New Anti-leukemia Compounds Fb2 Preclinical Pharmacokinetic Studies

FB2is a novel Abl/Src dual tyrosine kinase inhibitor which belongs to N-(thiazol-2-yl) pyrimidin-4-amine class. Pharmacology studies suggested that FB2inhibited not only the growth of imatinib-resistant CML cell lines (K562), but also the proliferation of Src over activated cells, such as human prostate cancer cells (DU145) and human breast cancer cells (MDA-MB-23). Furthermore, FB2potently prolonged the survival time of non-obese diabetic/severe combined immunodeficient mice harboured K562/G5.0cells and Balb/c mice bearing Ba/F3p210(WT and Y253F) cells. In addition, FB2also decreased the expression of autophosphorylation of Bcr/Abl, c-Src and Lyn kinases, and showed more potent inhibition on Src kinase activity than dasatinib in both Ba/F3WT cells and Ba/F3cells. Consequently, FB2is identified as a novel highly potent antileukemia compound, which can be potentially used to overcome imatinib resistance in CML,On the basis of the guidelines of preclinical pharmacokinetic study, the LC-MS/MS method for quantitative analysis of FB2and metabolites in biological sample has been developed, which provided a simple, reliable, sensitive and specific assay for the preclinical pharmacokinetic study of FB2. In the present study, the absorption, distribution, metabolism and excretion of FB2were determinated in male and female rats after oral and intravenous dosing of FB2. The phase Ⅰand phase Ⅱ enzymes responsible for the biotransformation of FB2and the metabolites of FB2in vivo and in vitro were identified. The interaction between FB2and drug metabolic enzymes/P-gp was also investigated. Additionally, pharmacokinetics of different formulations of FB2was compared in beagle dogs. Our studies will provide useful information for the rational drug use in clinic.The results were shown as follows:1. After oral administration of FB2(9,18and36mg/kg) to male and female rats, FB2and its metabolites (FB7and FB10) were detected in plasma at5min and were close to the limit of detection (LOD) at12～24hours postdose. The average maximum concentrations (Cmax) of FB2in male rats were83.62,245.60and202.8ng/mL at (Tmax)0.3,0.35and0.38h, AUC(o-t) were117.03,402.68and440.53μg/L*h, MRT (o-t) were2.07,4.11and4.32h, respectively. Cmax and AUC(o-t) of FB2were not raised in a dose-dependent manner, suggesting the saturation of the absorption of FB2in male rats. The Cmax of FB7were32.32,63.40and69.94ng/mL at (Tmax)1.20,0.45and0.85h, AUC (o-t) and MRT (o-t) were139.01,288.60,486.13μg/L*h and3.95,5.30,6.92h. The Cmax of FB10were156.80,310.20and277.60ng/mL at (Tmax)0.30,0.30and0.25h, AUC(o-t) and MRT(o-t)were535.30, 1160.90,1769.86μg/L*h and4.56,6.05,7.80h, respectively. The Cmax of FB7and FB10were not raised in a dose-dependent manner, but AUC(o-t) of FB7and FB10were increased dose-dependently (R2=0.9878and0.9613), the MRT (o-t) were obviously delayed. The results indicated the saturation of the elimination of FB2metabolites in male rats.After oral administration of FB2(9,18and36mg/kg) to female rats, the CmaX;AUC (o-t) and MRT of FB2were46.84,170.70,493.80ng/mL,144.29,528.09,1933.35gμ/L*h, and5.24,7.15and6.23h. The Cmax,AUC (o-t) and MRT (o-t) of FB7were22.60,57.84,110.56ng/mL,128.19,385.11,1054.63μg/L*h and5.63,6.95,6.71h. The Cmax, AUC (o-t) and MRT of FB10were119.96,209.40,339.20ng/mL,645.46,1344.85,2928.96μg/L*h and5.79,7.42,7.11h, respectively. The Cmax and AUC (o-t) of FB2, FB7and FB10were increased in a dose-dependent manner (R2=0.983-0.9991). The data above suggested that FB2, FB7and FB10had a linear pharmacokinetics in femal rats.The oral bioavailability of FB2(36mg/kg) was11.96%in females and3.04%in male rats.After oral administration of FB2to rats at low, middle and high dose. AUC (o-t) and MRT (o-t) of FB2, FB7and FB10were much higher in female rats than those in males. Therefore, there was a significant gender difference in FB2pharmacokinetics profiles in rats.The recoveries of FB2, FB7and FB10were13.56%.11.37%and69.93%of dose in feces of male rats, and those were6.89%.12.20%and64.19%in female rats after oral administration of FB2at9mg/kg. The excretions of FB2and metabolites in rat bile and urine were considerably low.FB2and its metabolites were widely distributed in tissues and organs of male and female rats after a single oral dosing at9mg/kg. In male rats, the concentration order of FB2in tissues at8min was stomach> intestine> spleen> liver> fat> adrenal gland> lung> epididymis> muscle> marrow> kidney> testis> plasma> heart> brain, the distribution of FB2in fatty tissues was obviously decreased at15min and2hour postdose comparing with early time point. The metabolites (FB7and FB10) were predominantly distributed in liver, spleen, adrenal gland and digestive tract at three time points postdose. In female rat, the concentration order of FB2in tissues at8min was stomach> liver> intestine> marrow> adrenal gland> muscle> lung> spleen> brain> ovary> womb> plasma> kidney> fat>brain, the distribution order at15min and2hour was similar with8min. The metabolites of FB2were mainly found in liver, adrenal gland and digestive tract, and the distribution trends were almost identical at each time point. FB2in marrow was relatively higher in female rats.FB2was bound to rat, dog, human and monkey plasma protein at a high degree in vitro and the binding rates were96.45～100%. No species difference in PPB of FB2was observed.2. When formulation NO.7was given to beagle dogs (12mg/kg), FB2was detected in plasma at15-30min after dosing and was still above the LOD at12～24hour postdose. The Cmax of FB2, FB7and FB10in male were91.15,66.98and7.41ng/mL, Tmax were3.67,3.67and2.33h. In female dogs, the Cmax of FB2, FB7and FB10were29.93,31.71and8.11ng/mL, Tmax were3,2and0.75h, respectively. The AUC(o-t), Tmax and MRT(o-t) of FB2in male were much higher and CL was remarkably lower than female, suggesting that the gender difference may existed. Except male dog (NO.1), head welling and spiritless were observed in dogs after45min postdose, and recovered1～2hours later.The PK profile for Formulation NO.8showed higher Cmax and AUC (o-t) and lower MRT(o-t), Tmax and tl/2z in dogs.The results suggested that formulation NO.8may have a better absorption, more rapid elimination, less gender difference and adverse effect in beagle dogs.3. FB2was decreased by17%,15%,96%and58%. after incubateion with dog. human, monkey and rat liver microsomes. After incubated with rat and human liver microsomes. FB2was extensively metabolized into different metabolites, such as oxidative products:FB7and FB10(m/z=610/472), UGT conjugate of FB2:G1(m/z=770/456). UGT conjugates of oxidative products:G2and G3(m/z=786/472). The metabolites in human liver microsomes were similar with those in rat, however, the abundances of metabolites were different. After oral administration of FB2at9mg/kg to rats, oxidative products (FB7and FB10, m/z=610/472), UGT conjugate of oxidative product (G4, m/z=786/472) and sulfate conjugate (S1, m/z=690/472) can be found in urine, bile, plasma and feces. Another oxidative product (FB3, m/z=610/472) was only found in urine, bile and plasma. Phase Ⅱ conjugates of parent drug were not detected in vivo. FB2was stable in dog, human, monkey and rat plasma.4. Rat liver CYP3A2and2E1were primarily responsible for the formation of FB7and FB10, CYP2C11,1A2,2C6and2D2partially contributed to the metabolism of FB2. Human liver CYP3A4was major enzyme responsible for FB7and FB10formation and2C9was partially involved. UGT1A1,1A4,1A9and2B7were found to participate in the formation of FB2-G. UGT1A4,1A9and2B7were found to catalyze FB7into FB7-G, UGT1A1and2B7converted FB10to FB10-G.5. The PappA-B of FB2(10μM) in Caco-2cell was13.99×10-6cm/s, showing that FB2was a high permeability drug. The PapPA-B values of FB2and its metabolites (FB7and FB10) were much higher than PapPB-A, the efflux ratio (PapPB-A/PappA-B) of FB2, FB7and FB10were all remarkably decreased in the presence of P-gp inhibitor LSN335984in Caco-2cell model, indicating that FB2, FB7and FB10were the substrates of P-gp, and FB2was further confirmed to be substrate of P-gp by MDCK and MDCK-MDR1cell model. In single-pass perfusion model, compared with control group, Fraction metabolized of FB2was slightly decreased, Pblood of FB2and cumulative amount of metabolites in mesenteric blood were significantly increased, when LSN339584was added. The Foral of FB2was increased to24.52%when orally coadministrated with verapamil, which was significantly higher than that (5.7%) by FB2(18mg/kg) alone in rats. The AUC(o-t) and Cmax of FB2metabolites (FB7and FB10) were also increased in the presence of verapamil. The results suggested that inhibition of P-gp could decrease the efflux of FB2and its metabolites, so as to promote their absorption in rat intestine. Consequently, the low bioavailability of FB2was mostly due to the effect of P-gp on the absorption and transport of FB2in intestine.6. The ER (PappB-A/PapPA-B) of digoxin was decreased by74%,71.6%and29.2%in Caco-2cells, when FB2. FB7and FB10were added at a concentration of10μM. The results suggested that FB2, FB7and FB10inhibited P-gp at different extents. The induction study showed that FB2(1-10μM) had not induction on P-gp, but significant inhibition on ER of digoxin (decreased43.8%,52%and83%). FB7(1-10μM) and FB10(1-5μM) showed weakly inductive effects on P-gp.7. The activities of rat liver CYP1A2,2C11,2D2,2C6and2E1were inhibited by21.81～52.95%when FB2(1-10μM) was added to the incubation. FB2(1-10μM) also inhibited the activities of human liver CYP2D6,2E1and3A4by17.58±54.55%, but the activities of CYP1A2,2C9and2C19were not affected. The activities of human liver UGT1A1,1A4,1A9and2B7were decreased by5.14～44.44%with1-10μM of FB2. FB7and FB10(1-10μM) inhibited the activities of UGT1A1,1A4,1A9and2B71by3.7%～53.44%, respectively.8. Multiple oral dosing of FB2(9,18,36mg/kg) did not show any significant influence on the enzyme activities of CYP450, UDPGT and GST in rats.