Adobe Flash Player is required to view this feature. If you are using an operating system that does not support Flash, we are working to bring you alternative formats. Original Article Influence of Immunogenicity on the Long-Term Efficacy of Infliximab in Crohn's Disease Filip Baert, M.D., Maja Noman, M.D., Severine Vermeire, M.D., Ph.D., Gert Van Assche, M.D., Ph.D., Geert D' Haens, M.D., Ph.D., An Carbonez, Ph.D., and Paul Rutgeerts, M.D., Ph.D. N Engl J Med 2003; 348:601-608 DOI: 10.1056/NEJMoa020888.

Results A mean of 3.9 infusions (range, 1 to 17) per patient were administered over a mean period of 10 months. Antibodies against infliximab were detected in 61 percent of patients. The presence of concentrations of 8.0 μg per milliliter or greater before an infusion predicted a shorter duration of response (35 days, as compared with 71 days among patients with concentrations of less than 8.0 μg per milliliter; P. Figure 2 Relation between Concentrations of Antibodies against Infliximab and the Use of Immunosuppressive Therapy among Patients without Fistulas (Panel A) and Patients with Fistulas (Panel B).

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The box plots show the median values and the first and third quartiles in each group. The T bars represent the rest of the data with a maximum of 1.5 times the interquartile range. Concentrations of antibodies against infliximab were significantly lower (P. Infliximab (Remicade, Centocor), a chimeric monoclonal IgG1 antibody against tumor necrosis factor, has been approved for the treatment of moderate-to-severe Crohn's disease in patients who have an inadequate response to conventional therapy and for the management of enterocutaneous fistulas.

A single intravenous infusion induced a response at four weeks in 50 to 81 percent of patients with refractory luminal disease and induced remission in 25 to 48 percent. The response can be maintained with repeated infusions. In patients with fistulizing disease, four weeks after the last of three infusions, at weeks 0, 2, and 6, 38 to 55 percent of fistulas had completely closed, and the median duration of response was 90 days. Clinicians often re-treat patients with infliximab after a relapse. Infliximab therapy can result in the formation of antibodies against infliximab. The presence of these antibodies has been associated with infusion reactions in 6.9 to 19 percent of patients. These antibodies may also have shortened the duration of effect of repeated infliximab treatments.

We studied the relation between antibodies against infliximab and postinfusion infliximab concentrations, the clinical effect of infliximab, and infusion-related side effects. In addition, we investigated risk factors for the formation of antibodies against infliximab. Patients We studied 125 consecutive patients with refractory luminal or fistulizing Crohn's disease who were starting treatment with infliximab between December 1998 and July 2000.

All patients signed an informed-consent form describing procedures for safety follow-up. The study was approved by the institutional review board. Patients were treated for active luminal disease with a single intravenous infusion of 5 mg of infliximab per kilogram of body weight and for draining fistulas with a series of three infusions of 5 mg per kilogram, at weeks 0, 2, and 6. If a patient had a response, therapy was repeated on relapse of the disease. The decision to re-treat was made by an experienced clinician when relapse was apparent as a result of increased diarrhea, increased abdominal pain, and decreased well-being.

Infusions were administered under close supervision by a specially trained nurse at an infusion facility. An infusion reaction was defined as any significant adverse event that occurred during the infusion or within two hours afterward. When an infusion reaction occurred, the infusion was stopped and restarted at a slower rate. If the symptoms recurred, 100 mg of intravenous hydrocortisone and 50 mg of intramuscular promethazine were given. This regimen was then given prophylactically 30 minutes before each subsequent infusion.

Concomitant medications including immunosuppressive agents and corticosteroids were used as indicated to control bowel disease. Treatment with immunosuppressive agents consisted of azathioprine (2.0 to 2.5 mg per kilogram per day), mercaptopurine (1.0 to 1.25 mg per kilogram per day), or methotrexate (15 mg intramuscularly once weekly) for a median of 9.5 months (range, 2 to 84) before infliximab therapy began.

Evaluations All patients were evaluated before and every four weeks after each infusion, and side effects, including early reactions (infusion reactions) and late reactions (rash, arthralgia, fatigue, myalgia, and influenza-like symptoms), were recorded. Concentrations of infliximab and antibodies against infliximab in serum were measured at each visit and before each infusion. Each serum sample was assessed for infliximab and antibodies against infliximab in duplicate in a blinded fashion by Prometheus Laboratories; the values reported are the means. The infliximab assay was a microplate enzyme-linked immunosorbent assay in which infliximab bound to immobilized tumor necrosis factor α is detected with horseradish peroxidase–conjugated antihuman IgG (Fc-specific).

The cutoff value, which was based on the mean (+3 SD) value in serum samples from 40 patients who had never received infliximab, was 1.40 μg per milliliter. Concentrations below the cutoff value are reported as negative. In the case of 140 samples, concentrations greater than 20 μg per milliliter were reported as 21 μg per milliliter. The assay for antibodies against infliximab was a microplate enzyme-linked immunosorbent assay based on the double-antigen format in which infliximab is used both during the solid phase to capture antibodies against infliximab and during the biotinylated detection phase with Neutravidin–horseradish peroxidase.

The value is reported in micrograms per milliliter on the basis of calibrations made with affinity-purified polyclonal rabbit antimouse IgG F(ab'). The mean cutoff value in serum samples from 40 patients who had never received infliximab was 1.69 μg per milliliter. Antibodies against infliximab were reported as negative when the concentration was less than 1.69 μg per milliliter and the serum infliximab concentration was less than 1.40 μg per milliliter and as indeterminate when the concentration was less than 1.69 μg per milliliter but the infliximab concentration was 1.40 μg per milliliter or greater. Because infliximab interferes with the assay, these values cannot be conclusively determined. The antibody test was considered to be positive when the concentration exceeded 1.69 μg per milliliter and the infliximab concentration was less than 1.40 μg per milliliter.

Statistical Analysis Patients were included in efficacy analyses if they had an initial clinical response and if they required further infusions for active luminal disease. Patients who underwent surgery and patients in whom the interval between infusions exceeded 20 weeks were excluded from the analysis. Patients with fistulas were included only if they required repeated infusions for active luminal disease after the initial three infusions. All serum samples with an indeterminate value for antibodies against infliximab were excluded from the analyses of antibodies against infliximab. The use of azathioprine, mercaptopurine, or methotrexate was considered as a single risk factor.

We used SAS software (version 6.12, SAS Institute) for all statistical analyses. Possible associations between two binary variables were assessed with use of the relative risk and corresponding 95 percent confidence interval. Analysis of variance was used for the analysis of continuous outcome variables with discrete exploratory variables. The Tukey multiple-comparison procedure was used to evaluate the statistical significance of a set of multiple comparisons. We used a generalized linear model to evaluate the simultaneous effect of two or more factors on the response variables.

In some subgroups, we applied a nonparametric procedure (nonparametric one-way analysis), because of the small number of patients. The Kruskal–Wallis test was used to evaluate whether groups came from the same population. To investigate the relation between the duration of response and the concentration of antibodies against infliximab as well as infliximab concentrations, we used multiple linear regression analysis. The manufacturer of infliximab had no input into the design of the study, data analysis, or manuscript preparation. The analysis of the manufacturer's safety data was performed independently by the authors. Antibodies against Infliximab At base line no patient tested positive for antibodies against infliximab.

After the fifth infusion, 76 patients (61 percent) had detectable antibodies ( Figure 1 Cumulative Incidence of Antibodies against Infliximab (Panel A) and of Infusion Reactions (Panel B) in the Study Cohort. Antibody concentrations were measured at base line and at regular intervals during follow-up, beginning after the first infusion among patients with luminal disease and the first series of three infusions among patients with fistulizing disease and before each subsequent infliximab infusion. The incidence did not increase further with repeated infusions. The serum concentrations of antibodies against infliximab had two clusters that could be separated with the use of 8.0 μg per milliliter as a cutoff value: 46 of the 125 patients (37 percent) had a titer of 8.0 μg per milliliter or greater. Patients who were taking immunosuppressive agents had a lower incidence of antibodies than patients who were not taking immunosuppressive agents: 43 percent (24 of 56), as compared with 75 percent (52 of 69) (P. Infliximab Concentrations A significant relation was found (R 2=0.34, P. Discussion Infliximab has become common treatment for refractory Crohn's disease.

Our data show that the development of antibodies against infliximab correlates with an increased risk of infusion reactions and with a shorter duration of response owing to lower infliximab concentrations. Our data suggest that treatment with immunosuppressive agents prevents infusion reactions and helps maintain clinical efficacy. Several factors could account for the high incidence of antibodies against infliximab in this cohort of patients. Since serial measurements were obtained in patients who were treated intermittently, the longer intervals between treatments afforded more opportunities to measure antibodies without the confounding presence of infliximab in the serum.

All patients in the Anti-Tumor Necrosis Factor Trial in Rheumatoid Arthritis with Concomitant Therapy study of rheumatoid arthritis and most patients in the ACCENT (A Crohn's Disease Clinical Trial Evaluating Infliximab in a New Long-Term Treatment Regimen) study of Crohn's disease received infliximab every eight weeks. Less than half the patients in our cohort were taking immunosuppressive agents, in contrast to the studies of rheumatoid arthritis, in which all patients were taking methotrexate maintenance therapy.

The assays in our studies were performed at Prometheus Laboratories and differed from the assay used by Centocor. A formal comparison of the two assays is warranted, but it is unlikely that differences in methods will account for the differences in the incidence of antibody positivity. The results of measurements of antibodies against infliximab in serum samples obtained between infliximab infusions are often indeterminate. We therefore investigated whether measurement of the infliximab concentration four weeks after an infusion provided more reliable results. Although there was a clear relation between infliximab concentrations and the duration of response, this correlation was not stronger than the correlation between the antibody concentrations and the duration of response. A clear message from our study is that immunosuppressive treatment prevents the formation of antibodies against infliximab, thus reducing the incidence of infusion reactions and increasing the duration of response. Infusion reactions are important immunologic events induced by the presence of a substantial concentration of antibodies against infliximab in the serum.

After an infusion reaction, infliximab disappears quickly from serum and is undetectable four weeks after an infusion. Once an infusion reaction occurred, the duration of the response to subsequent infusions decreased.

Since antibodies develop soon after the initial infusion in most patients, we think immunosuppressive therapy should be instituted before infliximab therapy is started to prevent the formation of antibodies and improve the duration of response to the drug. Whether any one of the immunosuppressive agents — azathioprine, mercaptopurine, or methotrexate — provides greater protection against immunogenicity remains to be determined. A drug interaction between methotrexate and infliximab has been proposed. Although the maximal serum concentrations of infliximab are the same with or without concomitant methotrexate therapy, the rate of disappearance of infliximab was slower in one study among patients who were taking methotrexate than among those who were receiving infliximab alone. To overcome the immunogenicity problem, humanized and human antibodies have been engineered and studied in several diseases, including Crohn's disease. Further studies will show whether these antibodies are associated with a reduced incidence of immunogenicity and an increased ratio of efficacy to safety. Presented in part at the American Gastroenterology Association Digestive Disease Week symposium in Atlanta, May 2001, and San Francisco, May 2002, and at the United European Gastroenterology Week in Amsterdam, October 2001.

Supported by a grant from the Fonds voor Wetenschappelijk Onderzoek–Vlaanderen. Van Assche, D' Haens, and Rutgeerts have reported serving as consultants to Centocor and Schering-Plough. D' Haens and Rutgeerts have reported serving as paid speakers for Centocor and Schering-Plough. Van Assche has reported serving as a paid speaker for Centocor. Rutgeerts has reported receiving grant support from Centocor and Schering-Plough. Baert and Noman contributed equally to the article.

We are indebted to our data managers (Caroline Swijsen, Katrien Asnong, and Isolde Aerden) for the collection of data and serum samples, to Eric Pauwels for invaluable help with the figures, and to Prometheus Laboratories (San Diego, Calif.) for the analyses of infliximab and antibodies against infliximab. References • 1 Targan SR, Hanauer SB, van Deventer SJH, et al. A short-term study of chimeric monoclonal antibody cA2 to tumor necrosis factor α for Crohn's disease. N Engl J Med 1997;337:1029-1035 • 2 Rutgeerts P, D'Haens G, Targan S, et al. Efficacy and safety of retreatment with anti-tumor necrosis factor antibody (infliximab) to maintain remission in Crohn's disease. Gastroenterology 1999;117:761-769 • 3 Present DH, Rutgeerts P, Targan S, et al.

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