We previously demonstrated that β-sitosterol (BSS) inhibits the expression of the chemokine IL-8 in CF bronchial epithelial cells exposed to P. aeruginosa. In the mouse model of lung chronic infection, herein shown, BSS significantly reduced leukocyte recruitment in the bronchoalveolar lavage fluid and decreased bacteria recovered in the airways. Treatment with BSS decreased the expression of key cytokines involved in immune response, mainly neutrophil chemotaxis, in the lung homogenate. This anti-inflammatory activity is accompanied by a beneficial protecting activity against infection and improvement of health status. Our data suggest that BSS has the potential to become a new drug to target the excessive neutrophil recruitment in lungs chronically infected by P. aeruginosa and encourage future investigations on mechanism of protection driven by BSS.

Drug repositioning and the relevance of orphan drug designation for β-thalassemia is reviewed. Drug repositioning and similar terms ('drug repurposing', 'drug reprofiling', 'drug redirecting', ‘drug rescue’, ‘drug re-tasking’ and/or 'drug rediscovery') have gained great attention, especially in the field or rare diseases (RDs), and represent relevant novel drug development strategies to be considered together with the “off-label” use of pharmaceutical products under clinical trial regimen. The most significant advantage of drug repositioning over traditional drug development is that the repositioned drug has already passed a significant number of short- and long-term toxicity tests, as well as it has already undergone pharmacokinetic and pharmacodynamic (PK/PD) studies. The established safety of repositioned drugs is known to significantly reduce the probability of project failure. Furthermore, development of repurposed drugs can shorten much of the time needed to bring a drug to market. Finally, patent filing of repurposed drugs is expected to catch the attention of pharmaceutical industries interested in the development of therapeutic protocols for RDs. Repurposed molecules that could be proposed as potential drugs for β-thalassemia, will be reported, with some of the most solid examples, including sirolimus (rapamycin) that recently has been tested in a pilot clinical trial.

In this review article, we present the fascinating story of rapamycin (sirolimus), a drug able to induce γ-globin gene expression and increased production of fetal hemoglobin (HbF) in erythroid cells, including primary erythroid precursor cells (ErPCs) isolated from β-thalassemia patients. For this reason, rapamycin is considered of great interest for the treatment of β-thalassemia. In fact, high levels of HbF are known to be highly beneficial for β-thalassemia patients. The story of rapamycin discovery began in 1964, with METEI, the Medical Expedition to Easter Island (Rapa Nui). During this expedition, samples of the soil from different parts of the island were collected and, from this material, an antibiotic-producing microorganism (Streptomyces hygroscopicus) was identified. Rapamycin was extracted from the mycelium with organic solvents, isolated, and demonstrated to be very active as an anti-bacterial and anti-fungal agent. Later, rapamycin was demonstrated to inhibit the in vitro cell growth of tumor cell lines. More importantly, rapamycin was found to be an immunosuppressive agent applicable to prevent kidney rejection after transplantation. More recently, rapamycin was found to be a potent inducer of HbF both in vitro using ErPCs isolated from β-thalassemia patients, in vivo using experimental mice, and in patients treated with this compound. These studies were the basis for proposing clinical trials on β-thalassemia patients.

The β-thalassemias are hereditary monogenic diseases characterized by a low or absent production of adult hemoglobin and excess in the content of α-globin. This excess is cytotoxic for the erythroid cells and responsible for the β-thalassemia-associated ineffective erythropoiesis. Therefore, the decrease in excess α-globin is a relevant clinical effect for these patients and can be realized through the induction of fetal hemoglobin, autophagy, or both. The in vivo effects of sirolimus (rapamycin) and analogs on the induction of fetal hemoglobin (HbF) are of key importance for therapeutic protocols in a variety of hemoglobinopathies, including β-thalassemias. In this research communication, we report data showing that a decrease in autophagy-associated p62 protein, increased expression of ULK-1, and reduction in excess α-globin are occurring in erythroid precursors (ErPCs) stimulated in vitro with low dosages of sirolimus. In addition, increased ULK-1 mRNA content and a decrease in α-globin content were found in ErPCs isolated from β-thalassemia patients recruited for the NCT03877809 clinical trial and treated with 0.5–2 mg/day sirolimus. Our data support the concept that autophagy, ULK1 expression, and α-globin chain reduction should be considered important endpoints in sirolimus-based clinical trials for β-thalassemias.

The β-thalassemias are hereditary monogenic diseases characterized by low or absent production of adult hemoglobin and excess in the content of α-globin. This excess is cytotoxic for the erythroid cells and responsible of the β-thalassemia associated ineffective erythropoiesis. Therefore, the decrease of α-globin excess is a relevant clinical effect for these patients and can be obtained by induction of fetal hemoglobin, by autophagy, or both. The in vivo effects of sirolimus (rapamycin) and analogues on induction of fetal hemoglobin (HbF) is of key importance for therapeutic protocols in a variety of hemoglobinopathies, including β-thalassemia. In this research communication, we report data showing that decrease of autophagy-associated p62 protein, increased expression of ULK-1 and reduction of the excess of α-globin is operating in erythroid precursors (ErPCs) stimulated in vitro with low dosages of sirolimus. In addition, increased ULK-1 mRNA content and decrease of α-globin content were found in ErPCs isolated from β-thalassemia patients recruited for the NCT03877809 clinical trial and treated with 0.5-2 mg/day sirolimus. Our data support the concept that autophagy, ULK1 expression and α-globin chain reduction should be considered important endpoints in sirolimus-based clinical trials for β-thalassemia.