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Background and Purpose AMG 139 is a human anti-IL-23 antibody currently in a phase II trial for treating Crohn's disease. To support its clinical development in humans, in vitro assays and in vivo studies were conducted in cynomolgus monkeys to determine the pharmacology, preclinical characteristics and safety of this monoclonal antibody. Experimental Approach The in vitro pharmacology, pharmacokinetics (PK), pharmacodynamics and toxicology of AMG 139, after single or weekly i.v. or s.c. administration for up to 26 weeks, were evaluated in cynomolgus monkeys. Key Results AMG 139 bound with high affinity to both human and cynomolgus monkey IL-23 and specifically neutralized the biological activity of IL-23 without binding or blocking IL-12. After a single dose, linear PK with s.c. bioavailability of 81% and mean half-life of 8.4–13 days were observed. After weekly s.c. dosing for 3 or 6 months, AMG 139 exposure increased approximately dose-proportionally from 30 to 300 mg·kg−1 and mean accumulation between the first and last dose ranged from 2- to 3.5-fold. Peripheral blood immunophenotyping, T-cell-dependent antigen responses and bone formation markers were not different between AMG 139 and vehicle treatment. No adverse clinical signs, effects on body weight, vital signs, ophthalmic parameters, clinical pathology, ECG, organ weights or histopathology were observed in the monkeys with the highest dose of AMG 139 tested (300 mg·kg−1 s.c. or i.v.). Conclusions and Implications The in vitro pharmacology, PK, immunogenicity and safety characteristics of AMG 139 in cynomolgus monkeys support its continued clinical development for the treatment of various inflammatory diseases.

AIMS AMG 181 pharmacokinetics/pharmacodynamics (PK/PD), safety, tolerability and effects after single subcutaneous (s.c.) or intravenous (i.v.) administration were evaluated in a randomized, double-blind, placebo-controlled study. METHODS Healthy male subjects (n= 68) received a single dose of AMG 181 or placebo at 0.7, 2.1, 7, 21, 70 mg s.c. (or i.v.), 210 mg s.c. (or i.v.), 420 mg i.v. or placebo. Four ulcerative colitis (UC) subjects (n= 4, male : female 2:2) received 210 mg AMG 181 or placebo s.c. (3:1). AMG 181 concentration, anti-AMG 181-antibody (ADA), α4β7 receptor occupancy (RO), target cell counts, serum C-reactive protein, fecal biomarkers and Mayo score were measured. Subjects were followed 3-9 months after dose. RESULTS Following s.c. dosing, AMG 181 was absorbed with a median tmax ranging between 2-10 days and a bioavailability between 82% and 99%. Cmax and AUC increased dose-proportionally and approximately dose-proportionally, respectively, within the 70-210 mg s.c. and 70-420 mg i.v. ranges. The linear β-phase t1/2 was 31 (range 20-48) days. Target-mediated disposition occurred at serum AMG 181 concentrations of less than 1 μgml-1. The PD effect on α4β7 RO showed an EC50 of 0.01 μgml-1. Lymphocytes, eosinophils, CD4+ T cells and subset counts were unchanged. AMG 181-treated UC subjects were in remission with mucosal healing at weeks 6, 12 and/or 28. The placebo-treated UC subject experienced colitis flare at week 6. No ADA or AMG 181 treatment-related serious adverse events were observed. CONCLUSIONS AMG 181 has PK/PD, safety, and effect profiles suitable for further testing in subjects with inflammatory bowel diseases.

Multidrug resistance protein 2 (MRP2, ABCC2) is an efflux membrane transporter highly expressed in liver, kidney and intestine with important physiological and pharmacological roles. The goal of this study was to investigate the functional significance of promoter region polymorphisms in ABCC2 and potential allele-specific expression. Twelve polymorphisms in the 1.6 kb region upstream of the translation start site were identified by resequencing 247 DNA samples from ethnically diverse individuals. Luciferase reporter gene assays showed that ABCC2 -24CT both alone and as part of a common haplotype (-24CT/-1019AG/-1549GA) increased promoter function 35% compared with the reference sequence (P0.0001). No other common variants or haplotypes affected ABCC2 promoter activity. Allele-specific expression was also investigated as a mechanism to explain reported associations of the synonymous ABCC2 3972CT variant with pharmacokinetic phenotypes. In Caucasian liver samples (n=41) heterozygous for the 3972CT polymorphism, the 3972C allele was preferentially transcribed relative to the 3972T allele (P0.0001). This allelic imbalance was particularly apparent in samples with haplotypes containing two or three promoter/untranslated region variants (-1549GA, -1019AG and -24CT). The observed allelic imbalance was not associated with hepatic or renal ABCC2 mRNA expression. Additional mechanisms will need to be explored to account for the interindividual variation in ABCC2 expression and MRP2 function.

ABCB1 descriptionABCB1 (MDR1) is one of many ubiquitous adenosine triphosphate (ATP)-binding cassette (ABC) genes present in all kingdoms of life that is responsible for cellular homeostasis [1–3]. ABC genes encode transporter and channel proteins possessing multiple membrane-spanning domains that f

The human ATP-binding cassette (ABC) transporter superfamily consists of 48 integral membrane proteins that couple the action of ATP binding and hydrolysis to the transport of diverse substrates across cellular membranes. Defects in 18 transporters have been implicated in human disease. In hundreds of cases, disease phenotypes and defects in function can be traced to nonsynonymous single nucleotide polymorphisms (nsSNPs). The functional impact of the majority of ABC transporter nsSNPs has yet to be experimentally characterized. Here, we combine experimental mutational studies with sequence and structural analysis to describe the impact of nsSNPs in human ABC transporters. First, the disease associations of 39 nsSNPs in 10 transporters were rationalized by identifying two conserved loops and a small α-helical region that may be involved in interdomain communication necessary for transport of substrates. Second, an approach to discriminate between disease-associated and neutral nsSNPs was developed and tailored to this superfamily. Finally, the functional impact of 40 unannotated nsSNPs in seven ABC transporters identified in 247 ethnically diverse individuals studied by the Pharmacogenetics of Membrane Transporters consortium was predicted. Three predictions were experimentally tested using human embryonic kidney epithelial (HEK) 293 cells stably transfected with the reference multidrug resistance transporter 4 and its variants to examine functional differences in transport of the antiviral drug, tenofovir. The experimental results confirmed two predictions. Our analysis provides a structural and evolutionary framework for rationalizing and predicting the functional effects of nsSNPs in this clinically important membrane transporter superfamily.

Objectives: ABCB1 encodes the efflux transporter P-glycoprotein (P-gp), which regulates the intracellular concentration of many xenobiotics, including several HIV protease inhibitors (PIs). Exposure to some xenobiotics, such as the antibiotic rifampicin, increases P-gp expression. In the present study, we investigated the effect of the HIV PIs saquinavir and atazanavir on the expression and function of ABCB1 and P-gp in primary and cultured lymphocytes, as well as the molecular interactions between these drugs and P-gp. ABCB1 and P-gp expression and function were examined in lymphocyte samples from healthy subjects before and after atazanavir-boosted saquinavir treatment. Expression and function were also studied in CEM cells following exposure to atazanavir and saquinavir. The inhibitory effects of these drugs were investigated in ABCB1-transfected HEK293T cells. Methods: P-gp expression and function were measured by flow cytometry. ABCB1 mRNA expression was evaluated using quantitative RT‐PCR. Results: There were no overall changes in ABCB1 or P-gp expression or function after saquinavir‐ atazanavir treatment in primary lymphocyte samples. However, there was considerable interindividual variability in baseline lymphocyte ABCB1 expression, as well as in the degree of change in ABCB1 expression after saquinavir‐atazanavir administration. In cell culture, 5 mM saquinavir increased ABCB1 levels, although it did not affect P-gp expression. Atazanavir inhibited P-gp function at concentrations above therapeutic levels. Conclusions: Differences in lymphocyte ABCB1 expression, which may be caused by genetic polymorphisms in ABCB1 or its regulatory partners, are a likely cause of interindividual variation in the disposition and efficacy of clinically relevant P-gp substrates, including HIV PIs.

Introduction Nerve growth factor plays a key role in the pathology of osteoarthritis (OA) related chronic pain. The aim of these studies was to evaluate the safety, tolerability, pharmacokinetics, and clinical response of AMG 403, a human anti-nerve growth factor monoclonal antibody, in healthy volunteers and subjects with knee OA. Methods Two phase I, randomized, placebo-controlled, double-blind studies were conducted. The single-ascending dose study randomized healthy volunteers (n = 48) 3:1 to receive AMG 403 (1, 3, 10, or 30 mg intravenously; or 10 or 30 mg subcutaneously; n = 8 per group) or placebo. The multiple-ascending dose study randomized knee OA subjects (n = 18) 3:1 to receive AMG 403 (3, 10, or 20 mg subcutaneously once monthly for four doses) or placebo. Safety, tolerability, and pharmacokinetics (PK) were assessed for both studies. Patient’s and physician’s disease assessments and total WOMAC score were determined in knee OA subjects. Results AMG 403 appeared to be well-tolerated after single and multiple doses, except for subject-reported hyperesthesia, pain, and paresthesia (mild to moderate severity). These treatment-emergent neurosensory events showed evidence of reversibility and a possible dose-dependence. Three serious adverse events were reported in AMG 403 treated subjects, but were not considered treatment related. AMG 403 PK was linear with an estimated half-life of 19.6 to 25.8 days. After multiple doses, AMG 403 PK showed modest accumulation (≤2.4-fold increase) in systemic exposure. Knee OA diagnosis, body weight, and anti-drug antibody development did not appear to affect AMG 403 PK. Patient’s and physician’s disease assessments and total WOMAC score showed improvement in AMG 403 treated knee OA subjects compared with placebo. Conclusions AMG 403 was generally safe and well-tolerated in both healthy volunteers and knee OA patients, and exhibited linear pharmacokinetics. Preliminary clinical efficacy was observed in knee OA subjects. Trial registration ClinicalTrials.gov NCT02348879. Registered 23 December 2014. Clintrials.gov NCT02318407. Registered 2 December 2014. Electronic supplementary material The online version of this article (doi:10.1186/s13075-015-0797-9) contains supplementary material, which is available to authorized users.

Membrane transporters play crucial roles in the cellular uptake and efflux of an array of small molecules including nutrients, environmental toxins, and many clinically used drugs. We hypothesized that common genetic variation in the proximal promoter regions of transporter genes contribute to observed variation in drug response. A total of 579 polymorphisms were identified in the proximal promoters (-250 to +50 bp) and flanking 5' sequence of 107 transporters in the ATP Binding Cassette (ABC) and Solute Carrier (SLC) superfamilies in 272 DNA samples from ethnically diverse populations. Many transporter promoters contained multiple common polymorphisms. Using a sliding window analysis, we observed that, on average, nucleotide diversity (pi) was lowest at approximately 300 bp upstream of the transcription start site, suggesting that this region may harbor important functional elements. The proximal promoters of transporters that were highly expressed in the liver had greater nucleotide diversity than those that were highly expressed in the kidney consistent with greater negative selective pressure on the promoters of kidney transporters. Twenty-one promoters were evaluated for activity using reporter assays. Greater nucleotide diversity was observed in promoters with strong activity compared to promoters with weak activity, suggesting that weak promoters are under more negative selective pressure than promoters with high activity. Collectively, these results suggest that the proximal promoter region of membrane transporters is rich in variation and that variants in these regions may play a role in interindividual variation in drug disposition and response.

Genetic variation in ABCB1, encoding P-glycoprotein (P-gp), is a potential cause of interindividual variation in drug response. Numerous studies have focused on the effects of coding region variants on P-gp expression and function, whereas few noncoding region variants have been investigated. The 3'-untranslated region (UTR) regulates mRNA levels or stability via RNA-protein interactions with mRNA degradation machinery. mRNA stability is a key regulatory step controlling ABCB1 mRNA expression that ultimately affects P-gp levels and function. We hypothesized that ABCB1 3'-UTR polymorphisms alter mRNA stability by disrupting RNA-protein interactions. An ethnically diverse panel of DNA samples was sequenced to identify 3'-UTR polymorphisms and determine allele frequencies. The three most common variants, along with reference ABCB1, were stably expressed in cells in order to measure mRNA half-life. The calculated half-life for ABCB1 reference in HEK293 cells was 9.4 +/- 1.3 h and was similar to that estimated for the 3'-UTR variants. Endogenous ABCB1 mRNA decay was similar in lymphoblastoid cell lines carrying 3'-UTR variant and reference alleles. Although the examined ABCB1 3'-UTR variants have no effect on ABCB1 mRNA stability, these data represent one of the first attempts to determine the influence of genetic variation in UTRs on ABCB1 mRNA levels.

Category: genotype Project: Pharmacogenetics of Membrane Transporters [Table 1][1] provides HUGO Gene Nomenclature Committee (HGNC) symbols, PharmGKB submission URLs, submission dates, and release dates. [Table 2][2] provides HGNC symbols, HGNC names, synonyms, GenBank accession numbers, and locus

Using our automated bioinformatics tool, we recently classified 40 point mutations in ABC transporters as “neutral” or “disease-associated,” followed by experimentally testing three of the predictions.1 Scanning the scientific literature, Varadi et al. found support for nine of our predictions, whereas suggesting that 10 were incorrect. However, the interpretation of the literature by Varadi et al. is itself not unambiguous, as illustrated by at least 6 of the 10 examples of presumed misprediction. The first five examples are likely pleiotropic mutations, with a different impact on a different function: Mutation Q141K in the breast cancer resistance protein BCRP does not significantly influence the disposition of HIV drug lamivudine,2 thus not supporting the unequivocal “disease-associated” classification by Varadi et al. Mutation P269S in BCRP is associated with a 40% decrease in vesicular uptake of [(3)H]estrone-3-sulfate and [(3)H]methotrexate compared to the wild-type form,3 which is inconsistent with the unambiguous “neutral” annotation. Mutation V444A in the bile salt export pump BSEP does not change taurocholate transport function in vitro;4 homozygous V444A mutations were prevalent in both progressive familial intrahepatic cholestasis patients and in noncholestatic patients;5 and this variant has also been observed in other healthy populations.6 Thus, a clearcut classification as “disease-associated” is not justified. Mutant V1251I of ABCB1 showed decreased transport of BODIPY-FL-paclitaxel, but increased transport of calcein-AM in an assay using transfected HEK293T cells,7 arguing against an unequivocal “disease-associated” classification. Mutation S1141T in ABCB1 showed either normal or increased transport in the yeast assay,8 and increased function in transfected HEK293T cells.7 Therefore, this mutation should not be classified simply as “disease-associated.” The final, sixth case is an example of the lack of evidence for the classification by Varadi et al.: Mutation A1291T in ABCC6 occurs simultaneously with a nonsense mutation (Y1069X) in the analyzed form of the transporter.9 Therefore, we do not yet know whether the nonsense mutation or the A1291T mutation (or both) is causative of the disease state, contrary to a definitive “disease-associated” classification. If one accepts the qualifications above, 5 of the 40 predictions by our automated tool are invalidated, including one by our experiments1 and four by the scientific literature cited by Varadi et al.; and 11 mutations are confirmed, including two by our experiments1 and nine by the literature cited by Varadi et al. The corresponding accuracy level (69%) is in fact comparable to that estimated by the random forest test (Fig. 4 in Ref.1). In conclusion, we agree with Varadi et al. that there are inherent difficulties in predicting the functional impact of point mutations. Moreover, as we illustrate above, there are also inherent difficulties in characterizing the functional impact of mutations by experiment, and in interpreting the corresponding scientific literature. Classification of mutations into only two classes (such as our “neutral” and “disease-associated” classes) is a serious limitation, given the complexities of protein localization, degradation, interactions with other small molecules and macromolecules, as well as function in general, resulting in the pleiotropy of some mutations.7 Nevertheless, the difficulty of the problem does not mean we should not address it as long as the progress is properly qualified, as we believe we did in our publication.

BACKGROUND/AIMS Efflux transporters in the ATP binding cassette (ABC) superfamily have been implicated in multidrug resistance (MDR). Characterization of ABC transporters at the site of drug action is essential to overcoming MDR. In the current study, we examined the expression of seven ABC transporters in humanPBMCs in response to administration of the HIV protease inhibitors atazanavir (ATZ) and saquinavir (SQV). METHODS Fourteen healthy volunteers received 1500 mg SQV and 200 mg ATZ orally twice daily for 11 days. PBMCs were isolated from each subject at baseline and on the 11th day of treatment. Quantitative RT-PCR was used to measure PBMC expression of ABCB1, ABCC1, ABCC2, ABCC3, ABCC4, ABCC6, and ABCG2. RESULTS Preliminary results show an overall increase in ABCB1 mRNA expression following SQV-ATZ administration. Interestingly, 86% of subjects demonstrated an increase in ABCB1 mRNA expression, while the remaining subjects exhibited a decrease. The effect of SQV-ATZ treatment on mRNA expression of ABCC1, ABCC2, ABCC3, ABCC4, ABCC6, and ABCG2 was variable, with induction of any gene observed in no more than 63% of individuals. (Table) Effect of SQV-ATZ administration on ABCB1 mRNA expression ABCB1 expression change N Mean change (%) Range (%) P-value Upregulated 12 55 1 to 131