The Clinical Drugs Palbociclib And Pimozide Suppress IgE?mediated Mast Cell Activation In Vitro And In Vivo

Background: Allergic diseases caused by hypersensitive immune reactions,including allergic asthma,allergic rhinitis,and atopic dermatitis,affect one in five people worldwide currently,and have been increasing with socioeconomic development.Mast cells(MCs),the principle effector cells of innate immunity,play a major role in allergy pathogenesis by releasing mediators,chemokines,and cytokines,and are thus a key target for suppression of allergic inflammation.Antihistamines and steroids are the main allergic disease therapies,but can produce adverse secondary effects.Although biologic anti-allergy medicines can be effective,especially in patients with recalcitrant conditions or those with intolerable secondary effects to standard medication,they require close monitoring.There remains a need for additional antiallergy strategy options.New pharmacotherapies may be derived from new drug development or drug repurposing.We have observed that the CDK inhibitor palbociclib and the dopaminergic receptor antagonist pimozide can inhibit MC activation.Palbociclib is an FDA-approved first-line treatment for advanced estrogen receptorpositive(ER+)/human epidermal growth factor-receptor 2 negative(HER2-)breast cancer.Pimozide is an FDA-approved treatment for psychosis,Tourette syndrome,and drug-resistant tics.Objectives: 1.To study the effects of palbociclib and pimozide on Ig E-mediated MC activation and degranulation in vitro.2.To investigate the ability of palbociclib and pimozide to suppress passive cutaneous anaphylaxis(PCA)and ovalbumin(OVA)-induced active systemic anaphylaxis(ASA)in vivo.3.To analyze the expression of MC-activation signaling molecules and explore the molecular mechanisms by which palbociclib and pimozide influence MC activation.Methods: Rat basophils(RBLs)and mouse bone marrow-derived mast cells(BMMCs)were sensitized with anti-dinitrophenol(DNP)immunoglobulin E(Ig E)antibody treatment,and then challenged with DNP-human serum albumin(HSA)antigen(Ag)with or without palbociclib/pimozide pretreatment.Release of the cellular mediator histamine and β-hexosaminidase and expression of the MC activation surface marker CD63 were measured.MC morphology,which changes upon activation,and granule release were examined with toluidine blue staining.Rearrangement of filamentous actin(F-actin)that enables MC degranulation was quantitated with phalloidin staining and fluorescence microscopy.PCA and OVA-induced ASA models were used to detect the effects of palbociclib and pimozide on allergic reactions in vivo.Immunoblotting was used to detect the expression of signal molecules related to MC activation.Results: The MTT results showed that the cell viabilities of RBLs and BMMCs were not affected by palbociclib at 100μM.Activated RBLs and BMMCs exhibited dramatic histamine and β-hexosaminidase release,which was reduced by palbociclib in a concentration-dependent manner(p < 0.05,p < 0.01 vs.the DNP-HSA group).In additionally,palbociclib inhibited expression of the MC activation marker CD63 in activated RBLs(p < 0.05,p < 0.01 vs the DNP-HSA group).Palbociclib inhibited granule release while preventing morphological changes(elongated shape maintained)and F-actin reorganization by toluidine blue staining and FITC-phalloidin staining(p < 0.05,p < 0.01,compared to the DNP-HSA group).Palbociclib pretreatment,prior to a DNP-HSA challenge,inhibited activation of Lyn as well as activation of MAPKs(p38,JNK,and ERK)in RBLs,as evidenced by decreased levels of p-Lyn,p-p38,p-JNK,and p-ERK1/2.Similarly,down-regulation of Lyn expression in RBLs did not affect palbociclib inhibition of mast cell activation,as evidenced by detection of β-hexosaminidase release.Similar results were obtained with the ERK inhibitor U0126,the JNK inhibitor SP600125,and the p38 inhibitor SB203580.Palbociclib inhibited dye solution extravasation and ear thickening in PCA mice dose-dependently(p < 0.05,p < 0.01 vs control).Palbociclib attenuated body temperature reduction and diminished total serum IL-4 and IL-10 levels in OVA-challenged ASA mice(p < 0.05,p < 0.01 vs control).Pimozide at 100μM showed no cell viabilities of RBLs and BMMCs compared to the control.Pimozide inhibited degranulation,reduced β-hexosaminidase release dosedependently in activated RBLs and BMMCs(p < 0.05 vs non-treated activated cells).Pimozide reduced MC degranulation,morphological changes and F-actin cytoskeletal changes by toluidine blue staining and FITC-phalloidin staining(p < 0.05 vs.activated cells without treatment).Ig E/Ag-induced migration was suppressed by pimozide dosedependently.Western blot analysis showed that pimozide pretreatment attenuated the phosphorylation of SYK,p38,ERK,and JNK in activated RBLs consistent with a dosedependent inhibitory effect(p <0.05 vs non-treated activated cells).Pimozide treatment decreased the PCA reaction markedly,as evidenced by a reduced ear thickness and diminished extrusion of dye from injected ears compared to the activated group.Pimozide treatment was associated with increased rectal temperature in OVAchallenged ASA mice,similar to the effect observed in the ketotifen(anti-allergy drug)positive control group.Additionally,pimozide decreased OVA-induced histamine release in serum.Furthermore,flow cytometry showed that following treatment with pimozide,the population of MCs among splenic cells was reduced significantly(p <0.05 vs activated group without pimozide or ketotifen pretreatment).Conclusions: Palbociclib and pimozide suppress Ig E-mediated MC activation in vitro and in vivo and should be considered for repurposing to suppress MC-mediated diseases.