| I Establishment of BCRP-MDCKII cells with high expression of human breast cancer resistance proteinBreast cancer resistance protein (BCRP), a member of the ABC transporter superfamily, is a 72 kDa protein encoded by the BCRP gene. BCRP serves as a key factor in conferring the multi-drug resistance (MDR) phenotype to cancer cells in clinical trial. Apart from cancer cells, BCRP is also highly expressed in the apical membrane of normal tissues and epithelial barriers such as liver, intestine, kidney, blood-brain and blood-placenta barriers. Therefore, the modulation of BCRP can affect the oral bioavailability, biliary or renal clearance, brain uptake of drugs and increase the risk of DDIs, which should be taken into the consideration for drugs with narrow therapeutic indexes such as mitoxantrone and topotecan particularly. Small changes in the systemic exposure of these drugs could result in substantial alterations in therapeutic effects and toxic outcomes.Due to the high expression of human BCRP and short culture time, BCRP-MDCKII cells transfected with the human BCRP gene have become an attractive model to study DDIs mediated by BCRP.In the present study, BCRP-MDCKII cell model was established by the transfection of a lentivirus carrying human BCRP gene into the parent MDCKII cells. The results were summarized as follows:1. The results of restriction analysis and DNA sequencing showed that the BCRP expressive strains pSIN4-EF2-ABCG2-IRES-Neo carrying a human BCRP gene was accurately amplified and enriched in the Soc medium.2. Lentivirus was produced after the transfection of plasmid pSIN4-EF2-ABCG2-IRES-Neo into HEK-293T cells. The viral titer was up to 5×104 pfu/mL.3. BCRP-MDCKII cells with high expression of human BCRP were established after MDCKII cells were infected with the lentivirus carrying a human BCRP gene and incubated with G418 (500 ng/mL) for two weeks. Western blotting analysis showed that the expression of human BCRP was stable and high in BCRP-MDCKII cells after expanding for 10 generations. Moreover, the decreased accumulation of Hoechest 33342 and enhanced efflux of mitoxantrone were observed in BCRP-MDCKII cells, indicating the high BCRP activity of BCRP-MDCKII cells.In conclusion, BCRP-MDCKII cells with high and stable expression of human BCRP were established in the present study, and it could be used as a valuable in vitro tool for the rapid screening of BCRP substrates/inhibitors and DDI mediated via BCRP. Ⅱ Preclinical pharmacokinetics of H002H002 is a novel agonist of sphingosine 1-phosphate receptor 1 (S1PR1). Unlike the traditional immune-suppressive agents (glucocorticoid, cyclosporine A, tacrolimus etc.), H002 reversibly phosphorylates to its active form H002-P, acting as a potent agonist towards SIPR1. Previous pharmacology studies showed that H002-P could induce receptor internalization and lymphocyte homing, and inhibit the egress of lymphocytes from lymph nodes in pathological animal models of rheumatoid arthritis and psoriasis. Additionally, the increasing number of periphery blood lymphocytes could be decreased to normal levels after discontinuation with less influence on normal immune function. Compared with FTY-720, the agonist activity (ratio of EC50) of H002 towards SIPR3 was 150 times over SIPR1, which was much higher than that of FTY720 (3 times), showing better selectivity for SIPR1 and less possibility on bradycardia.Based on the guideline of preclinical pharmacokinetic study, a simple, reliable, sensitive and specific LC-MS/MS method for the quantitative analysis of H002 and its major metabolites in biological samples was developed in the present study. The absorption, distribution, metabolism and excretion of H002 were determined in rats and beagle dogs after oral and intravenous administration of H002. The bio trans formation of H002 was studied and the related drug metabolizing enzymes and metabolic location were identified. The plasma protein binding of H002 and H002-P were investigated in rat, beagle dog, cynomolgus monkey and human blood and the effect of analytes on the activities of CYP450s and UDPGT were also assessed. The results provided the useful experimental basis for revealing the dynamic process of species and gender differences in vivo, understanding the metabolism and elimination pathways of H002 and prediction of potential drug-drug interactions. The results were shown as follows:1. Establishment of LC-MS/MS method for the determination of H002 and its metabolites in biological samples.In rat blood, the linear detection responses were obtained for H002, the phosphorylated metabolite H002-P and hydroxylated metabolite H002-M ranging from 0.2-100 ng/mL,0.25-100 ng/mL and 0.2-100 ng/mL, respectively; while no significantly matrix effect was observed in any of blood, tissue or excreta. The intra-and inter-day precisions (R.S.D.%) for QC samples (0.5,25,80 ng/mL for H002,1.5, 25,80 ng/mL for H002-P,0.5,25,80 ng/mL for H002-M) were within 11.76%, while the deviation of assay accuracies was within ±9.84%. The average recoveries and matrix effects were 92.02-108.18% and 95.01-104.08%, respectively. The samples were found to be stable after being placed on ice for 6 h, at room temperature for 24 h or auto-sampler for 48 h after preparation, stored at -80℃ for 7,30 days and 3 months or through three freeze-thaw cycles. Furthermore, no effect on samples dilution (2,5,10 or 20 times) with blank blood was observed.In beagle dog blood, the linear detection responses were obtained for H002, the phosphorylated metabolite H002-P and hydroxylated metabolite H002-M ranging from 0.5-500 ng/mL,0.25-100 ng/mL and 0.5-100 ng/mL, respectively; while no significantly matrix effect was observed in any of blood or excreta. The intra- and inter-day precisions (R.S.D.%) for QC samples (1.5,75,400 ng/mL for H002,0.6,25, 80 ng/mL for H002-P,1.5,25,80 ng/mL for H002-M) were within 9.30%, while the deviation of assay accuracies was within ±18.00% for QC1 and ±14.80% for QC2 and QC3. The average recoveries and matrix effects were 106.78-112.49% and 97.45-104.42%, respectively. The samples were found to be stable after being placed on ice for 6 h, at room temperature for 24 h or auto-sampler for 48 h after preparation, stored at-80℃ for 7,30 days and 6 months or through three freeze-thaw cycles. Furthermore, no effect on samples dilution (5,10 or 20 times) with blank blood was observed.2. Pharmacokinetic study of H002 in rats.(1) Pharmacokinetic study of H002 in rats after oral administrationH002, H002-P and H002-M can be detected in blood at 5 min after oral administration of H002 (1,3,10 and 30 mg/kg), while the blood concentrations of H002 and its metabolites were close to the limit of detection at 72-144 h post-dose in male and female rats.In male rats, the average Cmax of H002 at dose of 1,3,10 and 30 mg/kg were 5.87,22.20,55.30 and 267.60 ng/mL at 5.6,1.2,4.0 and 2.0 h, AUC(0-t) were 93.16, 125.79,630.27 and 3782.58 ng/mL×h, t1/2 were 9.12,13.42,10.41 and 18.37 h, MRT(0-t) were 12.05,10.42,11.37 and 14.38 h after oral dosing, respectively. The average Cmax of H002-P of 1,3,10 and 30 mg/kg were 24.02,45.76,161.22 and 567.20 ng/mL at 7.2,6.8,6.0 and 5.6 h, AUC (0-t) were 384.09,464.82,2348.96 and 10802.89 ng/mL×h, t1/2 were 8.14,12.43,12.65 and 16.00 h, MRT(0-t) were 14.75, 12.34,13.72 and 16.33 h after oral dosing, respectively. The average Cmax of H002-M of 1,3,10 and 30 mg/kg were 5.40,13.06,33.50 and 114.46 ng/mL at 7.2,4.8,6.8 and 6.0 h, AUC (0-t) were 82.35,127.40,427.18 and 2213.58 ng/mL×h, t1/2 were 8.74, 5.95,7.07 and 16.62 h, MRT(0-t) were 11.80,9.94,11.26 and 15.98 h after oral dosing, respectively.In female rats, the average Cmax of H002 at dose of 1,3,10 and 30 mg/kg were 7.74,21.38,69.50 and 435.4 ng/mL at 4.0,2.5,4.8 and 1.3 h, AUC(0-t) were 104.63, 192.08,987.84 and 5526.58 ng/mLxh, t1/2 were 5.04,16.21,11.57 and 15.02 h, MRT(0-t) were 10.72,11.42,11.39 and 20.58 h after oral dosing, respectively. The average Cmax of H002-P of 1,3,10 and 30 mg/kg were 28.68,56.08,235.00 and 770.60 ng/mL at 8.8,6.4,6.4 and 4.0 h, AUC (0-t) were 461.73,705.88,3723.55 and 16612.94 ng/mL×h, t1/2 were 9.74,12.31,13.42 and 17.13 h, MRT(0-t) were 13.26, 13.19,13.53 and 22.43 h after oral dosing, respectively. The average Cmax of H002-M of 1,3,10 and 30 mg/kg were 4.62,12.28,31.82 and 79.60 ng/mL at 7.2,5.2,5.6 and 10.4 h, AUC (0-t) were 65.90,130.42,472.68 and 2059.02 ng/mL×h, t1/2 were 8.75, 7.22,7.91 and 15.43 h, MRT(0-t) were 11.46,10.73,11.40 and 24.26 h after oral dosing, respectively.In male and female rats, the Cmax and AUC(0-t) of H002, H002-P and H002-M were all increased dose-dependently, presenting a linear pharmacokinetic manners in the range of 1-30 mg/kg. A slight delays of t1/2 and MRT(0-t) were observed in high dose group. The Cmax and AUC(0-t) values of H002-P were higher than the parent drug and H002-M in all tested doses. Additionally, higher AUC(0-t) values of H002 and H002-P were observed in female rats compared with that in males with no statistical difference.(2) Pharmacokinetic study of H002 in rats after intravenous administrationH002, H002-P and H002-M were detected in blood at 2 min after intravenous administration and blood concentration of H002 and its metabolites were close to the limit of detection at 48 h post-dose in male and female rats.In female and male rats, the average Cmax of parent drug were 348.6 and 304 ng/mL at 2 min after intravenous administration of H002 while the AUC(0-t) were 187.85 and 141.3 ng/mL×h, t1/2 were 7.73 and 7.43 h, MRT(0-t) were 6.71 and 5.83 h, respectively. The average Cmax of H002-P was 41.72 and 47.12 ng/mL at 0.93 and 0.41 h post-dose while the AUC(0-t) were 399.49 and 299.88 ng/mL×h, t1/2 were 7.47 and 6.95 h, MRT(0-t) were 9.56 and 8.37 h, respectively. The average Cmax of H002-M was 6.71 and 7.69 ng/mL at 0.69 and 0.10 h post-dose while the AUC(0-t) were 57.29 and 43.56 ng/mL×h, t1/2 were 9.7 and 10.33 h, MRT(0-t) were 11.54 and 11.37 h, respectively.The oral bioavailability of H002 (3 mg/kg) was 8.90% in males and 9.62% in females.(3) Tissue distribution of H002 and its major metabolites in rats after oral administrationH002 and its metabolites were widely distributed in rat tissues after a single oral dosing at 1 mg/kg. In male rats, the concentration order of H002 at 0.5 h post-dose was stomach> intestine> lymph-nodes> adrenal-gland> lung> liver> kidney> marrow> fat> spleen> muscle> epididymis> heart> thymus> testis> blood, while the concentration of H002 in brain was below LLOQ. In female rats, the concentration order of H002 at 0.5 h post-dose was lymph-nodes> stomach> intestine> adrenal-gland> lung> uterus> heart> ovary> liver> kidney> marrow> fat> spleen> muscle> thymus> brain> blood. The distribution order of H002 at 4 h and 18 h post-dose were similar with that of 0.5 h in male and female rats. The distribution orders of H002-P and H002-M were similar with the parent drug. H002 and its metabolites were mainly distributed in gastrointestinal tract and tissues with abundant blood flow. The concentrations of H002, H002-P and H002-M in most tissues were higher than that in blood at the same time points. No significant gender difference was observed.(4) The excretion of H002 through feces, urine and bile in rats after oral administrationH002, H002-P, H002-M and sulfate of H002-M (H002-OSO3) can be found in rat feces, urine and bile after a single oral dose of H002 at 1 mg/kg.In the bile of male and female rats, total excretion of H002, H002-M, H002-OSO3 and H002-P were 0.007%,0.375%,4.826%,0.008% and 0.011%, 0.642%,5.796%,0.060% of dose, respectively. The accumulative excretion of H002 and its metabolites were 5.216% and 6.509% during 72 h post-dose, respectively.In the feces of male and female rats, total excretion of H002, H002-M, H002-OSO3 and H002-P were 2.353%,15.233%,33.114%,0.047% and 1.627%, 12.825%,20.569%,0.052% of dose, respectively. The accumulative excretion of H002 and its metabolites were 50.747% and 35.073% during 6 d post-dose, respectively.In the urine of male and female rats, total excretion of H002, H002-M, H002-OSO3 and H002-P were 0.049%,5.576%,0.630%,0.010% and 0.012%, 7.197%,0.666%,0.005% of dose, respectively. The accumulative excretion of H002 and its metabolites were 6.265% and 7.880% during 6 d post-dose, respectively.The total recovery of H002 and its metabolites from feces and urine were 57.01% and 42.95% of dose in male and female rats. The results demonstrated that H002 was mainly excreted through the feces of rats.(5) Plasma protein binding of H002 among speciesH002 (30,300 and 1500 ng/mL) was found to highly bind to rat, human, dog and monkey plasma protein in vitro (98.13-99.97%). No significant species difference or dose-dependent effects were observed.3. Pharmacokinetic study of H002 in beagle dogs(1) Pharmacokinetic study of H002 in beagle dogs after a single oral administrationAfter oral administration of H002 (0.3,1.8 and 10 mg/kg) to male and female beagle dogs, H002, H002-P and H002-M can be detected in blood in 5-30 min and the blood concentration of H002 were close to the limit of detection at 384-1248 h post-dose. Multiple peaks were observed in the blood concentration-time curve.In female dogs, the average Cmax of H002 at dose of 0.3,1.8 and 10 mg/kg were 28.45,276.25 and 1562.5 ng/mL at 19.75,6.50 and 5.50 h, AUC(0-t) were 2951.13, 28030.93 and 291344.91 ng/mL×h, t1/2 were 82.95,97.63 and 160.88 h, MRT(0-t) Were 111.24,119.75 and 233.38 h after oral dosing, respectively. The average Cmax of H002-P of 0.3,1.8 and 10 mg/kg were 2.48,11.67 and 90.88 ng/mL at 19.50,21.00 and 34.50 h, AUC (0-t) were 401.53,1460.88 and 19964.51 ng/mLxh, t1/2 were 136.23, 89.91 and 143.52 h, MRT(0-t) were 146.70,112.51 and 237.31 h after oral dosing, respectively. The average Cmax of H002-M of 1.8 and 10 mg/kg were 5.74 and 44.35 ng/mL at 16.00 and 19.00 h, AUC (0-t) were 471.12 and 4877.23 ng/mLxh, t1/2 were 148.63 and 125.44 h, MRT(0-t)were 81.55 and 158.08 h after oral dosing, respectively.In male dogs, the average Cmax of H002 at dose of 0.3,1.8 and 10 mg/kg were 27.38,283.5 and 1685.00 ng/mL at 11.00,4.25 and 5.00 h, AUC(0-t) were 1347.83, 22169.09 and 108385.97 ng/mL×h, t1/2 were 46.71,89.83 and 168.56 h, MRT(0-t)were 72.80,98.46 and 170.17 h after oral dosing, respectively. The average Cmax of H002-P of 0.3,1.8 and 10 mg/kg were 1.72,10.09 and 93.20 ng/mL at 15.00,18.00 and 19.50 h, AUC (0-t) were 202.87,1092.17 and 10254.62 ng/mLxh, t1/2 were 106.53,68.50 and 111.27 h, MRT(0-t)V were 91.57,90.62 and 142.82 h after oral dosing, respectively. The average Cmax of H002-M of 1.8 and 10 mg/kg were 9.74 and 55.48 ng/mL at 18.00 and 20.00 h, AUC (0-t) were 518.04 and 4799.64 ng/mL×h, t1/2 were 82.66 and 93.08 h, MRT(0-t) were 120.57 and 117.51 h after oral dosing, respectively.In female and male beagle dogs, the Cmax and AUC(0-t) of H002, H002-P and H002-M were all increased dose-dependently, presenting a linear pharmacokinetic manners in the range of 0.3-10 mg/kg. The slight delays of t1/2 and MRT(o-t) were observed in high dose group. The Cmax and AUC(0-t) values of parent drug were much higher than H002-P and H002-M in all tested doses. Additionally, higher AUC(0-t) along with longer t1/2 and MRT(0-t)values of H002 and H002-P were observed in female dogs with no statistical difference.(2) Pharmacokinetic study of H002 in beagle dogs after intravenous administrationAfter intravenous administration of H002 to male and female beagle dogs, H002 and H002-P can be detected in blood at 2 min post dose, while H002-M was below LLOQ at most of time points. The blood concentrations of H002 and H002-P were close to the limit of detection at 22 d and 16 d post-dose, respectively.In female and male beagle dogs, the average Cmax of parent drug were 152.50 and 155.00 ng/mL at 2 min after intravenous administration, the AUC(0-t) were 3352.71 and 2238.90 ng/mL×h, t1/2 were 111.91 and 62.92 h, MRT(0-t) were 85.04 and 63.29 h, respectively. The average Cmax of H002-P were 1.13 and 1.23 ng/mL, AUC(0-t) were 159.02 and 103.02 ng/mL×h, t1/2 were 102.99 and 73.45 h, MRT(0-t) were 110.67 and 61.80 h, respectively.The oral bioavailability of H002 (1.8 mg/kg) was 46.4% in females and 55.0% in males.(3) Pharmacokinetic study of H002 in beagle dogs after multiple oral dosesAfter multiple oral doses of H002 (1.8 mg/kg/d × 12 d), H002, H002-P and H002-M reached steady state in dog blood. No significant gender difference was observed after multiple dosing. Compared with the single dosing, Cmax and AUC(0-t) of H002 and its metabolites increased remarkably after multiple dosing of H002. The Cmax of H002, H002-P and H002-M increased 3.06,3.14 and 4.24 times in females and 2.91,3.23 and 3.42 times in males. The AUC(0-t) increased 3.31,3.78,4.93 times and 3.03,3.30,3.68 times in female and male dogs, respectively. The results indicated that a potential of accumulation could happen in dogs after multiple oral dose of H002.(4) The excretion of H002 through feces and urine in beagle dogs after oral administrationH002, H002-P, H002-M and H002-OSO3 can be detected in feces and urine of beagle dog after a single oral dosing of H002 at 1.8 mg/kg.In the feces of male and female dogs, total excretions of H002, H002-M, H002-OSO3 and H002-P were 15.622%,5.945%,74.912%,0.269% and 16.797%, 5.733%,73.872%,0.250% of dose, respectively. The accumulative excretion of H002 and its metabolites were 96.748% and 96.652% during 30 d post-dose.In the urine of male and female dogs, total excretions of H002, H002-M, H002-OSO3 and H002-P were 0.549%,2.494%,1.499%,0.005% and 0.476%, 2.671%,2.140%,0.005% of dose, respectively. The accumulative excretion of H002 and its metabolites were 4.547% and 5.292% during 30 d post-dose.The total recovery of H002 from feces and urine were 101.30% and 101.94% of dose in female and male dogs within 30 d post-dose, respectively. The results demonstrated that H002 was excreted mainly through feces in dogs.4. The biotransformation of H002 in vitroSeveral metabolites of H002 can be detected in blood, feces, urine and bile of rats and beagle dogs after oral administration, including H002-P, H002-M and H002-OSO3. H002-P and H002-M could also be detected in liver microsomal incubations of rat, human, dog, monkey in vitro. A correlation metabolic pattern H002 was observed between in vivo and in vitro study.H002 was stable in simulated gastric/intestinal fluid, intestinal bacteria and rat plasma during 6 h incubation. The production of H002-P increased after 6 h incubation H002 with blood of rat, dog, monkey and human, suggesting that blood might be the metabolic site of H002 phosphorylation.After incubation of H002 with various rat tissue homogenates and blood, the order of H002-P production was blood> liver> spleen> brain> stomach> lymph-nodes> heart> kidney> lung> intestine> thymus. The order of H002-M production was liver> intestine> lung> spleen> lymph- nodes> kidney> brain> thymus> heart> stomach, which suggested that liver and intestine was the major sites of H002-M formation and H002-P was formed in blood.The results from incubation of H002 with red blood cells (RBC) of rat, dog, monkey and human demonstrated that RBC was the major site of H002-P production. The order of metabolic rate H002-P was rat> monkey> human> dog. CB5468139 (specific inhibitor of SP HK1), DMS (dual inhibitor of SPHK1 and SPHK2) and FTY720 (competitive inhibitor of SPHK2) could inhibit the production of H002-P in certain extends, indicating that both SPHK1 and SPHK2 might be participate in the phosphorylation of H002.Except H002-M, another hydroxylated metabolite, H002-M1, could be detected in rat, dog and human LMs incubations in vitro, while H002-M1 was found to be the major oxidative metabolite in monkey LMs. Incubation studies with selective chemical inhibitors and antibodies of CYPs and 14 recombinant enzymes demonstrated that CYP1A1, CYP2J2, CYP3A4 and CYP4F2 were found to be the major isoforms mediated in the formation of H002-M, following by CYP1A2, CYP2D6 and CYP4F12. CYP2J2, CYP4F2 and CYP4F3B mainly mediated in the formation of H002-M1. The results indicated that multiple isozymes participated in the hydroxylation of H002.5. Inhibition/induction of H002 and its metabolites on metabolizing enzymes.H002 (1-10 μM) showed 20.16%-40.33% inhibition on the activities of CYP1A2, CYP2C19, CYP4F2, CYP2C9, CYP2D6, CYP2J2 and CYP2E1 in rat, human, dog and monkey liver microsomes in vitro. H002-P (1-10 μM) and H002-M (1-10 μM) showed 26.10%-43.17% and 21.07%-56.69% inhibitions on the activity of CYP1A2, CYP2C19, CYP4F2, CYP2C9, CYP2J2 and CYP2E1, respectively. No significant effects of H002 on CYP2D6 and CYP3A4 were observed. The concentrations of H002, H002-P and H002-M used in the in vitro assay were higher than the Cmax in rat/dog in vivo pharmacokinetic studies and the inhibition were less potent compared with the selective inhibitors of CYPs.After multiple oral administration of H002 (3 mg/kg/d × 7 d), the moderate decrease (21.1%-33.3%) of the activity of CYP2E1 and CYP2J3/4 were observed in male rats, while 20.1%-44.1% of increase of CYP1A2, CYP2C11 and CYP2C6 along with 21.2% decrease in CYP4F1 were observed in female rats. Additionally, H002 showed slight induction on GST and inhibition on UDPGT in males but no significant influence on females.6. The transportation of H002 by Caco-2 and MDR1-MDCKII cellsH002 was transported directionally by Caco-2 and MDR1-MDCKI cells, which was reversed after addition of specific inhibitor of P-gp, PSC833, indicating that H002 could be a substrate of P-gp.Additionally, H002 showed a significant inhibition on the transport of digoxin (a substrate of P-gp) with an IC50 value of 0.29 μM.
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