Cystic fibrosis, regarded as rare disease, is a chronic hereditary disease with the highest incidence among Caucasians (more than 25,000 people in the European Union). Cystic Fibrosis is generated by mutations in the gene CFTR, a chloride and bicarbonate permeable transmembrane channel, that produce a defect on salt transport in epithelial cells, with serious consequences in several organs, like lungs, pancreas and intestine, causing mucus accumulation in these areas. This disease is potentially lethal and patients usually die from respiratory complications related to bacterial lung infections. Currently there is no cure, only treatments that improve symptoms and prolong life expectancy. Additionally, the drugs used currently only allow treating patients with a specific mutation. TAT-CF project evaluated small molecules capable of facilitating the transmembrane transport of anions such as chloride and bicarbonate and will thus enable CF treatment by replacing the missing CFTR anion permeation activity. This represents an unexplored path in the treatment of CF and a paradigm shift with respect to current strategies searching for a cure for CF. To achieve this goal we set up a comprehensive program to validate this research concept and complete the preclinical development of a new lead compound, making it ready for early clinical development.
TAT-CF was divided into 8 Work Packages:
This WP focused on the synthesis of new compounds capable of facilitating transmembrane transport of anions (anionophores). Based in the extensive background on small molecule anion transporters synthesis and design the first candidates were produced from the beginning of the project and over the lifetime of the project UBU and AVIDIN have synthesised around 200 novel compounds, based on different potential anionophores structures. Seven lead compounds were selected based on their transport activities in different cell lines as well as their toxicological profile.
•WP2 Compound Activity Screening.
Novel libraries of anionophores were considered for their ability to transport anions with different methods. The screening was initially done in phospholipid liposome models and then cell models and procedures with increasing complexity. The toxicity properties of the anionophores have also determined as a relevant point to consider in order to optimize analogs with improved features. On the basis of the data obtained, we have been able to select six lead anionophores showing high capacity of transport iodide, chloride and bicarbonate, potency and low cytotoxicity.
•WP3. Cell Biophysics and Physiology.
The central objective of this workpackage has been the characterization of the transport mechanism of anionophores, identifying those that have a reduced toxicity, so that they can be applied to cells or model systems of bronchial epithelium. Ultimately, it has been proven that the application of anionophores on the apical side of the epithelium induces a reduction in the viscosity of the mucus, probably related to the transport of anions. Regarding sensors, two Chinese Hamster Ovary (CHO) sensor cell lines detecting chloride transport and bicarbonate transport activity of anionophores were developed. SIG characterized several compounds using the developed chloride sensor cell as well as the bicarbonate sensor cell.
•WP 4. Medicinal Chemistry
Based on already described ionophore molecules and drug-like compounds identified in WP2, three pharmacophore structures were identified and the defined basic structures serve the basis for generating additional new and diverse scaffolds by using scaffold hopping. Pharmacophores were also used for hit identification and lead optimization. Continuous information on selected chemical structures and their cellular in vitro activity has enabled us to perform a detailed SAR analysis. This information has driven the optimization of lead compounds
•WP5: Preclinical Studies
We have obtained relevant preclinical information for the selected leads, including in vitro and in vivo efficacy and toxicology data. Innovative preclinical models, like lung epithelial cells from p.F508del iPS cells and organoids have been developed, helping to obtain more significant efficacy models. On a second step, acute and repeated dose toxicity, in oral and pulmonary delivery have been studied for the more relevant compounds in rodents, showing promising, low toxicity results. A pilot, limited study has been performed using knockout mice harboring CF mutation.
•WP6. Drug Delivery and Formulation
We have performed characterization of the substances in order to determine their drugability profiles. This has included stability, solubility, at different conditions. We have also developed different formualtions, including nanoformulations in lipid matrices in order to obtain final formulations for oral and pulmonary delivery, including nanoparticles freeze drying. The formulations have been successfully tested in animal models (oral and pulmonary delivery)
•WP7 – Dissemination and exploitation
Communication and dissemination actions have been developed during the project in order to maximize the visibility and awareness of TAT-CF results. Three scientific publications were released during the lifetime opf the project and TAT-CF results were presented in several key international conferences in the field such as the ECFS Basic Science Conference. In December 2018 a dissemination event held in Burgos attracted the interest from several patients associations and the lectures were recorded and made available in youtube. A patent application to protect the results of the project was filled in march 2019.
•WP8 – Coordination and management
A continuous technical, financial and administrative management has been carried out during the first period of the project. Seven project meetings have been held during this period in different locations. Finally, UBU as coordinator has carried out a regular monitoring of Quality Assurance and risk analysis procedures.
Our most important achievement is the demonstration of the viability of this therapeutic approach. The best in vitro preclinical model of CF disease are synthetic epithelium derived from primary human bronchial epithelial cells. We have proved that our compounds can correct CF epithelia function to normal values in terms of fluid reabsorption and mucus viscosity, two key parameters in the pathophysiology of CF patients. We have paved the way to develop therapies based in TAT-CF central idea, bypassing CFTR function using synthetic molecules facilitating an alternative pathway to anion permeation in epithelia. Although the current results in clinical trials of combinations of potentiators and correctors are very encouraging, unfortunately it is not expected that these developments would provide therapeutic options to all CF patients. In particular, there are several class of mutations, such as nonsense mutations, resulting in no CFTR being produced for which this strategy has no efficacy. Patients harboring these mutations are comparatively more prevalent in some European countries than anywhere in the world. TAT-CF results represent a new hope to all of them.