in both serum and tissue in patients treated with Cripec®.

Development of novel anti-CD19 antibody-drug conjugates for B-cell lymphoma treatment

B-cell lymphoma remains one of the most refractory tumors, and as such the development of novel treatment approaches, such as antibody-drug conjugates (ADCs), is required. To improve the stability and homogeneity of the ADCs, a humanized anti-CD19 monoclonal antibody (RC58) was developed in the present study. RC58 was based on the CD19 antigen as a potential molecular target of human B-cell lymphomas. RC58 has high CD19-binding affinity and can be internalized in CD19-positive cells through endocytosis. Furthermore, three types of RC58-based ADCs (ADC-1, ADC-2, and ADC-3) were generated using three kinds of Maleimide-PEG-based linkers with two different cytotoxins. The anti-tumor activities of the ADCs were confirmed by in vitro and in vivo experiments. The stability of the ADCs was also evaluated by incubation in human plasma for 10 days. In vitro experiments showed that the three ADCs had distinct inhibitory effects on several B-lymphoma cell lines. Meanwhile, a close correlation between efficacy and drug concentration was found in a nude mouse xenograft model of human B-cell lymphoma, after treatment with RC58-based ADCs. Our results suggest that ADC-1, with high efficiency, could be used as a potential therapeutic agent for human B-cell malignancies.

Metabolites Identification of Bioactive Compounds Daturataturin A, Daturametelin I, N-Trans-Feruloyltyramine, and Cannabisin F From the Seeds of Datura metel in Rats.

Datura metel L. is a widely used traditional herbal medicine, and withanolides and amides are the two groups of main bioactive constituents in Datura metel seeds. This study aimed to elucidate the metabolism of four representative bioactive compositions containing daturataturin A (1), daturametelin I (2), N-trans-feruloyltyramine (3), and cannabisin F (4) in rats. After separately oral administration of 20 mg/kg withanolides (1, 2) and amides (3, 4) to rats, a total of 12, 24, and 21 metabolites were detected in the plasma, urine, and fecal samples, respectively. Among them, three hydroxylated metabolites, 1-M3, 2-M2, and 3-M5, were detected in plasma and rat liver microsome incubation system in high abundance. Two metabolites of 1 and 2 were unambiguously identified by comparing with reference standards. Particularly, the methylated metabolite 27α-methoxy-(22R)-22,26-epoxy-27-[(β-D-glucopyranosyl)oxy]ergosta-2,4,6,24-tetraene-1,26-dione (daturametelin L) is a new compound. The withanolides could readily get hydroxylation or methylation metabolism. Meanwhile, the phase II metabolism (glucuronidation or sulfation) was the major reaction for the amides. This is the first study on in vivo metabolism of these active compounds in seeds of Datura metel.

Electrochemically deposited silver detection substrate for surface-enhanced Raman spectroscopy cancer diagnostics.

Surface-enhanced Raman spectroscopy (SERS) of blood plasma on an electrochemically prepared silver surface has been studied as a label-free, noninvasive diagnostic test for colorectal cancer. Indium tin oxide glass substrates were modified with 0.01 mol dm - 3 silver nitrate using the pulsed double-potentiostatic method. The prepared silver substrates were tested with Rhodamine 6G as a model analyte and the surface with the highest signal enhancement was selected. This silver dendritic surface was used as a diagnostic substrate for SERS measurements of human blood plasma. A group of oncological patients with declared colorectal carcinoma (n = 15) and the control group of healthy volunteers (n = 15) were compared. The biomolecular changes in chemical composition in the cancer samples were detected by statistical processing of the resulting SERS spectra. About 94% specificity and 100% sensitivity were achieved for the analysis by the ratio of the SERS peak intensity at 725 cm - 1 for adenine to the peak intensity at 638 cm - 1 for tyrosine and 100% specificity and sensitivity by using principal component analysis. This method of SERS diagnostics of colorectal cancer, which does not require the nanoparticle preparation, mixing, and incubation of plasma with a colloidal solution as in conventional tests, is a rapid, inexpensive method, which could be introduced as a primary diagnostic test.