Aisha L. Walker

Influence of Polymorphic OATP1B-Type Carriers on the Disposition of Docetaxel

Purpose: Docetaxel is extensively metabolized by CYP3A4 in the liver but mechanisms by which the drug is taken up into hepatocytes remain poorly understood. We hypothesized that (i) liver uptake of docetaxel is mediated by the polymorphic solute carriers OATP1B1 and OATP1B3 and (ii) inherited genetic defects in this process may impair systemic drug elimination. Experimental Design: Transport of docetaxel was studied in vitro using various cell lines stably transfected with OATP1B1*1A (wild-type), OATP1B1*5 [c.521T>C (V174A); rs4149056], OATP1B3, or the mouse transporter Oatp1b2. Docetaxel clearance was evaluated in wild-type and Oatp1b2-knockout mice as well as in two cohorts of patients with multiple variant transporter genotypes (n = 213). Results: Docetaxel was found to be a substrate for OATP1B1, OATP1B3, and Oatp1b2 but was not transported by OATP1B1*5. Deficiency of Oatp1b2 in mice was associated with an 18-fold decrease in docetaxel clearance (P = 0.0099), which was unrelated to changes in intrinsic metabolic capacity in mouse liver microsomes. In patients, however, none of the studied common reduced function variants in OATP1B1 or OATP1B3 were associated with docetaxel clearance (P > 0.05). Conclusions: The existence of at least two potentially redundant uptake transporters in the human liver with similar affinity for docetaxel supports the possibility that functional defects in both of these proteins may be required to confer substantially altered disposition phenotypes. In view of the established exposure–toxicity relationships for docetaxel, we suggest that caution is warranted if docetaxel has to be administered together with agents that potently inhibit both OATP1B1 and OATP1B3. Clin Cancer Res; 18(16); 4433–40. ©2012 AACR.

Epigenetic and molecular profiles of erythroid cells after hydroxyurea treatment in sickle cell anemia

Hydroxyurea has been shown to be efficacious for the treatment of sickle cell anemia (SCA), primarily through the induction of fetal hemoglobin (HbF). However, the exact mechanisms by which hydroxyurea can induce HbF remain incompletely defined, although direct transcriptional effects and altered cell cycle kinetics have been proposed. In this study, we investigated potential epigenetic and alternative molecular mechanisms of hydroxyurea-mediated HbF induction by examining methylation patterns within the (G)γ-globin promoter and miRNA expression within primary CD71(+) erythrocytes of patients with SCA, both at baseline before beginning hydroxyurea therapy and after reaching maximum tolerated dose (MTD). Using both cross-sectional analysis and paired-sample analysis, we found that the highly methylated (G)γ-globin promoter was inversely correlated to baseline HbF levels, but only slightly altered by hydroxyurea treatment. Conversely, expression of several specific miRNAs was significantly increased after hydroxyurea treatment, and expression of miR-26b and miR-151-3p were both associated with HbF levels at MTD. The significant associations identified in these studies suggest that methylation may be important for regulation of baseline HbF, but not after hydroxyurea treatment, whereas changes in miRNA expression may be associated with hydroxyurea-mediated HbF induction. This study was registered at ClinicalTrials.gov (NCT00305175).

Modulation of OATP1B-type Transporter Function Alters Cellular Uptake and Disposition of Platinum Chemotherapeutics

Expression of the human organic anion transporting polypeptides OATP1B1 and OATP1B3 has been previously believed to be restricted to hepatocytes. Here we show that the gene encoding OATP1B3, but not OATP1B1, is abundantly expressed in multiple human solid tumors that include hepatocellular, lung, and ovarian carcinomas. Surprisingly, OATP1B3 gene expression in a panel of 60 human tumor cell lines was linked with sensitivity to multiple cytotoxic agents, including the platinum anticancer drugs cisplatin, carboplatin, and oxaliplatin. In addition, overexpression of OATP1B3 in mammalian cells increased cellular accumulation of platinum agents and decreased cell survival. In mice with a targeted disruption of the ortholog transporter Oatp1b2, the liver-to-plasma ratio of cisplatin was significantly reduced compared with wild-type mice, without concurrent changes in expression profiles of other transporter genes. Our findings indicate an unexpected role for tumoral and host OATP1B-type carriers in the toxicity and disposition of platinum anticancer drugs, and may provide a foundation for understanding the extensive interindividual pharmacodynamic variability seen with these drugs in patients. Mol Cancer Ther; 12(8); 1537–44. ©2013 AACR.

Transcellular movement of hydroxyurea is mediated by specific solute carrier transporters

OBJECTIVE Hydroxyurea has proven laboratory and clinical therapeutic benefits for sickle cell anemia and other diseases, yet many questions remain about its in vivo pharmacokinetic and pharmacodynamic profiles. Previous reports suggest that hydroxyurea passively diffuses across cells, but its observed rapid absorption and distribution are more consistent with facilitated or active transport. We investigated the potential role of solute carrier (SLC) transporters in cellular uptake and accumulation of hydroxyurea. MATERIALS AND METHODS Passive diffusion of hydroxyurea across cell membranes was determined using the parallel artificial membrane permeability assay. SLC transporter screens were conducted using in vitro intracellular drug accumulation and transcellular transport assays in cell lines and oocytes overexpressing SLC transporters. Gene expression of SLC transporters was measured by real-time polymerase chain reaction in human tissues and cell lines. RESULTS Hydroxyurea had minimal diffusion across a lipid bilayer but was a substrate for five different SLC transporters belonging to the organic cation/carnitine transporters and organic anion transporting polypeptides (OATP) families of transporters and urea transporters A and B. Further characterization of hydroxyurea transport revealed that cellular uptake by OATP1B3 is time- and temperature-dependent and inhibited by known substrates of OATP1B3. Urea transporters A and B are expressed differentially in human tissues and erythroid cells, and transport hydroxyurea bidirectionally via facilitated diffusion. CONCLUSIONS These studies provide new insight into drug transport proteins that may be involved in the in vivo absorption, cellular distribution, and elimination of hydroxyurea. Elucidation of hydroxyurea transcellular movement should improve our understanding of its pharmacokinetics and pharmacodynamics, and may help explain some of the interpatient drug variability observed in patients with sickle cell anemia.

Nonhematopoietic Nrf2 dominantly impedes adult progression of sickle cell anemia in mice

The prevention of organ damage and early death in young adults is a major clinical concern in sickle cell disease (SCD). However, mechanisms that control adult progression of SCD during the transition from adolescence are poorly defined with no cognate prophylaxis. Here, we demonstrate in a longitudinal cohort of homozygous SCD (SS) mice a link between intravascular hemolysis, vascular inflammation, lung injury, and early death. Prophylactic Nrf2 activation in young SS mice stabilized intravascular hemolysis, reversed vascular inflammation, and attenuated lung edema in adulthood. Enhanced Nrf2 activation in endothelial cells in vitro concurred with the dramatic effect on vascular inflammation in the mice. BM chimeric SS mice lacking Nrf2 expression in nonhematopoietic tissues were created to dissect the role of nonerythroid Nrf2 in SCD progression. The SS chimeras developed severe intravascular hemolysis despite having erythroid Nrf2. In addition, they developed premature vascular inflammation and pulmonary edema and died younger than donor littermates with intact nonhematopoietic Nrf2. Our results reveal a dominant protective role for nonhematopoietic Nrf2 against tissue damage in both erythroid and nonerythroid tissues in SCD. Furthermore, we show that prophylactic augmentation of Nrf2-coordinated cytoprotection effectively impedes onset of the severe adult phenotype of SCD in mice.

Inflammatory targets of therapy in sickle cell disease

Sickle cell disease (SCD) is a monogenic globin disorder characterized by the production of a structurally abnormal hemoglobin (Hb) variant Hb S, which causes severe hemolytic anemia, episodic painful vaso-occlusion, and ultimately end-organ damage. The primary disease pathophysiology is intracellular Hb S polymerization and consequent sickling of erythrocytes. It has become evident for more than several decades that a more complex disease process contributes to the myriad of clinical complications seen in patients with SCD with inflammation playing a central role. Drugs targeting specific inflammatory pathways therefore offer an attractive therapeutic strategy to ameliorate many of the clinical events in SCD. In addition, they are useful tools to dissect the molecular and cellular mechanisms that promote individual clinical events and for developing improved therapeutics to address more challenging clinical dilemmas such as refractoriness to opioids or hyperalgesia. Here, we discuss the prospect of targeting multiple inflammatory pathways implicated in the pathogenesis of SCD with a focus on new therapeutics, striving to link the actions of the anti-inflammatory agents to a defined pathobiology, and specific clinical manifestations of SCD. We also review the anti-inflammatory attributes and the cognate inflammatory targets of hydroxyurea, the only Food and Drug Administration-approved drug for SCD.

Organic anion transporting polypeptide 1B transporters modulate hydroxyurea pharmacokinetics

Hydroxyurea is currently the only FDA-approved drug that ameliorates the pathophysiology of sickle cell anemia. Unfortunately, substantial interpatient variability in the pharmacokinetics (PK) of hydroxyurea may result in variation of the drugs efficacy. However, little is known about mechanisms that modulate hydroxyurea PK. Recent in vitro studies identifying hydroxyurea as a substrate for organic anion transporting polypeptide (OATP1B) transporters prompted the current investigation assessing the role of OATP1B transporters in modulating hydroxyurea PK. Using wild-type and Oatp1b knockout (Oatp1b(-/-)) mice, hydroxyurea PK was analyzed in vivo by measuring [(14)C]hydroxyurea distribution in plasma, kidney, liver, urine, or the exhaled (14)CO2 metabolite. Plasma levels were significantly reduced by 20% in Oatp1b(-/-) mice compared with wild-type (area under the curve of 38.64 or 48.45 μg·h(-1)·ml(-1), respectively) after oral administration, whereas no difference was observed between groups following intravenous administration. Accumulation in the kidney was significantly decreased by twofold in Oatp1b(-/-) mice (356.9 vs. 748.1 pmol/g), which correlated with a significant decrease in urinary excretion. Hydroxyurea accumulation in the liver was also decreased (136.6 vs. 107.3 pmol/g in wild-type or Oatp1b(-/-) mice, respectively) correlating with a decrease in exhaled (14)CO2. These findings illustrate that deficiency of Oatp1b transporters alters the absorption, distribution, and elimination of hydroxyurea thus providing the first in vivo evidence that cell membrane transporters may play a significant role in modulating hydroxyurea PK. Future studies to investigate other transporters and their role in hydroxyurea disposition are warranted for understanding the sources of variation in hydroxyureas PK.

Role of cell membrane transporters in the pharmacological treatment of Sickle cell anemia

T agarose migration test revealed the influence of temperature on seasonal fluctuation of locomotional activity of nuclear hemocytes of frogs of the genus Rana ridibunda Pall. It’s been shown that at different incubation temperatures, the spontaneous migration area of blood cells changed in spring and summer, and did not change during autumn and winter. It’s been found that the migratory activity of erythrocytes and leukocytes in the spring increased only at low incubation temperature, in the summer both at low and at high temperatures.M syndromes (MDS) are a group of heterogeneous clonal bone marrow disorders characterized by ineffective hematopoiesis, peripheral blood cytopenias, and potential for malignant transformation. Lower/intermediate risk MDS are associated with longer survival and high RBC transfusion requirements resulting in secondary iron overload. Recent data suggest that markers of iron overload portend a relatively poor prognosis and retrospective analysis demonstrates that iron chelation therapy is associated with prolonged survival in transfusion-dependent MDS patients. New data provides concrete evidence of irons adverse effects on erythroid precursors in vitro and in vivo. Renewed interest in the iron field was heralded by the discovery of hepcidin, the main serum peptide hormone negative regulator of body iron. Evidence from β-thalassemia suggests that regulation of hepcidin by erythropoiesis dominates regulation by iron. Since iron overload develops in some MDS patients who do not require RBC transfusions, the suppressive effect of ineffective erythropoiesis on hepcidin may also play a role in iron overload. We anticipate that additional novel tools for measuring iron overload and a molecular mechanism-driven description of MDS subtypes will provide a deeper understanding of how iron metabolism and erythropoiesis intersect in MDS and improve clinical management of this patient population.T objectives of the present study were to a) ascertain baseline data on haematochemical profile and b) to assess their variation under different seasons in adult Cholistani AI bulls (n=06) being reared at semen Production Unit (SPU), Karaniwala, Bahawalpur, Pakistan. To determine the variation, 4 seasons of 2 months duration each were defined viz. i) stress free autumn (October-November), ii) stressful winter (December-January), stressful dry summer (May-June) and iv) stressful wet summer (July-August). Blood collection was carried out fortnightly during the study period and a total of 16 blood/blood serum samples were collected per animal (24 samples per season). Amongst the red blood cell values, only haemoglobin (Hb) revealed a significant (P<0.05) effect of season, being lower in stressful winter season and higher in the remaining three seasons. Amongst the white blood cell values, Total Leukocytic Count (TLC) was found to be significantly higher (P<0.05) in dry summer. Serum chemistry analyses revealed that Na+ was significantly higher (P<0.05) and K+ was significantly lower (P<0.05) in stress free autumn. Cholesterol was significantly higher (P<0.05) in winter, whereas glucose was higher in dry summer. The present study revealed that the Cholistani AI bulls had an amazing tendency to maintain most of their haematochemical parameters at a near constant level during stress free or stressful times which is suggestive of their adaptability under harsh stressful climates without showing any signs of stress.T pharmacological management of any disease can be complex due the multitude of factors driving pharmacokinetic (PK) and pharmacodynamic (PD) properties of drugs. Sickle cell anemia (SCA) is particularly difficult to manage due to the complex pathophysiology that affects multiple organ systems. In order to improve current therapies and develop new drugs for the treatment of SCA, it is important to understand factors that modulate PK and PD. Recent studies have demonstrated that cell membrane transport proteins encoded by the family of ATP-binding cassette (ABC) genes and solute carrier (SLC) genes play an integral role in regulating PK and PD properties of oncology drugs. Transporters mediate cellular uptake and efflux of substrates which influence drug disposition as well as targeted and off-target drug effects. Variation in transporter function or expression contributes to inter-patientvariability in drug response and drug-drug or drug-food interactions. The oncology field is moving rapidly to further examine the impact of ABC and SLC transporters on drug distribution, toxicity, efficacy, and interactions for investigational and approved drugs. Likewise, pharmacological studies of commonly used drugs in hematology may help to improve treatment for SCA. Hydroxyurea, currently the only FDA-approved drug for the treatment of SCA, induces fetal hemoglobin expression and decreases sickle cell-related morbidity. However, PK and PD properties of hydroxyurea vary widely between patients, and the source of inter-patient variability remains largely unknown. Studies to elucidate PK and PD properties of hydroxyurea are needed. Data implicating transporters in the modulation of hydroxyurea pharmacology will be presented including (i) identification of specific transporters and (ii) demonstration of how transporter deficiency alters hydroxyurea PK. From these findings, it is evident that additional studies are warranted to investigate the role of transporters as determinants of hydroxyurea PK and PD responses. Knowledge gained from these and similar studies of hematological drugs may be particularly applicable for optimizing individual dosing regimens, predicting individual drug response, and avoiding drug-drug or drug-food interactions. All of which can help to improve the pharmacological treatment for SCA.A lymphoblastic leukemia (ALL) occurs in both children and adults with peak incidence between 2 and 5 years of age. Over the last decades, survival in pediatric ALL has improved to 85%~90% and around 40% in adults. Still the relapse remains major obstacle for the cure of ALL. In our previous study, we demonstrated that integrin alpha4 blockade using natalizumab, a humanized monoclonal antibody against integrin alpha4, reduced adhesion of pre-B ALL cells to bone marrow (BM) stroma and with combination of conventional chemotherapy eliminated resistant pre-B-ALL clones in a MRD mouse model. The present study explored the potential of Natalizumab as a novel T-ALL treatment modality. Natalizumab inhibited adhesion of patient-derived T-ALL cells to BM stroma and attenuated leukemia progression, resulting in prolonged survival of recipient NOD/SCID IL2R gamma-/mice of patient-derived T-ALL. Integrin alpha4 blockade interferes with adhesion of T-All cells to its ligand VCAM-1 and might be considered as a novel adjuvant strategy against T-ALL. Further study is needed to investigate at the molecular level to elucidate the effect of alpha4 blockade in T-ALL.Methods: A cross-sectional household survey was conducted in January 2011 on 423 samples of children, aged 6–14 years, selected by systematic random sampling. A pre-tested structured questioner was adopted and used to collectthe data. To measure hemoglobin level HaemoCue digital photometer was used. Anthropometric indicators were measured using WHO’s guideline. Data analysis was made using SPSS Version 16.0 and WHO AnthroPlus version 1.0.2.0. Multivariate analysis was mad to identify the predictors of anemia.T study was conducted to assess the prevalence of anaemia among school age children infected with Schistosoma mansoni (S. mansoni) in Northwest Ethiopia. School based cross sectional study was conducted from April 30, 2013 to June 30, 2013. Stool and blood samples were collected from 410 school age children. S. mansoni infection was determined using wet mount and Kato–Katz technique and hemoglobin concentration was measured by portable HemocueTM device (hemoglobinometer, Angelholm, Sweden). The prevalence of S. mansoni was 74.1%. Out of the 410 children examined, 12.43% were anaemic. Mean hemoglobin concentration was significantly lower among children infected with S. mansoni compared to those who were not (P<0.05). The difference in haemoglobin concentration was significant among children with light (mean 14.36 (±1.6 SD) g/dl), moderate (mean 13.71 (±1.5 SD) g/dl) and heavy intensity of infection (mean 13.87 (±1.7 SD) g/dl) (P<0.05). Furthermore, the level of hemoglobin was negatively correlated with intensity of S. mansoni infection (r=-0.6). The findings of this study demonstrated high prevalence of anaemia among school age children infected with S. mansoni in Northwest Ethiopia. Therefore, there is a need to design strategies that help to diagnose school age children for anaemia instead of testing for only S. mansoni infection.R years have seen significant developments in the treatment of several types of hematological cancers and in some settings, this has translated into improved clinical outcome. Arguably, the most dramatic of these improvements has been seen in the treatment of multiple myeloma. These developments have mostly been guided by advances in our understanding of the underlying biology and pathogenicity of these diseases. These advances have for example highlighted a series of driver mutations in key components of cellular signaling pathways that provide molecular targets for novel small molecule drugs. Parallel developments in gene and protein profiling technologies are beginning to allow the tailored employment of these drugs in personalized treatment protocols. Nonetheless, this approach has limitations, possibly the most important being the genetic instability of the tumor and the development of drug resistance. An alternate approach to improved cancer therapy is the use of targeted drug delivery systems. These are, in the first instance, reliant on the ability to identify a unique tumor cell surface component, for use as a specific “address” to deliver the drug. This approach has advantages in terms of target cell specificity and bypassing drug resistance pathways. This paper will first describe the advantages and limitations of both targeted molecular therapy and targeted drug delivery approaches and then make suggestions to amalgamate the two systems. Finally, the talk will present an update on our work to develop targeted drug delivery systems for Chronic Lymphocytic Leukemia and Multiple Myeloma.Methods: Study 1: Volunteers aged 20-40 years were randomized in two groups with the consumption of hipocaloric diets with 30% energy restriction during 8 weeks with a cod diet (3x150 g/week) or a salmon diet (3x150 g/week). Study 2: Portuguese sample-population mean age 50.4±12.7 years, was followed for 4 weeks. The volunteers ate their usual diet and supplemented it with two canned sardine per week. Fatty acid profile of erythrocyte (RBCs) phospholipids was determined at baseline and endpoint by GC-FID.