Metabolism And Clinical Pharmacokinetics Of Some Tyrosine Kinase Inhibitors

The overexpression,imbalances or mutations in protein kinases can lead to several diseases,especially developmental and metabolic disorders and cancer.Tyrosine kinase inhibitors?TKIs?have shown great therapeutic effects in clinical applications,which have the advantages of high selectivity and small adverse reactions,leading their therapeutic success for various cancers.The study focued on the metabolism and clinical pharmacokinetics?PK?of some TKIs.The main contents include the following four parts:?1?Pharmacokinetic drug interactions of apatinib with rifampin and itraconazole;?2?the pharmacokinetics,metabolism and CYP3A4 emzyme induction of alflutinib;?3?characterization of covalent bingding to human serum albumin by pyrotinib;?4?characterization of covalent binding to human plasma protein by selected covalent TKIs.1.Pharmacokinetic drug interactions of apatinib with rifampin and itraconazoleApatinib?YN-968D1?is an anti-angiogenesis inhibitor which targeted vascular endothelial cell growth factor receptor-2?VEGFR-2?.In 2014,apatinib was approved for treating patients with advanced gastric-esophageal junction cancer and gastric cancer in China.Previous study has shown that apatinib is extensively metabolized in human,and the main metabolites are E-3-hydroxy apatinib?M1-1?,Z-3-hydroxy apatinib?M1-2?,apatinib-25-N-oxide?M1-6?and E-3-hydroxy apatinib-O-glucuronic acid conjugation?M9-2?.In vitro experiments showed that apatinib is mainly metabolized by cytochrome P450?CYP?3A4/5.Thus,the pharmacokinetic interaction of rifampin?a strong inducer of CYP3A4?and itraconazole?a strong inhibitor of CYP3A?with apatinib were evaluated in healthy volunteers.The clearance?CL/F?of apatinib increased by 5.6-fold when coadministration with rifampin,and the t_1/2 of apatinib decreased from 11.2 h to 5.44 h.Rifampin reduced the C_max of Apatinib by 61%and the AUC by 83%.The results indicated that rifampin had a strong effect on the pharmacokinetics of apatinib.At the same time,rifampin also reduced the plasma exposure of the four metabolites significantly.The CL/F of apatinib was reduced by 41%,and the t_1/2 of apatinib increased from7.79 h to 10.0 h when coadministration with itraconazole.Itraconazole increased the C_max of apatinib by 1.34-fold and the AUC by 1.75-flod.The effects of itraconazole on the pharmacokinetics of apatinib are weak.At the same time,itraconazole also increased the plasma exposure of the four metabolites to varying degrees.In vitro experiments show that metabolites M1-1,M1-2 and M1-6 are substrates of CYP3A4/5,P-glycoprotein?P-gp?and breast cancer resistance protein?BCRP?.Rifampin is an inducer of CYP3A4 and P-gp,while itraconazole is an inhibitor of CYP3A,P-gp and BCRP.Rifampin or itraconazole could induce or inhibit the metabolism and excretion of metabolites M1-1,M1-2 and M1-6,respectively.Therefore,the plasma exposure of metabolites M1-1,M1-2 and M1-6 were reduced or increased,respectively.To determine whether these effects are of clinical significance and dose adjustments are needed,more information about the plasma concentration-safety and plasma concentration-efficacy relationship of apatinib are need.This study added the information of drug–drug interaction to the lable of apatinib,which can guide clinical application of apatinib.2.The pharmacokinetics,metabolism and CYP3A4 emzyme induction of alflutinibAlflutinib?AST2818?is a third-generation EGFR inhibitor targeting both EGFR-sensitive mutations and T790M mutations.It is at the new drug application stage and is intended to treat patients with locally advanced or metastatic non-small cell lung cancer?NSCLC?.N-desmethyl metabolite of alflutinib?AST5902?is the main metabolite of alflutinib and its antitumor activity is comparable to that of alflutinib.The study developed a LC-MS/MS method to determine the plasma concentration of alflutinib and AST5902.50?L plasma was uesd for sample pretreatment with acetonitrile.The linear ranges of alflutinib and AST5902 were 0.20–100 ng/mL and0.050–25.0 ng/mL,respectively.The validated method was successfully applied to pharmacokinetics study of alflutinib mesylate in patients with NSCLC.Alflutinib exhibited dose-proportional increases in exposure(C_max and AUC)across the dose range of 20 to 240 mg.After multiple dosing,alflutinib exhibited nonlinear pharmacokinetics and displayed a time-and dose-dependent increase in CL/F.In this study,we investigated the CYP isozymes involved in the metabolism of alflutinib and evaluated the enzyme inhibition and induction potential of alflutinib.The data showed that alflutinib in human liver microsomes?HLMs?was metabolized mainly by CYP3A4,which could catalyze the formation of AST5902.Alflutinib did not inhibit CYP isozymes in HLMs but could induce CYP3A4 in human hepatocytes.Rifampin is a known strong CYP3A4 inducer and is recommended by FDA as a positive control in the CYP3A4 induction assay.We found that the induction potential of alflutinib was comparable to that of rifampin.The E_max values of alflutinib in three lots of human hepatocytes were 9.24-,11.2-and 10.4-fold,while the fold-induction values of rifampin?10?M?were 7.22-,19.4-and 9.46-fold,respectively.The EC₅₀ of alflutinib-induced CYP3A4 mRNA expression was 0.25?M,which was similar to that of rifampin.In addition,AST5902 exhibited much weak CYP3A4 induction potential compared to alflutinib.Given the plasma exposure of alflutinib and AST5902,both are likely to affect the pharmacokinetics of CYP3A4 substrates.Considering that alflutinib is a CYP3A4 substrate and a potent CYP3A4 inducer,drug–drug interactions are expected during alflutinib treatment.3.Characterization of covalent binding to human serum albumin by pyrotinibPyrotinib is a novel irreversible TKI approved for the treatment of HER2+breast cancer in China.In a phase I clinical trial,unextractable radioactivity was observed in human plasma after a single oral administration of 402 mg?5.55 MBq?[¹⁴C]-pyrotinib to healthy male volunteers.In vitro,after incubating[¹⁴C]-pyrotinib with human plasma for 24 h at 37°C,the recovery of pyrotinib decreased to 46.7%.We speculated that the acrylamide of pyrotinib forms a covalent bond with human plasma proteins,which resulted in the low recovery of pyrotinib.The purpose of this study was to identify the binding sites of pyrotinib to human plasma proteins and to evaluate the reversibility of covalent binding.After incubating pyrotinib with 10X-diluted human plasma for 36 h at 37°C,it was detected by UPLC/Q-TOF MS.The molecular weight of pyrotinib-HSA adduct was67025 Da,which was 582 Da?molecular weight of pyrotinib?higher than the molecular weight of HSA?66443 Da?,indicating that pyrotinib can covalently bind to HSA.After incubating pyrotinib with HSA for 36 h at 37°C,it was hydrolyzed by pronase.Pronase is a non-specific enzyme that hydrolyzes proteins into single amino acid.In the full-scan mass spectrum,the molecular ion of the pyrotinib-lysine adduct was detected at m/z 729.The product ion was detected at m/z 583,which resulted from the cleavage of the C-N bond between lysine and pyrotinib.In addition,m/z 242 was detected,which is an adduct of partial structure of pyrotinib and lysine.No adducts of pyrotinib with other amino acids were detected,suggesting that pyrotinib only binds to the lysine of HSA.Human plasma incubated with[¹⁴C]-pyrotinib for 24 h at 37°C was hydrolyzed by HCl at 90°C for 2 h.The product profiles of digested[¹⁴C]-pyrotinib-treated plasma was determined using HPLC combined with dynamic flow radioactive detector.The[¹⁴C]-pyrotinib adducts were identified by UPLC/Q-TOF MS.The radioactive peaks were identified by mass spectral data to be amide hydrolyzed of[¹⁴C]-pyrotinib?m/z448.141?,[¹⁴C]-pyrotinib?m/z 585.225?,as well as a group of[¹⁴C]-pyrotinib adducts.These adducts included[¹⁴C]-pyrotinib-lysine?m/z 731?,[¹⁴C]-pyrotinib and oligopeptide adducts,such as glycine-lysine?m/z 788?,lysine-alanine?m/z 802?,glycine-lysine-alanine?m/z 859?,and lysine-alanine-serine?m/z 889?.The result indicated that pyrotinib can bind to the glycine-lysine-alanine-serine peptide.The glycine-lysine-alanine-serine peptide corresponds to amino acid residues 189-192 in HSA.These data indicated that the binding site of pyrotinib to HSA is Lys-190.In summary,the?-NH₂ of lysine attacked acrylamide of pyrotinib and formed pyrotinib-HSA adduct by Michael addition.The half-life of total plasma radioactivity is higher than that of pyrotinib and major metabolites,which is about 1/10 of the half-life of HSA.These data indicated that the covalent binding is reversible under physiological conditions.The adduct of pyrotinib and plasma proteins was spiked to PBS buffer?0.1 M,pH 7.4?.Pyrotinib was observed to be released,indicating that the covalent binding of pyrotinib to plasma proteins was reversible.The covalent binding mode between pyrotinib and human serum albumin was studied by molecular docking.The unextractable radioactivity in human plasma was the result of covalent binding via Michael addition between pyrotinib and Lys-190 of HSA,and the covalent binding is reversible under physiological condition.4.Characterization of covalent binding to human plasma proteins by selected covalent tyrosine kinase inhibitorsThe structures of covalent TKIs contain electrophilic warheads,which may covalently bind to human plasma proteins.Eight covalent TKIs were investigated to determine the characteristics of their covalent binding to plasma proteins.These drugs are afatinib,neratinib,osimertinib,olmutinib,ibrutinib,pyrotinib,alflutinib,and alflutinib metabolite AST5902.The covalent binding to plasma proteins of selected covalent TKIs were of species difference.The stability of osimertinib and neratinib in human plasma is much lower than that of other species.Pyrotinib,ibrutinib and afatinib exhibited the least stability in human plasma,while olmutinib showed the least stability in dog plasma.Alflutinib and AST5902 had the least stability in rat plasma,followed by mouse plasma.In conclusion,covalent TKIs tend to be more covalent bound to human plasma proteins.In addition to pyrotinib,neratinib and ibrutinib,the adducts of HSA with osimertinib,alflutinib,AST5902,afatinib,and olmutinib were also detected.Covalent TKIs were incubated with HSA and hydrolyzed by pronase.The data showed that all TKIs that tested can covalently bind to the lysine residue of HSA.The covalent TKIs-treated HSA samples were hydrolyzed with HCl at 90?C for 2 h.In addition to the parent drugs,we also detected amide hydrolysates of covalent TKIs and a series of covalent TKIs'adducts with oligopeptides,including lysine,glycine-lysine,lysine-alanine,glycine-lysine-alanine,and lysine-alanine-serine.In summary,in addition to the reported results on neratinib and pyrotinib,we confirmed that osimertinib,alflutinib,AST5902,and ibrutinib can bind to the Lys-190 of HSA.Through Induced Fit Docking and Covalent Docking,it is shown that covalent TKIs can form?-?stacking,?-cation,and hydrogen bonding with the amino acid residues in the pocket where Lys-190 is located.These non-covalent interactions stabilizd the complexes of covalent TKIs and HSA,which created the conditions for Michael addition.The half-life of covalent TKIs in HSA and human plasma were determined in vitro.Quantitative calculations and linear modeling methods were used to screen for high correlative features of interactions between TKIs and HSA or human plasma proteins.?-carbon local electrophilicity index,electrophilicity index,activation energy??Ea?and docking score of covalent docking are highly correlate with the half-life of covalent TKIs in HSA solution.Similarly,we found that the?-carbon Fukui Function,?-carbon local electrophilicity index,electrophilicity index and docking score of covalent docking are highly correlated with half-life of covalent TKIs in human plasmaReversibility study showed that the covalent binding of 8 drugs to human plasma proteins was reversible.Except for osimertinib,the covalent TKIs-HSA adducts were reversible in vitro.The adducts decomposed faster at higher temperature and concentrations in solution.5.ConclusionRifampin had a strong effect on the pharmacokinetics of apatinib,while itraconazole had a weak effect on the pharmacokinetics of apatinib.This study added the information of drug–drug interactions to the lable of apatinib,which can guide the clinical application of apatinib.A rapid and sensitive LC-MS/MS method was developed for the determination of alflutinib and AST5902 in human plasma,and a complete method validation was performed.This method was applied to the pharmacokinetics study of alflutinib and AST5902 in patients with NSCLC.After a single oral administration,alflutinib exhibited dose-proportional increases in exposure across the dose range of 20 to 240mg.After multiple administrations,the clearance of alflutinib increased in a time-and dose-dependent manner.Further studies showed that alflutinib is a substrate and a strong inducer of CYP3A4.The acrylamide of pyrotinib can covalently bind to the?-NH₂ of HSA Lys-190 by Michael addition.The covalent binding showed species difference and was reversible.Molecular docking explained the non-covalent interactions between covalent TKIs and the binding pocket,which can stabilize the pyrotinib-HSA complex and increase the hydrophobicity of pyrotinib in the hydrophobic pocket.Michael addition between Lys-190 and pyrotinib acrylamide are likely to occur under the circumstances.Eight covalent tyrosine kinase inhibitors?TKIs?were investigated to determine the characteristics of their covalent binding to plasma proteins.Covalent TKIs with acrylamide as a warhead can covalently bind to HSA through Michael addition,most of which are Lys-190 of HSA.The covalent binding is of species difference,which show great difference between animal and human.Molecular docking explained the binding mode of covalent TKIs and HSA.The electrophilicity of the acrylamide?-carbon is an important factor that affect the speed of covalent binding between covalent TKIs and human plasma protein.Except for osimertinib,covalent binding between TKIs and HSA are reversible.