Early drug and anti-infliximab antibody levels for prediction of primary nonresponse to infliximab therapy.

BACKGROUND: Primary nonresponse, defined as lack of clinical benefit during the induction phase, occurs in up to 30% of IBD patients treated with infliximab. The mechanisms underlying primary nonresponse have not yet been clearly defined. AIM: To evaluate the association of early (week 2 and week 6) induction infliximab and anti-infliximab antibody levels with primary nonresponse. METHODS: A retrospective observational case-control study of inflammatory bowel disease patients treated with infliximab and followed at Sheba Medical Center between 2009 and 2016 was performed. Pre-infusion infliximab and antibodies to infliximab (ATI) levels were measured by our previously described drug-tolerant ELISA assay. RESULTS: Thirty-five primary nonresponders have been identified and matched with 105 primary responders (1:3 ratios). Both week 2 and week 6 infliximab levels were significantly lower among primary nonresponders compared to responders (week 2, 6: median level 7.2, 2.2 µg/mL vs 13.5, 9.5 µg/mL, P = .0019, P < .0001 respectively). Antibodies to infliximab appeared more frequently (either week 2 or 6, 68% vs 28% prevalence, P = .0004) and at higher levels in nonresponders compared to responders (week 2, 6: median ATI 7.3, 10.8 µg/mL-eq vs 3.8, 4.4 µg/mL-eq, P = .005, P = .008 respectively). Moreover, week 2 infliximab levels <6.8 µg/mL (AUC = 0.68, P = .002, sensitivity 50%, specificity 86%) and antibodies to infliximab levels >4.3 µg/mL-eq (AUC = 0.78, P = .0004, sensitivity 77%, specificity 71%) were predictive of primary nonresponse. Among the other clinical and demographic variables, higher baseline ulcerative colitis clinical score, infliximab monotherapy, prior adalimumab therapy and previous Crohn's disease-related surgeries were also associated with an increased risk of primary nonresponse. CONCLUSIONS: Infliximab levels below 6.8 µg/mL and antibodies to infliximab levels above 4.3 µg/mL-eq before the second infusion are associated with primary nonresponse, especially among Crohn's disease patients.

Addition of an immunomodulator can reverse antibody formation and loss of response in patients treated with adalimumab.

BACKGROUND: Anti-adalimumab antibodies (AAA) are associated with loss of clinical response (LOR). Addition of an immunomodulator has been shown to reverse immunogenicity and regain response with infliximab monotherapy. Similar data on adalimumab are lacking. AIM: To study the impact of immunomodulator addition on the emergence of AAA and LOR among adalimumab therapy patients. METHODS: The databases of three tertiary medical centres were reviewed to identify patients who developed AAA during adalimumab monotherapy with resultant LOR, and received an immunomodulator as a salvage combination therapy. All sera were prospectively analysed using previously described ELISA assays. Clinical response was determined using appropriate clinical scores. Elimination of AAA, designated as 'sero-reversal', elevation of drug levels and regained clinical response were the sought outcomes. RESULTS: Twenty-three patients (21 Crohn's disease, and 2 ulcerative colitis) developed AAA with subsequent LOR and were thereafter prescribed an immunomodulator as salvage therapy (thiopurine n = 14, methotrexate n = 9). Eleven patients (48%) underwent sero-reversal with gradual elimination of AAA, increase in drug trough levels and restoration of clinical response (median time to sero-reversal 5 months). In 12 patients (52%), immunogenicity and loss of response could not be reversed. There was no difference between responders and nonresponders in the type of immunomodulators used or baseline clinical characteristics. CONCLUSIONS: In almost half of inflammatory bowel disease patients developing anti-adalimumab antibodies and loss of response, established immunogenicity of adalimumab can be gradually reversed by the addition of immunomodulator therapy with restoration of a clinico-biological response. However, these observations need to be confirmed with larger studies.

Induction infliximab levels among patients with acute severe ulcerative colitis compared with patients with moderately severe ulcerative colitis.

BACKGROUND: Infliximab is effective as salvage therapy for patients with steroid refractory acute severe ulcerative colitis (UC). Although current data suggest that the pharmacokinetics of infliximab are influenced by inflammatory burden in patients with acute severe UC, data comparing infliximab trough levels in patients with acute severe UC vs. moderately severe UC are scarce. AIM: To compare infliximab trough and anti-infliximab antibody levels at a standard fixed time-point during induction between patients with acute severe and moderately severe UC. METHODS: A multi-centre retrospective study comparing infliximab drug and antibody levels 14 days after the first infusion in hospitalised acute severe UC versus out-patients with moderately severe UC was performed. RESULTS: Sixteen acute severe UC patients, hospitalised between 2010-2015 and refractory to intravenous corticosteroids, were treated with infliximab 5 mg/kg salvage therapy. They were compared to 16 moderately severe UC out-patient controls. Mean infliximab trough levels at day 14 were significantly lower in patients with acute severe UC compared to moderately severe UC (7.15 ± 5.3 vs. 14.4 ± 11.2 µg/mL, P = 0.007). Seven patients (three acute severe and four moderate severe UC) were primary nonresponders to infliximab induction therapy. Infliximab level at day 14 did not differ between responders and nonresponders (9.8 ± 9 vs. 12.1 ± 10.6 µg/mL, respectively, P = N.S.). However, week 2 median antibody-to-infliximab levels were numerically higher among primary nonresponders (3.4 ± 5.7 vs. 1.2 ± 4 µg/mL-eq, respectively, P = 0.06). CONCLUSIONS: Infliximab trough levels at day 14 were lower in patients with acute severe UC compared to moderately severe UC, possibly due to a higher inflammatory burden and/or increased drug clearance. However, drug levels at day 14 were not lower among nonresponders compared with responders. Controlled trials are warranted to examine whether an a-priori-intensified infliximab induction protocol will lead to an improved outcome in acute severe UC.

Undetectable anti-TNF drug levels in patients with long-term remission predict successful drug withdrawal.

BACKGROUND: Low drug levels are associated with emerging loss of response to anti-TNF. However, this may not be the case in patients with long-term remission. AIM: To investigate the outcome of anti-TNF discontinuation in patients with long-term remission and incidental undetectable drug levels. METHODS: A retrospective cohort study examining the duration of relapse-free survival in IBD patients in remission who discontinued infliximab or adalimumab having undetectable drug levels. RESULTS: Forty eight patients who discontinued anti-TNF while in remission and had available drug levels were identified in two centres in France and Israel (infliximab-treated 35, adalimumab-13, Crohn's disease 30, ulcerative colitis 18, mean treatment duration of 22.7 ± 12.4 months). Endoscopy/MRE before stopping showed absence of active inflammation in 40/42 (95%) of evaluated patients, while inflammatory biomarkers (CRP and/or Calprotectin) were completely normal in only 31/48 (65%) of patients. During 12 months median follow-up, relapse occurred in 16/20 (80%) of patients who stopped anti-TNF while having measurable drug levels compared with 9/28 (32%) of patients who had undetectable drug levels (OR: 8.4, 95% CI: 2.2-32, P = 0.002). Relapse-free survival after anti-TNF cessation was significantly longer in patients with absent drug compared to those with detectable drug (P < 0.001, log rank test). On multivariate analysis, a patient's decision to stop therapy was weakly associated and abnormal inflammatory biomarkers and detectable drug levels were both strongly and independently associated with a higher risk of relapse after drug discontinuation. CONCLUSION: Incidental finding of undetectable anti-TNF drug levels in patients with stable long-term deep remission may identify a subset of patients whose clinical remission is no longer dependent on anti-TNF treatment.

Oral administration of non-absorbable delayed release 6-mercaptopurine is locally active in the gut, exerts a systemic immune effect and alleviates Crohn's disease with low rate of side effects: results of double blind Phase II clinical trial.

Therapy for Crohn's disease (CD) with thiopurines is limited by systemic side effects. A novel formulation of fixed-dose, delayed-release 6-mercaptopurine (DR-6MP) was developed, with local effect on the gut immune system and minimal absorption. The aim of this study was to evaluate the safety and efficacy of DR-6MP in patients with moderately severe CD compared to systemically delivered 6-mercaptopurine (Purinethol). Seventy CD patients were enrolled into a 12-week, double-blind controlled trial. The primary end-point was the percentage of subjects with clinical remission [Crohn's Disease Activity Index (CDAI) < 150] or clinical response (100-point CDAI reduction). Twenty-six (56·5%) and 13 (54·2%) subjects from the DR-6MP and Purinethol cohorts, respectively, completed the study. DR-6MP had similar efficacy to Purinethol following 12 weeks of treatment. However, the time to maximal clinical response was 8 weeks for DR-6MP versus 12 weeks for Purinethol. A higher proportion of patients on DR-6MP showed clinical remission at week 8. A greater improvement in Inflammatory Bowel Disease Questionnaire (IBDQ) score was noted in the DR-6MP group. DR-6MP led to a decrease of CD62(+) expression on T cells, implying a reduction of lymphocyte adhesion to site of inflammation. DR-6MP was safer than Purinethol, with significantly fewer adverse events (AEs). There was no evidence of drug-induced leucopenia in the DR-6MP group; the proportion of subjects who developed hepatotoxicity was lower for the DR-6MP. Non-absorbable DR-6MP is safe and biologically active in the gut. It is clinically effective, exerting a systemic immune response with low systemic bioavailability and a low incidence of side effects.

Adalimumab drug and antibody levels as predictors of clinical and laboratory response in patients with Crohn's disease.

BACKGROUND: Adalimumab is an effective treatment for Crohn's disease (CD). Anti-adalimumab antibodies (AAA) and low trough serum drug concentrations have been implicated as pre-disposing factors for treatment failure. AIMS: To assess adalimumab and AAA serum levels, and to examine their association and discriminatory ability with clinical response and serum C-reactive protein (CRP). METHODS: We performed a cross-sectional study using trough sera from adalimumab-treated CD patients. Demographical data, Montreal classification, treatment regimen and clinical status were recorded. Serum adalimumab, AAA and CRP were measured. Receiver operating characteristic analysis and a multivariate regression model were performed to find drug and antibody thresholds for predicting disease activity at time of serum sampling. RESULTS: One hundred and eighteen trough serum samples were included from 71 patients. High adalimumab trough serum concentration was associated with disease remission (Area Under Curve 0.748, P < 0.001). A cut-off drug level of 5.85 µg/mL yielded optimal sensitivity, specificity and positive likelihood ratio for remission prediction (68%, 70.6% and 2.3, respectively). AAA were inversely related with adalimumab drug levels (Spearman's r = -0.411, P < 0.001) and when subdivided into categorical values, positively related with disease activity (P < 0.001). High drug levels and stricturing vs. penetrating or inflammatory phenotype, but not AAA levels, independently predicted disease remission in a multivariate logistic regression model. CONCLUSIONS: Adalimumab drug levels were inversely related to disease activity. High levels of anti-adalimumab antibodies were positively associated with disease activity, but this association was mediated mostly by adalimumab drug levels.

The management of iron deficiency in inflammatory bowel disease--an online tool developed by the RAND/UCLA appropriateness method.

BACKGROUND: Iron deficiency is a common and undertreated problem in inflammatory bowel disease (IBD). AIM: To develop an online tool to support treatment choice at the patient-specific level. METHODS: Using the RAND/UCLA Appropriateness Method (RUAM), a European expert panel assessed the appropriateness of treatment regimens for a variety of clinical scenarios in patients with non-anaemic iron deficiency (NAID) and iron deficiency anaemia (IDA). Treatment options included adjustment of IBD medication only, oral iron supplementation, high-/low-dose intravenous (IV) regimens, IV iron plus erythropoietin-stimulating agent (ESA), and blood transfusion. The panel process consisted of two individual rating rounds (1148 treatment indications; 9-point scale) and three plenary discussion meetings. RESULTS: The panel reached agreement on 71% of treatment indications. 'No treatment' was never considered appropriate, and repeat treatment after previous failure was generally discouraged. For 98% of scenarios, at least one treatment was appropriate. Adjustment of IBD medication was deemed appropriate in all patients with active disease. Use of oral iron was mainly considered an option in NAID and mildly anaemic patients without disease activity. IV regimens were often judged appropriate, with high-dose IV iron being the preferred option in 77% of IDA scenarios. Blood transfusion and IV+ESA were indicated in exceptional cases only. CONCLUSIONS: The RUAM revealed high agreement amongst experts on the management of iron deficiency in patients with IBD. High-dose IV iron was more often considered appropriate than other options. To facilitate dissemination of the recommendations, panel outcomes were embedded in an online tool, accessible via http://ferroscope.com/.

Extended haplotype association study in Crohn's disease identifies a novel, Ashkenazi Jewish-specific missense mutation in the NF-κB pathway gene, HEATR3.

The Ashkenazi Jewish population has a several-fold higher prevalence of Crohn's disease (CD) compared with non-Jewish European ancestry populations and has a unique genetic history. Haplotype association is critical to CD etiology in this population, most notably at NOD2, in which three causal, uncommon and conditionally independent NOD2 variants reside on a shared background haplotype. We present an analysis of extended haplotypes that showed significantly greater association to CD in the Ashkenazi Jewish population compared with a non-Jewish population (145 haplotypes and no haplotypes with P-value <10(-3), respectively). Two haplotype regions, one each on chromosomes 16 and 21, conferred increased disease risk within established CD loci. We performed exome sequencing of 55 Ashkenazi Jewish individuals and follow-up genotyping focused on variants in these two regions. We observed Ashkenazi Jewish-specific nominal association at R755C in TRPM2 on chromosome 21. Within the chromosome 16 region, R642S of HEATR3 and rs9922362 of BRD7 showed genome-wide significance. Expression studies of HEATR3 demonstrated a positive role in NOD2-mediated NF-κB signaling. The BRD7 signal showed conditional dependence with only the downstream rare CD-causal variants in NOD2, but not with the background haplotype; this elaborates NOD2 as a key illustration of synthetic association.

A novel device to improve colon cleanliness during colonoscopy.

BACKGROUND AND STUDY AIM: Effective colonoscopy depends on adequate visualization of the intestine, which might be ensured by intraprocedural use of a cleansing device. We investigated the performance of a novel endoscopic device with regard to cleanliness, safety, and tolerability during colonoscopy, compared with standard cleansing. PATIENTS AND METHODS: At a single center, colonoscopy patients in whom the cecum was accessed and at least one bowel segment was inadequately cleansed were assigned to either use of a disposable catheter cleansing device (JetPrep), used through the endoscope working channel, or standard manual cleansing using a 50-ml syringe. The cleansing quality, for each segment and before and after irrigation, was recorded using a 4-point scale ranging from excellent (grade 1, no more than small bits of adherent feces) to poor (grade 4, large amount of fecal residue). RESULTS: 38 patients were included, 19 in each group. Reasons for referral included colorectal cancer screening (52 %), or blood loss (31 %). Each segment showed improvement after cleansing with JetPrep. Overall cleansing grade improved by a mean of 0.74 points (standard deviation [SD] 0.82) in the investigation group compared with 0.19 (0.40) in the control group (P < 0.0001), and right colon cleansing improved by 1.59 points (0.71) versus 0.31 (0.48) in the controls (P < 0.0001). There was no significant difference in procedure time between the groups. No adverse events or side effects were encountered. CONCLUSIONS: The JetPrep disposable catheter device is safe and efficient for intraprocedural cleansing of a suboptimally prepared colon, allowing higher quality colonoscopy.

The effect of once daily omeprazole and succinic acid (VECAM) vs once daily omeprazole on 24-h intragastric pH.

BACKGROUND: Parietal cell H(+)/K(+) ATPase activation is essential for optimal proton pump inhibitor (PPI) activity. Succinic acid (SA) was shown to induce gastric acid secretion. VECAM is a combination of omeprazole (OMP) and SA. To compare the effect of once daily bedtime dosing of VECAM 40 and VECAM 20 without food vs OMP 20 mg administered before breakfast on gastric acidity. METHODS: Open label, randomized, crossover study enrolling 36 healthy subjects comparing the study treatments at steady state for 24 h intragastric pH monitoring. KEY RESULTS: The median percent time intragastric pH > 4 demonstrated that VECAM 40 was superior to VECAM 20 (65.7%vs 49.1%P < 0.0001) and OMP 20 mg (65.7%vs 47.6%P = 0.005) during 24 h. VECAM 40 was superior to VECAM 20 (52.8%vs 38.8%P = 0.0079) and OMP 20 mg (52.8%vs 27.2%P < 0.0001), and VECAM 20 was superior to OMP 20 mg (38.8 vs 27.2 P = 0.0069) during the nighttime. VECAM 20 and OMP 20 mg were comparable during 24 h. CONCLUSIONS & INFERENCES: VECAM 40 and VECAM 20 were significantly better in maintaining intragastric pH > 4 during the nighttime than OMP 20 mg. Succinic acid eliminates the need for a subsequent meal for intragastric pH control by VECAM.

The decline of anti-drug antibody titres after discontinuation of anti-TNFs: implications for predicting re-induction outcome in IBD.

BACKGROUND: Anti-drug antibodies can be elicited by infliximab and adalimumab, but the rate of their decay after therapy is stopped is unknown. AIM: To investigate the decline of anti-drug antibody titre after anti-TNF cessation, and to evaluate the clinical utility of anti-drug antibody measurement before anti-TNF re-induction. METHODS: Inflammatory bowel disease (IBD) patients who stopped anti-TNF therapy and had measurable anti-drug antibodies were prospectively followed up by serial blood measurements of antibodies levels. The clinical outcome of a second cohort of patients who received re-induction by infliximab or adalimumab after a drug holiday >4 months was determined vis-à-vis their anti-drug antibodies status before re-induction. RESULTS: The first cohort included 22 patients with anti-drug antibodies who were prospectively followed up after cessation of anti-TNF. Sixteen had antibodies-to-infliximab (ATI) and six had antibodies-to-adalimumab (ATA). ATI titres declined within 12 months to below detection levels in 13/16 infliximab-treated patients, whereas ATA titres became undetectable in only 2/6 adalimumab-treated patients (P = 0.04). The second cohort comprised 27 patients who resumed anti-TNFs (24 infliximab, 3 adalimumab). Of these, 3/5 patients with measurable anti-drug antibodies before re-induction experienced severe hypersensitivity reaction and/or nonresponse mandating drug-discontinuation, compared to 11/22 patients who were re-induced without measurable anti-drug antibodies (OR = 1.5, 95% CI 0.2-11, P = 0.7). CONCLUSIONS: Antibodies to infliximab titres decline to undetectable levels within one year of cessation of infliximab in the majority of patients, whereas antibodies to adalimumab seem to persist longer after adalimumab discontinuation. Measuring antibodies to infliximab prior to infliximab re-induction is probably of little clinical utility, especially if more than a 12-month drug-holiday has elapsed.

Review article: loss of response to anti-TNF treatments in Crohn's disease.

BACKGROUND: Loss of response to anti-TNF agents in Crohn's disease is an emerging clinical problem. AIM: To review the causes, incidence and management approach of loss of response. METHODS: A search of medical database (PubMed) and of meetings' proceedings for definitions, causes and incidence of loss of response was carried out. Personal correspondence with principal investigators was conducted to retrieve missing data. RESULTS: Various definitions of loss of response abound, hampering the ability to assess accurately the magnitude and management of this clinical problem. We propose to distinguish between a clinical worsening on anti-TNF treatment and a true loss of response to anti-TNFs. Accordingly, loss of response to anti-TNFs at 12 months of therapy occurs in 23-46% of patients when judged by dose intensification, or 5-13% when gauged by drug discontinuation rates. The management of loss of response should allow for a period of watchful waiting as quite often the patients' symptoms may resolve without alteration of therapy. If they do not, then identifying the correct mechanism responsible for clinical deterioration is prudent. Once symptoms are ascertained to arise from inflammatory IBD activity, drug level and antidrug antibody measurement can then help distinguish between non-adherence to therapy, immunogenicity and non-immune clearance of anti-TNF, or an un-chequered inflammation despite adequate anti-TNF levels. The latter finding may be best addressed by a switch to another class of immunomodulators, whereas a low drug level should probably be managed by dose intensification or a switch to another anti-TNF. CONCLUSION: Studies defining how best to translate drug-level monitoring and other mechanistic considerations into clinical decisions are urgently needed.

Taking immunogenicity assessment of therapeutic proteins to the next level.

Therapeutic proteins provide innovative and effective therapies for numerous diseases. However, some of these products are associated with unwanted immunogenicity that may lead to clinical consequences such as reduced or loss of efficacy, altered pharmacokinetics (PK), general immune and hypersensitivity reactions, and neutralisation of the natural counterpart (e.g. the physiological hormone). Regulatory guidance on immunogenicity assessment needs to take into consideration a great diversity of products, indications and patient populations as well as constantly advancing manufacturing technologies. Such guidance needs to be sufficiently specific while, at the same time, allowing interactive discussion and adjusted benefit-risk weighing of each product on a case-by-case basis, e.g. for a unique treatment of a life threatening disease acceptable treatment risks may differ considerably from the ones in case of less serious disease. This theme was the focus of the international conference "Taking immunogenicity assessment of therapeutic proteins to the next level", held at the Paul-Ehrlich-Institut in Langen, Germany, on the 10-11. June 2010. The objectives of the conference were to highlight how the field could move from that of a mere description of risk factors to a system of risk assessment and mitigation, as well as an understanding of the impact of unwanted immunogenicity on the overall benefit/risk consideration for a medicinal product. More than 150 experts from industry, academia and regulatory authorities worldwide discussed the phenomenon of undesired immunogenicity from different perspectives. The conference focussed on issues relevant to three areas: (1) new European guidelines that are currently the subject of discussion; (2) testing strategies for immunogenicity assessment; and (3) scientific progress on the product-related factors that may contribute to the development of pathogenesis of immunogenicity, in particular in the field of protein aggregation and post-translational modifications. This report provides an overview of issues, insights, and conclusions that were discussed and achieved during the meeting.

The anti-inflammatory target A(3) adenosine receptor is over-expressed in rheumatoid arthritis, psoriasis and Crohn's disease.

The Gi protein associated A(3) adenosine receptor (A(3)AR) was recently defined as a novel anti-inflammatory target. The aim of this study was to look at A(3)AR expression levels in peripheral blood mononuclear cells (PBMCs) of patients with autoimmune inflammatory diseases and to explore transcription factors involved receptor expression. Over-expression of A(3)AR was found in PBMCs derived from patients with rheumatoid arthritis (RA), psoriasis and Crohn's disease compared with PBMCs from healthy subjects. Bioinformatics analysis demonstrated the presence of DNA binding sites for nuclear factor-kappaB (NF-kappaB) and cyclic AMP-responsive element binding protein (CREB) in the A(3)AR gene promoter. Up-regulation of NF-kappaB and CREB was found in the PBMCs from patients with RA, psoriasis and Crohn's disease. The PI3K-PKB/Akt signaling pathway, known to regulate both the NF-kappaB and CREB, was also up-regulated in the patients' PBMCs. Taken together, NF-kappaB and CREB are involved with the over-expression of A(3)AR in patients with autoimmune inflammatory diseases. The receptor may be considered as a specific target to combat inflammation.

Early preservation of effector functions followed by eventual T cell memory depletion: a model for the delayed onset of the effect of thiopurines.

OBJECTIVE: The onset of the effect of thiopurines is delayed for several months. The aim of this study was to investigate immune mechanisms for this delay. METHODS: The effects of thiopurines on human peripheral blood T cells and on lamina propria lymphocytes were investigated for apoptosis induction by Annexin V/propidium iodide (PI) and for cytokine secretion by intracellular staining and ELISA assays. To investigate the mechanism of the effect of thiopurines in vivo, Balb/C mice were co-immunised with HEL/OVA (hen egg lysozyme/ovalbumin) antigens, and then repeatedly challenged by HEL only, while being treated by mercaptopurine or vehicle alone for either 4 or 20 weeks. The memory response of CD4+ splenocytes towards HEL/OVA was then determined by CFSE (carboxyfluorescein succinimidyl ester) dilution. RESULTS: Thiopurines arrested the proliferation of stimulated T cells but did not enhance the apoptosis of either resting T cells or activated T cells until day 5 poststimulation. Despite the proliferation arrest, stimulated T cells successfully differentiated into effector cells, as evidenced by their capacity for proinflammatory cytokine secretion, potent adhesion and cytotoxicity. Prolonged mercaptopurine treatment of mice for 20 weeks selectively reduced the CD4+ memory response to a repeatedly encountered HEL antigen, but did not affect the T cell memory pool to the previously presented OVA antigen. A shorter, 4 weeks, treatment with mercaptopurine did not inhibit the memory response to either antigen. CONCLUSIONS: T cells arrested from cycling by thiopurines can still differentiate into potent effector cells capable of propagating the inflammatory process. Thiopurine treatment results in depletion of antigen-specific memory T cells, but this effect is dependent upon repeated encounters with the antigen over a prolonged time course.