Amber Dahlin

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.

Gene therapy for cancer using single-chain Fv fragments specific for 4-1BB

Monoclonal antibodies against the T-cell activation molecule 4-1BB have been effective in the treatment of established mouse tumors. To create a vaccine that stimulates the immune system similarly to the efficacious monoclonal anti-4-1BB antibody, 1D8, we constructed a vector encoding cell-bound single-chain Fv fragments from 1D8. We transfected the vector into cells from the K1735 melanoma, selected because of its low immunogenicity and very low expression of major histocompatibility complex class I. The transfected cells induced a strong type 1 T-helper cell response, for which CD4+ but not CD8+ T lymphocytes were necessary and that involved natural killer cells. Vaccinated mice rejected established wild-type K1735 tumors growing as subcutaneous nodules or in the lung. An analogous approach may be effective against micrometastases in human patients, including tumors whose expression of major histocompatibility complex class I is very low.

Cutting Edge: CD83 Regulates the Development of Cellular Immunity

We recently found that human CD83, a marker of mature dendritic cells, is an adhesion receptor that binds to resting monocytes and a subset of activated CD8+ T cells. We injected CD83-Ig into mice transplanted with the immunogenic P815 mastocytoma and showed that it significantly enhanced the rate of tumor growth and inhibited the development of cytotoxic T cells. In contrast, mice immunized with CD83-transfected K1735 cells, a poorly immunogenic melanoma, could prevent the outgrowth of wild-type K1735 cells. Studies performed in vitro with human PBL showed that coimmobilized CD83-Ig and anti-CD3 enhanced T cell proliferation and increased the proportion of CD8+ T cells. CD83-transfected B-lymphoblastoid T51 cells stimulated T cell proliferation more effectively than untransfected T51 cells in MLR cultures and increased the generation of cytolytic T cells. We conclude that CD83 is a functionally important receptor that can regulate the development of cellular immunity by interacting with its ligand(s).

The effect of novel promoter variants in MATE1 and MATE2 on the pharmacokinetics and pharmacodynamics of metformin.

Interindividual variation in response to metformin, first‐line therapy for type 2 diabetes, is substantial. Given that transporters are determinants of metformin pharmacokinetics, we examined the effects of promoter variants in both multidrug and toxin extrusion protein 1 (MATE1) (g.–66T→C, rs2252281) and MATE2 (g.–130G→A, rs12943590) on variation in metformin disposition and response. The pharmacokinetics and glucose‐lowering effects of metformin were assessed in healthy volunteers (n = 57) receiving metformin. The renal and secretory clearances of metformin were higher (22% and 26%, respectively) in carriers of variant MATE2 who were also MATE1 reference (P < 0.05). Both MATE genotypes were associated with altered post‐metformin glucose tolerance, with variant carriers of MATE1 and MATE2 having an enhanced (P < 0.01) and reduced (P < 0.05) response, respectively. Consistent with these results, patients with diabetes (n = 145) carrying the MATE1 variant showed enhanced metformin response. These findings suggest that promoter variants of MATE1 and MATE2 are important determinants of metformin disposition and response in healthy volunteers and diabetic patients.

Placental drug transporters.

Any treatment of a pregnant woman with medication (drugs) de facto results in the treatment of her unborn child, even when her unborn child is not the target of drug therapy. This is because, in most instances, the placenta is not a complete barrier to the passage of drugs from the maternal to the fetal compartment. This barrier is in part due to the presence of various efflux transporters in the placenta. The placenta is also richly endowed with influx transporters. In this article, we will review the physiological characteristics of the placenta and how it functions as a barrier to passage of drugs into the fetal compartment. In addition, we will review placental transporters that are important in modulating the exposure of the fetus to drugs and, therefore, the efficacy and toxicity of such drugs towards the fetus.

EXPRESSION AND IMMUNOLOCALIZATION OF THE PLASMA MEMBRANE MONOAMINE TRANSPORTER IN THE BRAIN

High affinity monoamine transporters efficiently terminate neurotransmission through synaptic reuptake of released neurotransmitter. We recently cloned and characterized a novel low-affinity, high capacity plasma membrane monoamine transporter (PMAT) that is strongly expressed in the human brain and efficiently transports 5-HT and dopamine (DA). In efforts to understand the physiological function of PMAT and its relevance in monoaminergic pathways, we cloned the PMAT homolog from the mouse brain, demonstrated its capability for transporting 5-HT and DA, and determined the regional and cellular localization of mouse plasma membrane monoamine transporter (mPMAT) in adult mouse brain by reverse-transcription polymerase chain reaction, non-radioactive in situ hybridization, and immunohistochemical methods. Our results showed that mPMAT mRNA and protein are broadly expressed in the mouse brain and are particularly abundant in forebrain cortex, olfactory tubercle, hippocampus, cerebellum and epithelial cells of the choroid plexus. Dual-immunofluorescence histochemistry with established phenotypic markers microtubule-associated protein (MAP2) and glial fibrillary acidic protein (GFAP) revealed that mPMAT is expressed in neuronal cells but not in astrocytes. mPMAT is co-expressed in many brain regions with the high affinity 5-HT transporter (SERT) and the dopamine transporter (DAT), but is also found in certain sites that receive monoamine innervation but lack significant expression of SERT or DAT. These findings suggest that mPMAT is a widely distributed, neuronally-expressed transporter, which may support the role of 5-HT and DA uptake under certain conditions.

Expression Profiling of the Solute Carrier Gene Family in the Mouse Brain

The solute carrier (Slc) superfamily is a major group of membrane transport proteins present in mammalian cells. Although Slc transporters play essential and diverse roles in the central nervous system, the localization and function of the vast majority of Slc genes in the mammalian brain are largely unknown. Using high-throughput in situ hybridization data generated by the Allen Brain Atlas, we systematically and quantitatively analyzed the spatial and cellular distribution of 307 Slc genes, which represent nearly 90% of presently known mouse Slc genes, in the adult C57BL/6J mouse brain. Our analysis showed that 252 (82%) of the 307 Slc genes are present in the brain, and a large proportion of these genes were detected at low to moderate expression levels. Evaluation of 20 anatomical brain subdivisions demonstrated a comparable level of Slc gene complexity but significant difference in transcript enrichment. The distribution of the expressed Slc genes was diverse, ranging from near-ubiquitous to highly localized. Functional annotation in 20 brain regions, including the blood-brain and blood-cerebral spinal fluid (CSF) barriers, suggests major roles of Slc transporters in supporting brain energy utilization, neurotransmission, nutrient supply, and CSF production. Furthermore, hierarchical cluster analysis revealed intricate Slc expression patterns associated with neuroanatomical organization. Our studies also revealed Slc genes present within defined brain microstructures and described the putative cell types expressing individual Slc genes. These results provide a useful resource for investigators to explore the roles of Slc genes in neurophysiological and pathological processes.

Genetic predictors associated with improvement of asthma symptoms in response to inhaled corticosteroids

BACKGROUND To date, genome-wide association studies (GWASs) of inhaled corticosteroid (ICS) response in asthmatic patients have focused primarily on lung function and exacerbations. OBJECTIVE We hypothesized that GWAS analysis could identify novel genetic markers predicting a symptomatic response to ICSs. METHODS We analyzed differences in asthma symptoms in response to ICSs in 124 white children from the Childhood Asthma Management Program (CAMP) trial using scores from diary cards. Of the 440,862 single nucleotide polymorphisms (SNPs) analyzed, the top 100 ranked SNPs were pursued for replication initially in subjects from the pediatric Childhood Asthma Research and Education trials (77 white children) and then in subjects from the adult Asthma Clinical Research Network (110 white adults) and Leukotriene Modifier or Corticosteroid or Corticosteroid-Salmeterol trials (110 white adults). RESULTS The lowest P value for GWAS analysis in the CAMP trial was 8.94 × 10(-8) (rs2388639). Of the 60 SNPs available in the Childhood Asthma Research and Education Network trials, rs1558726 (combined P = 1.02 × 10(-5)), rs2388639 (combined P = 8.56 × 10(-9)), and rs10044254 (combined P = 9.16 × 10(-8)) independently replicated. However, these 3 SNPs were not additionally replicated in the adult asthmatic patients of the remaining trials. rs10044254 lies in the intronic region of F-box and leucine-rich repeat protein 7 (FBXL7) and is associated with decreased expression in immortalized B cells derived from CAMP participants. CONCLUSIONS We have identified a novel SNP, rs10044254, associated with both decreased expression of FBXL7 and improved symptomatic response to ICSs in 2 independent pediatric cohorts. Our results suggest that there might be a specific genetic mechanism regulating symptomatic response to ICSs in children that does not carry over to adults.

Gene Expression Profiling of Transporters in the Solute Carrier and ATP-Binding Cassette Superfamilies in Human Eye Substructures

The barrier epithelia of the cornea and retina control drug and nutrient access to various compartments of the human eye. While ocular transporters are likely to play a critical role in homeostasis and drug delivery, little is known about their expression, localization and function. In this study, the mRNA expression levels of 445 transporters, metabolic enzymes, transcription factors and nuclear receptors were profiled in five regions of the human eye: cornea, iris, ciliary body, choroid and retina. Through RNA expression profiling and immunohistochemistry, several transporters were identified as putative targets for drug transport in ocular tissues. Our analysis identified SLC22A7 (OAT2), a carrier for the antiviral drug acyclovir, in the corneal epithelium, in addition to ABCG2 (BCRP), an important xenobiotic efflux pump, in retinal nerve fibers and the retinal pigment epithelium. Collectively, our results provide an understanding of the transporters that serve to maintain ocular homeostasis and which may be potential targets for drug delivery to deep compartments of the eye.

High Selectivity of the γ-Aminobutyric Acid Transporter 2 (GAT-2, SLC6A13) Revealed by Structure-based Approach

Background: GAT-2 is physiologically and pharmacologically important for regulating peripheral GABAergic mechanisms. Results: We identify GAT-2 ligands, including drugs, metabolites, and fragments, using comparative modeling, virtual screening, and experiments. Conclusion: GAT-2 is a high selectivity/low affinity transporter that is resistant to inhibition by typical GABAergic inhibitors. Significance: Our results explain pharmacological and physiological effects of GAT-2 ligands and identify specificity determinants in the SLC6 family. The solute carrier 6 (SLC6) is a family of ion-dependent transporters that mediate uptake into the cell of osmolytes such as neurotransmitters and amino acids. Four SLC6 members transport GABA, a key neurotransmitter that triggers inhibitory signaling pathways via various receptors (e.g., GABAA). The GABA transporters (GATs) regulate the concentration of GABA available for signaling and are thus targeted by a variety of anticonvulsant and relaxant drugs. Here, we characterize GAT-2, a transporter that plays a role in peripheral GABAergic mechanisms, by constructing comparative structural models based on crystallographic structures of the leucine transporter LeuT. Models of GAT-2 in two different conformations were constructed and experimentally validated, using site-directed mutagenesis. Computational screening of 594,166 compounds including drugs, metabolites, and fragment-like molecules from the ZINC database revealed distinct ligands for the two GAT-2 models. 31 small molecules, including high scoring compounds and molecules chemically related to known and predicted GAT-2 ligands, were experimentally tested in inhibition assays. Twelve ligands were found, six of which were chemically novel (e.g., homotaurine). Our results suggest that GAT-2 is a high selectivity/low affinity transporter that is resistant to inhibition by typical GABAergic inhibitors. Finally, we compared the binding site of GAT-2 with those of other SLC6 members, including the norepinephrine transporter and other GATs, to identify ligand specificity determinants for this family. Our combined approach may be useful for characterizing interactions between small molecules and other membrane proteins, as well as for describing substrate specificities in other protein families.