Abstract by Nina Mertz

Sustained drug release formulations for intra-articular (IA) administration constitutes a promising approach for local treatment of pain and inflammation associated with joint diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA). For this purpose, in situ forming drug delivery system based on the generation of inverse lyotropic non-lamellar liquid crystalline phases (LCPs) have been found of interest. The LCPs are formed through self-assembly of amphiphilic lipids in response to exposure to excess aqueous medium. For sustained drug delivery, the inverse bicontinuous cubic (Q₂) and inverse discontinuous hexagonal (H₂) phases appear most attractive, however, the high viscosity of these LCPs prevents their direct injection. To overcome this challenge, low viscous injectable preformulations can be prepared, from which the Q₂ and H₂ phases are formed in situ upon exposure to the biological environment. It is of interest to gain insight on the dynamic structural transitions taking place in the in situ forming process at biologically relevant conditions, as these events may influence the performance of the LCP depot.

Biologically relevant in vitro release models capable of mimicking the in vivo conditions of the synovial joint are needed to assist the development and evaluation of in situ forming LCP depot formulations for IA injection. Thus, to improve the understanding of events occurring during the in the in situ forming process, the aim of the present PhD project was to explore and develop in vitro methods for characterization of in situ forming LCP depots for IA administration. The investigations were performed employing the following in vitro methods: the Scissor (the Subcutaneous Injection-Site Simulator) system and an injection-cell setup applicable for spatially and time-resolved SAXS monitoring and UV-Vis imaging.

Lipid-based preformulations forming Q₂ and H₂ phases upon exposure to excess aqueous media were designed to be applied for evaluation of the developed in vitro methods. The preformulations were loaded with 18 mg/g diclofenac, a non-steroidal anti-inflammatory drug (NSAID), which is relevant for local treatment of pain and inflammation associated with OA and RA. In the studies involving the Scissor system, preformulations consisting of 76:4:10:10 % (w/w) Dimodan/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG)/ethanol/water or Dimodan/medium chain triglycerides (MCT)/ethanol/water were employed, whereas preformulations composed of 72:8:10:10 % (w/w) glycerol monooleate (GMO)/ 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DOPG)/ethanol/ water and 68:12:10:10 % (w/w) GMO/MCT/ethanol/water were used in the UV-Vis imaging and time-resolved SAXS studies.

The Scissor system, a commercially available two-compartment release model, was modified towards conditions relevant for IA administration, e.g., by incorporating biologically relevant matrix components (hyaluronic acid (HA) and human serum albumin (HSA)) and introducing an injection-step into the small donor compartment. As compared to the release from aqueous solution, sustained release of diclofenac from the in situ formed LCPs was observed in the Scissor system, and it was possible to discriminate between the initial diclofenac release from LCPs depots with different structural characteristics. The duration of the in vitro release experiments in the Scissor system was limited to 7 h due to the observed escape of HA and HSA from the donor compartment. The results presented here suggests that the modified Scissor system may be useful for in vitro studies of the initial release of in situ forming depot formulations for IA administration.

An injection-cell setup applicable for synchrotron SAXS and UV-Vis imaging was developed to combine real-time monitoring of the dynamic structural transitions occurring during the in situ formation of the LCP depots and the initial drug release upon injection of the diclofenac-loaded preformulations into bio-relevant media at clinically relevant volumes. The time-resolved SAXS measurements showed a fast hydration-triggered in situ formation of the LCP depots leading to the formation of LCP depots characterized by different structural features depending on the lipid composition of the preformulations. 2D spatial maps constructed from SAXS measurements at 70 positions of the injection-cell, showed structural heterogeneity of the in situ formed LCP depots. The UV-Vis images recorded at 300 nm and 520 nm enabled quantification of the LCP depot sizes during the in situ forming process, where differences in the depot sizes and shapes were found for the GMO:DOPG- and the GMO:MCT LCP depots. From the recorded UV-Vis absorbance maps, difference in the initial diclofenac release from the LCP depots formed in situ from the GMO:DOPG- and GMO:MCT-based preformulations was apparent. This was most likely attributed to the varying sizes and shapes observed for the depots generated in situ from preformulations with different lipid composition. The injection-cell setup utilizing the combination of time-resolved SAXS measurements and UV-Vis imaging may be an attractive approach for evaluating the in situ forming process of LCP depots at conditions, which are biologically relevant for IA administration. Future development of the UV-Vis imaging in vitro release method should seek to include low flow conditions in the injection-cell setup to allow long-term release testing.

The findings obtained using a combination of the developed in vitro methods constitute important steps on the way towards a better understanding of the events occuring during the in situ forming process of LCP depots including the involved dynamic structural transitions and the initial drug release properties.