Relationship between IGF-1 and body weight in inflammatory bowel diseases: Cellular and molecular mechanisms involved

Inflammatory bowel diseases (IBD), represented by ulcerative colitis (UC) and Crohn''s disease (CD), are characterized by chronic inflammation of the gastrointestinal tract, what leads to diarrhea, malnutrition, and weight loss. Depression of the growth hormone-insulin-like growth factor-1 axis (GH-IGF-1 axis) could be responsible of these symptoms. We demonstrate that long-term treatment (54 weeks) of adult CD patients with adalimumab (ADA) results in a decrease in serum IGF-1 without changes in serum IGF-1 binding protein (IGF1BP4). These results prompted us to conduct a preclinical study to test the efficiency of IGF-1 in the medication for experimental colitis. IGF-1 treatment of rats with DSS-induced colitis has a beneficial effect on the following circulating biochemical parameters: glucose, albumin, and total protein levels. In this experimental group we also observed healthy maintenance of colon size, body weight, and lean mass in comparison with the DSS-only group. Histological analysis revealed restoration of the mucosal barrier with the IGF-1 treatment, which was characterized by healthy quantities of mucin production, structural maintenance of adherers junctions (AJs), recuperation of E-cadherin and ß-catenin levels and decrease in infiltrating immune cells and in metalloproteinase-2 levels. The experimentally induced colitis caused activation of apoptosis markers, including cleaved caspase 3, caspase 8, and PARP and decreases cell-cycle checkpoint activators including phosphorylated Rb, cyclin E, and E2F1. The IGF-1 treatment inhibited cyclin E depletion and partially protects PARP levels. The beneficial effects of IGF-1 in experimental colitis could be explained by a re-sensitization of the IGF-1/IRS-1/AKT cascade to exogenous IGF-1. Given these results, we postulate that IGF-1 treatment of IBD patients could prove to be successful in reducing disease pathology. © 2021 The Author

Evaluación preclínica de la eficacia de la Azatioprina en el tratamiento de tumores sólidos

Premio Extraordinario de Doctorado de la UAH en 2015Pérez Gisbert, Francisco Javier, codir.La Azatioprina (AZA) es una molécula que pertenece a la familia de las tiopurinas. Es un inmunosupresor muy utilizado en el tratamiento de enfermedades autoinmunes y para evitar el rechazo en los trasplantes. El cáncer de hígado es el sexto cáncer más común en el mundo con 692000 muertes al año siendo el tercero más letal en hombres y el sexto en mujeres. El sistema de clasificación BCLC permite identificar claramente el estadio del tumor, lo cual es esencial para conocer el mecanismo molecular involucrado en la oncogénesis para conseguir una diagnosis correcta y así poder aplicar un tratamiento personalizado combinando fármacos clásicos y nuevos. Los tumores sólidos muestran resistencia a la irradiación y a la quimioterapia debido a la estimulación de bucles autocrinos (por ejemplo de IGF-1) y a los altos niveles de antioxidantes (por ejemplo la elevada producción de glutation). Por lo tanto, el uso de fármacos que disminuyan estos efectos podrían ser de utilidad. OBJETIVOS Los objetivos de esta tesis son evaluar la eficacia y la toxicidad de la AZA en el tratamiento de tumores sólidos (cáncer de hígado) y estudiar el mecanismo molecular de acción de la AZA que tiene lugar en estos. MATERIALES Y MÉTODOS Para estudiar la eficacia, la toxicidad y el mecanismo molecular de la AZA se utilizaron métodos clásicos de bioquímica y biología molecular tanto in vitro como in vivo: a) in vitro, se emplearon diferentes líneas celulares en cultivo (HepG2, HuH6, Hep3B, HuH7, Chang, hepatocitos de neonatos de ratón, RKO, SW-480, SW-48 y LoVo); b) in vivo, utilizando un modelo de cáncer xenograft en ratones atímicos. RESULTADOS Y CONCLUSIÓN Los experimentos llevados a cabo en esta tesis han determinado que AZA induce autofagia en un modelo de hepatoblastoma humano (células HepG2). Este efecto se produjo mediante activación sostenida de p70S6K a través de la ruta de señalización Ras/Raf/MEK/ERK/TSC-2/mTOR, lo cual induce la degradación de IRS-1 en el proteosoma y la subsecuente desensibilización de la célula al IGF-1. La resistencia al IGF-1 podría ser la causa de la activación de la autofagia, de la parada del ciclo y del aumento de la senescencia celular observadas después del tratamiento con AZA. Sin embargo, las células no mueren como consecuencia de el tratamiento con el fármaco; pero cuando la tiopurina es combinada con bafilomicina A1 (BAF), un inhibidor específico de la enzima ATPasa lisosomal dependiente de protones, las células murieron por apoptosis porque no pudieron degradar las estructuras celulares dañadas. Además, cuando las células cancerosas fueron tratadas con una combinación de AZA y L-butionina-[S,R]-sulfoximina (BSO), un inhibidor específico de la síntesis de glutation, las células murieron por necrosis, independientemente de caspasa-3 pero de manera dependiente de la activación de las quinasas del estrés (p38 y JNK) y de la integridad de la membrana mitocondrial. Estos datos sugieren un mecanismo de muerte celular regulado conocido como necroptosis. Cuando las células cancerosas (HepG2) fueron introducidas subcutáneamente en los ratones inmunodeprimidos para desarrollar el tumor, se observó que el volumen del mismo se incrementaba con el tiempo. El tratamiento intratumoral con AZA y BSO produjo una disminución significativa del volumen del tumor con respecto a los animales tratados con sólo el excipiente (DMSO). Los estudios de seguridad, llevados a cabo por métodos bioquímicos, mostraron efectos moderados de la combinación de AZA y BSO con respecto al efecto del DMSO sólo. En conclusión, la AZA en combinación con inhibidores de la autofagia (por ejemplo, BAF) o con depletores de glutation (por ejemplo, BSO) podría ser útil para el tratamiento de tumores sólidos de hígado u origen gastrointestinal

Evaluación preclínica de la eficacia de la Azatioprina en el tratamiento de tumores sólidos

Premio Extraordinario de Doctorado de la UAH en 2015Pérez Gisbert, Francisco Javier, codir.La Azatioprina (AZA) es una molécula que pertenece a la familia de las tiopurinas. Es un inmunosupresor muy utilizado en el tratamiento de enfermedades autoinmunes y para evitar el rechazo en los trasplantes. El cáncer de hígado es el sexto cáncer más común en el mundo con 692000 muertes al año siendo el tercero más letal en hombres y el sexto en mujeres. El sistema de clasificación BCLC permite identificar claramente el estadio del tumor, lo cual es esencial para conocer el mecanismo molecular involucrado en la oncogénesis para conseguir una diagnosis correcta y así poder aplicar un tratamiento personalizado combinando fármacos clásicos y nuevos. Los tumores sólidos muestran resistencia a la irradiación y a la quimioterapia debido a la estimulación de bucles autocrinos (por ejemplo de IGF-1) y a los altos niveles de antioxidantes (por ejemplo la elevada producción de glutation). Por lo tanto, el uso de fármacos que disminuyan estos efectos podrían ser de utilidad. OBJETIVOS Los objetivos de esta tesis son evaluar la eficacia y la toxicidad de la AZA en el tratamiento de tumores sólidos (cáncer de hígado) y estudiar el mecanismo molecular de acción de la AZA que tiene lugar en estos. MATERIALES Y MÉTODOS Para estudiar la eficacia, la toxicidad y el mecanismo molecular de la AZA se utilizaron métodos clásicos de bioquímica y biología molecular tanto in vitro como in vivo: a) in vitro, se emplearon diferentes líneas celulares en cultivo (HepG2, HuH6, Hep3B, HuH7, Chang, hepatocitos de neonatos de ratón, RKO, SW-480, SW-48 y LoVo); b) in vivo, utilizando un modelo de cáncer xenograft en ratones atímicos. RESULTADOS Y CONCLUSIÓN Los experimentos llevados a cabo en esta tesis han determinado que AZA induce autofagia en un modelo de hepatoblastoma humano (células HepG2). Este efecto se produjo mediante activación sostenida de p70S6K a través de la ruta de señalización Ras/Raf/MEK/ERK/TSC-2/mTOR, lo cual induce la degradación de IRS-1 en el proteosoma y la subsecuente desensibilización de la célula al IGF-1. La resistencia al IGF-1 podría ser la causa de la activación de la autofagia, de la parada del ciclo y del aumento de la senescencia celular observadas después del tratamiento con AZA. Sin embargo, las células no mueren como consecuencia de el tratamiento con el fármaco; pero cuando la tiopurina es combinada con bafilomicina A1 (BAF), un inhibidor específico de la enzima ATPasa lisosomal dependiente de protones, las células murieron por apoptosis porque no pudieron degradar las estructuras celulares dañadas. Además, cuando las células cancerosas fueron tratadas con una combinación de AZA y L-butionina-[S,R]-sulfoximina (BSO), un inhibidor específico de la síntesis de glutation, las células murieron por necrosis, independientemente de caspasa-3 pero de manera dependiente de la activación de las quinasas del estrés (p38 y JNK) y de la integridad de la membrana mitocondrial. Estos datos sugieren un mecanismo de muerte celular regulado conocido como necroptosis. Cuando las células cancerosas (HepG2) fueron introducidas subcutáneamente en los ratones inmunodeprimidos para desarrollar el tumor, se observó que el volumen del mismo se incrementaba con el tiempo. El tratamiento intratumoral con AZA y BSO produjo una disminución significativa del volumen del tumor con respecto a los animales tratados con sólo el excipiente (DMSO). Los estudios de seguridad, llevados a cabo por métodos bioquímicos, mostraron efectos moderados de la combinación de AZA y BSO con respecto al efecto del DMSO sólo. En conclusión, la AZA en combinación con inhibidores de la autofagia (por ejemplo, BAF) o con depletores de glutation (por ejemplo, BSO) podría ser útil para el tratamiento de tumores sólidos de hígado u origen gastrointestinal

Immunogenicity of Therapeutic Antibodies: Monitoring Antidrug Antibodies in a Clinical Context

One of the factors that may impact drug levels of therapeutic antibodies in patients is immunogenicity, with potential loss of efficacy. Nowadays, many immunogenicity assays are available for testing antidrug antibodies (ADA). In this article, we discuss different types of immunogenicity assays and their clinical relevance in terms of drug tolerance, relation with pharmacokinetics (PK), neutralizing antibodies, potential adverse events associated with ADA, and prediction of ADA production. Drug-tolerant assays can provide insight into the process of immunogenicity, but for clinical management, these assays do not necessarily outperform drug-sensitive assays. The usefulness of any ADA assay for clinical decision making will be larger when drug concentrations are also measured, and this is true, in particular, for drug-tolerant assay

Dendritic Nanotheranostic for the Delivery of Infliximab: A Potential Carrier in Rheumatoid Arthritis Therapy

Antibodies are macromolecules that specifically recognize their target, making them good candidates to be employed in various therapies. The possibility of attaching a drug to an immunoglobulin makes it possible to release it specifically into the affected tissue as long as it overexpresses the target. However, chemical coupling could affect the functionality (specificity and affinity) of the antibody. It has been observed that the use of intermediaries, such as dendrimers, could resolve this issue. Because carbosilane dendrimers have aroused great interest in the field of biomedicine, this report describes the synthesis of an anionic carbosilane dendrimer with a fluorochrome on its surface that then forms a conjugate with an antibody. It has been used as immunoglobulin and infliximab, whose target is TNF-α, which is a cytokine that is overexpressed in the inflamed area or even in the blood of patients with autoimmune diseases, such as rheumatoid arthritis. In addition, the integrity and functionality of the antibody has been studied to see if they have been affected after the chemical coupling process

Low Serum BAFF Concentration Is Associated with Response to TNF Inhibitors in Seropositive Patients with Rheumatoid Arthritis

We investigated B-cell-activating factor (BAFF) in relation to response to treatment with TNF inhibitors (TNFis) in rheumatoid arthritis (RA). This was a longitudinal study including 158 patients with RA treated with TNFis and followed up for 6 months. Clinical response at 6 months of treatment was defined according to the EULAR criteria for good responders (GRs). BAFF concentration was measured in serum samples, collected at baseline and at 6 months. Associations with EULAR response were evaluated using univariable and multivariable logistic regression models. ROC analysis was performed to determine the optimal threshold of serum BAFF concentration associated with good EULAR response to treatment. After 6 months of TNFi treatment, 24% of patients were GRs. They had a lower BMI, lower baseline DAS28 and lower baseline serum BAFF concentration than non-responders. After 6 months of TNFi treatment, autoantibody-positive patients who attained GR had significantly lower serum BAFF concentrations compared with patients who did not. Serum BAFF < 968 pg/mL at 6 months represented the concentration likely to best discriminate between GR and non-GR at 6 months of TNFi treatment. Autoantibody-seropositive patients who had serum BAFF < 968 pg/mL at 6 months demonstrated a more than four-fold increased probability to be GRs compared with patients with higher BAFF concentrations. In conclusion, serum BAFF concentrations were associated with response to TNFis in seropositive RA patients, corroborating the importance of the B-cell compartment in RA

Therapeutic drug monitoring of biopharmaceuticals in inflammatory rheumatic and musculoskeletal disease: A systematic literature review informing EULAR points to consider

The objectives of this review were to collect and summarise evidence on therapeutic drug monitoring (TDM) of biopharmaceuticals in inflammatory rheumatic and musculoskeletal diseases and to inform the EULAR Task Force for the formulation of evidence-based points to consider. A systematic literature review (SLR) was performed, covering technical aspects and (clinical) utility of TDM, to answer 13 research questions. MEDLINE, Embase and Cochrane were searched until July 2020. American College of Rheumatology and EULAR abstracts were also considered for inclusion. Data were extracted in evidence tables and risk of bias assessment was performed. For the search on technical aspects, 678 records were identified, of which 22 papers were selected. For the clinical utility search, 3846 records were identified, of which 108 papers were included. Patient-related factors associated with biopharmaceutical blood concentrations included body weight, methotrexate comedication and disease activity. The identification of a target range was hampered by study variability, mainly disease activity measures and study type. Evidence was inconsistent for multiple clinical situations in which TDM is currently applied. However, for some particular scenarios, including prediction of future treatment response, non-response to treatment, tapering and hypersensitivity reactions, robust evidence was found. There is currently no evidence for routine use of proactive TDM, in part because published cost-effectiveness analyses do not incorporate the current landscape of biopharmaceutical costs and usage. This SLR yields evidence in favour of TDM of biopharmaceuticals in some clinical scenarios, but evidence is insufficient to support implementation of routine use of TDM

Association between concomitant csDMARDs and clinical response to TNF inhibitors in overweight patients with axial spondyloarthritis

Abstract Background The aim of our study was to investigate the influence of conventional synthetic disease-modifying anti-rheumatic drugs (csDMARDs) and body mass index (BMI) on circulating drug levels and clinical response to tumour necrosis factor inhibitor (TNFi) therapy in axial spondyloarthritis (axSpA) patients. Methods Prospective observational study during 1 year with 2 cohorts (Madrid and Amsterdam) including 180 axSpA patients treated with standard doses of infliximab or adalimumab. Patients were stratified by BMI, being 78 (43%) normal weight (18.5–24.9 kg/m2) and 102 (57%) overweight/obese (≥ 25.0 kg/m2). After the first year of treatment, TNFi trough levels were measured by capture ELISA. Clinical response to TNFi was defined as ∆BASDAI ≥ 2 and clinical remission as BASDAI < 2 and CRP ≤ 5 mg/L. Logistic regression models were employed to analyse the association between concomitant csDMARDs and BMI with drug levels and clinical response. Results Seventy-nine patients (44%) received concomitant csDMARDs. The administration of concomitant csDMARDs (OR 3.82; 95% CI 1.06–13.84) and being normal weight (OR 18.38; 95% CI 2.24–150.63) were independently associated with serum TNFi drug persistence. Additionally, the use of concomitant csDMARDs contributed positively to achieve clinical response (OR 7.86; 95% CI 2.39–25.78) and remission (OR 4.84; 95% CI 1.09–21.36) in overweight/obese patients, but no association was found for normal-weight patients (OR 1.10; 0.33–3.58). Conclusions The use of concomitant csDMARDs with TNFi may increase the probability of achieving clinical response in overweight/obese axSpA patients. Further research studies including larger cohorts of patients need to be done to confirm it

Preclinical evaluation of azathioprine plus buthionine sulfoximine in the treatment of human hepatocarcinoma and colon carcinoma

AIM: To evaluate the efficacy and the safety of azathioprine (AZA) and buthionine sulfoximine (BSO) by localized application into HepG2 tumor in vivo

Association between concomitant csDMARDs and clinical response to TNF inhibitors in overweight patients with axial spondyloarthritis

Abstract Background The aim of our study was to investigate the influence of conventional synthetic disease-modifying anti-rheumatic drugs (csDMARDs) and body mass index (BMI) on circulating drug levels and clinical response to tumour necrosis factor inhibitor (TNFi) therapy in axial spondyloarthritis (axSpA) patients. Methods Prospective observational study during 1 year with 2 cohorts (Madrid and Amsterdam) including 180 axSpA patients treated with standard doses of infliximab or adalimumab. Patients were stratified by BMI, being 78 (43%) normal weight (18.5–24.9 kg/m2) and 102 (57%) overweight/obese (≥ 25.0 kg/m2). After the first year of treatment, TNFi trough levels were measured by capture ELISA. Clinical response to TNFi was defined as ∆BASDAI ≥ 2 and clinical remission as BASDAI < 2 and CRP ≤ 5 mg/L. Logistic regression models were employed to analyse the association between concomitant csDMARDs and BMI with drug levels and clinical response. Results Seventy-nine patients (44%) received concomitant csDMARDs. The administration of concomitant csDMARDs (OR 3.82; 95% CI 1.06–13.84) and being normal weight (OR 18.38; 95% CI 2.24–150.63) were independently associated with serum TNFi drug persistence. Additionally, the use of concomitant csDMARDs contributed positively to achieve clinical response (OR 7.86; 95% CI 2.39–25.78) and remission (OR 4.84; 95% CI 1.09–21.36) in overweight/obese patients, but no association was found for normal-weight patients (OR 1.10; 0.33–3.58). Conclusions The use of concomitant csDMARDs with TNFi may increase the probability of achieving clinical response in overweight/obese axSpA patients. Further research studies including larger cohorts of patients need to be done to confirm it