Development and biological evaluation of pNIPAM-based nanogels as vaccine carriers.

"Smart" nanogels are an attractive tool for the development of new strategies of immunization in veterinary medicine. Here, we reported the synthesis and physicochemical characterization of thermoresponsive nanogels based on poly(N-isopropylacrylamide) (pNIPAM) and theirin vitro, ex vivoand in vivo (mice model) performance. Smart nanogels of ca. 250 nm, with a transition temperature of 32 °C were obtained by precipitation polymerization. Assays to evaluatepNIPAM nanogels cytotoxicity were performed in different cell lines showing high biocompatibility (>70 %). The efficient internalization of the system was studied by confocal microscopy as well as flow cytometry. The ability to protect and deliver antigens was analyzed using the outer membrane lipoprotein A (OmlA), an important virulence factor ofActinobacillus pleuropneumoniae(App)cause of porcine pleuropneumonia. This lipoprotein was synthesized by recombinant technology and its technique was also described. The biodistribution ofpNIPAM nanogels administered intranasally was performedinvivo and ex vivo through Pearl Imaging System, which showed that nanogels were kept mostly in the lungs during the evaluated time. Besides, the efficacy of the proposal nanogel-based vaccine was studiedin vivoby measuring the antibody titers of BALB/c mice inoculated with OmlA encapsulated intopNIPAM nanogels compared to OmlA plus aluminum hydroxide adjuvant. The results proved the ability of nanogels to stimulate a humoral immune response. Therefore, we have demonstrated thatpNIPAM nanogels can be used as an efficient platform for vaccine nanocarriers.

Non-clinical safety assessment and in vivo biodistribution of CoviFab, an RBD-specific F(ab')2 fragment derived from equine polyclonal antibodies.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has required the urgent development of new therapies, among which passive immunotherapy is contemplated. CoviFab (INM005) is a RBD-specific F(ab')2 fragment derived from equine polyclonal antibodies. We investigate their preclinical security and biodistribution by in vivo and ex vivo NIR imaging after intravenous administration of a dose of 4 mg/kg at time 0 and 48 h. Images were taken at 1, 12, 24, 36, 48, 49, 60, 72, 84, 96, 108, 120, 132 and 144 h after the first intravenous injection. At 96 and 144 h, mice were sacrificed for haematology, serum chemistry, clinical pathology, histopathology and ex vivo imaging. The biodistribution profile was similar in all organs studied, with the highest fluorescence at 1 h after each injection, gradually decreasing after that each one and until the end of the study (144 h). The toxicology study revealed no significant changes in the haematology and serum chemistry parameters. Further, there were no changes in the gross and histological examination of organs. Nonclinical data of the current study confirm that CoviFab is safe, without observable adverse effects in mice. Furthermore, we confirm that bioimaging studies are a useful approach in preclinical trials to determine biodistribution.

Desarrollo y validación de técnicas analíticas para la determinación de biomarcadores sanguíneos en ensayos preclínicos. ¿Por qué y para qué? / Development and validation of analytical techniques for the determination of blood biomarkers in preclinical trials. Why and what for?

Resumen El descubrimiento de un nuevo principio activo farmacéutico implica estudios preclínicos, que tienen como objetivo demostrar que es eficaz y seguro para un posterior ensayo en seres humanos. Esto conduce a la necesidad de desarrollar tecnologías que aprovechen las nuevas herramientas analíticas disponi bles dentro de un contexto donde los resultados de las pruebas realizadas, estén plenamente documentados, bajo sistemas de buenas prácticas de laboratorio auditables. En esta revisión se actualizan y describen algunos de los ensayos realizados en la etapa preclínica del desarrollo de un nuevo fármaco y el estado actual de la tecnología analítica empleada para el dosaje de diferentes biomarcadores sanguíneos de interés. Se analizaron los biomarcadores más relevantes, las normativas de validación de las técnicas analíticas empleadas para su determinación y los problemas que se presentan al tratar de aplicarlas.

Abstract New drug discovery involves preclinical studies to demonstrate its effectivity and safety for further tests in humans. This leads to the need to develop technologies that take advantage of the new analytical tools available within a context where the results of the tests carried out are fully documented, under auditable systems of good laboratory practice. This review updates and describes some of the tests carried out in the preclinical stage of the development of a new drug and the current state of the analytical technology used to measure different blood biomarkers of interest. Biomarker parameters were analyzed at the physiological level, considering both the validation regulations of the analytical techniques used for their determination as the problems that arise when trying to apply them, since many of these biomarkers are endogenous compounds in the used matrices.

[Development and validation of analytical techniques for the determination of blood biomarkers in preclinical trials. Why and what for?] / Desarrollo y validación de técnicas analíticas para la determinación de biomarcadores sanguíneos en ensayos preclínicos. ¿Por qué y para qué?

New drug discovery involves preclinical studies to demonstrate its effectivity and safety for further tests in humans. This leads to the need to develop technologies that take advantage of the new analytical tools available within a context where the results of the tests carried out are fully documented, under auditable systems of good laboratory practice. This review updates and describes some of the tests carried out in the preclinical stage of the development of a new drug and the current state of the analytical technology used to measure different blood biomarkers of interest. Biomarker parameters were analyzed at the physiological level, considering both the validation regulations of the analytical techniques used for their determination as the problems that arise when trying to apply them, since many of these biomarkers are endogenous compounds in the used matrices.

El descubrimiento de un nuevo principio activo farmacéutico implica estudios preclínicos, que tienen como objetivo demostrar que es eficaz y seguro para un posterior ensayo en seres humanos. Esto conduce a la necesidad de desarrollar tecnologías que aprovechen las nuevas herramientas analíticas disponibles dentro de un contexto donde los resultados de las pruebas realizadas, estén plenamente documentados, bajo sistemas de buenas prácticas de laboratorio auditables. En esta revisión se actualizan y describen algunos de los ensayos realizados en la etapa preclínica del desarrollo de un nuevo fármaco y el estado actual de la tecnología analítica empleada para el dosaje de diferentes biomarcadores sanguíneos de interés. Se analizaron los biomarcadores más relevantes, las normativas de validación de las técnicas analíticas empleadas para su determinación y los problemas que se presentan al tratar de aplicarlas.

New inhibitor targeting Acyl-CoA synthetase 4 reduces breast and prostate tumor growth, therapeutic resistance and steroidogenesis.

Acyl-CoA synthetase 4 (ACSL4) is an isoenzyme of the fatty acid ligase-coenzyme-A family taking part in arachidonic acid metabolism and steroidogenesis. ACSL4 is involved in the development of tumor aggressiveness in breast and prostate tumors through the regulation of various signal transduction pathways. Here, a bioinformatics analysis shows that the ACSL4 gene expression and proteomic signatures obtained using a cell model was also observed in tumor samples from breast and cancer patients. A well-validated ACSL4 inhibitor, however, has not been reported hindering the full exploration of this promising target and its therapeutic application on cancer and steroidogenesis inhibition. In this study, ACSL4 inhibitor PRGL493 was identified using a homology model for ACSL4 and docking based virtual screening. PRGL493 was then chemically characterized through nuclear magnetic resonance and mass spectroscopy. The inhibitory activity was demonstrated through the inhibition of arachidonic acid transformation into arachidonoyl-CoA using the recombinant enzyme and cellular models. The compound blocked cell proliferation and tumor growth in both breast and prostate cellular and animal models and sensitized tumor cells to chemotherapeutic and hormonal treatment. Moreover, PGRL493 inhibited de novo steroid synthesis in testis and adrenal cells, in a mouse model and in prostate tumor cells. This work provides proof of concept for the potential application of PGRL493 in clinical practice. Also, these findings may prove key to therapies aiming at the control of tumor growth and drug resistance in tumors which express ACSL4 and depend on steroid synthesis.