Pgp Functional Activity Research Articles

Effect of ethylmethylhydroxypyridine succinate on the functional activity of the transporter P-glycoprotein in the blood-brain barrier of rats under normal conditions and under hypoxic hypoxia

Relevance. Ethylmethylhydroxypyridine succinate (EMHPS) is a reference domestic drug with pronounced antioxidant and antihypoxic activity. P-Glycoprotein (Pgp) is an ATP-dependent transport protein localized in tissue barriers and protecting cells and organs from the effects of xenobiotics. Being expressed in the blood-brain barrier (BBB), Pgp limits the penetration of drugs and toxic substances into the brain tissue. Aim – to evaluate the effect of EMHPS on the functional activity of Pgp in the BBB of rats in normal conditions and in acute hypoxic hypobaric hypoxia in the experiment. Methods. The studies were carried out on male Wistar rats weighing 200–250 g, which were divided into 4 groups: group 1 (control, n = 30) – intact rats; Group 2 (control of hypoxia, n = 30) – rats, which were simulated hypoxia and before that they were once injected with water for injection; Group 3 (n = 30) – intact animals, which were injected intravenously with EMHPS at a dose of 50 mg / kg body weight; Group 4 (n = 30) – rats, which were injected intravenously with EMHPS at a dose of 50 mg/kg body weight before modeling hypoxia. 30 minutes after injection, animals of groups 2 and 4 were simulated acute hypoxic hypoxia for 30 minutes by ascending to an altitude of 8000 m with an ascent and descent speed of 50 m/s. 3 h after descent animals of groups 2 and 4 and 30 min after intravenous injection in animals of groups 1 and 3, the functional activity of Pgp in the BBB was assessed by the penetration of fexofenadine, a marker substrate of Pgp, into the brain tissue. For this, fexofenadine was injected into the tail vein of rats at a dose of 10 mg/kg of body weight. After 5, 10, 15, 30, 45, 60 minutes after administration, they were euthanized, at least 4 ml of blood was taken from the abdominal aorta into heparinized tubes and the cortex of the frontal lobes of the brain. The concentration of fexofenadine in biosamples was analyzed by HPLC-UV according to original methods. Results. In the course of the study, it was shown that a single intravenous injection of EMHPS at a dose of 50 mg/kg of body weight causes an increase in the content of fexofenadine in the cerebral cortex of rats, which indicates a decrease in the activity of the Pgp transporter protein. Simulation of acute hypoxic hypoxia was also accompanied by an increase in the permeability of the transport protein substrate into the brain tissue. At the same time, the prophylactic administration of EMHPS before hypoxic exposure did not significantly affect the BBB permeability, which remained significantly higher than the control and did not differ from the permeability during isolated hypoxic exposure. Conclusions: EMHPS with a single intravenous injection at a dose of 50 mg/kg body weight reduces the activity of Pgp in the BBB in normal conditions and does not significantly affect the penetration of the transporter substrate – fexofenadine into the brain tissue in acute hypoxic hypoxia

INFLUENCE OF POLYSACCHARIDE COMPLEXES OF PLANTS OF CENTRAL RUSSIA ON THE P-GLYCOPROTEIN ACTIVITY IN VITRO

The purpose of this work was to study the effect of polysaccharide complexes isolated from tansy flowers (Tanacetum vulgare L., fam. Asteraceae) and melilotus herb (Melilotus officinalis L., fam. Fabaceae) on P-glycoprotein (Pgp, ABCB1 protein) activity in vitro.
 On Caco-2 cell line the effect of polysaccharide complex isolated from tansy flowers and melilotus herb on Pgp activity was studied. In vitro Pgp activity was assessed by the transport of fexofenadine in the transwell system. High performance liquid chromatography with UV detection at wavelength 220 nm was used to determine fexofenadine concentration in the transport medium.
 It was revealed that when polysaccharide isolated from tansy flowers was added to the transport medium in concentrations 10 and 100 μM the ratio of the apparent permeability coefficients of fexofenadine b-a/a-b decreased by 1.81 and 2.65 times, respectively, compared with the series of isolated transport of fexofenadine, which indicated decreased Pgp functional activity under the polysaccharide action. The polysaccharide complex of the melilotus herb did not change the b-a/a-b ratio in any of the applied concentrations, thus it did not affect the activity of this transporter. It is advisable to continue the study of tansy flower polysaccharide complex as an inhibitor of Pgp transporter protein in order to assess the possibility of its clinical use for the treatment of pharmacoresistant forms of cancer by overcoming the phenomenon of multidrug resistance of cells.

Novel Intrinsic Mechanisms of Active Drug Extrusion at the Blood-Brain Barrier: Potential Targets for Enhancing Drug Delivery to the Brain?

The blood-brain barrier (BBB) limits the pharmacotherapy of several brain disorders. In addition to the structural and metabolic characteristics of the BBB, the ATP-driven, drug efflux transporter P-glycoprotein (Pgp) is a selective gatekeeper of the BBB; thus, it is a primary hindrance to drug delivery into the brain. Here, we review the complex regulation of Pgp expression and functional activity at the BBB with an emphasis on recent studies from our laboratory. In addition to traditional processes such as transcriptional regulation and posttranscriptional or posttranslational modification of Pgp expression and functionality, novel mechanisms such as intra- and intercellular Pgp trafficking and intracellular Pgp-mediated lysosomal sequestration in BBB endothelial cells with subsequent disposal by blood neutrophils are discussed. These intrinsic mechanisms of active drug extrusion at the BBB are potential therapeutic targets that could be used to modulate P-glycoprotein activity in the treatment of brain diseases and enhance drug delivery to the brain.

HPLC methods of fexofenadine quantitative analysis in rabbits’ liver

The investigation of pharmacokinetics of marker substrates of carrier protein P-glycoprotein (Pgp, ABCB1-protein) including fexofenadine, is one of the methods of its functional activity evaluation.The aim of the study was to work out the HPLC methods of the quantitative determination of fexofenadine in rabbits’ liver.Materials and methods. The quantitative determination of fexofenadine was performed using Stayer chromatographic system (Akvilon, Russia) with UVV 104 ultraviolet detector. Reverse-phased chromatographic column Luna C18 100Å (250*4.6) was used with 5 µm granulation at 45°С. The сoncentration of fexofenadine was determined by methods of absolute peak area calibration.Results. The work was conducted in the isocratic mode. The composition of the mobile phase consisted of deionized water, acetonitrile and glacial acetic acid at the ratio of 267.4:120:4.33 brought to pH=6.7 with triethylamine. The sample processing was in the form of homogenization of 500 mg of ground liver in 500 µl of purified water with the subsequent centrifugation (1750 g) and selection of the supernatant. The proteins were precipitated by acetonitrile (2.5 ml) acidified with 375 µl of hydrochloric acid by shaking at 500 rev/min. The supernatant was transported into a separate test tube, where methylene chloride, diethyl ether and ethyl acetate were added (2 ml each). Then the solution was again shaken for 10 minutes (500 rev/min). After that, the solution was centrifuged (1750 g) and the supernatant was evaporated on a rotor-vacuum evaporator at 50°С. 300 µl of the mobile phase was added to the dry residue, and 100 µl was injected into the chromatograph. The method was validated in the linear range from 3 to 60 µg/g of fexofenadine with the acceptable intra- and intercycle accuracy, precision and stability. The method was tested on rabbits after the intravenous administration of fexofenadine at the dose of 11 mg/kg.Conclusion. The HPLC methods of fexofenadine quantitative determination in the hepatic tissue of rabbits has been worked out. It can be used for the evaluation of the functional activity of Pgp in preclinical studies.Abbreviations: Pgp – P-glycoprotein, HPLC – high performance liquid chromatography, rev/min – revolutions per minute

Functional activity of p-glycoprotein in blood-brain barrier during experimental par-kinson's syndrome

Background. P-glycoprotein (Pgp, ABCB1-protein) is a membrane transporter with broad substrate specificity that is localized in hepatocytes, enterocytes, epithelial renal tubules, and also in tissue barriers, including blood-brain barrier (BBB). Increased Pgp activity in BBB is one of the reasons for the pharmacoresistance of a number of CNS diseases.
 Aim. Analysis of Pgp functional activity in BBB during experimental Parkinson's syndrome.
 Materials and Methods. The work was performed on 90 Wistar rats, divided into 3 series (n=30 in each). The 1 series (control) was subcutaneously injected sunflower oil once a day for 7 days, and Pgp activity in BBB was assessed on the 8th day. The 2 and 3 series (pathology control) - were administered rotenone at a dose of 2.5 mg/kg once a day for 7 and 28 days respectively to simulate parkin-sonism. At the end of the experiment Pgp activity was estimated. To confirm Parkinson's syndrome, in addition to the clinical picture, level of dopamine in midbrain and striatum was determined using enzyme-linked immunosorbent assay. Pgp functional activity in BBB was assessed by the degree of penetration of its marker substrate fexofenadine into the brain after its intravenous administration at a dose of 10 mg/kg. The content of fexofenadine in the blood plasma and in brain tissue was estimated by the area under pharmacokinetic curve of the substance (in the blood or brain tissues) - AUC0-t(plasma) or AUC0-t(brain) respectively. To assess the BBB permeability the ratio AUC0-t(brain) / AUC0-t(plasma) was calculated.
 Results. Rotenone administration led to the development of parkinsonism typical picture: muscle stiffness, hypokinesia, gait instability. There was a decrease in dopamine level in the striatum after 7 days by 69.6% (p=0.095), after 28 days - by 93.9% (p=0.008), in midbrain - by 72.7% (p=0.095) and 68.7% (p=0.032) respectively. Fexofenadine AUC0-t(plasma) and AUC0-t(brain) after its intravenous administration to control rats were 266.2 (246.4; 285.6) μg/ml*min and 5.9 (5.8;6.6) µg/g*min respectively, AUC0-t(brain) /AUC0-t(plasma) - 0.020 (0.019; 0.022). When rotenone was for 7 days administered - fexofenadine AUC0-t(brain) increased 2.02 times (p=0.0163), AUC0-t(brain) / AUC0-t(plasma) - 2.4 times (p=0.0283). 28 days administration of rotenone led to augmentation of AUC0-t(brain) of fexofenadine by 1.75 times (p=0.0283), AUC0-t(brain) / AUC0-t(plasma) - by 2.27 times (p=0.0163).
 Conclusions. The development of Parkinson’s syndrome, caused by the administration of rotenone, inhibits Pgp functional activity in BBB, which is confirmed by the accumulation in the brain marker substrate of the transporter - fexofenadine.

P-glycoprotein functional activity in «soviet chinchilla» rabbits during pregnancy

P-glycoprotein (Pgp, ABCB1-protein) is a membrane transporter protein that plays the key role in pharmacokinetics of drugs with a broad spectrum of action. Substrates of this transporter are some medical drugs (antibacterial, antiretroviral, hypotensive) that are prescribed to pregnant women for long-term intake, sometimes throughout the whole gestation period.
 Aim to study the activity of Pgp on the organism level in rabbits of Soviet Chinchilla breed in pregnancy.
 Materials and Methods. The study was performed on 21 Soviet Chinchilla female rabbits (3000-3500 g). The animals were divided into 3 series. The first series (n=6) included rabbits with 7 days of pregnancy; the second series (n=5) - animals with 14 days of pregnancy; the third series (n=10) - rabbits with 21 days of pregnancy. 7 Days before the study and in the indicated gestation periods, functional activity of Pgp was assessed by the pharmacokinetics of marker transporter substrate – fexofenadine, after its single oral introduction (67.5 mg/kg). Besides, serum concentrations of progesterone, estradiol, testosterone and prolactin were determined by radio immune method
 Results. In all the studied gestational periods, serum concentrations of estradiol, testosterone and prolactin did not significantly differ from those before pregnancy, but the level of progesterone in blood serum was significantly elevated above the norm. On the 7th day of pregnancy pharmacokinetic parameters of fexofenadine did not show any reliable changes as compared to the initial va-lues. On the 14th day of pregnancy a reliable increase in Cmax, AUC0-t, T1/2 of fexofenadine was noted as compared to the parameters before pregnancy, which indicates a decrease in Pgp functional activity on the organism level. On the 21st day of pregnancy Cmax of fexofenadine remained elevated. Other pharmacokinetic parameters of fexofenadine did not show reliable changes. Conclusion. Reduction in Pgp functional activity, determined by the pharmacokinetics of its marker substrate (fexofenadine), was noted in rabbits of Soviet Chinchilla breed on the 14th and 21st days of pregnancy with the underlying significant increase in progesterone level.

Vegetative polysaccharides: new target and direction of clinical use

P-glycoprotein (Pgp, ABCB1-protein) is a membrane efflux protein transporter located in hepatocytes, enterocytes, epithelial cells of the proximal renal tubules, histogematic barriers, and also in tumor cells, which releases the substrates from cells and plays an important role in pharmacokinetics of drugs. Inhibition of Pgp functional activity is promising for increasing the effectiveness of drug treatment of oncological diseases and pharmacoresistant epilepsy, but today no synthetic inhibitor of the transporter can be used in clinical practice because of side effects development. The aim of this review is to prove the relevance to use polysaccharides of plant origin, their derivatives and chemical modifications as effective, safe and cost-effective inhibitors of the transporter protein. The article describes the chemical structure of Pgp substrates and inhibitors, the ability to predict the drugs belonging to the transporter protein substrates or inhibitors. Some results of investigations of the belonging of oligo- and polysaccharides to Pgp substrates and inhibitors are presented. They are premises for carrying out appropriate experiments for polysaccharides of plant origin both in vitro and in vivo.

Experimental Studies of the Effects of Progesterone on the Functional Activity of P-Glycoprotein

Experiments on female Chinchilla rabbits addressed the effects of progesterone on the functional activity of P-glycoprotein (Pgp). Animals were divided into four groups: animals of group 1 underwent sham operations; group 2 underwent ovariectomy; groups 3 and 4 underwent ovariectomy followed 15 days later by p.o. treatment with progesterone (2 and 15 mg/rabbit respectively). The functional activity of Pgp was determined seven days before the experiment started and on post-operative days 14, 28, and 42 by analysis of the pharmacokinetics of its marker substrate fexofenadine and serum sex hormone concentrations. At the end of the study, animals of each group were harvested by air embolism and specimens of large intestine were collected for immunohistochemical analysis. Ovariectomy led to a decrease in the functional activity of Pgp and a decrease in the staining intensity of the apical and lateral membranes of enterocytes, providing indirect evidence of a decrease in the expression of the transporter protein. Administration of progesterone at a dose of 2 mg/rabbit on the background of ovariectomy increased the functional activity of Pgp from the level seen in the ovariectomy group, though its activity remained decreased compared with the baseline level. This group showed no changes in the staining intensity of enterocyte membranes as compared with the ovariectomy group. Use of progesterone at a dose of 15 mg/rabbit after ovariectomy increased the functional activity of Pgp and the staining intensity of enterocyte apical and lateral membranes from the level seen in the ovariectomy group, restoring the level of transporter protein activity to that of intact animals, probably because of increased expression.

Влияние прогестерона на функциональную активность гликопротеина-P в эксперименте

В исследовании на кроликах-самках породы шиншилла изучено влияние прогестерона на функциональную активность гликопротеина-P (Pgp). Животные были разделены на 4 группы: первой группе животных была проведена «ложная операция»; второй группе – овариоэктомия; третьей и четвертой группе проводили овариоэктомию и с 15 сут после операции per os вводили прогестерон (2 и 15 мг/кролик соответственно). За 7 сут до начала опыта, на 14, 28 и 42 сут после операции определяли функциональную активность Pgp по анализу фармакокинетики его маркерного субстрата фексофенадина и сывороточные концентрации половых гормонов. В конце исследования животных каждой группы выводили из эксперимента методом воздушной эмболии и забирали образец тощей кишки для иммуногистохимического анализа. Овариоэктомия приводила к снижению функциональной активности Pgp и уменьшению интенсивности окрашивания апикальной и латеральных мембран энтероцитов, косвенно свидетельствующей о снижении экспрессии белка-транспортера. Введение прогестерона в дозе 2 мг/кролик на фоне овариоэктомии повышало функциональную активность Pgp по сравнению с показателями группы овариоэктомии, однако его активность оставалась сниженной по сравнению с исходными данными. Также в данной группе не выявлено изменения интенсивности окрашивания мембран энтероцитов в сравнении с серией овариоэктомии. Применение прогестерона в дозе 15 мг/кролик при овариоэктомии повышало функциональную активность Pgp и интенсивность окрашивания апикальной и латеральных мембран энтероцитов по сравнению с показателями группы овариоэктомии, восстанавливая активность белка-транспортера до показателей интактных животных, вероятно, за счет повышения его экспресии.

Influence of female hormones on the P-glycoprotein functioning

The P-glycoprotein (Pgp, ABCB1-protein) is a transport protein localized in the membrane of hepatocytes, small and large intestine enterocytes, renal tubular epitheliocytes, endothelial cells of histohematic barriers, and tumor cells.
 Aim — to study the effect of estradiol and progesterone physiological concentrations on the Pgp functional activity on the whole body.
 Material and methods. The work was performed on 17 adult female chinchilla rabbits. The first group of rabbits underwent a “false operation” (n=5); the second group (n=6) underwent ovariectomy. The third group (n=6) was ovariectomized and received estradiol (0.5 mg/rabbit) for 14 days (starting on the 14th postoperative day), followed by combined administration of estradiol (0.5 mg/rabbit) and progesterone (5 mg/rabbit) for 14 days. The Pgp functional activity was determined in rabbits of all groups using HPLC analysis of fexofenadine pharmacokinetics on day 7 before the study onset and on day 14, 28, and 42 after the operation. Accumulation of fexofenadine in rabbits indicates Pgp inhibition, and a decrease in its content means Pgp induction.
 Results. Ovariectomy led to a decrease in the Pgp functional activity. The estradiol introduction for 14 days after ovariectomy did not significantly affect the transport protein functional activity. Combined administration of estradiol and progesterone for 14 days resulted in an increase in the Pgp activity, compared to that in the ovariectomy series and baseline values.
 Conclusions. The dependence of the transport protein activity on the dynamics of physiological estradiol and progesterone concentrations suggests that the effectiveness of pharmacotherapy with Pgp substrates may depend on the menstrual cycle phase, and drug dose correction may be used to increase the pharmacotherapy effectiveness.

Effects of Testosterone on the Functional Activity of P-Glycoprotein

The effects of testosterone undecanoate on the functional activity of P-glycoprotein (ABCB1 protein) were studied in male Chinchilla rabbits. Testosterone was given as single intramuscular doses of 12 or 24 mg/kg. The functional activity of P-glycoprotein was determined in terms of the pharmacokinetics of its marker substrate - fexofenadine - on days 7, 14, and 21 after hormone administration. Administration of testosterone at a dose of 12 mg/kg (n = 7) increased the C max of fexofenadine on days 7 and 14 and AUC 0-t on day 14 and decreased total clearance on day 14. Use of testosterone at a dose of 24 mg/kg (n = 7) led to increases in the C max of fexofenadine on day 7 and AUC 0-t on days 7, 14, and 21 and decreased total clearance on days 7 and 14. These changes in the pharmacokinetics of fexofenadine provide evidence of decreases in the functional activity of Pgp on the background of testosterone administration. Correlational relationships were found between measures of the pharmacokinetics of fexofenadine and serum testosterone levels.

The role of testosterone in the regulation of P-glycoprotein functioning

The functional activity of P-glycoprotein (Pgp) was studied in Chinchilla rabbits after orchiectomy and administration of testosterone undecanoate after surgery. The functional activity of Pgp was determined by the pharmacokinetics of fexofenadine, its marker substrate. In addition, the localization of the transport protein was assessed in the liver, kidney, and jejunum by the immunohistochemistry. It was found that a decrease in the serum testosterone concentration after orchiectomy leads to an increase in Pgp activity on the 14th and 21st days after the surgery, which is accompanied by an increase in the staining intensity of Pgp-positive epithelium membranes of the proximal and distal renal tubules. It was found that the administration of testosterone at a dose of 6 mg/kg body weight in the period after the surgery neutralizes these changes.

Influence of different hypoxia types on p-glycoprotein functional activity and expression

The review of literature is devoted to researches in vitro and in vivo in which functioning changes P-glycoprotein (Pgp) under the influence of hypoxia of different types are shown. In the majority of scientific works activation of expression and functional activity of Pgp is shown that authors connect with activation of HIF-1 and Sp1 which are described in more detail. However a number of researches disproves the conclusion about induction of activity of Pgp in the conditions of hypoxia that testifies about difficult mechanisms involved in regulation of Pgp activity, their dependence on a type and duration of hypoxia, redox status and other parameters.

Cluster of Differentiation 44 Targeted Hyaluronic Acid Based Nanoparticles for MDR1 siRNA Delivery to Overcome Drug Resistance in Ovarian Cancer.

Approaches for the synthesis of biomaterials to facilitate the delivery of "biologics" is a major area of research in cancer therapy. Here we designed and characterized a hyaluronic acid (HA) based self-assembling nanoparticles that can target CD44 receptors overexpressed on multidrug resistance (MDR) ovarian cancer. The nanoparticle system is composed of HA-poly(ethyleneimine)/HA-poly(ethylene glycol) (HA-PEI/HA-PEG) designed to deliver MDR1 siRNA for the treatment of MDR in an ovarian cancer model. HA-PEI/HA-PEG nanoparticles were synthesized and characterized, then the cellular uptake and knockdown efficiency of HA-PEI/HA-PEG/MDR1 siRNA nanoparticles was further determined. A human xenograft MDR ovarian cancer model was established to evaluate the effects of the combination of HA-PEI/HA-PEG/MDR1 siRNA nanoparticles and paclitaxel on MDR tumor growth. Our results demonstrated that HA-PEI/HA-PEG nanoparticles successfully targeted CD44 and delivered MDR1 siRNA into OVCAR8TR (established paclitaxel resistant) tumors. Additionally, HA-PEI/HA-PEG nanoparticles loaded with MDR1 siRNA efficiently down-regulated the expression of MDR1 and P-glycoprotein (Pgp), inhibited the functional activity of Pgp, and subsequently increased cell sensitivity to paclitaxel. HA-PEI/HA-PEG/MDR1 siRNA nanoparticle therapy followed by paclitaxel treatment inhibited tumor growth in MDR ovarian cancer mouse models. These findings suggest that this CD44 targeted HA-PEI/HA-PEG nanoparticle platform may be a clinicaly relevant gene delivery system for systemic siRNA-based anticancer therapeutics for the treatment of MDR cancers.

ORAL HORMONAL CONTRACEPTIVE "YARINA" INFLUENCE ON P-GLYCOPROTEIN FUNCTIONAL ACTIVITY

In the research on rabbits influence of combined oral contraceptive "Yarina" on the functional activity of the protein efflux transporter P-glycoprotein (Pgp) was studied. Pgp activity was assessed by pharmacokinetic analysis of marker substrate fexofenadine. It was found that administration of the drug "Yarina" for 14 and 21 days resulting in inhibition of Pgp functional activity.

Abstract LB-13: Hyaluronic acid-based CD44 targeted nanoparticle delivery of combination MDR1 siRNA/paclitaxel to overcome drug resistance in ovarian cancer

Abstract A major obstacle in the success of chemotherapy in ovarian cancer is the emergence of multidrug resistance (MDR). Overexpression of the MDR1 gene and corresponding P-glycoprotein (Pgp) efflux pumps is one of the best characterized MDR mechanisms. Although MDR1 siRNA based strategies are emerging as highly promising approaches to reverse MDR, the systemic delivery still remains a great challenge. In the present study, CD44 targeting hyaluronic acid (HA) based self-assembling nanoparticle systems were designed with MDR1 siRNA to evaluate its delivery efficiency and combination anticancer therapeutic efficacy with paclitaxel in vitro and in vivo. The results showed that HA-poly(ethyleneimine)/HA-poly(ethylene glycol) (HA-PEI/HA-PEG) nanoparticle successfully delivered MDR1 siRNA into MDR ovarian cancer cell lines, SKOV-3TR and OVCAR8TR. Western blot analysis, calcein AM retention assay, and MTT assay further demonstrated that HA-PEI/HA-PEG nanoparticles loaded with MDR1 siRNA efficiently down-regulated the expression of MDR1 and inhibited the functional activity of Pgp, and subsequently increase cell sensitivity to paclitaxel. HA-PEI/HA-PEG/MDR1 siRNA nanoparticle therapy followed by paclitaxel treatment produced a significant inhibitory effect on the drug-resistant tumor growth as compared with control groups in MDR ovarian cancer xenograft mouse models. These results indicated CD44-targeted HA-PEI/HA-PEG nanoparticle platform is an effective siRNA delivery system for potential siRNA-based anticancer therapeutics in ovarian cancer. Citation Format: Xiaoqian Yang, Arun lyer, Francis hornicek, Mansoor Amiji, Zhenfeng Duan. Hyaluronic acid-based CD44 targeted nanoparticle delivery of combination MDR1 siRNA/paclitaxel to overcome drug resistance in ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-13. doi:10.1158/1538-7445.AM2014-LB-13

Oral hormonal contraceptive "janine" influence on P-glycoprotein functional activity

In the research on rabbits influence of combined oral contraceptive, "Janine" on the functional activity of the protein efflux transporter P-glycoprotein (Pgp) was studied. Pgp activity was assessed by pharmacokinetic analysis of marker substrate fexofenadine. It was found that administration of the drug "Janine" for 14 and 21 days, resulting in inhibition of Pgp functional activity.

FINASTERIDE INFLUENCE ON P-GLYCOPROTEIN FUNCTIONAL ACTIVITY

In the research on rabbits influence of finasteride on the functional activity of the protein efflux transporter P-glycoprotein (Pgp) was studied. Pgp activity was assessed by pharmacokinetic analysis of it’s marker substrate fexofenadine. It was found that the 14-days course of finasteride administration leads to tissue-specific induction of Pgp functional activity in liver and kidney.

Drug Transporters in Imatinib Resistance: A New Therapeutic Targeted Approach In CML

Abstract 5010Chronic myeloid leukaemia (CML) is a mieloproliferative disorder characterized by the presence of the BCR-ABL gene fusion, which encodes an oncoprotein with a deregulated tyrosine kinase activity. This oncoprotein became the main therapeutic target of the disease. In fact, the first-line treatment is Imatinib, a specific tyrosine inhibitor that blocks BCR-ABL tyrosine kinase activity. Although, the good results with Imatinib, the development of drug resistance is a reality and can be mediated by different pathways, besides the BCR-ABL mutations, like the unbalance between influx and efflux drug transporters expression. Therefore, the knowledge of these changes would enable the use of new therapeutics approaches in CML.The aims of this study are to evaluate the role of drug influx and efflux transporters in Imatinib resistance and the potential therapeutic of Reversin 205 in overcoming the resistance to Imatinib. For this purpose, we used a CML cell line sensitive to Imatinib, the K562 cells, and generated two sub-cell lines resistant to Imatinib, the K562-RC and K562-RD cells. To obtain these resistant cells, K562 cells were exposed to the drug following two strategies: continuous exposure of increasing concentrations of Imatinib (K562-RC cells) and discontinuous exposure, with interchanged mediums with and without Imatinib (K562-RD cells). Drug influx and efflux transporters expression levels, namely OCT1, OCTN2, PgP and BCRP, were evaluated by flow cytometry (FC) using monoclonal antibodies labeled with fluorescent probes. Functional activity of PgP was assessed by a kinetic study using the radiosensitizer 99mTc Sestamibi. To evaluate the effect of Imatinib and Reversin 205, a P-gP inhibitor, on cell viability, all cell lines were treated in the absence and presence of different drug’s concentrations and analyzed by the resazurin assay. Cell death was analyzed by optical microscopy (May-Grünwald staining) and by FC using the Annexin V and Propidium Iodide double staining and caspases expression levels.Our results show that the half maximal inhibitory concentration (IC50) of Imatinib in K562 sensitive cells is 75nM, whereas in K562-RC cells this value is increased eight times (to 605nM) and in K562-RD cells is around eighteen times higher (1389nM). On the other hand, we observed a decrease in OCT1 and OCT2 expression levels and an increase in PgP and BCRP expression in resistant cells compared with the sensitive cell line, which may contribute to the resistance to Imatinib. Besides the alterations in proteins expression levels we found, in resistant cell lines, a lower uptake profile of the radiosensitizer, demonstrating the functional activity of the MDR protein efflux transporter, Pgp. Concordantly with these results, we observed that when resistant cell lines were exposed to the combination of Reversin 205 (5μM), plus increasing concentrations of Imatinib, the IC50 decreased to 170nM in K562 RC and to 367nM in K562 RD. These results suggest that Reversin 205 may circumvent resistance to imatinib in CML cells. On the other hand, at higher doses, this compound revealed a cytotoxic effect in sensitive and resistant cells, and induces cell death by apoptosis in a time- and dose- dependent manner. The IC50 at 48 hours of exposure was of 12μM to K562, 18,9 μM to K562-RD and 18,7 μM to K562-RC cells.In conclusion, our results suggest that discontinuous drug exposure induces higher resistance levels and drug's transporters are involved in the acquisition of resistance to Imatinib. The use of Reversin 205 or other drug efflux transporters inhibitors might circumvent this resistance and could be used as a new potential therapeutic targeted approach in CML. Disclosures:No relevant conflicts of interest to declare.

Differential metabolic responses to pluronic in MDR and non-MDR cells: A novel pathway for chemosensitization of drug resistant cancers

A synthetic amphiphilic block copolymer, Pluronic, is a potent chemosensitizer of multidrug resistant (MDR) cancers that has shown promise in clinical trials. It has unique activities in MDR cells, which include a decrease in ATP pools and inhibition of P-glycoprotein (Pgp) resulting in increased drug accumulation in cells. This work demonstrates that Pluronic rapidly (15 min) translocates into MDR cells and co-localizes with the mitochondria. It inhibits complex I and complex IV of the mitochondria respiratory chain, decreases oxygen consumption and causes ATP depletion in MDR cells. These effects are selective and pronounced for MDR cells compared to non-MDR counterparts and demonstrated for both drug-selected and Pgp-transfected cell models. Furthermore, inhibition of Pgp functional activity also abolishes the effects of Pluronic on intracellular ATP levels in MDR cells suggesting that Pgp contributes to increased responsiveness of molecular “targets” of Pluronic in the mitochondria of MDR cells. The Pluronic-caused impairment of respiration in mitochondria of MDR cells is accompanied with a decrease in mitochondria membrane potential, production of ROS, and release of cytochrome c. Altogether these effects eventually enhance drug-induced apoptosis and contribute to potent chemosensitization of MDR tumors by Pluronic.