Abstract
Chemical modification of sickle hemoglobin (Hb S) to form stable high affinity Schiff-base adducts has been an attractive approach towards finding a potential therapeutic option for sickle cell disease (SCD). An ideal candidate drug should rapidly enter the bloodstream, permeate red blood cell membrane, bind specifically with intracellular Hb S and inhibit cell sickling with minimal adverse effect. In an effort to find drugs that satisfy these criteria, we recently designed, synthesized and studied three novel benzaldehydes (INN-296, INN-298 and INN-312) with enhanced potency. The compounds are pyridyl derivatives of benzaldehyde, and hence, combine structural features of two previously determined antisickling agents: vanillin and pyridoxal. All three compounds shifted the allosteric equilibrium of Hb S toward the oxy- or R-state by destabilizing the deoxy- or T-state. The results of in vitro studies of the antisickling effects of a representative compound (INN-312) are reported. Upon incubation of suspensions of sickle erythrocytes (SS cells) with 0.5, 1 or 2 mM of INN-312 under hypoxia (4% O2 :96% N2) at 37°C, sickling of SS cells was inhibited in a dose-dependent manner (15 ± 2, 44 ± 10 and 81 ± 8% inhibition, respectively). Cation-exchange HPLC analysis of lysates from the pre-incubated SS cells revealed a new peak in addition to the original Hb S peak, indicative of formation of Schiff-base adducts of Hb. Oxygen equilibrium curves (OECs) of SS-cell suspensions and lysates were shifted toward the left in a dose-dependent manner. X-ray crystal structures of these derivatives revealed their symmetric binding to the two N-terminal αVal1 of Hb S, and seem to indicate that their superior antisickling activity may arise from effector-induced interference with Hb S polymerization, as well as shifting the OEC to the high affinity state. In vitro studies on INN-296 and INN-298 showed similar results.
Studies in vivo were performed using transgenic sickle mice (3 mice per group). The mice were treated intraperitoneally with single doses of 50, 100 or 150 mg/kg of INN-312. To study pharmacokinetic profiles of INN-312 in treated mice, blood samples (~20 μl each) were collected under anesthesia via retro-orbital venipuncture into EDTA tubes at 30 min, 1 h and every hour afterwards for 5 hours. Plasma from each sample was de-proteinized and analyzed by reversed-phase HPLC for quantification of INN-312 present in the blood. A non-compartmental pharmacokinetic model with first-order elimination rate was used to determine the plasma concentration-time data using PK Solutions 2.0 software (SUMMIT Research Services, Montrose, CO, USA). The area under the plasma concentration curve (AUC) increased in a dose-dependent manner (314 ± 22 μg/ml/min, 648 ± 33 μg/ml/min and 1044 ± 63 μg/ml/min in mice treated with 50, 100 and 150 mg/kg, respectively). The terminal half-life (T1/2= 0.75 ± 0.15 h), peak concentration time (Tmax= 0.5 h), and mean resident time (MRT= 1.2 ± 0.2 h) values were consistent for all three dosage groups. The observed maximum plasma concentration (Cmax)was also increased in a dose-dependent manner. These novel pyridyl derivatives of benzaldehyde shifted the position of Hb OEC toward the left most strongly among various compounds reported to date. Further detailed studies are necessary to validate this approach to developing better antisickling agents.
Substitution of lanthanide ions for calcium ions in the activation of bovine prothrombin by activated factor X. High affinity metal-binding sites of prothrombin and the derivatives of prothrombin activation.