Physiological Plasma Concentrations Research Articles

Effects of electro-mechanical uncouplers, hormonal stimulation and pacing rate on the stability and function of cultured rabbit myocardial slices.

Introduction: Recent advances have enabled organotypic culture of beating human myocardial slices that are stable for weeks. However, human myocardial samples are rare, exhibit high variability and frequently originate from diseased hearts. Thus, there is a need to adapt long-term slice culture for animal myocardium. When applied to animal cardiac slices, studies in healthy or genetically modified myocardium will be possible. We present the culture of slices from rabbit hearts, which resemble the human heart in microstructure, electrophysiology and excitation-contraction coupling. Methods: Left ventricular myocardium from New Zealand White rabbits was cut using a vibratome and cultured in biomimetic chambers for up to 7days (d). Electro-mechanical uncoupling agents 2,3-butanedione monoxime (BDM) and cytochalasin D (CytoD) were added during initiation of culture and effects on myocyte survival were quantified. We investigated pacing rates (0.5Hz, 1Hz, and 2Hz) and hormonal supplements (cortisol, T3, catecholamines) at physiological plasma concentrations. T3 was buffered using BSA. Contractile force was recorded continuously. Glucose consumption and lactate production were measured. Whole-slice Ca2+ transients and action potentials were recorded. Effects of culture on microstructure were investigated with confocal microscopy and image analysis. Results: Protocols for human myocardial culture resulted in sustained contracture and myocyte death in rabbit slices within 24h, which could be prevented by transient application of a combination of BDM and CytoD. Cortisol stabilized contraction amplitude and kinetics in culture. T3 and catecholaminergic stimulation did not further improve stability. T3 and higher pacing rates increased metabolic rate and lactate production. T3 stabilized the response to β-adrenergic stimulation over 7d. Pacing rates above 1Hz resulted in progredient decline in contraction force. Image analysis revealed no changes in volume fractions of cardiomyocytes or measures of fibrosis over 7d. Ca2+ transient amplitudes and responsiveness to isoprenaline were comparable after 1d and 7d, while Ca2+ transient duration was prolonged after 7d in culture. Conclusions: A workflow for rabbit myocardial culture has been established, preserving function for up to 7d. This research underscores the importance of glucocorticoid signaling in maintaining tissue function and extending culture duration. Furthermore, BDM and CytoD appear to protect from tissue damage during the initiation phase of tissue culture.

β2-glycoprotein I promotes the clearance of circulating mitochondria.

β2-glycoprotein I (β2-Gp1) is a cardiolipin-binding plasma glycoprotein. It is evolutionarily conserved from invertebrates, and cardiolipin-bound β2-Gp1 is a major target of antiphospholipid antibodies seen in autoimmune disorders. Cardiolipin is almost exclusively present in mitochondria, and mitochondria are present in circulating blood. We show that β2-Gp1 binds to cell-free mitochondria (CFM) in the circulation and promotes its phagocytosis by macrophages at physiological plasma concentrations. Exogenous CFM had a short circulation time of less than 10 minutes in mice. Following infusion of CFM, β2-Gp1-deficient mice had significantly higher levels of transfused mitochondria at 5 minutes (9.9 ± 6.4 pg/ml versus 4.0 ± 2.3 pg/ml in wildtype, p = 0.01) and at 10 minutes (3.0 ± 3.6 pg/ml versus 1.0 ± 0.06 pg/ml in wild-type, p = 0.033, n = 10). In addition, the splenic macrophages had less phagocytosed CFM in β2-Gp1-deficient mice (24.4 ± 2.72% versus 35.6 ± 3.5 in wild-type, p = 0.001, n = 5). A patient with abnormal β2-Gp1, unable to bind cardiolipin, has increased CFM in blood (5.09 pg/ml versus 1.26 ± 1.35 in normal) and his plasma induced less phagocytosis of CFM by macrophages (47.3 ± 1.6% versus 54.3 ± 1.3, p = 0.01) compared to normal plasma. These results show the evolutionarily conserved β2-Gp1 is one of the mediators of the clearance of CFM in circulation.

3D printed, personalized sustained release cortisol for patients with adrenal insufficiency

The standard of care for patients with Adrenal Insufficiency (AI) is suboptimal. Administration of hydrocortisone three times a day produces plasma cortisol fluctuations associated with negative health outcomes. Furthermore, there is a high inter-individual variability in cortisol need, necessitating a personalized approach. It is hypothesized that a personalized, sustained release formulation would enhance the pharmacotherapy by mimicking the physiological cortisol plasma concentration at a higher level. Therefore, a novel 24 h sustained release 3D printed (3DP) hydrocortisone formulation has been developed (M3DICORT) by coupling hot-melt extrusion with fused deposition modeling. A uniform drug distribution in the 3DP tablets is demonstrated by a content of 101.66 ± 1.60 % with an acceptance value of 4.01. Furthermore, tablets had a stable 24 h dissolution profile where the intra-batch standard deviation was ± 2.8 % and the inter-batch standard deviation was ± 6.8 %. Tablet height and hydrocortisone content were correlated (R2 = 0.996), providing a tool for easy dose personalization. Tablets maintained critical quality attributes, such as dissolution profile (f2 > 60) and content uniformity after process transfer from a single-screw extruder to a twin-screw extruder. Impurities were observed in the final product which should be mitigated before clinical assessment. To our knowledge, M3DICORT is the first 3DP hydrocortisone formulation specifically developed for AI.

Olfactory dysfunction: prevalence, diagnosis and treatment

Topicality: Odors affect the vital body functions, the general psychophysiological state. The sense of smell plays an important role in regulating human behavior. For example, the olfactory impulse reaches the brain faster than the impulse of pain, and therefore very effectively regulates us, regardless of awareness. Odors can change the work of various body systems: they affect the rhythm of our breathing, the excitability of muscles, the work of the brain and the entire nervous system. The sense of smell affects the limbic system, the part of the brain that controls hunger, feelings of aggression, sexual emotions and other "animal" feelings of a person. OD violates the informational and controlling roles of odorous substances (attractants, repellents, pheromones), therefore disorganizing the performance of nutritional, sexual, protective, orientation functions. For people of some specialties (taster, perfumer, cook), the decrease in the sense of smell can mean job loss and social maladaptation. Complaints about problems with taste perception more often reflect violations of the sense of smell, since the taste of a number of foods (chocolate, coffee, vanilla, strawberries, etc.) in reality depends on the stimulation of the sense of smell by volatile components that enter the nasopharynx when swallowing. Aim: To offer an otorhinolaryngologist guidance in the information stream regarding the problem of olfactory dysfunction, its prevalence, diagnosis and treatment. Materials and methods: The Sniffin’ Sticks test (CST) has been certified in the EU since 1995, and is used in Germany, Austria, Switzerland, and Italy. Odors are offered in the form of markers, in which the odorous substance takes the place of the coloring one. There is a version with 16 odors from the extended test [extendid] or with 12 odors [screening]. The test is well validated, can, unlike CCSIT, be used repeatedly, the shelf life is 0.5-1 year, depending on the intensity of application. The test can be used by patients independently. The study consists of 3 stages: threshold test (THR) – determination of the minimum odor concentration that the patient feels; discrimination test (D) is aimed at identifying the patient's ability to distinguish odors; identification test (I) for odor recognition. "University of Pennsylvania Smell Identification Test" (UPSIT) – the test is used exclusively to identify 40 odors (some of them are little known outside of North America). Odorous substances are microcapsulated on paper in the form of a rectangle and are released as a result of friction when held over it with a simple pencil. The patient is given 4 possible answers. Normally, a healthy man correctly identifies 34-40 odors, a healthy woman – 35-40. If the subject scores 18 or less, it is considered that he has a complete loss of sense of smell. The test system is a single-use and expensive. Digital volumetric tomography (CT). The visualization of the olfactory cleft, the upper nasal concha, and the configuration of the olfactory groove (OJ) turned out to be informative. Results and discussions: The review discusses the unique properties of OE and the potential use of olfactory epithelial grafts to restore olfactory function. Permanent population of multipotent stem cells proliferating throughout the life of the organism is located in the mammal OE. The cells formed during division go through several stages of differentiation and replace the dying olfactory receptor neurons. Neural stem cells were isolated from the olfactory region of the mucous membrane of humans, rats and mice. It is this population of neural stem cells that is being investigated as an autologous material for transplantation and the future use of olfactory transplants as a potential method of restoring the sense of smell. OE survives when transplanted to various areas of the brain, can be grafted directly to OL. Conclusions: A new direction in the treatment of OD is associated with cell therapy technologies, for example, using platelet-rich plasma (PRP), plasma in which the concentration of platelets is several times higher than normal. The term PRP is legitimate at a concentration of 700 thousand to 1 million platelets in 1 ml of plasma (the physiological plasma concentration of platelets is (180-360) x109 per liter). PRP is used to accelerate tissue regeneration, reduce scarring, stimulate angiogenesis, as well as a local antiseptic. The spectrum of pharmacological action of PRP is determined by growth factors: platelet growth factor (PDGF), transforming growth factor (TGF-β), vascular endothelial growth factor (VEGF), epithelial growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF). The active secretion of these growth factors by platelets begins within 10 minutes after activation (activation can be stimulated by almost any environmental disturbance, up to a simple mechanical stress), while more than 95% of the previously synthesized growth factors are secreted within 1 hour.

Influence of Bisphosphonates or Recombinant Human Parathyroid Hormone on in Vitro Chemotherapy Sensitivity in Acute Lymphoblastic Leukemia Cells

BACKGROUNDOsteonecrosis and low bone mineral density (BMD) are serious osteogenic side effects of acute lymphoblastic leukemia (ALL) treatment. Bisphosphonates and recombinant human parathyroid hormone (rPTH) tend to be used to ameliorate osteonecrosis-related symptoms as well as to enhance bone mineral density in children with ALL and severe bone fragility. Only one preclinical study on the safety of bisphosphonates during ALL treatment is available, which raises concerns about their impact on leukemic drug sensitivity. Here, we assessed the influence of various bone-modifying agents (zoledronate, pamidronate and rPTH) on in vitro cytotoxicity of chemotherapeutic agents (vincristine (VCR), daunorubicin (DNR), dexamethasone (DEXA), 6-mercaptopurine (6-MP), PEG-asparaginase (PEG-ASP)) and prednisone (PRED) that are commonly used in ALL treatment.METHODSPotential cytotoxic effects of the bone-modifying agents on leukemia cell viability and on in vitro cytotoxic responses of chemotherapeutic agents were tested in various T-cell and B-cell leukemia cell lines using methyl-thiazol-tetrazolium (MTT) assays. Bone-modifying agents were added at concentrations up to a five-fold of their physiological peak plasma concentration. For each assay, 50th percentile of maximal inhibitory concentration was determined. To quantify the combined effects of the bone-modifying agents on chemotherapeutic agent-induced cytotoxicity, median (interquartile range) combination indexes (CI) were calculated. We considered a median CI of < 0.8 as synergism and > 1.2 as antagonism (based on the method of Chou).RESULTSZoledronate, pamidronate or rPTH in combination with DNR, 6-MP and PEG-ASP showed median CI values between 0.8 and 1.2. Variable inconclusive results were obtained in combination with VCR. Only the combination of a five-fold peak plasma concentration of zoledronate or pamidronate with DEXA resulted in median CI values of 1.15 (range, 1.08-1.48), and 1.34 (range, 1.07-1.62), respectively. Additional experiments using DEXA as well as PRED in combination with one-, three- or five-fold physiological peak plasma concentrations of zoledronate or pamidronate revealed that median CI values stay within 0.8 and 1.2, except for DEXA exposed leukemia cells in combination with a five-fold physiological peak plasma concentration of pamidronate which repeatedly showed a median CI value above 1.2 (1.34, range 1.04-1.86).CONCLUSIONSZoledronate, pamidronate or rPTH do not seem to influence drug sensitivity of DNR, 6-MP or PEG-ASP, even at a five-fold physiological peak plasma concentration. Nevertheless, our findings suggest a minimal effect of pamidronate on DEXA-induced leukemia cell death. This suggests that even though zoledronate or pamidronate do not seem to negatively influence DEXA- or PRED- induced toxicity in expected physiological concentrations (one- to three-fold physiological peak plasma concentrations), these bone-modifying agents may only be considered with caution in individual cases, and preferably in clinical trial settings before being applied on a large scale in children with ALL. DisclosuresNo relevant conflicts of interest to declare.

Human Prothrombin Variants with Modifications in the Active Site S4 Subpocket Demonstrate Resistance to Dabigatran

Chymotrypsin-like serine proteases are hallmarked by a protease domain comprising the catalytic triad residues His57, Asp102, and Ser195 (chymotrypsinogen numbering) situated in the active site cleft. While the catalytic triad in conjunction with the oxyanion hole residues regulate substrate cleavage, the active site subpockets (S1-4) control substrate recognition and binding. The high structural homology of the serine protease domains allows for analogous strategies in drug design, which is underscored by the direct oral anticoagulants (DOACs) for the prophylactic management of stroke in atrial fibrillation and prevention and treatment of venous thrombosis. DOACs inhibit coagulation serine proteases by reversibly engaging the active site with high affinity. To expand the repertoire of DOAC-specific reversal agents we have previously successfully modified the S4 active site subpocket of human factor Xa to prevent DOAC binding while preserving catalytic activity [Verhoef 2017 Nature Commun.]. To explore whether an analogous strategy can be applied to create DOAC resistance in the serine protease thrombin, specific substitutions or sequences in or around the dabigatran-binding S4 subsite derived from naturally occurring serine proteases or plasma proteins were introduced in prothrombin.A panel of prothrombin variants was generated and transfected into HEK293 cells to allow for stable protein expression. In some of the generated prothrombin variants comprising insertions in amino acid sequence 91-99 that is directly adjacent to the S4 subsite protein expression was severely impaired. This indicates that exchange of any surface-exposed serine protease or plasma protein region into the prothrombin protease domain is not necessarily compatible with protein expression. In contrast, exchange of the human prothrombin 91-99 sequence for that of human kallikrein 3 or targeted amino acid replacement of S4 subsite residue Ile174 resulted in prothrombin protein expression levels similar to wild-type prothrombin.Following expression, prothrombin variants were purified to homogeneity using the CaptureSelect tm affinity matrix that selects for fully gamma-carboxylated prothrombin. The specific prothrombin clotting activity analyses of the purified prothrombin variants KL3 (0.7 ± 0.2 U/mg), I174A (0.8 ± 0.2 U/mg), and I174F (0.8 ± 0.3 U/mg) demonstrated an overall ~10-fold reduced specific activity relative to wild-type prothrombin (7.5 ± 0.1 U/mg). As such, modification of the S4 subsite likely interferes with the binding and subsequent conversion of fibrinogen by thrombin. To determine whether the prothrombin variants supported tissue factor-initiated thrombin formation in human plasma, prothrombin-deficient plasma was supplemented with increasing plasma concentrations of prothrombin variant (90-180 ug/mL). Consistent with their reduced specific clotting activity, 180 ug/mL prothrombin variant was required to obtain substantial thrombin generation but with reduced thrombin generation parameters (peak thrombin, ETP) relative to supplementation with plasma concentrations of wild-type prothrombin (90 ug/mL). This calibrated automated thrombin generation assay set-up was used to assess the DOAC-resistance of the prothrombin variants. While thrombin formation reached half-maximum inhibition at 532 ± 58 nM dabigatran in wild-type prothrombin-supplemented plasma, addition of the prothrombin variants displayed a ~2-fold reduced sensitivity to dabigatran inhibition (IC50: 1186 ± 136 nM prothrombin-KL3; 851 ± 97 nM prothrombin-I174F; 772 ± 80 nM prothrombin-I174A). This demonstrates that the S4 subsite-modified prothrombin variants are able to support thrombin generation in the presence of physiological plasma concentrations of inhibitor.Collectively, our findings indicate that human prothrombin variants comprising a single point mutation at position Ile174 in the S4 subsite or at a region directly adjacent to the S4 subsite are able to generate thrombin in plasma inhibited by dabigatran. Hence, serine proteases with S4 subpocket modifications have the potential to bypass DOAC therapy and could provide a generic strategy in the development of novel DOAC-bypassing agents. [Display omitted] DisclosuresReitsma: VarmX. B.V.: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Patents & Royalties. Verhoef: VarmX. B.V.: Current Employment, Current holder of individual stocks in a privately-held company. Bos: VarmX B.V.: Research Funding; uniQure Biopharma B.V.: Research Funding.

Positive GABAA receptor modulating steroids and their antagonists: Implications for clinical treatments.

GABA is the main inhibitory neurotransmitter in the brain and GABAergic transmission has been shown to be of importance for regulation of mood, memory and food intake. The progesterone metabolite allopregnanolone (Allo) is a positive GABAA receptor modulating steroid with potent effects. In humans, disorders such as premenstrual dysphoric disorder (PMDD), hepatic encephalopathy and polycystic ovarian syndrome are associated with elevated Allo levels and increased negative mood, disturbed memory and increased food intake in some individuals. This is surprising because Allo shares many properties with benzodiazepines and is mainly considered to be anxiolytic and anti-depressant. However, it is well established that, in certain individuals, GABAA receptor activating compounds could have paradoxical effects and thus be anxiogenic in low physiological plasma concentrations but anxiolytic at high levels. We have demonstrated that isoallopregnanolone (Isoallo), the 3β-OH sibling of Allo, functions as a GABAA receptor modulating steroid antagonist (GAMSA) but without any effects of its own on GABAA receptors. The antagonistic effect is noted in most GABAA subtypes investigated in vitro to date. In vivo, Isoallo can inhibit Allo-induced anaesthesia in rats, as well as sedation or saccadic eye velocity in humans. Isoallo treatment has been studied in women with PMDD. In a first phase II study, Isoallo (Sepranolone; Asarina Pharma) injections significantly ameliorated negative mood in women with PMDD compared with placebo. Several GAMSAs for oral administration have also been developed. The GAMSA, UC1011, can inhibit Allo induced memory disturbances in rats and an oral GAMSA, GR3027, has been shown to restore learning and motor coordination in rats with hepatic encephalopathy. In humans, vigilance, cognition and pathological electroencephalogram were improved in patients with hepatic encephalopathy on treatment with GR3027. In conclusion GAMSAs are a new possible treatment for disorders and symptoms caused by hyperactivity in the GABAA system.

Phe174-Mutated Human Factor X As Bypassing Agent to the Direct FXa Inhibitors

The direct thrombin and factor Xa (FXa) inhibiting oral anticoagulants have emerged as the preferred anticoagulant therapy, having major benefits for the patient's quality of life due to similar or lower risks of bleeding compared to the vitamin K antagonists. Unfortunately, in the case of bleeding-related adverse effects, no reversal agent is available to counteract the FXa-targeted anticoagulant therapy and restore hemostasis.The direct FXa inhibitors effectively block its catalytic activity by reversible high affinity occupation of the FXa active site. X-ray crystal structures of human FXa in complex with the FXa inhibitors apixaban or rivaroxaban revealed their specific binding mode and demonstrated occupation of the FXa substrate binding S1 and S4 subsites. To better understand the molecular requirements for direct inhibitor binding in FXa, we performed 750 ns Molecular Dynamics (MD) simulations of the FXa-apixaban complex. During MD, the narrow distribution of close range contacts (≤ 0.5 nm) between the side chain atoms of the S4 pocket residues Tyr99 and Phe174 (chymotrypsinogen numbering) and apixaban confirmed stabilization of apixaban in the S4 subsite. To directly assess the contribution of S4 pocket residues Tyr99 and Phe174 to apixaban binding, we generated human FX(a) variants comprising either Tyr99Ala, Phe174Ala, or both. While substitution of either one of these residues reduced the sensitivity for apixaban by ~8-fold, replacement of both increased the IC50 >100-fold (5 nM wt-FXa; 34 nM Y99A-FXa; 45 nM F174A-FXa; 616 nM Y99A/F174A-FXa). The FX clotting activity, on the other hand, was drastically reduced by substituting Tyr99 (6% residual activity) and essentially lost with both replacements. In contrast, following Phe174 replacement full FX clotting activity was maintained. Since this points to Phe174 substitution as a promising target for abrogated inhibitor binding while maintaining FX activity, we continued with assessing the efficacy of Phe174-mutated FX variants as bypassing agent for the direct FXa inhibitors.To do so, the human variants FX-F174A and FX-F174S were stably expressed in HEK293 cells and purified to homogeneity using ion-exchange chromatography. Evaluation of the specific FX clotting activity revealed that the Phe174-substituted FX variants exhibited a similar clotting activity (84 mU/µg FX-F174A; 122 mU/µg FX-F174S) relative to human wild-type (wt-)FX (105 mU/µg). This indicates that substitution of Phe174 does not interfere with the binding and subsequent conversion of the FXa substrate. Inhibition of the RVV-X-activated FX variants employing peptidyl substrate conversion revealed a reduced sensitivity to apixaban, demonstrated by a 10-fold increase in IC50 (2 nM wt-FX; 19 nM FX-F174A; 20 nM FX-F174S). Consequently, a decrease in sensitivity was also observed for inhibition by rivaroxaban (3-8-fold) and edoxaban (4-7-fold). To determine whether the FX variants supported tissue factor-initiated (6 pM) thrombin formation in normal human plasma, we performed FXa inhibitor titrations using the calibrated automated thrombin generation assay. While thrombin formation reached half-maximum inhibition at 55 nM apixaban in wt-FX-spiked plasma, plasma spiked with the FX variants displayed a ~10-fold reduced sensitivity to apixaban inhibition (IC50: 640 nM FX-F174S; 405 nM FX-F174A). A similar loss of inhibitor-sensitivity was observed for inhibition by rivaroxaban (6-10-fold) and edoxaban (17-fold), demonstrating that the Phe174-mutated FX variants are able to support thrombin generation in the presence of physiological plasma concentrations of inhibitor. Furthermore, at supra-physiological inhibitor concentrations (1 µM), little if any thrombin was formed upon adding up to 60 µg/ml wt-FX. In contrast, supplementation with 30-45 µg/ml Phe174-mutated FX restored the thrombin peak up to 80% in edoxaban-treated plasma, while the thrombin peak was fully restored in the presence of apixaban or rivaroxaban.Collectively, our findings indicate that human FX variants comprising a single point mutation at position Phe174 in the S4 subsite are able to restore hemostasis in plasma inhibited by the direct FXa inhibitors. As such, these variants have the potential to serve as rescue therapeutic agents to overcome the effect of the direct FXa inhibitors in case of potential life-threatening bleeding events or emergency surgical interventions. DisclosuresVerhoef:Leiden University Medical Center: Patents & Royalties: co-inventor on a patent application related to this abstract. Reitsma:Leiden University Medical Center; VarmX B.V.: Equity Ownership, Patents & Royalties: co-inventor on a patent application related to this abstract. Bos:Leiden University Medical Center: Patents & Royalties: co-inventor on a patent application related to a submitted abstract; Bayer: Other: Fee for Presenting at a Bayer Conference; Bayer Hemophilia Awards: Research Funding.

Allysine and α-Aminoadipic Acid as Markers of the Glyco-Oxidative Damage to Human Serum Albumin under Pathological Glucose Concentrations.

Understanding the molecular basis of the disease is of the utmost scientific interest as it contributes to the development of targeted strategies of prevention, diagnosis, and therapy. Protein carbonylation is a typical feature of glyco-oxidative stress and takes place in health disorders such as diabetes. Allysine as well as its oxidation product, the α-amino adipic acid (α-AA) have been found to be markers of diabetes risk whereas little is known about the chemistry involved in its formation under hyperglycemic conditions. To provide insight into this issue, human serum albumin was incubated in the presence of FeCl3 (25 μM) and increasing glucose concentrations for 32 h at 37 °C. These concentrations were selected to simulate (i) physiological fasting plasma concentration (4 mM), (ii) pathological pre-diabetes fasting plasma concentration (8 mM), and pathological diabetes fasting plasma concentration (12 mM) of glucose. While both allysine and α-AA were found to increase with increasing glucose concentrations, the carboxylic acid was only detected at pathological glucose concentrations and appeared to be a more reliable indicator of glyco-oxidative stress. The underlying chemical mechanisms of lysine glycation as well as of the depletion of tryptophan and formation of fluorescent and colored advanced glycation products are discussed.

Leucine decreases intramyocellular lipid deposition in an mTORC1-independent manner in palmitate-treated C2C12 myotubes.

Higher intramyocellular lipid (IMCL) deposition in skeletal muscle is commonly observed in patients with obesity, resulting in mitochondrial damage. Palmitic acid, a saturated fatty acid, has been reported to induce obesogenic conditions in C2C12 myotubes. Leucine has been shown to improve obesity-related metabolic signatures; however, evidence for the effect of leucine on IMCL and the underlying mechanisms are still lacking. The objective of this study was to determine the effect of leucine on IMCL deposition and identify the potential mechanisms. Palmitate-treated C2C12 myotubes were used as an in vitro model of obesity. Two doses of leucine were used: 0.5 mM (postprandial physiological plasma concentration) and 1.5 mM (supraphysiological plasma concentration). Rapamycin was used to determine the role of mammalian target of rapamycin complex 1 (mTORC1) in leucine's regulation of lipid deposition in C2C12 myotubes. One-way ANOVA followed by Tukey's post hoc test was used to calculate differences between treatment groups. Our results demonstrate that leucine reduces IMCL deposition in an mTORC1-independent fashion. Furthermore, leucine acts independently of mTORC1 to upregulate gene expression related to fatty acid metabolism and works through both mTORC1-dependent and mTORC1-independent pathways to regulate mitochondrial biogenesis in palmitate-treated C2C12 myotubes. In agreement with increased mitochondrial biogenesis, increased mitochondrial content, circularity, and decreased autophagy are observed in the presence of 1.5 mM leucine. Taken together, the results indicate leucine reduces IMCL potentially through an mTORC1-independent pathway in palmitate-treated C2C12 myotubes.

Short-Term Effects of Branched-Chain Amino Acids–Enriched Dialysis Fluid on Branched-Chain Amino Acids Plasma Level and Mass Balance: A Randomized Cross-Over Study

The hemodialysis (HD) session per se is a catabolic event contributing to protein-energy wasting via several mechanisms including nutrient losses. Amino acids (AAs) losses in the dialysate are estimated from 6 to 10g per treatment. The HD patient plasma AA concentration is usually lower than in normal subjects. This is even more marked in patients with long dialysis vintage or malnutrition. The aim of the study was to evaluate the effect on mass balance of a branched-chain AA (BCAA)-enriched (valine, isoleucine, leucine) dialysis fluid in a group of 6 stable HD anuric patients, fasting since 12hours. The specific choice of BCAA relied on their key role on protein and muscle anabolism and their usual decreased plasma concentration in HD patients. Each patient was prescribed in a cross-over design and random order, either receiving a standard high-flux HD or an HD treatment using a BCAA-enriched acid concentrate designed to achieve a physiological plasma concentration of BCAAs. HD prescription remained unchanged during the 2 phases of study. Dialysate electrolytes prescription was kept constant for each individual patient, as well as dialysate glucose concentration (5.5 mmol/L). Pre- and post-dialysis BCAAs concentrations were measured by Ion-Exchange Liquid Chromatography. Postdialysis concentrations were corrected for hemoconcentration, and net mass transfer was calculated. Six stable prevalent end-stage kidney disease patients were studied. They consisted of 5 men and 1 woman, aged 69.9years, with body mass index of 25.2kg/m2. Treatment schedule consisted of treatment time 4hours, high-flux polysulfone membrane (1.8m2), blood flow 350mL/minute, and dialysate/blood flow ratio at 1.5. The average BCAAs concentration in dialysate was targeted to physiological levels and assessed in 6 different samples, respectively for plasma valine, isoleucine, and leucine at 271, 78, and 145 μmol/L. With standard dialysate, plasma valine decreased from 204.5 to 130.8 (P=.0014). Plasma isoleucine and leucine changes were not significant, respectively from 65.7 to 59.3 μmol/L and 110.3 to 113.4 μmol/L. When using the BCAA-enriched dialysis fluid, plasma valine increased from 197.2 to 269.2 μmol/L (P=.0001), plasma isoleucine and leucine respectively from 63.2 to 84.7 (P=.0022) and from 107.2 to 161.6 μmoles/L (P=.0002). Dialysis dose estimated from KT/V did not differ between the sessions. The mass transfer with BCAA-enriched dialysate was +115, +16, and+83 μmol per session for leucine, isoleucine, and valine, respectively. In conclusion, the addition of BCAAs at physiological concentration in the dialysis fluid contributes to restore physiological plasma concentrations for valine, isoleucine, and leucine at the end of a dialysis session. As BCAAs are essential to muscle balance, this could help to limit losses of BCAAs, restore physiological BCAAs concentrations, and decrease muscle catabolism observed during the HD treatment. Further outcome-based studies are needed to confirm this hypothesis on a larger scale and longer treatment time.

P-208 - Inhibition of secretory sphingomyelinase by epicatechin-3-O-gallate and other antioxidants

Objective Secretory sphingomyelinase (sSMase) is one of the key enzymes involved in oxidative stress-related diseases [Biol. Pharm. Bull, 2013]. The elevation of its activity resulted in an increase of ceramide that is the bioactive lipid inducing cellular apoptosis, proliferation, and differentiation. In this study, we searched for novel sSMase inhibitors during some antioxidants which are expected to ameliorate oxidative stress-related diseases. Methods Each of several antioxidants was incubated with and sSMase derived from rats in the assay mixture (pH 5.0) and analyzed their inhibitory activity by using HPLC. Results Epicatechin 3-O-gallate (ECg) significantly inhibited sSMase activity as compared with non-gallate type catechins. In addition, Anti-Oxidant Biofactor (AOB) extract that is the mixture of fermented grain foods and catechins, markedly inhibited sSMase activity. Conclusion ECg and some gallate type catechins had a strong inhibitory effect on sSMase in a physiological plasma concentration (IC50=1.7–25.7 µM). This inhibition may be due to their specific structures such as galloyl residue, and it depends on the reducing activity. Moreover, antioxidant mixture such as AOB might inhibit synergistically sSMase. It will be further discussed.

Bortezomib, carfilzomib and ixazomib do not mediate relevant transporter-based drug-drug interactions.

In order to optimize the clinical application of an increasing number of proteasome inhibitors, investigations into the differences between their respective pharmacodynamic and pharmacokinetic profiles, including their ability to act as a perpetrator in drug-drug interactions, are warranted. Therefore, in the present in vitro study, it was investigated whether bortezomib, carfilzomib and ixazomib are able to alter the expression, and/or the activity, of specific drug transporters generally relevant for pharmacokinetic drug-drug interactions. Through induction experiments, the current study demonstrated that the aforementioned three proteasome inhibitors do not induce mRNA expression of the transporter genes ATP binding cassette (ABC)B1, C1, C2 and G2 in the LS180 cell line, which was used as a model for systemic induction. By contrast, in certain myeloma cell lines, ixazomib provoked minor alterations in individual transporter gene expression. None of the proteasome inhibitors tested relevantly inhibited drug transporters within the range of physiological plasma concentrations. Taken together, transporter-based drug-drug interactions are unlikely to be a primary concern in the clinical application of the tested compounds.

Tin Selenide Quantum Dots Electrochemical Biotransducer for the Determination of Indinavir - A Protease Inhibitor Anti-Retroviral Drug

Biocompatibility of tin selenide quantum dots was achieved by the incorporation of 3-mercaptopropionic acid (3-MPA) as a capping agent, which also improved the stability and the solubility of the material. The UV-Vis spectrophotometric analysis of the quantum dots revealed a broad absorption band at ~ 330 nm (with a corresponding band gap, Eg, value of 3.75 eV), which is within the range of values expected for quantum dots materials. The 3-mercaptopropionic acid-capped tin selenide (3-MPA-SnSe) quantum dots were used to develop an electrochemical biosensor for indinavir, which is a protease inhibitor antiretroviral (ARV) drug. The biosensor was prepared by the self-assembly of L-cysteine on a gold electrode that was functionalised with 3-MPA-SnSe quantum dots, followed by cross-linking with cytochrome P450-3A4 (CYP3A4) using 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The electrocatalytic properties of the biosensor included a characteristic cyclic voltammetric reduction peak at-380 mV, which was used to detect the response of the biosensor to indinavir. The sensor performance parameters included response time and limit of detection (LOD) values of 11 s and 3.22 pg/mL, respectively. The test concentration range studied (0.014 – 0.066 ng/mL) gave a linear calibration plot for indinavir, and it was lower than the physiological plasma concentration index (i.e. maximum plasma concentrations, Cmax,) of indinavir (5 - 15 ng/mL) normally observed 8 h after intake. This indicates that the biosensor can be very useful in the case of ultra-rapid metabolisers where very low Cmax values are expected

Tin Selenide Quantum Dots Electrochemical Biotransducer for the Determination of Indinavir - A Protease Inhibitor Anti-Retroviral Drug

Biocompatibility of tin selenide quantum dots was achieved by the incorporation of 3-mercaptopropionic acid (3-MPA) as a capping agent, which also improved the stability and the solubility of the material. The UV-Vis spectrophotometric analysis of the quantum dots revealed a broad absorption band at ~ 330 nm (with a corresponding band gap, Eg, value of 3.75 eV), which is within the range of values expected for quantum dots materials. The 3-mercaptopropionic acid-capped tin selenide (3-MPA-SnSe) quantum dots were used to develop an electrochemical biosensor for indinavir, which is a protease inhibitor antiretroviral (ARV) drug. The biosensor was prepared by the self-assembly of L-cysteine on a gold electrode that was functionalised with 3-MPA-SnSe quantum dots, followed by cross-linking with cytochrome P450-3A4 (CYP3A4) using 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The electrocatalytic properties of the biosensor included a characteristic cyclic voltammetric reduction peak at-380 mV, which was used to detect the response of the biosensor to indinavir. The sensor performance parameters included a response time and sensitivity values of 11 s and 0.221 μA/nM, respectively. The test concentration range studied (0.014 – 0.066 ng/mL) gave a linear calibration plot for indinavir, and it was lower than the physiological plasma concentration index (i.e. maximum plasma concentrations, Cmax,) of indinavir (5 - 15 ng/mL) normally observed 8 h after intake. This indicates that the biosensor can be very useful in the case of ultra-rapid metabolisers where very low Cmax values are expected

Cardiac N-methyl D-aspartate Receptors as a Pharmacological Target.

This study focuses on characterization of the cardiac N-methyl D-aspartate receptors (NMDARs) as a target for endogenous and synthetic agonists and antagonists. Using isolated perfused rat hearts, we have shown that intracoronary administration of the NMDAR agonists and antagonists has a pronounced effect on autonomous heart function. Perfusion of rat hearts with autologous blood supplemented with NMDAR agonists was associated with induction of tachycardia, sinus arrhythmia, and ischemia occurring within physiological plasma concentration range for glutamate and glycine. Intracoronary administration of the NMDAR antagonists exerted an antiarrhythmic effect and resulted in bradycardia and improvement of capillary perfusion. Action of antagonists eliprodil, Ro25-6981, memantine, ketamine, and MK-801 on autonomous heart function diverged strikingly from that of L-type Ca channel blockers. Cardiac NMDAR subunit composition differed from that of neuronal receptors and was age specific and chamber specific. Transcripts of the GluN3A and GluN2D were found in all heart chambers, whereas expression of GluN1 and GluN2A and 2C was restricted to the atria. Expression of the GluN2B protein in ventricles increased markedly with age of the animals. The obtained data reveal that NMDARs are expressed in rat heart contributing to the autonomic heart rate regulation and the function of the cardiac conduction system.

Differentiation with elaidate tends to impair insulin-dependent glucose uptake and GLUT4 translocation in 3T3-L1 adipocytes

Development of type 2 diabetes mellitus and insulin resistance is associated with a quality of dietary fatty acids such as saturated and unsaturated fatty acids. Dietary fatty acids also include transform of unsaturated fatty acids and intake of transform of oleate (elaidate) is associated with cardiovascular disease. However, little is known about the roles of elaidate in insulin responsiveness. We show here that elaidate impairs insulin-dependent glucose uptake in adipocytes. Differentiation with 10 μM elaidate, which is close to physiological plasma concentration, reduces insulin-dependent glucose uptake. Furthermore, insulin-dependent GLUT4 translocation is disturbed in adipocytes differentiated with elaidate. In addition, analysis of lipolysis and gene expression shows that deteriorative effects of elaidate on insulin responsiveness are limited but not general. Thus, our findings reveal that differentiation with elaidate tends to affect insulin-dependent glucose uptake through alternation of GLUT4 translocation from cytosol to the plasma membrane.

Strong Inhibition of Secretory Sphingomyelinase by Catechins, Particularly by (-)-Epicatechin 3-O-Gallate and (-)-3'-O-Methylepigallocatechin 3-O-Gallate.

Sphingomyelinases (SMases) are key enzymes involved in many diseases which are caused by oxidative stress, such as atherosclerosis, diabetes mellitus, nonalcoholic fatty liver disease, and Alzheimer's disease. SMases hydrolyze sphingomyelin to generate ceramide, a well-known pro-apoptotic lipid. SMases are classified into five types based on pH optimum, subcellular localization, and cation dependence. Previously, we demonstrated that elevation of secretory sphingomyelinase (sSMase) activity increased the plasma ceramide concentration under oxidative stress induced by diabetes and atherosclerosis in murine models. These results suggest that sSMase inhibitors can prevent the progress of these diseases. The present study demonstrated that sSMase activity was activated by oxidation and inhibited by reduction. Furthermore, we examined whether catechins inhibited the sSMase activity in a physiological plasma concentration. Among catechins, (-)-epicatechin 3-O-gallate (ECg) exhibited strong inhibitory effect on sSMase (IC50=25.7 μM). This effect was attenuated by methylation at the 3″- or 4″-position. On the other hand, (-)-epigallocatechin 3-O-gallate (EGCg) and (-)-catechin 3-O-gallate (Cg) exhibited weaker inhibitory activity than ECg, and (-)-epicatechin and (-)-epigallocatechin did not affect sSMase activity. Additionally, one synthetic catechin, (-)-3'-O-methylepigallocatechin 3-O-gallate (EGCg-3'-O-Me), showed the strongest inhibitory effect (IC50=1.7 μM) on sSMase. This phenomenon was not observed for (-)-4'-O-methylepigallocatechin 3-O-gallate. These results suggest that the reduction potential, the presence of the galloyl residue at the C-3 position, and the steric requirement to interact with sSMase protein are important for effective inhibition of sSMase.

The Marine-Derived Fungus Clonostachys rosea, Source of a Rare Conjugated 4-Me-6E,8E-hexadecadienoic Acid Reducing Viability of MCF-7 Breast Cancer Cells and Gene Expression of Lipogenic Enzymes.

A marine-derived strain of Clonostachys rosea isolated from sediments of the river Loire estuary (France) was investigated for its high lipid production. The fungal strain was grown on six different culture media to explore lipid production changes. An original branched conjugated fatty acid, mainly present in triglycerides and mostly produced when grown on DCA (23% of total fatty acid composition). It was identified as 4-Me-6E,8E-hexadecadienoic on the basis of spectroscopic analyses. This fatty acid reduced viability of MCF-7 breast cancer cells in a dose dependent manner (up to 63%) at physiological free fatty acid human plasma concentration (100 μM). Reduction of gene expression of two lipogenic enzymes, the acetyl CoA carboxylase (ACC) and the fatty acid synthase (FAS) was evaluated to explore the mechanisms of action of 4-Me-6E,8E-16:2 acid. At 50 μM, 50% and 35% of mRNA gene expression inhibition were observed for ACC and FAS, respectively.

Design Of a Most Efficient Inhibitor Of TFPI By Molecular Fusion Of Two Inhibitory Peptides

TFPI is an important inhibitor of the extrinsic coagulation pathway. It efficiently inhibits TF-FVIIa and FXa by quaternary complex formation. Plasma contains various truncated forms of TFPI which are poor inhibitors, and full length (fl)TFPI (0.3 – 0.5 nM) which is the most active TFPI in plasma. flTFPI is released from platelets upon activation, and increases flTFPI concentrations locally up to 30-fold. Most intravascular TFPI (∼80%) is associated with endothelial cells. Both endothelial forms, TFPIa and TFPIb, are similarily effective inhibitors of FX activation on the endothelial cell surface. Inhibition of TFPI in hemophilia models with blocking antibodies, aptamers or peptide inhibitors improves hemostasis and may become an option to treat hemophilia. Recently, we presented peptide inhibitors of TFPI that enhance coagulation in hemophilia models. Two optimized peptides, JBT-A7 and JBT-B5, efficiently blocked inhibitory activity of TFPI and bound to distinct binding sites.We demonstrated the crystal structure of JBT-A7, a linear TFPI inhibitory peptide composed of 20 amino acids, bound to NtermK1 (TFPI 1-83). JBT-B5, a cyclic TFPI inhibitory peptide of 23 amino acids, co-crystallized with TFPI KD1-KD2 (TFPI 22-150). Overlaying the KD1 structure in the KD1-KD2/JBT-B5 and the NTermK1/JBT-A7 complex provided atomic details for linking the two peptide entities. Binding of peptides to TFPI and TFPI fragments was studied by BioCore. The TFPI inhibitory potential of the resulting fusion peptide was tested in model systems (FXa inhibition and TF-FVIIa catalyzed FX activation) and global hemostatic assays (TF-triggered thrombin generation) using hemophilia plasma. To model situations of increased TFPI concentration, both model and plasma assays were carried out at TFPI concentrations up to 10 nM, which is 40-50-fold higher than the physiological flTFPI plasma concentration. To characterize the inhibition of platelet TFPI, we used platelets isolated from blood samples and platelet rich plasma from different donors. Binding of a biotinylated fusion peptide on living HUVE cells was assessed by fluorescence activated cell sorting (FACS) and fluorescence microscopy. Inhibition of cell surface TFPI was analyzed on cultivated HUVECs stimulated with TNFa for TF expression. We monitored FXa generation by the TFPI-dependent cell surface FX activation complex by conversion of an FXa-specific fluorogenic substrate.The overlay of the crystal structures of KD1-KD2/JBT-B5 and the NTermKD1/JBT-A7 complexes revealed non-overlapping epitopes and close proximity of the termini of both peptides. The distance could be bridged by an approximately ten amino acid linker. A fusion peptide with a 10-serine-linker was synthesized and showed highly improved dissociation in Biacore experiments and most efficiently inhibited TFPI activity in the model assays. In contrast, single peptides only partially inhibit TFPI especially at high TFPI concentrations. In thrombin generation assays using hemophilia plasma, the fusion peptide showed a substantially higher ability than the single peptides to increase the thrombin peak even at elevated TFPI. The fusion peptide efficiently inhibited TFPI released from platelets and improved thrombin generation in TFPI deficient plasma reconstituted with platelets as the only source of TFPI released upon platelet activation. The fusion peptide was also shown to bind TFPI on the surface of living HUVECs. This is consistent with its binding epitopes on KD1 and KD2 which result in inhibition of cell surface TFPI in a cell based FX activation assay. Thus, we demonstrate that a molecular fusion peptide most efficiently inhibits all physiologic forms of TFPI.X-ray structures of binary and ternary peptide TFPI complexes provided atomic details for linking two single peptides to generate a fusion peptide that most efficiently blocks TFPI in plasma, released from platelets and associated with endothelial cells. It most efficiently neutralizes TFPI even at substantially elevated concentrations occurring at sites of platelet activation. Our observations support the notion that targeting TFPI with TFPI inhibitors is a promising novel strategy to mitigate the bleeding risk in hemophilia patients. Disclosures:Dockal:Baxter Innovations GmbH, Vienna, Austria: Employment. Hartmann:Baxter Innovations GmbH, Vienna, Austria: Employment. Polakowski:3B Pharmaceuticals, Berlin, Germany: Employment. Brandstetter:Baxter Innovations GmbH, Vienna, Austria: Research Funding. Kammlander:Baxter Innovations GmbH, Vienna, Austria: Employment. Panholzer:Baxter Innovations GmbH, Vienna, Austria: Employment. Redl:Baxter Innovations GmbH, Vienna, Austria: Employment. Osterkamp:3B Pharmaceuticals, Berlin, Germany: Employment. Rosing:Baxter Innovations GmbH, Vienna, Austria: Consultancy, Research Funding. Scheiflinger:Baxter Innovations GmbH, Vienna, Austria: Employment.