Contradistinction between doxorubicin and epirubicin: in-vitro interaction with blood components

The molecular structure and anti-tumour activity of doxorubicin and epirubicin are similar. However, the incidence of their cardiotoxicity occurs at different cumulative dose concentrations. The purpose of this study was to investigate the in-vitro interaction of these two drugs with different blood components, namely intact erythrocytes, haemoglobin and erythrocyte ghosts. Plasma protein binding was also evaluated. The intended goal was to identify the most relevant samples among total blood, plasma or blood cells for pharmacokinetic analysis. The methodology involved the incubation of each of the blood components (the intact erythrocytes, erythrocyte ghosts, haemoglobin and plasma proteins) at physiological pH and temperature with different concentrations of each drug, followed by measurement by HPLC and fluorometry at excitation and emission wavelengths of 480 and 580 nm, respectively. The results indicated that the binding of doxorubicin and epirubicin to plasma proteins, erythrocyte ghosts and intact erythrocytes was essentially the same. However, the binding of both compounds to intact erythrocytes was significantly different from erythrocyte ghosts, which indicates that haemoglobin plays an important role in the binding to and uptake by erythrocytes. The isotherms of binding to haemoglobin revealed that the maximum binding of doxorubicin was approximately 0.42 microg mg(-1) haemoglobin; for epirubicin this value was ten times greater than for doxorubicin. The Scatchard plot of binding of both drugs to haemoglobin exhibited two distinct binding sites for each drug. The constant of association of high affinity and low capacity binding sites was significantly greater for epirubicin, whereas the constant of association of low affinity and high capacity binding sites was significantly higher for doxorubicin. The number of high affinity binding sites per mg of haemoglobin was estimated to be 0.072 for doxorubcin and 0.030 for epirubicin. The number of low affinity binding sites was significantly greater for epirubicin (1.963) than for doxorubicin (0.305). Since the combined number of binding sites for epirubicin was more than doxorubicin, and the total uptake by erythrocytes remained the same for both drugs, it was concluded that epirubicin, being a more lipophilic compound, may diffuse more freely into the cells. Therefore, it binds more to haemoglobin, whereas doxorubicin remains more adsorbed on the surface of the cells due to its self-association property. It was concluded that the interaction of both drugs with erythrocytes, although it appears to be similar, is significantly different due to the interaction with haemoglobin. The difference in this interaction is expected to influence the disposition of both drugs in-vivo.