Inhaled JAK inhibitor GDC-0214 reduces exhaled nitric oxide in patients with mild asthma: A randomized, controlled, proof-of-activity trial.

BACKGROUND: The Janus kinase (JAK) pathway mediates the activity of many asthma-relevant cytokines, including IL-4 and IL-13. GDC-0214 is a potent, inhaled, small-molecule JAK inhibitor being developed for the treatment of asthma. OBJECTIVE: We sought to determine whether GDC-0214 reduces fractional exhaled nitric oxide (Feno), a JAK1-dependent biomarker of airway inflammation, in patients with mild asthma. METHODS: We conducted a double-blind, randomized, placebo-controlled, phase 1 proof-of-activity study in adults with mild asthma and Feno higher than 40 parts per billion (ppb). Subjects were randomized 2:1 (GDC-0214:placebo) into 4 sequential ascending-dose cohorts (1 mg once daily [QD], 4 mg QD, 15 mg QD, or 15 mg twice daily). All subjects received 4 days of blinded placebo, then 10 days of either active drug or placebo. The primary outcome was placebo-corrected percent reduction in Feno from baseline to day 14. Baseline was defined as the average Feno during the blinded placebo period. Pharmacokinetics, safety, and tolerability were also assessed. RESULTS: Thirty-six subjects (mean age, 28 years; 54% females) were enrolled. Mean Feno at baseline across all subjects was 93 ± 43 ppb. At day 14, placebo-corrected difference in Feno was -23% (95% CI, -37.3 to -9) for 15 mg QD and -42% (95% CI, -57 to -27.4) for 15 mg twice daily. Higher plasma exposure was associated with greater Feno reduction. No dose-limiting adverse events, serious adverse events, or treatment discontinuations occurred. There were no major imbalances in adverse events or laboratory findings, or evidence of systemic JAK inhibition. CONCLUSIONS: GDC-0214, an inhaled JAK inhibitor, caused dose-dependent reductions in Feno in mild asthma and was well tolerated without evidence of systemic toxicity.

Effect of genetic variation in the organic cation transporter 1 (OCT1) on metformin action.

Metformin is among the most widely prescribed drugs for the treatment of type 2 diabetes. Organic cation transporter 1 (OCT1) plays a role in the hepatic uptake of metformin, but its role in the therapeutic effects of the drug, which involve activation of AMP-activated protein kinase (AMPK), is unknown. Recent studies have shown that human OCT1 is highly polymorphic. We investigated whether OCT1 plays a role in the action of metformin and whether individuals with OCT1 polymorphisms have reduced response to the drug. In mouse hepatocytes, deletion of Oct1 resulted in a reduction in the effects of metformin on AMPK phosphorylation and gluconeogenesis. In Oct1-deficient mice the glucose-lowering effects of metformin were completely abolished. Seven nonsynonymous polymorphisms of OCT1 that exhibited reduced uptake of metformin were identified. Notably, OCT1-420del (allele frequency of about 20% in white Americans), previously shown to have normal activity for model substrates, had reduced activity for metformin. In clinical studies, the effects of metformin in glucose tolerance tests were significantly lower in individuals carrying reduced function polymorphisms of OCT1. Collectively, the data indicate that OCT1 is important for metformin therapeutic action and that genetic variation in OCT1 may contribute to variation in response to the drug.

Genome-wide association studies, field synopses, and the development of the knowledge base on genetic variation and human diseases.

Genome-wide association studies (GWAS) have led to a rapid increase in available data on common genetic variants and phenotypes and numerous discoveries of new loci associated with susceptibility to common complex diseases. Integrating the evidence from GWAS and candidate gene studies depends on concerted efforts in data production, online publication, database development, and continuously updated data synthesis. Here the authors summarize current experience and challenges on these fronts, which were discussed at a 2008 multidisciplinary workshop sponsored by the Human Genome Epidemiology Network. Comprehensive field synopses that integrate many reported gene-disease associations have been systematically developed for several fields, including Alzheimer's disease, schizophrenia, bladder cancer, coronary heart disease, preterm birth, and DNA repair genes in various cancers. The authors summarize insights from these field synopses and discuss remaining unresolved issues -- especially in the light of evidence from GWAS, for which they summarize empirical P-value and effect-size data on 223 discovered associations for binary outcomes (142 with P < 10(-7)). They also present a vision of collaboration that builds reliable cumulative evidence for genetic associations with common complex diseases and a transparent, distributed, authoritative knowledge base on genetic variation and human health. As a next step in the evolution of Human Genome Epidemiology reviews, the authors invite investigators to submit field synopses for possible publication in the American Journal of Epidemiology.

PharmGKB and the International Warfarin Pharmacogenetics Consortium: the changing role for pharmacogenomic databases and single-drug pharmacogenetics.

PharmGKB, the pharmacogenetics and pharmacogenomics knowledge base (www.pharmgkb.org) is a publicly available online resource dedicated to the dissemination of how genetic variation leads to variation in drug responses. The goals of PharmGKB are to describe relationships between genes, drugs, and diseases, and to generate knowledge to catalyze pharmacogenetic and pharmacogenomic research. PharmGKB delivers knowledge in the form of curated literature annotations, drug pathway diagrams, and very important pharmacogene (VIP) summaries. Recently, PharmGKB has embraced a new role--broker of pharmacogenomic data for data sharing consortia. In particular, we have helped create the International Warfarin Pharmacogenetics Consortium (IWPC), which is devoted to pooling genotype and phenotype data relevant to the anticoagulant warfarin. PharmGKB has embraced the challenge of continuing to maintain its original mission while taking an active role in the formation of pharmacogenetic consortia.

PharmGKB: an integrated resource of pharmacogenomic data and knowledge.

The PharmGKB is a publicly available online resource that aims to facilitate understanding how genetic variation contributes to variation in drug response. It is not only a repository of pharmacogenomics primary data, but it also provides fully curated knowledge including drug pathways, annotated pharmacogene summaries, and relationships among genes, drugs, and diseases. This unit describes how to navigate the PharmGKB Web site to retrieve detailed information on genes and important variants, as well as their relationship to drugs and diseases. It also includes protocols on our drug-centered pathway, annotated pharmacogene summaries, and our Web services for downloading the underlying data. Workflow on how to use PharmGKB to facilitate design of the pharmacogenomic study is also described in this unit.

Molecular determinants of specificity for synthetic nucleoside analogs in the concentrative nucleoside transporter, CNT2.

Members of the concentrative nucleoside transporter (CNT) family (SLC28) mediate the transport of naturally-occurring nucleosides, and nucleoside analog drugs across the plasma membrane of epithelial cells. Each of the three CNT family members has a distinct specificity for naturally occurring nucleosides, and residues that contribute to the specificity of each transporter have been identified. In contrast, the molecular determinants of specificity for synthetic nucleoside analogs are not known. In this study, we take advantage of the large species difference that exists between human and rat CNT2 (hCNT2 and rCNT2) in their ability to transport the nucleoside analog drug cladribine, 2CdA, (rCNT2 > > > hCNT2) to identify the critical domains and amino acid residues that contribute to the observed difference in specificity between CNT2 orthologs. Using chimeric proteins of human and rat CNT2, we determined that the C-terminal half of CNT2 contained the determinants of 2CdA selectivity. We replaced key residues in the C terminus of hCNT2 with the equivalent residue in rCNT2. One residue in the C-terminal portion of CNT2 was found to significantly contribute to 2CdA selectivity: hCNT2-S354A. This mutant caused an increase of 5-6-fold over hCNT2. The 2-chloro pharmacophore, rather than the 2'-deoxyribose was responsible for the reduced 2CdA uptake by hCNT2. Our data are consistent with a model in which an increased capability for hydrogen bonding in critical amino acids that reside in the C terminus of rCNT2 contributes to its enhanced selectivity for 2CdA.

Functional characterization and haplotype analysis of polymorphisms in the human equilibrative nucleoside transporter, ENT2.

The equilibrative nucleoside transporter 2 (ENT2; SLC29A2) is a bidirectional transporter that is involved in the disposition of naturally occurring nucleosides as well as a variety of anticancer and antiviral nucleoside analogs. The goal of the current study was to evaluate the function of genetic variants in ENT2 in cellular assays and to determine the haplotype structure of the coding and flanking intronic region of the gene. As part of a large study focused on genetic variation in membrane transporters (Leabman et al., 2003), DNA samples from ethnically diverse populations (100 African-Americans, 100 European-Americans, 30 Asians, 10 Mexicans, and 7 Pacific Islanders) were screened for variants in membrane transporters, including SLC29A2. Fourteen polymorphic sites in SLC29A2 were found, including 11 in the coding region. Five protein-altering variants were identified: three nonsynonymous variants, and two deletions. Each of the protein-altering variants was found at a very low frequency, occurring only once in the sample population. The nonsynonymous variants and the deletions were constructed via site-directed mutagenesis and were subsequently characterized in Xenopus laevis oocytes. All variants were able to take up inosine with the exception of ENT2-Delta845-846, which resulted in a frameshift mutation that prematurely truncated the protein. ENT2 showed very infrequent variation compared with most other transporter proteins studied, and it was found that five haplotypes were sufficient to describe the entire sample set. The low overall genetic diversity in SLC29A2 makes it unlikely that variation in the coding region contributes significantly to clinically observed differences in drug response.

Genetic analysis and functional characterization of polymorphisms in the human concentrative nucleoside transporter, CNT2.

The concentrative nucleoside transporter CNT2 (SPNT1; SLC28A2) plays a role in the absorption and disposition of naturally occurring nucleosides, as well as nucleoside analog drugs. The aim of the present study was to characterize genetic variation in SLC28A2, the gene encoding CNT2, and to functionally analyse non-synonymous variants of CNT2, as a first step towards understanding whether genetic variation in this nucleoside transporter contributes to variation in response to nucleoside analogs. As part of a larger study, DNA samples from an ethnically diverse population (100 African-Americans, 100 European-Americans, 30 Asians, 10 Mexicans and seven Pacific Islanders) were screened and 10 coding region variants of CNT2 were identified. The non-synonymous variants were then constructed and characterized in Xenopus laevis oocytes. Six non-synonymous variants were identified, and all were able to transport guanosine. The four common variants (>1% in the sample population) were further characterized with the anti-viral nucleoside analog drug ribavirin. No differences were observed among the four common variants in the uptake kinetics of 3H-ribavirin (Km in microM: 35.6+/-9.27 for CNT2-reference, 40.7+/-6.47 for CNT2-P22L, 31.2+/-15.8 for CNT2-S75R, 26.7+/-6.13 for CNT2-S245T and 49.9+/-14.6 for CNT2-F355S). The variant CNT2-F355S exhibited a change in specificity for the naturally occurring nucleosides, inosine and uridine. All non-synonymous variants of CNT2 took up guanosine, and the four variants examined showed no significant difference in ribavirin kinetics. However, CNT2-F355S (3% allele frequency in the African-American sample) was found to alter specificity for naturally occurring nucleosides, which may have implications for nucleoside homeostasis.

The concentrative nucleoside transporter family, SLC28.

The SLC28 family consists of three subtypes of sodium-dependent, concentrative nucleoside transporters, CNT1, CNT2, and CNT3 (SLC28A1, SLC28A2, and SLC28A3, respectively), that transport both naturally occurring nucleosides and synthetic nucleoside analogs used in the treatment of various diseases. These subtypes differ in their substrate specificities: CNT1 is pyrimidine-nucleoside preferring, CNT2 is purine-nucleoside preferring, and CNT3 transports both pyrimidine and purine nucleosides. Recent studies have identified key amino acid residues that are determinants of pyrimidine and purine specificity of CNT1 and CNT2. The tissue distributions of the CNTs vary: CNT1 is localized primarily in epithelia, whereas CNT2 and CNT3 have more generalized distributions. Nucleoside transporters in the SLC28 and SLC29 families play critical roles in nucleoside salvage pathways where they mediate the first step of nucleotide biosynthesis. In addition, these transporters work in concert to terminate adenosine signaling. SLC28 family members are crucial determinants of response to a variety of anticancer and antiviral nucleoside analogs, as they modulate the entry of these analogs into target tissues. Further, this family is involved in the absorption and disposition of many nucleoside analogs. Several CNT single nucleoside polymorphisms (SNPs) have been identified, but have yet to be characterized.

Functional and genetic diversity in the concentrative nucleoside transporter, CNT1, in human populations.

The concentrative nucleoside transporter, CNT1 (SLC28A1), mediates the cellular uptake of naturally occurring pyrimidine nucleosides and many structurally diverse anticancer and antiviral nucleoside analogs. As a first step toward understanding whether genetic variation in CNT1 contributes to variation in the uptake and disposition of clinically used nucleoside analogs, we determined the haplotype structure and functionally analyzed all coding region variants of CNT1 identified in ethnically diverse populations (100 African Americans, 100 European Americans, 30 Asians, 10 Mexican Americans, and 7 Pacific Islanders) (Leabman et al., 2003). A total of 58 coding region haplotypes were identified using PHASE analysis, 44 of which contained at least one amino acid variant. More than half of the coding region haplotypes were population-specific. Using site-directed mutagenesis, 15 protein-altering CNT1 variants, including one amino acid insertion and one base pair (bp) deletion, were constructed and expressed in Xenopus laevis oocytes. All variant transporters took up [3H]thymidine with the exception of CNT1-Ser546Pro, a rare variant, and CNT1-1153del, a single bp deletion found at a frequency of 3% in the African American population. The bp deletion results in a frame-shift followed by a stop-codon. The anticancer nucleoside analog gemcitabine had a reduced affinity for CNT1-Val189Ile (a common CNT1 variant found at a frequency of 26%) compared with reference CNT1 (IC50=13.8 +/- 0.60 microM for CNT1-reference and 23.3 +/- 1.5 microM for CNT1-Val189Ile, p<0.05). These data suggest that common genetic variants of CNT1 may contribute to variation in systemic and intracellular levels of anti-cancer nucleoside analogs.