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Effects of Btk inhibitors on platelet activation
tion; namely no effect at low doses with blockade at high doses. For ibrutinib, we have shown that inhibition of aggregation correlates strongly with loss of phosphoryla- tion at Src Y418. However, this is not altered by acalabru- tinib demonstrating an as yet unidentified off-target action. Bye et al.13 also showed that ibrutinib dose-depen- dently inhibits phosphorylation of Src Y418. However, in a different study they found that both low-dose ibrutinib and acalabrutinib potentiated Src Y418 phosphorylation.29 We were not able to replicate this latter finding.
We have shown that, despite acalabrutinib’s more favorable selectivity to Btk over other Src, Syk and Tec kinases in in vitro kinase assays, the window between Btk inhibition and blockade of GPVI-induced aggregation in vitro is similar to that of ibrutinib. Despite this, acalabru- tinib, but not ibrutinib, fails to block GPVI-mediated platelet activation ex vivo. We propose that this is because of the differential dosing and pharmacodynamics of the two Btk inhibitors. Acalabrutinib is used at a dose of 1.5 mg/kg twice daily17,30 and ibrutinib at a single daily dose of 6 mg/kg in CLL or 8 mg/kg in mantle cell lymphoma. Pharmacokinetic studies have shown that ibrutinib achieves Btk occupancy of >95% at doses of 2.5 mg/kg but that doses of 6 mg/kg are required to maintain this over 24 h.31 Acalabrutinib at 1.5 mg/kg twice daily also achieves full Btk occupancy over 24 h.17 The peak unbound plasma concentration of ibrutinib in patients is 0.5 mM11 and that of acalabrutinib 1.3 mM.17 The initial and terminal half-lives of ibrutinib are 2-3 h and 4-8 h, respectively.31 The half-life of acalabrutinib is 1 h.17 Despite the peak concentration of acalabrutinib being approximately 2-fold higher than the concentration of ibrutinib, the 5-fold lower potency of acalabrutinib as an inhibitor of Btk30 means that, in potency terms, it is dosed at a lower level consistent with the lack of inhibition of GPVI. This implies that ibrutinib could be used at a lower concentration to achieve Btk blockade. Indeed, there is retrospective clinical evidence that doses less than 6 mg/kg are as effective as 6 mg/kg for treating CLL32 and a prospective clinical trial using doses as low as 2.5 mg/kg is being undertaken.33
It is important to consider the incidence of minor and major bleeding in patients taking ibrutinib for CLL or at the higher dose for mantle cell lymphoma. In reported studies involving patients treated with ibrutinib for man- tle cell lymphoma, minor and major bleeding was seen in 9-15% and 1-5% of patients, respectively.34-36 In the study with the largest cohort of patients with mantle cell lym- phoma, the rate of major hemorrhages was 5%. This is comparable to the 4-8% major hemorrhage rate seen in patients taking ibrutinib for CLL.9 Thus, there is no increase in bleeding rates with higher doses of ibrutinib. This implies that the inhibitory effect of 420 mg ibrutinib on platelets is at a physiological maximum.
During the writing of this manuscript, Bye et al. reported thrombus instability on collagen in a flow adhesion assay in blood treated in vitro with high doses of ibrutinib and ex vivo in patients treated with ibrutinib.29 This is consistent with our findings that Btk kinase function is not required for platelet adhesion to collagen under flow, but that off- target effects of ibrutinib seen with higher doses mediate this inhibition. They also reported complete blockade of platelet aggregation in response to supramaximal concen- trations of collagen in patients receiving ibrutinib or acal-
abrutinib29 in contrast to the findings of this study. Bye et al. used the Optimul 96-well microtiter assay to measure aggregation rather than the widely used light transmission aggregometry. We have shown that Optimul is a more sensitive assay than light transmission aggregometry.37,38 We suggest that the delay in onset of aggregation observed using light transmission aggregometry with con- centrations of ibrutinib or acalabrutinib that just block Btk manifest as complete blockade in the Optimul assay. Bye et al. also concluded that the increased bleeding observed with ibrutinib is due to blockade of Src family kinases (SFK).29 We agree that bleeding caused by ibrutinib is due to off-target actions, and that acalabrutinib has a greater selectively to Btk over SFK relative to ibrutinib. However, our results show that a similar fold increase in the concen- tration of ibrutinib and acalabrutinib causes inhibition of platelet aggregation in response to CRP but without con- comitant SFK blockade in the acalabrutinib-treated platelets. Thus, the off-target action of ibrutinib and acal- abrutinib that inhibits aggregation cannot be explained solely by differential blockade of SFK.
The results of our study explain the lack of major bleed- ing side effects experienced by patients taking acalabruti- nib and suggest that the bleeding side effect of ibrutinib can potentially be abolished by reducing the dose. Furthermore, this study also shows that the bleeding caused by ibrutinib is not due to an irreversible action. This predicts that the GPVI blockade wears off over a peri- od of 24 h as the drug is cleared.31 We hypothesize that, in the event of a major bleed, there may be no need to use expensive and potentially harmful platelet transfusions to correct the signaling deficit. Nevertheless, each clinical scenario should be judged on its own merits and individ- ual clinicians’ discretion is crucial.
In conclusion, the present study shows that inhibition of Btk kinase activity causes only partial inhibition of GPVI signaling in platelets and provides evidence that Btk supports GPVI signaling by functioning as an adapter pro- tein as well as a kinase. The excessive bleeding induced by ibrutinib relative to acalabrutinib is likely to reflect a non- Tec family kinase off-target inhibitory effect of ibrutinib, probably on Src.
Acknowledgments
This work was supported by British Haeart Foundation (BHF) Programme grants (RG/13/18/30563), a BHF clinical fellowship to PLRN (FS/17/20/32738), an AMS springboard grant to AYP (SBF002\1099) and BHF studentship to ATH, the University of Birmingham’s Institute of Translation Medicine and Institute of Cardiovascular Sciences; SPW holds a BHF Chair (CH03/003).
We would like to thank Alex Bye and Jon Gibbins for their expertise on the Ca2+ mobilization assay. We would also like to thank Vicky Simms, Natalie Poulter and Steve Thomas for their help with the flow adhesion assay and Mark Crowther, Nick Pemberton, Salim Shafeek, Kate Arthur, Gaynor Pemberton, Lesley Candlin and Rebekah Hart at Worcestershire Royal Hospital, Shankara Paneesha, Alison Hardy and Melanie Kelly at Birmingham Heartlands Hospital and Tina McSkeane, Gillian Marshall and Michelle Harry at the Queen Elizabeth Hospital for provision of patients’ samples. Finally, we would like to thank Andrew Wilkinson and Robert Neely from the School of Chemistry at the University of Birmingham for their help with the chemical analysis of ibrutinib and acalabrutinib.
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