Rhodamine 123 Requires Carrier-Mediated Influx for Its Activity as a P-Glycoprotein Substrate in Caco-2 Cells

Purpose. The purpose of this work was to elucidate transport pathways of the P-glycoprotein (P-gp) substrates rhodamine 123 (R123) and doxorubicin across Caco-2 cells. Methods. Experiments were designed to identify saturable and nonsaturable transport processes and transport barriers for R123 and doxorubicin transport across Caco-2 cells. Confocal laser scanning microscopy (CLSM) imaged R123 transport under normal conditions and in the presence of the P-gp inhibitor, GW918 (used to abolish P-gp-mediated efflux activity). Results. R123 secretory P _app (P _app,BA) showed concentration dependence, whereas R123 absorptive P _app (P _app,AB) did not. Inhibition of P-gp efflux revealed that P-gp-mediated efflux had no effect on R123 or doxorubicin P _app,AB, but enhanced R123 and doxorubicin P _app,BA. In calcium-free medium, R123 P _app,AB increased 15-fold, indicating intercellular junctions are a barrier to R123 absorption. CLSM of R123 fluorescence during absorptive transport under normal conditions and in the presence of GW918 was identical, and was limited to paracellular space, confirming that P-gp is not a barrier to R123 absorption. CLSM revealed that R123 fluorescence during secretory transport under normal conditions and in the presence of GW918 was localized intracellularly and in paracellular space. R123 and doxorubicin uptake across Caco-2 cells basolateral membrane was saturable. Conclusions. R123 absorptive transport occurs primarily by paracellular route, whereas R123 secretory transport involves influx across BL membrane mediated solely by a saturable process followed by apically directed efflux via P-gp. Doxorubicin utilizes similar transport pathways to cross Caco-2 cells.     

环胞素A和粉防己碱对牛脑微血管内皮细胞Rh-123摄取的影响

目的　研究环胞素A和粉防己碱对脑毛细血管内皮细胞上P 糖蛋白 (P glycoprotein ,P gp)的外排转运系统的作用。方法　利用荧光分光光度法研究环胞素A和粉防己碱对体外培养牛脑毛细血管内皮细胞罗丹明 12 3摄取的影响。结果　当给予P gp抑制剂环胞素A后 ,Rh 12 3在牛脑毛细血管内皮细胞 (braincapillaryendothelialcell,BCEC)单层的积累量明显增加。粉防己碱能够明显增加BCEC的Rh 12 3积累 ,并且呈剂量依赖性。结论　研究表明 ,此种方法是一个敏感 ,可靠的研究方法 ;粉防己碱是一个很有前途的血脑屏障上P gp的抑制剂     

P糖蛋白表达对HIV-1蛋白酶抑制剂在体内吸收与分布的影响

目的 通过介绍体内各屏障高度表达的P糖蛋白外排作用对HIV-1蛋白酶抑制剂的影响,探讨如何通过有效调节P糖蛋白,优化对艾滋病患者的治疗。方法 查阅国外文献进行总结和归纳。结果 综述了P糖蛋白表达对HIV-1蛋白酶抑制剂在体内吸收、分布的影响。 结论 P糖蛋白一定程度地阻碍了HIV-1蛋白酶抑制剂在体内的吸收和分布,通过抑制P糖蛋白的活性,可以增加HIV-1蛋白酶抑制剂的化学疗效,未来艾滋病的治疗,P糖蛋白抑制剂的选择和使用将是一项艰巨的任务。     

Age-dependent Expression of P-Glycoprotein gp17O in Caco-2 Cell Monolayers

Purpose. To determine whether the expression and activity of the P-glycoprotein (P-GP) drug efflux pump vary with the culture age of Caco-2 cell monolayers. Methods. Caco-2 cell monolayers were grown for 3–27 days on tissue culture-treated Transwells. P-GP efflux function was determined by measuring transmonolayer fluxes of cyclosporin A (CsA) and verapamil, while P-GP expression level was evaluated by Western blot analysis using monoclonal antibody C219. Results. The apparent permeability coefficient (Papp) of CsA (0.5 µM) in the basolateral-to-apical (B A) direction increased with culture age and was higher than the apical-to-basolateral (A B) direction at all times. Net secretory Papp significantly increased from day 17 onward compared to that observed during day 3 through 13. Verapamil (100 µM) significantly inhibited CsA transport in the B A direction from day 17 to 27, while elevating CsA transport in the A B direction from day 6 to 27. Interestingly, the Papp of verapamil (0.5 µM) in the B A direction was significantly higher than in the A B direction from day 6 to 27, rendering increases in net secretory Papp of verapamil with culture age. Western analysis revealed that P-GP expression level was in the order of 4 weeks 1 week > 3 weeks > 2 weeks at equal loading of cell proteins. Conclusions. P-GP is continuously expressed throughout the culture period, but it may not be fully functional at an early age. Caco-2 cell monolayers of day 17 to 27 appear to be a good model to evaluate the functional role of P-GP in drug efflux.     

Effect of P-Glycoprotein Modulator,Cyclosporin A,on the Gastrointestinal Excretion of Irinotecan and Its Metabolite SN-38 in Rats

Purpose. The purpose of this work was to investigate the role of the hepatic and intestinal P-glycoprotein (P-gp) and canalicular multispecific organic anion transporter /multidrug resistance-associated protein 2 (cMOAT/MRP2) on both biliary excretion and intestinal exsorption of irinotecan hydrochloride (CPT-11) and its metabolite, SN-38, in the lactone and carboxylate forms. Cyclosporin A (CsA) was used to modulate P-gp and cMOAT/MRP2. Methods. The transcellular transport of CPT-11 and SN-38 was examined by using LLC-PK1 derivative cell lines transfected with murine mdr1a both in the absence or in the presence of CsA. The excretions of the compounds through the biliary and intestinal membrane routes were investigated by in situ perfusion technique. Results. Basolateral-to-apical transport of CPT-11 lactone in L-mdr1a cells was significantly decreased by CsA (10 M). The trans- cellular transport of SN-38 lactone showed similar behaviors as those of CPT-11 lactone. The biliary excretion and the intestinal exsorption of both forms of CPT-11 and SN-38 were significantly inhibited when the drug was co-administered with CsA. Conclusions. The transports of CPT-11 and SN-38 via the biliary route seem to be essentially related with cMOAT/MRP2, whereas those of both compounds via the intestinal membrane seem to be related with P-gp.     

Assessment of the First and Second Generation Antihistamines Brain Penetration and Role of P-Glycoprotein

Purpose The sedating effect of first generation H₁-antihistamines has been associated with their ability to penetrate the blood-brain barrier (BBB) and lack of efflux by P-glycoprotein (Pgp). Second generation H₁-antihistamines are relatively free of sedation and their limited brain penetration has been suggested to arise from Pgp-mediated efflux. The objective of this work was to evaluate the role of Pgp in brain penetration of first and second generation antihistamines. Methods Potential of antihistamines to be Pgp substrates was tested in vitro using Madin Darby canine kidney cells transfected with human Pgp. The role of Pgp in limiting brain penetration of antihistamines was tested by using the in situ brain perfusion technique. Results Majority of antihistamines were Pgp substrates in vitro. Following in situ brain perfusion, the first generation antihistamines substantially penetrated into rat brain independently from Pgp function. The second generation antihistamines terfenadine and loratadine, achieved substantial brain penetration, which was further enhanced by Pgp inhibition by cyclosporin A (CSA). In contrast, fexofenadine and cetirizine, penetrated brain poorly regardless of CSA administration. Conclusions Antihistamines greatly differ in their ability to cross the BBB as well as in the role of Pgp in limiting their transport into the CNS in vivo.     

Human Jejunal Permeability of Cyclosporin A: Influence of Surfactants on P-Glycoprotein Efflux in Caco-2 Cells

Purpose. The purpose of this work was to determine the jejunal permeability of cyclosporin A (CsA) in humans and whether formulation variables modulate the effects of P-glycoprotein (P-gp) on the permeability of CsA in Caco-2 cells. Methods. A solution containing CsA, phenylalanine, propranolol, polyethyleneglycol (PEG) 400, and PEG 4000 was perfused through a 10-cm jejunal segment in 12 subjects. Caco-2 transport studies were performed using previously reported methodology. Results. The mean P _eff (±SD) of CsA in humans was 1.65 (0.53). The mean permeabilities for phenylalanine, propranolol, and PEG 400 were 4.54 (2.39), 2.90 (1.28), and 0.83 (0.51) × 10^-4 cm/s, respectively. The presence of surfactants significantly decreased the permeabilities of CsA in both directions in Caco-2 cells. Conclusions. The results suggest that the effects of surfactants via micellar solubilization and inhibition of P-gp efflux on CsA transport in Caco-2 cells are significant. CsA can rightly be classified as a low solubility-high permeability Class II BCS drug and its highly variable absorption from Sandimmune® oral formulations is the result of poor dissolution characteristics.     

黄连素对大鼠体内罗丹明123药动学的影响

目的：探讨盐酸黄连素（berberine hydrochloride,BBR）是否影响P糖蛋白（P-glycolprotein,P-gp）底物罗丹明123（rhodamine 123,Rh 123）在大鼠体内的代谢.方法：以生理盐水作为阴性对照,以P-gp抑制药维拉帕米为阳性对照.不同组别大鼠分别给予不同剂量BBR或维拉帕米（4 mg · kg-1）连续灌胃10 d后,单次灌胃Rh123（5 mg·kg-1）,自尾静脉采血,HPLC法测定大鼠血浆中Rh123的浓度,研究Rh123在大鼠体内的代谢.结果：建立的大鼠血浆中Rh123的HPLC检测方法完全符合Rh123大鼠血浆样本分析测试的要求,具有较高的灵敏度和特异性.大鼠给予不同药物后各组Rh 123的主要药动学参数：AUC（0-t）（μg·h·L-1）分别为：阴性对照组（48.36±6.4）、BBR 50 mg·kg-1组（59.58±13.37）、BBR 100 mg·kg-1组（77.51±6.84）、BBR 200 mg·kg-1组（95.49±15.99）、维拉帕米4 mg·kg-1组（93.01±13.07）;Cmax（μg·L-1）分别为：阴性对照组（4.41±0.45）、BBR50mg· kg 1组（10.18±5.59）、BBR 100 mg·kg-1组（11.78±3.19）、BBR 200 mg·kg-1组（16.25±8.65）、维拉帕米4 mg·kg-1组（11.39±2.76）.BBR 100,200 mg·kg-和维拉帕米4 mg·kg-1均能够显著升高Rh 123的AUC（0-t） （P ＜0.01）;BBR 50,100,200 mg·kg-1和维拉帕米4 mg·kg-1均能够显著升高Rh 123的Cmax（P＜0.05）且呈剂量依赖性.结论：黄连素可以显著抑制Rh123的代谢,该抑制作用很有可能与BBR抑制了P-gp的活性有关.     

Cyclosporine A Formulation Affects Its Ocular Distribution in Rabbits

Pharmaceutical Research -     

Inhibition of P-Glycoprotein: Rapid Assessment of Its Implication in Blood-Brain Barrier Integrity and Drug Transport to the Brain by an In Vitro Model of the Blood-Brain Barrier

Purpose. The objective of this work was to assess, in vitro, the passage of P-glycoprotein dependent drugs across brain capillary endothelial cells, when these drugs are associated with a reversing agent. Methods. An in vitro model of the blood-brain barrier consisting of a coculture of brain capillary endothelial cells and astrocytes was used. Results. We demonstrate that P-glycoprotein expression is upregulated by the presence of astrocytes. Uptake in the cells and transport across endothelial cell monolayers of vincristine, cyclosporin A and doxorubicin were studied. Using S9788 or verapamil as reversing agents, we found an increase in vincristine transport across the endothelial cell monolayers. On the other hand, the association of S9788 or verapamil with cyclosporin A failed to increase the transport of this drug. An increase in the transport of doxorubicin from luminal to abluminal compartment was also observed, due to endothelial cell monolayer breakdown. Conclusions. Using this model, it is possible to predict the passage of a P-glycoprotein dependent drug to the brain or its sequestration in brain capillary endothelial cells when this drug is associated with a reversing agent, or its toxicity on the blood-brain barrier integrity.     

Identification of P-Glycoprotein and Transport Mechanism of Paclitaxel in Syncytiotrophoblast Cells

When chemotherapy is administered during pregnancy, it is important to consider the fetus chemotherapy exposure, because it may lead to fetal consequences. Paclitaxel has become widely used in the metastatic and adjuvant settings for woman with cancer including breast and ovarian cancer. Therefore, we attempted to clarify the transport mechanisms of paclitaxel through blood-placenta barrier using rat conditionally immortalized syncytiotrophoblast cell lines (TR-TBTs). The uptake of paclitaxel was time- and temperature-dependent. Paclitaxel was eliminated about 50% from the cells within 30 min. The uptake of paclitaxel was saturable with K_m of 168 μM and 371 μM in TR-TBT 18d-1 and TR-TBT 18d-2, respectively. [³H]Paclitaxel uptake was markedly inhibited by cyclosporine and verapamil, well-known substrates of P-glycoprotein (P-gp) transporter. However, several MRP substrates and organic anions had no effect on [³H]paclitaxel uptake in TR-TBT cells. These results suggest that P-gp may be involved in paclitaxel transport at the placenta. TR-TBT cells expressed mRNA of P-gp. These findings are important for therapy of breast and ovarian cancer of pregnant women, and should be useful data in elucidating teratogenicity of paclitaxel during pregnancy.     

A Compartmental Model for the Ocular Pharmacokinetics of Cyclosporine in Rabbits

Studies were conducted in rabbits to determine the ocular distribution and elimination of cyclosporine, with the objective of developing a comprehensive pharmacokinetic model. Following a bolus dose into the anterior chamber, drug levels were measured in the aqueous humor, cornea, iris/ciliary body, lens, sclera, and conjunctiva. Cyclosporine was rapidly eliminated from the aqueous, but drug levels in ocular tissues persisted for in excess of 48 hours, particularly in the cornea and iris/ciliary body. The terminal elimination half life from these tissues was 45 hr and 30 hr, respectively, providing evidence that these tissues could act as a reservoir for the drug. It was found that a compartmental model accurately described the experimental data. A single compartment was used for each of the tissues and fluids sampled, except for the cornea, which was subdivided into two compartments, representing its tissue and aqueous regions.     

A Human Lymphocyte Based Ex Vivo Assay to Study the Effect of Drugs on P-glycoprotein (p-Gp) Function1

Purpose. The effect of drugs on P-glycoprotein (P-gp) is normally studied in transfected or overexpressing cell lines derived from tumor cells or animal tissue. We wanted to develop an assay using normal healthy human tissue to study and characterize the drug-transporter interaction.Methods. Lymphocytes were isolated from healthy human blood. The effect of inhibitors of P-gp (cyclosporine, tacrolimus, verapamil, quinidine, vinblastine) and of other transporters (indomethacin, probenecid, sulfinpyrazone) on intracellular accumulation of rhodamine 123 was evaluated by flow cytometry.Results. The efflux of rhodamine 123 was inhibited by P-gp inhibitors in a saturable, concentration-dependent manner. The potency of inhibition of P-gp was cyclosporine > tacrolimus > quinidine > verapamil > vinblastine. Vinblastine inhibited P-gp at lower concentrations, whereas at high concentrations, there was an activation of rhodamine 123 efflux from lymphocytes. The multidrug resistance associated protein (MRP) inhibitors, sulfinpyrazone and probenecid, did not have any significant effect on intracellular accumulation of rhodamine 123, but indomethacin caused a concentration-dependent increase in retention of rhodamine 123, indicating the involvement of other uncharacterized transporters.Conclusions. Lymphocytes can serve as a model tissue for studying modulation of P-gp activity by drugs. Both inhibitors and inducers of P-gp activity can be evaluated.     

Pharmacokinetics of Uridine Following Ocular,Oral and Intravenous Administration in Rabbits

The pyrimidine nucleoside uridine has recently been reported to have a protective effect on cultured human corneal epithelial cells, in an animal model of dry eye and in patients. In this study, we investigate the pharmacokinetic profile of uridine in rabbits, following topical ocular (8 mg/eye), oral (450 mg/kg) and intravenous (100 mg/kg) administration. Blood and urine samples were serially taken, and uridine was measured by high-performance liquid chromatography-tandem mass spectrometry. No symptoms were noted in the animals after uridine treatment. Uridine was not detected in either plasma or urine after topical ocular administration, indicating no systemic exposure to uridine with this treatment route. Following a single intravenous dose, the plasma concentration of uridine showed a bi-exponential decay, with a rapid decline over 10 min, followed by a slow decay with a terminal half-life of 0.36 ± 0.05 h. Clearance and volume of distribution were 1.8 ± 0.6 L/h/kg and 0.58 ± 0.32 L/kg, respectively. The area under the plasma concentration-time curves (AUC) was 59.7 ± 18.2 μg·hr/ml, and urinary excretion up to 12 hr was ~7.7% of the dose. Plasma uridine reached a peak of 25.8 ± 4.1 μg/ml at 2.3 ± 0.8 hr after oral administration. The AUC was 79.0 ± 13.9 μg·hr/ml, representing ~29.4% of absolute bioavailability. About 1% of the oral dose was excreted in the urine. These results should prove useful in the design of future clinical and nonclinical studies conducted with uridine.     

卡维地洛对慢性心力衰竭兔心肌应变及应变率的影响

目的探讨卡维地洛对慢性心力衰竭兔心肌应变及应变率的作用,以评价其对心肌局部功能的影响和意义。方法30只日本大耳兔随机分为3组,分别为空白对照组（CTL组,6只）、慢性心力衰竭组（CHF组,12只）和卡维地洛干预心衰组（CVD组,12只）。CHF组和CVD组兔每周2次耳缘静脉注射盐酸阿霉素溶液（浓度：1mg·mL^-1）1mL·kg^-1,共计8周;对照组同期耳缘静脉注射等量生理盐水。对照组和模型组兔自第12周始每日以生理盐水（5mg·kg^-1·d^-1）灌胃,CVD组兔以等量卡维地洛生理盐水（浓度：1mg·mL^-1）灌胃,共8周。分别于第12,20周测定3组兔血清B型钠尿肽（BNP）水平,心脏超声测定左心室舒张末期内径（LVEDD）、左室射血分数（LVEF）和缩短分数（FS）等,同时应用心肌应变率显像（SRI）技术检测3组兔左室侧壁和室间隔基底部心肌的收缩期和舒张早期峰值应变率（SRs和SRe）、收缩期和舒张期峰值应变（PSS和PDS）的变化。结果①CHF组和CVD组兔在12周时,其血清BNP水平较CTL组明显升高、LVEDD扩大、LVEF和FS下降,左室侧壁和室间隔基底部心肌SRs,SRe,PSS,PDS明显降低。②20周时,CVD组兔较12周时血清BNP水平和LVEDD明显下降、LVEF和FS明显升高,SRs,SRe,PSS,PDS显著增高;CHF组和对照组上述指标与第12周比较无明显变化。结论慢性心衰时局部心肌收缩期和舒张期峰值应变和应变率降低;卡维地洛可以改善心力衰竭兔整体心肌收缩功能、改善心脏重构;也可以增加收缩期和舒张期峰值应变及应变率,改善局部心肌收缩和舒张功能。     

Role of P-Glycoprotein in Restricting Propranolol Transport in Cultured Rabbit Conjunctival Epithelial Cell Layers

Purpose. To determine the role of P-glycoprotein (P-gp) in propranololtransport in cultured rabbit conjunctival epithelial cell layers (RCEC). Methods. The localization of P-gp in the cultured RCEC as well asin the excised conjunctiva was determined by immunofluorescencetechnique. The role of P-gp in transepithelial transport and uptake ofpropranolol in conjunctival epithelial cells cultured on Transwell filterswas evaluated in the presence and absence of P-gp competing substrates, ananti-P-gp monoclonal antibody (4E3 mAb), or a metabolicinhibitor, 2, 4-dinitrophenol (2,4-DNP). Results. Immunofluorescence studies revealed positive staining in theapical membrane of cultured RCEC and in the apical surface of thesuperficial cell layers in the excised conjunctiva, but not the basolateralmembrane of cultured RCEC. Transport of propranolol showed preferencein the basolateral-to-apical direction. The net secretory flux wassaturable with a K_m of 71.5 ± 24.0 nM and a J_max of 1.45 ± 0.17pmol/cm²/hr. Cyclosporin A, progesterone, rhodamine 123, verapamil,4E3 mAb and 2,4-DNP all increased apical 50 nM propranolol uptakeby 43% to 66%. On the other hand, neither -blockers (atenolol,metoprolol, and alprenolol) nor organic cation transporter substrates(tetraethylammonium (TEA) and guanidine), affected apical 50 nMpropranolol uptake. Conclusions. The energy-dependent efflux pump P-gp appears to bepredominantly located on the apical plasma membrane of the conjunctivalepithelium. It may play an important role in restricting the conjunctivalabsorption of some lipophilic drugs.     

Comparative Studies on in Vitro Methods for Evaluating in Vivo Function of MDR1 P-Glycoprotein

Purpose. MDR1 P-glycoprotein (P-gp) plays an important role in determining drug disposition. The purpose of the present study was to establish in vitro models to predict the in vivo function of P-gp. Methods. As an in vitro method, the transcellular transport of 12 compounds across the monolayer of Caco-2- and MDR1-transfected cells was examined. The ability of these compounds to stimulate the ATP hydrolysis was also determined using the isolated membrane fraction expressing P-gp. As a parameter to describe the in vivo P-gp function, we calculated the brain-to-plasma concentration ratio of compounds in mdr1a/1b knockout mice divided by the same ratio in wild type mice. Results. A good correlation was observed between the in vitro flux ratio across the monolayer and in vivo P-gp function for 12 compounds. Although all compounds that stimulated ATP hydrolysis were significantly transported by P-gp, some compounds were transported by P-gp without significantly affecting ATP hydrolysis. Conclusion. Collectively, the in vitro flux ratio across monolayers of P-gp-expressing cells may be used to predict in vivo P-gp function. The extent of ATP-hydrolysis in vitro may also be a useful parameter for in vivo prediction, particularly for eliminating P-gp substrates in high-throughput screening procedures.     

Scintigraphic Evaluation of the Ocular Disposition of 18F-Imirestat in Rabbits

Pharmaceutical Research -