Identification, purification and biological studies of the lead compound from Chinese herbs for the reactivation of fetal hemoglobin expression

Hemoglobinopathies such as the sickle cell anemia and thalassemias are among the most prevalent serious genetic disorders affecting human populations and represent a major health burden worldwide. Inheritance of defected globin genes results in a clinical condition. Regular red cell transfusions and iron chelating therapy remain the basis of therapy for this disease for the majority of patients. However, in most of these patients, the upstream gamma globin genes are intact and fully functional. If these could be reactivated, functional hemoglobin synthesis could be maintained during adulthood, ameliorating the severity of the disease. For many years, the search for treatment aimed at reduction of globin chain imbalance in patients with thalassemia has focused on the pharmacological manipulation of fetal hemoglobin (alpha2gamma2; HbF).; In our project, a rational strategy was established by discovering new lead compounds to reactivate fetal hemoglobin in traditional Chinese herbal medicines. A previously established K562 cell screen model was adopted for random screening. Total 67 kinds of herbs were screened and one herb was discovered to have significant in vitro efficacy on inducing hemoglobin expression.; The chemical components of the herb were analyzed. It was found the biological activity was induced by a single lead compound, namely LC978. The lead compound LC978 was isolated and purified from the original herbal extract by newly established separation protocols. Its purity was analyzed by peak purity angle and threshold plot.; The in vitro pharmacological assessment of LC978 was performed on both K562 cells and normal human erythroid progenitor cells. Encouraging results were got from these analyses. LC978 could significantly induce different kinds of globin gene expression in K562 cells. The efficacy of LC978 is better than hydroxyurea, while the ED50 of LC978 is 12.5ng/ml, about 2000--3000 fold lower than hydroxyurea. The optimal dosage of LC978 is distant from its cytotoxic dosage, while for hydroxyurea it is very close.{09}Preliminary assessment on primary cultures of normal human erythroid progenitor cells showed similar results. LC978 achieved predominant in vitro performance, which involved its better biological activity and lower toxicity, comparing with other known drugs.; Preliminary molecular biological analysis also indicated that LC978 reactivate fetal hemoglobin expression through a signal transduction pathway that is likely different from hydroxyurea inducing pathway. Therefore, the discovery of LC978 also provided a promising chance for obtaining further insights on globin gene regulation mechanisms.