不同性激素对P-糖蛋白表达水平及甲磺酸伊马替尼转运的影响

目的：探讨在ABCB1（1199G/A）重组细胞模型中不同性激素对P-糖蛋白（P-gp）表达水平及甲磺酸伊马替尼转运的影响。方法：将构建的ABCB1_1199G/wt和ABCB1_1199A/mut重组质粒稳定转染入HEK293细胞中,采用RT-PCR和Western Blot分别检测不同性激素对P-gp mRNA和蛋白的表达水平;采用HPLC检测性激素对甲磺酸伊马替尼累积量的影响。结果：浓度梯度的性激素（雌酮、雌三醇雌二酮、黄体酮和睾酮）刺激ABCB1_1199G/wt 和ABCB1_1199A/mut重组细胞后,雌酮和雌三醇能刺激P-gp mRNA和蛋白表达水平,且雌三醇上调ABCB1_1199A/mut mRNA的表达水平显著高于ABCB1_1199G/wt;雌酮和雌三醇可降低甲磺酸伊马替尼在细胞内的累积量,产生对伊马替尼的耐药性。结论：雌酮和雌三醇增加了细胞内P-gp的表达,并促进P-gp介导甲磺酸伊马替尼的转运能力。     

ABCB1(1199G>A)基因多态性对多西他赛转运影响的分子机制

目的：在体外研究ABCB1（1199G>A）基因多态性对多西他赛转运影响的分子机制。方法：将ABCB1（1199G>A）野生型和突变型基因分别导入HEK293细胞,研究2种重组细胞株对多西他赛的摄取以及跨膜转运的差异。结果：在细胞毒性分析中,ABCB1_1199A/mut细胞对多西他赛表现出更强的耐药性。多西他赛在2种重组细胞中的含量均显著性的低于对照组细胞,证实了多西他赛是由P-糖蛋白介导转运的,并且在ABCB1_1199A/mut细胞中跨膜转运更高。ABCB1_1199A/mut细胞介导转运多西他赛的P-糖蛋白的活性较ABCB1_1199G/wt细胞的更强。结论：ABCB1（1199G>A）基因多态性能够显著性影响P-糖蛋白转运多西他赛的能力,由ABCB1突变型基因编码的P-糖蛋白能够更有效的转运多西他赛。因此ABCB1（1199G>A）基因多态性可能会影响P-糖蛋白的活性,并对药物的分布和消除产生影响,从而影响药物的治疗作用。     

甲磺酸伊马替尼治疗中晚期高危胃肠间质瘤的疗效和安全性分析

目的 分析甲磺酸伊马替尼治疗中晚期高危胃肠间质瘤的疗效和安全性。方法 回顾2008年10月-2014年10月到武汉商职医院诊治的中晚期高危胃肠间质瘤患者共96例,根据甲磺酸伊马替尼治疗时间分为两组,观察组48例患者治疗时间≥ 3年,对照组48例患者治疗时间<3年,比较两组患者疗效。结果 观察组患者1、2、3、4、5年无进展生存率均明显高于对照组,差异有统计学意义（P<0.05）。性别、年龄和治疗时间是影响甲磺酸伊马替尼治疗有效率的重要因素（P<0.05）。治疗后观察组患者血清基质金属蛋白酶-9（MMP-9）蛋白及蛋白酶激活受体（PAR-2） mRNA水平均明显低于对照组,差异有统计学意义（P<0.05）。治疗期间两组患者血细胞减少、肝肾功能受损、胃肠道反应、乏力及皮肤黏膜水肿等不良反应发生率均未表现出明显差异。结论 延长甲磺酸伊马替尼治疗时间至3年以上能够有效提高中晚期高危胃肠间质瘤患者无进展生存率,且不良反应发生率并未明显升高,安全性好,建议临床推广应用。     

甲磺酸伊马替尼联合塞来昔布对胃肠间质瘤细胞增殖及凋亡的影响

目的观察甲磺酸伊马替尼和塞来昔布联合对胃肠间质瘤细胞增殖及凋亡的影响。方法甲磺酸伊马替尼及塞来昔布单独或联合处理胃肠间质瘤细胞株GIST-T1,MTT法检测药物对胃肠间质瘤细胞株的生长抑制,Western blot检测Bax及Bcl-2蛋白表达。结果甲磺酸伊马替尼与塞来昔布联合应用显著增强甲磺酸伊马替尼对胃肠间质瘤细胞增殖;和甲磺酸伊马替尼及塞来昔布单用相比,二者联用后胃肠间质瘤细胞Bax表达增高及Bcl-2表达降低。结论甲磺酸伊马替尼和塞来昔布联合应用显著抑制胃肠间质瘤细胞增殖,可能和促肿瘤细胞凋亡有关,其机制可能与Bax表达上调及Bcl-2表达下调有关。     

甲磺酸伊马替尼增加顺铂对耐药卵巢癌细胞作用的研究

目的探讨靶向治疗药物甲磺酸伊马替尼(Imatinib mesylate)对耐顺铂人卵巢腺癌细胞株SKOV3/DDP的增殖抑制和诱导凋亡作用以及与顺铂合用的效果。方法 MTT法检测不同浓度甲磺酸伊马替尼、DDP以及联合用药对人卵巢癌耐顺铂细胞株SKOV3/DDP的增殖抑制情况,采用双染色流式细胞术检测甲磺酸伊马替尼对SKOV3/DDP的诱导凋亡作用。结果 MTT法检测出不同药物浓度的甲磺酸伊马替尼作用于SKOV3/DDP细胞呈现细胞生长抑制作用,甲磺酸伊马替尼与不同浓度顺铂联合用药对细胞生长抑制率分别为38.63%、51.55%、66.54%,明显高于DDP单用药组的8.30%、16.68%、30.83%,两组比较差异有统计学意义(P<0.05)。两药合用后的IC50为9.57,是单用顺铂组的4.13倍。甲磺酸伊马替尼与顺铂合用后,早期凋亡率由1.25%增加到12.31%(P<0.05)。结论甲磺酸伊马替尼可以抑制卵巢癌耐DDP细胞株SKOV3/DDP的增殖和诱导细胞凋亡,并显著增强其对DDP的敏感性。     

五味子乙素在Caco 2细胞模型中的转运研究

摘 要 目的：通过经典的体外细胞模型探讨五味子乙素对P 糖蛋白的影响。方法: 以Caco-2细胞模型为载体,选取罗丹明123和环孢素-为P-gp底物,采用高效液相色谱法（HPLC）和液体闪烁计数法检测不同条件下五味子乙素、罗丹明123及环孢素 跨膜转运的变化,计算表观渗透系数、表观渗透率等参数。结果: 五味子乙素浓度在20 ,40 ,80 μg·mL^-1时双向跨膜转运的速率相似,无选择性差异。五味子乙素浓度为20~160 μg·mL^-1时可显著抑制罗丹明123和环孢素A在Caco-2细胞模型中BL→AP定向转运(P＜0.05),且呈剂量相关性。结论:五味子乙素为P-gp抑制药,但不是P-gp底物。     

临床药师参与阿帕替尼致肾小球损害治疗实践

目的 为发生甲磺酸阿帕替尼致肾小球损害的小细胞肺癌患者的用药监护提供参考。方法 对1例口服甲磺酸阿帕替尼片后出现肾小球损害患者的临床资料进行回顾性分析,结合临床药学专业知识,从患者出现肾脏损害的原因、治疗措施等方面进行分析,并复习相关文献,提出口服阿帕替尼出现肾脏损害的解决措施,实施全程药学监护。结果 甲磺酸阿帕替尼引起的肾小球损害可能与其导致内皮细胞损伤有关,出现肾脏损害后应及时停药,并根据不良反应缓解程度调整剂量,临床药师参与后患者肾脏损伤改善明显。结论 阿帕替尼导致的肾小球损害较少见,在用药过程中临床药师需注意监护,及时调整用药方案并给予对症治疗,减少不良反应的发生率。     

小檗碱对有机阳离子药物转运体抑制作用研究

目的 研究小檗碱对人有机阳离子转运蛋白（OCTs）—OCT1、OCT2、OCT3、OCTN1和OCTN2的抑制作用。方法 应用由转染试剂Lipo 3 000介导的动物细胞转基因方法、经筛选得到各药物转运体过表达细胞株S2-OCT1、S2-OCT2、S2-OCT3、S2-OCTN1和S2-OCTN2;通过检测OCTs介导的放射性探针底物的跨膜转运,建立OCTs体外评价模型;以野生型（WT）细胞为对照组,应用各转运体抑制剂验证其活性;应用上述方法观察小檗碱对各转运体的抑制作用,并计算小檗碱对各药物转运体活性的半数抑制浓度（IC₅₀）。结果 各转运体细胞组与各自WT细胞株比较,转运活性均提高了5倍多,加入抑制剂后,转运活性均明显下降;小檗碱对OCT1、OCT2、OCT3和OCTN1抑制作用较强,对OCTN2的抑制作用相对较弱,IC₅₀分别为7.63、6.80、2.25、4.66和210.34 μmol/L。结论 小檗碱对这5种有机阳离子转运体均有抑制作用,其中对OCT1、OCT2、OCT3、OCTN1的抑制作用较强,发生由其介导的DDI的可能性较大,对OCTN2的抑制作用相对较弱。     

甲磺酸伊马替尼对K562细胞bcr/abl、SHIP-2基因表达的影响及意义

目的研究甲磺酸伊马替尼对K562细胞bcr/abl、SHIP2的表达影响及意义。方法采用定量PCR方法检测甲磺酸伊马替尼干预K562细胞后bcr/abl、SHIP2基因表达变化。采用MTT及Western blot方法分别检测细胞增殖与AKT磷酸化水平的变化。结果随着甲磺酸伊马替尼浓度增加及作用时间的延长bcr/abl基因的表达下降（P〈0.01）,SHIP2的表达增加（P〈0.01）。Akt磷酸化水平降低,细胞增殖能力降低。结论甲磺酸伊马替尼能下调bcr/abl基因的表达;SHIP2表达可能受bcr/abl基因的调节,并可能通过调节PI3K/AKT途径影响细胞的增殖。     

“4＋7”带量采购对内蒙古自治区人民医院口服抗肿瘤药原研药和仿制药使用情况影响

目的 分析“4＋7”带量采购政策对内蒙古自治区人民医院口服抗肿瘤药品原研药和仿制药使用的影响，为临床药学服务和医院的药事管理提供依据。方法 采用Excel软件计算口服抗肿瘤药品的使用数量、金额、价格、用药频度（DDDs）、限定日费用（DDC）、占比、增幅、降幅，及仿制药替代率、费用节省率进行分析。结果 “4＋7”带量采购实施后，原研药和仿制药价格出现了不同幅度的降价，其中中选品种来曲唑单品价格降幅最大，降幅超过60%，原研药中价格降幅最大的是阿斯利康制药有限公司生产的吉非替尼，达30%。“4＋7”带量采购实施后，甲磺酸伊马替尼、替吉奥、来曲唑、阿那曲唑、卡培他滨、替莫唑胺仿制药DDDs均增加，而销售金额均下降。吉非替尼仿制药DDDs增幅最大，达74.17%，销售金额则增加57.38%。甲磺酸伊马替尼原研药受带量采购政策影响，DDDs和销售金额均增加，而其他相应的原研药的DDDs和销售金额均减少。“4＋7”带量采购的实施进一步提高仿制药的使用率，为医保费用节省899万元，费用节省率为49.99%。结论 带量政策实施后，药品费用支出减少，切实减轻了部分患者的用药负担。     

Impacts of ABCB1 (G1199A) polymorphism on resistance,uptake, and efflux to steroid drugs

1.?P-glycoprotein (P-gp) substrates, including steroid drugs, involve in the inter-individual differences in resistant phenotype. This study was performed to evaluate whether G1199A polymorphism in ABCB1 gene can alter the sensitivity, accumulation, and transepithelial efflux to steroids in LLC-PK1 cells.

2.?The stable recombinant LLC-PK1 cell lines transfected with ABCB1 1199G and ABCB1 1199A were used to assess the sensitivity, accumulation, and transepithelial permeability to steroids.

3.?The cells transfected with 1199A allele displayed stronger resistance to aldosterone, dexamethasone, and cortisol (2.5-, 2.0-, and 1.6-fold, respectively) than cells overexpressing 1199G allele, while the two types of recombinant cells showed a similar resistance to corticosterone. The accumulation of aldosterone, dexamethasone, and cortisol in recombinant 1199A cells were significantly decreased when compared to 1199G cells (2.9-, 4.4-, and 3.9-fold, respectively). The net efflux ratios of P-gp-mediated aldosterone, dexamethasone, and cortisol in cells expressing 1199A allele were apparently greater than cells transfected with 1199G allele (3.3-, 3.5-, and 4.0-fold, respectively).

4.?The impacts of ABCB1 (G1199A) single nucleotide polymorphism on the efflux of P-gp substrates presented as drug-specific. Overall, the transport ability of P-gp-dependent steroid drugs in recombinant model overexpressing variant 1199A allele is stronger in comparison to cells overexpressing wild-type 1199G allele. Therefore, the ABCB1 (G1199A) polymorphism may affect effective steroids concentration in target cells by regulating the drug transport and distribution.     

Impact of ABCB1 (MDR1) gene polymorphism and P-glycoprotein inhibitors on digoxin serum concentration in congestive heart failure patients

Digoxin, a drug of narrow therapeutic index, is a substrate for common transmembrane transporter, P-glycoprotein, encoded by ABCB1 ( MDR1 ) gene. It has been suggested that ABCB1 polymorphism, as well as co-administration of P-glycoprotein inhibitors, may influence digoxin bioavailability. The aim of the present study was to evaluate the effects of ABCB1 gene polymorphism and P-gp inhibitor co-administration on steady-state digoxin serum concentration in congestive heart failure patients. Digoxin concentrations as well as 3435C > T and 2677G > A,T ABCB1 single nucleotide polymorphisms, were determined in 77 patients administered digoxin (0.25 mg daily) and methyldigoxin (0.50 mg daily), some of them co-medicated with known P-glycoprotein (Pgp) inhibitors. Significant differences were noted in digoxin serum concentrations (C(min,ss)) between patients co-administered and not co-administered P-gp inhibitors: 0.868 +/- 0.348 and 0.524 +/- 0.281 for digoxin (p < 0.002), as well as 1.280 +/- 0.524 and 0.908 +/- 0.358 for methyldigoxin (p < 0.02), respectively. No influence of ABCB1 2677G > A,T and C3435C > T polymorphisms on digoxin concentration was noted. Although some of the previous studies have shown that digoxin pharmacokinetics might be affected by ABCB1 genetic polymorphism, those modest changes are probably clinically irrelevant, and digoxin dose adjustment should include P-gp inhibitor co-administration rather than ABCB1 genotyping.     

Structure-activity relationships and in silico models of P-glycoprotein (ABCB1) inhibitors

Abstract

1. The efflux pump p-glycoprotein (P-gp/ABCB1) has received enormous attention in drug (xenobiotic) disposition due to its role in modulation of the drug availability and in protection of sensitive organs.

2. P-gp mediated efflux is one of main mechanisms for multidrug resistance in cancer cells. A main approach to reverse the resistance and restore the drug efficacy is to use specific inhibitors of P-gp that suppress the efflux activity.

3. This review summarizes the binding capabilities of known chemical inhibitors based on the analyses of structure–activity relationships, and computational modeling of the inhibitors as well as the binding site of P-gp protein.

4. The molecular models will facilitate the design of lead inhibitors as drug candidates. Also, it helps scientists in early drug discovery phase to synthesize chemical series with better understanding of their P-gp binding liabilities.     

ABCB1 G1199A polymorphism and ovarian cancer response to paclitaxel

P-glycoprotein (P-gp), encoded by the ABCB1 gene, confers multi-drug resistance to a variety of antineoplastic agents, for example, paclitaxel. Recently, the G1199T/A polymorphism in the ABCB1 gene was shown to be important for the function of P-gp as well as for the resistance to several chemotherapeutic agents in vitro. We analyzed the allelic distribution of the G1199T/A and other polymorphisms in exons 11 and 12 of the ABCB1 gene in ovarian cancer patients treated with paclitaxel and carboplatin in order to evaluate their predictive value in vivo. The SNPs C1236T, G1199T/A, and A1308G were determined using Pyrosequencing in 51 patients with advanced ovarian cancer and correlated to the progression free survival. The G1199T/A SNP was found to affect the progression free survival. Although only two heterozygous (G/A) patients were found their mean progression free survival was only 2 months as compared to 19 months for the wild-type patients. This is in accordance with the higher resistance for the 1199A genetic variant found in vitro. Genotyping of the ABCB1 gene may be important for determining the tumor resistance to paclitaxel and provide useful information for individualized therapy.     

Hyaluronated imatinib liposomes with hybrid approach to target CD44 and P-gp overexpressing MDR cancer: an in-vitro,in-vivo and mechanistic investigation

Abstract

Multi Drug Resistance (MDR) of cancer cells is a constant threat to the clinically used drugs as well as new drug development. In present work, we aimed to assess in-vitro as well as in-vivo efficacy of the developed Imatinib loaded liposomes in MDR cancer. An array of tests was also carried out to comprehensively understand the bio-mechanism that enable these nanocarriers to modulate P-gp activity. Hyaluronic acid coated, Imatinib mesylate containing lipsomes (HA-LIPO-IM) were analysed through in-vitro and in-vivo studies in MDR cancer cells and tumour models. Effect of developed hyaluronated liposomes on various biomolecular mechanisms was also evaluated. Around 3.5 times lower IC₅₀ for HA-LIPO-IM in comparison to drug solution in HT-29 and Colo-320 cells proved the enhanced action of the drug in MDR cells. HA-LIPO formulations were demonstrated to have inhibitory effect on ATPase enzyme. Molecular masking of Imatinib mesylate and CD-44 mediated endocytosis were also found responsible for anti-MDR effect. In-vivo studies revealed the prolonged tumour accumulation and 4-fold increase in tumour regression efficacy of HA-LIPO-IM in comparison to free drug solution. The work demonstrated the target specific accumulation of HA-LIPO-IM in CD-44 overexpressing cancer cells through P-gp modulation.     

A novel MDR1 G1199T variant alters drug resistance and efflux transport activity of P-glycoprotein in recombinant Hek cells

The human multidrug resistance gene MDR1 encodes the protein product P-glycoprotein (P-gp). P-gp is an integral membrane protein which mediates ATP-dependent substrate efflux. We recently discovered a novel G --> T variant at 1199 nucleotide position of MDR1 which exhibits a 2.3% allelic frequency in leukemia patients. The functional effects of this MDR1-G1199T variant were evaluated with recombinant HEK cells that stably express the wild-type, G1199A, or G1199T variant of the MDR1 protein, P-gp, at comparable levels. A panel of cytotoxic P-gp substrates comprising doxorubicin, vinblastine, vincristine, paclitaxel, or topotecan (a poor P-gp substrate) was used to evaluate the functional impact of G1199 variations. Compared to MDR1(wt), MDR1(G1199A) exhibited an increased resistance to doxorubicin, paclitaxel, vinblastine, and vincristine. In contrast, MDR1(G1199T) reduced resistance to (1/4) that of MDR1(wt) for all drugs except topotecan. Expression of MDR1 exhibits some degree of resistance to topotecan, but 1199 variation has no impact. These data were consistent with the variation in intracellular doxorubicin concentrations measured in MDR1 recombinant cells. Our results suggest that patients with the novel MDR1-G1199T variant may exhibit a lower degree of MDR1 dependent chemoresistance, and those with the G1199A polymorphism may exhibit a higher degree of resistance, compared with MDR1 wild-type patients.     

Structure,function and regulation of P-glycoprotein and its clinical relevance in drug disposition

1. P-glycoprotein (P-gp/MDR1), one of the most clinically important transmembrane transporters in humans, is encoded by the ABCB1/MDR1 gene. Recent insights into the structural features of P-gp/MDR1 enable a re-evaluation of the biochemical evidence on the binding and transport of drugs by P-gp/MDR1.

2. P-gp/MDR1 is found in various human tissues in addition to being expressed in tumours cells. It is located on the apical surface of intestinal epithelial cells, bile canaliculi, renal tubular cells, and placenta and the luminal surface of capillary endothelial cells in the brain and testes.

3. P-gp/MDR1 confers a multi-drug resistance (MDR) phenotype to cancer cells that have developed resistance to chemotherapy drugs. P-gp/MDR1 activity is also of great clinical importance in non-cancer-related drug therapy due to its wide-ranging effects on the absorption and excretion of a variety of drugs.

4. P-gp/MDR1 excretes xenobiotics such as cytotoxic compounds into the gastrointestinal tract, bile and urine. It also participates in the function of the blood–brain barrier.

5. One of the most interesting characteristics of P-gp/MDR1 is that its many substrates vary greatly in their structure and functionality, ranging from small molecules such as organic cations, carbohydrates, amino acids and some antibiotics to macromolecules such as polysaccharides and proteins.

6. Quite a number of single nucleotide polymorphisms have been found for the MDR1 gene. These single nucleotide polymorphisms are associated with altered oral bioavailability of P-gp/MDR1 substrates, drug resistance, and a susceptibility to some human diseases.

7. Altered P-gp/MDR1 activity due to induction and/or inhibition can cause drug–drug interactions with altered drug pharmacokinetics and response.

8. Further studies are warranted to explore the physiological function and pharmacological role of P-gp/MDR1.     

Influence of Passive Permeability on Apparent P-glycoprotein Kinetics

Purpose. The objectives of this work were to evaluate the importance of moderate passive permeability on apparent P-glycoprotein (P-gp) kinetics, and demonstrate that inspection of basolateral to apical and apical to basolateral (BL-AP/AP-BL) permeability ratios may result in a compound being overlooked as a P-gp substrate and inhibitor of another drug's transport via P-gp inhibition. Methods. The permeability ratios of nicardipine, vinblastine, cimetidine, and ranitidine were determined across Caco-2 monolayers that express P-gp, in the presence and absence of the specific P-gp inhibitor, GF120918. In addition, the permeability ratio of vinblastine was studied after pretreatment of Caco-2 monolayers with nicardipine, ranitidine, or cimetidine. Similar studies were repeated with hMDR1-MDCK monolayers. Results. The permeability ratios for cimetidine and vinblastine were >2. The permeability ratios for nicardipine and ranitidine were close to unity, and were not affected by the addition of GF120918. Based solely on ratios, only compounds with moderate transcellular permeability (vinblastine and cimetidine) would be identified as P-gp substrates. Although the permeability ratios appeared to be unity for nicardipine and ranitidine, both compounds affected the permeability of vinblastine, and were identified as substrates and inhibitors of P-gp. Studies performed in hMDR1-MDCK cells confirmed these experimental results. Data were explained in the context of a kinetic model, where passive permeability and P-gp efflux contribute to overall drug transport. Conclusions. Moderate passive permeability was necessary for P-gp to reduce the AP-BL drug permeability. Inspection of the permeability ratio after directional transport studies did not effectively identify P-gp substrates that affected the P-gp kinetics of vinblastine. Because of the role of passive permeability, drug interaction studies with known P-gp substrates, rather than directional permeability studies, are needed to elucidate a more complete understanding of P-gp kinetics.