None of the BSc2118-treated animals (n = 7) demonstrated any signs of toxicity that could be identified by observation. No weight loss, diarrhea, hair loss, or neurological symptoms (like tremor, ataxia, paralysis) were observed in BSc2118-treated mice, even if mice were treated daily with a 60 mg/kg dose for 7 consecutive days. In mice treated
with bortezomib the drug was administered at 1 Venetoclax nmr mg/kg every second day. Animals that were given higher daily doses of bortezomib died on day three after start of treatment (data not shown). Concluding, BSc2118 inhibits the proteasome activity In Vivo to a similar extend as bortezomib (Figure 3), but is better tolerated and less toxic in spite of daily administration. Some proteasome inhibitors like MG132 become instable in presence of microsomal fractions due to various mechanisms involving binding or modification. To analyze the resistance of BSc2118 against microsomal proteins, measurement of 20S inhibitory activity in the presence/absence of liver microsomes was performed. As shown in Figure 4, BSc2118 is stable for up to 4 hours of incubation with microsomes. After 8 hours of incubation with liver microsomes, there is an observed 30% loss of inhibitory activity. Thus, BSc2118 turned out to be more stable than MG132, which lost its activity at 4 hours in a dose and time dependent manner, i.e., 50% of activity loss at 4
hours and 70% of activity loss at 8 hours, After 24 hours of treatment, the activity loss for both MG132 and BSc2118 was comparative. To track the biodistribution of selleck BSc2118, the Bodipy-labeled BSc2118 (BSc2118-FL) was directly analyzed in tissue sections detecting spontaneous fluorescence by means of confocal microscopy. In order to achieve sufficient direct fluorescence signal, dosages of BSc2118-FL had to be at least 10 mg/kg body weight. Although dosages of 5 mg/kg body weight significantly inhibited 20S activities and induced accumulation of polyubiquitinated proteins, spontaneous fluorescence signal was too weak for direct observations (data not shown). The fluorescence within erythrocytes mounted after 1 hour or 24 hours post
i.p. injection (10 C59 clinical trial mg/kg) is depicted in (Figure 5A). Erythrocytes (0 h) from vehicle injected mice served as control to visualize the autofluorescence signal of hemoglobin. The inhibitor-specific fluorescence was most pronounced in animals at 1 hour after inhibitor injection. After 24 hours the fluorescence intensity was approximating the baseline due to inhibitor fluorescence instability or to the low reversibility of Bodipy-BSc2118. In organs, bright specific BSc2118-FL fluorescence was observed exemplarily in intestine (I), heart (II) and in proximal tubules of kidney (III) ( Figure 5B). Remarkably, no fluorescence was observed in intact brain (not shown), confirming the 20S activity data of brain lysates included in Figure 3.