The objective of this research line is to develop and validate new strategies that facilitate safer and more effective pharmaceutical development by using liver cell models to investigate drug metabolism and hepatotoxicity. Research involves developing highly differentiated cellular models and multi-parametric screening tests to predict and classify the toxicity of new compounds based on the simultaneous evaluation of different biomarkers indicative of different cellular functions and/or mechanisms of toxicity. Essentially, two main approaches are used: transcriptomic and metabolomic analysis, and high-content microscopy image analysis techniques. Furthermore, the research in this area aims to develop models and strategies that allow for further investigation of the various mechanisms of hepatotoxicity, including cholestasis, steatosis, phospholipidosis, mitochondrial disruption, oxidative stress, metabolic idiosyncrasy, and inactivation. It also aims to identify biomarkers for clinical diagnosis, monitoring, prevention, prognosis and treatment of drug-induced hepatotoxicity.

The research group has successfully applied metabolomic analysis techniques to drug-induced liver toxicity studies. By measuring the metabolic changes that occur in hepatocytes after toxic exposure, it is possible to relate the effects observed in vitro to clinical data and to determine the mechanisms of toxicity involved. Several studies using model hepatotoxins support the use of serum or urine metabolomics analysis as a powerful approach to search for new biomarkers of DILI. Metabolomics analysis has proven to be a powerful tool for accurately describing the phenotype of liver damage, estimating the degree of metabolic recovery of the liver, and for the clarification of the mechanisms of DILI.

The main objective of this line of research is to develop non-invasive strategies and tools to monitor the extent of non-alcoholic fatty liver disease (NAFLD), mainly based on metabolomic and miRNomic analysis of patient serum. Bariatric surgery patients who are morbidly obese often have comorbidities related to obesity, such as NAFLD. They provide an excellent clinical model for studying the disease, as clinical samples (serum and liver biopsy) can be obtained during the intervention. The metabolome and miRNome can then be studied to monitor the disease's progression and the patient’s response to treatment.

The main hypothesis of the project suggests that multiple transcriptional regulatory pathways are involved in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). The overall aim is to investigate new transcriptional mechanisms involved in the development and progression of NAFLD and, in particular, the toxicogenomic effects of steatotic drugs. Additionally, the group aims to identify specific biomarkers (e.g. microRNAs, metabolites, etc.) that can differentiate between metabolic and drug-induced steatosis. Currently, a clinical study is being conducted in paediatric patients treated with valproate, a drug that, as shown by in vitro studies, can cause fat accumulation in hepatocytes and is suspected of causing silent steatosis in paediatric patients. This clinical study aims to identify epileptic paediatric patients at risk of iatrogenic hepatic steatosis by applying steatosis biomarkers (metabolomic and miRNomic) discovered in studies of fatty liver patients (both serum and liver) and in human hepatocyte culture studies treated with valproate.         

La finalitat última d'esta línia d'investigació és determinar l'estat metabòlic d'un òrgan de donant cadavèric previ al seu implant a través de l'anàlisi metabolómico d'una mostra de teixit hepàtic. La presencia/absència de determinats metabòlits és un bon predictor del grau d'èxit funcional del fetge implantat. I d'esta manera es pretén disposar d'un indicador que, més enllà dels aspectes morfològics i histològics del fetge, assenyale la seua capacitat funcional i l'èxit del trasplantament. L'estudi inicialment desenvolupat a l'hospital La FE ha donat pas a un estudi multicèntric amb la participació de 5 unitats de Trasplantament hepàtic i prop de 1000 trasplantaments estudiats.

This research area focuses on the development of scoring systems that help predict the long-term outcomes of both the liver transplant patients and the graft itself. Previous research has enabled the design and implementation of new scoring systems to asses a patient's progress following a liver transplant. The model uses normal clinical parameters. It has been validated and compared with other scoring systems. New models are currently being developed that combine the above parameters with others to provide a more accurate prediction of a patient's clinical outcome after transplantation. The test is also being adapted to assess liver function in patients undergoing major liver resection.

The group aims to explore and advance liver cell therapy with hepatocytes and adults as well as liver progenitors for the treatment of liver diseases. Another objective is to explore the clinical utility of other cell lines such as reprogrammed cells (iPSCs: direct and indirect conversion of fibroblasts into iHepSCs) or embryonic pluripotent stem cells (hESC). In this area, the use of iPSCs with genomic editing technology for personalised medicine is being considered as a realistic treatment for certain congenital metabolic diseases.

One of the main limitations of in vitro studies on drug-induced liver toxicity or other pathologies is their predominantly idiosyncratic nature, making it difficult to reproduce the unique characteristics of hepatocytes from such patients in vitro. Due to the impossibility of obtaining hepatocytes from these patients and maintaining them in culture to observe the pathological phenomenon in question, a strategy has been developed and implemented in the laboratory that generates hepatocytes of a given patient through direct reprogramming of somatic cells. The direct reprogramming process, which bypasses the pluripotent cell stage, involves the use of vectors that encode three essential hepatic transcription factors: HNF4a, HNF1a and FOXA3. With this triple infection, or with a polycistronic vector encompassing all three, liver-like cells (iHeps) can be generated rapidly. All three hepatic factors are preceded by a doxycycline-inducible promoter developed by our team. These cells are transfected with another vector containing the hTERT gene, which prevents entry into cellular senescence, and the rtTA required for the expression of promoter-regulated genes upon the addition of doxycycline. Thus, liver cells can be obtained from a patient without the need for biopsy. A clinical study is currently underway to demonstrate to what extent these iHEPs behave like liver hepatocytes. The study compares the behaviour, when exposed to different drugs, of hepatocytes obtained from a surgical liver biopsy and that of iHEPs generated from fibroblasts, mesenchymal and epithelial cells from the urinary tract of the same patients.

A phase I/II clinical trial has been launched in order to establish a simple test for determining the liver's metabolic capacity for xenobiotics through a minimally invasive procedure. The trial  involves measuring the levels of a combination of drugs and their metabolites in the patient's serum or urine after oral administration. By using drug/metabolite ratios and blood and urine kinetics, it is possible to determine the activity of liver enzymes involved in the biotransformation of the xenobiotics. The cocktail of drugs administered to the patients includes caffeine, paracetamol, dextrometorfene and clorfenilamine. Following oral administration, the presence of the drugs and their primary metabolites are detected in the subject's serum and urine and measured after one and two hours, respectively. The study was conducted on patients scheduled for liver surgery and was carried out one day prior to the procedure. A sample of liver tissue is taken during the surgical procedure to determine the enzymatic activities of drug biotransformation. These data are used to develop a model that estimates enzyme activities in the liver from serum/urine measurements and incorporates computer modelling of PBPK models.

Various strategies are being pursued to promote the use and application of liver cell transplantation. For instance, one approach involves generating induced hepatocytes (iHeps) for transplantation, while another approach, specific to X-linked diseases in heterozygous patients, takes advantage of the spontaneous phenomenon of silencing of one of the X alleles. This approach involves selecting fibroblast cells expressing the non-defective X allele and then reprogramming them into functional hepatocytes in order to treat metabolic diseases.  This technique enables the use of the patient's own cells for autologous transplantation, reducing the risk of immunological rejection.