Toshihisa Ishikawa

Membrane transporters in drug development

Membrane transporters can be major determinants of the pharmacokinetic, safety and efficacy profiles of drugs. This presents several key questions for drug development, including which transporters are clinically important in drug absorption and disposition, and which in vitro methods are suitable for studying drug interactions with these transporters. In addition, what criteria should trigger follow-up clinical studies, and which clinical studies should be conducted if needed. In this article, we provide the recommendations of the International Transporter Consortium on these issues, and present decision trees that are intended to help guide clinical studies on the currently recognized most important drug transporter interactions. The recommendations are generally intended to support clinical development and filing of a new drug application. Overall, it is advised that the timing of transporter investigations should be driven by efficacy, safety and clinical trial enrolment questions (for example, exclusion and inclusion criteria), as well as a need for further understanding of the absorption, distribution, metabolism and excretion properties of the drug molecule, and information required for drug labelling.

ITC Recommendations for Transporter Kinetic Parameter Estimation and Translational Modeling of Transport‐Mediated PK and DDIs in Humans

This white paper provides a critical analysis of methods for estimating transporter kinetics and recommendations on proper parameter calculation in various experimental systems. Rational interpretation of transporter‐knockout animal findings and application of static and dynamic physiologically based modeling approaches for prediction of human transporter‐mediated pharmacokinetics and drug–drug interactions (DDIs) are presented. The objective is to provide appropriate guidance for the use of in vitro, in vivo, and modeling tools in translational transporter science.

Inhibition by Cyclosporin A of Adenosine Triphosphate- Dependent Transport From the Hepatocyte Into Bile

BACKGROUND Immunosuppressive treatment with cyclosporin A may be associated with impaired hepatobiliary elimination of bile salts and with cholestasis. Inhibition by cyclosporin A of the primary-active adenosine triphosphate (ATP)-dependent transport systems responsible for excretion of bile salts and cysteinyl leukotrienes across the hepatocyte canalicular membrane into bile may explain the cholestatic side effect. METHODS ATP-dependent transport of bile salt and of cysteinyl leukotrienes was studied in human liver plasma membrane vesicles and additionally in rat liver plasma membrane vesicles enriched in canalicular membranes. RESULTS Inhibition of ATP-dependent taurocholate transport in human liver by 50% was measured at 3 mumol/L cyclosporin A and at 4 mumol/L fujimycin. Kinetic analyses in rat liver indicated non-competitive inhibition by cyclosporin A with respect to ATP and competitive inhibition with respect to taurocholate with inhibition constant (Ki) values of 1.0 and 0.3 mumol/L, respectively. CONCLUSIONS The ATP-dependent export carriers for bile salts and cysteinyl leukotrienes in the hepatocyte canalicular membrane are novel targets for inhibitory side effects of cyclosporin A. Inhibition of ATP-dependent bile salt transport may induce cholestasis.

Pivotal roles of peptide transporter PEPT1 and ATP-binding cassette (ABC) transporter ABCG2 in 5-aminolevulinic acid (ALA)-based photocytotoxicity of gastric cancer cells in vitro

BACKGROUND Recently, 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) is being widely used in cancer therapy owing to the tumor-specific accumulation of photosensitizing protoporphyrin IX (PpIX) after the administration of ALA. In the present study, by focusing on genes involved in the porphyrin biosynthesis pathway, we aimed to explore biomarkers that are predictive for the efficacy of ALA-PDT. METHODS We used five lines of human gastric cancer cells to measure the ALA-based photocytotoxicity. ALA-induced production of PpIX in cancer cells was quantified by fluorescence spectrophotometry. To examine the potential involvement of PEPT1 and ABCG2 in the ALA-PDT sensitivity, stable cell lines overexpressing PEPT1 were established and ABCG2-specific siRNA used. RESULTS We observed that three cell lines were photosensitive, whereas the other two cell lines were resistant to ALA-based photocytotoxicity. The ALA-based photocytotoxicity was found to be well correlated with intracellular PpIX levels, which suggests that certain enzymes and/or transporters involved in ALA-induced PpIX production are critical determinants. We found that high expression of the peptide transporter PEPT1 (ALA influx transporter) and low expression of the ATP-binding cassette transporter ABCG2 (porphyrin efflux transporter) determined ALA-induced PpIX production and cellular photosensitivity in vitro. CONCLUSION PEPT1 and ABCG2 are key players in regulating intracellular PpIX levels and determining the efficacy of ALA-based photocytotoxicity against gastric cancer cells in vitro. Evaluation of the expression levels of PEPT1 and ABCG2 genes could be useful to predict the efficacy of ALA-PDT. Primers specific to those target genes are practical and useful biomarkers for predicting the photo-sensitivity to ALA-PDT.

Gefitinib enhances the efficacy of photodynamic therapy using 5-aminolevulinic acid in malignant brain tumor cells

BACKGROUND Since the ATP-binding cassette transporter ABCG2 plays a physiologically significant role of porphyrin efflux from living cells, the expression of ABCG2 may influences the efficacy of photodynamic therapy (PDT). In this study, we evaluated the effect of gefitinib, a potent ABCG2 inhibitor, on 5-aminolevulinic acid (5-ALA)-PDT in brain tumor cell lines in vitro. MATERIALS AND METHODS Four human glioma cell lines (U87MG, U118MG, A172, and T98G) and a malignant meningioma cell line (IOMM-Lee) were incubated with gefitinib (0.01-1.0μM) before incubation with 5-ALA (1mM). The effects gefitinib on intracellular protoporphyrin IX (PpIX), mRNA and protein expression of ABCG2, and PDT were evaluated, in vitro. RESULTS At concentrations of 0.1μM or higher, gefitinib enhanced intracellular levels of PpIX in a dose-dependent manner in all those cell lines. Gefitinib decreased mRNA and plasma membrane protein expression of ABCG2, and efficiently enhanced the effect of 5-ALA-PDT in malignant brain tumor cells. CONCLUSION Gefitinib can inhibit ABCG2-mediated PpIX efflux from malignant brain tumor cells to increase the intracellular PpIX and thereby enhance the PDT effect.

Critical role of ABCG2 in ALA-photodynamic diagnosis and therapy of human brain tumor.

Primary brain tumors occur in around 250,000 people per year globally. Survival rates in primary brain tumors depend on the type of tumor, patients age, the extent of surgical tumor removal, and other factors. Photodynamic diagnosis (PDD) is a practical tool currently used in surgical operation of aggressive brain tumors, such as glioblastoma and meningiomas, whereas clinical application of photodynamic therapy (PDT) to brain tumor therapy has just recently started. Both PDD and PDT are achieved by a photon-induced physicochemical reaction, which is induced by the excitation of porphyrins exposed to light. In fluorescence-guided gross-total resection, PDD can be achieved by the administration of 5-aminolevulinic acid (5-ALA) as the precursor of protoporphyrin IX (PpIX). Exogenously administered ALA induces biosynthesis and accumulation of PpIX, a natural photosensitizer, in cancer cells. However, ATP-binding cassette transporter ABCG2 plays a critical role in regulating the cellular accumulation of porphyrins in cancer cells and thereby its expression and function can affect the efficacy of PDD and PDT. In response to the photoreaction of porphyrins leading to oxidative stress, the nuclear factor erythroid-derived 2-related transcription factor can transcriptionally upregulate ABCG2, which may reduce the efficacy of PDD and PDT. On the other hand, certain protein kinase inhibitors potentially enhance the efficacy of PDD and PDT by blocking ABCG2-mediated porphyrin efflux from cancer cells. In this context, it is of great interest to develop ABCG2 inhibitors that can be applied to PDD or PDT for the therapy of brain tumor and other tumors.

Recent Advances in Pharmacogenomic Technology for Personalized Medicine

Genetic polymorphisms and mutations in drug metabolizing enzymes, transporters, receptors, and other drug targets (e.g., toxicity targets) are linked to inter-individual differences in the efficacy and toxicity of many medications as well as risk of genetic diseases. Validation of clinically important genetic polymorphisms and the development of new technologies to rapidly detect clinically important variants are critical issues for advancing personalized medicine.

Pharmacogenomics of drug transporters and clinical applications

Each year, nearly 1.4 million Americans are diagnosed with ca ncer, another 600,000 die from it. The medicines used as treatments have clear limitations. Chemotherapy, radiation, and surgery-all big three treatments–all wreak havoc on hea lthy cells and tissues as well as cancerous ones. If there is a case to be made for personalized medicine, “cancer is it”. Current literature strongly supports the need for a great scient ific effort to be made in this field. Notably, the lack of identified molecular biomarkers tha t fully explain the heritability of complex traits, and the inability to pinpoint causative genetic effects in some complex tumors, m aking it a very difficult for target recognition and treatme nt. However, for developing safe and effective medicines to ca ncer that raised hopes, nanomedicine “is not just one more too l, it is an entire field benefiting from the use of the modern cutting ed e molecular tools. Remarkably, they are enabling academ ic researchers and oncologists to combine all advances, cr ating nanosized particles that contain drugs targeting speci fic cell surface receptors and other potent molecules designed to kill cancerous cells. Targeting the most appropriate pati ents might be a way of using newer medicines in the most effective manner essentially devoid of side effects. Scientific advances in the use of genetic information to estimate disease risk, assess prognosis, and manage pat ient c re. By rigorous target validation, identifying risk factors, prognost ic factors, and predictive factors, hope is that interven tions can be tailored to maximize patient benefit and minimize toxicity , he primary goal of personalized nanomedicine. But, ma ny outstanding questions remain.The prevalence of hypertension and pre-hypertension in the pediatric population are increasing (Ellis & Miyashita, 2011). A significant cardiovascular risk factor the development of primary hypertension earlier in life increases the chances of cardiovascular morbidity and mortality later in life. Studies have already shown that 34 to 38% of children and adolescents with mild untreated hypertension already show signs of left ventricular hypertrophy (Hanevold C. et al, 2004). It is critically important that hypertension and pre-hypertension are recognized and appropriately managed to decrease the risk of CAD, CVD, heart failure, CHD, and sleep apnea. Evidence shows that this problem is under-recognized due to a number of factors including lack of provider knowledge regarding the AHA blood pressure recommendations and the National High Blood Pressure Educational Program (NHBPEP) guidelines. Another issue is the complexity of factors involved in diagnosing HTN in this population delineated by the (NHBPEP) guidelines. The blood pressure tables include 1,904 variables based on gender, age, and height percentile delineated from the CDC development charts. To more easily detect hypertension in children and adolescents, a Smartphone application, Pedia BP, was designed and developed. The existing tables from The Fourth Report on the Diagnosis, Evaluation and Treatment of High Blood Pressure in Children and Adolescents, which contain hundreds of normal and abnormal blood pressure values based on gender, age, and height percentile, were analyzed and streamlined into a much simplified single user interface Smartphone application. Pedia BP makes it easy to identify abnormal blood pressure values in almost any potential care or screening setting.Current prostate cancer treatment remains ineffective primarily due to ineffectual therapeutic strategies and numerous tumorassociated physiological barriers which hinder efficacy of anticancer agents. Therefore, the focus of this study was to investigate a new combination therapy approach simultaneously targeting the androgen-androgen receptor (AR) and X-linked inhibitor of apoptosis protein (XIAP) signaling pathways for treating prostate cancer. The effect of bicalutamide (antiandrogen), embelin (XIAP inhibitor) and their derivatives on the growth of prostate cancer cells in vitro and in vivo was first examined. Using a combination of MTT assay and isobologram analyses, combination of bicalutamide, embelin and their derivatives were observed to synergistic in their ability to inhibit cell growth. This effect was found to be cell line and schedule dependent. Since bicalutamide and embelin are extremely hydrophobic, polymeric micelles were fabricated using lactic acid and carbonate based copolymers specifically designed to improve drug solubility. Polymeric micelle formulations were found to increase the aqueous solubility of bicalutamide and embelin several fold. Additionally, the potential benefit of antiandrogen and XIAP inhibitor combination was confirmed in animal studies. Tumor growth was effectively regressed upon simultaneous treatment with antiandrogen and XIAP inhibitor formulated in polymeric micelles.O of the most enduring views held by mainstream medicine is the close association between circulating cholesterol and cardiovascular disease and in particular coronary heart disease. Except under rare circumstances, a prerequisite precursor to acute coronary or cardiovascular events of an ischemic nature is atherosclerosis, which is known to start in some individuals at an early age and the prevalence is strongly age dependent. It is widely believed that cholesterol drives atherosclerosis, a notion that can be found in the introductions of hundreds of papers. The advent of non-invasive coronary artery imaging techniques such as electron beam computed tomography and coronary computed tomographic angiography have allowed medical scientists to inadvertently test this hypothesis by direct observation of the prevalence and progression of calcified, mixed and non-calcified coronary plaque. The results of a large number of studies involving a broad spectrum of age, ethnic background and both genders fail to support this hypothesis and in fact find a total lack of correlation between circulating total or LDL cholesterol and coronary plaque burden and progression in individuals with subclinical atherosclerosis, including type 2 diabetics who have well documented elevated risk of acute cardiovascular events. Furthermore, a number of studies have consistently found that lipid lowering, especially with the statin class of drug, fails to impact either the prevalence or progression of subclinical coronary atherosclerosis in either non-diabetics or diabetics. This apparent falsification of the hypothesis that cholesterol drives coronary atherosclerosis continues to be ignored. Furthermore, in true primary prevention trials using statin therapy to prevent acute coronary events in both non-diabetic and diabetic individuals, almost all generate absolute risk reductions that are very small, some would say not clinically significant, and there is no impact on mortality. The belief that this approach represents significant preventive therapy, especially for those who are asymptomatic but judged of high risk, including diabetics, is based on relative risk reductions which have the well recognized and intrinsic potential for exaggerating benefit and misleading clinical practice. Large numbers needed to treat to prevent one acute event suggests the need for a new paradigm.Ann-Marie Chacko completed her Ph.D. at the University of Pennsylvania in Pharmacology with a focus in Radiopharmaceutical Chemistry. She continued her postdoctoral training at Penn and is currently a Research Associate in Penn Radiology, leading translational efforts in bringing macromolecular radiopharmaceuticals (i.e., proteins and nanoparticles) as diagnostics and therapeutics to the clinic. She is a recipient of a KL2 Career Development Award from the Penn Clinical and Translational Research Award (CTSA), and is currently pursuing her master’s in Translational Research (MTR). Guiding translational medicine with immunotargeted probes for PET imaging of the cancer biomarker TEM1/EndosialinO personal genome potentially contains tremendous amount of valuable information for our health but we are currently using only a very small portion of them. Especially for polygenic diseases, it is important to make use of the data on multiple SNPs derived from GWAS, processing these integrated data into genetic risk scores, which are then returned to the individuals. Type 2 diabetes mellitus (T2DM) is one of the representative polygenic diseases with its prevalence currently increasing worldwide. T2DM is preventable as long as the preemptive cares begin at the very early phase or even before onset. Values of genetic tests, though not satiable compared to those of laboratory tests in disease prediction at a certain point in given time, are indicated to have power in evaluating the lifetime onset. Several lines of evidence show the effectiveness of risk assessment by the post-test probability using genotype-based likelihood ratio for the T2DM management in healthy persons. Such risk assessment method has been developed originally based on SNP genotype-disease database of Caucasian population. Taking into consideration the significant differences in risk allele frequency and risk effects between Caucasian and Japanese, we thought it inevitable that we build a Japanese version of SNP genotype-T2DM database. As expected, the SNP combination used for risk calculation for Japanese turned out to be different from the Caucasian set. Pre-evaluation of our risk assessment in a Japanese local cohort (derived from Takahata district) revealed that our risk scoring has better discrimination ability (C-statistics, 0.70; 95% CI, 0.66 to 0.74) than the simple risk allele count scoring (C-statistics, 0.62; 95% CI, 0.57-0.67). Future prospective cohort study should be taken to validate this risk assessment method. Furthermore it will also be important to assess the influence brought by the disclosure of the genetic risk score to the individuals on their behaviors in a sense of prevention of the disease.A are widely distributed especially in hospital environment. Acinetobacter poses very little risk to healthy human, on the contrary the microbes cause problems typically in Intensive Care Unit (ICU) and Healthcare setting especially those housing very ill patients. Among all Acinetobacter, A. baumannii accounts about 80% of reported infections. A.baumannii strains are often resistant to many antimicrobial drugs giving difficulty in therapy. We analyzed the susceptibility pattern of all A.baumannii isolated from all kind of specimens that collected from hospitalized patients in ICU in one private hospital in West Jakarta and one public hospital in Central Jakarta, Indonesia, in 2010-2012. Data analysis were using WHONET 5.6 and CLSI breakpoin criteria were applied. A total of 328 A. baumannii isolates from ICU patients of both hospitals showed highly resistance to all antibiotic tested except for Tigecycline and Amikacin. A. baumannii susceptible to Tigecycline were 82% (n=23) in 2010 followed by 50% (n=58) and 34% (n=50) in 2011 and 2012, while for amikacin i.e. 87%, 75%, 50% for the same isolates, came from private hospital. In public hospital the Tigecycline susceptibility pattern were i.e: 80% (n=26), 82% (n=72), 86% (n=99) and for amikacin i.e. 40%, 51%, 25%, in consecutive year, respectively. Those profiles showed Tigecycline and amikacin being less susceptible to A. baumannii isolates by year, but Tigecycline in public hospital showed consistent in susceptibility. A study to identify the resistance genes of A. baumannii in advance was considered to conduct.Nan Hu has completed his Ph.D. in 2010 from Department of Biostatistics at the University of Washington, School of Public Health. He is now an Assistant Professor of Medicine at the University of Utah, and an investigator at Huntsman Cancer Institute. He has published more than 10 papers in reputed Statistics journals and Medical journals and served as Statistics reviewers for Statistic Sinica and several medical journals. Evaluating the over-time prognostic performance of biomarkers for cancer prognoses using time-dependent receiver operating characteristic (ROC) curveJingfang Ju, Ph.D. is Co-Director of Translational Research Laboratory at Stony Brook University. He received his Ph.D. in Biochemistry/Molecular biology from University of Southern California and postdoctoral fellow at the Cancer Center of Yale University. His group was the first to discover that non-coding microRNAs are part of the p53 tumor suppressor network. Currently, he is focusing on elucidating the molecular mechanism of miRNA mediated cancer stem cell chemoresistance. His group made the initial systematic discovery that miRNA is stable in archival FFPE specimens (RNA, 2007); this has open the opportunity for miRNA based biomarker discovery. Chemo resistance mediated by micro RNAs in cancerS muscular atrophy (SMA), the leading genetic cause of infant mortality, is characterized by the loss of spinal motor neurons controlling locomotion. However, the pathogenic mechanism of SMA remains largely unknown. Using two SMA mouse models and induced pluripotent stem (iPS) cells from SMA patients’ fibroblasts, we show here that the activity of cyclin-dependent kinase (Cdk) is specifically up-regulated in SMA spinal motor neurons, leading to the phosphorylation of histone deacetylase 5 (HDAC5) and its cytoplasmic accumulation in vivo. The up-regulation of HDAC5 phosphorylation occurs before detectable SMA pathology, suggesting it may be one of the early initiators of the disease. In SMA motor neurons, Cdk phosphorylates HDAC5 on a novel phosphorylation site, Serine 279 (S279), within the nuclear localization sequence. This phosphorylation event results in decreased nuclear localizations of HDAC5 and its associating pro-survival transcription factor Myocyte Enhancer Factor 2 (MEF2), leading to motor neuron death in SMA. Intriguingly, drug inhibition of the Cdk signaling pathway diminishes disease symptoms in human SMA iPS cell-derived motor neurons. Together, our results reveal a mechanism regulating motor neuron degeneration in SMA through phosphorylation-dependent nuclear export of a chromatin-remodeling enzyme, and suggest a new therapeutic strategy for treating SMA and other neurodegenerative disorders.The goal of pharmacogenomics is to aid in individualized treatment (precision medicine). Pharmacogenomics now plays an increasingly important role in the process of drug development. Pharmacogenomics-informed treatment has the potential to increase the efficacy of commonly used therapeutics in hospital settings, while greatly reducing adverse drug reactions. Over the past few years, genetic polymorphisms in proteins involved in metabolism or transport have now been identified as clinically relevant, explaining differences in efficacy and impacting clinical care. Single nucleotide polymorphisms (SNPs) are the most commonly observed variants, and are responsible for greater than ninety percent of all genetic variation in the human genome. The Clinical Center at the National Institutes of Health has recently implemented pharmacogenetics testing in patients receiving certain medications. It is therefore important to review and understand the molecular and physiological basis for gene-drug interactions, and appreciate the impact on drug therapy and precision medicine. Biography Price received his B.S. degree from the University of Arkansas, his Ph.D. from Penn State University, and was a Howard Hughes Medical Institute post-doctoral fellow at Emory University. After 6 years as the Director of Molecular Biology at the Carolinas Medical Center, Dr. Price joined the National Cancer Institute in 2000 continuing his research efforts on prostate cancer in the Medical Oncology Branch. Dr. Price has over 80 publications and has been presented an NIH Award of Merit.The vitamin D receptor (VDR) is a ligand-activated transcription factor sharing its 3D fold with other nuclear receptors. VDR’s natural hormone, (1,25D) regulates over 200 genes associated with calcium/phosphorus homeostasis, immune responses and cellular growth, differentiation or apoptosis. During the last ten years many VDR complexes have been successfully crystallized in the absence/presence of peptides which mimic the coactivator sequences. Superimposition of those complexes shows that, even when the ligands differ drastically in their biological potency, vitamin D analogs anchor to the receptor cavity in a similar fashion to 1,25D. To facilitate structure comparison of VDR complexes we developed two programs suited for tracking structure changes due to ligand-protein interactions [http://www.biocomp.chem.uw.edu.pl]. CCOMP identifies differently oriented amino acids, whereas MSITE tracks how structure changes are transmitted from the binding pocket to the protein surface. Comprehensive CCOMP/MSITE analysis of VDR crystal structures shows that 1,25D analogs predominantly change side chain orientation of amino acids residing far away from the VDR binding cavity. Many of those residues are exposed to the protein’s surface and are known to interact with cofactors modulating VDR functions. We believe that residues identified by sequential CCOMP-MSITE analysis determine specific action of 1,25D analogs. Biography Wanda Sicinska and Dominik Gront are members of Laboratory of Theory of Biopolymers (LTB). The laboratory research interest revolves around computer aided drug design, computer modeling of protein folding pathway simulations, software for large scale molecular modeling and computational analysis of experimental data on biomacromolecules, bioinformatics and biological statistics.Sevtap Savas obtained her Ph.D. in Molecular Biology and Genetics in 1999 from the Bogazici University, Turkey. She was trained as a post-doctoral fellow and Research Associate in Louisiana State University (USA), Mount Sinai Hospital Research Institute (Canada) and Princess Margaret Hospital/Ontario Cancer Institute (Canada). Since 2008, she has been an Assistant Professor at Discipline of Genetics, Memorial University of Newfoundland (Canada). Her research program currently focuses on genetic prognostic studies in colorectal cancer using genetic, epidemiological, biostatistical and computational approaches and development of public databases. She also serves as a reviewer, academic editor or editorial board member for several journals. Can inclusion of genetic polymorphisms improve the prognostic models in colorectal cancer?

Transport of Cysteinyl Leukotrienes

The cysteinyl leukotrienes comprise the metabolites formed from the glutathione conjugate leukotriene C4 (LTC4) in the mercapturate pathway as well as the ω- and s-oxidized catabolites derived from the cysteine conjugate LTE4 and from the —acetylcysteine conjugate N-acetyl—LTE4. The cysteinylglycine derivative LTD4 is the most biologically potent mediator (Lewis and Austen, 1984), whereas the ω- and s-oxidized catabolites are inactive (Samhoun et al., 1989). The cysteinyl leukotrienes, with the relative potencies LTD4>LTC4>LTE4>N—acetyl—LTE4, contract at nanomolar concentrations respiratory, vascular, and intestinal smooth muscles, and are involved in inflammatory and anaphylactic reactions (Lewis and Austen, 1984; Samuelsson et al., 1987). The parent molecule LTC4 is formed in a limited number of cell types including mast cells, basophils, eosinophils, monocytes, and macrophages (Austen and Soberman, 1988). Export of LTC4 from a leukotriene-synthesizing cell into the extracellular space (Fig. 1) is a prerequisite for interaction with receptors located on the surface of target cells. Moreover, the ectoenzyme-catalyzed removal of the γ-glutamyl moiety from LTC4 by γ-glutamyltransferase in the extracellular space is a prerequisite for generation of the more potent LTD4 (Fig. 1).