*2.2. In Vitro Assessment of Lipomatrix Containing* SRO *Gastric Resistance*

One of the three aliquots (triplicate) coming from the gastric-resistance experiment with Lipomatrix powder containing SRO described in Materials and Methods (Section 3.2.4) and the one coming from the same experiment assessed on equivalent amount of not formulated SRO contained in two common gelatin soft-gel capsules have been collected and visually compared each other. An oily layer on the GSF surface is appreciable only in the beaker containing SRO from the soft-gel capsules, while no oily separation is appreciable in the beaker containing Lipomatrix powder. This provides a significant evidence that no SRO release occurred throughout the GSF during the test (Figure 3).

**Figure 3.** Visual comparison of the liquids coming from disaggregation test in GSF between Lipomatrix containing SRO (left beaker) and soft-gel SRO contained in two soft-gel capsules (right beaker). It is clearly appreciable the presence of an oily layer of SRO in the right beaker relating to the soft-gel capsules and the absence of the oily layer in the surface of the GSF liquid relating to Lipomatrix powder.

GC-MS analysis confirmed the absence of SRO derived fatty acids in GSF with Lipomatrix, suggesting that the technology guarantees a complete gastric resistance of SRO at full therapeutic dosage (Figure 4). The major explanation of this observed behavior is that ASP remains unionized in the stomach since low pH does not permit its enediolic ionization (pH < pKa) and this phenomenon contributes, in synergy with MDGFA, to the overall gastric-resistance of the powder (see the supplemental file section for more detailed explanation).

**Figure 4.** GC-MS overlapping chromatograms of SRO raw material and one of the aliquots of GSF tested (in triplicate, see the supplementary file). The upper graph shows the peculiar picks of fatty acids present in SRO (raw material) that completely disappeared in the lower graph (Lipomatrix specimen), clearly indicating that no SRO release from Lipomatrix has occurred.
