In terms of cytotoxicity studies under two
In terms of cytotoxicity studies under two-dimensional cell culture conditions, only two of the above-mentioned publications with liposomal formulations of tyrosine kinase inhibitors reported viability data as compared to respective free drugs. On the one hand, liposomal gefitinib exerted strongly reduced activity compared to free gefitinib (with a fold change between 15 and 20) while, on the other hand, liposomal cabozantinib exhibited a much stronger cytotoxicity compared to the free inhibitor (fold change between 0.02 and 0.18). Usually, PEGylated liposomal formulations should reduce the biological activity compared to the free drugs.51, 52, 53 As such, so-called “stealth liposomes” hamper endocytic uptake by macrophages.54, 55 Our liposomal formulations exhibited a double-edged behavior on a panel of FGFR-driven lung cancer cell lines. At low concentrations similar activities compared to the free drugs were observed, but at concentrations higher than 1 μM the activity of the liposomal formulations was distinctly lower. To further investigate this Clonidine HCl effect, the uptake kinetics were analyzed by flow cytometry exploiting the intrinsic fluorescence properties of the three FGFR inhibitors ( and unpublished data). Indeed, quite similar uptake dynamics (at least for nintedanib and PD173074) were observed at 1 μM, while the one of ponatinib was slower in case of the nanoformulations. In contrast, the uptake was strongly reduced compared to the free drugs at 10 μM for all three TKIs in line with the lower cytotoxic activity. Concerning NCI-H1703 cells, the very strong sensitivity of this cell line towards FGFR inhibition may account for the unaltered cytotoxicity profiles of liposomal as compared to free drugs even despite reduced intracellular uptake levels. Importantly, longer drug exposure times (168 h) led to a comparable activity profile between free and liposomal ponatinib in vitro. This observation may reflect the desired temporary detargeting effect of liposomal drug encapsulation, leading to an initial latency phase (up to 72 h at higher concentrations), but eventually to an assimilation of anticancer activity compared to the free drug at later time points. Finally, the liposomal formulation with the highest EE (liposomal ponatinib) was investigated in vivo against the K7M2 osteosarcoma allograft in BALB/c mice. K7M2 cells express high levels of FGFR1 and exhibit exquisite sensitivity against FGFR inhibitors in vitro. Off-target toxicity represents a major obstacle in systemic anticancer therapy. In case of molecularly-designed TKIs, this is based on expression of target RTKs also in normal tissues. Due to the high conservation of tyrosine kinase domains, TKIs often exhibit non-exclusive specificity profiles. This also applies for ponatinib, which – besides Abl kinase and FGFR – also targets VEGFR and PDGFR family members. These receptors are highly expressed in endothelial cells and, as such, are crucial factors in the maintenance of blood vessel integrity. Consequently, ponatinib administration has been observed to pose a considerable risk for vascular toxicity.58, 59 In the clinical situation, commonly observed ponatinib adverse effects include cutaneous toxicity such as non-specific rash or dryness. Moreover, several cases of pityriasis rubra pilaris-like dermatitis as well as folliculocentric, ichthyosiform and seborrhoeic eruptions have been documented in patients. The skin-associated side-effects observed in our study in mice treated with free ponatinib likely recapitulated these clinical symptoms. The absence of such side effects of liposomal ponatinib demonstrates that a nanoparticulate formulation is highly favorable with respect to its safety profile as compared to the free drug. We assume that this improved toxicity profile is a consequence of a reduced non-specific biodistribution. Proof of concept of this assumption and in-depth analysis of differences in pharmacokinetics such as tissue distribution as well as histopathological effects between free and liposomal ponatinib are pending in a current follow-up study.