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Ibrutinib and acalabrutinib: effects on platelet functions
bition and that the effect of the efflux pump inhibitors was visible at 30 min and maximal at 1 h in the LS group. These data are in agreement with those from Nicolson et al.27 and suggest that the intra-platelet concentration of the drug cor- relates with the aggregation defect.
Thus, with regard to collagen-induced platelet aggrega- tion in the normal population, two groups of individuals were distinguished based on their in vitro sensitivity to ibru- tinib, as previously found in CLL patients treated with this drug.10,11
Since acalabrutinib could be an option for patients requir- ing a switch from ibrutinib therapy, we analyzed its effect at the clinically relevant dose of 2 mM in the two groups. Acalabrutinib was less efficient than ibrutinib on maximal platelet aggregation induced by collagen (Figure 1C, D). The ibrutinib LS donors were not affected by acalabrutinib and a large proportion of ibrutinib HS donors were not or only weakly affected. In a small percentage of donors (10%) both drugs strongly inhibited collagen-induced platelet aggregation. Dose-dependent curves illustrate the lack of effect of acalabrutinib in the LS group and its relatively weak effect in the HS group (Figure 1E). However, while acalabrutinib was less efficient than ibrutinib on maximal platelet aggregation, it consistently delayed the aggregation response (Figure 1D). This is illustrated by a decrease in the area under the aggregation curve (Figure 1C, D). This effect was dose-dependent and more pronounced in the ibrutinib HS group (Figure 1E). It is noteworthy that acalabrutinib had no impact on platelet aggregation induced by thrombin receptor activating peptide (TRAP), the thromboxane A2 analog U46619 or ADP but did affect to some extent platelet aggregation induced by the GPVI agonist, collagen- related peptide, particularly in the HS group (Online Supplementary Figure S3). Of note, the drug efflux pump inhibitors alone or in combination did not significantly amplify the effect of acalabrutinib on maximal platelet aggregation but tended to increase its impact on the delay of aggregation in response to collagen (3.3 mg/mL) in the LS group (data not shown).
The effect of acalabrutinib on collagen-induced platelet aggregation was also tested in vitro in PRP from 16 Btk inhibitor-naïve CLL patients. The maximal platelet aggrega- tion evoked by collagen was reduced compared to that of healthy donors but two groups, ibrutinib LS (n=4) and ibru- tinib HS (n=12), were again identified (Figure 2A). While acalabrutinib had no significant effect in ibrutinib LS CLL patients, it significantly decreased the maximal platelet aggregation in ibrutinib HS CLL patients (Figure 2B). In the ibrutinib HS group, only one patient was not sensitive to acalabrutinib.
Acalabrutinib is less efficient than ibrutinib at inhibiting signaling events downstream of GPVI
We then compared the impact of acalabrutinib and ibru- tinib on tyrosine phosphorylation events downstream of GPVI using washed platelets. Compared with PRP, in which plasma proteins are known to bind and sequester the drug, acalabrutinib was more efficient at inhibiting colla- gen-induced aggregation of washed platelets. In ibrutinib LS donors the half maximal inhibitory concentration (IC50) was 1.07 ± 0.35 mM, while it was 0.69 ± 0.44 mM in HS donors (Figure 3A). Both ibrutinib and acalabrutinib strong- ly inhibited Btk autophosphorylation in ibrutinib LS and HS groups (Figure 3B). Ibrutinib was very efficient at blocking PLCγ2 phosphorylation on the Btk-dependent phosphory-
lation site Tyr753 in both groups. Acalabrutinib at 1 or 2 mM significantly inhibited PLCγ2 Tyr753 phosphorylation in both groups (Figure 3B). The observed stronger effect of ibrutinib on PLCγ2 phosphorylation would be consistent with an off-target effect of this drug on Tec and possibly Src kinases. Indeed, consistent with previous reports,10,24 ibruti- nib significantly affected Src activation as assessed by the intensity of its tyrosine 418 phosphorylation. The effect of ibrutinib was more pronounced in the ibrutinib HS group (44 ± 6% inhibition in the HS group vs. 15 ± 5% in the LS group, P<0.01, n=7 for HS and n=10 for LS). Interestingly, while ibrutinib inhibited Src particularly in the HS group, acalabrutinib had no or very little effect on Src activation in both groups. The improved profile of acalabrutinib over ibrutinib on global tyrosine phosphorylation events was confirmed by western blot analysis of the pan-tyrosine phosphorylation pattern in response to collagen stimulation (Online Supplementary Figure S4).
Weak impact of acalabrutinib on thrombus formation on collagen under flow
Ibrutinib has been shown to affect thrombus formation and stability on a collagen matrix under flow12,24 and firm platelet adhesion on von Willebrand factor.10 Given the dif- ference of effects of acalabrutinib observed in the two groups of healthy donors, we performed platelet adhesion and thrombus formation assays under an arterial shear rate to mimic the in vivo situation. Whole blood, treated or not
A
B
Figure 2. Effect of ibrutinib and acalabrutinib on collagen-induced platelet aggregation in vitro in patients with chronic lymphocytic leukemia. Platelet- rich plasma from 16 Bruton kinase inhibitor-naïve patients with chronic lympho- cytic leukemia was treated or not with ibrutinib or acalabrutinib (ACP) for 1 h at 37°C and stimulated with collagen 3.3 mg/mL. Platelet aggregation was assessed by turbidimetry during 10 min. (A) Four patients were identified as having low sensitivity (LS) to ibrutinib (reduction of maximal platelet aggrega- tion <50%, blue) and 12 as having high sensitivity (HS) to ibrutinib (reduction of maximal platelet aggregation >50%, red). (B) Results, expressed as percentage of maximal aggregation in the two groups, are mean ± standard error of mean. **P<0.01, ***P<0.001 according to one-way analysis of variance.
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