New potential nanotechnology-based therapies for the treatment of rheumatoid arthritis

Doctoral Thesis NEW POTENTIAL NANOTECHNOLOGYBASED THERAPIES FOR THE TREATMENT OF RHEUMATOID ARTHRITIS Nataliya Storozhylova Doctoral Program in Nanomedicine and Pharmaceutical Innovation Faculty of Pharmacy SANTIAGO DE COMPOSTELA 2018 2 3 UNIVERSIDAD DE SANTIAGO DE COMPOSTELA FACULTAD DE FARMACIA DEPARTAMENTO DE FARMACOLOGÍA, FARMACIA Y TECNOLOGÍA FARMACÉUTICA UNIVERSITÉ DE NANTES FACULTÉ DES SCIENCES ET TECHNIQUES Doctoral Thesis NEW POTENTIAL NANOTECHNOLOGYBASED THERAPIES FOR THE TREATMENT OF RHEUMATOID ARTHRITIS Nataliya Storozhylova Doctoral Program in Nanomedicine and Pharmaceutical Innovation SANTIAGO DE COMPOSTELA 2018 4 5 Prof. María José Alonso Fernández, Full Professor at the Department of Pharmacology, Pharmacy and Pharmaceutical Technology in the University of Santiago de Compostela, Spain Dr. José Crecente Campo, Research Scientist at the Department of Pharmacology, Pharmacy and Pharmaceutical Technology in the University of Santiago de Compostela, Spain Dr. Cyrille Grandjean, Chargé de Recherche CNRS – Unité Fonctionnalité et Ingénierie des Protéines (UFIP), UMR 6286, Université de Nantes, France / Researcher at French National Centre for Scientific Research (CNRS), UFIP, UMR 6286, University of Nantes, France. Report: That the experimental dissertation entitled: “New potential nanotechnology-based therapies for the treatment of rheumatoid arthritis” presented by Nataliya Storozhylova was conducted under their supervision at the Department of Pharmacology, Pharmacy and Pharmaceutical Technology at the University of Santiago de Compostela and at the Faculty of Sciences and Techniques, Unit of Functionality and Proteins Engineering, UMR CNRS 6286 at the University of Nantes, France. Being completed, they authorize its presentation, evaluation by the assigned jury members, considering that it meets the requirements demanded in article 34 of the Regulation of Doctoral Studies of the USC, and that as supervisors of this thesis they do not incur in the abstention causes established by the law 40/2015. And for the record, they issue and sign the present certificate in Santiago de Compostela, February th and Nantes, February th 2018. Prof. María José Alonso Fernández Dr. José Crecente Campo Dr. Cyrille Grandjean 6 7 DECLARACIÓN DO AUTOR/A DA TESE New potential nanotechnology-based therapies for the treatment of rheumatoid arthritis D./Dna. Nataliya Storozhylova Presento a miña tese, seguindo o procedemento axeitado ao Regulamento, e declaro que: 1) A tese abarca os resultados da elaboración do meu traballo. 2) De selo caso, na tese faise referencia ás colaboracións que tivo este traballo. 3) A tese é a versión definitiva presentada para a súa defensa e coincide coa versión enviada en formato electrónico. 4) Confirmo que a tese non incorre en ningún tipo de plaxio doutros autores nin de traballos presentados por min para a obtención doutros títulos. En Santiago de Compostela, 05 de febrero de 2018 Asdo. . 8 9 To the memory of my Godmother, who has always put her faith in me and who has passed away before I could find a medicine to her. “I мертвим, і живим, і ненарожденним землякам моїм в Украйні і не в Украйні моє дружнєє посланіє…” Поема-послання Тарас Шевченко, 1845 Acknowledgements 16 As a Fellowship holder of the Nanofar: European Doctorate in Nanomedicine and pharmaceutical innovation, I would also like to extend my gratitude and great appreciation to the financial support from the European Commission, Education, Audiovisual and Culture Executive Agency (EACEA), Erasmus Mundus programme and pay obeisance to our consortium: the University of Angers (UA) and the University of Nantes (UN), The University of Louvain (UCL) and the University of Liège (ULg), the University of Nottingham (UNott), the University of Santiago de Compostela (USC). 17 Contents Abstract …………………………………………………………………... 19 Resumen…………………………………………………………………... 23 Abbreviation list………………………………………………………….. 27 RESUMEN IN EXTENSO Nuevas terapias basadas en la nanotecnología para el tratamiento de la artritis reumatoide………………………………………………………… 29 RÉSUMÉ De nouvelles thérapies basées sur les nanotechnologies pour le traitement de l'arthrite rhumatoïde……………………………………………………. 49 INTRODUCTION Intra-articular therapies for an effective treatment of rheumatoid arthritis... 67 Background, Hypothesis and Objectives……………………………….. 115 Chapter 1 An injectable, in situ hyaluronic acid-fibrin hydrogel containing nanocapsules for prolonged intra-articular drug delivery ……………… 123 Chapter 2 New galectin-3 inhibitor as a lead compound for anti-inflammatory drug candidates………………………………………………………………… 177 Overall discussion………………………………………………………… 239 Conclusions………………..………………..………………..………….... 263 Annex (permissions and reprints).………………..………….................. 267 18 19 Abstract Some other trifling particulars might be mentioned, but they are all of slight importance and have nothing to do with the true relation of the history; and no history can be bad so long as it is true. Miguel de Cervantes Saavedra, 1605 20 Abstract 21 ABSTRACT The local administration of drugs into the intra-articular cavity is beneficial for the treatment of a number of joints pathologies. Unfortunately, despite this interest, the efficient intra-articular delivery of drugs represents a challenge, which is mainly associated to the fast drug elimination from the synovial cavity. Therefore, there is a clear need to develop advanced controlled release technologies that may overcome this limitation. At the same time, the discovery of novel immunotherapeutic targets has prompted the design and development of highly specific antagonists for effective therapies. This is the case of the extracellular protein galectin-3 (Gal-3), which has been recently identified as a primer trigger of the pro-inflammatory cascade in arthritis. The inhibition of Gal-3 could block the whole inflammatory cascade, prevent further joints degradation and alleviate the inflammatory joint diseases. In this work we have developed a novel drug delivery system (DDS) composed of an in situ forming hydrogel combined with hyaluronic acid (HA) nanocapsules (NCs). HA NCs consisting of an olive oil core surrounded by a HA shell were prepared by the solvent displacement technique. The NCs exhibited a particle size of 135 ± 9 nm, a negative surface charge and a modest capacity to encapsulate dexamethasone, which was used as a model drug. Thereafter, two injectable in situ hydrogels systems composed of HA-fibrin and fortified HA-fibrin (with crosslinker factor XIII and α2-antiplasmin) were developed. Both gels displayed easily adjustable gelation time and mechanical properties. These NCscontaining hydrogels showed the capacity to control the release of dexamethasone in simulated synovial fluid for up to 72 h. On the other hand, an antagonist of Gal-3 was designed, synthesized and characterized. It was based on its natural ligand type II lactosamine [Gal(1→4)-GlcN] core, modified with aromatic substituents to greatly increase its affinity and specificity. Gal-3 inhibitor revealed high affinity to Gal-3 (Kd = 0.59 µM at 4 °C) and selectivity among Gal-1/ Gal-3/Gal-7. This compound was further encapsulated within HA NCs (531 ± 5 µg/mL), dispersed within the in situ HA-fibrin hydrogel and tested in a carrageenan-induced acute knee joint synovitis rat model. Both, groups with drug-loaded NCs alone and in the gel, administrated intra-articularly at microgram scale doses (200 and 55 μg/kg), showed a remarkable suppression of inflammation compared with the non-treated control. These findings highlight the potential of Gal-3 inhibitor as a lead compound for the treatment of rheumatoid arthritis. In addition, the in vitro and in vivo experiments showed the interest of the nanotechnology-based DDS for intra-articular application. 22 23 Resumen Otras algunas menudencias había que advertir, pero todas son de poca importancia y que no hacen al caso a la verdadera relación de la historia, que ninguna es mala como sea verdadera. Miguel de Cervantes Saavedra, 1605 24 Resumen 25 RESUMEN La administración local de fármacos en la cavidad intra-articular puede ser beneficiosa para el tratamiento de ciertas patologías asociadas a las articulaciones. Sin embargo, a pesar de su interés, la liberación intra-articular de fármacos eficiente representa un desafío, principalmente debido a su rápida eliminación de la cavidad sinovial. Por lo tanto, hay una clara necesidad de desarrollar tecnologías de liberación controlada avanzadas que puedan superar estas dificultades. Al mismo tiempo, el descubrimiento de nuevas dianas inmunoterapéuticas ha suscitado el diseño y desarrollo de antagonistas altamente específicos con el fin de obtener terapias efectivas. Este es el caso de la proteína extracelular galectina-3 (Gal-3), que ha sido identificada recientemente como un iniciador principal de la cascada proinflamatoria en la artritis. La inhibición de Gal-3 podría bloquear toda la cascada inflamatoria, prevenir una mayor degradación de la articulación y aliviar las enfermedades inflamatorias de las articulaciones. En este trabajo hemos desarrollado un sistema de liberación de fármacos compuesto por un hidrogel, que se forma in situ, combinado con nanocápsulas (NCs) de ácido hialurónico (HA). Las NCs de HA consisten en un núcleo oleoso de aceite de oliva rodeado por una cubierta de HA y fueron preparadas por el método de desplazamiento de disolvente. Las NCs presentan un tamaño de 135 ± 9 nm, una carga superficial negativa y una capacidad modesta de encapsular dexametasona, usada en este estudio como fármaco modelo. Posteriormente, se desarrollaron dos sistemas tipo hidrogel formados por HA-fibrina y HA- fibrina reforzado (con el entrecruzante factor XIII y α2-antiplasmina). Ambos geles muestran tiempos de gelificación y propiedades mecánicas fácilmente modulables. Los hidrogeles cargados de NCs permitieron la liberación controlada de dexametasona en fluido sinovial simulado durante 72 h. Por otro lado, se ha diseñado, sintetizado y caracterizado un antagonista de la Gal-3. Dicho antagonista se basa en su ligando natural, con un núcleo tipo II de lactosamina [Gal(1→4)-GlcN], modificado con sustituyentes aromáticos para incrementar en mayor medida su afinidad y especificidad. El inhibidor de Gal-3 mostró elevada afinidad por la Gal- 3 (Kd = 0.59 µM a 4 °C) y selectividad entre Gal-1/Gal-3/Gal-7. Posteriormente, este compuesto fue encapsulado en NCs de HA (531 ± 5 µg/mL), que a su vez fueron dispersadas en un hidrogel de HA-fibrina y el conjunto evaluado en ratas, en un modelo de sinovitis aguda de rodilla inducida por carragenano. Tanto el grupo tratado con las NCs cargadas de fármaco como el grupo tratado con dichas NCs dispersadas en el gel, administrados intra- Resumen in extenso 32 Simultáneamente, los sistemas de liberación controlada pueden tener una papel de viscosuplementación, para mejorar la homeostasis de la rodilla, y servir de andamiajes celulares in situ para la regeneración del tejido del cartílago. Al mismo tiempo, el descubrimiento de nuevas dianas terapéuticas ha suscitado grandes esfuerzos dirigidos hacia el diseño y desarrollo de pequeñas moléculas antagonistas altamente específicas para conseguir terapias eficaces (en particular para diferentes tipos de cáncer, enfermedades autoinmunes, diabetes, etcétera).20–23 Este es el caso de la proteína extracelular galectina-3 (Gal-3), que ha sido recientemente identificada como desencadenante de la inflamación.24,25 Además, esta proteína favorece la secreción de citocinas pro-inflamatorias, quimiocinas24 y metaloproteinasas de la matriz extracelular,26 que están involucradas en la remodelación del cartílago y la desmineralización ósea.5,27 2. Hipótesis 1. El uso de nanocápsulas (NCs) cargadas con fármacos dispersas en hidrogeles puede aumentar sustancialmente el tiempo de retención intra-articular de dichos fármacos. Las NCs pueden actuar como reservorios de fármacos lipófilos, permitiendo su liberación controlada, mientras que el hidrogel formado in situ podría proteger a las NCs de una rápida descomposición, endocitosis y eliminación de la cavidad sinovial, modulando también la liberación del fármaco. Dicha formulación inyectable de NCs combinadas con un hidrogel también podría contribuir a la homeostasis de la rodilla, debido al núcleo de aceite de oliva de las NCs y al recubrimiento de ácido hialurónico (HA) de 700 kDa, ambos con propiedades antiinflamatorias.28–31 Además, el hidrogel 3D de fibrina fortalecida con ácido hialurónico podría actuar como viscosuplemento, con propiedades elásticas adecuadas para su permanencia en la cavidad articular y como soporte para la regeneración del cartílago.32–34 2. Dado que el bloqueo tradicional de COX-2 y las citocinas pro-inflamatorias TNF-α, IL-1 e IL-6, ha demostrado una efectividad limitada en las enfermedades inflamatorias de las articulaciones,2,4,35–37 planteamos la hipótesis de que la inhibición de la diana galectina-3, podría bloquear toda la cascada inflamatoria y evitar la posterior degradación de las articulaciones. Resumen in extenso 33 3. Objetivos Basándonos en los antecedentes bibliográficos y en las hipótesis anteriormente expuestas, el objetivo de esta tesis ha sido doble, por un lado, el de desarrollar un nuevo hidrogel que se forma in situ tras la inyección y que posee nano-reservorios que permiten la liberación prolongada de fármacos en la cavidad intraarticular; por otro lado, un objetivo adicional ha sido el de sintetizar un nuevo antagonista de la galactina-3 y su incorporación en el hidrogel antes mencionado para una evaluación in vivo. Con este propósito, los objetivos generales se dividen en los siguientes objetivos específicos: 1) Desarrollo y evaluación in vitro de un nuevo sistema de liberación de fármacos destinado a la administración intra-articular En el desarrollo de este objetivo se han cubierto las siguientes fases de trabajo: 1. Formulación de NCs de ácido hialurónico, cargadas con dexametasona (fármaco modelo), y posterior caracterización con respecto a sus propiedades fisicoquímicas, estabilidad en líquido sinovial simulado y estabilidad en almacenamiento. 2. Diseño y desarrollo de un hidrogel destinado a gelificar in situ tras la administración intraarticular, y estudio de la influencia de los parámetros que pueden modificar su tiempo de gelificación, propiedades mecánicas y evaluar su capacidad de ser inyectado. 3. Estudio de la formación del hidrogel en presencia de las NCs, análisis de su capacidad de carga y de la modificación de las propiedades reológicas del hidrogel en presencia de las NCs. Caracterización del comportamiento reológico tanto de los hidrogeles blancos como de los cargados con NCs, su estructura superficial y porosidad. 4. Optimización y caracterización de dos hidrogeles cargados con NCs, que difieren en el grado de entrecruzamiento de la red del hidrogel, diseñados para tratar la inflamación aguda y crónica de las articulaciones usando modelos in vivo de rata. 5. Evaluación del perfil de liberación in vitro de la dexametasona a partir del gel cargado con NCs. Resumen in extenso 34 Los resultados correspondientes a estos objetivos se presentan en el Capítulo 1: “An injectable, in situ forming hydrogel containing nanocapsules for intra-articular drug delivery”. 2) Síntesis de un nuevo compuesto anti-inflamatorio, incorporación en el sistema de liberación de fármacos desarrollado y evaluación de su actividad in vivo En el desarrollo de este objetivo se han cubierto las siguientes fases de trabajo: 6. Diseño y síntesis un nuevo candidato a fármaco antiinflamatorio – un antagonista de la galectina-3 extracelular (Gal-3), usando como núcleo su ligando natural, la lactosamina de tipo II [Gal(1→4)-GlcN], modificado con sustituyentes aromáticos para aumentar en mayor medida su afinidad y especificidad. 7. Caracterización del inhibidor de Gal-3 obtenido, a través de la valoración de su afinidad y selectividad entre Gal-1/Gal-3/Gal-7, comparación con el primer inhibidor de bajo peso molecular de la Gal-3 comercializado (TD139) y selección del inhibidor sintético más potente para su posterior evaluación in vivo. 8. Incorporación del inhibidor de Gal-3 en las NCs cargadas en el hidrogel, posterior caracterización de sus propiedades fisicoquímicas y evaluación de su actividad antiinflamatoria in vivo en un modelo en rata con sinovitis aguda de rodilla inducida por carragenano. Los resultados relacionados con este trabajo se presentan en el Capítulo 2: “New galectin-3 inhibitor as a lead compound for anti-inflammatory drug candidates”. Resumen in extenso 35 4. Resultados y discusión 1. Gal-3i como compuesto líder candidato a fármaco en el tratamiento de la artritis reumatoide Teniendo en cuenta su participación en la fisiopatología de la artritis reumatoide, la galectina-3 (Gal-3) se presenta como una nueva diana inmunoterapéutica,24,27,38–40 cuyo bloqueo podría prevenir toda la cascada inflamatoria y la posterior degradación de las articulaciones. Con este objetivo, se han diseñado, sintetizado y caracterizado antagonistas de bajo peso molecular de la Gal-3 extracelular. Los compuestos se basaron en el núcleo que posee el ligando natural de Gal-3: una lactosamina tipo II [Gal(1→4)-GlcN], modificado con sustituyentes aromáticos para aumentar de manera considerable su afinidad y especificidad. Sus estructuras, denominadas oxazolina y Gal-3i, se comparan en la Figura 1 con otros inhibidores de la Gal-3 que han sido evaluados en ensayos clínicos para el tratamiento de diferentes patologías.41–48 Gal-3i, oxazolina y TD139 (el primer antagonista de Gal-3 de bajo PM aprobado por la FDA), testados en un ensayo de polarización de fluorescencia directo y competitivo, revelaron selectividad entre Gal-1/Gal-3/Gal-7. Gal-3i demostró una afinidad 7,5 veces mayor a la Gal-3 (Kd = 0,59 μM a 4 °C y 2,99 μM a 25 °C) que la oxazolina (Kd = 4,4 μM a 4 °C y 23 μM a 25 °C) y se seleccionó para su evaluación in vivo. Figura 1. Estructuras de dos inhibidores comerciales de Gal-3: GM-CT-01 (DAVANAT®) y TD139; junto a la oxazolina y el Gal-3i sintetizados en este trabajo. Resumen in extenso 36 2. Desarrollo y caracterización de un sistema de liberación de controlada de fármacos para la aplicación intraarticular Para evitar los dos principales desafíos que afrontan los sistemas de administración IA de fármacos, la internalización celular y la rápida eliminación de la cavidad sinovial,2,15 se desarrolló un sistema compuesto de NCs dispersadas en un hidrogel formado in situ (Figura 2). Figura 2. Ilustración esquemática de la estructura de una articulación sinovial y la inyección intraarticular de un hidrogel formado in situ combinado con nanocápsulas cargadas con un fármaco lipofílico. Adaptada y modificada con el permiso,15 Copyright© 2009, Taylor & Francis. Las NCs, preparadas mediante la técnica de desplazamiento de disolvente, están compuestas de aceite de oliva (OO), tensioactivos biodegradables y biocompatibles: lecitina de soja (Lec) y oleilamina (OAm) y, finalmente, HA de 700 kDa como polímero de cubierta. Nuestro objetivo era desarrollar NCs con un tamaño de ~130 nm, que no se espera que causen daño en el cartílago u otros daños mecánicos en la articulación11 y que permitan encapsular la cantidad suficiente de fármacos lipofílicos (un fármaco modelo – dexametasona [DXM] y el nuevo compuesto Gal-3i). En la Tabla 1 se resumen el tamaño de partícula, el potencial zeta y la eficacia de encapsulación de las formulaciones. Las NCs fueron estables en condiciones de almacenamiento a 4 °C durante un período de al menos 1 mes. Sin embargo, la estabilidad de las NCs se vio comprometida tanto en PBS como en líquido sinovial simulado. Por lo tanto, desarrollamos un hidrogel inyectable, formado en presencia de NCs, para mejorar su estabilidad y aumentar su retención en la Resumen in extenso 37 cavidad articular (evitando la internalización celular y su rápida eliminación de la articulación). Al mismo tiempo, el hidrogel permitiría la liberación prolongada del fármaco. Para la preparación del gel se seleccionaron los biopolímeros HA y fibrina que forman una red 3D interpenetrante (IPN),33,49–52 debido a la afinidad del HA de alto PM por la fibrina.32,53 La formación del hidrogel se basa en la activación enzimática del fibrinógeno por la trombina (Thr), y permite diseñar por etapas una IPN en presencia de HA de diferentes PM, de agentes entrecruzantes de fibrina (el factor XIII y la α2-antiplasmina) y de NCs cargadas con el fármaco. Tabla 1. Propiedades fisicoquímicas de las NCs de HA de 700 kDa blancas y cargadas con fármaco (media ± SD, n = 6). OO = aceite de oliva, Lec = lecitina de soja, OAm = oleilamina, PDI = índice de polidispersión Tipo de NCs OO (mg) Lec (mg) OAm (mg) Tamaño de partícula (nm) PDI Potencial ζ (mV) Concentración de fármaco (mg/mL) Blancas prototipo 1 15 3,75 0,75 128 ± 13 0,2 −27 ± 3 n/a prototipo 2 22 5,625 1,125 121 ± 10 0,2 −30 ± 3 n/a Cargadas con el fármaco DXM prototipo 1 15 3,75 0,75 160 ± 12 0,2 −20 ± 4 0,75 ± 0,13 prototipo 2 22 5,625 1,125 135 ± 9 0,2 −31 ± 5 5,60 ± 0,40 Gal- 3i prototipo 2 22 5,625 1,125 122 ± 11 0,2 −29 ± 5 0,53 ± 0,05 La formación de los geles se monitorizó mediante análisis de turbidez (λ = 350 nm) y estudios reológicos que permitieron identificar la fase de retardo, el punto de gelificación (Figura 3) y la evolución del gel en el tiempo. Resumen in extenso 38 Figura 3. Resumen de la determinación del punto de gelificación mediante reología en los diferentes sistemas: geles blancos de "HA-fibrina" y de "HA-fibrina fortalecido" y estos geles con NCs al 30%. La línea de trazos y puntos refleja la fase de retardo requerida de 2 minutos. Las mediciones se llevaron a cabo a 37 ºC. Expresado como media ± SD; n = 3. La modificación de la concentración de distintos compuestos, como la Thr, HAs, NCs, Ca2+, factor XIII y α2-antiplasmina han permitido una modificación controlada de la microarquitectura del gel y de su tiempo de gelificación, obteniendo un tiempo de retardo de la gelificación adecuado para los estudios in vivo. Los geles permitieron albergar un 30% (v/v) de NCs en su estructura durante su autoensamblaje, con una distribución homogénea dentro del gel y con poros bien definidos (7,29 ± 1,23 μm y 4,88 ± 1,12 μm, respectivamente para los geles de HA-fibrina y HA-fibrina reforzado con 30% (v/v) de NCs). Los valores iniciales de viscosidad de la mezcla física HA-fibrina (sin adición la Thr) fueron 118,9 mPa·s y 81,3 mPa·s a 20 °C y 37 °C, respectivamente, significativamente más bajos que los viscosuplementos comerciales,54,55 por lo que se puede anticipar una fácil inyección, previa a la formación in situ del hidrogel, en los estudios in vivo. El hidrogel formado presentó características reológicas tanto de material elástico como de tipo sólido, adecuado para sostener las deformaciones intra-articulares. En resumen, hemos desarrollado dos sistemas de liberación controlada: "un gel de HA-fibrina con 30% de NCs" y "un gel reforzado de HA- fibrina con 30% de NCs" (Figura 3), que podrían ser adecuados para tratar inflamaciones articulares agudas y crónicas, respectivamente. Resumen in extenso 39 El perfil de liberación in vitro de la dexametasona desde el gel de HA-fibrina con 30% de NCs se prolongó hasta 72 h en fluido sinovial simulado, lo que podría ser altamente ventajoso para la administración intraarticular del fármaco (Figura 4). Figura 4. Liberación de dexametasona (DXM) desde el hidrogel de ácido hialurónico-fibrina con 30% de NCs en líquido sinovial simulado, 37 ºC, 72 h. 3. Actividad antiinflamatoria in vivo del inhibidor de Gal-3 Para evaluar la actividad biológica del Gal-3i, se empleó un modelo de inflamación aguda inducida por carragenano en ratas,56 ampliamente usado para testar nuevos fármacos antiinflamatorios.57 Los animales con sinovitis inducida se distribuyeron en tres grupos de acuerdo con el tratamiento recibido (100 μL, IA): NCs blancas utilizadas como vehículo control (grupo 1), NCs cargadas con Gal-3i a una dosis de 200 μg/kg (grupo 2) y NCs cargadas con Gal-3i dispersadas (30% v/v) en el gel de HA-fibrina reforzado a una dosis de 55 μg/kg (grupo 3). La actividad del fármaco se controló a las 4 horas después de la inducción de la sinovitis, cuando se observa el pico de la inflamación.56 Los datos de las mediciones de la hinchazón de la rodilla, los análisis de sangre, el análisis histopatológico y la cuantificación de los marcadores pro-inflamatorios del plasma sanguíneo, demostraron una actividad antiinflamatoria pronunciada del Gal-3i y la mejora del estado de las articulaciones. Los datos de hinchazón de la rodilla (Figura 5) mostraron una reducción del diámetro de la rodilla en: 1,6 Resumen in extenso 40 veces (grupo 1), 3,5 veces (grupo 2) y 7 veces (grupo 3) en comparación con el control de carragenano. Figura 5. Efecto del tratamiento con el inhibidor de Gal-3 en el modelo de inflamación de la articulación inducido por carragenano usando las NCs y las NCs en gel comparado con NCs blancas y el control de carragenano (n = 5 para los grupos 1-3, n = 1 para el control de carragenano). Δ es la diferencia en el diámetro de la rodilla en diferentes momentos antes y después de la inyección de carragenano, expresada en mm. Datos expresados como media ± SEM. Este hallazgo resulta más notable si se considera que la dosis del grupo 3 (NCs cargadas con Gal-3i en el gel a una dosis de 55 μg/kg), es considerablemente más baja que en el grupo 2 (NCs cargadas con Gal-3i a una dosis de 200 µg/kg). Posiblemente, el hidrogel de HA-Fibrina mantiene de manera efectiva las NCs dentro de la cavidad articular, modula la liberación y, por sí mismo, tiene un efecto beneficioso en la curación de las articulaciones, incluso en el modelo de inflamación aguda en ratas. Los resultados obtenidos en este ensayo preliminar in vivo demostraron una notable supresión de la inflamación debida al Gal-3i encapsulado en el hidrogel y administrado de manera intraarticular en dosis de microgramos. Los resultados presentan al Gal-3i como un compuesto líder candidato a fármaco inmunoterapéutico para las enfermedades de la rodilla, en particular para la artritis reumatoide. Es de destacar, que el hidrogel con NCs sin fármaco también demostró una tendencia a contribuir a la curación de las articulaciones y a reducir la inflamación sinovial, gracias, probablemente, al núcleo de aceite de oliva y al ácido hialurónico presente en su composición.28,29,58 Al mismo tiempo, el hidrogel de ácido hialurónico-fibrina podría, potencialmente, funcionar como un viscosuplemento, con Resumen in extenso 41 propiedades elásticas adecuadas para la permanencia intra-articular y como una estructura adhesiva in situ con alto grado de citocompatibilidad para la regeneración del cartílago. 5. Conclusiones El objetivo de esta tesis ha sido el diseño y desarrollo de un nuevo sistema inyectable destinado a prolongar el tiempo de residencia intraarticular y lograr una liberación controlada de fármacos. El sistema consiste en un hidrogel con nanocápsulas dispersas en él, sirviendo como multireservorio para diferentes fármacos antiinflamatorios y lipófilos, tales como i) la dexametasona, empleada en este estudio como fármaco modelo y ii) un nuevo candidato a fármaco – un antagonista de la galectina-3 (Gal-3). Los resultados experimentales obtenidos condujeron a las siguientes conclusiones: 1) En este estudio, se desarrolló y sintetizó un nuevo inhibidor monovalente altamente afín, potente y selectivo de la Gal-3, formado por dos restos diferentes ensamblados a un núcleo de lactosamina modificado. Su Kd hacia la Gal-3, medido en un ensayo de polarización de fluorescencia directo y competitivo, fue de 0,59 μM a 4 °C (2,99 μM a 25 °C), mientras que el inhibidor de Gal-3 marcado con fluoresceína mostró una afinidad muy alta hacia la Gal-3, Kd = 14 nM a 4 °C (82 nM a 25 °C), ciertamente más alta que la del inhibidor comercial de Gal-3 DAVANAT®. Una característica importante de este compuesto es la capacidad de discriminar entre Gal-1/Gal-3/Gal-7, mientras que DAVANAT® difícilmente discrimina entre Gal-1 y Gal-3. Por otro lado, se ha confirmado experimentalmente que un primer antagonista de Gal-3 de bajo PM TD139, aprobado por la FDA, con el núcleo de tio-digalactósido modificado, medido en el mismo ensayo, tiene una alta afinidad, con una Kd (Gal-3) = 0,036 μM a 4 °C (0,166 μM a 25 °C) y alta selectividad entre Gal-1/Gal-3/Gal-7. Teniendo en cuenta los datos de Kd prometedores del compuesto que hemos desarrollado y sintetizado, consideramos interesante estudiar in vivo la eficacia de este inhibidor de la Gal-3 de bajo PM. 2) Se prepararon exitosamente nuevas nanocápsulas (NCs) con núcleo de aceite de oliva y recubrimiento de ácido hialurónico, utilizando para ello el método de desplazamiento de disolvente. Estas NCs tienen la capacidad de encapsular dexametasona (5,6 ± 0,4 mg/mL) Resumen in extenso 48 58. Cecerale, S. in Olive oil - Constituents, Health Properties and Bioconversions (INTECH, 2011). 49 RÉSUMÉ De nouvelles thérapies basées sur les nanotechnologies pour le traitement de l'arthrite rhumatoïde La seule chose qui compte, c'est l’effort. Le Petit Prince, Antoine de Saint-Exupéry, 1943 50 Resumen in extenso 51 1. Introduction La plupart des articulations, en raison de leurs caractéristiques anatomiques, se prêtent aux injections intra-articulaires (IA). Ces dernières années, il a été démontré que l'administration locale de médicaments avec un mode d'action direct pouvait grandement améliorer l'efficacité des traitements des maladies articulaires.1–4 Les avantages de l'administration intra-articulaire comparée aux voies orales ou intraveineuses sont les suivantes : (i) biodisponibilité accrue, (ii) réduction de la toxicité du médicament, (iii) l’obtention d'une concentration efficace avec une dose minimale de médicament, (iv) réduction de l'exposition systémique et des effets indésirables associés, (v) diminution de l’apparition de résistance, et (vi) réduction du coût total des médicaments. Les principaux médicaments utilisés pour le traitement des arthropathies et l’amélioration de l’hémostase du genou qui sont actuellement disponibles sur le marché sont la cyclooxygénase-2 (COX-2), les antagonistes des TNF-α, IL-1 et IL-6. Les principales limites de ces médicaments sont le manque de spécificité pour les articulations et l'apparition d'effets secondaires considérables2,5–7 désignant l'administration IA comme une alternative intéressante. Les progrès dans le domaine biopharmaceutique au cours des dernières décennies ont permis le développement de systèmes de délivrance des médicaments (SDMs) les rendant moins toxiques tout en offrant une libération contrôlée et prolongée des drogues directement au site d’action.2,4 Cependant, l'administration de médicaments IA dans l’articulation reste complexe, principalement en raison d'efflux rapide dans la cavité synoviale allié à la rotation rapide du fluide synovial (FS), l'internalisation cellulaire et du caractère invasif de cette route d’administration.2 Différents SDMs ont été testés pour le traitement local des arthropathies à partir de nano-/microparticules et d’hydrogels.2,4,6,8 Les formulations les plus prometteuses et avancées portent sur une combinaison des deux systèmes.9–14 Ces SDMs permettent une rétention suffisante et la libération prolongée de médicaments de faible masse moléculaire (MM), connus pour avoir un temps de séjour court dans les articulations2,15 lorsqu’ils sont administrés en solution IA. Le cartilage articulaire a une capacité limitée pour l'auto-régénération.16–18 En outre, le FS a largement perdu ses fonctions intrinsèques dans des conditions pathologiques des articulations.19 Avec cela à l'esprit, le recours à des transporteurs de médicaments multifonctionnels devrait contribuer à la guérison des pathologies diartrosiques. Outre la Resumen in extenso 52 libération contrôlée et prolongée de médicament, ces systèmes composés de matériaux résistants à la déformation peuvent contribuer au maintien de la rotule et à la lutte contre les dégradations enzymatiques. Simultanément, les SDMs pourraient fonctionner comme agents de viscosupplémentation pour améliorer l'homéostase du genou et, comme échafaudages cellulaires pour la régénération in situ du tissu cartilagineux. En même temps, la découverte de nouvelles cibles immunitaires a conduit à d’intenses efforts pour la conception et le développement de petites molécules antagonistes pour des thérapies efficaces (en particulier pour les différents types de cancer, les maladies auto-immunes, le diabète…).20–23 Tel est le cas de la protéine galectine-3 (Gal-3), qui a récemment été identifié comme favorisant l’apparition de la réponse inflammatoire.24,25 Entre autre, cette protéine favorise la sécrétion de cytokines proinflammatoires, chimiokines par les macrophages,24 et l’expression de métalloprotéinases de la matrice,26 qui sont impliquées dans le remodelage du cartilage et de la déminéralisation de l'os.5,27 2. Hypothèse 1. L’utilisation de nanocapsules (NCs) chargées avec des médicaments dans des hydrogels dispersés in situ peut sensiblement augmenter le temps de rétention intraarticulaire des drogues. Les NCs peuvent agir comme réservoirs pour de multiples drogues lipophiles, et permettre leur libération contrôlée, tandis que l'hydrogel in situ pourrait protéger les NCs d’une décomposition rapide, l'endocytose et l'efflux de la cavité synoviale tout en contribuant également à moduler la libération des drogues. De telles compositions injectables pourraient aussi contribuer à l’hémostase du genou si un noyau d'huile d'olive et un revêtement d'acide hyaluronique (HA) 700 kDa, connus pour posséder des propriétés anti-inflammatoires, sont utilisés.28–31 Nous avons aussi imaginé qu’un l'hydrogel de fibrine en 3D renforcé avec de l'acide hyaluronique devrait servir de viscosupplément (chaînes réticulées de HA) avec des propriétés élastiques pour persister dans l’ariculation et servir de support pour la régénération du cartilage.32–34 2. Etant donné que le blocage traditionnel de la COX-2 et les cytokines proinflammatoires TNF-α, IL-1 et IL-6 n'a pas conduit à un soulagement adéquat des maladies articulaires inflammatoires,2,4,35–37 nous avons émis l'hypothèse que le Resumen in extenso 53 ciblage d’une nouvelle cible, la galectine-3, pourrait aider à bloquer toute la cascade inflammatoire et prévenir la dégradation des articulations ultérieures du système immunitaire. 3. Objectifs Sur la base des hypothèses décrites, l'objectif de cette thèse était double, d'une part, de développer un nouvel d'hydrogel avec des nano réservoirs injectables pour la libération prolongée de médicaments lipophiles dans la cavité intra-articulaire; d'autre part, la synthèse d'un nouvel antagoniste de la galectine-3 et son incorporation dans l'hydrogel mentionné cidessus pour l'évaluation in vivo. La démarche adoptée pour atteindre les objectifs finaux a été décomposée comme suit : 1) Développement et validation de SDMs injectables 1. Formuler, optimiser et caractériser de nouveaux NCs, vides ou chargés avec de la dexaméthasone (en tant que médicament modèle), en termes de propriétés physicochimiques, de stabilité dans un liquide synovial reconstitué et de stabilité au stockage. 2. Concevoir et développer un nouvel hydrogel injectable intra-articulaire et étudier l'influence des paramètres qui peuvent modifier leur temps de gélification, les propriétés mécaniques et évaluer leur seringabilité. 3. La formation d'hydrogel en présence de NCs, la capacité de charge et la modification de ses propriétés rhéologiques. Caractériser le comportement rhéologique des hydrogels formulés en résence ou en absence de NCs, en termes de surface et de tailles. 4. Optimiser et caractériser deux hydrogels in situ formulés avec des NCs destinés à étudier leur effet dans un modèle d'inflammation aiguë chez le rat induit par le carraghénane. 5. Test le profil de libération in vitro de dexaméthasone du gel in situ formulé avec les NCs. Resumen in extenso 54 Les résultats correspondant à ces tâches sont présentés au Chapitre 1: “An injectable, in situ forming hydrogel containing nanocapsules for intra-articular drug delivery”. 2) Synthèse du médicament anti-inflammatoire, l'incorporation dans le SDM développé et évaluation de l'activité in vivo 6. Concevoir et synthétiser un nouveau « lead » pour le traitement de l’inflammation – une petite molécule inhibitrice de la forme extracellulaire de la galectine-3 (Gal-3), dérivée de la lactosamine de type II [Gal(1→4)-GlcN], motif naturel reconnu par cette lectine, décorée avec des substituants aromatiques en vue d’augmenter son affinité et sa spécificité. 7. Caractériser l’inhibiteur Gal-3 obtenu, vérifier son affinité et sa sélectivité vis-à-vis d’un panel de galectines, en comparaison avec une petite molécule inhibitrice dérivée de sucre connue, le bis-3-[(3-fluorophényl)triazolyl] thiodigalactoside (souvent désigné TD139) qui vient d’être favorablement évalué en étude clinique de phase II pour le traitement de la fibrose pulmonaire. 8. Ajouter l'inhibiteur Gal-3 dans le SDM développé, caractériser les propriétés physicochimiques de la formulation obtenue et tester l'activité anti-inflammatoire de cet inhibiteur in vivo dans un modèle de synovite aigüe de l’articulation du genou induite par la carraghénane chez de rat. Les résultats liés à ce travail sont présentés au Chapitre 2: “New galectin-3 inhibitor as a lead compound for anti-inflammatory drug candidates.” 4. Résultats et discussion 1. Gal-3i en tant que composé principal pour de nouveaux médicaments candidats pour le traitement de l'arthrite rhumatoïde Compte tenu de son implication dans la physiopathologie de l'arthrite rhumatoïde, la galectine-3 (Gal-3) apparaît comme une nouvelle cible potentielle immunothérapeutique,24,27,38–40 dont le blocage peut empêcher/éviter la cascade inflammatoire et la dégradation des articulations qui l’accompagne. Nous avons donc conçu Resumen in extenso 55 et réalisé la synthèse de petites molécules antagonistes de la galectine-3. Les composés ont été obtenus à partir d’un ligand naturel de la Gal-3, la lactosamine de type II [Gal(1→4)- GlcN], modifié avec des substituants aromatiques. Leurs structures présentées dans la Figure 1, appelées oxazoline et Gal-3i, par rapport à d'autres inhibiteurs de Gal-3 sont comparées à d’autres structures qui font l’objet d'essais cliniques pour le traitement de diverses maladies.44–51 Gal-3i, oxazoline et TD139 (un premier antagoniste Gal-3 de faible poids moléculaire approuvé par la FDA), testé dans des essais de polarisation de fluorescence compétitive ont révélé une sélectivité entre Gal-1/Gal-3/Gal-7. Gal-3i a montré une affinité 7,5 fois plus grande pour Gal-3 (Kd = 0,59 μM à 4 °C et 2,99 μM à 25 °C) que l'oxazoline (Kd = 4,4 μM à 4 °C et 23 μM à 25 °C) et a été sélectionné en tant que composé principal pour évaluer l'inhibition de Gal-3 in vivo. Figure 1. Les structures des deux inhibiteurs commerciaux Gal-3 : GM-CT-01 (DAVANAT®) TD139, oxazoline et Gal-3i synthétisé. 2. Développement et caractérisation d’une application SDM intra-articulaire Afin de contourner deux des principaux écueils auxquels les systèmes délivrance de médicament IA doivent faire face, l'internalisation cellulaire et l'élimination rapide de la cavité synoviale,2,15 nous avons développé un SDM composé de NCs dispersées dans un hydrogel in situ (Figure 2). Resumen in extenso 56 Figure 2. Représentation schématique de la structure d'une articulation synoviale et de l’injection intra-articulaire d’un hydrogel formé in situ, combiné à des nanocapsules chargées d'un médicament lipophile. Adapté et modifié avec l'autorisation de 15, Copyright© 2009, Taylor & Francis. Les NCs reportés dans ce travail de thèse ont été préparées par une technique de déplacement de solvant et sont composées d'huile d'olive (OO), d’agents tensio-actifs biodégradables et biocompatibles - lécithine de soja (Lec) et oléylamine (OAm) et, finalement, d’acide hyaluronique (HA 700 kDa) en tant que polymère d'enveloppe. Nous avons développé des NCs ayant une taille d'environ ~130 nm propres à ne pas créer de dommages à l'articulation11 tout en permettant d’encapsuler des drogues lipophiles en quantité suffisante (un anti-inflammatoire modèle – la dexaméthasone [DXM] et Gal-3i). La taille des particules, le potentiel zéta et l'efficacité d'encapsulation des formulations sont décrites dans le Tableau 1. Les NCs sont stables dans des suspensions aqueuses stockées à 4 °C pendant une période d'au moins 1 mois. Les NCs maintiennent leur stabilité dans l'eau mais agrègent dans du PBS ou dans le liquide synovial reconstitué. Par conséquent, nous avons développé un hydrogel injectable in situ, en présence des NCs préformées pour améliorer la stabilité, avec maintien dans la cavité articulaire et une libération prolongée des drogues encapsulées. Les gels ont été formés à partir de deux biopolymères HA et de la fibrine capables de former un réseau interpénétrant 3D (IPN)33,52–55, en raison de l'affinité de HA de haut poids moléculaire pour la fibrine.32,56 La formation d'hydrogel est basée sur l'activation enzymatique du fibrinogène par la thrombine (Thr), et permet de concevoir un IPN pas à pas, en présence de HA de différents poids moléculaires, d’agents renforçant la fibrine (facteur XIII et α2-antiplasmine) et les NCs chargées avec la drogue. Resumen in extenso 57 Tableau 1. Propriétés physico-chimiques des NCs vides ou chargées en drogue préparée avec HA 700 kDa (moyenne ± SD, n = 6). OO: d'huile d'olive, Lec: lécithine de soja, OAm: oléylamine, PDI: indice de polydispersité. Type de NCs OO (mg) Lec (mg) OAm (mg) Taille de particule (nm) PDI ζpotentiel (mV) La concentration de drogue (mg/mL) Blanc prototype 1 15 3,75 0,75 128 ± 13 0,2 −27 ± 3 n/a prototype 2 22 5,625 1,125 121 ± 10 0,2 −30 ± 3 n/a Chargé avec la drogue DXM prototype 1 15 3,75 0,75 160 ± 12 0,2 −20 ± 4 0,75 ± 0,13 prototype 2 22 5,625 1,125 135 ± 9 0,2 −31 ± 5 5,60 ± 0,40 Gal- 3i prototype 2 22 5,625 1,125 122 ± 11 0,2 −29 ± 5 0,53 ± 0,05 L'auto-assemblage des gels a été contrôlée par analyse de turbidité (λ = 350 nm) et des études rhéologiques qui identifient le retard de phase, déterminent le point de gel (Figure 3) et permettent d’observer l'évolution dans le temps du gel. Figure 3. Figure 3. Résumé de la détermination du point de gélification par des mesures rhéologiques dans des systèmes différents: gels blancs "HA-fibrine" et "HA-fibrine renforcées" et ces gels 30% NCs. La ligne en pointillés représente le retard de phase requis 2 minutes. 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C. & Wang, Y. J. A novel fibrin gel derived from hyaluronic acid-grafted fibrinogen. Biomed. Mater. 6, 25009 (2011). 55. Lee, F. & Kurisawa, M. Formation and stability of interpenetrating polymer network Resumen in extenso 66 hydrogels consisting of fibrin and hyaluronic acid for tissue engineering. Acta Biomater. 9, 5143–5152 (2013). 56. Weigel, P. H., Fuller, G. M. & LeBoeuf, R. D. A model for the role of hyaluronic acid and fibrin in the early events during the inflammatory response and wound healing. J. Theor. Biol. 119, 219–234 (1986). 57. Instruction For Use Synvisc® (hylan G-F 20). (2014). 58. Eymard, F. et al. Predictors of response to viscosupplementation in patients with hip osteoarthritis: results of a prospective, observational, multicentre, open-label, pilot study. BMC Musculoskelet. Disord. 18, 1–8 (2017). 59. Ekundi-Valentim, E. et al. Differing effects of exogenous and endogenous hydrogen sulphide in carrageenan-induced knee joint synovitis in the rat. Br. J. Pharmacol. 159, 1463–1474 (2010). 60. Santos, J. M. et al. The role of human umbilical cord tissue-derived mesenchymal stromal cells (UCX®) in the treatment of inflammatory arthritis. J. Transl. Med. 11, 18 (2013). 61. Cecerale, S. in Olive oil - Constituents, Health Properties and Bioconversions (INTECH, 2011). 67 Introduction Intra-articular therapies for the treatment of rheumatoid arthritis Life can only be lived forwards, but must be understood backwards. Søren Kierkegaard 68 Introduction 69 1. Intra-articular therapies and local delivery of drugs Over the last decades it has become clear that the local delivery of drugs in the intraarticular (IA) cavity can greatly improve the treatment schemes and effectiveness of jointassociated pathologies.1–4 The current systemic therapies for these pathologies, administered by the oral or the intravenous routes, can be classified by the nature of the drug, as i) nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen;5 ii) corticosteroid antiinflammatory drugs (e.g. dexamethasone);6 iii) disease-modifying anti-rheumatic drugs (DMARDs), e.g. methotrexate;7,8 iv) monoclonal antibodies (e.g. infliximab);9 and v) natural agents, such as oils with anti-inflammatory and anti-oxidant properties.10,11 Unfortunately, none of these drugs exhibit joint specificity.2,12,13 Therefore, medication has considerable risks to humans due to the high doses required, with the subsequent systemic adverse effects (e.g. the cardiovascular, gastrointestinal complications, kidney dysfunction, skin and muscle atrophy, glaucoma, premature mortality).12,14,15 Furthermore, after prolonged treatment, joints often become resistant to the systemic treatment.1–3,16 Within this context, the intra-articular modality of administration offers the possibility to concentrate the drug locally,2,17 and reduce the systemic toxicity.2,12,18,19 Nevertheless, IA therapy still remains challenging due to a possible induction of septic arthritis (a risk controlled by careful handling of IA injections);2,20,21 rapid drug elimination from the joint cavity,2,22,23 and crystal-induced synovitis.18,22,24 The latter two could be overcome by the development of drug delivery systems (DDSs) that display sufficient IA residence time with controlled and prolonged drug release profile.2,16 Anatomically, the human knee is a diarthrodial joint filled with 2 - 3.5 mL of synovial fluid (SF), a viscous liquid that plays an essential role in the knee homeostasis3 (Figure 1), providing mechanical, nutritional and immune functions in the knee.25 A key component of SF is high molecular weight (MW) hyaluronic acid (HA), present at an approximate concentration of 3 mg/mL.26,27 In addition, HA reduces friction by lubricating the articulating joints and adsorbing shocks. Finally, the immune cells presented in the SF, mediated by HA, keep the immune control due to their high capacity of phagocytosis.28–32 Introduction 70 Figure 1. A schematic illustration of a knee joint. Reprinted with permission from The McGrow-Hill company, Inc, Copyright © 2009. Given that in inflammatory conditions swollen knees accumulate between two and five times higher volumes of SF, the aspiration to remove the excess of fluid remains the first line treatment.33–36 Besides, the administration of viscosupplementation agents, mainly HA or HA derivatives, has been widely used for improving the normal joints performance.37,38 These products restore joint lubrication, provide elasticity and viscosity, improve the mobility, reduce pain, and protect the cartilage.2,39–42 However, a limitation of this therapy is related to their high viscosity, causing intense IA pressure and pain in the knee joint.43 The marketed viscosupplements can be divided into non-crosslinked HA, that are normally highly viscous solutions, and crosslinked HA substances, usually injected as preformed hydrogels (Table 1).2,44–54 These HA-based medical devices have shown significant improvement of viscoelastic properties of SF, increasing joint mobility, relieving the pain and inducing chondroprotective effect on osteoarthritis (OA).55 Generally, crosslinked HA has been reported to have a more prolonged residence time in the articular cavity, compared to the unmodified molecule.2,48,56 As a result, these viscosupplements have shown long-lasting effects on OA (up to 26 weeks)52–54,57–60 and, in some cases, have also been shown to alleviate rheumatoid arthritis (RA) symptoms, although they were not specifically designed for this pathology.61 The longest therapeutic effect at OA was achieved by RenehaVis® (a course of 1-3 injections of a mixture of 1 and 2 MDa HAs, 12 months) and Crespine® Gel (a single injection of crosslinked HA, up to 6-8 months). Introduction 71 Table 1. Marketed intraarticular viscosupplements for arthropathies treatment. Product name Composition Volume, dose and MW of HA Number of injections per course Company Duration of therapeutic effect Ref. Unmodified-HA viscosupplements Hyalgan® 0.5 – 0.73 MDa HA 2 mL, 20 mg 3 Sanofi-Aventis, USA 3 months 45,62 Supartz® 0.6 – 1.2 MDa HA 2.5 mL, 25 mg 3 – 5 Seikagaku Corp., Japan 3 – 6 months 42,63 Euflexxa® 2.4 – 3.6 MDa HA 2 mL, 20 mg 3 Ferring Pharmaceuticals Inc., USA 6 months 45,64 Orthovisc® 1.0 – 2.9 MDa HA 2 mL, 30 mg 3 – 4 Anika Therapeutics, Inc., USA 6 months 65,66 RenehaVis® 1 MDa (2.2%) and 2 MDa (1%) HA 0.7 mL, 15.4 mg (1 MDa), 0.7 mL, 7 mg (2 MDa) 1 – 3 MTD Int’l s.a., Switzerland 12 months 46,67–70 Crosslinked HA hydrogels viscosupplements Synvisc- One® Hylan(a) G-F20: a mixture of 80% (v/v) of Hylan A fluid, lightly crosslinked by formaldehyde and 20% (v/v) of Hylan B gel, crosslinked by sulfonyl-bis-ethyl 6 mL, 48 mg 6 MDa HA 1 Sanofi Biosurgery Inc, USA Over 6 months 47,48, 71 Gel-One® HA crosslinked by dimers of cinnamic acid 3 mL, 30 mg High MW (not specified) 1 Seikagaku Corporation, Japan Over 3 months 44,72, 73 Durolane® HA with 1,4-butane-diol diglycidyl ether crosslinker 3 mL, 60 mg High MW (not specified) 1 Bioventus Coöperatief U.A., Netherlands Over 3 months (up to 9 months in some patients) 49,74, 75 Introduction 72 Monovisc® Crosslinked HA (not disclosed) 4 mL, 88 mg 1 – 2.9 MDa HA 1 Anika Therapeutics, Inc., USA Over 6 months 50,51 Hymovis® (HYADD®4) A non-chemically crosslinked hexadecylamide derivative of HA 3 mL, 24 mg 500 – 730 kDa HA 2 Fidia Farmaceutici SpA, Italy Over 6 months 52– 54,59, 60 Crespine® Gel A mixture of non- and crosslinked HA by Covalent Reticulated Matrix (CRM®) technology HA 2 mL, 30 mg (MW not reported) 1 Biopolymer GmbH, Germany 6-8 months 51,76 (a) Hyalans (hyaluronan derivates) are produced by chemical crosslinking hyaluronan chains, without affecting the carboxylic and N-acetyl groups.48,77 Introduction 73 When the degenerative illness has progressed, cartilage/osteochondral implants for hyaline cartilage or bone repair/substitution are the preferred treatments.78–81 A variety of injectable in situ hydrogel/gel implants based on natural,82–91 synthetic92–99 and combined materials100–107 were evaluated in vivo for articular tissues repair. Despite successful outcomes at the research level,80,81,108 currently only one in situ hydrogel based on chitosan and glycerol phosphate (BST-CarGel®; Biosyntech, Canada)109 is being tested on Phase IV clinical trials.109–111 2. Advanced drug delivery systems for intra-articular delivery Drug-polymer conjugates constitute a more advanced treatment for IA pathologies compared with viscosupplements, which are actually considered as medical devices. They are designed to achieve combined effects: the action of the drug and the viscosupplementation of the polymer. In this regard, HA is the most used polymer in these products, although other examples are found, such as N-(2-hydroxypropyl)methacrylamide (HPMA)- dexamethasone.63,112–115 It is worth mentioning the case of HA-methotrexate (DK226), designed specifically for the treatment of RA, which combines viscosupplementation agent with the methotrexate anti-inflammatory properties, showing a long lasting effect (24 days) in several RA rat models.116,117 The work in the area of IA drug delivery systems, aiming at prolonging the retention and controlling the drug release in the articular cavity, is currently moving in two principal directions: the development of nano-/microsystems and hydrogels (Figure 2). Figure 2. Different types of systems designed for IA administration. Reproduced with permission from112 Copyright © 2014 Janssen et al., Creative Commons Attribution License. Introduction 80 Photosensitizers Anionic photosensitizers: 1) Tetra-phenyl- porphyrin-tetra- sulfonate; 2) tetra-phenyl- chlorin-tetra- carboxylic acid; 3) Chlorin e6 Chitosan-based nanogels decorated with hyaluronate, 40–140 nm Mouse antigen-induced arthritis Selective delivery of photosensitizers to macrophages and photodynamic destruction of macrophages. Efficient retention and decreasing the clearance of photosensitizers from the inflamed articular joints. For up to 24 h they were retained inside the cells and even at day 8 showed after light exposure showed efficacy of such a photodynamic therapy 168 Model drug Fluorescent probe A tetrapeptide labeled with fluorescein isothiocyanate Eudragit RL100 NPs, 130 nm Healthy rat NPs injected to the synovial cavity form ionically crosslinked hydrogel with endogenous HA, allowing 7 days retention 124 1,1′-dioctadecyl- 3,3,3′,3′ tetramethylindotri carbocyanine iodide PLGA/Eudragit RL NPs, 170 nm Healthy mice Microscopic imaging showed improved retention time in the knee joint with the filamentous NPs/HA aggregates, with over 50% preservation of the fluorescent signal 28 days after injection 161 Introduction 81 2.2. Hydrogels Hydrogels are insoluble crosslinked 3D structures of polymer chains with water or biological fluid swollen inside them. They can be designed with an adequate elasticity, mechanical strength, permeability and diffusivity, being versatile DDSs for arthropathies. Such hydrogels display highly porous networks, allowing the entrapment of drugs, nano-/ microcarriers or cells inside their pores and, depending on the material, the gels may support cell adhesion, proliferation, and differentiation.112,169–172 Hydrogels used for IA drug delivery are injectable pre-formed173 or in situ forming hydrogels174 with physically mixed173 or chemically bound drugs.175 For instance, a hydrogel with pre-mixed dexamethasone (DXM) has been shown to significantly reduced the inherent DXM toxicity in a rat OA model, providing an anti-inflammatory and chondroprotective effect at 12 weeks after the injection.173 A different strategy has relied on the design of in situ forming hydrogels that are easy to inject and exhibit a prolonged residence time in the articular cavity. Among them, it is worth mentioning a radiotherapeutical pH responsive Holmium-166-chitosan hydrogel,176 in Phase II clinical trials for the treatment of knee synovitis typical for RA.177 Another example, is a thermosensitive triblock poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) copolymer hydrogel, which has been proposed for the controlled delivery of methotrexate in rat joints. The complete drug clearance was monitored during 24 h in plasma and occurred slower compared to free methotrexate.178 2.3. Nano-/microsystems incorporated into hydrogels Recent attention has been paid to the combination of drug loaded nano-/microsystems with hydrogels aiming at improving the residence time in the synovial cavity and the drug release profile.80,179,180 These nano-/microcarrier-loaded hydrogels have been found to control the delivery of the associated drugs for about a month174,181,182 or even longer.183 Nevertheless, overall, the reported work of in situ-forming hydrogels containing drug-loaded nano-/microparticles is at a very early stage and only a few of these formulations have been tested in vivo (Table 4).145,163,184 In most of the cases these combined systems were tested in healthy animals what did not allow to adequately estimate the residence time and prolonged release at inflamed joints. However, radio-labelled liposomes (235 nm) incorporated into Carbopol 940/carboxymethylcellulose gel evaluated at rabbit induced arthritis model demonstrated 67% retention in the articular cavity at 24 h after injection.145,163 At healthy rat joints the Introduction 82 maximal sustained-released reached 42 days post IA injection by using amphotericin B precipitates/crystals suspended in glyceryl monooleate-HA hydrogel.183 An intra-articular DDS should fulfil a number of properties, such as biocompatibility, and biodegradability, in addition to an effective and prolonged disease improvement.185 Another distinctive feature is bioadhesiveness,81 which allows to increase the residence time of DDS in the synovial cavity and impart the chondroprotective effect.186 Given the complexity of the joints anatomy, drug-loaded in situ hydrogels combined with nano- /microsystems might receive a significant benefit for IA drug delivery. Introduction 83 Table 4. Nano-/microsystems-loaded hydrogels for IA applications tested in vivo. Drug Drug delivery systems In vivo model Key observation Ref. Celecoxib (NSAID) Precipitates/crystals (size non specified) dispersed in a poly(ɛ-caprolactone-co-lactide)- b-poly(ethylene glycol)-b-poly(ɛ-caprolactone- co-lactide) (PCLA-PEG-PCLA) gel network (in situ) Healthy rat The degradation and the release profiles were tested by different routes of administration: 1) In the articular cavity the DDS degraded between week 3 and 4. Release was not evaluated. 2) At subcutaneous injection in rats gel degradation happened in ~12 weeks, ∼30 % celecoxib released during the first 3 days followed by a sustained release up to 4-8 weeks 174,181 Ibuprofen (NSAID) Polyethylene glycol (PEG)-microspheres (40– 100 µm) crosslinked by short PLGA chains to a hydrogel. Two populations: slow degradable and non-degradable Healthy sheep shoulder joint At 4 weeks non-degradable MS and not completely degraded MS were found, in synovial fluid and in the synovium. Degradable MS caused low inflammation compared to the non-degradable ones 182,187 Amphotericin B (Antifungal) Precipitates/crystals (size non defined) suspended in glyceryl monooleate-HA hydrogel (in situ) Healthy rat This formulation showed sustained-released during 42 days after IA injection 183 Dexamethasone (Corticosteroid) Monomethyl poly(ethylene glycol)-poly(εcaprolactone) (MPEG-PCL) micelles (25 nm) incorporated into thermosensitive in situ PEG-PCL-PEG hydrogel (in situ) Rat repeatedinjury adhesion model The gel biodegradation was evaluated upon subcutaneous injection and degraded in ~20 days 184 Diclofenac Sodium (Corticosteroid) Lipogelosomes: Radio-labelled liposomes DMPC–CHOL–DCP (235 nm) incorporated into Carbopol 940/carboxymethylcellulose gel Rabbit antigeninduced arthritis Scintigraphic imaging showed 67% retention of the formulations in the articular cavity at 24 h after injection 145,163 Introduction 84 3. Galectin-3 role in pathophysiology of rheumatoid arthritis Galectins are attracting more and more attention of scientists due to their presence in most of the tissues and their crucial role in biological processes and pathologies. Galectins are proteins able to bind to β-galactoside motifs (noteworthy, lactosamine [Gal(1→3/4)- GlcN] types, so-called LN1 and LN2) by one or two conserved carbohydrate-recognition domains (CRDs) and lack of enzymatic activity.188–190 Galectins are widely distributed in the organism and they are continuously produced by different cell lines.189,191 Their dual localization, intracellular (in nuclear and cytoplasmic compartments) and extracellular (at the cell membrane and extracellular matrix),192,193 is associated with a wide range of biological processes, notably in cell cycle,194195–197 cell migration,198–201 inflammation, and immune response202–207 (Figure 3). Overall, galectins synthesis can represent up to 1% of total protein in the producer cells.207 However, during pathological processes the level of galectins can raise considerably. For instance, galectins local concentration at the inflammation site increases up to 40 mg/kg (of wet tissue), leaving the immune cells immersed in a sea of different galectins.207 As a consequence, the deregulation of the galectins expression have been directly or indirectly associated with at least 137 pathologies headed by different cancer types, heart failure and RA, showing the potential of lectins as therapeutic targets.208 Currently, galectin family is represented by 15 members in mammals, which according to their architecture can be classified as: a) proto-, b) tandem-repeat-, and c) chimera-types (Figure 3).191,209,210 Proto-type galectins have one CRD and exist as monomers, but in vivo they are able to dimerize via non-covalent interactions to form homodimers. The tandem-repeat type is composed of two distinct, but homologous CRDs covalently linked to a dimer by a functional peptide. Finally, chimera-type galectins are made of one CRD that enables multimerization and N-terminal domain, including intermediate lateral collagen-like sequence, which implements alternative multimerization upon binding glycan ligands, mostly forming trimers and pentamers207,211–213 (Figure 3). Galectin-3 (Gal-3) is a key member of galectin family and a unique representative of the chimera-type.202 It is present in vertebrates, protochordates, invertebrates, mushrooms, and viruses.209 Human Gal-3 was found to be expressed in various tissues (gastrointestinal tract,214 lung,215,216 kidney,217 skeletal tissues and brain218) and cell lines (immune Introduction 85 cells,206,207,213 osteoblasts,219,220 chondrocytes,221,222 fibroblasts,223 epithelial,224–226 and endotelial cells).227 Figure 3. Intra- and extracellular located galectins and their functions. Proto-type: Galectin- 1, -2, -5, -7, -10, -11, -13, -14, and -15; chimera-type: Galectin-3; tandem-repeat type: Galectin-4, -6, -8, -9, and -12. Functional diversification of galectins: (1) Galectin transcripts are translated in the cytoplasm, and the proteins can be translocated into the nucleus (2) where they can associate with ribonucleoproteins. Via unconventional mechanism(s), galectins can be secreted to the extracellular space (3) where they can function as pattern recognition receptors for microbial glycans (4), bind to the host cell surface glycans (5), and cross-link them with ECM glycans (6) thereby, for example, promoting cell migration. Galectins can also cross-link cell surface glycans and induce clustering of microdomains and lattice formation at the cell surface (7) that can trigger signaling cascades, or cross-link neighbouring cells (8) and promote cell–cell interactions/adhesion. Reproduced with permission from,228 Copyright © 2012, Vasta et al., Creative Commons Attribution License. 3.1. Galectin-3 intracellular functions Multiple functions of Gal-3 greatly comply with the noticeable phenomenon of dual localization.213 Intracellularly, at the nucleus compartment, Gal-3 regulates pre-mRNA- splicing213 and stabilizes protein-DNA interactions facilitating transcription,207 contributing to gene expression and regulation.229 But shuttling across nuclear pores to the cytoplasm Gal-3 has a post-transcriptional role in the stabilization of mature mucin 4 mRNAs,230 Introduction 86 controls cell growth and has anti-apoptotic activity by the interaction with the apoptosis repressor Bcl-2.196,231 Besides, Gal-3 inhibits the apoptosis by preventing the mitochondrial cytochrome C release.207 Other Gal-3 functions were described in different stages of the cell cycle: cell differentiation,208 intracellular trafficking,225,232 and cell proliferation233 (Figure 4). Figure 4. The intracellular functions of galectin-3. Red arrows indicate positive effects, blue lines – negative effects. Akt – the serine/threonine kinase Akt, Ask-1 – apoptosis signalregulating kinase 1, CBP70 – carbohydrate binding protein 70, Chrp – cysteine- and histidine-rich protein, CREB – cAMP-response element-binding protein, ERK – extracellular signal-regulated kinase, Gal-3 – galectin-3, GTP – guanosine triphosphate, JNK – c-Jun NH2-terminal kinase, MEK – mitogen-activated protein/ERK kinase, P – phosphate, PI3K – phosphatidylinositol 3-kinase, Raf-1 – the serine/threonine kinase Raf-1, Tcf-4 – T cell factor 4, TF – transcription factor, TTF-1 – thyroid-specific transcription factor. Reprinted with permission from,234 Copyright © 2006, Elsevier. 3.2. Galectin-3 extracellular functions Extracellularly Gal-3 releases in vesicles, likely through exosomes or by direct translocation and stays on the cell membrane, in the vicinity of the cell or bounds to the Introduction 87 extracellular matrix.207 Being abundantly expressed in different tissues, Gal-3 modulates distinct cellular processes such as cell-cell and cell-matrix interactions through specific binding to extracellular sugar ligands.208 In such a manner, this protein takes part in multiple biological processes: cell adhesion,226,235 cell migration,200,236 transmembrane signalling,237,238 immune response and inflammation204,212,239,240 (Figure 5). Figure 5. The extracellular functions of galectin-3. Red arrows indicate positive effects. AGE – advanced glycation end products, C4.4A – the GPI-anchoredglycoprotein C4.4A, CEA – carcinoembryonic antigen, CRD – carbohydrate-recognition domain, EGFR – epidermal growth factor receptor, FcεR – Fcε receptor, Gal-3 – galectin-3, IgE – immunoglobulin E, L1 – neural adhesion molecule L1, Lamp 1/2 – lysosome associated membrane protein 1/2, LPS – lipopolysaccharide, Mac-2BP – Mac-2 binding protein, MAG – myelin associated glycoprotein, N-CAM – neural cell adhesion molecule, NCA-160 – non-specific cross-reacting antigen 160, ND – N-terminal domain, NG2 – the transmembrane chondroitin sulfate proteoglycan NG2, TβR – transforming growth factor β receptor, TCR – T cell receptor. Reprinted with permission from,234 Copyright © 2006, Elsevier. One of the most important extracellular functions of Gal-3 is supporting the control of immune and inflammatory response.207,212,241 The immune system cells are Gal-3 rich and Introduction 88 involved basically in each process: from controlling the B cells maturation in the bone marrow to activation or inhibition of B and T cells apoptosis.207 Although resting B and T cells do not express it.213 Gal-3 is expressed in neutrophils, basophils, eosinophils, macrophages (at all the stages from monocytes differentiation to macrophages and dendritic cells),220,242–244 mast cells,213 and has also considerable effects on both innate and adaptive immunity.207 Innate immune functions of Gal-3 are cell activation, adhesion, migration, and promotion of pathogens phagocytosis (including mycobacteria, fungi, and parasites)207 as well as autophagy.245 It is both pro-inflammatory and anti-inflammatory mediator, working as a chemoattractant for monocytes, macrophages, followed by macrophages polarization,246 and increasing the amount of neutrophils.207 In adaptive immune system Gal-3 exposes immunosuppressive effect, restricting T cell receptor recruitment into immune synapses, prevents T cell proliferation, differentiation, and directly promotes T cell apoptosis,207 resulting to prolonged inflammation.247 On the other hand, Gal-3 anti-inflammatory activity includes phagocytosis of dead cells and stimulation of wound healing.207 3.3. Galetin-3 deregulation in RA Noticeably, Gal-3 expression is up-regulated in synovial tissues in RA,213 its concentration in SF increases from about 50 ng/mL up to 130-300 ng/mL212 and being a proinflammatory regulator, it amplifies the inflammation.247 As reported, Gal-3 stimulates synovial fibroblasts as well as infiltrated immune cells to release the pro-inflammatory cytokines (TNF-α, IL-6, IL-17) and chemokines (CCL2, CCL3, CCL5, CXCL8),212 the expression of matrix metalloproteinases MMP-3,248 MMP-9249 and A Disintegrin And Metalloproteinase with Thrombospondin Motifs 5 (ADAMTS5).212 These enzymes are involved in the degradation of the ECM248 and type I-III collagen and tissue remodeling.30,250 They also inhibit the osteocalcin production220 leading to bone demineralization,212 and, eventually, causing joint degradation.19,251 In addition, IA injection of recombinant galectin-3 in mice induced cartilage and subchondral bone lesions.220,247 Taking into account the involvement of Gal-3 into the pathophysiology of RA this protein appears as a novel potential immunotherapeutic target. 3.4. Galectin-3 structure Human Gal-3 is a 30-35 kDa protein coded by the LGALS3 gene,234 composed by 250 amino acids (aa) organized into a secondary structure, mostly forming anti-parallel β-sheets and only in few regions assemble into α-helixes.213,252,253 The protein is divided into 3 regions: N-terminal peptide (1-18 aa) with two phosphorylation sites involved in Gal-3 Introduction 89 secretion;254–256 a lateral collagen-like sequence (19-111 aa), which includes consensus sequence of nine PGAYP repeats,213,254 which can give rise to multimerization; and the carbohydrate recognition domain – CRD (112-250 aa) that includes highly conserved amino acids, termed the carbohydrate-binding cassette, critical for glycan recognition and complexation.207,252,257,258 Gal-3 CRD domain has a structure typical for galectin family members with a canonical amino acid sequence,259 suggesting that all the Gal-CRDs are able to bind βgalactoside motifs with similar Kd values for these ligands.190,256,260 It is composed of two antiparallel β-sheets of six (S1-S6) and five (F1-F5) strands (Figure 6A) and includes highly conserved residues (e.g. His158, Asn160, Arg162, Glu165, Asn174, Trp181, Glu184) responsible for complexation with carbohydrate ligands and salt-bridge network (Arg162- Glu165-Glu184-Arg186), determining LN2 binding preference over LN1 core (Figure 6B).190,252,259,261 Structural alignments and superimposition of CRD domains fragments (S4-S6) of human Gal-3 CRD (hGal-3-CRD), Gal-1 (hGal-1) and Gal-7 (hGal-7) CRDs are shown in Figure 6C and D.190 These similarities explain the low specificity of some synthetic Gal-3 inhibitors that have dissociation constants of the same order for these three galectins.262 It is important to emphasize that two arginine residues (Arg144 and Arg186), adjacent to the binding site of Gal-3 CRD, do not participate in natural ligands complexation,259,263 but offer an opportunity to establish new binding contacts for selective targeting of Gal-3190,252 (Figure 7A). 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Most diarthrodial joints, due to their anatomical features, are well suited and studied for intra-articular (IA) injections. The main benefit of IA administration over oral or intravenous drug delivery is related to the increased drug concentration at the target place and, a consequent reduction of its systemic toxicity.1–4 The main drugs used for the treatment of arthropathies and improvement of knee homeostasis that are currently available on the market are COX-2, TNF-α, IL-1 and IL-6 antagonists administrated systematically. Only very few products are available for local delivery. 2. The advances in the biopharmaceutical field along the past decades have led to the development of drug delivery systems (DDSs) that release the drug in a controlled manner in the articular cavity, thereby improving the efficacy/toxicity balance of the drug.3,5 Nevertheless, IA drug delivery remains challenging mainly due to the premature elimination of the DDS from the synovial cavity along with fast synovial fluid (SF) turnover.3 Among the DDSs developed for the local treatment of arthropathies are nano-/microcarriers and hydrogels,2,3,5,6 being the most promising and advanced formulations a combination of both systems.7–12 Such DDSs allow increased retention and prolonged release of the drugs in the joints compared with the same drugs administered in solution.3,13 3. Articular cartilage has a limited ability for self-regeneration.14–16 Besides, SF greatly loses its intrinsic functions at pathological joints conditions.17 Having this in mind, multifunctional drug delivery carriers should contribute to diarthrosis healing. They should not only provide a controlled and prolonged drug release, but also be composed of deformation-resistant materials that may have a prolonged retention in the articulation. Simultaneously, such a nanotechnology-based platform should ideally work as a viscosupplementation agent for improving the knee homeostasis, and as an in situ cell scaffold for cartilage tissue regeneration. 4. The discovery of novel immune targets is driving research towards the development of highly specific antagonists for more effective therapies.18–21 This is the case of the extracellular protein galectin-3 (Gal-3), which was recently identified as a primer trigger of inflammation.22,23 Moreover, this protein favors the secretion of macrophage pro-inflammatory cytokines, chemokines,22 and matrix metalloproteinases,24 which are involved in cartilage remodeling and bone demineralization.1,25 Background, Hypothesis and Objectives 117 Hypothesis 1. The use of drug-loaded nanocapsules (NCs) dispersed in in situ hydrogels, composed by hyaluronic acid (HA) and fibrin, can substantially increase intra-articular drug retention time. NCs can act as multireservoirs for lipophilic drugs, and will allow controlled release, while in situ hydrogel might protect NCs from the fast decomposition, endocytosis and efflux from the synovial cavity contribution to the modulation of drug release as well. Such an injectable composition of NCs combined with in situ hydrogel could also have a role in maintaining the knee hemostasis. This is supported by the fact that both, olive oil core and 700 kDa HA coating of the NCs that have anti-inflammatory properties.26–29 Besides, HA-fortified fibrin 3D hydrogel should serve as a viscosupplement with elastic properties for IA persistence and a support for cartilage regeneration.30–32 2. Since blocking of traditional COX-2 and pro-inflammatory cytokines TNF-α, IL-1, IL-6 has not led to the adequate alleviation of the inflammatory joint diseases,3,5,33–35 we hypothesize that targeting to the novel immune target galectin-3,22 could help to block more efficiently the inflammatory cascade and prevent further joints degradation. Objectives Based on the background information and outlined hypothesis, the objective of this thesis has been double, on the one hand, to develop a new injectable hydrogel containingnano-reservoirs for the prolong release of lipophilic drugs to the intra-articular cavity; on the other hand, to synthesize a new galactin-3 antagonist and its incorporation in the before-mentioned hydrogel for in vivo evaluation. For this purpose overall aims have been aligned with the following experimental activities: 1) Development and in vitro assessment of a new DDS intended for intra-articular administration Background, Hypothesis and Objectives 118 1. To develop new dexamethasone (as a model drug)-loaded NCs and to characterize them with regard to their physicochemical properties, stability in simulated synovial fluid and stability upon storage. 2. To design and develop a novel injectable intra-articular hydrogel, and study the influence of its composition on the gelation time as well as on its rheological and mechanical properties. 3. To study the hydrogel formation in the presence of the NCs and the modification of its rheological properties and its microscopic architecture. 4. To optimize and characterize two DDS made of NCs combined with in situ hydrogels designed for acute- and chronic-joint inflammatory rat models differed by additional fortification of the interpenetrating network. 5. To test the in vitro release profile of dexamethasone from the NCs loaded in the gel. The results corresponding to these objectives are presented in Chapter 1: “An injectable, in situ hyaluronic acid-fibrin hydrogel containing nanocapsules for prolonged intra-articular drug delivery”. 2) Anti-inflammatory drug synthesis, incorporation into the developed DDS and in vivo activity assessment 6. To design and synthesise a small-molecule-antagonist of extracellular galectin-3 (Gal- 3), using its natural ligand type II lactosamine [Gal(1→4)-GlcN] core, modified with aromatic substituents to greatly amplify its affinity and specificity. 7. To characterise the obtained Gal-3 inhibitor, test its affinity and selectivity among Gal-1/Gal-3/Gal-7, compare with the first commercial low MW Gal-3 inhibitor (TD139) and select the most potent synthetic inhibitor for in vivo evaluation. 8. To incorporate Gal-3 inhibitor in the developed DDS, characterise its physicochemical properties, and test the in vivo anti-inflammatory activity in a carrageenan-induced acute knee joint synovitis rat model. The results related to this work are presented in Chapter 2: “New galectin-3 inhibitor as a lead compound for anti-inflammatory drug candidates”. Background, Hypothesis and Objectives 119 References: 1. Quan, L.-D., Thiele, G. M., Tian, J. & Wang, D. The Development of Novel Therapies for Rheumatoid Arthritis. Expert Opin. Ther. Pat. 18, 723–738 (2008). 2. Kapoor, B., Singh, S. K., Gulati, M., Gupta, R. & Vaidya, Y. Application of liposomes in treatment of rheumatoid arthritis: Quo vadis. Sci. World J. 2014, (2014). 3. Evans, C. H., Kraus, V. B. & Setton., L. A. Progress in intra-articular therapy. Nat. Rev. Rheumatol. 10, 11–22 (2015). 4. Butoescu, N., Jordan, O. & Doelker, E. Intra-articular drug delivery systems for the treatment of rheumatic diseases: A review of the factors influencing their performance. Eur. J. Pharm. Biopharm. 73, 205–218 (2009). 5. Kang, M. L. & Im, G.-I. Drug delivery systems for intra-articular treatment of osteoarthritis. Expert Opin. Drug Deliv. 11, 269–82 (2014). 6. Evans, C. H., Kraus, V. B. & Setton, L. a. Progress in intra-articular therapy. Nat. Rev. Rheumatol. 10, 11–22 (2014). 7. Vorvolakos, K., Isayeva, I. S., do Luu, H. M., Patwardhan, D. V. & Pollack, S. K. Ionically cross-linked hyaluronic acid: Wetting, lubrication, and viscoelasticity of a modified adhesion barrier gel. Med. Devices Evid. Res. 4, 1–10 (2011). 8. Turker, S. et al. Gamma-irradiated liposome/noisome and lipogelosome/niogelosome formulations for the treatment of rheumatoid arthritis. Interv. Med. Appl. Sci. 5, 60–69 (2013). 9. Morgen, M. et al. Nanoparticles for improved local retention after intra-articular injection into the knee joint. Pharm. Res. 30, 257–268 (2013). 10. Kim, S. R. et al. Cationic PLGA/eudragit RL nanoparticles for increasing retention time in synovial cavity after intra-articular injection in knee joint. Int. J. Nanomedicine 10, 5263–5271 (2015). 11. Wu, Q. et al. Thermosensitive hydrogel containing dexamethasone micelles for preventing postsurgical adhesion in a repeated-injury model. Sci. Rep. 5, 13553 (2015). 12. Webber, M. J., Matson, J. B., Tamboli, V. K. & Stupp, S. I. Controlled release of dexamethasone from peptide nanofiber gels to modulate inflammatory response. Biomaterials 33, 6823–6832 (2012). 13. Burt, H. M., Tsallas, A., Gilchrist, S. & Liang, L. S. Intra-articular drug delivery systems: Overcoming the shortcomings of joint disease therapy. Expert Opin. Drug Deliv. 6, 17–26 (2009). 14. Spiller, K. L., Maher, S. a & Lowman, A. M. Hydrogels for the repair of articular cartilage defects. Tissue Eng. Part B. Rev. 17, 281–99 (2011). Background, Hypothesis and Objectives 120 15. Ahearne, M., Buckley, C. T. & Kelly, D. J. A growth factor delivery system for chondrogenic induction of infrapatellar fat pad-derived stem cells in fibrin hydrogels. Biotechnol. Appl. Biochem. 58, 345–352 (2011). 16. Jiang, Y., Chen, J., Deng, C., Suuronen, E. J. & Zhong, Z. Click hydrogels, microgels and nanogels: Emerging platforms for drug delivery and tissue engineering. Biomaterials 35, 4969–4985 (2014). 17. Lipowitz, A. J. in Textbook of Small Animal Orthopaedics (1985). 18. Klyosov, A. A. in Galectin Therapeutics (2012). 19. Roy, R., Murphy, P. V. & Gabius, H. J. Multivalent carbohydrate-lectin interactions: How synthetic chemistry enables insights into nanometric recognition. Molecules 21, (2016). 20. Oberg, C. T., Leffler, H. & Nilsson, U. J. Inhibition of galectins with small molecules. Chimia (Aarau). 65, 18–23 (2011). 21. St-Pierre, C. et al. Galectin-1-specific inhibitors as a new class of compounds to treat HIV-1 infection. Antimicrob. Agents Chemother. 56, 154–162 (2012). 22. Hu, Y., Yéléhé-Okouma, M., Ea, H. K., Jouzeau, J. Y. & Reboul, P. Galectin-3: A key player in arthritis. Jt. Bone Spine 84, 15–20 (2017). 23. Janelle-Montcalm, A. et al. Extracellular localization of galectin-3 has a deleterious role in joint tissues. Arthritis Res. Ther. 9, R20 (2007). 24. Palmer, M., Stanford, E. & Murray, M. M. The effect of synovial fluid enzymes on the biodegradability of collagen and fibrin clots. Materials (Basel). 4, 1469–1482 (2011). 25. Page-McCaw, A., Ewald, A. J. & Werb, Z. Matrix metalloproteinases and the regulation of tissue. Nat Rev Mol Cell Biol. 8, 221–233 (2007). 26. Fezai, M., Senovilla, L., Jemaà, M., Ben-Attia, M. & Ben-Attia, M. Analgesic, Anti- Inflammatory and Anticancer Activities of Extra Virgin Olive Oil. J. Lipids 2013, 1–7 (2013). 27. Ghosh, P. & Guidolin, D. Potential mechanism of action of intra-articular hyaluronan therapy in osteoarthritis: Are the effects molecular weight dependent? Semin. Arthritis Rheum. 32, 10–37 (2002). 28. Aly, M. N. S. Intra-articular drug delivery: a fast growing approach. Recent Pat. Drug Deliv. Formul. 2, 231–7 (2008). 29. Mehta, D. P., Shodhan, K., Modi, R. I. & Ghosh, P. K. Sodium hyaluronate of defined molecular size for treating osteoarthritis. Curr. Sci. 92, 209–213 (2007). Background, Hypothesis and Objectives 121 30. LeBoeuf, R. D., Raja, R. H., Fuller, G. M. & Weigel, P. H. Human fibrinogen specifically binds hyaluronic acid. J. Biol. Chem. 261, 12586–12592 (1986). 31. Zhang, Y., Heher, P., Hilborn, J., Redl, H. & Ossipov, D. a. Hyaluronic acid-fibrin interpenetrating double network hydrogel prepared in situ by orthogonal disulfide cross-linking reaction for biomedical applications. Acta Biomater. 38, 23–32 (2016). 32. Snyder, T. N., Madhavan, K., Intrator, M., Dregalla, R. C. & Park, D. A fibrin/hyaluronic acid hydrogel for the delivery of mesenchymal stem cells and potential for articular cartilage repair. J. Biol. Eng. 8, 10 (2014). 33. Li, S., Yu, Y., Koehn, C. D., Zhang, Z. & Su, K. Galectins in the pathogenesis of rheumatoid arthritis. J Clin Cell Immunol 4, 164 (2013). 34. De Oliveira, F. L. et al. Galectin-3 in autoimmunity and autoimmune diseases. Exp. Biol. Med. (2015). doi:10.1177/1535370215593826 35. Chen, H. Y., Liu, F.-T. & Yang, R.-Y. Roles of galectin-3 in immune responses. Arch. Immunol. Ther. Exp. (Warsz). 53, 497–504 (2005). Chapter 1 128 Table 1. Marketed formulations or in clinical trials based on nano-/microsystems for joints pathologies treatment by IA route. Product name Drug Drug delivery system Indication Phase of development Ref. Lipotalon® Dexamethasone 21-palmitate (Corticosteroid anti-inflammatory) Liposomes, ~200 nm Inflammatory OA Market, 2004 11–13 Zilretta™ (FX006) Triamcinolone acetonide (Corticosteroid anti-inflammatory) poly(D,L- lactic/glycolic acid) (PLGA) microspheres 9.7–10.3 μm OA Market, 2017 19–23 Clodrosome® Clodronate disodium salt (Antiosteoporotic cytotoxic) Liposomes, ~200 nm RA, osteoporosis Preliminary clinical trial on patients. Marketed for non-human RA model studies 16,18,24 The commercialization of these products has encompassed a quite active research activity in the field of liposomes and nano-/microparticles for intra-articular application made of a variety of biomaterials.25–30 The most important recent advances with these formulations in vivo are summarized in Chapter Introduction, Table 3. Overall, these liposomal or polymeric formulations containing anti-inflammatory or disease-modifying anti-rheumatic drugs have shown expected PK/PD changes and increased drug residence time and, consequently, an enhancement of their efficacy and a reduction of their systemic toxicity. The drugs that have been more extensively studied are corticosteroids (i.e dexamethasone (DXM), betamethasone, prednisolone), NSAIDs (i.e. diclofenac, indomethacin), DMARDs cytotoxic drugs (i.e. methotrexate, paclitaxel, clodronate), and others, such as kartogenin and actarit.9,31–48 With regard to the composition of the nanosystems, in most cases they have been made of lipids (liposomes), polyesters (e.g. PLGA, PLA nanoparticles) and polysaccahrides (e.g. chitosan nanoparticles). Despite of the positive initial in vivo data obtained with these formulations, their therapeutic responses have not been as long-lasting as expected. In fact, the longest response has been reported for the thermo-responsive Pluronic F127/chitosan nanospheres for co-delivery of kartogenin and diclofenac (to achieve a dual chondroprotective and anti-inflammatory effect), and showed a- Chapter 1 129 2 week retention time in joints.48,49 A strategy to prolong the dwelling time of the nanoparticulate systems has relied on making them interactive with the surrounding environment. For example, cationic (Eudragit RL100 or PLGA/Eudragit RL) NPs, with the capacity to interact with endogenous HA, formed an in situ gel once injected to the rat joint cavity.50,51 As a consequence, the NPs remained in the articular joints up to 28 days after the injection.51 1.2. Hydrogels Due to their rheological properties hydrogels have received significant attention as IA drug delivery systems. As a result of the intense research in that field some formulations has already reached the market. This is the case of viscosupplementation agents, consisting of crosslinked high MW HA52–63 or HA derivatives, an example of which is Hymovis® that contains a hexadecylamide derivative of HA.64–66 The prolonged residence time of these hydrogels in the articular cavity5,60,67 has resulted in a long-lasting effect on OA patients (up to 26 weeks),60,66 and RA animal models (up to 8 weeks).67 The longest therapeutic effect for a HA-based hydrogel has been reported for Crespine® (6-8 months), which is indicated for the treatment of OA. However, a limitation of these hydrogel therapies is related to their high viscosity, which often leads to an increase of the IA pressure and causes pain in the knee joint.70 In addition to the viscosupplementation properties, these hydrogels have also been used for the controlled delivery of drugs. For example, a crosslinked HA gel with pre-mixed DXM has been shown to significantly extend the drug release in an OA rat model. As a consequence, the gel significantly reduced the inherent DXM toxicity providing an antiinflammatory and chondroprotective effect at 12 weeks after the injection.71 A different strategy has relied on the design of in situ forming hydrogels that are easy to inject and exhibit a prolonged residence time in the articular cavity. Among them, it is worth mentioning a radiopharmaceutical pH responsive Holmium-166-chitosan hydrogel72 tested in Phase II clinical trials for the treatment of knee synovitis in RA.73 Another example, so far only tested in rats, is a thermosensitive triblock poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) copolymer hydrogel, which has been proposed for the controlled delivery of methotrexate. However, the drug release from this hydrogel could only be controlled in vivo for up to 24 h.74 Chapter 1 130 1.3. Nano-/microsystems-loaded hydrogels Recent attention has also been paid to the combination of drug loaded nano- /microsystems with hydrogels aiming at improving the residence time in the synovial cavity and the drug release profile.75–77 These nano-/microcarrier-containing hydrogels were reported to control the delivery of the associated drugs for about a month78–80 or even longer.81 Nevertheless, overall, the work of in situ-forming hydrogels containing drug-loaded nano-/microparticles is at a very early stage and only a few of these formulations have been tested in vivo (Table 2).78,79,81,82 In most of the cases these combined systems were tested in healthy animals and their residence time and prolonged release at inflamed joints remains unknown. An exception is represented by the radio-labelled liposomes (235 nm) incorporated into a Carbopol 940/carboxymethylcellulose gel, which was evaluated in a rabbit induced arthritis model, and was shown to be 67% retained in the articular cavity at 24 h after injection.34,83 In healthy rat joints the maximal sustained-released reached 42 days post IA injection by using amphotericin B precipitates/crystals suspended in glyceryl monooleate-HA hydrogel.81 Chapter 1 131 Table 2. Nano-/microsystems loaded into hydrogels for IA applications tested in vivo. Drug Drug delivery system In vivo model Key observation Ref. Celecoxib (NSAID) Precipitates/crystals (size non defined) dispersed in a poly(ɛ-caprolactone-co-lactide)- b-poly(ethylene glycol)-b-poly(ɛ-caprolactone- co-lactide) (PCLA-PEG-PCLA) gel network (in situ) Healthy rat The degradation and the release profiles were tested by different routes of administration: 3) In the articular cavity the DDS degraded between week 3 and 4. Release was not evaluated. 4) At subcutaneous injection in rats gel degradation happened in ~12 weeks, ∼30 % celecoxib released during the first 3 days followed by a sustained release up to 4-8 weeks 78,79 Ibuprofen (NSAID) Polyethylene glycol (PEG)-microspheres (40– 100 µm) crosslinked by short PLGA chains to a hydrogel. Two populations: slow degradable and non-degradable Healthy sheep shoulder joint At 4 weeks non-degradable MS and not completely degraded MS were found, in synovial fluid and in the synovium. Degradable MS caused low inflammation compared to the non-degradable ones 80,84 Amphotericin B (Antifungal) Precipitates/crystals (size non defined) suspended in glyceryl monooleate-HA hydrogel (in situ) Healthy rat This formulation showed sustained-released during 42 days after IA injection 81 Dexamethasone (Corticosteroid) Monomethyl poly(ethylene glycol)-poly(ε - caprolactone) (MPEG-PCL) micelles (25 nm) incorporated into thermosensitive in situ PEG-PCL-PEG hydrogel (in situ) Rat repeatedinjury adhesion model The gel biodegradation was evaluated upon subcutaneous injection and degraded in ~20 days 82 Diclofenac Sodium (Corticosteroid) Lipogelosomes: Radio-labelled liposomes DMPC–CHOL–DCP (235 nm) incorporated into Carbopol 940/carboxymethylcellulose gel Rabbit antigeninduced arthritis Scintigraphic imaging showed 67% retention of the formulations in the articular cavity at 24 h after injection 34,83 Chapter 1 132 1.4. Rational design and selection of the DDS components An intra-articular DDS should fulfil a number of properties, such as biocompatibility, and biodegradability, in addition to an effective and prolonged disease improvement.85 Another distinctive feature is bioadhesiveness,86 which allows to increase the residence time of DDS in the synovial cavity and impart the chondroprotective effect.87 Taking these criteria into account, we have developed a novel in situ HA-fibrin hydrogel containing HA nanocapsules (NCs). NCs serve as multireservoirs for lipophilic drugs, i.e. DXM, used as a model anti-inflammatory drug in this study. The hydrogel prevents the fast and uncontrolled biodistribution of the NCs, thereby extending the effect of the encapsulated drug (Figure 1). Figure 1. Schematic illustration showing the intra-articular injection of an in situ forming hydrogel. Adapted and modified from8 with permission, Copyright© 2009, Taylor & Francis. HA was selected as a biomaterial to form the hydrogel due to it essential role in diarthrodial joints homeostasis.55,88–91 High MW HA is a key component of articular cartilage92,93 and SF,94 working as a physical barrier, providing the joint with adequate lubrication in addition to a chondroprotective effect.3,89,90 Furthermore, HA provides viscoelasticity of SF and viscoinduction (promoting the endogenous HA synthesis); it also has anti-inflammatory and antinociceptive properties.88,90,95 The other key component of the hydrogel, fibrin, is a biomaterial derived from human blood plasma, marketed and widely used as a glue sealant Tisseel® (Baxter Healthcare Chapter 1 133 corporation, USA) for postsurgical tissue healing.96–98 It has been used to prepare different hydrogels, in combination with other polymers,86,99–103 noteworthy, with HA that has received major breakthrough for testing at different biomedical application for tissue engineering.99,101,104–107 Hence, we exploited the affinity of high MW HA to fibrin108–110 and their capacity to form interpenetrating 3D networks (IPN),99,101,104 through covalent and ionic interactions.108,110 The synergistic effect of HA and fibrin is expected to provide the resulting hydrogel with improved mechanical properties and resistance against intra-articular deformations.101,111 Aiming at extending the residence time in the articular cavity, fibrin was fortified by factor XIII (FXIII),112,113 its natural covalent crosslinker,96,114 and α2-antiplasmin (α2AP) in order to reduce the degradation by enzymes of pathological synovial fluid.115–118 Additionally, HA, as the main component of hyaline cartilage, together with fibrin are essential for chondrocytes-ECM interactions, migration, proliferation, and differentiation what can be an advantage for regeneration of the damaged tissue if they are included in a DDS.105,106 Besides, the biodegradability,97,99,119 bioadhesiveness103,120,121 and cytocompatibility97,99,105 of the components could contribute to an adequate joint healing. In order to validate the potential of our in situ-forming hydrogel we selected DXM, a potent synthetic anti-inflammatory corticosteroid,122 widely used for the treatment of arthropathies.71 Within the context of this work it is important to mention that RegenoGel™, composed of fibrinogen chemically linked to a high molecular weight HA, has been recently approved as a medical device of next generation by the Israeli Ministry of Health for degenerative joint diseases, primary for OA.123 Moreover, its modified form (RegenoGel- OSP™ that uses autologous patient’s plasma, instead of fibrinogen) now in Phase I/II clinical trials, has also shown an unprecedented response in long pain relief, superior stability and mechanical integrity, along with a strong chondrogenic effect. No adverse effects, neither local nor systemic were observed for both products.123–125 Chapter 1 134 2. MATERIALS AND METHODS 2.1. Materials Fibrinogen and thrombin, both from human blood plasma (premium quality grade) were purchased from Sigma-Aldrich, Germany. Sodium hyaluronate (research grade) of different average molecular weights 51 kDa, 752 kDa and 1.38 MDa, with trade names 40 kDa, 700 kDa, 1.5 MDa, correspondingly, were acquired from Lifecore Biomedical, USA. Factor XIII (FXIII) from the human blood plasma (>95% pure) was obtained from Haematologic Technologies, Inc, USA. Alpha-2 antiplasmin (α2AP) from human blood plasma (>95% pure) was bought from Lee Biosolutions, USA. Dexamethasone (97.2%) was purchased from Acofarma, Spain. The stabilizing surfactants, phosphatidylcholine enriched soy lecithin (EPIKURON® 145 V) was a kind gift from Cargill, Spain, and oleylamine and Tween 80 were bought from Sigma-Aldrich, Germany, all of high-purity grade. Labrafac™ Lipophile WL1349 was acquired from Gattefossé, France, Poloxamer-407 (Kolliphor® P 407) was obtained from Basf, Germany, Lipoid PEG-2000 was purchased from Lipoid, Germany, all of pharmaceutical grade. Virgin olive oil (extra pure) was purchased from Acros Organics, Belgium. Nile red (technical grade), HEPES, BSA (≥98%), sodium chloride, calcium chloride dihydrate, potassium chloride, sodium phosphate dibasic and potassium phosphate monobasic of ≥99% purity, were acquired from Sigma-Aldrich, Germany. Human synovial fluid (rheumatoid arthritis) was purchased from SeraLab, UK. Ultrapure (Milli-Q) or endotoxin-free water was used throughout the experiments and organic solvents were of HPLC grade. 2.2. Preparation and characterization of HA NCs 2.2.1. Preparation of HA NCs HA NCs were produced by the solvent displacement technique, adapting the procedure earlier described by our group.126 Briefly, lecithin (Lec, 5.625 mg), oleylamine (OAm, 1.125 mg) and olive oil (OO, 30 mg) were dissolved in 5 mL of ethanol. In the case of drug loaded NCs, DXM (8.1 mg or 2.5 mg) was incorporated to the ethanol, and for labeled NCs, Nile Red (0.125 mg) was also included to this phase. Then, the organic phase was Chapter 1 135 injected under pressure through a needle (23G) to 10 mL of aqueous phase containing 2.5 mg of HA 700 kDa, and kept under magnetic stirring at 900 rpm during 10 minutes at room temperature. The elimination of organic solvent was performed by evaporation under vacuum (Rotavapor Heidolph, Germany) and the final volume was adjusted to 5 mL with ultrapure water. Thereafter, the NCs were isolated by ultracentrifugation (Avanti® J-E, Ultracentrifuge, Beckman Coulter, USA) at 30,000×g for 1 h at 15 °C. 2.2.2. Physicochemical and morphological properties of HA NCs The mean size and polydispersity index (PDI) of the HA NCs were measured after dilution (100×) with ultrapure water by dynamic light scattering (DLS) at 25 °C with an angle detection of 173°. The zeta potential (ζ) was measured by laser Doppler anemometry (LDA) after diluting the samples (100×) with ultrapure water. Both DLS and LDA analysis were performed in a Zetasizer®, NanoZS, Malvern Instruments, Malvern, UK. Particle size distribution and morphology were evaluated by transmission electron microscopy (TEM) using a JEOL JEM-2010 microscope, 200 kV, resolution: 0.23 nm (Tokyo, Japan). Samples for TEM analysis were diluted (20×), deposited on a copper grid, stained with a phosphotungstic acid solution (2% w/v) and allowed to dry overnight prior to analysis. The pH of nanoformulations was measured at Sartorius Basic Meter PB-11, Sartorius AG, Germany. 2.2.3. Determination of dexamethasone concentration in the formulation The dexamethasone (DXM) concentration was determined after the isolation of the NCs by ultracentrifugation at 30,000×g for 1 h at 15 °C (Avanti® J-E, Ultracentrifuge, Beckman Coulter, USA). The amount of the free drug in the undernatant was quantified by high-performance liquid chromatography (HPLC), described below. For the extraction of DXM 0.1 mL of sample were mixed with 0.9 mL ethanol/acetonitrile (ACN) and kept under magnetic stirring at a high speed overnight to obtain a clear solution. The samples were analyzed by HPLC, using a method adapted from the literature.127–129 The HPLC system consisted of a VWR-Hitachi LaChrom Elite® system equipped with a UV detector L-2400 set at 239 nm and a reverse phase SunFire column 186002560, 100Å, C18 (4.6 ID × 250 mm, pore size 5 μm), Waters, USA. The mobile phase was a mixture of methanol and ultrapure water (65 % : 35 % v/v), isocratic, and the flow rate was 1 mL/min. The column was set at Chapter 1 136 30 °C and the injection volume was 20 μL. The standard calibration curves of DXM were linear in the range of 1-1000 μg/mL (r2 = 0.999). Samples were transferred into auto-sampler vials, capped and placed in the HPLC auto-sampler. The concentration of DXM in the aliquots was used to calculate the free amount of DXM and, indirectly, its concentration in the final system. 2.3. Preparation of blank and NCs-loaded HA-fibrin in situ forming hydrogels 2.3.1. Preparation of HA-fibrin in situ forming hydrogels The HA-fibrin in situ hydrogels matrixes were obtained by thrombin (Thr)-activated enzymatic polymerization. For this purpose the following stock solutions were prepared. Lyophilized powder of fibrinogen (Fg) from human blood plasma was dissolved in 0.15 M solution of NaCl at 37 °C to a final concentration of 25 mg/mL, aliquoted and stored at -20 °C. Before each experiment, an aliquot was thawed at 37 °C for 30 min and was maintained at 37 °C no more than 2 hours.130 Lyophilized powder of thrombin from human blood plasma was dissolved in cooled to 4 °C PBS buffer (0.01 M Na2HPO4, 0.0018 M KH2PO4, 0.137 M NaCl, and 0.0027 M KCl, pH 7.2) to a final concentration of 100 NIH-U/mL. Thrombin activity is usually expressed in NIH-U/mL, established by National Institute of Health standard for the calibration of commercial thrombin reagents,131 taken 1 NIH-U = 0.324 ± 0.073 μg (9.0 nM). Additionally, 1% (w/v) solution of BSA was added as stabilizing agent. Thrombin was aliquoted and stored at -20 °C. Before each experiment the aliquot was thawed on an ice plate. Sodium hyaluronate (HA) of different MW 40 kDa, 700 kDa and 1.5 MDa was diluted in warmed to 37 °C “polymerization buffer 10-4 M Ca2+” (0.02 М HEPES, 0.1 М NaCl, 10-4 М CaCl2, pH 7.4) or “polymerization buffer 10-3 M Ca2+” (0.02 М HEPES, 0.1 М NaCl, 10-3 М CaCl2, pH 7.4) to 0.1%, 0.25%, 0.5% and 0.65% (w/v) solutions. The solutions were incubated at 37 °C under shaking during 2 hours to dissolve HA and were stored at 4 °C. Additionally, HA mixed solutions 0.5% and 0.65% (w/v) made from HA 700 kDa : HA 1.5 MDa = 1:1 were prepared. Factor XIII from human blood plasma was aliquoted and kept in 50% glycerol, 0.5 mM EDTA at concentration 5.1 mg/mL at -80 °C. Chapter 1 137 Lyophilized powder of α2-antiplasmin (α2AP) from human blood plasma was dissolved in PBS buffer (0.01 M Na2HPO4, 0.0018 M KH2PO4, 0.137 M NaCl, and 0.0027 M KCl, pH 7.2) + 1% (w/v) BSA as carrier and stored at concentration 0.1 mg/mL at -20 °C. The hydrogel preparation was started with testing the gelation time of Fg by the action of Thr. For that purpose, stock solution of Fg was added to the “polymerization buffer 10-4 M Ca2+” to final concentration 1 mg/mL and by the action of Thr in the range of concentrations 0.1-2 NIH-U/mL was converted to the gel. Later, we tested the influence of HA on fibrin gelation and obtained so-called HA- fibrin hydrogel. Fg at a fixed concentration of 1 mg/mL was mixed with HA solutions of 40 kDa, 700 kDa and 1.5 MDa and theirs mixtures at concentrations of 0.1%, 0.25%, 0.5% (w/v) in “polymerization buffer 10-4 M Ca2+” gelated upon addition of Thr in the range of concentrations 0.1-1 NIH-U/mL. Finally, 0.5% (w/v) mixture of HA 700 kDa : HA 1.5 MDa = 1:1 was selected at the optimized third component to enable syringeability due to moderate viscosity and postponed gelation time. To formulate fortified HA-fibrin hydrogel FXIII at final concentration 10 µg/mL and α2AP at final concentration 0.14 µg/mL were added to the Fg (1 mg/mL) and HAs 0.5% (w/v) mixture 700 kDa : 1.5 MDa = 1:1 in “polymerization buffer 10-4 M Ca2+”. Thr was added as the last component at 0.75-1 NIH-U/mL to initiate the polymerization. 2.3.2. Loading of HA NCs into the hydrogel The maximum loading of isolated HA NCs to in situ hydrogels was performed for non-fortified and fortified HA-fibrin hydrogels. Prior the experiments Fg samples, 0.5% (w/v) HAs Mix 1:1 = 700 kDa : 1.5 MDa solutions in “polymerization buffer 10-4 M Ca2+”, 0.5% (w/v) HAs Mix 1:1 = 700 kDa : 1.5 MDa solutions in “polymerization buffer 10-3 M Ca2+” and blank or drug loaded HA NCs were warmed up at 37 °C for 30 minutes. For the samples preparation, we used fibrin at concentration 1 mg/mL and 1.5 mg/mL, 0.5% (w/v, final concentration) mixture of HA 700 kDa : HA 1.5 MDa in = 1:1 in “polymerization buffer 10-4 M Ca2+” or in “polymerization buffer 10-3 M Ca2+”, and FXIII 10 µg/mL, α2AP 0.14 µg/mL (in case of fortified HA-fibrin hydrogels), as summarized in Table 3. The reaction was initiated by Thr (1-2 NIH-U/mL). In situ hydrogel NCs loading capacity in the range of 10 - 50% was tested. Chapter 1 144 surfactant/cationic surfactant/shell polymer 4:1:0.2:0.5 yielded stable 100 – 200 nm NCs. The injection under pressure of the organic phase over the aqueous phase was applied in order to decrease the NCs size. The particle size, zeta potential and encapsulation efficiency of two different formulations are summarized in Table 5. The first prototype with encapsulated DXM was prepared with a ratio slightly modified of DXM/OO/Lec/OAm/HA = 1:6:1.5:0.3:1 (prototype 1) to better control the NCs physicochemical properties, however, the DXM EE% was low (Table 5). On the other hand, in order to increase the DXM loading a new prototype of NCs (prototype 2) with a ratio DXM/OO/Lec/OAm/HA = 3.2:8.8:2.3:0.45:1 was prepared, with higher amount of oil and surfactants in an attempt to increase the drug content in the suspension (Figure 2). Table 5. Physicochemical characteristics of the nanocapsules (NCs) prepared with 700 kDa HA (mean ± SD; n = 6). PDI: polydispersity index. Type of NCs OO (mg) Lec (mg) OAm (mg) Particle size (nm) PDI ζ-potential (mV) Drug concentration (mg/mL) Blank prototype 1 15 3.75 0.75 128 ± 13 0.2 −27 ± 3 n/a prototype 2 22 5.625 1.125 121 ± 10 0.2 −30 ± 3 n/a DXM - loaded prototype 1 15 3.75 0.75 160 ± 12 0.2 −20 ± 4 0.75 ± 0.125 prototype 2 22 5.625 1.125 135 ± 9 0.2 −31 ± 5 5.6 ± 0.4 Chapter 1 145 Figure 2. Representation of the structure of HA nanocapsule. The size of the NCs, obtained by DLS, was compared with TEM data. Images from electronic microscopy (Figure 3) revealed the spherical shape of NCs with considerably smaller particle size values compared to DLS data (Figure 3B and C). These results can be explained considering that TEM requires detection of NCs in a dry state, whereas DLS analyzes NCs in aqueous suspension, measuring the hydrodynamic diameter.148 A B Figure 3. TEM images of HA blank nanocapsules: (A) at magnification 50,000×; (B) and (C) at 500,000×. Chapter 1 146 3.2. Preparation and characterization of in situ forming hydrogels In order to increase the residence time of NCs in the articular cavity, we developed an in situ forming hydrogel, which in addition to having good rheological properties and high resistance to deformation after IA injection, would serve as a reservoir for the NCs. For its preparation we used a combination of HA and fibrin with crosslinking agents, i.e. factor XIII (FXIII) and α2-antiplasmin (α2AP). For the production of fibrin gel, we started with the precursor fibrinogen (Fg), at a concentration of 1 mg/mL, and investigated the time required for its polymerization by the action of different concentrations of thrombin (Thr, 0.1-2 NIH-U/mL) (Figure 4A and B). The optimal gelation speed at physiological conditions was achieved at 1-5 NIH-U/mL.149 A B Figure 4. (A) Influence of thrombin (0.1 or 1 NIH-U/mL) on the fibrin gelation time. (B) Fibrin gelation points by the action of thrombin (0.1 - 2 NIH-U/mL) on fibrinogen. The range marked by dot-dash line represents the thrombin activity at which the hydrogels revealed the best stability profiles. The polymerization kinetics was determined by turbidity analysis during 20 minutes at λ = 350 nm. Fibrinogen was always taken at concentration 1 mg/mL. For thrombin (Thr), 1 NIH-U = 0.324 ± 0.073 μg (9.0 nM). Expressed as mean ± SD; n = 3. In the first stage of polymerization, shown as the “fluid” phase in Figure 4A, fibrin is presented in the form of soluble fibrin-monomers. The second stage of polymerization130 is characterized by the lateral association of protofibrils to fibrils and their subsequente organization in the form of a 3D-network hydrogel.150,151 As shown in Figure 4A this process is characterized by a constant increase of the absorbance until the gel is completely formed Chapter 1 147 and, hence is named as “gel” phase. The times at which fibrin starts to polymerize (gelation points) were calculated from the kinetics curves of polymerization by the method described by Chernysh et al.152 (Figure 4B). Ca2+ ions are among the key factors controlling the speed of polymerization and final gel rheology, rigidity and porosity. Based on this, the buffer for hydrogel formation was made with a Ca2+ concentration that was one order of magnitude lower to the one found in plasma (1-1.1×10-3 M),130 with a composition of 0.02 М HEPES, 0.1 М NaCl, 10-4 М CaCl2, pH 7.4. Taking advantage of the affinity between HA and fibrin’s positively charged E- domain and αC-domains150,151 (Kd ~ 45 × 10-9 M),108,109 we explored the formation of HA- fibrin hydrogels. We used HA polymers of different MW to study the influence of the macromolecule length in the polymerization process. We tested HA of three MW (40 kDa, 700 kDa, 1.5 MDa) in different concentrations. The gelation point of the HA-fibrin system was calculated from the kinetics curves of polymerization, as described earlier for fibrin kinetics. For these experiments, 0.1%, 0.25%, and 0.5% (w/v) solutions of HA of each MW (40 kDa, 700 kDa and 1.5 MDa) and a mixture of 700 kDa and 1.5 MDa were used (Table 6). Table 6. Screening of compositions to optimize HA-fibrin gel formation. Fg: fibrinogen, Thr: Thrombin, HA: hyaluronic acid, FXIII: factor XIII, α2AP: α2-antiplasmin. System Components concentrations Fg (mg/mL) Thr (NIH- U/mL) HA FXIII (µg/mL) α2AP (µg/mL) w/v MW Fibrin gel 1 0.1 - 2 - - - - HA-fibrin gel 1 0.5 - 1 0.1 - 0.5% 40 kDa - - 0.1 - 0.5% 700 kDa 0.1 - 0.5% 1.5 MDa 0.5% Mix 1:1 of 700 kDa : 1.5 MDa Fortified HA- fibrin gel 1 0.75 - 1 0.5% Mix 1:1 of 700 kDa : 1.5 MDa 10 0.14 As shown in Figure 5, in general, the higher the concentration of HA the higher the inhibition of the gelation process is. Namely, in the case of 700 kDa and 1.5 MDa HA, an Chapter 1 148 increase in HA concentration from 0.1% to 0.5% led to a significant increase in the gelation time. However, in the case of 40 kDa HA, the influence of the HA concentration had only a minor impact on the gelation process. On the other hand, 0.5% 1.5 MDa HA solution was highly viscous and, thus, difficult to inject. For that reason, a mixture of HAs (700 kDa : HA 1.5 MDa = 1:1, 0.5%), with moderate viscosity, was selected to enable proper injection into the knee. In addition, the use of a mixture of HA of different molecular weights allowed us to modulate the gelation time to the one required for the adequate syringeability. This composition was also found to be good for viscosupplementations purposes.153–155 In this regard, it is worth noting that the marketed product RenehaVis®, which involves a combination of 1 MDa (2.2%, w/v) and 2 MDa (1%, w/v) HAs, is highly used for the long term treatment (12 months) of OA.156–159 Furthermore, HA with a MW of 0.5-1 MDa has been found to be effective in reducing synovial inflammation,55,90,160 while 1.3-1.7 MDa HAs are able to restore the rheological properties of arthritic synovial fluid.55,89,90 Figure 5. Comparative studies of the influence of different hyaluronic acid (HA) on fibrin polymerization kinetics (indicated as “without HA”). HA, 0.5% (w/v) of 40 kDa, 700 kDa, 1.5 MDa and HA mix solution of 700 kDa : 1.5 MDa = 1:1 were added to the “polymerization buffer 10-4 M Ca2+”. The polymerization kinetics was recorded by turbidity analysis during 20 minutes at λ = 350 nm. Fibrinogen was always taken at 1 mg/mL concentration, activity of thrombin (Thr) = 0.5 - 1 NIH-U/mL. The gelation point was calculated from the kinetics curves of polymerization. The numbers above the bars represent in how many times the gelation is inhibited compared to fibrin polymerization alone. Expressed as mean ± SD; n = 3. Chapter 1 149 To increase the in vivo half-life of the developed HA-fibrin hydrogel we incorporated FXIII and α2AP as fortifying components of the gel. FXIII is a natural fibrin covalent crosslinker that fortifies and stabilizes fibrin mesh increasing the resistance against the gel degradation by 5 times.112,113 Whereas, α2AP, incorporated into fibrin network, greatly reduces the degradation of crosslinked fibrin gels by plasmin, normally found in rheumatic SF.115,117,118 Hence, by adding FXIII and α2AP to in situ HA-fibrin hydrogel a fortified HA- fibrin IPN was engineered (Figure 6). This combination of materials is expected to resist the action of hyaluronidase, which degrades non-crosslinked HA at RA condition.161,162 Figure 6. The schematic illustration of the fortified HA-fibrin gel formation, activated by thrombin. HA: hyaluronic acid, FXIII: factor XIII, α2AP: α2-antiplasmin. We investigated the influence of FXIII and α2AP on the hydrogel formation by turbidity analysis. The results gathered in Figure 7 indicate that, as expected, the gelation time is reduced upon addition of FXIII. Overall, we could control the polymerization speed and the rheological properties of the developed hydrogel by adjusting different parameters, namely the temperature, the amount of thrombin, the MW and concentration of HA, Ca2+ and FXIII. 3.3. Loading of NCs into the in situ forming hydrogel We investigated the capacity of the hydrogel to load NCs in the range of 10 – 50% (v/v) (blank or drug-loaded NCs concentrated to 1 mL) in HA-fibrin and fortified HA-fibrin systems at the concentrations gathered in Table 3. NCs showed a significant inhibition of the Chapter 1 150 gel formation process (> 20 min) and even no gelation was observed when the NCs/hydrogel v/v ratio was higher than 30% (fibrinogen 1 mg/mL, thrombin 1 NIH-U/mL and HAs 0.5% (w/v) mixture 700 kDa : 1.5 MDa = 1:1 in a “polymerization buffer 10-4 M Ca2+”). Thus, the next step was the adjustment of the hydrogel formation conditions (fibrin, thrombin, Ca2+ and HAs concentrations) for 30% NCs loaded gels. The optimal time for the formation of a HA- fibrin gel with adequate rheological properties was achieved at a concentration of 1.5 mg/mL of fibrinogen, 2 NIH-U/mL of thrombin, 0.5% (w/v) mixture 700 kDa : 1.5 MDa HAs and 10-3 M Ca2+. In the case of the fortified HA-fibrin hydrogel, 10 µg/mL of FXIII and 0.14 µg/mL α2AP were additionally used. Figure 7 shows the summary of polymerization kinetics observed by turbidity analysis, the lag time was calculated based on spectrophotometric determinations, as described in Section 3.2. The amount of HA in the gel was envisaged to be ~3 mg/mL, bearing in mind that this is the HA physiological concentration found in healthy SF.94 Figure 7. Comparative studies for the gelation of fibrin (Fn) (without HA), HA-fibrin, and fortified HA-fibrin systems, driven by different Ca2+ ions concentration in blank and 30% NCs loaded gels. The polymerization kinetics was recorded by turbidity analysis during 20 minutes at λ = 350 nm. Fibrinogen was always taken at 1 mg/mL concentration, activity of thrombin (Thr) = 0.5 - 1 NIH-U/mL for blank gels and NCs loaded gels. The gelation point was calculated from the kinetics curves of polymerization. The numbers above the bars represent in how many times the gelation is inhibited compared to Fn polymerization alone. Expressed as mean ± SD; n = 3. Chapter 1 151 3.4. Rheological behavior of in situ forming the hydrogels A basic principle for the development of an in situ gelling system is to have a low vicosity of the components mixture in order to ensure its good syringeability. The viscosity of the HA-fibrin mixture (with no thrombin addition) at 20 °C and 37 °C is 118.9 mPa·s and 81.3 mPa·s, respectively. As these values are much lower than those of commercial viscosupplements,59,154 the mixed solutions necessary to form the in situ hydrogel are expected to be easily injected intra-articularly in vivo. Oscillatory rheological experiments are known to be a useful tool to determine the macro- and micro-structural rearrangements that influence the rheological behavior of gels with time.163,164 The gelling point can be detected by monitoring the gelation process with an oscillatory time sweep. The temporal evolution of the elastic or storage modulus (G'), which measures the elastic nature of the material, and the viscous or shear loss modulus (G''), which measures the viscous nature of the material was monitored upon the tests. As an example, Figure 8A and B gathers the gelation process of HA-fibrin and fortified HA-fibrin hydrogels with 30% loaded NCs. It can be observed thatthe viscous/shear loss modulus (G'') is higher than the elastic/storage modulus (G'). This is logical if we take into account that the gel samples are in liquid state, where the viscous properties predominate. As soon as the blank or NCs-loaded gels begin to form a crosslinked network the two indicated moduli rise. Nevertheless, the increase of G' is faster than that of G'', because the elastic nature of the gelling hydrogel starts to dominate. Hence, a crossover point appears in the curves is the gelation time of the sample. These gelling times are compared with those obtained for HA-fibrin and fortified HA-fibrin blank gels (Figure 9). The results of the rheology studies allowed us to detect the starting gelation point, by observing the gel evolution in time and the endpoint at which the gel is completely formed. Chapter 1 152 A B Figure 8. Gelation point detection by the response of elastic (G’) and viscous (G”) moduli in HA-fibrin with 30% loaded NCs (A) and fortified HA-fibrin hydrogels with 30% loaded NCs (B). Fibrinogen was always taken at 1.5 mg/mL concentration, activity of thrombin (Thr) = 2 NIH-U/mL, 0.5% HAs mix solution 1:1 (1.5 MDa : 700 kDa), 10-3 M Ca2+, 10 µg/mL of factor XIII and 0.14 µg/mL α2-antiplasmin at 37 °C. Expressed as mean ± SD; n = 3. To provide an adequate syringeability of in situ hydrogel for in vivo experiments, our approach was to delay the gel formation for at least a 2 min (lag-time) after mixing the gel components in non- and fortified HA-fibrin blank and 30% NCs loaded gels. The resulting gelation times are shown in Figure 9. Figure 9. Summary of gelation time point determination by rheological measurements in different systems: HA-fibrin (HA-Fn) and fortified HA-Fn blank and 30% NCs loaded gels. For blank gels fibrinogen was always taken at 1 mg/mL concentration, activity of thrombin (Thr) = 1 NIH-U/mL. For 30% NCs loaded gels fibrinogen was always taken at 1.5 mg/mL concentration, activity of Thr = 2 NIH-U/mL. The dash-and-dot line reflects the 2 minutes lag-phase requirements. The measurements were carried out at 37 °C. Expressed as mean ± SD; n = 3. Chapter 1 153 To evaluate the deformation profiles we measured the behavior of the G' and G'' moduli of the formed blank and NCs-loaded HA-fibrin and fortified HA-fibrin gels by oscillatory strain and frequency sweeps. As an example, Figure 10 shows the elastic and viscous moduli of the non- and fortified HA-fibrin hydrogel loaded with 30% NCs as a function of the deformation. Initially, the linear viscoelastic region, i.e. the region in which deformations are not large enough to destroy material structure, was identified through strain sweeps ranging from 0.1% to 1,000 or 10,000% depending on the parameters of the crossover point. At strains lower than 10- 15% (critical strain), the value of elastic and viscous moduli is constant and independent of the deformation, G’>G”. In this linear viscoelastic region the deformation is completely recoverable and the material exhibits an elastic “solid-like” behavior. As mentioned earlier, HA and fibrin significantly contribute to rheological properties of each other: e.g. high hydrophilicity of HA prevents the compression of the fibrin network and improves it mechanical properties,101 while fibrin provides desired elasticity and resistance to stretching for HA gels.96 The crossover point of G’ and G” modulus, indicating a sample deformation point in this experiment, for NCs loaded fortified HA-fibrin gels was observed at a strain of 630%, while the NCs loaded non-fortified HA-fibrin gels destroyed at a strain of 100%. Thus, fortified NCs loaded gels reveled 6.3 times higher resistance to deformations compared to non-fortified loaded gels. Other authors also showed that fibrin hydrogels crosslinked by FXIII under physiological conditions are more stable and shows 8 times higher Young’s (elastic) modulus values than non-crosslinked one.96 Furthermore, FXIII increases the rigidity and strength of the fibrin hydrogel and protects it against shear stress in the physiological environment.112,113,165 Under larger deformations an overshoot phenomenon can be observed in the G’ and G” moduli of the fortified HA-fibrin hydrogel loaded with 30% NCs (Figure 10B). This response is typical for materials with resistance to a permanent deformation, which is the evidence of FXIII contribution to the stability profile. Lorand and Muszbek et al. have also observed increase in viscous and elastic moduli of fibrin gel crosslinked by FXIII as a measure of clot stability against shear stress.113,114 At the higher strains applied the values of two moduli decreased as the strain increases corresponding to thinning behavior until the breakdown of the material structure. Overall discussion 256 models, serving as a temporary matrix for in vivo cells encapsulation.35 In vivo gel half-life time in rat joint was not evaluated in this study, but might be interesting for further exploration of DDS performance. Such a composite might result to a synergistic effect in tissue self-healing properties – HA-fibrin matrix plays a major role in the subsequent tissue regeneration processes.37 In summary, this work highlights the development of a new nanotechnology-based platform for an efficient treatment of joint pathologies (e.g. rheumatoid arthritis). An intraarticular injectable in situ hydrogel consisting of an IPN of HA and fibrin crosslinked with FXIII and α2-AP, containing drug loaded NCs (30%). The system presents well-organized structure and rheological behavior typical for stable gel networks, with features of both elastic and solid-like materials desired for IA application. HA NCs, dispersed in the gel, serve as multireservoirs for lipophilic anti-inflammatory drugs and they control and prolong the release of the encapsulated drug. The physicochemical evaluation of the drug delivery system was performed for DXM, as a model drug, and later on applied to a synthetized highly affine and specific Gal-3i. The results obtained in a preliminary in vivo acute knee synovitis rat model showed a remarkable suppression of inflammation by Gal-3 inhibitor encapsulated within hydrogel and administrated intra-articular at microgram scale doses. Treated rats showed improvement on the knee swelling, at the histological and at the white cells levels. These findings present a promising strategy for the treatment of inflammatory knee diseases by the inhibition of galectin-3 and Gal-3 inhibitor as a lead compound for immunotherapeutic drug candidate in particular for RA. Efficiency of Gal-3 inhibitor could be further enhanced by preparation of multivalent inhibitors using synthetic scaffolds mimicking lactose units. Apart from joints pathologies, Gal-3 mono- or multivalent inhibitors could be applied for the treatment of other inflammatory conditions, e.g. cancer, inflammatory bowel disease, etc. increasing the range of therapeutic possibilities for these new compounds. At the same time, we showed the potential of a new in situ hydrogel as an IA DDS. 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Biochem. 58, 345–352 (2011). 263 Conclusions If you can’t explain it simply, you don’t understand it well enough. Albert Einstein 264 Conclusions 265 CONCLUSIONS This thesis project comprised the design, development and characterization of a novel injectable nanotechnology-based system intended to prolong and control the release of the associated drug in the intra-articular (IA) cavity. The system is composed of an in situ forming hydrogel loaded with polymeric nanocapsules, which serve as multireservoirs for different lipophilic anti-inflammatory drugs, namely i) a model drug – dexamethasone; and ii) a novel synthesized drug candidate – galectin-3 (Gal-3) antagonist. The obtained experimental results led to following conclusions: 1) A novel highly affine, potent and selective monovalent Gal-3 inhibitor, composed of two different moieties assembled to a modified lactosamine core was developed and synthetized in this study. Its Kd to Gal-3, measured by direct and competitive fluorescence polarization assay, was 0.59 µM at 4 °C (2.99 µM at 25 °C), while fluorescein-labelled Gal-3 inhibitor displayed very high affinity to Gal-3, Kd = 14 nM at 4 °C (82 nM at 25 °C), certainly higher than a reported commercial Gal-3 inhibitor DAVANAT® has. An important feature of this compound is the ability to discriminate between Gal-1/Gal-3/Gal-7, while DAVANAT® could hardly discriminate between Gal-1 and Gal-3. On the other hand, a first low MW Gal-3 antagonist TD139, approved by FDA, with modified thio-digalactoside core, measured in the same assay, has been experimentally confirmed to have high affinity Kd(Gal-3) = 0.036 µM at 4 °C (0.166 µM at 25 °C) and high selectivity among Gal- 1/Gal-3/Gal-7. Considering the success of TD139, the low MW Gal-3 inhibitor that we developed might receive future pharmaceutical advancement. 2) Polymeric NCs consisting of an olive oil core and a HA coating were successfully prepared using a simple solvent displacement method. The NCs can be loaded with dexamethasone (5.6 ± 0.4 mg/mL) or Gal-3 inhibitor (531 ± 5 µg/mL), have nanometric size (135 ± 9 nm and 122 ± 11 nm, correspondingly), negative surface charge and regular spherical shape. 3) Two injectable in situ forming hydrogel systems were developed. One of them is composed of HA and fibrin interpenetrating network, and the other one contains HA and fibrin additionally crosslinked and fortified with factor XIII and α2-antiplasmin in 272 Permission to use to use graphical material as Figure 2, Chapter Introduction. 273 Permission to use graphical material as Figure 3, Chapter Introduction. 274 Permission to use graphical material as Figure 4-5, Chapter Introduction. 275 Permission to use graphical material as Figure 6, Chapter Introduction. 276 Permission to use graphical material as Figure 7A, Chapter Introduction. 277 Permission to use graphical material as Figure 7B, Chapter Introduction. 278 Permission to use graphical material as Figure 1 in Chapter 2. 279 Permission to use graphical material as Figure 2 in Chapter 2. 280 CONFLICT OF INTEREST I declare no conflict of interest regarding the context of this thesis, entitled “New potential nanotechnology-based therapies for the treatment of rheumatoid arthritis”. Sgd. Nataliya Storozhylova 28 Downstream end for further modulation Envisaged modifications at Gal 3’-C position Envisaged modifications at GlcN 2-C position (A) (C) (B) a) Bu2SnO [MeOH], reflux, < 65-67 °C, 5 h. b) 3,5- diMeOBnB r/ NaphtylMe Br [DMF], RT, 48 h, 63% TES-Cl; [DMF], imidazole, RT, 12 h, 95% 1. Acetylatio n: AcOH, Ac2O, HClO; RT, 1-2 h 2. Brominati on: HBr; [AcOH], 0 °C, 1 h, 94% 3. Reduction : Zn, [AcOH/H2O ]; 0 °C, 48 h, 72%. 4. Deacetylat ion: dry Na2CO3; [MeOH], RT, 12 h, 79% 4 7a 6a 7b 6b TES-Cl; [DMF], imidazole, RT, 12 h, 95% Ph2Se2, PhI(AcO)2, TMSN3 [DCM], -20 °C (10 min), -10 °C, 1-2 h, 56% TBAF [THF], 0 °C RT, 8 h, 95% a) Bu2SnO. [MeOH], reflux< 65- 67 °C, 5 h. b) 3,5- diMeOBnB r [DMF], RT, 48 h, 63% Ac2O [DCM], DIEA, DMAP 0 °C - RT, 12 h, 96% Acetylatio n: AcOH, Ac2O, HClO4; RT, 1-2 h Brominati on: HBr; [AcOH], 0 °C, 1 h, 94% Reduction : Zn, [AcOH/H2O ]; 0 °C, 48 h, 72%. Deacetylat ion: dry Na2CO3; [MeOH], RT, 12 h, 79% 4 10 9 12 11 Br2 [DCM], -20 °C, 1 h 61% 15 16 (CH3)3P [THF], RT, 12 h 92.5% 3,5- dimethoxyb enzoyl-Cl [DCM], DIEA, DMAP 0-30 °C, 14 h 67% 13 14 • Bu2S nO. [MeOH], reflux, < 65-67 °C, 5 h. b) NaphtylMeBr [DMF], 40 °C, 12 h 50% Ac2O [DCM], RT, 24 h 85% 18 19 17 a) Ac2O/H2SO4 [DCM], -15 °C  4 °C, 12 h b) piperidine THF, RT, 12 h, c) Cl3CCN, DBU cat, [DCM], RT, overall yield 73% 21 a) TBDMSOTf, [CH2Cl2], -30 °C, 1 h; b) HF-py, [ACN], 0 °C, 2 h, 32% c) MeONa, MeOH, RT,2 h, 75% a) Ethylenediami ne [MeOH], 50 °C, 5 h, 97% b) 3- Methoxybenz oyl-Cl [DCM], Et₃N, RT, 6 h, 80% c) AcCl, Py THF, -20°C, 5 h, 76% 26 a) PMe3, [THF], H2O, 80% b) FITC, DiPEA, [DMF], 55% 22 28 24