Development of Ternary Amorphous Solid Dispersions Manufactured by Hot-Melt Extrusion and Spray-Drying─Comparison of In Vitro and In Vivo Performance.

Producing amorphous solid dispersions (ASDs) by hot-melt extrusion (HME) is favorable from an economic and ecological perspective but also limited to thermostable active pharmaceutical ingredients (APIs). A potential technology shift from spray-drying to hot-melt extrusion at later stages of drug product development is a desirable goal, however bearing the risk of insufficient comparability of the in vitro and in vivo performance of the final dosage form. Hot-melt extrusion was performed using API/polymer/surfactant mixtures with hydroxypropyl methylcellulose acetate succinate (HPMCAS) as the polymer and evaluated regarding the extrudability of binary and ternary amorphous solid dispersions (ASDs). Additionally, spray-dried ASDs were produced, and solid-state properties were compared to the melt-extruded ASDs. Tablets were manufactured of a ternary ASD lead candidate comparing their in vitro dissolution and in vivo performance. The extrudability of HPMCAS was improved by adding a surfactant as plasticizer, thereby lowering the high melt-viscosity. d-α-Tocopheryl polyethylene glycol succinate (TPGS) as surfactant showed the most similar solid-state properties between spray-dried and extruded ASDs compared to those of poloxamer 188 and sodium dodecyl sulfate. The addition of TPGS, however, barely affected API/polymer interactions. The in vitro dissolution experiment and in vivo dog study revealed a higher drug release of tablets manufactured from the spray-dried ASD compared to the melt-extruded ASD; this was attributed to the different particle size. We could further demonstrate that the drug release can be controlled by adjusting the particle size of melt-extruded ASDs leading to a similar release profile compared to tablets containing the spray-dried dispersion, which confirmed the feasibility of a technology shift from spray-drying to HME upon drug product development.

Enhancing the Stabilization Potential of Lyophilization for Extracellular Vesicles.

Extracellular vesicles (EV) are an emerging technology as immune therapeutics and drug delivery vehicles. However, EVs are usually stored at -80 °C which limits potential clinical applicability. Freeze-drying of EVs striving for long-term stable formulations is therefore studied. The most appropriate formulation parameters are identified in freeze-thawing studies with two different EV types. After a freeze-drying feasibility study, four lyophilized EV formulations are tested for storage stability for up to 6 months. Freeze-thawing studies revealed improved colloidal EV stability in presence of sucrose or potassium phosphate buffer instead of sodium phosphate buffer or phosphate-buffered saline. Less aggregation and/or vesicle fusion occurred at neutral pH compared to slightly acidic or alkaline pH. EVs colloidal stability can be most effectively preserved by addition of low amounts of poloxamer 188. Polyvinyl pyrrolidone failed to preserve EVs upon freeze-drying. Particle size and concentration of EVs are retained over 6 months at 40 °C in lyophilizates containing 10 mm K- or Na-phosphate buffer, 0.02% poloxamer 188, and 5% sucrose. The biological activity of associated beta-glucuronidase is maintained for 1 month, but decreased after 6 months. Here optimized parameters for lyophilization of EVs that contribute to generate long-term stable EV formulations are presented.

Phenotypic drug screening in a human fibrosis model identified a novel class of antifibrotic therapeutics.

Fibrogenic processes instigate fatal chronic diseases leading to organ failure and death. Underlying biological processes involve induced massive deposition of extracellular matrix (ECM) by aberrant fibroblasts. We subjected diseased primary human lung fibroblasts to an advanced three-dimensional phenotypic high-content assay and screened a repurposing drug library of small molecules for inhibiting ECM deposition. Fibrotic Pattern Detection by Artificial Intelligence identified tranilast as an effective inhibitor. Structure-activity relationship studies confirmed N-(2-butoxyphenyl)-3-(phenyl)acrylamides (N23Ps) as a novel and highly potent compound class. N23Ps suppressed myofibroblast transdifferentiation, ECM deposition, cellular contractility, and altered cell shapes, thus advocating a unique mode of action. Mechanistically, transcriptomics identified SMURF2 as a potential therapeutic target network. Antifibrotic activity of N23Ps was verified by proteomics in a human ex vivo tissue fibrosis disease model, suppressing profibrotic markers SERPINE1 and CXCL8. Conclusively, N23Ps are a novel class of highly potent compounds inhibiting organ fibrosis in patients.

Formulation, process, and storage strategies for lyophilizates of lipophilic nanoparticulate systems established based on the two models paliperidone palmitate and solid lipid nanoparticles.

Lyophilization formulation and process development for lipophilic nanoparticle (NPs) products is highly challenging as the NPs have a low colloidal stability. We compared two different NP types, pure paliperidone palmitate nanocrystals and trimyristin solid lipid nanoparticles regarding formulation, process, and storage stability aspects. Freeze-thaw studies were conducted to investigate the basic formulation aspects such as buffer type, pH, and ionic strength as well as different cryoprotectants. In freeze-drying conventional ramp freezing was performed and compared to freezing with an annealing step added or with controlled ice nucleation. Different formulations were lyophilized and tested for short-term storage stability up to 6 weeks. Samples were analyzed for particle size, subvisible particle number, specific surface area, residual moisture, crystallinity, and glass transition temperature. Sucrose significantly better stabilized both NP types against freeze-thaw stress compared to mannitol demonstrating the importance of a fully amorphous matrix. While the impact of buffer type and pH was negligible, the aggregation propensity of NPs was reduced in presence of NaCl. The freezing step also impacted NP aggregation but the effect was less important than the formulation design. Surfactants did not necessarily improve the colloidal stability but resulted in a lower glass transition temperature of the lyophilizates and may cause phase separation which limits storage stability. This hurdle can be overcome by using a hydroxypropyl-ß-cyclodextrin/ sucrose mixture as cryoprotectant. In general, we could show a similar freeze-drying behavior of the two NP types. Thus, we established a formulation and process approach to achieve stable lyophilizates of lipophilic NPs based on two different types of NPs. The general rules should be transferable to other NPs facilitating lyophilization development.

Freeze-thaw stability of aluminum oxide nanoparticles.

The use of inorganic nanoparticles (NPs) gains interest for pharmaceutical applications, e.g. as adjuvants or drug delivery vehicles. Colloidal stability of NPs in aqueous suspensions is a major development challenge. Both frozen and lyophilized liquids are alternative presentations to liquid dispersion. To improve the basic understanding, we investigated the freeze-thawing stability of model α-Al2O3 NPs. Freeze-thawing was conducted in three different buffer types at pH5 and 8 without and with additives to determine fundamental formulation principles. Before freeze-thawing, α-Al2O3 NPs could be stabilized in sodium citrate buffer at pH5 and 8, and in sodium or potassium phosphate at pH8. Particles revealed low zeta potential values in phosphate buffers at pH5 indicating insufficient electrostatic stabilization. After freeze-thawing, an increase in NP size was strongly reduced in potassium phosphate and sodium citrate buffers. Subsequent pH measurements upon freezing revealed a drastic acidic pH shift in sodium phosphate which was further demonstrated to destabilize NPs. The ionic stabilizers gelatin A/B, Na-CMC, and SDS, were suitable to improve colloidal stability in phosphate buffers at pH5 highlighting the importance of charge stabilization. Freeze-thawing stability was best in presence of gelatin A/B, followed by PVA, mannitol, or sucrose. Depletion and steric stabilization were insufficient using PEG and surfactants respectively. Thus, we could identify the fundamental formulation principles to preserve inorganic NPs upon freezing: i) sufficient charge stabilization, ii) a maintained pH during freezing, and iii) the addition of a suitable stabilizer, preferably gelatin, not necessarily surfactants. This forms the basis for future studies, e.g. on lyophilization.

Freeze-drying of nanoparticles: How to overcome colloidal instability by formulation and process optimization.

Lyophilization of nanoparticle (NP) suspensions is a promising technology to improve stability, especially during long-term storage, and offers new routes of administration in solid state. Although considered as a gentle drying process, freeze-drying is also known to cause several stresses leading to physical instability, e.g. aggregation, fusion, or content leakage. NPs are heterogeneous regarding their physico-chemical properties which renders them different in their sensitivity to lyophilization stress and upon storage. But still basic concepts can be deducted. We summarize basic colloidal stabilization mechanisms of NPs in the liquid and the dried state. Furthermore, we give information about stresses occurring during the freezing and the drying step of lyophilization. Subsequently, we review the most commonly investigated NP types including lipophilic, polymeric, or vesicular NPs regarding their particle properties, stabilization mechanisms in the liquid state, and important freeze-drying process, formulation and storage strategies. Finally, practical advice is provided to facilitate purposeful formulation and process development to achieve NP lyophilizates with high colloidal stability.