Iria Torres Terán

• SC administration is used for delivering drugs which cannot be administered via the oral route. Yet, the absorption profile after SC administration is difficult to predict from preclinical studies. One of the main reasons for this poor prediction may be the differences in the physiology that preclinical species and humans have, but there is a lack of information about the SC tissue composition and, particularly, about the fluid that perfuses this area, the ISF. Better knowledge on the composition and physicochemical properties of the ISF may provide better insights into in vivo drug performance and even allow for developing simulated ISF to be used in in vitro models, to further investigate in vivo drug performance. To date, a comparison of ISF from different preclinical species and humans has not been published. Furthermore, few simulated ISF media have been proposed. Yet, those are literature-based and most of them cannot be regarded as biorelevant since non-physiological components are part of the composition. In this context, this study aimed to investigate the composition and physicochemical properties of the ISF from preclinical species and humans, to discern interspecies differences and, ultimately, to develop SISFs for in vitro predictive assays. As a first step, ISF was isolated from the SC tissue of preclinical species and humans using the centrifugation method. This technique results in undisturbed ISF, a critical factor to consider given the invasiveness of the isolation process. Secondly, the isolated ISF samples were characterized using the same techniques across all samples, to enable data comparison. The characterization studies constituted a considerable contribution towards the understanding of the ISF composition across species. Similar values for pH, electrolyte content and osmolality were found for the ISF samples from the different species, while significant differences were observed for some parameters, particularly for albumin content. For instance, non-human primates ISF samples showed significantly higher albumin content than rodents ISF samples, while being in the same range than human ISF samples. After the isolation and characterization of ISF samples, the resulting data were utilized to develop simulated ISF media. These SISFs aimed to replicate as closely as possible, the experimental characterization data, while keeping overall composition and preparation of the media simple to ensure easy implementation in the laboratory. pH, osmolality, and electrolyte and albumin content were set as target parameters. Meanwhile, buffer capacity, colloid osmotic pressure and surface tension were defined as reference parameters, i.e., were only assessed after preparation. Initially, a buffer was prepared, considering the target pH, electrolyte content, and osmolality, as these parameters were consistent across all species. The physicochemical properties of this buffer were measured, and the buffer was designated as Blank SISF. Different albumin concentrations were incorporated into the Blank SISF for representing ISF of mice and rats (17.5 g/L) and non-human primates and humans (30.0 g/L), developing in this way two media: the SMR-ISF and the SHM-ISF. After preparation, the SISFs were screened in the same manner as the ISF samples, and the resulting data were compared. At this step, slight adjustments were made in the electrolyte composition to better approximate the desired physicochemical properties. Finally, in vitro experiments with the SISFs were conducted to analyze the influence of their composition, particularly their albumin content, on drug diffusion from the interstitial to the intravascular space. Liraglutide was selected as the model drug due to its high affinity for albumin. For these experiments, the Artificial Subcutaneous Tissue assay was utilized. The latter consists of a transwell plate with a gel-like artificial matrix mimicking key components of the extracellular matrix, such as collagen and hyaluronic acid. For these studies, SISF was added on the matrix prior to the incorporation of the formulation to be tested and PBS was used as receiver medium. Since PBS does not contain albumin, it was defined as Blank SPlasma. In a second step, Blank SPlasma was spiked with albumin to mimic the composition of the plasma samples determined in the characterization studies (25 g/L for the SMR-Plasma and 60 g/L for the SHM-Plasma). In this way, the SMR-ISF was used in conjunction with the SMR-Plasma in one setup, and the SHM-ISF was used together with the SHM-Plasma in a second setup. Higher diffusion rates were observed when the respective SISF at the apical side was combined with Blank SPlasma at the basolateral side, i.e., in absence of albumin in the receiver media. The samples analysis revealed the diffusion of both liraglutide and albumin through the membrane until equilibrium was achieved. Therefore, the results obtained with Blank SPlasma may be more representative of the physiological diffusion of liraglutide than those obtained with SPlasma containing albumin. Overall, these new media are promising tools in the context of developing new biorelevant models aiming to explain the differences in the in vivo performance of SC injectables in preclinical species and humans. This is because biorelevant components such as albumin significantly impact the physicochemical properties of the media. However, other aspects, such as the fat content difference in the SC tissue or even the exact injection point, may also influence in vivo performance and could be considered for future investigations. In conclusion, this study has provided essential insights into the composition and properties of the SC ISF, clarifying previously limited data. The findings suggest that, among the studied preclinical species, the ISF of non-human primates is the most similar to human ISF. Yet, further research is needed to understand which differences between non-human primates and humans influence the bioavailability profile disparities. The ISF characterization data were utilized to develop SISFs. The addition of albumin at targeted concentrations resulted in physicochemical properties closer to those of the reference ISF data. The results presented here indicate that these media offer several advantages over those previously described in literature. Principally, the SISFs provide a better representation of the ISF composition and physicochemical properties, while also accounting for the differences among species. Although the in vitro model used for the first in vitro experiments still requires refinement, the SISFs will undoubtedly help to better understand the in vivo performance of subcutaneously injected formulations. Furthermore, the SISFs can provide insights into how results obtained in preclinical species can be extrapolated to humans and, more importantly, avoid unnecessary animal testing. Overall, the work presented in this research therefore marks a significant step forward in developing in vitro tools to investigate the behavior of SC injectables and should inspire further investigation and refinement for future applications.