Pump P-glycoprotein Research Articles

A search for BACE inhibitors reveals new biosynthetically related pyrrolidones, furanones and pyrroles from a southern Australian marine sponge, Ianthella sp.

Fractionation of a southern Australian marine sponge, Ianthella sp., yielded sixteen metabolites including a new class of pyrrolidone, ianthellidones A-F (1-6), a new class of furanone, ianthellidones G-H (7-8), new and known lamellarins, lamellarins O1 (9), O2 (10), O (11) and Q (12), plus the known 4-hydroxybenzaldehyde (13), 4-hydroxybenzoic acid (14), 4-methoxybenzoic acid (15) and ethyl 4-hydroxybenzoate (16). Structures for all Ianthella metabolites were determined by detailed spectroscopic analysis, supported by a plausible biosynthetic relationship. The ianthellidones were non-cytotoxic towards two human colon cancer cell lines (SW620 and SW620 Ad300), as well as Gram +ve and Gram -ve bacteria, and a fungus. Ianthellidone F (6) and lamellarins O2 (10) and O (11) displayed modest BACE inhibitory properties (IC(50) > 10 μM), while lamellarin O1 (9) was more potent (IC(50) < 10 μM). Lamellarin O (11) exhibited modest cytotoxicity towards SW620 and SW620 Ad300 cell lines (IC(50) > 22 μM), was an inhibitor of the multi-drug resistance efflux pump P-glycoprotein, and displayed selective growth inhibitory activity against the Gram +ve bacterium Bacillus subtilis (ATCC 6633) (IC(50) 2.5 μM).

Dorsal root ganglia sensory neuron transporters and their role in drug-induced neurotoxicity

Drug transporters have been widely studied in cancer cells, mostly due to their importance in drug resistance. Given their clinical relevance, the role of drug transporters in chemotherapy-induced peripheral neurotoxicity (CIPN) is of particular interest, but knowledge is largely incomplete in dorsal root ganglia (DRG) sensory neurons. This critical aspect of CIPN has never been addressed until the observation in a single in vivo study that copper transporters are present and active in rat DRG neurons [1]. However, evidence is still missing regarding several crucial aspects of drug transporters, such as i) the possible co-localization of more than one transporter, ii) their relevance to each class of neurotoxic drugs, iii) their functionality and iv) the possibility to reduce toxicity through their pharmacological modulation in vivo. The precise mechanisms of cellular uptake of DNA-intercalating platinum drugs are partly unknown, although it is commonly supposed that both passive diffusion as well as facilitated or active transport mechanisms play substantial roles. Cisplatin transporters suggested to be involved in modulation of platinum accumulation are MRP2, the copper transporters CTR1, ATP7A and ATP7B. Moreover, a recent notion is that various anticancer drugs can enter mammalian cells by facilitated transport via solute carriers (SLCs). Among all the SLCs transporters, organic cationic transporter 2 (OCT2) is a critical determinant in the uptake and cytotoxicity of cisplatin in non-neuronal cells, and its activity modulation has been associated with reduced toxicity [2]. The antitubulin alkaloid paclitaxel is a substrate of the energy-dependent drug efflux pump P-glycoprotein (P-gp). P-gp is a member of the ATP-binding cassette (ABC) family of transporters widely expressed in many human cancers and the enhanced efflux of paclitaxel has been observed in drug-resistant tumor cells that overexpress P-gp. The role of P-gp in paclitaxel efflux has been confirmed by employing membrane modulating agents (such as verapamil and cyclosporin A) in combination with paclitaxel. The distribution of this transporter in the central nervous system is well known, but it has never been investigated in peripheral neurons [1]. Recently, paclitaxel has also been recognized as a substrate for the copper transporter ATP7A. The proteasome inhibitor bortezomib has only recently been described to be a substrate of ABCG2, another member of the ABC family, but only in human leukemia cells. Other cellular transportes might also be implicated in neuronal damage in CIPN. We recently performed a thorough investigation on glutamate transporters derived from the observation that glutamate production inhibition reduced in rat models the neurotoxicity of cisplatin, paclitaxel and bortezomib [3]. We demonstrated the presence in the DRG of the transporters, their functionality and the possibility to modulate this functionality using pharmacological inhibitors (DL-threo- -hydroxy-aspartate, dihydrokainate and L-serine-O-sulfate) in in vitro neuronal systems [4,5]. Preliminary results have already been obtained regarding OCT2 demonstrating the presence at the mRNA level and the possibility to modulate in vitro cisplatin cytotoxicity in DRG neurons using the transporter competitor cimetidine. Based on these observations, the possible role of the different families of drug transporters and their possible relevance to CIP onset and severity will be here revised.

Interactions between antidepressants and P‐glycoprotein at the blood–brain barrier: clinical significance of in vitro and in vivo findings

The drug efflux pump P-glycoprotein (P-gp) plays an important role in the function of the blood-brain barrier by selectively extruding certain endogenous and exogenous molecules, thus limiting the ability of its substrates to reach the brain. Emerging evidence suggests that P-gp may restrict the uptake of several antidepressants into the brain, thus contributing to the poor success rate of current antidepressant therapies. Despite some inconsistency in the literature, clinical investigations of potential associations between functional single nucleotide polymorphisms in ABCB1, the gene which encodes P-gp, and antidepressant response have highlighted a potential link between P-gp function and treatment-resistant depression (TRD). Therefore, co-administration of P-gp inhibitors with antidepressants to patients who are refractory to antidepressant therapy may represent a novel therapeutic approach in the management of TRD. Furthermore, certain antidepressants inhibit P-gp in vitro, and it has been hypothesized that inhibition of P-gp by such antidepressant drugs may play a role in their therapeutic action. The present review summarizes the available in vitro, in vivo and clinical data pertaining to interactions between antidepressant drugs and P-gp, and discusses the potential relevance of these interactions in the treatment of depression.

Receptor mechanisms of antipsychotic drug action in bipolar disorder – focus on asenapine

The atypical antipsychotic drugs are considered a first-line treatment for mania in bipolar disorder with many having a proven superiority to the classical mood stabilisers. This review addresses the pharmacological mechanisms underlying this therapeutic efficacy, as well as those mechanisms considered responsible for the adverse effects of antipsychotic drugs, with a particular focus on the recently introduced asenapine. The high efficacy in bipolar mania of haloperidol, a relatively selective dopamine D2-like receptor antagonist, indicates that the one common receptor mechanism underlying antipsychotic effects on mania is antagonism at the D2 receptor. Serotonin receptors are implicated in antidepressant response, and relief of depressed mood in mixed states is likely to involve drug effects at one, or more likely several interacting, serotonin receptors. Asenapine shows a unique breadth of action at these sites, with potential effects at clinical doses at 5HT1A, 1B, 2A, 2C, 6 and 7 receptors. Antagonism at alpha2 adrenoceptors may also be involved. Adverse effects include those classically associated with dopamine D2 receptor blockade, the extrapyramidal side effects (EPS), and which are relatively diminished in the atypical (in comparison with the conventional) antipsychotics. A variety of protective mechanisms against EPS associated with different drugs include low D2 affinity, D2 partial agonism, high 5-HT2A and 2C antagonism. Similar effects at the D2 and 5-HT2C receptors may underlie the low propensity for hyperprolactinaemia of the atypicals, although the strong prolactin-elevating effect of risperidone reflects its relatively high blood/brain concentration ratio, a consequence of it being a substrate for the p-glycoprotein pump. Weight gain is a further concern of antipsychotic treatment of bipolar disorder which is particularly severe with olanzapine. Histamine H1, alpha1 adrenergic and particularly 5-HT2C receptors are implicated in this effect, although the lower propensity for weight gain shown by asenapine which, like olanzapine, binds to these receptors, indicates that other protective receptor mechanisms, or subtle differences in the 5-HT2C receptor-mediated effects, may be important. Of other peripheral and central effects, the pharmacological basis of sedation (H1 receptors) and postural hypotension (alpha1 adrenoceptors) are rather better understood. The relative benefits of atypical antipsychotics like asenapine can be understood from their receptor pharmacology, and this understanding is key to the future development of improved treatment for bipolar disorder.

Itraconazol Inhibits the P-Glycoprotein Pump and Increases Vincristine Induced Toxicity in Patients with Acute Lymphoblastic Leukemia Carrying a Genetic Variant of the MDR1 Gene

Abstract 231One of the most well known drug interactions in pediatric oncology concerns the interaction of vincristine, one of the cornerstones in treatment of acute lymphoblastic leukemia( ALL), and the antifungal agent itraconazole. Several studies have shown that the co-administration of itraconazole with vincristine can induce increased neurotoxicity. It is believed that this increased neurotoxicity is mainly caused by the inhibitory effect of itraconazole on the CYP3A subfamily enzymes system (e.g. CYP3A4/5) and that, depending on the genetic variation, some patients are more prone to exhibit this neurotoxicity. However, itraconazole can also inhibit the efflux pump P-glycoprotein (Pg) but so far, data on the role of the Pg pump on vincristine toxicity are lacking. We therefore performed a pharmacogenetic analysis in 103 Caucasian pediatric patients with ALL analyzing both the MDR1 gene and the CYP3A5 gene in the germ-line. CYP3A5 was not associated with vincristine-related toxicity in our cohort. However, one SNP in MDR1 (3435C>T) was significantly associated with developing peripheral neuropathy (Odds Ratio [OR] = 3.8, 95% confidence interval [CI] 1.2 – 11.9, p=0.026). It is known that this genetic variant of the MDR gene (3435T/2677T), leads to a reduced expression of the Pg-pump. Peripheral neurotoxicity in patients carrying this SNP was present even without the co-administration of itraconazole. A subgroup of these patients did receive itraconazole, and the patients with the genetic variant 3435T/2677T of MDR1 showed not only increased peripheral neurotoxicity, but also developed central nervous toxicity (OR=6.4, 95% CI 1.1 – 37.4 p=0.038) upon combined VCR-itraconazole therapy.To gain more insight in the mechanisms of increased vincristine induced toxicity during azole treatment, in vitro experiments using the LLC-PK1-MDR cell line that over-expresses the MDR1 gene and has no expression of the CYP3A4, were performed. The LLC-PK1-MDR cells were resistant to vincristine exposure in vitro, but became sensitive for VCR after inhibition of the Pg-pump bij PSC833. In contrast, LLC-PK1-MDR1 cells exposed to vincristine in the presence of itraconazole (0.5 and 5 microgram/ml) showed significantly decreased survival in a dose dependent way (LD50 at a dose of 10 −4M and 10−7 M vincristine at dose respectively of 0.5 and 5.0 microgram/ml itraconazole), in contrast to a cell survival of 100% with the same dose of vincristine itraconazole (p< 0.001).These results indicate that itraconazol indeed inhibits the efflux Pg-pump which may explain the CNS toxicity that occurred in our patient group during the period in which itraconazole was co-administerd during vincristine treatment. Based on these data, we conclude that the Pg- pump plays a dominant role in increased vincristine toxicity during azole treatment. Furthermore, those patients carrying the genetic variant 3435T/2677T of the MDR gene are at increased risk for this toxicity due to reduced expression of the efflux pump Pg. Disclosures:No relevant conflicts of interest to declare.

Investigation into the biological properties of the olive polyphenol, hydroxytyrosol: mechanistic insights by genome-wide mRNA-Seq analysis

The medicinal properties of the leaves and fruit of Olea Europaea (olive tree) have been known since antiquity. Numerous contemporary studies have linked the Mediterranean diet with increased health. In particular, consumption of olive oil has been associated with a decreased risk of cardiovascular disease and certain cancers. Increasingly, there has been an interest in the biological properties of polyphenols, which are minor constituents of olive oil. For example, hydroxytyrosol has been shown to be a potent antioxidant and has anti-atherogenic and anti-cancer properties. The overall aim of this study was to provide insights into the molecular mechanisms of action of hydroxytyrosol using genome-wide mRNA-Seq. Initial experiments were aimed at assessing cytotoxicity, apoptosis and cell cycle effects of hydroxytyrosol in various cell lines. The findings indicated a dose-dependent reduction in cell viability in human erythroleukemic K562 and human keratinocytes. When comparing the viability in parental CEM-CCRF and R100 cells (which overexpress the P-glycoprotein pump), it was determined that the R100 cells were more resistant to effects of hydroxytyrosol suggesting efflux by the multi-drug resistance pump. By comparing the uptake of Hoechst 33342 in the two cell lines that had been pretreated with hydroxytyrosol, it was determined that the polyphenol may have P-glycoprotein-modulating activity. Further, initial studies indicated modest radioprotective effects of relatively low doses of hydroxytyrosol in human keratinocytes. Analysis of mRNA sequencing data identified that treatment of keratinocytes with 20μM hydroxytyrosol results in the upregulation of numerous antioxidant proteins and enzymes, including heme oxygenase-1 (15.46-fold upregulation), glutaredoxin (1.65) and glutathione peroxidase (1.53). This may account for the radioprotective activity of the compound, and reduction in oxidative stress suggests a mechanism for chemoprevention of cancer by hydroxytyrosol. Alteration in the expression of transcription factors may also contribute to the anti-cancer effects described in numerous studies. These include changes in the expression of STAT3, STAT6, SMAD7 and ETS-1. The telomerase subunit TERT was also found to be downregulated in K562 cells. Overall, our findings provide insights into the mechanisms of action of hydroxytyrosol, and more generally, we identify potential gene candidates for further exploration.

Acute Exposure to Doxorubicin Results in Increased Cardiac P-glycoprotein Expression

Doxorubicin is a frequently used anticancer drug, but its use is restricted due to the occurrence of severe side effects, namely strong cardiotoxicity. It is known from cancer cells that doxorubicin enhanced the expression of its efflux pump P-glycoprotein (P-gp), which may modulate local drug concentrations. We therefore studied the cardiac expression of P-gp in doxorubicin-treated mice. Mice were treated with doxorubicin, and P-gp expression was studied after 1, 3, and 5 days. Thereby, we could show a significant upregulation of abcb1a (162 ± 15% of control) and abcb1b (418 ± 110% of control) mRNA transcripts after 3 days. On protein level, western blot analysis and immunofluorescence staining revealed a similar finding 5 days after doxorubicin administration. In addition, these results could be confirmed by in vitro models using primary rat cardiomyocytes and the murine cardiomyocyte-like HL-1 cells. Besides an enhanced mRNA and protein expression, doxorubicin-treated HL-1 cells also demonstrated an enhanced P-gp function as assessed by a daunorubicin accumulation assay. Our in vivo and in vitro results demonstrate a cardiac upregulation of P-gp in doxorubicin-treated mice on expression and functional level. This finding may be relevant for cardiac tissue concentrations of P-gp substrates and may represent a mechanism in cardiac self-protection against xenobiotics. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:3951–3958, 2011

The Role of P-Glycoprotein in Psychiatric Disorders: A Reliable Guard of the Brain?

A major component in the protection of the brain against blood-borne toxic influences is the multispecific efflux pump P-glycoprotein. This pump, a 170 kD protein, located at the luminal side of the capillary endothelial cells, has a large capacity and is capable of extruding a wide array of structurally divergent substrates. The brain uptake of the majority of antidepressants and antipsychotics, as well as many other psychotropic drugs and endogenous compounds is hampered by the activity of P-glycoprotein. In this review we discuss the current state of knowledge concerning the role of Pglycoprotein on pharmacokinetics of psychiatric drugs and the impact of modulation of P-glycoprotein on major psychiatric disorders. Relevant issues in reference to the function of P-glycoprotein and other efflux pumps in the blood-brain barrier related to mood disorders and schizophrenia are addressed, such as a possible role of P-glycoprotein as a susceptibility factor in depressive disorders and psychotic disorders.

Superhigh-magnetization nanocarrier as a doxorubicin delivery platform for magnetic targeting therapy

The aim of this study describes the creation of superhigh-magnetization nanocarriers (SHMNCs) comprised of a magnetic Fe 3O 4 (SHMNPs) core and a shell of aqueous stable self-doped poly[ N-(1-one-butyric acid)]aniline (SPAnH), which have a high drug loading capacity (∼27.1 wt%) of doxorubicin (DOX). The SHMNCs display superparamagnetic property with a magnetization of 89.7 emu/g greater than that of Resovist (a commercial contrast agent used for magnetic resonance imaging; 73.7 emu/g). Conjugating the anticancer drug DOX to these nanocarriers enhances the drug’s thermal stability and maximizes the efficiency with which it is delivered by magnetic targeting (MT) therapy to MGH-U1 bladder cancer cells, in part by avoiding the effects of p-glycoprotein (P-gp) pumps to enhance the intracellular concentration of DOX. The high R2 relaxivity (434.7 mM −1s −1) of SHMNCs not only be a most effective MT carrier of chemotherapeutic agent but be an excellent contrast agent of MRI, allowing the assessment of the distribution and concentration of DOX in various tissues and organs. This advanced drug delivery system promises to provide more effective MT therapy and tumor treatment using lower therapeutic doses and potentially reducing the side effects of cardiotoxicity caused by DOX.

Overexpression of the ABC transporter AvtAB increases avermectin production in Streptomyces avermitilis

Avermectins are 16-membered macrocyclic polyketides with potent antiparasitic activities, produced by Streptomyces avermitilis. Upstream of the avermectin biosynthetic gene cluster, there is the avtAB operon encoding the ABC transporter AvtAB, which is highly homologous to the mammalian multidrug efflux pump P-glycoprotein (Pgp). Inactivation of avtAB had no effect, but increasing the concentration of avtAB mRNA 30-500-fold, using a multi-copy plasmid in S. avermitilis, enhanced avermectin production about two-fold both in the wild-type and in a high-yield producer strain on agar plates. In liquid industrial fermentation medium, the overall productivity of avermectin B1a in the engineered high-yield producer was improved for about 50%, from 3.3 to 4.8g/l. In liquid YMG medium, moreover, the ratio of intracellular to extracellular accumulation of avermectin B1a was dropped from 6:1 to 4.5:1 in response to multiple copies of avtAB. Additionally, the overexpression of avtAB did not cause any increased expression of the avermectin biosynthetic genes through RT-PCR analysis. We propose that the AvtAB transporter exports avermectin, and thus reduces the feedback inhibition on avermectin production inside the cell. This strategy may be useful for enhancing the production of other antibiotics.

Collateral sensitivity of multidrug-resistant cells to the orphan drug tiopronin.

A major challenge in the treatment of cancer is multidrug resistance (MDR) that develops during chemotherapy. Here we demonstrate that tiopronin (1), a thiol-substituted N-propanoylglycine derivative, was selectively toxic to a series of cell lines expressing the drug efflux pump P-glycoprotein (P-gp, ABCB1) and MRP1 (ABCC1). Treatment of MDR cells with 1 led to instability of the ABCB1 mRNA and consequently a reduction in P-gp protein, despite functional assays demonstrating that tiopronin does not interact with P-gp. Long-term exposure of P-gp-expressing cells to 1 sensitized them to doxorubicin and paclitaxel, both P-gp substrates. Treatment of MRP1-overexpressing cells with tiopronin led to a significant reduction in MRP1 protein. Synthesis and screening of analogues of tiopronin demonstrated that the thiol functional group was essential for collateral sensitivity while substitution of the amino acid backbone altered but did not destroy specificity, pointing to future development of targeted analogues.

Nitric oxide and P-glycoprotein modulate the phagocytosis of colon cancer cells

The anticancer drug doxorubicin induces the synthesis of nitric oxide, a small molecule that enhances the drug cytotoxicity and reduces the drug efflux through the membrane pump P-glycoprotein (Pgp). Doxorubicin also induces the translocation on the plasma membrane of the protein calreticulin (CRT), which allows tumour cells to be phagocytized by dendritic cells. We have shown that doxorubicin elicits nitric oxide synthesis and CRT exposure only in drug-sensitive cells, not in drug-resistant ones, which are indeed chemo-immunoresistant. In this work, we investigate the mechanisms by which nitric oxide induces the translocation of CRT and the molecular basis of this chemo-immunoresistance. In the drug-sensitive colon cancer HT29 cells doxorubicin increased nitric oxide synthesis, CRT exposure and cells phagocytosis. Nitric oxide promoted the translocation of CRT in a guanosine monophosphate (cGMP) and actin cytoskeleton-dependent way. CRT translocation did not occur in drug-resistant HT29-dx cells, where the doxorubicin-induced nitric oxide synthesis was absent. By increasing nitric oxide with stimuli other than doxorubicin, the CRT exposure was obtained also in HT29-dx cells. Although in sensitive cells the CRT translocation was followed by the phagocytosis, in drug-resistant cells the phagocytosis did not occur despite the CRT exposure. In HT29-dx cells CRT was bound to Pgp and only by silencing the latter the CRT-operated phagocytosis was restored, suggesting that Pgp impairs the functional activity of CRT and the tumour cells phagocytosis. Our work suggests that the levels of nitric oxide and Pgp critically modulate the recognition of the tumour cells by dendritic cells, and proposes a new potential therapeutic approach against chemo-immunoresistant tumours.

Targeting SVCT for enhanced drug absorption: Synthesis and in vitro evaluation of a novel vitamin C conjugated prodrug of saquinavir

In order to improve oral absorption, a novel prodrug of saquinavir (Saq), ascorbyl-succinic-saquinavir (AA-Su-Saq) targeting sodium dependent vitamin C transporter (SVCT) was synthesized and evaluated. Aqueous solubility, stability and cytotoxicity were determined. Affinity of AA-Su-Saq towards efflux pump P-glycoprotein (P-gp) and recognition of AA-Su-Saq by SVCT were studied. Transepithelial permeability across polarized MDCK-MDR1 and Caco-2 cells were determined. Metabolic stability of AA-Su-Saq in rat liver microsomes was investigated. AA-Su-Saq appears to be fairly stable in both DPBS and Caco-2 cells with half lives of 9.65 and 5.73 h, respectively. Uptake of [ 3H]Saquinavir accelerated by 2.7 and 1.9 fold in the presence of 50 μM Saq and AA-Su-Saq in MDCK-MDR1 cells. Cellular accumulation of [ 14C]AA diminished by about 50–70% relative to control in the presence of 200 μM AA-Su-Saq in MDCK-MDR1 and Caco-2 cells. Uptake of AA-Su-Saq was lowered by 27% and 34% in the presence of 5 mM AA in MDCK-MDR1 and Caco-2 cells, respectively. Absorptive permeability of AA-Su-Saq was elevated about 4-5 fold and efflux index reduced by about 13-15 fold across the polarized MDCK-MDR1 and Caco-2 cells. Absorptive permeability of AA-Su-Saq decreased 44% in the presence of 5 mM AA across MDCK-MDR1 cells. AA-Su-Saq was devoid of cytotoxicity over the concentration range studied. AA-Su-Saq significantly enhanced the metabolic stability but lowered the affinity towards CYP3A4. In conclusion, prodrug modification of Saq through conjugation to AA via a linker significantly raised the absorptive permeability and metabolic stability. Such modification also caused significant evading of P-gp mediated efflux and CYP3A4 mediated metabolism. SVCT targeted prodrug approach can be an attractive strategy to enhance the oral absorption and systemic bioavailability of anti-HIV protease inhibitors.

Nocardioazines: A Novel Bridged Diketopiperazine Scaffold from a Marine-Derived Bacterium Inhibits P-Glycoprotein

An Australian marine sediment-derived isolate, Nocardiopsis sp. (CMB-M0232), yielded a new class of prenylated diketopiperazine, indicative of the action of a uniquely regioselective diketopiperazine indole prenyltransferase. The bridged scaffold of nocardioazine A proved to be a noncytotoxic inhibitor of the membrane protein efflux pump P-glycoprotein, reversing doxorubicin resistance in a multidrug resistant colon cancer cell.

Abstract 1693: Mrp7 as a breast cancer resistance factor

Abstract Breast cancer is the most common cancer and the second leading cause of cancer deaths in American women. Breast cancer kills more than 40,000 women every year in the United States. Taxanes and epothilone B are used to treat breast cancer, but cellular resistance to chemotherapeutic agents is a major obstacle. Therapy-induced overexpression of ATP-binding cassette (ABC) drug efflux pumps promotes resistance in many drug-treated cancers. While early studies focused on overexpression and activity of the P-glycoprotein (Pgp) pump, a structurally distinct group of ABC efflux pumps, known as the Multidrug Resistance Proteins (MRPs), have been gaining increasing attention as alternative sources of resistance. There are 9 MRP family members (MRP1-9), with each family member preferentially transporting somewhat overlapping groups of substrates. We have previously shown that MRP7 is distinct from other MRPs because MRP7 confers resistance to a wide variety of anticancer agents including taxanes, vinca alkaloids and nucleoside-based analogs. We also have evidence that Mrp7 null mice are sensitized to taxanes (submitted). We bred a mammary tumor model to our Mrp7 null mouse to investigate Mrp7's in vivo activities regarding breast cancer resistance mechanisms. To better understand the resistance mechanisms of MRP7 the expression level of Mrp7 and expression levels of other transporters was determined in these cell lines using qRT-PCR. In addition, cell lines derived from mammary tumors are being evaluated to determine the impact Mrp7 has on sensitization to agents used in the treatment of breast cancer: epothilone B, docetaxel and paclitaxel. Further, the growth properties of these mammary tumor cell lines will be characterized. Lastly, to better understand the physiological role of this efflux pump, MRP7 cellular localization and polarity are being investigated. Further research into previously mentioned aims and specific MRP7 inhibitors will aid in combating cellular resistance to chemotherapeutic agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1693. doi:10.1158/1538-7445.AM2011-1693

Abstract 4508: Nonclinical pharmacokinetics of TAK-441, a hedgehog pathway inhibitor

Abstract TAK-441 is a potent and selective smoothened (Smo) antagonist that blocks hedgehog (Hh) signaling. TAK-441 is being developed for the treatment of advanced nonhematologic and hematologic malignancies. In vivo pharmacokinetic (PK) studies using rats and dogs and various in vitro studies were conducted to describe the PK of TAK-441 to support its clinical development. After oral administration of TAK-441 and/or [14C]TAK-441 to rats and dogs, plasma, urine, feces and bile were collected at designated time points. The concentrations of TAK-441 and total radioactivity in the biological samples were measured by high performance liquid chromatography / tandem mass spectrometry and liquid scintillation counter, respectively. The PK of TAK-441 was characterized in rats and dogs by low plasma clearance (397.9 and 161.3 mL/hr/kg), moderate plasma volume of distribution at steady state (681.6 and 2181.3 mL/kg), a moderate terminal elimination half-life (t1/2) (1.7 and 9.8 hours), and an oral bioavailability of 31.7% and 90.3%, respectively. A potential food effect was observed in dogs with an approximate 2-fold increase in the extent of absorption when TAK-441 was dosed to fed dogs (compared with fasted dogs), producing both a higher maximal drug concentration (Cmax) and area under the plasma concentration versus time curve from 0 to 24 hours (AUC0-24h). At 1 μg/mL, TAK-441 had high plasma protein binding in mice (99.7%) and rats (96.2%) and had lower plasma protein binding in dogs (79.6%) and humans (87.7%). No metabolites unique to humans were detected after incubation in hepatic microsomes. CYP3A4/5 was the major CYP isozyme contributing to the metabolism of TAK-441, followed by CYP2C19 and 2C9. The rank order of contribution of the five major human CYP isozymes to TAK-441 metabolism (determined using chemical inhibitors) was 3A4/5 (80.3%) &amp;gt;2C19 (14.4%) &amp;gt;2C9 (5.2%) &amp;gt;1A2 and 2D6 (&amp;lt;1%). TAK-441 showed no appreciable induction of CYP1A2 or CYP3A4/5 activity in human primary hepatocytes. TAK-441 was a weak inhibitor of CYP2C8 (IC50 of 25 μM) and had no inhibitory effect on the other CYP isoforms. TAK-441 was not a time-dependent CYP inhibitor in human liver microsomes. Taken together, there is a low potential for TAK-441 to affect the PK of other concomitantly administered CYP substrates. However, the PK of TAK-441 could be affected by concomitantly administered strong CYP3A4/5 inhibitors and inducers. TAK-441 was mainly excreted in the feces as metabolites. Urinary excretion of the parent TAK-441 was quite low (LOQ and &amp;lt;1% of dose in rats and dogs, respectively). TAK-441 has high permeability in Caco-2 cells, is a poor substrate for efflux pump P-glycoprotein (P-gp), and is a weak inhibitor of P-gp (IC50 of 6.59 μM). In conclusion, TAK-441 has a favourable nonclinical absorption, distribution, metabolism, and excretion (ADME) profile for clinical evaluation and development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4508. doi:10.1158/1538-7445.AM2011-4508

Abstract 1703: Importance of pregnane X receptor (PXR) and P-glycoprotein (MDR1) to tamoxifen response and endoxifen disposition

Abstract Tamoxifen, a widely prescribed anti-estrogen for treating estrogen receptor positive breast cancer, is a prodrug which requires bioactivation by cytochrome P450 (CYP) enzymes 2D6 and 3A4 to generate the active metabolite, endoxifen. One obstacle to optimal tamoxifen therapy is the emergence of acquired endocrine resistance during therapy. Although resistance may be multifactorial, studies have shown reduced intra-tumour tamoxifen concentrations in patients with acquired resistance, suggesting the involvement of drug transporters. Tamoxifen is known to induce the expression of the nuclear hormone receptor pregnane X receptor (PXR), which we have shown to be upregulated in breast tumour cells compared with normal adjacent tissue. PXR is also known to regulate the expression of drug transporters including the efflux pump P-glycoprotein (MDR1). Together, we hypothesize that an individual's response to tamoxifen therapy may depend on the interplay between PXR and MDR1. Although, MDR1 does not transport tamoxifen, its role in mediating transport of endoxifen is unknown. Therefore, we assessed the ability of MDR1 to transport endoxifen in vitro by comparing endoxifen flux using a polarized model system of LLCPK cells and LLCPK cells over-expressing human MDR1. In contrast to LLCPK cells, we observed a markedly higher transport of endoxifen in the basal to apical direction of LMDR1 cells, which was abrogated in the presence of the potent and specific MDR1 inhibitor LY335979. To validate the in vivo importance of MDR1 to endoxifen disposition, plasma and tissue concentrations were determined in Mdr1a deficient mice after oral administration of endoxifen. Plasma endoxifen levels did not significantly differ between wildtype and Mdr1a deficient mice. However, brain concentrations of endoxifen were nearly 20-fold higher in Mdr1a deficient mice compared to wildtype mice. Together, these data indicate that endoxifen is a substrate of MDR1. As MDR1 can be overexpressed in some breast tumours, variation in expression and function of this transporter may alter the attained intra-tumour concentration of endoxifen, which may manifest as aberrant clinical response to tamoxifen. We are further investigating the contribution of PXR to the transcriptional regulation of MDR1 and its impact on cellular retention of endoxifen in breast cancer cells. To do this we will utilize the ability of tamoxifen to induce MDR1 expression through PXR activation in breast cancer cell lines and correlate the effect of MDR1 on the intracellular retention of tamoxifen and endoxifen. Finally, we plan to analyze breast tumour tissue from tamoxifen treated and non-treated patients to better understand the role of PXR and MDR1 to clinical response. Accordingly, activation of PXR by tamoxifen and subsequent induction of MDR1 may be an important prognostic factor for patients at risk for tamoxifen resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1703. doi:10.1158/1538-7445.AM2011-1703

Profile of Paul Roepe

Profile of Paul Roepe

Clinical Development of Cabazitaxel for the Treatment of Castration-Resistant Prostate Cancer

Castration resistant prostate cancer has historically been considered chemotherapy insensitive. However, the approval of estramustine phosphate, mitoxantrone, and docetaxel, over the past few decades, has challenged this notion. Despite these advances, until recently, only docetaxel had been shown to improve survival in patients with castration-resistant disease, and there has been no standard treatment options available for men with disease progression on docetaxel. In the last year, cabazitaxel, a novel taxane with decreased affinity for ATP-dependent drug efflux pump P-glycoprotein, became the first cytotoxic agent to demonstrate an improvement in survival in men with docetaxel-refractory disease, and has received regulatory approval for treatment in this setting. In this review, we examine the clinical development of cabazitaxel for the treatment of castration-resistant prostate cancer, as well as rationale and direction of future therapeutic investigation.

Molecular mechanism of glucocorticoid resistance in inflammatory bowel disease

Natural and synthetic glucocorticoids (GCs) are widely employed in a number of inflammatory, autoimmune and neoplastic diseases, and, despite the introduction of novel therapies, remain the first-line treatment for inducing remission in moderate to severe active Crohn's disease and ulcerative colitis. Despite their extensive therapeutic use and the proven effectiveness, considerable clinical evidence of wide inter-individual differences in GC efficacy among patients has been reported, in particular when these agents are used in inflammatory diseases. In recent years, a detailed knowledge of the GC mechanism of action and of the genetic variants affecting GC activity at the molecular level has arisen from several studies. GCs interact with their cytoplasmic receptor, and are able to repress inflammatory gene expression through several distinct mechanisms. The glucocorticoid receptor (GR) is therefore crucial for the effects of these agents: mutations in the GR gene (NR3C1, nuclear receptor subfamily 3, group C, member 1) are the primary cause of a rare, inherited form of GC resistance; in addition, several polymorphisms of this gene have been described and associated with GC response and toxicity. However, the GR is not self-standing in the cell and the receptor-mediated functions are the result of a complex interplay of GR and many other cellular partners. The latter comprise several chaperonins of the large cooperative hetero-oligomeric complex that binds the hormone-free GR in the cytosol, and several factors involved in the transcriptional machinery and chromatin remodeling, that are critical for the hormonal control of target genes transcription in the nucleus. Furthermore, variants in the principal effectors of GCs (e.g. cytokines and their regulators) have also to be taken into account for a comprehensive evaluation of the variability in GC response. Polymorphisms in genes involved in the transport and/or metabolism of these hormones have also been suggested as other possible candidates of interest that could play a role in the observed inter-individual differences in efficacy and toxicity. The best-characterized example is the drug efflux pump P-glycoprotein, a membrane transporter that extrudes GCs from cells, thereby lowering their intracellular concentration. This protein is encoded by the ABCB1/MDR1 gene; this gene presents different known polymorphic sites that can influence its expression and function. This editorial reviews the current knowledge on this topic and underlines the role of genetics in predicting GC clinical response. The ambitious goal of pharmacogenomic studies is to adapt therapies to a patient's specific genetic background, thus improving on efficacy and safety rates.