NHERF, a 55 kDa PDZ-containing protein, binds receptors and ion transporters to mediate signal transduction at the plasma membrane. Recombinant NHERF demonstrated an apparent size of 150 kDa on gel filtration, which could be reduced to approximately 55 kDa by protein denaturing agents, consistent with the formation of NHERF dimers. Biosensor studies established the time-and concentration-dependent dimerization of NHERF. Overlays of recombinant NHERF fragments suggested that NHERF dimerization was principally mediated by the N-terminal PDZ-I domain. In PS120 cells, reversible protein phosphorylation modulated NHERF dimerization and suggested a role for NHERF dimers in hormonal signaling. ß

BACKGROUND & AIMS: The a 4 b 7 integrin is a validated target in inflammatory bowel disease. This randomized, phase 2b, placebo-controlled, double-blind study evaluated the efficacy and safety of the anti-a 4 b 7 antibody abrilumab in patients with moderate-to-severe ulcerative colitis despite treatment with conventional therapies. METHODS: Patients (total Mayo Score 6-12, recto-sigmoidoscopy score !2) with inadequate response or intolerance to conventional therapies were randomized to receive subcutaneous abrilumab (7, 21, or 70 mg) on day 1, weeks 2 and 4, and every 4 weeks; abrilumab 210 mg on day 1; or placebo. The primary end point was remission (total Mayo Score 2 points, no individual sub-score >1 point) for the 2 highest dosages at week 8. Key secondary end points were response and mucosal healing (centrally read) at week 8. RESULTS: For 354 patients who received !1 dose of investigational product (placebo, n ¼ 116; 7 mg, n ¼ 21; 21 mg, n ¼ 40; 70 mg, n ¼ 98; 210 mg, n ¼ 79), non-adjusted remission rates at week 8 were 4.3%, 13.3%, and 12.7% for the placebo and abrilumab 70-mg and 210-mg groups, respectively (P < .05 for 70 and 210 mg vs placebo); odds of achieving remission were significantly greater with abrilumab 70 mg (odds ratio 3.35; 90% CI 1.41-7.95; P ¼ .021) and 210 mg (odds ratio 3.33; 90% confidence interval 1.34-8.26; P ¼ .030) than with placebo. Response and mucosal healing rates with these dosages also were significantly greater than with placebo. Higher baseline a 4 b 7 levels on naïve CD4 þ T cells were a prognostic indicator for overall outcome, but not a predictive biomarker of abrilumab response. There were no cases of progressive multifocal leukoencephalopathy or deaths. CONCLUSIONS: Abrilumab treatment for 8 weeks induced remission, clinical response, and mucosal healing in patients with moderate-to-severe ulcerative colitis. ClinicalTrials.gov, number NCT01694485.

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 μg ml−1. The PD effect on α4β7 RO showed an EC50 of 0.01 μg ml−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.

Prior studies have indicated a requirement for the PDZ domain-containing protein, Na Inhibition of the renal apical membrane Na ϩ /H ϩ exchanger 3 (NHE3) 1 by cAMP-dependent protein kinase (PKA) requires the presence of an additional protein co-factor called the Na ϩ /H ϩ Exchanger Regulatory Factor (NHERF) (1). NHERF contains two tandem PSD-95/Dlg/ZO-1 (PDZ) domains and functions in a signal-complex of proteins, including ezrin, PKA, and NHE3 (1-3). This multiprotein complex facilitates the phosphorylation of NHE3 and the acute down-regulation of its activity (1-3). Subsequent to the elucidation of the role of NHERF in the regulation of NHE3, a role for this protein in the regulation of the activity of other transporters has been proposed, and it has been suggested that the signal-complex model of regulation of renal transport proteins may be more common than currently appreciated (4). In the renal proximal tubule, there is an intimate relation between the apical membrane Na ϩ /H ϩ exchanger and the basolateral membrane sodium bicarbonate co-transporter (NBC). These two transporters are regulated in parallel in response to a variety of experimental maneuvers, hormones, and second messengers (5-9). The coordinated regulation of NHE3 and NBC appears to be the result of specific regulatory mechanisms and not merely the consequence of the availability of ion substrates for the transporters. NBC, like NHE3, is regulated by stimuli that raise intracellular cAMP (8, 9). In association with Bernardo et al. (10), we have previously provided evidence that PKA-mediated inhibition of renal NBC requires NHERF. It is not known, however, if NHERF functions to form a signal-complex that regulates NBC activity or if NBC itself is phosphorylated by PKA. To study the relation between NBC and NHERF, wild-type, truncated, and mutated forms of NHERF were expressed in BSC-1 cells to determine if NHERF binds NBC, if NHERF is required for the phosphorylation of NBC, if the phosphorylation of NHERF is required for NBC regulation, and if NBC requires tethering to the actin cytoskeleton to be regulated by cAMP. The results indicate that the interaction between NHERF and NBC differs from the interactions between NHERF and NHE3. Although the ezrin-radixin-moesin (ERM) binding domain of NHERF is required for cAMP regulation of NBC, NHERF does not bind to NBC or facilitate cAMP-mediated phosphorylation of NBC. Moreover, 15-min treatment of the cells with cAMP does not affect the cell surface expression NBC. It is suggested that cAMP-mediated inhibition of NBC is the result of a biochemical modification of the NBC transporter by a process that requires NHERF but is not associated with phosphorylation of the transporter itself. EXPERIMENTAL PROCEDURESConstruction and Use of NHERF Expression Plasmids-Studies were performed using BSC-1 cells that express endogenous NBC activity but

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