Design of smart linkers and their applications in controlled-release drug delivery systems

Design of smart linkers and their applications in controlled-release drug delivery systems

Antibody drug conjugates (ADC) represents an interesting strategy in tumour targeted therapy . The approach is based on the combination of the high affinity of an antibody towards its antigen and the high cytotoxicity of a drug, leading to a selective therapuetic agent with improved efficacy and saf...

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Autor principal: Ferhati, Xhenti
Otros Autores: Corzana López, Francisco (null)
Formato: text (thesis)
Lenguaje:eng
Publicado: Universidad de La Rioja (España) 2019
Acceso en línea:https://dialnet.unirioja.es/servlet/oaites?codigo=252736
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description Antibody drug conjugates (ADC) represents an interesting strategy in tumour targeted therapy . The approach is based on the combination of the high affinity of an antibody towards its antigen and the high cytotoxicity of a drug, leading to a selective therapuetic agent with improved efficacy and safety. When considering the development of an ADC, several factors are of great importance. On the one hand, conjugation chemistry defines the properties of the ADC, such as antibody to drug ratio (DAR) and is fundamental to yield stable and homogeneus conjugates. On the other hand, the linker used to connect the drug with the antibody plays an important role in the stability of the final conjugate and its efficiency in terms of cell killing. Considering the importance of these two factors in ADC development, this Thesis has been focused on conjugation reactions and linker chemistry. First, new conjugation strategies have been proposed for the design of homogeneous, stable and efficaceus ADCs. The use of carbonyl acrylamide derivatives has allowed the irreversible cysteine-selective protein modification. Thus, when combined with Thiomab technology, an ADC with DAR 2 was obtained in high yield, using stoichiometric amount of the reagent and under mild conditions. Similarly, a new, ultrafast reagent, based on quaternised vinyl pyridinium scaffold, has been described. After proving that the compound is also cysteine selective in proteins and antibodies, the synthesis of a vinyl pyridinium bearing an alkyne tag for further functionalization was optimized. This approach allowed the introduction of a drug, and the resulting derivative was efficiently used for ADC synthesis. Concerning the linker, we have demonstrated that acetals represent an interesting cleavable moiety for the preparation of ADCs as well as small molecules drug conjugates (SMDC). We have prepared acetal linkers featuring coumarin as fluorophore and a duocarmycin analogue as an example of cytotoxic drug. Markedly, it represents the first example a duocarmycin analogue is protected with an acid cleavable moiety. Kinetic studies were performed on these linkers and showed that they are stable in plasma, while being rapidly cleaved under acidic conditons. This methodology was applied then to the design of a small molecule drug conjugate (SMDC), as well as an antibody drug conjugate (ADC). Of note, interesting outcomes emerged from the stability studies performed on the ADC. In fact, our results showed that the stability of the ADC depends not only on the conjugation site, but also different payloads can affect the stability of the acetal linker, depending on the 3D disposition they adopt in the antibody pocket. Finally, we have exploited the use of the Grob fragmentation to design ‘self-immolative’ linkers for controlled drug release. Altough the reaction mechanism is well know and presents several synthethic application, no biological application has been described to date. The screening of different substrates allowed the identification of 3-aminocyclohexanol scaffold as a suitable moiety for fragmentation reaction under biological mimicking conditions. The novel methodology has been applied to the controlled release of Crizotinib, a drug used for the treatment of metastatic lung cancer. Thus, blocking the Grob fragmentation pathway, the pro-drug is stable and intact; at the same time, activation of the pathway by removing the amine protecting group of 3-aminocyclohexanol derivative, results in drug release.
author2 Corzana López, Francisco (null)
author_facet Corzana López, Francisco (null)
Ferhati, Xhenti
format text (thesis)
author Ferhati, Xhenti
spellingShingle Ferhati, Xhenti
Design of smart linkers and their applications in controlled-release drug delivery systems
author_sort Ferhati, Xhenti
title Design of smart linkers and their applications in controlled-release drug delivery systems
title_short Design of smart linkers and their applications in controlled-release drug delivery systems
title_full Design of smart linkers and their applications in controlled-release drug delivery systems
title_fullStr Design of smart linkers and their applications in controlled-release drug delivery systems
title_full_unstemmed Design of smart linkers and their applications in controlled-release drug delivery systems
title_sort design of smart linkers and their applications in controlled-release drug delivery systems
publisher Universidad de La Rioja (España)
publishDate 2019
url https://dialnet.unirioja.es/servlet/oaites?codigo=252736
work_keys_str_mv AT ferhatixhenti designofsmartlinkersandtheirapplicationsincontrolledreleasedrugdeliverysystems
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spelling oai-TES00000229322020-02-05Design of smart linkers and their applications in controlled-release drug delivery systemsFerhati, XhentiAntibody drug conjugates (ADC) represents an interesting strategy in tumour targeted therapy . The approach is based on the combination of the high affinity of an antibody towards its antigen and the high cytotoxicity of a drug, leading to a selective therapuetic agent with improved efficacy and safety. When considering the development of an ADC, several factors are of great importance. On the one hand, conjugation chemistry defines the properties of the ADC, such as antibody to drug ratio (DAR) and is fundamental to yield stable and homogeneus conjugates. On the other hand, the linker used to connect the drug with the antibody plays an important role in the stability of the final conjugate and its efficiency in terms of cell killing. Considering the importance of these two factors in ADC development, this Thesis has been focused on conjugation reactions and linker chemistry. First, new conjugation strategies have been proposed for the design of homogeneous, stable and efficaceus ADCs. The use of carbonyl acrylamide derivatives has allowed the irreversible cysteine-selective protein modification. Thus, when combined with Thiomab technology, an ADC with DAR 2 was obtained in high yield, using stoichiometric amount of the reagent and under mild conditions. Similarly, a new, ultrafast reagent, based on quaternised vinyl pyridinium scaffold, has been described. After proving that the compound is also cysteine selective in proteins and antibodies, the synthesis of a vinyl pyridinium bearing an alkyne tag for further functionalization was optimized. This approach allowed the introduction of a drug, and the resulting derivative was efficiently used for ADC synthesis. Concerning the linker, we have demonstrated that acetals represent an interesting cleavable moiety for the preparation of ADCs as well as small molecules drug conjugates (SMDC). We have prepared acetal linkers featuring coumarin as fluorophore and a duocarmycin analogue as an example of cytotoxic drug. Markedly, it represents the first example a duocarmycin analogue is protected with an acid cleavable moiety. Kinetic studies were performed on these linkers and showed that they are stable in plasma, while being rapidly cleaved under acidic conditons. This methodology was applied then to the design of a small molecule drug conjugate (SMDC), as well as an antibody drug conjugate (ADC). Of note, interesting outcomes emerged from the stability studies performed on the ADC. In fact, our results showed that the stability of the ADC depends not only on the conjugation site, but also different payloads can affect the stability of the acetal linker, depending on the 3D disposition they adopt in the antibody pocket. Finally, we have exploited the use of the Grob fragmentation to design ‘self-immolative’ linkers for controlled drug release. Altough the reaction mechanism is well know and presents several synthethic application, no biological application has been described to date. The screening of different substrates allowed the identification of 3-aminocyclohexanol scaffold as a suitable moiety for fragmentation reaction under biological mimicking conditions. The novel methodology has been applied to the controlled release of Crizotinib, a drug used for the treatment of metastatic lung cancer. Thus, blocking the Grob fragmentation pathway, the pro-drug is stable and intact; at the same time, activation of the pathway by removing the amine protecting group of 3-aminocyclohexanol derivative, results in drug release.Los derivados fármaco- anticuerpo representan una interesante estrategia en la terapia antitumoral dirigida. Dicha estrategia combina la gran afinidad que los anticuerpos presentan por sus correspondientes antígenos con la presencia de fármacos altamente citotóxicos. Como resultado se obtiene un agente terapéutico selectivo, a la vez que altamente eficaz y seguro. En el diseño de estos derivados fármaco-anticuerpo existen muchos factores para tener en cuenta. Así, por ejemplo, la reacción química utilizada en la conjugación define las propiedades del compuesto final (conjugado), así como el número de fármacos presentes por anticuerpo, y es decisiva para obtener un anticuerpo estable y homogéneo. Además, el linker que conecta el fármaco con el anticuerpo juega un papel importante en la estabilidad del conjugado final y en su eficacia respecto a la toxicidad en células tumorales. Teniendo en cuenta la importancia de estos dos factores, esta tesis doctoral se ha centrado en el desarrollo de nuevas estrategias de conjugación y en el diseño de nuevos linkers. En primer lugar, se han propuesto metodologías de conjugación alternativas para el diseño de nuevos conjugados. Para ello, se ha descrito la síntesis de derivados de fármacos que llevan una carbonil acrilamida, la cual reacciona selectivamente con los residuos cisteína presentes en las proteínas o anticuerpos. Así, combinado esta estrategia con un Thiomab se han preparado conjugados estables y que presentan dos unidades de fármaco por anticuerpo. De forma similar, se ha descrito el uso de vinil piridinas cuaternizadas, las cuales pueden reaccionar también de forma selectiva con las cisteínas presentes en la proteína. Con respecto al linker, en esta Tesis se ha demostrado que los acetales pueden ser un grupo funcional interesante para la preparación de ADCs y de pequeñas moléculas (SMDC), donde el fluoróforo o el fármaco al que están unidos estas entidades se liberan en medio ácido gracias a la hidrólisis del acetal. En este sentido, se han preparado acetales unidos a cumarina y a un análogo de duocarmicina. Este último compuesto representa el primer ejemplo de un análogo de duocarmicina que está protegido con un linker sensible al medio ácido. En paralelo, se han realizado estudios cinéticos con estos linkers y se ha demostrado que, aunque son estables en el plasma, se hidrolizan rápidamente en condiciones ácidas. Es de destacar que la estabilidad del ADC con un linker tipo acetal depende no sólo del sitio de la conjugación, sino que también de la naturaleza del fármaco o fluoróforo unido a este conector. Finalmente, hemos explotado el uso de la fragmentación de Grob para diseñar linkers "auto-inmolativos" para con el objetivo de liberar fármacos de forma controlada. Aunque el mecanismo de esta reacción es bien conocido y presenta varias aplicaciones sintéticas, hasta la fecha no se ha descrito ninguna aplicación en el ámbito de la biológica. El cribado de diferentes sustratos permitió identificar al 3-aminociclohexanol como una molécula adecuada para llevar a cabo la reacción de fragmentación en condiciones que imitan a un medio biológico. La nueva metodología se ha aplicado a la liberación controlada de crizotinib, un fármaco utilizado para el tratamiento del cáncer de pulmón metastásico. Así, bloqueando la fragmentación de Grob, el profármaco es estable y no toxico. Sin embargo, la activación de la reacción mediante la eliminación del grupo protector de amina del derivado 3-aminociclohexanol, resulta en la liberación del fármaco.Universidad de La Rioja (España)Corzana López, Francisco (null)Bernardes, Gonçalo (null)2019text (thesis)application/pdfhttps://dialnet.unirioja.es/servlet/oaites?codigo=252736engLICENCIA DE USO: Los documentos a texto completo incluidos en Dialnet son de acceso libre y propiedad de sus autores y/o editores. Por tanto, cualquier acto de reproducción, distribución, comunicación pública y/o transformación total o parcial requiere el consentimiento expreso y escrito de aquéllos. Cualquier enlace al texto completo de estos documentos deberá hacerse a través de la URL oficial de éstos en Dialnet. Más información: https://dialnet.unirioja.es/info/derechosOAI | INTELLECTUAL PROPERTY RIGHTS STATEMENT: Full text documents hosted by Dialnet are protected by copyright and/or related rights. 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