Discovery of Clinical Candidate ACT-777991, a Potent CXCR3 Antagonist for Antigen-Driven and Inflammatory Pathologies.

The CXCR3 chemokine receptor is a G protein-coupled receptor mainly expressed on immune cells from the lymphoid lineage, including activated T cells. Binding of its inducible chemokine ligands CXCL9, CXCL10, and CXCL11 leads to downstream signaling events and the migration of activated T cells to sites of inflammation. Herein, we report the third part of our CXCR3 antagonist program in the field of autoimmunity, culminating in the discovery of the clinical compound ACT-777991 (8a). A previously disclosed advanced molecule was exclusively metabolized by the CYP2D6 enzyme, and options to address the issue are described. ACT-777991 is a highly potent, insurmountable, and selective CXCR3 antagonist that showed dose-dependent efficacy and target engagement in a mouse model of acute lung inflammation. The excellent properties and safety profile warranted progress in the clinics.

Incorporation of mRNA in Lamellar Lipid Matrices for Parenteral Administration.

Insertion of high molecular weight messenger RNA (mRNA) into lyotropic lipid phases as model systems for controlled release formulations for the mRNA was investigated. Low fractions of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were used as an anchor to load the mRNA into a lamellar lipid matrix. Dispersions of zwitterionic lipid in the aqueous phase in the presence of increasing fractions of mRNA and cationic lipid were prepared, and the molecular organization was investigated as a function of mRNA and cationic lipid fraction. Insertion of both cationic lipid and mRNA was clearly proven from the physicochemical characteristics. The d-spacing of the lipid bilayers, as determined by small-angle X-ray scattering (SAXS) measurements, responded sensitively to the amount of inserted DOTAP and mRNA. A concise model of the insertion of the mRNA in the lipid matrices was derived, indicating that the mRNA was accommodated in the aqueous slab between lipid bilayers. Depending on the DOTAP and mRNA fraction, a different excess of water was present in this slab. Results from further physicochemical characterization, including determination of free and bound mRNA, zeta potential, and calorimetry data, were in line with this assumption. The structure of these concentrated lipid/mRNA preparations was maintained upon dilution. The functionality of the inserted mRNA was proven by cell culture experiments using C2C12 murine myoblast cells with the luciferase-encoding mRNA. The described lipid phases as carriers for the mRNA may be applicable for different routes of local administration, where control of the release kinetics and the form of the released mRNA (bound or free) is required.

Predicting the in vivo release from a liposomal formulation by IVIVC and non-invasive positron emission tomography imaging.

This study aimed to predict the in vivo performance from the in vitro release of a low-molecular weight model compound, [(18)F]-2-fluoro-2-deoxy-d-glucose ([(18)F]FDG), from liposomes and by means of positron emission tomography (PET). Liposomes composed of hydrogenated phosphatidylcholine (HPC) were prepared by a freeze-thaw method. Particle size distribution was measured by dynamic light scattering (DLS). In vitro release was examined with a dispersion method detecting the radioactivity of [(18)F]FDG. In vivo release of [(18)F]FDG, following i.p. injection of the liposomes in rats, was determined by using a Micro-PET scanner. Convolution was performed to predict the in vivo profiles from the in vitro data and to establish an in vitro-in vivo correlation (IVIVC). The in vivo predictions slightly underestimated the experimentally determined values. The magnitude of the prediction errors (13% and 19%) displayed a satisfactory IVIV relationship leaving yet room for further improvement. This study demonstrated for the first time the use of PET in attaining an IVIVC for a parenterally administered modified release dosage form. It is therefore possible to predict target tissue concentrations, e.g., in the brain, from in vitro release experiments. IVIVC using non-invasive PET imaging could thus be a valuable tool in drug formulation development, resulting in reduced animal testing.

Hyperbranched polyglycerol-based lipids via oxyanionic polymerization: toward multifunctional stealth liposomes.

We describe the synthesis of linear-hyperbranched lipids for liposome preparation based on linear poly(ethylene glycol) (PEG) and hyperbranched polyglycerol (PG). Molecular weights were adjusted to values around 3000 g/mol with varying degrees of polymerization of the linear and the branched segments in analogy to PEG-based stealth lipids; polydispersities were generally low and below 1.3. The hydrophobic anchors were introduced into the lipid structures as initiators for the anionic polymerization of ethylene oxide and are either based on cholesterol or on different aliphatic glyceryl ethers. Complete incorporation of the apolar initiators was evidenced by MALDI-ToF analysis at all stages of the reaction. The linear-hyperbranched polyether lipid is incorporated as the polyfunctional shell in liposome formulations together with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The resulting liposomes were subsequently characterized via dynamic light scattering (DLS) and small angle neutron scattering (SANS) as well as transmission electron microscopy (TEM), demonstrating the formation of unilamellar liposomes in the size range of 40 to 50 nm.