W1266 Confocal Laser Endomicroscopy (CLE) Reveals Mucosal Accumulation of Plga- Nanoparticles in Ulcerous Lesions of Patients With Inflammatory Bowel Diseases Carsten Schmidt, Eva M. Collnot, Christian Bojarski, Michael Schumann, Joerg D. Schulzke, Claus M. Lehr, Andreas Stallmach The standard treatment of IBD involves the application of anti-inflammatory drugs. These drugs become systemically bioavailable and bear a potential of strong adverse effects. Thus, targeting the inflamed areas of the intestine can reduce adverse effects. Polymeric particles have turned out to be a promising tool for the targeted delivery of drugs. We aimed to investigate the distribution of fluoresceine-labelled nanoparticles (NP) and microparticles (MP) in the rectal mucosa of patients with IBD by use of CLE. Fluoresceinamine was covalently bound to poly(L-lactide-co-glycolide) (PLGA). From this modified polymer, nanoparticles of approximately 300 nm size and microparticles of 3.5 μm size were prepared. After bowel preparation a 30 ml enema containing 10E9 MP or 10E13 NP, resp., has been applied to patients with rectal Crohns disease (CD) or ulcerative colitis (UC). After two hours endoscope based CLE has been performed to visualize nanoparticles in areas of different stages of inflammation. Biopsies have been obtained from IBD patients to evaluate mucosal transport processes in miniaturized Ussing chambers by confocal laser endoscopy. We examined 16 patients with IBD (8 patients with CD, 10 patients with (UC), 7 of whom received NP while 9 received MP. No particles have been visualized on intact mucosal surfaces. In areas of mild to moderate inflammation only minor amounts of particles were visible. In contrast, we observed a marked accumulation of MP rather than NP in ulcerous lesions. Preliminary results from Ussing chamber experiments suggest an influx of particles into the gut mucosa through epithelial lesions. Representative images are shown in fig. 1. This is the first study demonstrating mucosal accumulation of microparticles and nanoparticles in intestinal lesions of patients with IBD. Drug containing particles may have a great potential to target these lesions more specifically in order to maximize therapeutic efficacy and minimize potential side effects. CLE is a useful tool to investigate drug delivery to mucosal surfaces. Fig. 1: PLGA-MP in a patient with UC. Only single particles in mild inflammation (left) but pronounced accumulation in a rectal ulcer (right). W1267 Thiopurine-Induced Peripheral T Cells Apoptosis and Drug Response in Patients With Crohn's Disease Andrea Cossu, Livia Biancone, Francesco Pallone, Monica Boirivant Background and Aim: in Crohn's disease(CD), both azathioprine(AZA)and 6-mercaptopur- ine(6-MP) showed proven efficacy in induction of remission. The response increases after 17 weeks, thus suggesting that there is a minimum length of time for trial of thioupurine therapy. Up to 20% of patients treated with thiopurines need to discontinue therapy due to the occurrence of adverse events. Therefore, besides their proven efficacy, the relatively long period necessary to obtain the full pharmacological effect and the possibility of adverse reactions, do not encourage the use of thioupurine as early therapeutic option. The develop- ment of tests to predict responsiveness to thiopurine represents a major attempt in the clinical management of CD patients. It has been demonstrated that AZA is able to induce apoptosis of T cells. Aim of the present study was to analyze the “In Vitro” thiopurine- induced peripheral T cells apoptosis in a group of CD patients with known response to a previous treatment with either AZA or 6-MP. Results were then analyzed according to the response to previous treatment. Methods: A heparinised peripheral blood sample was obtained from a total of 11 CD patients previously treated with thiopurines during a scheduled visit. CD4+ T cells were obtained from density-gradient isolated PBMC by immunomagnetic selection. Cells were stimulated “In Vitro” for 4-5 days with aCD3/28 Abs in complete medium + 20U/ml of hrIL-2 in the presence or absence of AZA or 6-MP or 6-thioguanine at 5μM concentration, and apoptosis was assessed using Annexin V staining and analyzed by cytofluorimeter. IFN-γ production was evaluated in supernatants by ELISA. Results: among the 11 CD patients enrolled, 6 were not responsive to a previous treatment with thiopurines. The % of apoptosis after aCD3/aCD28 stimulation was not different between responders patients (R) and not responders patients (NR) (R: 16.7 (10.8-38.8); NR: 15.6 (8.1-58.3) median (range). All the patients showed an increase in the % of apoptotic cells after aCD3/aCD28 stimulation in the presence of AZA (p<0.05 by Wilcoxon test). To quantify the ability of AZA to increase the apoptosis, we conveyed the data as apoptosis stimulation index (ASI). NR patients showed a significant reduced ASI when compared to R patients (NR: 1.62 (1.37-1.93); R: 2.45 (2.19-2.65) median (range) p=0.004 by Mann Whitney test). Incubation with AZA reduced the IFN-γ production in R to a greater extent when compared to NR. Conclusions: evaluation of apoptosis stimulation index of peripheral CD4+T cell after incubation with AZA might represent a parameter useful for a proper selection of CD patients candidate to thiopurine treatment. S-687 AGA Abstracts W1268 The Course of Infliximab Discontinuation After Long-Term Maintenance Treatment in Crohn's Disease Alessandro Armuzzi, Marina Rizzi, Rita Monterubbianesi, Manuela Marzo, Michele Cicala, Luisa Guidi, Cosimo Prantera, Anna Kohn INTRODUCTION: Although there is strong evidence supporting the short-term efficacy and safety of infliximab (IFX) in Crohn's disease (CD), few studies have examined the effects of long-term maintenance therapy. In particular, the question of whether IFX maintenance treatment should be continued or interrupted after prolonged remission is still under debate. AIMS & METHODS: To evaluate the course of IFX discontinuation after long-term mainten- ance treatment with prolonged remission and to assess the risk of relapse after IFX withdrawal. Medical records of CD patients in sustained clinical benefit under scheduled long-term (at least 1 year) IFX treatment and in whom IFX was somehow discontinued were retrospectively reviewed in three Italian Centres. Details on demographic, clinical and endoscopic character- istics and adverse events were recorded. 115 patients (53 males, mean age 36±13 years, mean duration of disease 7±7 years) were identified. RESULTS: At baseline, 82% and 33% were on concomitant immunosuppressants or steroid respectively and 39% had previous surgery. CD patients (85 luminal, 30 perianal) received a median number of infusions of 12 (range 8-31) in the course of 24 months (range 12-88) median treatment. 34% required dose flexibility. IFX was discontinued in 69 patients (60%) because of prolonged remission off steroids, in 18 patients (16%) because of adverse events and in 28 patients (24%) because of loss of response (7% underwent elective surgery). Complete mucosal healing rates before IFX discontinuation amounted to 49.5%. 43.8% of patients in whom IFX was discontinued because of prolonged remission relapsed after 13 months (range 3-63) median follow up, while 56.2% maintains sustained clinical benefit after 14 months (range 3-79) median follow up. Survival analysis identified mucosal healing as predictor of sustained clinical benefit after long-term IFX discontinuation (HR 2.7, 95%CI 1.3-6.6); P=0.009). 10 out of 11 patients who relapsed after IFX discontinuation are in remission without adverse events after IFX re-treatment. CONCLUSION: In this real-life experience, the majority of CD patients discon- tinued long-term IFX scheduled maintenance treatment because of prolonged remission. Sustained clinical benefit was observed in more than half of patients one year after IFX discontinuation. Complete mucosal healing before IFX discontinuation predicted long-term maintenance of remission. IFX re-treatment after relapse was effective and safe in most of patients. W1269 Lower Lymphocyte Counts and Azathioprine Efficacy in Inflammatory Bowel Disease Sharmilla Subramaniam, Chris J. Hawkey INTRODUCTION: Azathioprine (AZA) is an effective immunosuppressant in inflammatory bowel disease (IBD). Although, the exact mode of action of AZA is not fully understood. Lymphocyte suppression may be involved. We aimed to determine if the lymphocyte count may be used as to predict AZA efficacy in IBD. METHODS: We studied 75 men and women with IBD [41 Ulcerative Colitis (UC), 34 Crohn's Disease), maintained on AZA. Episodes of relapse on AZA were identified in these patients and the lymphocyte count at the visit immediately preceding the relapse recorded, as well as the relapse value itself. These were compared with values recorded during randomly selected visits with maintained remission. Other corresponding clinical data such as neutrophil, platelet, mean cell volume (MCV) counts and AZA dose were also noted. RESULTS: Mean lymphocyte count before AZA was 1.926 (95% CI 1.707 to 2.144) x 109/L. This fell to 1.491 (1.427 to 1.556) x 109/L whilst patients were on AZA (p<0.0001). Lymphocyte counts in patients taking AZA varied significantly according to clinical course. At visits immediately prior to or at relapse, the lymphocyte count was 1.618 (1.488 to 1.749) x 109/L and 1.676 (1.534 to 1.818) x 109/ L versus 1.435 (1.371 to 1.498) x 109/L at visits with maintained remission (p=0.000). Of patients on AZA with a lymphocyte count below median values (1.5 x 109/L) 57.8% were in remission compared with 37.8% of patients with higher values. (p<0.001). Similar relationships between lymphocyte counts and clinical course were noted in both UC and Crohn's disease separately. Other parameters also varied according to clinical course. MCV on treatment was significantly higher in patients in remission than prior to or at relapse [92.544 (91.739 to 93.358) fL versus 90.118 (88.497 to 91.739)fL and 89.992 (88.309 to 91.682)fL]. In addition, on treatment neutrophil and platelet counts were significantly elevated prior to or at relapse. Significant differences in lymphocyte counts were also related to AZA dose. Doses >2mg/kg were associated with lower lymphocyte counts [1.486 (1.339 to 1.633) x 109/L] compared with 1.695 (1.618 to 1.772) x 109/L on lower doses. (p= 0.039). CONCLUSION: In patients with IBD, on AZA, a lower lymphocyte count is associated with a lower incidence of disease relapse. Variation in other parameters such as neutrophil, platelet and MCV counts may also predict clinical course on AZA. Studies on dose escalation according to lymphocyte count will be a useful step towards achieving better therapeutic response on AZA. W1270 Discriminating Between Response Types in Infliximab-Treated Patients With Crohn's Disease: Sensitivity and Specificity of Combined Assessment of Infliximab Trough Levels and Anti-Drug Antibodies Casper Steenholdt, Ole O. Thomsen, Jorn Brynskov, Klaus Bendtzen, Mark A. Ainsworth Introduction: The TNF-blocker infliximab (IFX) is effective in Crohn's disease (CD). However, not all patients respond, and a considerable fraction of initial responders lose efficacy over time. Using recently developed radioimmunoassays (RIA), we have shown that the average antidrug- antibody (ADA) levels are higher, and serum (s)-IFX trough levels lower, in CD patients who lose response compared to those who maintain it.1 However, precise cut-off levels for clinically significant ADA and s-IFX levels were not determined in the precedent pilot study.1 Aim: To establish cut-off levels for both ADA and s-IFX which provide optimal sensitivity and specificity in terms of discrimination between different types of clinical AGA Abstracts