(M1330-02-11) Artificial Extracellular Matrices as Tools for In Vivo Injectable Formulation Analysis

Purpose: Striking differences have been observed in measured bioavailability between pre-clinical models and human clinical outcomes following subcutaneous delivery, particularly in the case of biopharmaceutical drugs.¹ To this end, the subcutaneous injection site simulator (SCISSOR) was designed and validated to better mimic the human subcutaneous extracellular matrix (ECM) environment with a series of monoclonal antibodies.² To expand on this body of work we present data for a range of pharmaceutical motifs (small molecules, peptides, proteins, and monoclonal antibodies) examined in the SCISSOR with artificial ECMs.

Methods: Material characteristics of artificial extracellular matrix - The rheological characteristics of the SCISSOR artificial extracellular matrix (ECM) and extended-release artificial extracellular matrix (ECM-XR) were analyzed before and after assay using an Anton-Paar® MCR102e rheometer. ECM and ECM-XRs were deposited between 25mm parallel plates and frequency scans were conducted from 100-0.01 s^-1 at 1% strain. SCISSOR assay of therapeutics - Multiple formulations including caffeine, insulin, denosumab, and superpositive (+) and supernegative (-) green fluorescent protein (GFP) were analyzed using the SCISSOR® system. Concentrations of API in the receiving chambers were monitored in real-time using in situ fiber optic dip probes connected to the Rainbow® UV-Vis spectrometer (Pion Inc.) or offline analysis was carried out with an Agilent A1100 HPLC after sampling

Results: ECM and ECM-XR material characteristics – After >100 hour assays the ECM showed a ~99% loss in complex viscosity, from 1.99 ±> 0.7 to 0.004 ± 0.001 Pa·s, while the ECM-XR conserved viscosity with a drop from 2.0 ± 0.1 to 0.6 ± 0.4 Pa·s. The conserved viscosity will allow for longer analysis within SCISSOR. (Table 1.) Caffeine injections – Caffeine release profiles were collected using the ECM and ECM-XR (50uL injection, N=3). The caffeine release from both models indicated complete release at 24 hours, with 1 trial exhibiting early release. (Figure 1) Superpositive (+) and supernegative (-) (GFP) injections – Release profiles within the ECM and ECM-XR of 50 uL +GFP and -GFP injections (N=1) were monitored over 20 hours. +GFP had 10% release over 15 hours in the ECM, while the ECM-XR released 0%. The -GFP had 100% release over 20 hours within the ECM, while the ECM-XR plateaued at 25% release at 5 hours. Denosumab injections – Release of denosumab injections (50 uL, N=3) were monitored over 1 week. ECM injections released to 100% within 72 ± 6 hrs, where the ECM-XR release plateaued at 73 ± 10% at 160 hours. (Figure 2.) Insulin injections – Rapid and basal insulin (50 uL, N=2) were injected into the ECM and ECM-XR and monitored over 4 days. In both the ECM and ECM-XR, rapid insulin reached 100% release in < 1 day. Basal insulin plateaued at < 20% release over the duration of the experiment.

Conclusion: Two artificial ECMs (ECM & ECM-XR) were evaluated using 5 model injectables within Pion’s subcutaneous injection site simulator (SCISSOR). The release profiles were compared to show how each model released over smaller time scales, while the ECM-XR could sustain release over 1 week. Caffeine injections into each model showed similar behavior over short time scales for both the ECM and ECM-XR, demonstrating the ECM-XR’s analogous behavior in shorter release studies. Injections of +/- GFP showed that the ECM-XR could appropriately complex +GFP for days, as opposed to the ECM release over the same time frame, demonstrating the expanded stability [BS1] of the ECM-XR. Lastly, commercially available formulations of insulin analogs and denosumab were injected to elucidate peptide and monoclonal antibody release behavior within the ECM-XR. In conclusion, although both the ECM and ECM-XR correctly model the environment of the human subcutaneous space, the ECM-XR demonstrated prolonged release profiles – up to 1 week.

References: 1. Kinnunen, H. M., Sharma, V., Contreras-Rojas, L. R., Yu, Y., Alleman, C., Sreedhara, A., . . . Mrsny, R. J. (2015). A novel in vitro method to model the fate of subcutaneously administered biopharmaceuticals and associated formulation components. Journal of Controlled Release, 94-102.
2. Bown, H. K., Bonn, C., Yohe, S., Yadav, D. B., Patapoff, T. W., Daugherty, A., & Mrsny, R. J. (2018). In vitro model for predicting bioavailability of subcutaneously injected monoclonal antibodies. J Control Release, 13-20.

Figure 1. Caffeine release profiles from the ECM and ECM-XR.



Figure 2. Relative denosumab release profiles from ECM (green) and ECM-XR (orange), N=3.



ECM and ECM-XR rheological parameters before and after >100 hour assays, 1% strain @ 0.9 s-1.