Pump P-glycoprotein Research Articles

ZD6474 reverses multidrug resistance by directly inhibiting the function of P-glycoprotein

P-glycoprotein (P-gp) pumps multiple types of drugs out of the cell, using energy generated from ATP, and confers multidrug resistance (MDR) on cancer cells. ZD6474 is an orally active, selective inhibitor of the vascular endothelial growth factor receptor, epidermal growth factor receptor, and rearranged during transfection tyrosine kinases. This study was designed to examine whether ZD6474 reverses P-gp-mediated MDR in cancer cells. Here, we show that clinically achievable levels of ZD6474 reverse P-gp-mediated MDR of the P-gp-overexpressing cell lines derived from breast cancer, MCF-7/adriamycin (ADR), and human oral epidermoid carcinoma, KBV200 to ADR, docetaxel, and vinorelbine. This ability to reverse the P-gp-mediated resistance is comparable to that of another frequently used reversal agent known as verapamil. ZD6474 itself moderately inhibits the proliferation of both MCF-7 and MCF-7/ADR cells with almost equal activity, but its inhibitory effect is not altered by co-incubation with verapamil, suggesting that ZD6474 may not be a substrate of P-gp. In addition, ZD6474 increases the intracellular accumulation of the P-gp substrate, rhodamine-123, and ADR, by enhancing the uptake and/or decreasing the efflux of these compounds in resistant cells. Further studies show that ZD6474 stimulates ATPase activity in a dose-dependent manner, which is required for the proper function of P-gp. In contrast, ZD6474 does not inhibit the expression level of P-gp. Our results suggest that ZD6474 is capable of reversing MDR in cancer cells by directly inhibiting the function of P-gp, a finding that may have clinical implications for ZD6474.

TC-99m MIBI Spect Imaging in Patients With Lung Carcinoma

Multidrug-resistance (MDR) phenotype concerns altered membrane transport that results in lower cell concentrations of cytotoxic drug in many cancer types, including lung cancer, and is related to the overexpression of a variety of proteins that act as adenosine triphosphate-dependent extrusion pumps. Tc-99m Sestamibi (MIBI) is a transport substrate for P-glycoprotein (Pgp) pump. In this study, we assessed the uptake and clearance of technetium-99m-2-hexakis 2-methoxyisobutylisonitrile (Tc-99m MIBI) from the tumor and its correlation with messenger RNA (mRNA) levels of Pgp, MDR-associated protein (MRP1), and lung resistance protein (LRP) in lung carcinoma. This study was carried out on 19 patients (mean age, 60.1 +/- 2.07 years) with advanced-stage lung carcinoma. The tumor samples obtained by bronchoscopy were assessed to estimate the levels of Pgp, MRP1, and LRP expression on mRNA level by quantitative real-time reverse-transcription polymerase chain reaction. Tc-99m MIBI chest imaging was performed 15 and 180 minutes after injection of 740 MBq Tc-99m MIBI. The early (T/Be) and delayed (T/Bd) Tc-99m MIBI uptakes and washout rate (WR) of Tc-99m MIBI from the tumor were measured. No correlation was found between the T/Be Tc-99m MIBI uptake of tumors (T/Be) and the levels of Pgp mRNA, MRP1 mRNA, and LRP mRNA by reverse-transcription polymerase chain reaction. There was a correlation between the mean T/Bd Tc-99m MIBI uptake and Pgp expression of the tumors (P = 0.001, Spearman rho = - 0.702). There was a correlation between the WR of Tc-99m MIBI from the tumor and Pgp expression of the tumor (P = 0.000, Spearman rho = 0.875). Washout rate of Tc-99m MIBI was not related to the levels of MRP1 mRNA (P = 0.93, Spearman rho = 0.02) or LRP mRNA (P = 0.47, Spearman rho = 0.177). Increased WR of Tc-99m MIBI is related in Pgp over expression of the tumor. Tc-99m MIBI single photon emission computed tomography imaging may be a functional probe of overexpression of Pgp in patients with lung carcinoma. However, Tc-99m MIBI single photon emission computed tomography imaging cannot be used to identify the MDR involved in the MRP1 or LRP in these patients.

Pharmacogenetics of Tacrolimus and Sirolimus in Renal Transplant Patients: From Retrospective Analyses to Prospective Studies

The promises of pharmacogenetics are to elucidate the inherited basis of differences between individual responses to drugs in order to identify the right drug and dose for each patient. The recent identification of genetic polymorphisms in drug-metabolizing enzymes and drug transporters led to the hypothesis that genetic factors may be implicated in the interindividual variability of the pharmacokinetic or pharmacodynamic characteristics of immunosuppressive drugs, major side effects, and efficacy. The purpose of this study was to provide a short overview of recent results obtained in the field of pharmacogenetics of tacrolimus and sirolimus, both substrates of the cytochrome P450 3A ( CYP3A) enzymes and of the efflux pump P-glycoprotein, the product of the Multidrug Resistance-1 ( MDR1) genes. A number of retrospective studies that demonstrated a link between the polymorphisms governing the CYP3A5 protein expression, with more conflicting results with the MDR1 gene polymorphisms, related to the daily dose necessary to achieve adequate blood tacrolimus levels. The CYP3A5 polymorphisms have also been associated with sirolimus pharmacokinetics. One challenge is to investigate the combined effect of a number of different polymorphisms in various genes to define genetic backgrounds with different pharmacokinetic profiles using high throughput technologies. Another challenge is to move toward prospective randomized studies to explore whether a pharmacogenetic approach, taking into account a limited number of polymorphisms prior to drug treatment, could be used on an individual basis to guide initial dosing of a given drug. The last challenge is based on “target” pharmacogenetics to investigate the role of the polymorphisms of other genes implicated in the efficacy and/or safety of the drug.

Photochemical internalisation of chemotherapy potentiates killing of multidrug-resistant breast and bladder cancer cells

Multidrug resistance (MDR) is the major confounding factor in adjuvant solid tumour chemotherapy. Increasing intracellular amounts of chemotherapeutics to circumvent MDR may be achieved by a novel delivery method, photochemical internalisation (PCI). PCI consists of the co-administration of drug and photosensitiser; upon light activation the latter induces intracellular release of organelle-bound drug. We investigated whether co-administration of hypericin (photosensitiser) with mitoxantrone (MTZ, chemotherapeutic) plus illumination potentiates cytotoxicity in MDR cancer cells. We mapped the extent of intracellular co-localisation of drug/photosensitiser. We determined whether PCI altered drug-excreting efflux pump P-glycoprotein (Pgp) expression or function in MDR cells. Bladder and breast cancer cells and their Pgp-overexpressing MDR subclones (MGHU1, MGHU1/R, MCF-7, MCF-7/R) were given hypericin/MTZ combinations, with/without blue-light illumination. Pilot experiments determined appropriate sublethal doses for each. Viability was determined by the 3-[4,5-dimethylthiazolyl]-2,5-diphenyltetrazolium bromide assay. Intracellular localisation was mapped by confocal microscopy. Pgp expression was detected by immunofluorescence and Pgp function investigated by Rhodamine123 efflux on confocal microscopy. MTZ alone (0.1–0.2 μg ml−1) killed up to 89% of drug-sensitive cells; MDR cells exhibited less cytotoxicity (6–28%). Hypericin (0.1–0.2 μM) effects were similar for all cells; light illumination caused none or minimal toxicity. In combination, MTZ /hypericin plus illumination, potentiated MDR cell killing, vs hypericin or MTZ alone. (MGHU1/R: 38.65 and 36.63% increase, P<0.05; MCF-7/R: 80.2 and 46.1% increase, P<0.001). Illumination of combined MTZ/hypericin increased killing by 28.15% (P<0.05 MGHU1/R) compared to dark controls. Intracytoplasmic vesicular co-localisation of MTZ/hypericin was evident before illumination and at serial times post-illumination. MTZ was always found in sensitive cell nuclei, but not in dark resistant cell nuclei. In illuminated resistant cells there was some mobilisation of MTZ into the nucleus. Pgp expression remained unchanged, regardless of drug exposure. Pgp efflux was blocked by the Pgp inhibitor verapamil (positive control) but not impeded by hypericin. The increased killing of MDR cancer cells demonstrated is consistent with PCI. PCI is a promising technique for enhancing treatment efficacy.

Irradiation of rat brain reduces P-glycoprotein expression and function

The blood–brain barrier (BBB) hampers delivery of several drugs including chemotherapeutics to the brain. The drug efflux pump P-glycoprotein (P-gp), expressed on brain capillary endothelial cells, is part of the BBB. P-gp expression on capillary endothelium decreases 5 days after brain irradiation, which may reduce P-gp function and increase brain levels of P-gp substrates. To elucidate whether radiation therapy reduces P-gp expression and function in the brain, right hemispheres of rats were irradiated with single doses of 2–25 Gy followed by 10 mg kg−1 of the P-gp substrate cyclosporine A (CsA) intravenously (i.v.), with once 15 Gy followed by CsA (10, 15 or 20 mg kg−1), or with fractionated irradiation (4 × 5 Gy) followed by CsA (10 mg kg−1) 5 days later. Additionally, four groups of three rats received 25 Gy once and were killed 10, 15, 20 or 25 days later. The brains were removed and P-gp detected immunohistochemically. P-gp function was assessed by [11C]carvedilol uptake using quantitative autoradiography. Irradiation increased [11C]carvedilol uptake dose-dependently, to a maximum of 20% above non irradiated hemisphere. CsA increased [11C]carvedilol uptake dose-dependently in both hemispheres, but more (P<0.001) in the irradiated hemisphere. Fractionated irradiation resulted in a lost P-gp expression 10 days after start irradiation, which coincided with increased [11C]carvedilol uptake. P-gp expression decreased between day 15 and 20 after single dose irradiation, and increased again thereafter. Rat brain irradiation results in a temporary decreased P-gp function.

Minocycline and riluzole brain disposition: interactions with p‐glycoprotein at the blood–brain barrier

Amyotrophic lateral sclerosis is a neurodegenerative fatal disease. The only drug recognized to increase the survival time is riluzole(RLZ). In animal models, minocycline (MNC) delayed the onset of the disease and increased the survival time (in combination with RLZ). The objective of our work was to study the interactions between RLZ, MNC and the efflux pump p-glycoprotein (p-gp) at the blood-brain barrier. We investigated these two drugs as: (i) p-gp substrates by comparing their brain uptake in CF1 mdr1a (-/-) and mdr1a (+/+) mice, (ii) p-gp modulators by studying their effect on the cerebral uptake of digoxin. mdr1a (-/-) mice showed higher brain uptake of MNC and RLZ than mdr1a (+/+) (in a 1.6- and 1.4-fold, respectively); and in mdr1a (+/+) mice pre-treated with repeated doses of MNC, brain uptake of digoxin was increased. When both drugs were administrated to mdr1a (+/+) mice, MNC increased the brain uptake of RLZ in a 2.1-fold. In conclusion, MNC and RLZ are both p-gp substrates. MNC is also a p-gp inhibitor and increases the brain diffusion of RLZ. In vitro experiments with the GPNT cell line confirmed these results. These interactions should be taken into account in the design of future clinical trials.

Minimal effect of MDR1 and CYP3A5 genetic polymorphisms on the pharmacokinetics of indinavir in HIV‐infected patients

The protease inhibitor indinavir is characterized by an important interindividual pharmacokinetic variability, which results from the actions of the metabolizing enzymes cytochrome P450 (CYP) 3A and the multidrug efflux pump P-glycoprotein (P-gp), encoded by MDR1. Using a population pharmacokinetic approach, we investigated the effect of several MDR1 and CYP3A5 polymorphisms on the pharmacokinetic parameters of indinavir in HIV-infected patients. Twenty-eight patients receiving indinavir alone or together with ritonavir were included. Indinavir pharmacokinetics were studied over a 12 h interval. Genetic polymorphisms were assessed by real-time PCR assays and direct sequencing for MDR1 and by PCR-SSCP analysis for CYP3A5. The pharmacokinetics of indinavir were best described by a one-compartment model with first-order absorption. In the final model, the MDR1 C3435T genotype and ritonavir were identified as statistically significant covariates (P </= 0.001) for the absorption rate constant (95% confidence interval on the difference between CC and CT genotype 0.37, 5.53) and for clearance (95% confidence interval on the difference 5.8, 26.2), respectively. Patients with the CYP3A5*3/*3 genotype receiving indinavir alone had a 31% decrease in the indinavir clearance rate compared with patients carrying the CYP3A5*1/*3 genotype. The MDR1 C3435T genotype affects the absorption constant of indinavir suggesting that P-gp may be implicated in its pharmacokinetic variability. Through its inhibition of CYP3A and P-gp, ritonavir could attenuate the pharmacokinetic variability linked to genetic differences, reducing significantly the interindividual variability of indinavir. However, genotyping MDR1 and/or CYP3A5 to optimize protease inhibitor boosted regimens does not seem clinically relevant.

Pharmacokinetics and pharmacodynamics of the oral direct thrombin inhibitor ximelagatran co-administered with different classes of antibiotics in healthy volunteers

To study the effects of amoxicillin, doxycycline, ciprofloxacin, azithromycin, and cefuroxime on the pharmacokinetics and pharmacodynamics of melagatran, the active form of the oral direct thrombin inhibitor ximelagatran, which is a substrate for the P-glycoprotein pump (P-gp) transporter but is not metabolized by the cytochrome P450 (CYP450) enzyme system. Five parallel groups of 16 healthy volunteers received two sequential treatments. The first treatment was a single 36-mg dose of ximelagatran. During the second treatment period, one of the above antibiotics was given on days 1-5 after a washout of at least 2 days. A single 36-mg oral dose of ximelagatran was given on the mornings of days 1 and 5 of the second treatment period. No pharmacokinetic interactions were detected between ximelagatran and amoxicillin, doxycycline, or ciprofloxacin as the least-squares geometric mean treatment ratio of ximelagatran with-to-without antibiotic fell within the intervals of 0.80-1.25 for the area under the curve (AUC) and 0.7-1.43 for C(max). After co-administration with azithromycin, the least square mean ratio with-to-without antibiotic for AUC of melagatran was 1.60 (90% CI, 1.40-1.82) on day 1 and 1.41 (90% CI, 1.24-1.61) on day 5. For melagatran C(max), the corresponding ratios were 1.63 (90% CI, 1.38-1.92) and 1.40 (90% CI, 1.18-1.66). After co-administration with cefuroxime, the ratios were 1.23 (90% CI, 1.07-1.42) and 1.16 (90% CI, 0.972-1.38) for AUC and 1.33 (90% CI, 1.07-1.66) and 1.19 (90%CI, 0.888-1.58) for C(max) of melagatran. Co-administration with the antibiotics did not change mean time to C(max), half-life, or renal clearance of melagatran. The melagatran plasma concentration-response relationship for activated partial thromboplastin time (APTT) prolongation was not altered by any of the studied antibiotics, but the increased plasma concentrations of melagatran after co-administration of ximelagatran with azithromycin resulted in a minor increase in the mean maximum APTT of about 15%. The pharmacokinetics of ximelagatran were not affected by amoxicillin, doxycycline, or ciprofloxacin. Melagatran exposure was increased when ximelagatran was co-administered with azithromycin and, to a lesser extent, with cefuroxime. APTT was not significantly altered by any of the antibiotics.

Multi‐drug resistance gene‐1 (MDR‐1) haplotypes and the CYP3A5*1 genotype have no influence on ciclosporin dose requirements as assessed by C0 or C2 measurements

The intestinal efflux pump P-glycoprotein (P-gp), the product of the multi-drug resistance-1 (MDR-1) gene, significantly influences the pharmacokinetics of several drugs. Ciclosporin is a substrate for P-gp and is metabolized by cytochrome P450 (CYP) 3A enzymes. P-gp activity is affected by several known single nucleotide polymorphisms (SNPs) and haplotypes. MDR-1 genotypes of SNPs C1236T, G2677T/A and C3435T, as well as haplotypes C-G-C and T-T-T and CYP3A5*1 genotype (predictive of CYP3A5 expression), were related to ciclosporin blood concentrations measured at both 0 and 2 h after drug dosing in 197 stable renal transplant patients. Significant differences (of a magnitude unlikely to be relevant clinically) in dose-normalized blood ciclosporin concentrations were found only between MDR-1 genotypes of the C1236T SNP and between haplotype groups C-G-C and T-T-T in patients that were expressers of CYP3A5. MDR-1 SNPs and haplotypes and also CYP3A5*1 genotype, do not appear to have a major influence on ciclosporin pharmacokinetics.

Ability to Acquire Drug Resistance Arises Early during the Tumorigenesis Process

Resistance to chemotherapy is one of the principal causes of cancer mortality and is generally considered a late event in tumor progression. Although cellular models of drug resistance have been useful in identifying the molecules responsible for conferring drug resistance, most of these cellular models are derived from cell lines isolated from patients at a late stage in cancer progression. To ask at which stage in the tumorigenic progression does the cell gain the ability to acquire drug resistance, we generated a series of pre-tumorigenic and tumorigenic cells from human embryonic skin fibroblasts by introducing, sequentially, the catalytic subunit of telomerase, SV40 large T and small T oncoproteins, and an oncogenic form of ras. We show that the ability to acquire multidrug resistance (MDR) can arise before the malignant transformation stage. The minimal set of changes necessary to obtain pre-tumorigenic drug-resistant cells is expression of telomerase and inactivation of p53 and pRb. Thus, the pathways inactivated during tumorigenesis also confer the ability to acquire drug resistance. Microarray and functional studies of drug-resistant pre-tumorigenic cells indicate that the drug efflux pump P-glycoprotein is responsible for the MDR phenotype in this pre-tumorigenic cell model.

Increased??Absorption??of??Digoxin from??the??Human??Jejunum Due??to??Inhibition??of??Intestinal Transporter-Mediated Efflux

The contribution of transport in the small intestine by the apically located efflux pump P-glycoprotein to variable drug absorption in humans is still poorly understood. We therefore investigated whether inhibition of intestinal P-glycoprotein-mediated efflux by quinidine leads to increased absorption of the P-glycoprotein substrate digoxin. Using a multilumen perfusion catheter, we investigated the impact of P-glycoprotein inhibition on absorption of two compounds: the P-glycoprotein substrate digoxin and the marker for passive transcellular absorption antipyrine. Two 20cm adjacent jejunal segments were isolated with the multilumen perfusion catheter in seven healthy subjects. Unlabelled and deuterated digoxin and antipyrine, respectively, were simultaneously infused into either of the intestinal segments. One of the segments was additionally perfused with the P-glycoprotein inhibitor quinidine. Intestinal perfusates were collected for 3 hours, and drug concentrations were determined in the intestinal perfusates, plasma and urine. Quinidine did not affect the disposition of antipyrine. In contrast, coadministration of quinidine into one jejunal segment caused a considerable increase in the amount of digoxin absorbed from this segment compared with the absorption from the other quinidine-free segment (22.3 +/- 8.9% vs 55.8 +/- 21.2% of the dose; p < 0.05). Accordingly, the area under the plasma concentration-time curve and the maximum plasma concentration of digoxin were considerably higher when luminal quinidine was coadministered (p < 0.05 and p < 0.001, respectively). Differences in digoxin absorption from the two intestinal segments were also reflected by pronounced differences in urinary digoxin elimination (5.5 +/- 3.3% vs 19.2 +/- 8.1% of the dose; p < 0.01). P-glycoprotein inhibition in enterocytes increases systemic exposure of orally administered drugs that are P-glycoprotein substrates. These data highlight the importance of the small intestine as an active barrier against xenobiotics.

P-Glycoprotein Function in Peripheral Blood Mononuclear Cells of Myasthenia Gravis Patients Treated with Tacrolimus

Tacrolimus hydrate (FK506) reduces the symptoms of myasthenia gravis (MG) due to its immunosuppressive properties. A drug efflux pump P-glycoprotein (P-gp) actively transports FK506 out of target cells, thereby reducing their efficacy. We investigated the influence of FK506 therapy on the P-gp function of peripheral-blood mononuclear cells (PBMCs) in MG patients. Six MG patients treated with FK506 (MG(FK+)), four MG patients treated without FK506 administration (MG(FK-)), and 18 healthy subjects were included in this study. P-gp function was estimated by transporter activity that was inferred from a decrease in fluorescent P-gp substrate Rhodamine 123 (Rh123) and its inhibition by cyclosporine A (CsA). The P-gp efflux function in MG (FK+) patients assessed by the Kolmogorov-Smirnov (KS) statistic D was lower than in the healthy subjects (p=0.0084). However, PBMC sensitivity to FK506 in MG (FK+) patients was significantly higher compared to that of the healthy subjects (p=0.02). There was a significant correlation between the Rh123 efflux activity and PBMC sensitivity to FK506 in vitro (p=0.011). The data raise the possibility that FK506 treatment attenuated P-gp function in the PBMCs of the MG patients.

Herbal medicines and acute medical emergency admissions to hospital

Herbal medicines and acute medical emergency admissions to hospital

Differential partitioning of Gαi1 with the cellular microtubules: a possible mechanism of development of Taxol resistance in human ovarian carcinoma cells

BackgroundTaxol binds to the cellular microtubules and suppresses their dynamic instability. Development of tumor cell resistance to taxol is typically associated with increased expression of the drug efflux pump P-glycoprotein and/or alterations in the microtubules. Recently, changes in the dynamic instability of the microtubules have also been associated with development of taxol resistance in a lung cancer cell line. We have established a 250-fold taxol-resistant human ovarian carcinoma subline (2008/13/4) that does not display the typical alterations associated with development of drug resistance.ResultsUtilizing the mRNA differential display technique, we observed increased expression of an alpha subunit of the guanine nucleotide-binding protein, Gαi1, in the taxol-resistant human ovarian carcinoma cell lines compared to the parental 2008 cells. Several isoforms of the α-subunit of the G protein have been identified and the Gαi (inhibitory) are so named because they inhibit the activity of adenylate cyclase leading to inactivation of the cAMP-dependent protein kinase A (PKA) pathway. In addition, Gαi1 is also known to bind to microtubules and activates their GTPase activity and thus induces depolymerization of the microtubules. In the present study we demonstrate that the intracellular level of cAMP and the PKA activity were higher in the taxol-resistant 2008/13/4 and the 2008/17/4 cells despite the increased expression of Gαi1 in these cells. Moreover, Gαi1 was found to be localized not on the cell membrane, but in intracellular compartments in both the taxol-sensitive and -resistant human ovarian carcinoma cells. Interestingly, increased association of the Gαi1 protein and the microtubules in the taxol-resistant cells compared to the parental 2008 cells was observed, both prior to and after treatment of these cells with taxol.ConclusionBased on the opposing effects of taxol and the Gαi1 protein on the microtubule dynamic instability (taxol suppresses microtubule dynamic instability whilst the Gαi1 protein inhibits the suppression) our results indicate the operation of a novel pathway that would enable the cells to escape the cytotoxic effects of taxol.

ABCB1 gene polymorphisms are not associated with treatment outcome in elderly acute myeloid leukemia patients

The classical multidrug resistance (MDR) gene MDR1 (ABCB1) encodes for the drug efflux pump P-glycoprotein (P-gp). P-gp expression is an adverse prognostic factor for treatment outcome in acute myeloid leukemia (AML) and is more frequently observed in older patients. Single-nucleotide polymorphisms of the ABCB1 gene, C1236T, G2677T, and C3435T, have been associated with altered drug metabolism and treatment outcome. We prospectively determined these single-nucleotide polymorphisms in AML blasts in a cohort of patients aged 60 years or older with AML and evaluated their relevance with regard to P-gp function and expression, ABCB1 messenger ribonucleic acid (mRNA) expression, and clinical outcome. We have analyzed purified bone marrow-derived leukemic blasts, obtained at diagnosis, in 150 patients who were treated within a multicenter, randomized, phase 3 trial of elderly patients with AML. The significance of the allelic ABCB1 variants of C1236T, G2677T, and C3435T was evaluated with respect to P-gp expression and function in leukemic blasts and ABCB1 mRNA expression levels, and these values were correlated with treatment outcome. P-gp function and expression in leukemic blasts and ABCB1 mRNA levels in patients with AML did not vary significantly among any of the allelic variants of ABCB1. None of these allelic variations predicted a difference in complete response rate and survival endpoints. In AML patients aged 60 years or older, allelic ABCB1 variations of C1236T, G2677T, or C3435T are not associated with altered P-gp function or with MDR1 expression at the transcriptional or translational level in leukemic blasts, and they do not significantly affect clinical prognosis.

P‐Glycoprotein Effects of Cyclic Urea HIV Protease Inhibitor DMP 323 in Competitional Absorption Studies

Cyclic ureas form a perspective class of non-peptidic HIV-1 protease inhibitors with major bioavailability problems. Low absorption rates of DMP 323 as one of the first representatives led to investigations whether transport efflux pump P-glycoprotein (P-gp) within the intestine may be partly responsible for the low absorption rates. DMP 323 proved to be a P-gp substrate in competition studies with P-gp inhibitor ritonavir and H17 as a representative of another class of non-peptidic HIV-1 protease inhibitors. Intestinal DMP 323 absorption was mainly increased under co-application of both ritonavir and H17. DMP 323 used as a membrane efflux pump inhibitor itself showed little affinities to P-gp compared to H17 as strong P-gp inhibitor. So P-gp proved to play a decisive role in the low intestinal absorption of the cyclic urea representative DMP 323.

Shell-crosslinked Pluronic L121 micelles as a drug delivery vehicle

Pluronic block copolymers (PBCs) have been shown to reverse multidrug resistance (MDR) by inhibiting the P-glycoprotein (P-gp) pump in cancer cells. One of the problems encountered with the use of PBCs is that the micelles disassociate at low concentrations. The study focused on the stabilization of PBC L121 micelles by the formation of crosslinks within their outer shells. To form crosslinks, the two terminal alcohols on L121 were first chemically converted into aldehydes (L121-CHO) using the Dess–Martin periodinane. Diamine compounds were then used to bridge the converted aldehyde termini on L121-CHO via conjugated Schiff bases. After crosslinking, the morphology of the L121 micelles remained spherical in shape and the mean particle sizes of the micelles before and after crosslinking were comparable (100 nm). After exposure of MDR KBv cells to free rhodamine-123 (R123), the accumulation of R123 in cells was limited due to the function of P-gp. In contrast, crosslinking of L121 micelles within their outer shells significantly reduced their critical micelle concentration and greatly enhanced their stability, while maintaining their ability to inhibit P-gp function in resistant cells. The results indicated that the L121 micelles with shell crosslinks may be useful as a drug delivery vehicle for cancer chemotherapy.

EFFECTS OF GENETIC POLYMORPHISMS OF CYP3A4, CYP3A5 AND MDR1 ON CYCLOSPORINE PHARMACOKINETICS AFTER RENAL TRANSPLANTATION

1. The calcineurin inhibitor cyclosporine is widely used to prevent allograft rejection after solid organ transplantation. It has a narrow therapeutic index and shows considerable interindividual differences in its pharmacokinetics. Interindividual differences in the activity and expression of the metabolising enzymes cytochrome P450 (CYP) 3A4 and 3A5 and the multidrug efflux pump P-glycoprotein (P-gp) contribute considerably to cyclosporine pharmacokinetics. Variability in the activity of CYP3A4, CYP3A5 and P-gp could be considered to result from genetic polymorphisms encoding their genes. 2. The aim of the present study was to evaluate retrospectively the effects of genetic polymorphisms of CYP3A4, CYP3A5 and MDR1 on cyclosporine dose adjusted trough blood concentration during the early period after renal transplantation in Chinese patients. 3. One hundred and six renal transplant recipients in China were genotyped by polymerase chain reaction-restriction fragment length polymorphism for CYP3A4*18A, CYP3A5*3 and MDR1 C3435T. Cyclosporine whole blood levels were measured by fluorescence polarization immunoassay. Dose-adjusted trough blood concentrations (C(0)) were determined and compared among the different genotype groups. 4. The frequency of the CYP3A4*18A, CYP3A5*3 and MDR1 C3435T variant alleles were 0.005 (95% confidence interval (CI) 0.048, 0.0049), 0.783 (95% CI 0.781, 0.785) and 0.528 (95% CI 0.526, 0.531), respectively, and these alleles exhibited incomplete linkage disequilibrium. The median cyclosporine dose-adjusted C(0) in CYP3A5*1/*1 genotype subjects (n = 6) was 14.8 ng/mL per mg per kg (range 11.1-26.8 ng/mL per mg per kg), in CYP3A5*1/*3 patients (n = 34) it was 23.7 ng/mL per mg per kg (range 9.0-61.0 ng/mL per mg per kg) and for CYP3A5*3/*3 patients (n = 66) it was 26.4 ng/mL per mg per kg (range 9.8-85.8 ng/mL per mg per kg; P = 0.012, Kruskal-Wallis test). Accordingly, cyclosporine dose-adjusted C0 was larger in CYP3A5 non-expressors than expressors in the first week after renal transplantation. In addition, wild-type homozygotes (n = 21) for MDR1 C3435T had a slight but significantly lower dose-adjusted C0 compared with heterozygotes (n = 58): 17.7 (10.3-60.8) versus 26.4 (9.0-67.3) ng/mL per mg per kg, respectively (P = 0.014, Mann-Whitney U-test). 5. In conclusion, the present study shows that genetic polymorphisms in CYP3A5 may be responsible, in part, for the large interindividual variability of cyclosporine pharmacokinetics during the early phase after renal transplantation in Chinese patients. Patients with the CYP3A5*3 variant genotype require a low dose of cyclosporine to reach target levels compared with those with the CYP3A5*1 allele.

MDR1 gene polymorphisms are associated with neuropsychiatric adverse effects of mefloquine

Mefloquine, a drug used for treatment and prophylaxis of malaria, is known for its neuropsychiatric adverse effects. We hypothesized that neuropsychiatric adverse effects of mefloquine are associated with polymorphisms in the MDR1/ABCB1 gene that encodes for the efflux pump P-glycoprotein. The association between MDR1 C1236T, G2677T, and C3435T single-nucleotide polymorphisms and the occurrence of neuropsychiatric adverse effects was examined in a prospective cohort study of 89 healthy white travelers taking mefloquine. Of the subjects, 27 (28%) reported neuropsychiatric adverse effects, women significantly more frequently than men. Allele frequencies of the C1236T, G2677T, and C3435T polymorphisms were similar to those found in other white populations, and there was no significant association between any of the individual polymorphisms and neuropsychiatric adverse effects. However, women with the 1236TT, 2677TT, and 3435TT genotypes had a higher risk of neuropsychiatric adverse effects than the reference groups of women with heterozygous and homozygous CC or GG genotypes, with odds ratios of 6.3 (95% confidence interval [CI], 1.1-36.9), 10.5 (95% CI, 1.1-100.6), and 5.4 (95% CI, 1.1-30.0), respectively. The association for women homozygous for the 1236-2677-3435 TTT haplotype was even stronger (P = .004) than the effect of any of the individual polymorphisms. No associations with mefloquine blood levels were observed. In this study the MDR1 1236TT, 2677TT, and 3435TT genotypes, along with the 1236-2677-3435 TTT haplotype, were associated with neuropsychiatric adverse effects of mefloquine in women. MDR1 polymorphisms may play an important role in predicting the occurrence of neuropsychiatric adverse effects of mefloquine, particularly in female travelers.

Cyp3A4, Cyp3A5, and MDR-1 genetic influences on tacrolimus pharmacokinetics in renal transplant recipients

The immunosuppressive drug tacrolimus requires strict therapeutic monitoring due to its narrow therapeutic index and great inter-individual variability. Cytochrome P450 3A4 (Cyp3A4) and Cyp3A5 are the most important contributors to tacrolimus metabolism while the P-glycoprotein pump (MDR-1) modulates its bioavailability. The objective was to investigate the association between Cyp3A4, Cyp3A5, and MDR-1 polymorphisms and tacrolimus pharmacokinetics in the early period after renal transplantation. Forty-four renal transplant recipients were genotyped for 8 Cyp3A4, 7 Cyp3A5, and 5 MDR-1 genetic variants affecting the proteins' expression and/or function. Dose-adjusted tacrolimus though levels were determined during the first week after transplantation and correlated with corresponding genotype. We found no correlation between Cyp3A4 polymorphism and tacrolimus pharmacokinetics. Patients who do not carry both Cyp3A5*3 alleles achieved lower mean dose-adjusted tacrolimus blood concentrations (p<0.001) and needed a longer time to reach the target concentration (10-12 ng/ml; p<0.001) compared to Cyp3A5*3 homozygotes. Patients with less than three copies of MDR-1 (T-129C, C3435T and G2677T) polymorphisms, associated with reduced expression of P-glycoprotein, had also lower dose-adjusted tacrolimus blood concentrations compared to patients having equal to or greater than three copies of MDR-1 genetic variants (P=0.003). There was no difference in the rate of biopsy-confirmed acute rejection among groups during the first 3 months after transplantation. The complete absence of Cyp3A5*3 allele and the accumulation of less than three copies of MDR-1 (T-129C, C3435T and G2677T) polymorphisms are associated with lower tacrolimus blood levels identifying these genotypes as markers for patients requiring higher tacrolimus doses.