Understanding the In Vitro-In Vivo Nexus: Advanced correlation models predict clinical performance of liposomal doxorubicin.

In the century of precision medicine and predictive modeling, addressing quality-related issues in the medical supply chain is critical, with 62 % of the disruptions being attributable to quality challenges. This study centers on the development and safety of liposomal doxorubicin, where animal studies alone often do not adequately explain the complex interplay between critical quality attributes and in vivo performances. Anchored in our aim to elucidate this in vitro-in vivo nexus, we compared TLD-1, a novel liposomal doxorubicin delivery system, against the established formulations Doxil® and Lipodox®. Robust in vitro-in vivo correlations (IVIVCs) with excellent coefficients of determination (R2 > 0.98) were obtained in the presence of serum under dynamic high-shear conditions. They provided the foundation for an advanced characterization and benchmarking strategy. Despite the smaller vesicle size and reduced core crystallinity of TLD-1, its release behavior closely resembled that of Doxil®. Nevertheless, subtle differences between the dosage forms observed in the in vitro setting were reflected in the bioavailabilities observed in vivo. Data from a Phase-I clinical trial facilitated the development of patient-specific IVIVCs using the physiologically-based nanocarrier biopharmaceutics model, enabling a more accurate estimation of doxorubicin exposure. This advancement could impact clinical practice by allowing for more precise dose estimation and aiding in the assessment of the interchangeability of generic liposomal doxorubicin.

Cross-species complementation of bacterial- and eukaryotic-type cardiolipin synthases.

The glycerophospholipid cardiolipin is a unique constituent of bacterial and mitochondrial membranes. It is involved in forming and stabilizing high molecular mass membrane protein complexes and in maintaining membrane architecture. Absence of cardiolipin leads to reduced efficiency of the electron transport chain, decreased membrane potential, and, ultimately, impaired respiratory metabolism. For the protozoan parasite Trypanosoma brucei cardiolipin synthesis is essential for survival, indicating that the enzymes involved in cardiolipin production represent potential drug targets. T. brucei cardiolipin synthase (TbCLS) is unique as it belongs to the family of phospholipases D (PLD), harboring a prokaryotic-type cardiolipin synthase (CLS) active site domain. In contrast, most other eukaryotic CLS, including the yeast ortholog ScCrd1, are members of the CDP-alcohol phosphatidyltransferase family. To study if these mechanistically distinct CLS enzymes are able to catalyze cardiolipin production in a cell that normally expresses a different type of CLS, we expressed TbCLS and ScCrd1 in CLS-deficient yeast and trypanosome strains, respectively. Our results show that TbCLS complemented cardiolipin production in CRD1 knockout yeast and partly restored wild-type colony forming capability under stress conditions. Remarkably, CL remodeling appeared to be impaired in the transgenic construct, suggesting that CL production and remodeling are tightly coupled processes that may require a clustering of the involved proteins into specific CL-synthesizing domains. In contrast, no complementation was observed by heterologous expression of ScCrd1 in conditional TbCLS knockout trypanosomes, despite proper mitochondrial targeting of the protein.

RFT1 Protein Affects Glycosylphosphatidylinositol (GPI) Anchor Glycosylation.

The membrane protein RFT1 is essential for normal protein N-glycosylation, but its precise function is not known. RFT1 was originally proposed to translocate the glycolipid Man5GlcNAc2-PP-dolichol (needed to synthesize N-glycan precursors) across the endoplasmic reticulum membrane, but subsequent studies showed that it does not play a direct role in transport. In contrast to the situation in yeast, RFT1 is not essential for growth of the parasitic protozoan Trypanosoma brucei, enabling the study of its function in a null background. We now report that lack of T. brucei RFT1 (TbRFT1) not only affects protein N-glycosylation but also glycosylphosphatidylinositol (GPI) anchor side-chain modification. Analysis by immunoblotting, metabolic labeling, and mass spectrometry demonstrated that the major GPI-anchored proteins of T. brucei procyclic forms have truncated GPI anchor side chains in TbRFT1 null parasites when compared with wild-type cells, a defect that is corrected by expressing a tagged copy of TbRFT1 in the null background. In vivo and in vitro labeling experiments using radiolabeled GPI precursors showed that GPI underglycosylation was not the result of decreased formation of the GPI precursor lipid or defective galactosylation of GPI intermediates in the endoplasmic reticulum, but rather due to modifications that are expected to occur in the Golgi apparatus. Unexpectedly, immunofluorescence microscopy localized TbRFT1 to both the endoplasmic reticulum and the Golgi, consistent with the proposal that TbRFT1 plays a direct or indirect role in GPI anchor glycosylation in the Golgi apparatus. Our results implicate RFT1 in a wider range of glycosylation processes than previously appreciated.