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  • br Moreover AFC inhibited the ATP


    Moreover, AFC5261 inhibited the ATP-triggered IL-1β release from LPS-primed human THP1 TAK-242 in nanomolar concentrations, IC50 = 100.9 nM (Fig. 1A and B). AFC5261 displayed high CNS per-meability (brain/plasma ratio: 0.91) after oral administration and a half-life of approximately 1.5 h (data not shown).
    To validate sufficient in vivo concentration for effective P2X7R in-hibition, blood plasma concentrations were analyzed 2 h after admin-istration on day 6 and day 13, hence following 1 and 7 days of BID administration with 50 mg/kg, 100 mg/kg or 300 mg/kg AFC5261. All AFC5261 treated groups had plasma concentrations 3 to > 40 fold higher than the IC50 value (0.1 µM) for receptor inhibition, Fig. 2A and B. However this is only known for the respective timepoints 2 h after administration. Compound levels are not known for later timepoints.
    3.3. Acute P2X7R inhibition has no effect on cancer-induced bone pain
    Limb use deficit and weight-bearing displacement were assessed as measures of movement-evoked and background pain. Animals were observed daily until reaching a limb use score of 1 or 0. At this time, a pre-administration value was determined, followed by administration of vehicle, 300 mg/kg AFC5261 or morphine. The animal was retested 
    60 min after administration. Acute treatment with AFC5261 had no effect on neither movement-evoked nor background pain, (P > 0.05), Fig. 3A and B. Since no effect was observed for 300 mg/kg AFC5261, lower doses was not tested. Morphine was included as a positive con-trol, and in contrast to the AFC5261-treated animals, and significant increase in both limb use score and weight-bearing ratio was observed in the morphine-treated animals (P < 0.05 for each), Fig. 3A and B.
    3.4. Chronic P2X7R inhibition exacerbates cancer-induced bone pain
    In a chronic treatment approach, animals were administrated with 50 mg/kg, 100 mg/kg, 300 mg/kg or vehicle BID from day 5. The high dose of 300 mg/kg AFC5261 exacerbated the pain-related behavior, demonstrated by an earlier onset of both limb use and weight-bearing deficits. In contrast, the lower doses of 50 mg/kg and 100 mg/kg had no effect. From day 11 the high dose AFC5261-treated cancer animals displayed a significantly lower limb use score compared to the sham group (P < 0.05), Fig. 4A. In contrast the 100 mg/kg, 50 mg/kg AFC5261 and vehicle-treated animals, did not develop significant limb use deficits compared to the sham group until day 14 or 17 (P < 0.05 or P < TAK-242 0.01). The exacerbation of movement-evoked pain in the high dose AFC5261-treated group was supported by an earlier onset of weight-bearing displacement. The high dose AFC5261-treated group demonstrated a significantly lower weight-bearing ratio already on day
    Fig. 4. 300 mg/kg chronic BID administration with AFC5261 significantly exacerbated both limb use and weight-bearing demonstrated as an earlier onset of pain-related behavior. In contrast, 50 mg/kg and 100 mg/kg had no effect of the pain-related behavior compared to vehicle treatment (A, B). The exacerbation did not translate to a significantly earlier time point for reaching humane endpoint (C). n = 8–14.
    11 compared to sham animals (P < 0.05), whereas the middle, low and vehicle-treated cancer groups did not develop significantly lower weight-bearing ratio compared to sham until day 14 (P < 0.01–0.0001), Fig. 4B. Although a tendency was observed, the exacerbation in pain-related behavior did not translate to a significantly earlier time point for reaching the humane endpoint (P = 0.062), Fig. 4C.
    3.5. Chronic P2X7R inhibition has no effect on bone degradation
    The effect of chronic AFC5261-treatment on bone degradation was evaluated by x-ray densitometry. Overall, all cancer-bearing animals demonstrated similar bone degradation over time. Animals treated with vehicle or middle dose of AFC5261 had a significantly lower relative bone density compared to their own baseline on day 8 (P < 0.05), Fig. 5A, whereas the low and high dose AFC5261-treated cancer ani-mals displayed a significantly lower relative bone density compared to their own baseline from day 11 (P < 0.0001 for all), Fig. 5A. However, no difference was found between the AFC5261-treatet cancer groups at any time point, and the variation on day 8 is therefore most likely a reflection of normal inter-animal variation.