Die vorliegende Arbeit untersucht die strukturellen Grundlagen agonistischer und invers agonistischer Eigenschaften von Liganden am ADP-Rezeptor P2Y12. Dazu wurde eine Bibliothek systematisch veränderter Purinverbindungen am Wildtyp-P2Y12 (WT) mit und ohne ADP und an 28 konstitutiv aktiven P2Y12-Mutanten getestet. Dies ermöglichte die pharmakologische Zuordnung der Substanzen als Agonist, Antagonist oder inverser Agonist. Die Untersuchungen wurden in einem Hochdurchsatz-Hefe-Expressionssystem in Hefen durchgeführt. Als agonistische Liganden am P2Y12 Rezeptor konnten verschiedene ATP und ATP-Derivate identifiziert werden. Ihre agonistische Potenz am ADP-Rezeptor reihte sich wie folgt: 2-(methylthio)-ADP > 2-(methylthio)-ATP > ADP > ATP. In Dockingstudien wurde mittels eines komparativen Computermodels des P2Y12 für diese ATP-Derivate eine Bindungsstelle nachgewiesen, die von den Transmembrandomänen (TM) 3, 5, 6 und 7 gebildet wird. Die Aminosäuren Y105, E188, R256, Y259 und K280 besitzen in der Ligandeninteraktion einen besonderen Stellenwert. Zudem konnten einige Liganden identifiziert werden, die invers agonistische Eigenschaften an konstitutiv aktiven P2Y12-Mutanten zeigten. So führte eine N-Methyl-anthraniloyl-(mant) Modifizierung an der 3’-OH Gruppe der 2’-Deoxyribose (mant-dATP, mant-dADP) zu Liganden mit invers agonistischen Eigenschaften an 10 konstitutiv aktiven P2Y12-Mutanten. Diese Wirkung konnte mittels verschiedener funktioneller Tests in Säugerzellsystemen ebenfalls für den WT-Rezeptor bestätigt werden. Basierend auf den Ergebnissen computerassistierter Dockingstudien schienen inverse Agonisten und Agonisten dieselbe Bindungstasche zu nutzen. Eine mechanistische Erklärung für ihren funktionellen Unterschied am WT konnte das Modell jedoch nicht liefern. Zusammenfassend wurde gezeigt, dass der als ADP-Rezeptor bezeichnete P2Y12 mit einer etwas geringeren Potenz auch ATP als natürlichen Agonist erkennt und dass mant-modifiziertes dATP und dADP neue inverse Agonisten mit potenziellem therapeutischem Nutzen sind.

Commercial microbial milk clotting enzyme preparations were purified by immunoaffinity chromatography using purified antibody covalently coupled to porous glass beads as the column matrix. Commercial enzyme preparation diluted in 1 mM sodium acetate buffer at pH 5.0 was then biospecifically adsorbed to the column matrix by end-over-end mixing of the glass-antibody complex in the enzyme solution for 12 h at 5°C. The antibody bound enzyme adsorbed glass beads were soaked in .2 M glycine or ethanolarnine at pH 7.0 to block uncoupled reactive sites on the matrix. Following this, the column was washed with 1 mM sodium acetate buffer at pH 7 .0, followed by additional wash with .5 M NaCl, until absorbance at 280 nm returned to baseline. Elution of adsorbed enzyme was achieved with .2 M sodium acetate at pH 3.0, .2 M acetate at pH 3.5, containing .15 M NaCl and .5 M acetate at pH 4.0 containing .5 M NaCL At the same protein concentration, immunoaffinity chromatography purified enzymes had higher clotting activity than the commercial enzyme preparations. Amino acid analysis and OPA proteolysis tests of TCA soluble peptides liberated from casein hydrolysis showed purified enzymes to exhibit lower general proteolytic activity. Immunological reactivity of Mucor enzymes with calf rennet was determined with antibodies produced by intramuscular injections of Mucor miehei protease, Mucor pusillus protease and calf rennet emulsified in Freund's adjuvant into three New Zealand White rabbits . Harvested antisera were heated at 56°C for 30 min to inactivate complement factors and contaminating proteins then centrifuged at 1700 x g for 30 min. Ouchterlony double immunodiffusion method was used to test for presence of antibodies in the antisera, and for cross immunoreactivity. Antibodies against M. miehei were cross reactive with M. pusillus antigen and M. pusillus antibodies cross reacted with M. miehei antigen. Immunodiffusion assay did not show cross reactivity of calf rennet antibodies with either M. miehei antigen or M. pusillus antigen. Antibodies against the Mucor enzymes did not show cross reactivity with calf rennet antigen. Although their actions in milk differ, proteolytic enzyme preparations from M. miehei and M. pusillus are both used as calf rennet substitutes in cheese manufacture. Differences in the characteristics of the two Mucor enzyme preparations exist, even though they exhibit some immunological homology . From our results, at least one antigenic factor is common to both enzyme preparations.

No description available.

<p>The management of thrombosis in the presence of thrombocytopenia is challenging because the inherent risk of bleeding associated with anticoagulant use may increase due to low platelet counts. Guidelines regarding anticoagulant use in this situation are based mainly on expert opinions and anecdotal data. We developed an <em>in-vitro</em> model to study the effect of anticoagulants on plasma clot formation in the presence of low platelet counts. We used thromboelastography (TEG) to measure global viscoelastic properties of clot formation and scanning electron microscopy (SEM) to observe and quantify changes in the fibrin clot structure. Experiments were conducted in plasma with varying platelet concentrations from <10 >– 150 × 10⁹/L. Clotting was activated with tissue factor (TF) and calcium, in the presence of factor XIIa inhibitor, corn trypsin inhibitor. One of the following anticoagulants at therapeutic concentration was added to the mixture: unfractionated heparin (UFH), dalteparin, fondaparinux, rivaroxaban or dabigatran. We found clotting had different sensitivity to TF concentration depending on the anticoagulant present. Effects on TEG parameters varied at a fixed TF concentration with each anticoagulant. UFH had the greatest influence, delaying clotting significantly at low platelet counts. The factor-specific anticoagulants had the least impact on TEG parameters. SEM revealed that UFH had the greatest impact on clot structure. UFH caused significant increase in porosity and fibrin widths and had significantly less fibers when platelets decreased. In conclusion, this study may provide fundamental data to understand clot formation in the presence of anticoagulants at low platelet counts. At low platelets the anticoagulants can jeopardize clot formation, especially UFH. The mechanism of each anticoagulant may contribute to the variation in response to TF initiated clotting. AT-dependent anticoagulants compromised plasma clotting more than the newer factor specific anticoagulants, possibly related to the multiple, non-specific inhibition of coagulation factors.</p> / Master of Science (MSc)

O Fator IX (FIX) da coagulação sanguínea é uma proteína dependente de vitamina K de grande valor farmacêutico no tratamento da hemofilia B, o qual é baseado na administração de FIX derivado de plasma humano ou FIX recombinante. A terapia baseada nestas abordagens apresenta limitações como os altos custos e a baixa disponibilidade para a população de hemofílicos. O FIX é uma proteína complexa com grande variedade de modificações pós-traducionais e, por isso, apresenta baixa atividade específica e produtividade. A produção de um FIX recombinante com aumento na sua atividade coagulante pode contribuir para terapias de reposição e gênica mais eficazes nas quais quantidades menores das moléculas cataliticamente melhoradas serão necessárias para se obter os benefícios terapêuticos do FIX. Essa infusão diminuída poderá reduzir ou eliminar a geração de inibidores de FIX, minimizando os efeitos colaterais e reduzindo os custos do tratamento, sendo um dos desafios atuais no tratamento da hemofilia B. Nesse contexto, a engenharia genética tem se tornado uma ferramenta importante na modificação de proteínas de interesse farmacêutico visando a melhora das suas propriedades bioquímicas e biofísicas. Este trabalho teve como objetivo desenvolver novas variantes de FIX recombinante humano com aumento da sua atividade biológica. Para isso, fizemos um estudo detalhado da estrutura da proteína FIX e geramos três variantes FIX-YKALW, FIX-ALL e FIX-LLW, utilizando a técnica de mutagênese sítio dirigida. Essas variantes foram expressas em células humanas SK-Hep-1 tratadas com vitamina K e as proteínas foram caracterizadas in vitro e in vivo. A quantidade de FIX total secretada no sobrenadante celular foi similar entre as variantes FIX-YKALW e FIX-LLW (3,2 ?g/mL), enquanto FIX-ALL apresentou a menor expressão (1,8 ?g/mL), quantidades 2-3 vezes menores que de FIX controle (FIX-WT). Entretanto, a atividade biológica das variantes foi, em média, 6,2 vezes maior que FIX-WT, com atividade específica 11 vezes maior para FIX-YKALW e FIX-LLW e 24 vezes maior para FIX-ALL. FIX-YKALW apresentou a melhor geração de trombina in vitro dentre as variantes e destacou-se na avaliação da eficiência hemostática em camundongos hemofílicos B, na qual uma dose 5 vezes menor que a recomendada de FIX foi suficiente para promover uma resposta pró-coagulante. Neste trabalho foram desenvolvidas 3 novas variantes de FIX recombinante humano com maior atividade específica que o fator disponível no Sistema Único de Sáude. O FIX-ALL apresentou-se como a proteína mais potente in vitro descrita até o momento e FIX-YKALW como a mais promissora, dentre as mutantes desenvolvidas, na proteção hemostática in vivo. Estudos complementares são necessários para transpor a barreira da pesquisa para a utilização desses novos fatores de coagulação no tratamento da hemofilia B. Entretanto, esse trabalho apresenta novas proteínas com potencial de serem usadas na combinação com a terapia gênica bem como na terapia de reposição / Blood coagulation factor IX is a vitamin K-dependent protein, and it has become a valuable pharmaceutical in the treatment of hemophilia B which is based on the plasma-derived coagulation factor or recombinant protein. Coagulation therapy based on these approaches has limitations as high costs and low availability to hemophilic population. FIX is a complex protein with a wide variety of post-translational modifications and, therefore, presents low specific activity and productivity. The production of recombinant FIX with enhanced coagulant activity may contribute to more effective protein replacement and gene therapies in which reduced amounts of catalytically improved molecules will be required to obtain the therapeutic benefits of FIX. This fewer infusion may reduce or eliminate the generation of FIX inhibitors, minimizing side effects and reducing treatment costs. It is one of the current challenges for hemophilia B treatment. In this context, bioengineering has become an important tool for pharmaceutical interest protein modification to improve its biochemical and biophysical properties. This study focused on development of new recombinant human FIX variants with augmented clotting activity. We performed a detailed study of FIX protein structure and generated three variants, FIX-YKALW, FIX-ALL and FIX-LLW, using site-directed mutagenesis. Such variants were expressed in SK-Hep-1 cells treated with vitamin K and they were characterized in vitro and in vivo. The amount of FIX secreted in the cell supernatant was similar between FIX-YKALW and FIX-LLW (3.2 ?g/mL), whereas FIX-ALL displayed the lowest expression (1.8 ?g/mL), 2-3 times lower than FIX-wild-type (FIX-WT). However, the clotting activities of FIX variants were 6.2 times greater than FIX-WT. FIX-YKALW e FIX-LLW showed 11-fold higher specific activity and FIX-ALL showed 24-fold higher specific activity. FIX-YKALW displayed the greatest generation of thrombin in vitro among variants and was highlighted in the evaluation of haemostatic efficiency in hemophiliac B mice, in which a dose 5 times lower than the recommended FIX replacement therapy was enough to promote a procoagulant response. We generated 3 novel recombinant human FIX variants with enhanced specific activity than FIX available on the health system. FIX-ALL with higher in-vitro potency than any other known hyperfunctional FIX variant and FIX-YKALW as the most promising variant, among the generated mutants, for in-vivo haemostatic protection. Further studies are required to overcome the barrier to research for application of new coagulation factors on hemophilia B treatment. However, we have presented new proteins with potential for therapeutic use combined with gene therapy as well as in protein replacement therapy.

Ziele dieser Arbeit zum Tränkemanagement bei neonataler Kälberdiarrhoe waren, die Auswirkungen von oralen Rehydratationslösungen (ORL) auf die abomasale Milchgerinnung und den Labmagendurchmesser zu prüfen sowie die Wirksamkeit von unterschiedlich zubereiteten ORL bei bestehender Dehydratation zu vergleichen. Dazu wurden die folgenden zwei Untersuchungen durchgeführt: Die erste Untersuchung an gesunden und durchfallkranken Kälbern sollte mittels Ultraschall zeigen, ob die Einmischung eines bicarbonathaltigen Elektrolytpulvers in die Tränke deren abomasales Gerinnungsverhalten beeinträchtigt. Zeitgleich wurde der ventrodorsale Labmagendurchmesser erfasst, um daraus Rückschlüsse auf die abomasale Entleerung ziehen zu können. Diese Arbeit untersuchte erstmals die Milchgerinnung im Labmagen von spontan an Durchfall erkrankten Kälbern. In der zweiten Untersuchung sollten die Effekte der Fütterung von Milchaustauscher (MAT) sowie von in Wasser und in MAT zubereiteter ORL auf den Flüssigkeits- und Säuren-Basen-Haushalt experimentell dehydrierter Kälber ermittelt werden. Material und Methoden: Bei gesunden (n = 28) sowie durchfallkranken Kälbern (n = 15) wurde das abomasale Gerinnungsverhalten sowie der ventrodorsale Labmagendurchmesser (= Labmagenhöhe) vor und nach Fütterung von Milch bzw. MAT sowie nach Zusatz eines bicarbonathaltigen Elektrolytpulvers zur jeweiligen Tränke ultrasonografisch dargestellt. Im zweiten Untersuchungsteil wurden sechs Kälber nach einem modifizierten Protokoll von WALKER et al. (1998a) experimentell dehydriert. Im Anschluss wurden diese Tiere entweder mit MAT oder mit einer ORL, welche in Wasser (Wasser-ORL) oder MAT (MAT-ORL) zubereitet wurde, gefüttert. In einem weiteren Versuchsdurchlauf verblieben die mittel- bis hochgradig dehydrierten Probanden nüchtern. Nach einem definierten Schema wurden während der Versuchsphase venöse Blutproben vor und nach Induktion einer Dehydratation sowie vor und nach Fütterung entnommen. Es wurden Parameter des Flüssigkeits- und Säuren-Basen-Haushaltes zu den verschiedenen Untersuchungszeitpunkten bestimmt. Ergebnisse: Nach Gabe von Milch konnte mittels Ultraschall immer eine vollständige Zweiphasentrennung in Koagulum und Molke detektiert werden, wohingegen diese nach Fütterung des MAT nur unvollständig voneinander separiert waren. Die kombinierte Fütterung von Milch oder MAT und einer ORL, welche 62 bzw. 93 mmol/l Bicarbonat enthielt, führte zu keinen Unterschieden auf den ultrasonografischen Bildern des Labmageninhaltes im Vergleich zu denen der jeweiligen nativen Tränke. Des Weiteren war die abomasale Milchgerinnung nicht aufgrund eines Durchfallgeschehens gestört. Die unvollständige Gerinnung des MAT resultierte nicht in dessen schnellerer abomasaler Passage, sondern anhand des statistisch signifikant größeren Labmagendurchmessers ab vier Stunden nach MAT-Fütterung scheint es, dass die Entleerung des MAT aus dem Labmagen im Vergleich zu Milch leicht verzögert war. Innerhalb der beiden Versuchstiergruppen konnten keine statistisch signifikanten Unterschiede in Bezug auf den abomasalen Durchmesser zwischen den Tränken mit und ohne ORL-Zusatz festgestellt werden. Die statistisch signifikanten Differenzen des Labmagendurchmessers zwischen den gesunden und durchfallkranken Kälbern nach Fütterung der identischen Tränken weisen darauf hin, dass die Entleerung des Labmagens bei an Diarrhoe erkrankten Kälbern verzögert stattfindet. Bei den experimentell dehydrierten Probanden erhöhte sich das Plasmavolumen statistisch signifikant nach Aufnahme einer Tränkemahlzeit, wohingegen dieses ohne Behandlung konstant blieb. Die Rate der Plasmavolumenexpansion war nach Fütterung von MAT im Vergleich zu Wasser-ORL oder MAT-ORL vermindert. Die Zunahme des Plasmavolumens war bei den dehydrierten Kälbern nach Aufnahme von Wasser-ORL stärker ausgeprägt als nach Fütterung von MAT-ORL. Außerdem war nach Gabe der hypertonen MAT ORL die Plasmaosmolalität statistisch signifikant erhöht. Der Säuren-Basen-Status der Tiere verbesserte sich infolge der Absorption von Flüssigkeit. Dieser Effekt war allerdings weniger offensichtlich, da das Versuchsprotokoll eine hochgradige Dehydratation aber nur eine gering- bis maximal mittelgradige metabolische Azidose induzieren konnte. Schlussfolgerungen: Die unvollständige Gerinnung eines MAT im Labmagen scheint zu keiner schnelleren Entleerung zu führen. Die abomasale Milchgerinnung ist nicht beeinträchtigt, wenn die Milchfütterung mit einer 93 mmol/l Bicarbonat enthaltenden ORL kombiniert wird. Darüber hinaus resultiert aus einer Durchfallerkrankung keine Störung der Milchgerinnung im Labmagen. Die Einmischung eines bicarbonathaltigen Elektrolytpulvers in Milch oder MAT hat keine schnellere abomasale Passage der Ingesta zur Folge. Im Gegensatz zu gesunden Kälbern findet die Entleerung des Labmagens bei durchfallkranken Tieren verzögert statt. Es sind weitere Untersuchungen erforderlich, welche die Ursachen für die verlangsamte abomasale Passage bei an Durchfall leidenden Kälbern bestimmen. Aus den Ergebnissen der vorliegenden Arbeit kann geschlussfolgert werden, dass die gemeinsame Verabreichung von Milch bzw. MAT mit einem bicarbonathaltigen Elektrolytpulver weder die Milchgerinnung noch die abomasale Entleerung der Tränke bei durchfallkranken Kälbern beeinflusst. Folglich ist die Einmischung einer ORL in eine caseinhaltige Tränke möglich. Jedoch zeigen die Ergebnisse der zweiten Untersuchung, dass die Fütterung einer hypertonen MAT-ORL weniger effektiv bei der Erhöhung des Plasmavolumens dehydrierter Kälber ist als das in Wasser zubereitete Äquivalent (Wasser-ORL). Genau genommen erhöht die Verabreichung einer hypertonen MAT-ORL die Plasmaosmolalität bei dehydrierten Tieren, was möglicherweise bei durchfallkranken Kälbern zu einer akuten Kochsalzvergiftung führen könnte. In einer Folgeuntersuchung zu dieser Arbeit konnte gezeigt werden, dass die Gabe von hypertoner Milch-ORL in Kombination mit freiem Zugang zu Wasser eine effektive Behandlungsmaßnahme durchfallkranker Kälber darstellt, da die hohen Elektrolytgaben die Wasseraufnahme der Kälber stimulieren und keine Gefahr einer Hypernatriämie besteht (WENGE et al. 2014). Anhand der beiden Arbeiten kann geschlussfolgert werden, dass durchfallkranke Kälber, denen kein freier Zugang zu Wasser gewährt wird, wasserbasierte, isotone ORL erhalten sollten. / Aims of the present studies on oral rehydration management of calf diarrhoea were to reveal the effects of oral rehydration solutions (ORS) on abomasal milk clotting and abomasal diameter, as well as to compare the effectiveness of differently prepared ORS in calves with experimentally induced dehydration. For this purpose, two experiments were conducted: The first investigation in healthy and diarrhoeic calves should demonstrate via ultrasound whether the incorporation of bicarbonate-containing electrolyte powder into ‘milk meals’ impairs the abomasal coagulation of milk protein. At the same time, the ventrodorsal diameter of the abomasum was measured to outline abomasal emptying. This study is the first in which milk clotting in the abomasum of spontaneously diarrhoeic calves was investigated. The second investigation examined the effects of feeding milk replacer (MR), as well as ORS prepared in water or in MR on the fluid and acid-base balance of experimentally dehydrated calves. Materials and methods: Abomasal curd formation, as well as ventrodorsal diameter (= abomasal height), were ultrasonographically imaged in healthy (n = 28) and diarrhoeic calves (n = 15) before and after feeding milk, MR and ORS containing bicarbonate prepared in milk or MR, respectively. In the second investigation six calves were experimentally dehydrated according to a modified protocol of WALKER et al. (1998a). Subsequently, these calves were fed with either milk replacer (MR) or an ORS prepared in either water (water-ORS) or MR (MR-ORS). In one experiment, the dehydrated calves remained fasting. During the experimental period, venous blood samples were taken according to a defined schedule before and after induction of dehydration, as well as before and after feeding. Parameters of fluid and acid-base balance were determined at various timepoints. Results: After milk-feeding, a complete separation of curd and whey was always detected via ultrasound; whereas after MR-feeding, separation was incomplete. Feeding mixtures of milk or MR with ORS containing 62 - 93 mmol/L bicarbonate did not cause any differences in the ultrasonographic images of abomasal content compared to those of milk or MR. Moreover, abomasal milk clotting was not disturbed due to diarrhoea. Inadequate milk clotting of MR did not result in its faster abomasal passage but according to the significantly larger abomasal diameter starting from 4 h after MR-feeding gastric emptying of MR was slightly decreased when compared to milk. Within the two groups of experimental animals no statistically significant differences could be determined with respect to the abomasal diameter between the diets with and without addition of ORS. Statistically significant differences of abomasal diameter between healthy and diarrhoeic calves after feeding the same diet indicate that abomasal emptying is delayed in calves suffering from diarrhoea. Plasma volume increased significantly following the intake of a ‘fluid meal’ in experimentally dehydrated calves, whereas it remained constant in the absence of treatment. The rate of plasma volume expansion was reduced by feeding MR relative to water-ORS or MR-ORS. In dehydrated calves, the expansion of plasma volume was more pronounced following the intake of water-ORS compared to the feeding MR-ORS. Moreover, plasma osmolality increased significantly following the ingestion of hypertonic MR-ORS. The acid-base status of animals was corrected as a result of fluid absorption, but this effect was less obvious as the experimental protocol resulted in severe dehydration and only mild to moderate metabolic acidosis. Conclusions: Inadequate curd formation of an MR in the abomasum does not result in faster abomasal passage. Milk clotting in the abomasum is not affected when combining milk feeding with ORS containing 93 mmol/L of bicarbonate. Furthermore, abomasal curd formation is not disturbed due to diarrhoea. The addition of an bicarbonate-containing ORS in milk or MR does not result in faster abomasal passage of ingesta. In contrast to healthy calves, abomasal emptying is prolonged in diarrhoeic calves. Hence, further studies are needed to determine reasons for decelerated abomasal passage in calves suffering from diarrhoea. According to the results of the present study it can be concluded that combined feeding of milk/MR with an bicarbonate-containing ORS does not affect either milk clotting or abomasal emptying of the diet in diarrhoeic calves. Consequently, the addition of ORS to milk meal is possible. However, the results of the second investigation indicate that the feeding of hypertonic MR-ORS is less effective in increasing plasma volume of dehydrated calves than the water-based equivalent (water-ORS). In fact, administration of hypertonic MR-ORS increases plasma osmolality in dehydrated calves, potentially causing acute hypernatraemia in diarrhoeic calves. In a follow-up study to the present investigation, it could be demonstrated that feeding hypertonic milk-ORS combined with ad libitum access to water is an effective method of treating diarrhoeic calves because the high electrolyte content stimulates water intake of calves and there is no risk of hypernatraemia (WENGE et al. 2014). Based on these two studies, it can be concluded that diarrhoeic calves without free access to water should receive isotonic water-based ORS.

No description available.

Introduction : L’hémophilie B (HB) est une maladie hémorragique héréditaire caractérisée par un déficit en facteur IX (FIX) de la coagulation. La thérapie génique de l’HB par injection de virus adéno-associés (AAV) montre des résultats prometteurs, mais entraine une toxicité hépatique à forte dose. La création de nouveau transgène de FIX permettant d’injecter de moindres doses d’AAV est un réel enjeu. Matériel et Méthodes : Des transgènes thérapeutiques exprimant une protéine humaine de FIX à demi-vie prolongée par fusion à l’albumine (hFIX-Alb) ou exprimant un FIX une activité spécifique augmentée, le hFIX-E410H, ont été créés et injectés à des modèles murins. Une nouvelle molécule recombinante de hFIX à demi-vie prolongée par fusion à la sous-unité B du FXIII via un linker clivable par le facteur X activé (hFIX-LXa-FXIIIB) a été crée, produite et caractérisée. Résultats : Le transgène hFIX-Alb n’accumulait pas le niveau plasmatique du FIX par rapport au FIX sauvage. Des expériences ont été entreprises pour comprendre les mécanismes responsables du défaut d’expression. Le transgène hFIX-E410H, montrant une activité spécifique augmentée in vitro et in vivo chez les souris HB, permettait de diminuer les doses d’AAV d’environ 2,5 fois. La molécule hFIX-LXa-FXIIIB était fonctionnelle, corrigeait la génération de thrombine chez les souris HB, et présentait une demi-vie augmentée 3,9 fois chez la souris et 2,3 fois chez le rat. Conclusion : Nous avons développé et caractérisé de nouveaux transgènes de FIX modifiés et une nouvelle molécule de FIX à demi-vie prolongée, qui pourraient constituer de nouvelles perspectives thérapeutiques de l’HB / Introduction: Haemophilia B (HB) is an inherited bleeding disorder due to coagulation factor IX (FIX) deficiency. Adeno-associated virus (AAV)-based gene therapy for HB has shown promising results but can cause liver toxicity after administration of high dose of AAV vectors.The design of new transgene expressing modified FIX that would allow injecting fewer doses of AAV is a real challenge. Materials & Methods: Therapeutic transgene expressing human FIX with prolonged half-life due to fusion to mature albumin (hFIX-Alb) or expressing FIX with improved specific activity, hFIX-E410H, were designed and injected to murine animal model. A novel recombinant FIX molecule exhibiting enhanced half-life through fusion to the FXIIIB subunit via activated factor X-cleavable linker was design, produced and characterised. Results: The hFIX-Alb transgene did not increase the plasma FIX clotting activity compared to the transgene expressing wild-type hFIX. Experiments were undertaken to understand the mecanisms responsible for lower expression. The hFIX-E410H transgene, which showed improved specific activity in vitro and in vivo in HB mice, allowed injecting a 2.5-fold lower dose of AAV. The hFIX-LXa-FXIIIB molecule was functional, corrected the generation capacity in HB mice, and exhibited a 3.9-fold and 2.2-fold enhanced half-life in mice and in rats, respectively, compared to wild-type FIX. Conclusion: We have developed and characterised new transgenes expressing modified FIX, and a novel FIX molecule with prolonged half-life, which could become interesting perspectives for the treatment of HB

The purpose of this study was to evaluate the stability of vitamin K1 oral liquids in Sterile Water for Injection when stored in amber glass bottles and amber plastic syringes under refrigerated conditions. Four 100-mL batches of vitamin K1 in Sterile Water for Injection were prepared in amber glass bottles to protect from light. One of the batches was divided into 1-mL aliquots, using amber plastic oral syringes, and capped. The prepared bottles and syringes were stored in a laboratory refrigerator. On each day of sampling, 1-mL aliquots were removed from each bottle and mixed with an equal volume of ethanol. Likewise, the contents of sample syringes were mixed with ethanol to achieve an assay concentration of 0.5 mg/mL. Recovery of vitamin K1 in the compounded samples was quantified against a United States Pharmacopeia reference standard. Quantification was achieved using a stability-indicating high-performance liquid chromatography with ultraviolent light detection method. Product stability is defined as 90% to 110% of the initial concentration. The percent recovery in the Sterile Water for Injection preparations in glass bottles remained above 90% for the 105-day duration of the study, but some samples stored in amber plastic syringes fell below 90% on day 21. Furthermore, a statistically significant difference (2-way ANOVA, P < 0.0001) emerged between syringes at day 0 and day 30, and this trend continued through the day 60, 90, and 105 samples. The only statistically significant difference found within the bottle-stored samples occurred on day 105 (versus zero, P = 0.0465), but the recovery on day 105 still exceeded 90%. Vitamin K1 in Sterile Water for Injection, stored in a refrigerated amber glass bottle, is stable for 105 days. This preparation can also be stored in amber plastic syringes, but this decreases the beyond-use date to 14 days.

Neuropsychological impairment following cardiopulmonary bypass (CPB) remains a serious consequence of otherwise successful surgery. The incidence of neuropsychological decline is poorly understood due to varied measurement intervals, and perhaps more importantly the use of unreliable detection and classification methods. The reported incidence varies considerably, ranging anywhere from 30% to 90% of subjects. While the nature of this impairment has not been fully elucidated, recent evidence suggests that microembolism during surgery may be the principal causative agent of postoperative cerebral dysfunction. The work described in this thesis investigates one possible source of microembolism leading to postoperative decline, namely thromboembolism arising from excessive activation of the haemostatic mechanism. Crucial to the accurate detection of significant decline in individual patients, this work also focuses on the development and use of meaningful criteria to be used when describing change in neuropsychological performance measures. The strong haemostatic activation during CPB is controlled by heparin anticoagulation. The clinical performance of the Hepcon heparin-monitoring instrument was compared to the activated clotting time (ACT), which is used in most cardiac centres. An analysis of samples from 42 elective coronary artery bypass grafting (CABG) patients shows that the ACT does not detect the significant decline in heparin concentration seen upon connection to CPB, in comparison to the Hepcon. The Hepcon appears to be in satisfactory agreement with laboratory anti-Xa analysis of heparin concentration, with the mean difference for the Hepcon at -0.46 U/ml, and the limits of agreement +/- 1.12 U/ml. Further analysis shows that that for 95% of cases, the Hepcon will give values that are between 0.53 and 1.27 times the value for anti-Xa. The loss of relationship between ACT and heparin concentration was further investigated by converting ACT values to heparin concentration. The results provide data on the degree of prolongation in ACT times brought about by factors associated with CPB. A methodology is presented by which users can adjust for the loss of relationship between ACT and heparin. This work also demonstrates that under normal usage of the ACT, the user may obtain values up to 3 times appropriate for the plasma heparin concentration. The computer-administered neuropsychological testing tool (the MicroCog) was validated using 40 age-matched control subjects. Using a two-week interval, the summary score correlation coefficients ranged from .49 to .84, with all scores demonstrating significant practice effects. Also presented are retest normative data that may be used to determine significant change in a homogeneous sample using both reliable change and regression models of analysis. The performance of four different models of change analysis was then analysed using data from the clinical group. The regression technique of analysis was shown to be the most useful prediction model as it provides correction for both practice effects and regression toward the mean in each individual. A novel statistical rationale is presented for the choice of criteria in the identification of patients that may be defined as overall impaired when using a battery of test scores. When using one-tailed prediction models for decline, the binomial distribution of scores was shown to be a useful descriptive statistic providing an estimate of change due to chance. When applied to a suitable selection of scores that minimise shared variance, a value +/- 20% of test scores used was demonstrated to be a rational cut-off for an individual to be classified as impaired. Using this methodology, 32.7% of patients were identified as significantly deteriorated in neuropsychological test function immediately prior to discharge from hospital. Patient age was shown to be a significant predictor of neuropsychological decline following CPB. No significant relationship was identified between thrombin generation and neuropsychological change scores, however problems with patient recruitment and retention limited the statistical power of this study. An intriguing relationship with heparin concentration was noted that might warrant further investigation. This work highlights the complex nature of post-bypass neuropsychological dysfunction and the complexities in assessing decline. The regression-based model was shown to be highly useful in the analysis of data from a suitably validated neuropsychological testing tool. The argument that no suitable criterion exists for the identification of patients as overall impaired has been challenged with the development of a rational cut-off based on the likely distribution of change scores across a series. The work presented here confirms the need for standardised testing methods based on sound statistical criteria. This work also highlights the problems associated with current methods for monitoring anticoagulation therapy during bypass surgery. Methodology is presented that allows adjustment of ACT results to account for CPB-induced prolongation of clotting times. Current techniques for heparin monitoring overestimate heparin levels on bypass by up to threefold, which may predispose to subclinical coagulation and increased delivery of protamine.