Lyfjahvörf ondansetrons í fóstrum

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Physiological and pharmacokinetic properties of Placental Protein 13 (PP13) In vivo and in vitro studies in animals

Inngangur: Þegar kona verður þunguð, eiga sér stað miklar breytingar á æðakerfi hennar, til að mæta aukinni blóðþörf til fylgju og fósturs og tryggja eðlilegan vöxt fóstursins. Til að þetta sé hægt, þarf stækkun á æðum sem flytja blóð til fóstursins að eiga sér stað. Ef þessar breytingar á æðakerfi ná ekki að myndast, getur það leitt til ýmissa meðgöngutengdra sjúkdóma, eins og meðgöngueitrun. Tíðni meðgöngueitrunar er um 2-5% þungana í Evrópu og lýsir sér sem nýtilkominn háþrýstingur og því fylgir mælanlegt prótein í þvagi. Fylgjuprótein 13 (PP13, placental protein 13) er prótein sem er eingöngu seytt af fylgju og mælist í blóði mæðra frá fimmtu viku meðgöngu. Konur sem eru í áhættuhópi reynast vera með mun lægra magn af PP13 í sermi, en konur með heilbrigða meðgöngu. Því hefur verið lagt til að próteinið megi nýtast til greiningar á konum sem eru líklegri að þróa með sér meðgöngueitrun. Markmið: 1) Að skoða skammtíma- og langtímaáhrif proteins á æðakerfið í kringum legið (Grein III). 2) Að skilgreina verkunarmáta próteinsins á einangraðar legæðar úr rottum (Grein I). 3) Að meta áhrif próteinsins á æðakerfið í kringum legið og fósturvöxt í rottum. 4) Að meta lyfjahvörf próteinsins í kanínum (Grein II). Aðferðir og niðurstöður: Osmótískar dælur voru ígræddar í óþungaðar rottur sem losuðu um 10 µl/klst og tæmdu sig á sjö dögum. Sum dýranna (n=11) voru aflifuð eftir sjö daga meðferð til að meta skammtímaáhrifin, en önnur (n=16) voru aflifuð eftir 13 daga til að meta langtímaáhrif próteinsins á æðakerfið. Þvermál slag- og bláæða úr legi voru borin saman á milli hópa. Marktækur munur sást á öllum æðum í bæði langtíma- og skammtímahópnum. Til að skilgreina verkunarhátt fylgjupróteins 13, voru legslagæðar einangraðar og settar í sérhæfðan æðamæli (e. arteriograph). Próteinið reyndist hafa æðavíkkandi áhrif (38-50%) sem hægt var að hemja með því að hinda aðgengi nituroxíðs og arakídónsýru. Áhrifin hurfu ef æðaþelið var fjarlægt. EC50 mældist undir 1pM. Áhrif próteinsins í þunguðum rottum með ígræddar osmótískar dælur sem fengu nituroxíðhindra í drykkjarvatni leiddi í ljós marktækt minni unga samanborið við viðmiðunarhópinn. Þessi áhrif snertu einnig meginlegslagæð (MUA) og legbláæðar (RUV). Lyfjahvörf fylgjupróteins 13, sem fóru fram í kanínum, voru skoðuð með því að gefa þrjá mismunandi styrki af próteininu (5 ng/mL, 10 ng/mL og 50 ng/mL) í æð (I.V.), og síðan einn styrkur (50 ng/mL) var gefinn undir húð (S.C.). Blóðþéttni var mæld með ELISA prófi og lyfjahvarfafræðileg heðgun próteinsins reiknuð út, sem fylgdi svokölluðu tveggja-hólfa kerfi. Helmingunartíminn var marktækt hærri hjá S.C. hópnum (p<0.01), en það kom í ljós að bæði dreifirúmmál og flatarmálið undir ferlinum (AUC) reyndust vera skammtaháð milli I.V. hópanna. Ályktanir: Niðurstöðurnar sýndu að fylgjuprótein 13 hefur veruleg æðavíkkandi áhrif, sem jafnframt hefur jákvæð áhrif á vöxt fylgju og fósturs. Þetta prótein gæti verið lykilþáttur í undirbúningi æðakerfisins fyrir aukið blóðflæði síðar á meðgöngunni. Lyfjahvarfafræðilegar athuganir sýna að magn próteinsins sem er losað úr fylgju gæti verið mun hærri en áður var talið. Frekari rannsókna er þörf til að fá dýpri innsýn í áhrif þessa próteins á æðakerfi manna. Niðurstöður okkar er mikil hvatning fyrir áframhaldandi rannsóknum til að meta fylgjuprótein 13 sem mögulegan lyfjasprota til að fyrirbyggja meðgöngueitrun og aðra meðgöngutenda sjúkdóma sem tengjast ófullnægjandi blóðflæði til legs og fylgju.Background: During pregnancy extensive hemodynamic changes are required for optimal fetal growth and utero-placental circulation adaptation to increased blood volume. If those changes are altered or fail to occur they may lead to pathological pregnancies and preeclampsia. Preeclampsia is an obstetrical syndrome associated with high blood pressure and altered organ function that affects 2-5% of pregnant women. Placental protein 13 (PP13) is secreted solely by placenta and has been proposed as a potential biomarker whilst its serum levels in early pregnancy in women at risk are much lower than those in healthy pregnancies. Aims: First, to study the long and short term effects (Paper III), and evaluate overall effect of PP13 exposure on uterine vasculature, and the fetal outcome in rats. Second aim is to define the mechanism of action in isolated rat arteries in vitro (Paper I). The final aim is to evaluate the pharmacokinetic profile of PP13 in rabbits (Paper II). Methods and Main Results: Slow-release osmotic pums loaded with protein or saline were implanted in periscapular region in non-pregnt rats, releasing its content for seven days. Some animals (n=11) were sacrified after seven days with the pump (short-term), and some animals (n=16) were sacrified after thirteen days (long-term). The diameter of arteries and veins were compared between groups, resulting in significant vascular expansion of all vessels in rPP13 group both in short and long-term. Histidine tagged PP13 variant (his-PP13), caused significant vessel expansion in radial arteries, and only in short-term. Study conducted in pregnant rats, by inserting the osmotic pump at the time of placentation, in which some rats were treated with L-NAME via drinking water in order to inhibit the main vasodilation pathway (eNOS). The results showed that rPP13 treated animals had heavier pups and bigger placentas, in comparison to the control. All L-NAME treated groups resulted in significantly smaller pups and placentas in comparison to the control. Main uterine artery (MUA) and radial uterine veins (RUV) expansion in L-NAME treated animals were significatntly lower compared to the control. To study the mechanism of action uterine vessels were isolated and cannulated in arteriograph maintained at constant pressure of 50 mmHg. Vessels were then preconstricted and exposed to increasing concentrations of rPP13 (10-13 to 10-8 M). rPP13 elicited 38-50% arterial vasodilation mediated via endothelial signaling pathways of nitric oxide and arachidonic acid, with half maximal response (EC50) of approx. 1pM. In order to establish pharmacokinetic profile of PP13, three different concentrations of the protein (5 ng/mL, 10 ng/mL and 50 ng/mL) were administrated intravenously (I.V.) and one concentration (50 ng/mL) was administrated subcutaneously (S.C.) in New Zealand White rabbits. The serum levels of the protein were determined by enzyme-linked immunosorbent assay (ELISA). The pharmacokinetic profile is described as two-compartment model. The elimination half-life was found to be different between the groups (p<0.01), and volume of distribution and area under the curve were found to be dose dependent. Conclusion: The results indicate that PP13 is a potent vasodilator, has a positive effect on fetal growth and may be a key factor in preconditioning the uterine vasculature in order to accommodate the blood flow increase during pregnancy. In addition pharmacokinetic studies indicate that the concentration of total PP13 released into maternal circulation may be much higher than previously estimated. Further studies are required in order to get an insight into the effect on human vasculature, however, these results encourage evaluation of PP13 as a potential therapeutic agent for obstetric syndromes characterized by insufficient uteroplacental blood flow.The project was funded by: Icelandic Centre for Research (RANNIS), European Union (FP7) through the ASPRE project (601852) and Hananja ehf

placental protein 13 pp13 induced vasodilation of resistance arteries from pregnant and nonpregnant rats occurs via endothelial signaling pathways

ABSTRACTPlacental protein 13 (PP13) induces hypotension in rats. This study aims to evaluate PP13 effects on isolated uterine arteries from nonpregnant and mid-pregnant rats. Vessels were isolated, cannulated, and pressurized to 50 mmHg within an arteriograph, preconstricted and exposed to increasing PP13 concentrations (10−13–10−8 M). PP13 elicited 38–50% arterial vasodilation with half-maximum response (EC50) = 1 pM. The relaxation was mediated by activating the endothelial-signaling pathways of prostaglandin and nitric oxide (NO). Accordingly, these results encourage evaluation of PP13 as a possible therapy for gestational diseases characterized by insufficient uteroplacental blood flow and/or maternal hypertension

Pharmacokinetics of placental protein 13 after intravenous and subcutaneous administration in rabbits

Introduction: Human placental protein 13 (PP13) is a galectin predominantly expressed by the placenta. Low serum concentrations of PP13 in early pregnancy indicate a higher risk of developing preeclampsia. Methods: The pharmacokinetic disposition and bioavailability of PP13 were determined by single intravenous and subcutaneous administration to 12 healthy New Zealand White rabbits. The serum pharmacokinetic values were determined by enzyme-linked immunosorbent assay, and are best described by a two-compartment model. Results: Both volume of distribution and the area under the curve were dose dependent for the intravenous group (p < 0.01). PP13 elimination half-life was also found to be different between the groups (p < 0.01). The bioavailability of PP13 following subcutaneous administration was found to be 57%. Conclusion: This study shows that the concentration of total PP13 released into the maternal circulation during pregnancy might be much higher than previously estimated.The authors thank Hy Laboratories for providing PP13 to this study through support provided by the European Union through the ASPRE project (# 601852). This study was mainly sponsored by Hananjaehf and the Icelandic Research Fund (Rannis; grant number 163403-052).Peer Reviewe

Galectin 13 (PP13) facilitates remodeling and structural stabilization of maternal vessels during pregnancy

Funding Funding: This research was funded by Daniel Turnberg Fellowship, UK Academy of Medical Sciences and the EU COST action CA16113 – CkiniMark to M.S. This study was also sponsored in part by the European Union (FP7) through the ASPRE project (601852) to H.M., S.G. and T.D. were sponsored by Hananja ehf, and Icelandic Research Fund (Rannís), grant no. 163403-052. Publisher Copyright: © 2019 by the authors. Licensee MDPI, Basel, Switzerland.Galectins regulate cell growth, proliferation, differentiation, apoptosis, signal transduction, mRNA splicing, and interactions with the extracellular matrix. Here we focus on the galectins in the reproductive system, particularly on a group of six galectins that first appears in anthropoid primates in conjunction with the evolution of highly invasive placentation and long gestation. Of these six, placental protein 13 (PP13, galectin 13) interacts with glycoproteins and glycolipids to enable successful pregnancy. PP13 is related to the development of a major obstetric syndrome, preeclampsia, a life-threatening complication of pregnancy which affects ten million pregnant women globally. Preeclampsia is characterized by hypertension, proteinuria, and organ failure, and is often accompanied by fetal loss and major newborn disabilities. PP13 facilitates the expansion of uterine arteries and veins during pregnancy in an endothelial cell-dependent manner, via the eNOS and prostaglandin signaling pathways. PP13 acts through its carbohydrate recognition domain that binds to sugar residues of extracellular and connective tissue molecules, thus inducing structural stabilization of vessel expansion. Further, decidual PP13 aggregates may serve as a decoy that induces white blood cell apoptosis, contributing to the mother’s immune tolerance to pregnancy. Lower first trimester PP13 level is one of the biomarkers to predict the subsequent risk to develop preeclampsia, while its molecular mutations/polymorphisms that are associated with reduced PP13 expression are accompanied by higher rates of preeclampsia We propose a targeted PP13 replenishing therapy to fight preeclampsia in carriers of these mutations.Peer reviewe

Placental protein 13 (PP13) stimulates rat uterine vessels after slow subcutaneous administration.

To access publisher's full text version of this article click on the hyperlink belowReduced concentrations of placental protein 13 (PP13) during the first trimester of human pregnancy are associated with elevated risk for the subsequent development of preeclampsia, which is one of the deadliest obstetrical complications of pregnancy. Previous studies by our group have shown that PP13 lowers blood pressure in pregnant rats, increases the size and weight of pups and placentas, and induces vasodilation of resistance arteries through endothelial signaling pathways involving endothelial nitric oxid synthase and prostaglandin. In the present study, the effect of PP13 was investigated in nonpregnant female Sprague Dawley rats (n=27). Osmotic pumps were surgically implanted subcutaneously that released a constant dose of PP13 or saline over 7 days. Most animals were sacrificed 6 days after the end of PP13 release (on day 13), while some were sacrificed immediately at the end of day 7 (the last PP13 release day), to compare the short- and long-term impact of PP13 on vessels' growth and size. The uterine vessels were significantly expanded in the group exposed to recombinant PP13 (rPP13) compared to the control (saline) group. Both veins and arteries were significantly expanded by rPP13 with a more pronounced effect after 13 days compared to the corresponding vessels after 7 days. Furthermore, the long-term effect of treatment by rPP13 was more pronounced in the veins compared to the corresponding arteries. The effect of a PP13 variant with a histidine-tag (His-PP13) remained the same between 7 and 13 days. In conclusion, PP13 might play a key role in the expansive remodeling of the uterine vessels, reflecting what would happen if the rat was pregnant, preparing the uterine vascu-lature for the increase in uteroplacental blood flow, which is necessary for normal pregnancy. We suggest that PP13 could act by NO signaling pathways, a hypothesis that requires future study.European Union through ASPRE project Icelandic Research Fund (Rannis) Hananja eh

Þróun katjóniskra sýklódextrín pólýrótaxan/hýalúronsýru samsettra nanóagna sem kjarnsýruferjur

Background: Nanotechnology is a fast growing area that raises new prospects in the improvement of diagnosis and treatment of multiple diseases. The goal of nanotechnology in drug delivery is to improve the biokinetics and bioavailability of the drug in particular at the target tissue or in the target cells, with a goal to reduce side effects and toxicity. Cationic polyrotaxanes (PRx) for gene delivery has been previously used for functional siRNA delivery. In this study hyaluronic acid (HA), was used for stabilization and for make the nanoplexes more tolerable. Objectives: 1) To produce and characterize cationic nanoplexes of polyrotaxane (PRx) in combination with HA and either pDNA or siRNA with different N/P ratios. 2) To test cytotoxicity of the nanoplexes and perform luciferase transfection studies. Methods: The nanometric polyplex formation with small interfering RNA (siRNA) or alternative plasmid DNA was examined. The nanoplexes were optimized for size and stability using dynamic light scattering and NTA. As a control sample the cationic PEI was used, due to its known transfection efficacy. Drug loading was analyzed with agarose gel electrophoresis. Then the best nanoplexes of each polymer and nucleotide type were used in cytotoxicity tests on two different cell lines A549 and RAW264.7. Transfection assays using luciferase reporter gene and model siRNA were also performed on both cell lines. Results: Optimized PEI-HA control nanoplexes were obtained at N/P ratio 7. The two PRx-HA nanoplexes were successful in formation at the N/P ratio 2. For the achievement of stable complexes, however, these polyplexes needed pDNA, and part of it was replaced with HA. It was observed that polyrotaxane nanoplexes were better tolerated than PEI nanoplexes. RAW264.7 cell line was far more sensitive for the particle exposure, resulting in lower cell viability. Conclusion: The different nanoplex formulations were characterized in respect to size and PdI and nucleic acid loading capacity tested and cytotoxicity studies performed. However, this pilot transfection studies have been unsuccessful due to the high particle concentration used and therefore need further optimization.Bakgrunnur: Nanótækni er ört vaxandi vísindasvið þar sem nýjir möguleikar til bætingar á greiningu og meðhöndlun margra sjúkdóma blasa við okkur. Markmið nanótækni er að bæta lífhvarfafræði og aðgengi lyfs til ákveðinna líkamsvefja eða markfrumna, með minnstu mögulegu aukaverkunum og eitrunaráhrifum. Katjóniskar pólýrótaxanfjölliður (PRx) til gena flutninga sem áður hefur verið notuð sem ferja til siRNA flutnings. Í þessari rannsón hefur hýalúronsýru (HA) verið bætt við í nanófléttur til stöðgunar og til að auka þolanleika frumna gagvart nanóögnum. Markmið: 1) Að framleiða og skilgreina sérkenni katjóniskra pólýrótaxan nanófléttna (PRx) í samsetningu með hýalúrónsýru (HA) og annað hvort pDNA eða siRNA með mismunandi N/P hlutfalli. 2) Að athuga frumudrepandi áhrif nanófléttna og að framkvæma lúsíferasa tilfærslu tilraunir á þeim. Aðferðir: Fjölfléttur (e.polyplex), á nanóskala mynduð með siRNA eða pDNA voru rannsakaðar. Stærð og stöðugleiki agna var athugað með DLS og NTA tækni. Til samanburðar rannsókna var notuð PEI fjölliða sem er þekkt fyrir framúrskarandi tilfærslu eiginleika. Föngun kjarnsýru innan í samsetningunum var staðfest með aragrósa gel rafdrætti. Agnirnar sem skiluðu bestu niðurstöðum voru prófaðar fyrir frumudrepandi áhrifum og til þess voru notaðar tvær mismunandi frumulínur A549 og RAW264.7. Tilfærslutilraunir voru framkvæmdar með því að nota lúsíferasa vísigen og líkan siRNA, og voru framkvæmdar í báðum frumulínum. Niðurstöður: Bestu niðurstöður með PEI nanófléttum var náð með N/P hlutfalli 7 og pólýrótaxan mynduðu fullnægjandi nanófléttur með N/P hlutfalli 2. Til þess að öðlast stöðugrar samsetningar þurfti einnig að nota pDNA í samsetningu við fjölliður, en reynt var að skipta hluta þess með HA. Að frumudrepandi tilraunum loknum kom í ljós að frumurnar þoldu PRx betur en PEI nanóflækjur. Frumulína RAW264.7 var mun viðkvæmari þegar útsett fyrir nanóögnum sem skilaði sig sem lægri prósenta lifandi frumna. Ályktanir: Mismunandi sametningar voru prófaðar og bestu agnir valdar með tilliti til stærðar og stöðugleika. Einnig voru frumudrepandi áhrif nanófléttna framkvæmdar og bornar saman. Samt sem áður skiluðu þessar for tilfærslutilraunir ekki árangri vegna hárs styrks agna sem frumurnar voru útsettar fyrir og því er þörf á frekari rannsóknum

Placental protein 13 (PP13)-induced vasodilation of resistance arteries from pregnant and nonpregnant rats occurs via endothelial-signaling pathways

ABSTRACTPlacental protein 13 (PP13) induces hypotension in rats. This study aims to evaluate PP13 effects on isolated uterine arteries from nonpregnant and mid-pregnant rats. Vessels were isolated, cannulated, and pressurized to 50 mmHg within an arteriograph, preconstricted and exposed to increasing PP13 concentrations (10−13–10−8 M). PP13 elicited 38–50% arterial vasodilation with half-maximum response (EC50) = 1 pM. The relaxation was mediated by activating the endothelial-signaling pathways of prostaglandin and nitric oxide (NO). Accordingly, these results encourage evaluation of PP13 as a possible therapy for gestational diseases characterized by insufficient uteroplacental blood flow and/or maternal hypertension