Design, synthesis and biological evaluation of new polymer-drug conjugates based on polyglutamic acid and 5-Fluorouracil for the treatment of advanced colorectal cancer

Abstract

[spa] El agente 5-fluorouracilo (5-FU) es el tratamiento quimioterapéutico sistémico esencial para el tratamiento del cáncer colorectal. Sin embargo, la supervivencia global y superación de la enfermedad de pacientes tratados con 5-FU como primera línea de tratamiento es sólo del 10-15%. El uso de conjugados de polímero-fármaco (PDC) ha atraído gran atención en el campo de la administración controlada de fármacos para el tratamiento del cáncer. Éstos mejoran la acumulación de agentes citotóxicos en tejidos tumorales, aprovechando las características en la vascularización de los tumores, por consiguiente reduciendo la toxicidad en los tejidos sanos. Se han estudiado tres tipos diferentes de PDC basados en el polímero biodegradable ácido poly-(L-glutámico)(PGA): (i) Conjugado de PGA-5-FU; (ii) Conjugados de PGA-MMPpept-5FU utilizando enlazadores escindibles enzimáticamente, especialmente péptidos sensibles a MMP-7, ya que los niveles de algunas MMP aumentan a medida que el progresa el CRC; y (iii) conjugados de PGA-5FU-SN38 para evaluar la sinergia entre 5-FU y SN-38 conjugado en un solo vehículo. El conjugado PGA-5-FU mostró actividad terapéutica in vitro en las líneas celulares HCT-116.Fluc2-C9 y HT-29.FlucC4 e internalización mediante endocitosis, mediante la técnica de microscopía confocal. Además, experimentos de biodistribución in vivo confirmaron la acumulación en el tumor y la excreción vía renal y hepática. Con el conjugado PGA-MMP7-5FU se confirmó que la importancia del tipo de enlace entre el péptido MMP7 sensible (AHX-RPLALWRS-AHX) y el agente 5-FU; ya que se observó que la unión carbamato era demasiado estable in vitro, en comparación con la unión éster. Los estudios de citotxicidad in vitro con sobreexpresión de MMP7, confirmaron que la conformación en solución resultó muy accesible para la enzima MMP7. Finalmente, se sintetizó una familia de conjugados PGA-5FU-SN38 con diferentes proporciones de fármacos. Se estudió la sinergia entre ambos mediante el cálculo del índice de combinación, y se confirmó que la conjugación de SN-38 y 5-FU en un solo vehículo polimérico mostraba una fuerte sinérgica entre ambos fármacos en comparación con los PDC cargados con una única droga.

[eng] Although survival rates of colorectal cancer (CRC) treated with surgery and conventional chemotherapy are high, metastatic CRC still shows acute mortality rates. Current chemotherapeutic treatment involves high doses of cytotoxic drugs, particularly adjuvant combinations of 5-Fluorouracil (5-FU) and Irinotecan (prodrug of SN-38). However, these treatments cause undesirable effects to the patients, which can negatively contribute to their survival. The use of polymer-drug conjugates (PDC) has attracted great attention in the field of controlled drug delivery for cancer treatment, improving the ratio of cytotoxic drugs in tumour tissues, taking advantage of the enhanced permeability retention effect, and consequently reducing the toxicity in healthy tissues. In this thesis the poly-(L-glutamic acid) (PGA) biodegradable polymer has been chosen as the carrier of the designed polymer-drug conjugates. By reason of the chemotherapeutic agent 5-FU is the mainstay of cytotoxic therapy against advanced CRC, in all PDC studied 5-FU has been conjugated to PGA. Three different types of PGA-based PDC have been studied: (i) PGA-5-FU conjugates; (ii) PGA-MMPpept-5FU conjugates using enzymatically-cleavable linkers, particularly MMP-sensitive peptides since some MMPs levels increase as CRC progress; and (iii) PGA-5FU-SN38 conjugates to evaluate the synergism between 5-FU and SN-38 conjugated in a single PDC. Regarding the PGA-5-FU conjugate, it was demonstrated that the conjugation of 5-FU to a PGA carrier through an ester bond showed therapeutic activity in vitro in the HCT-116.Fluc2-C9 and HT-29.FlucC4 CRC cell lines. Cellular uptake experiments performed with PGA-5-FU labelled with carboxyfluoresceine fluorescent probe indicated that the internalization of the PDC was through the endocytic pathway. In addition, in vivo biodistribution experiments of PGA-5-FU labelled with an AlexaFluor dye confirmed that the accumulation in tumor was maximal at 4 h post-adminstration, confirming also that the product was excreted through the kidney, but also removed by the liver in smaller amounts. In the study of a new PDC sensitive to MMP7 to deliver 5-FU we confirmed that it is of significant importance the type of link between the MMP7-sensitive peptide (AHX-RPLALWRS-AHX) and the bioactive agent 5-FU. It was confirmed through cytotoxic experiments of the peptide-drug unit that the carbamate union was too stable in vitro, opposite to the ester bond behavior, resulting as an undesirable linkage for their use in polymeric nanoconjugates designed to deliver drugs gradually. Then, in vitro therapeutic efficacy studies performed in HCT-116.Fluc2-C9 and HT-29.Fluc-C4 cells overexpressing MMP7, showed that the conjugation of 5-FU linked to AHX-RPLALWRS-AHX peptide through an ester bond to a PGA carrier, lead a PDC that adopted a conformation in solution that resulted very accessible for the MMP7 enzyme. When comparing PGA-MMP7pept-5FU with the PGA-5-FU system, it was confirmed that the nature and length of the peptide linker is of significant relevance on the final conformation of the conjugate, therefore on the kinetic release of the drug. Finally, a family of PGA-5FU-SN38 conjugates through an ester bond carrying different proportions of drugs was synthesized using PGA carrier with a molecular weight of 15 KDa. In vitro studies showed an improvement of the cytotoxic activity in the HCT-116.Fluc2-C9 and HT-29.Fluc4 CRC cells of the conjugates carrying both agents in a ratio of SN-38/5-FU of 1:40 and 1:300 in comparison with the behavior observed with the combined drugs as single agents at the same ratio. It was studied the synergy through the calculation of the Combination Index, and we confirmed that the conjugation of SN-38 and 5-FU in a single polymeric vehicle was traduced in a strong synergic interaction between both drugs when compared to the single PDC.