Prazosin induced lysosomal tubulation interferes with cytokinesis and the endocytic sorting of the tumour antigen CD98hc.

The quinazoline based drug prazosin (PRZ) is a potent inducer of apoptosis in human cancer cells. We recently reported that PRZ enters cells via endocytosis and induces tubulation of the endolysosomal system. In a proteomics approach aimed at identifying potential membrane proteins with binding affinity to quinazolines, we detected the oncoprotein CD98hc. We confirmed shuttling of CD98hc towards lysosomes and upregulation of CD98hc expression in PRZ treated cells. Gene knockout (KO) experiments revealed that endocytosis of PRZ still occurs in the absence of CD98hc - suggesting that PRZ does not enter the cell via CD98hc but misroutes the protein towards tubular lysosomes. Lysosomal tubulation interfered with completion of cytokinesis and provoked endoreplication. CD98hc KO cells showed reduced endoreplication capacity and lower sensitivity towards PRZ induced apoptosis than wild type cells. Thus, loss of CD98hc does not affect endocytosis of PRZ and lysosomal tubulation, but the ability for endoreplication and survival of cells. Furthermore, we found that glutamine, lysomototropic agents - namely chloroquine and NH4Cl - as well as inhibition of v-ATPase, interfere with the intracellular transport of CD98hc. In summary, our study further emphasizes lysosomes as target organelles to inhibit proliferation and to induce cell death in cancer. Most importantly, we demonstrate for the first time that the intracellular trafficking of CD98hc can be modulated by small molecules. Since CD98hc is considered as a potential drug target in several types of human malignancies, our study possesses translational significance suggesting, that old drugs are able to act on a novel target.

Impact of simulated lung fluid components on the solubility of inhaled drugs and predicted in vivo performance.

Orally inhaled products (OIPs) are gaining increased attention, as pulmonary delivery is a preferred route for the treatment of various diseases. Yet, the field of inhalation biopharmaceutics is still in development phase. For a successful correlation between various in vitro data obtained during formulation characterization and in vivo performance, it is necessary to understand the impact of parameters such as solubility and dissolution of drugs. In this work, we used in vitro-in silico feedback-feedforward approach to gain a better insight into the biopharmaceutics behavior of inhaled Salbutamol Sulphate (SS) and Budesonide (BUD). The thorough characterization of the in vitro test media and the impact of different in vitro fluid components such as lipids and protein on the solubility of aforementioned drugs was studied. These results were subsequently used as an input into the developed in silico models to investigate potential PK parameter changes in vivo. Results revealed that media comprising lipids and albumin were the most biorelevant and impacted the solubility of BUD the most. On the contrary, no notable impact was seen in case of SS. The use of simple media such as phosphate buffer saline (PBS) might be sufficient to use in solubility studies of the highly soluble and permeable drugs. However, its use for the poorly soluble drugs is limited due to the greater potential for interactions within in vivo environment. The use of in silico tools showed that the model response varies, depending on the used media. Therefore, this work highlights the relevance of carefully selecting the media composition when investigating solubility and dissolution behavior, especially in the early phases of drug development and of poorly soluble drugs.