2  Lysosomal ion-trapping
If the pH of the molecular environment is lower (more acidic), more nitrogens are protonated, which in turn hinders the now-charged moieties from crossing membranes. This is particularly relevant for the intracellular distribution of basic drugs crossing between the cytosol (pH approx. 7.4) and the acidic lysosomal space (pH approx. 5.0). Fig. 2 shows this distribution conceptually. Basic compounds are in an equilibrium of a less polar unionized form (B) that can easily cross membranes, and a polar protonated form (BH+) that cannot easily cross membranes. As the unionized drug enters the acidic environment of the lysosome, it will be protonated and ‘trapped’ in the lysosome as the protonated form BH+ cannot easily diffuse back into the cytosol. As a result, high concentrations of the compound can accumulate in lysosomes. This concept of “ion-trapping” has been described and reviewed [7,8]. The magnitude of this accumulation depends on the mathematical relationship of the pKa of the compound of interest, its permeability, and the pH gradient between the two environments (e.g. cytosol [pH 7.4] and lysosome [pH 5.0]). If there is no permeability limitation, the expected ratio or concentration gradient can be calculated based on the well-known Henderson-Hasselbalch equation [8]. If the compound of interest has only one basic group, the ratio between the concentrations will be:(1) R=C1C2=H1+KaH2+Kawhere H1 and H2 are the respective proton concentrations (=10−pH) of the two environments (pH 5 and 7.4) and Ka is the dissociation constant (=10−pKa). Fig. 3 A shows the magnitude of the resulting accumulation as a function of the pKa value of the compound. Accumulation of up to 250-fold higher concentrations in lysosomes can be explained by the described mechanism.
Fig. 2 Concept of lysosomal ion-trapping. Basic compounds are in an equilibrium of a less polar unionized form (B) that can easily cross membranes, and a polar protonated form (BH+) that cannot easily cross membranes. As the unionized drug enters the acidic environment of a lysosome, it will be protonated and ‘trapped’ in the lysosome as the protonated form BH+ cannot easily diffuse back into the cytosol. As a result, high concentrations of the compound can accumulate in the lysosomes.
Fig. 3 Magnitude of lysosomal ion-trapping depending on the pKa of the compound of interest. For a monobasic compound (A), up to 250-fold higher concentrations are possible depending on pKa. For a dibasic compound (B), the accumulation can be over 60 000-fold. These simulations assume a pH gradient of 5.0 (lysosomes) and 7.4 (cytosol).
However, in cases where there are two basic centers in the molecule, this effect is considerably potentiated. There are two different monobasic species that are produced by protonation of the respective nitrogens, and neither of these can easily diffuse from the lysosome back into the cytosol. Furthermore, both species are in equilibrium with the biprotonated species, which is also trapped in the lysosome. Therefore, there are three different forms of the molecule (two monoprotonated and one biprotonated) that cannot easily diffuse back to the cytosol. This tremendously magnifies the ion-trapping effect. The expected accumulation ratio under these conditions is calculated as follows:(2) R=C1C2=H12+Ka1*H1+Ka1*Ka2H22+Ka1*H2+Ka1*Ka2where H1 and H2 are the respective proton concentrations (=10−pH) of the two environments (pH 5 and 7.4) and Ka1 and Ka2 are the dissociation constant (=10−pKa). Fig. 3B shows the magnitude of the resulting accumulation as a function of the pKa value of the compound, assuming equal values for pKa1 and pKa2. Accumulation of up to 60 000-fold higher concentrations in the lysosomes can be explained by the described mechanism.