BCL11A-targeted γ-globin gene induction by triterpenoid glycosides of Fagonia indica: A preclinical scientific validation of indigenous herb for the treatment of ß-hemoglobinopathies.

Pharmacological induction of fetal hemoglobin has proven to be a promising therapeutic intervention in ß-hemoglobinopathies by reducing the globin chain imbalance and inhibiting sickle cell polymerization. Fagonia indica has shown therapeutic relevance to ß-thalassemia. Therefore, we study the ethnopharmacological potential of Fagonia indica and its biomarker compounds for their HbF induction ability for the treatment of ß-thalassemia. Here, we identify, compound 8 (triterpenoid glycosides) of F. indica. as a prominent HbF inducer in-vitro and in-vivo. Compound 8 showed potent erythroid differentiation, enhanced cellular proliferation, ample accumulation of total hemoglobin, and a strong notion of γ-globin gene expression in K562 cultures. Compound 8 treatment also revealed strong induction of erythroid differentiation and fetal hemoglobin mRNA and protein in adult erythroid precursor cells. This induction was associated with simultaneous downregulation of BCL11A and SOX6, and overexpression of the GATA-1 gene, suggesting a compound 8-mediated partial mechanism involved in the reactivation of fetal-like globin genes. The in vivo study with compound 8 (10 mg/kg) in ß-YAC mice resulted in significant HbF synthesis demonstrated by the enhanced level of F-cells (84.14 %) and an 8.85-fold increase in the γ-globin gene. Overall, the study identifies compound 8 as a new HbF-inducing entity and provides an early "proof-of-concept" to enable the initiation of preclinical and clinical studies in the development of this HbF-inducing agent for ß-thalassemia.

IVS I-5 (G > C) is associated with changes to the RBC membrane lipidome in response to hydroxyurea treatment in ß-thalassemia patients.

The red blood cell membrane loses its integrity during hemoglobinopathies like ß-thalassemia and sickle cell disease. Various mutations have been associated with ß-thalassemia, the most prevalent of which is the IVS-1-5 (G > C) mutation. It is associated with poor prognosis of the disease with a dependency on transfusion. Here, we have investigated the effect of IVS mutation and the administration of hydroxyurea on the red blood cell membrane lipidome isolated from patients using a liquid chromatography coupled to tandem mass spectrometry based approach to identify changes in the red blood cell membrane lipidome of patients with/without the mutation and being/not being administered hydroxyurea. A total of 50 patients, with/without hydroxyurea treatment, were recruited and 62 lipid species were identified in all groups after statistical analyses using fold change analysis, ANOVA and lipids with higher VIP values extracted from the OPLS-DA loading plot. The presence of the IVS mutation showed altered expression levels of various lipid species as compared to non-IVS individuals, such as phosphatidylcholines, steroids, phenol lipids and fatty acids. Significant changes were though found with the administration of hydroxyurea where both the IVS and non-IVS groups showed a marked increase in complex lipids of the membrane, while a decrease was observed in those without hydroxyurea administration showing degradation of these membrane lipids. This study is the first to report changes incurred by IVS mutation and hydroxyurea administration in red blood cell membranes extracted from ß-thalassemia patients. Hydroxyurea administration has been perceived to improve the lipid profile of the red blood cell membrane in both IVS and non-IVS patients.

In Vitro and In Vivo Studies for the Investigation of γ-Globin Gene Induction by Adhatoda vasica: A Pre-Clinical Study of HbF Inducers for ß-Thalassemia.

Fetal hemoglobin (HbF) is a potent genetic modifier, and the γ-globin gene induction has proven to be a sustainable therapeutic approach for the management of ß-thalassemia. In this study, we have evaluated the HbF induction ability of A. vasica in vitro and in vivo, and the identification of potential therapeutic compounds through a bioassay-guided approach. In vitro benzidine-Hb assay demonstrated strong erythroid differentiation of K562 cells by A. vasica extracts. Subsequently, an in vivo study with an aqueous extract of A. vasica (100 mg/kg) showed significant induction of the γ-globin gene and HbF production. While in the acute study, the hematological and biochemical indices were found to be unaltered at the lower dose of A. vasica. Following the bioassay-guided approach, two isolated compounds, vasicinol (1) and vasicine (2) strongly enhanced HbF levels and showed prominent cellular growth kinetics with ample accumulation of total hemoglobin in K562 cultures. High HbF levels were examined by immunofluorescence and flow cytometry analysis, concomitant with the overexpression in the γ-globin gene level. Compound 1 (0.1 µM) and compound 2 (1 µM) resulted in a greater increase in F-cells (90 and 83%) with marked up (8-fold and 5.1-fold) expression of the γ-globin gene, respectively. Molecular docking studies indicated strong binding affinities of (1) and (2) with HDAC2 and KDM1 protein that predict the possible mechanism of compounds in inhibition of these epigenetic regulators in the γ-globin gene reactivation. Altogether, these observations demonstrated the therapeutic usefulness of A. vasica for fostering HbF production in clinical implications for blood disorders.

XMN polymorphism along with HU administration renders alterations to RBC membrane lipidome in ß-thalassemia patients.

RBCs membrane loses its integrity during hemoglobinopathies such as ß-thalassemia and sickle cell disease. The severity of ß-thalassemia has been historically linked to the presence of XMN polymorphism which is believed to ameliorate the severity. Here, we investigate the effect of XMN polymorphism on RBC membrane lipidome isolated from patients, using LC-MS/MS based approach. A total of 50 patients were recruited and 28 lipid species were identified in all groups after statistical analyses using volcano plot and ANOVA-SCA, and lipids with higher VIP values extracted from OPLS-DA loading plot. Alteration in lipid levels specifically the membrane lipids such as PC and fatty acids were observed. Samples with XMN polymorphism exhibited up-regulation of lipids involved in membrane stability such as cholenoic acid while PC (O-41:1) was down-regulated when compared to non-XMN samples. Additionally, HU administration to samples also had profound effect on the lipids of patients in both groups. A trend of improvement in the membrane lipids was observed in patients with XMN polymorphism. HU administration has proven to further improve the membrane integrity by upregulating certain membrane lipids in such patients. The study presents a comprehensive analysis of RBC membrane lipidome with respect to the genetic variation and HU administration.

Monoterpenes as therapeutic candidates to induce fetal hemoglobin synthesis and up-regulation of gamma-globin gene: An in vitro and in vivo investigation.

Pharmacologically induced production of fetal hemoglobin (HbF) is a pragmatic therapeutic strategy for the reduction of globin chain imbalance and improving the clinical severities of patients with ß-hemoglobinopathies. To identify highly desirable new therapeutic HbF-inducing agents, we screened functionally diverse ten monoterpenes, as molecular entities for their potent induction and erythroid differentiation ability in human erythroleukemia cell line (K562) and transgenic mice. Benzidine hemoglobin staining demonstrated six compounds to have significantly induced erythroid differentiation of K562 cells in a dose and time-dependent manner. This induction paralleled well with the optimal accumulated quantity of total hemoglobin in treated cultures. The cytotoxic studies revealed that three (carvacrol, 3-carene, and 1,4-cineole) of the six compounds with their maximal erythroid expansion ability did not affect cell proliferation and were found non-toxic. Four compounds were found to have high potency, with 4-8-fold induction of HbF at both transcriptional and protein levels in vitro. Subsequently, an in vivo study with the three active non-cytotoxic compounds showed significant overexpression of the γ-globin gene and HbF production. Carvacrol emerged as a lead HbF regulator suggested by the increase in expression of γ-globin mRNA content (5.762 ± 0.54-fold in K562 cells and 5.59 ± 0.20-fold increase in transgenic mice), accompanied by an increase in fetal hemoglobin (F-cells) levels (83.47% in K562 cells and 79.6% in mice model). This study implicates monoterpenes as new HbF inducing candidates but warrants mechanistic elucidation to develop them into potential therapeutic drugs in ß-thalassemia and sickle cell anemia.