Anti-inflammatory Therapy With Anakinra in Newly Diagnosed Type 1 Diabetes
Condition: Type 1 Diabetes Mellitus
MeSH terms: Diabetes Mellitus; Diabetes Mellitus, Type 1
Study type: Interventional
Phase: Phase 1/Phase 2

Summary: 
      The purpose of this study is to determine whether control of inflammatory pathways mediated
      by IL-1 beta using the IL-1 receptor antagonist anakinra will yield measurable decreases in
      expression of genes that are otherwise overexpressed as a consequence of IL-1 beta effects in
      children with newly diagnosed type 1 diabetes. Ultimately, we believe that control of IL-1
      beta pathways will be associated with preserved insulin secretory capacity.
    

Detailed description: 
      Type 1 diabetes mellitus (T1D) is caused by autoimmune and autoinflammatory destruction of
      the insulin-producing beta cells in the pancreatic islets of Langerhans. Historically,
      treatment for this condition has consisted of insulin replacement therapy and dietary
      modification. Recent studies have demonstrated the potential benefits of immunomodulatory
      therapy in patients with newly diagnosed T1D to prevent further immune-mediated damage. Thus
      far, there has been a paucity of completed research using agents that target pro-inflammatory
      cytokines dysregulated in T1D.

      IL1B, the gene encoding the proinflammatory cytokine interleukin-1β (IL-1β) is significantly
      overexpressed in peripheral blood mononuclear cells (PBMC) in patients with newly diagnosed
      T1D compared to healthy controls. There is ample precedent to support the involvement of
      IL-1β in the pathogenesis of diabetes. In particular, incubation of human or animal islets or
      insulinoma cell lines with IL-1β inhibits insulin secretion and leads to apoptosis of beta
      cells.

      Additionally, the IL-1 receptor antagonist protein, anakinra, improves glycemic control and
      insulin secretory capacity in patients with type 2 diabetes.However, no data have been
      published regarding efficacy of agents targeting IL-1β in modifying the course of the disease
      in patients with T1D. Anti-Interleukin-1 in Diabetes Action (AIDA) is a randomized, placebo
      controlled clinical trial of anakinra in 160 adults with newly diagnosed type 1 diabetes.
      This trial is currently recruiting subjects. Review of clinicaltrials.gov demonstrates two
      other planned trials of IL-1 agents in newly diagnosed T1D, one using canakinumab, a
      monoclonal antibody directed at IL-1β, and another using rilonacept, a cytokine trap
      targeting IL-1β. To assess the effectiveness of these drugs in future studies and in clinical
      practice, it will be valuable to identify biomarkers that allow us to monitor their
      therapeutic effects. However, to the best of our knowledge, the pattern of changes in gene
      expression induced by IL-1β in normal PBMC has not been systematically studied, making it
      difficult to identify candidate biomarkers for validation studies.

      In this exploratory study, we aimed to determine which of the characteristic changes in gene
      expression from patients with newly diagnosed T1D are IL-1β-mediated, using both in vitro and
      in vivo approaches. We also determined the effect size of a short course of anakinra therapy
      on glycemic control, insulin dosing, and C-peptide area under the curve during mixed-meal
      tolerance testing (MMTT). Finally, we evaluated the tolerability of anakinra in children and
      adolescents with newly diagnosed T1D.

      Methods In vitro studies To determine the effects of IL-1β on gene expression in peripheral
      blood mononuclear cells (PBMC), blood samples were collected from 7 healthy adult volunteers
      under an IRB-approved protocol.

      Isolating serum. Blood was collected in EDTA tubes (BD, Franklin Lakes, NJ) and centrifuged
      at 2500 rpm for 15 minutes. The plasma layer was treated with topical thrombin (5000 U/ml,
      King Pharmaceuticals, Bristol, TN) equaling 5% total volume and incubated at 38° for 20
      minutes. The resulting clot was removed from the serum and discarded.

      Cell culture. PBMC were isolated from the cellular fraction by centrifugation using
      Lymphocyte Separation Medium (Mediatech, Manassas, VA) and washed with PBS (Mediatech,
      Manassas, VA). Cells were plated at 3 x 106 per well in 6 well plates in RPMI 1640
      (Mediatech) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville,
      GA), 10% autologous human serum, 5.5 mM glucose, 100 U penicillin, 100 µg/ml streptomycin, 50
      µmol/l 2-mercaptoethanol and 5% HEPES. IL-1β (15 ng/ml final concentration, Abcam, Cambridge,
      MA), or 9.3 µg/ml S100b (Sigma, St. Louis, MO) were added to some wells, All experimental
      conditions were plated in triplicate. Cells were collected after 24 hours of incubation at
      37°C in a 5% CO2 atmosphere.

      Validation by RT-PCR. Total RNA was isolated using RNA-Stat 60 (Tel-Test, Friendswood, TX).
      The High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA) was used
      with 750 ng RNA to create cDNA. Real time PCR was performed on the Roche LightCycler 480
      system using 5 ul of the cDNA sample, the Roche LightCycler Probes Master kit and human IL1B
      primers (Applied Biosystems). RNA was quantitated by delta Ct values using GADPH as the
      internal control (Applied Biosystems).

      Microarray analysis. Triplicate samples for each experimental condition were pooled for
      further analysis. From 2-5 µg of total RNA, double-stranded cDNA containing the T7-dT(24)
      promoter sequence were generated using GeneChip® One-Cycle cDNA Synthesis Kits (Invitrogen,
      Santa Clara, CA). Synthesis of cRNA used 200ng of cDNA for in vitro transcription,
      amplification and labeling steps according to the manufacturer's instructions using the
      Illumina RNA amplification kit (Ambion Inc, Austin, TX). 1.5 µg of amplified biotin-labeled
      cRNA was subsequently hybridized to Illumina Human HT-12 v3.0 Beadchip microarrays according
      to standard protocols. at the Baylor Institute for Immunology Research, Dallas, TX.

      Slides were scanned on an Illumina Beadstation 500 and data processed with Beadstudio
      software. For each chip, raw intensity data were normalized to the mean intensity of all
      measurements on that chip. Data were imported into Genespring GX11 (Agilent) for further
      analysis. Probe sets were selected if "Present" or "Marginal" in at least 50% of samples in
      any group. Principal component analysis was performed to detect outliers. Class comparisons
      were performed using t-tests after log transformation with the Type 1 error rate controlled
      using Benjamini-Hochberg false discovery rates (FDR).

      In vivo studies Subjects. The study was approved by the Institutional Review Board of the
      University of Texas Southwestern Medical Center. Written informed consent was obtained from
      parents or legal guardians, and assent for participation was obtained from subjects aged 10
      years and older. Patients at Children's Medical Center Dallas between ages 6 and 18 years
      with Type 1 diabetes within one week of diagnosis were eligible. Exclusion criteria included
      treatment with systemic or inhaled corticosteroids or any other immunomodulatory drug, active
      infection, history of mycobacterial disease, pregnancy or lactation, use of a live vaccine
      within 90 days of study enrollment, and severe comorbidities (such as chronic kidney disease,
      heart failure, or uncontrolled hypertension). Patients were excluded if it was unclear
      whether they had Type 1 or Type 2 diabetes.

      Data collected from the medical charts included age, gender, race/ethnicity, weight, height,
      hemoglobin A1c (HbA1c) and beta-hydroxybutyrate at diagnosis. A blood sample was obtained at
      study entry; a portion was analyzed for screening labs including alanine aminotransferase
      (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatinine, complete
      blood count (CBC) with differential, and serum pregnancy test for all menstruating females
      and any female over age 10 years. Additionally, 20 mL was processed for microarray analysis.

      Diabetes care At diagnosis, all subjects were placed on a basal-bolus insulin regimen with
      glargine and either lispro or aspart. For the duration of the study, insulin doses were
      adjusted per standard clinic protocol with target glucose of 80-140 mg/dL fasting and 80-180
      mg/dL before meals. At each study visit, we recorded the subject's current insulin doses and
      weight to allow calculation of the total daily dose (units/kg/day).

      Anakinra After study enrollment, all subjects started anakinra (Kineret; Amgen, Thousand
      Oaks, CA) as a subcutaneous daily injection. Subjects weighing more than 25 kg at the time of
      enrollment received 100 mg daily whereas those weighing 25 kg or less received 50 mg daily.
      Anakinra was continued for a total of 28 days with no dose adjustment.

      Diabetes autoantibodies Per standard protocol, all patients were tested for antibodies to
      insulin, protein tyrosine phosphatase receptor type N (insulinoma-associated antigen (IA-2)),
      and glutamic acid decarboxylase (ARUP Laboratories, Salt Lake City, UT). The results of these
      studies were typically not available at the time of study enrollment and therefore were not
      used as criteria for study entry. However, we excluded patients with negative results for all
      three antibodies from further analysis.

      Surveillance laboratory tests Upon completion of anakinra therapy (4-5 weeks after
      diagnosis), a blood sample was collected and sent for ALT, AST, BUN, creatinine, and CBC with
      differential. Per standard protocol, all subjects had point-of-care hemoglobin A1c (DCA
      Vantage Analyzer, Siemens Healthcare Diagnostics, Deerfield, IL) and capillary glucose tested
      at clinic visits 4-5 weeks after diagnosis, 4 months after diagnosis, and 7 months after
      diagnosis.

      Adverse event monitoring During the 28 days of anakinra therapy, study personnel called
      subjects weekly to document frequency of hypoglycemia and presence of rash, injection site
      reactions (swelling, erythema, and pain at the site of anakinra administration), headaches,
      fevers, and any other adverse events. After the completion of anakinra therapy, patients were
      assessed for adverse events at each subsequent study visit.

      Mixed meal tolerance tests (MMTTs) MMTTs were conducted at the University of Texas
      Southwestern Medical Center Clinical Translational Research Center (CTRC). Subjects underwent
      MMTTs essentially as described (8) at 3-4 weeks after diagnosis and again at 7 months after
      diagnosis. C-peptide analyses were performed in the laboratory of Dr. Philip Raskin (UT
      Southwestern Medical Center, Dallas, TX).

      Microarray blood sample processing Microarray blood samples were collected in EDTA tubes (BD
      Vacutainer) at study enrollment, 3-4 weeks after diagnosis, and upon completion of anakinra
      therapy. PBMC were isolated and stored at -80°C within 4 hours of the blood draw. Cells were
      lysed in RLT lysis buffer containing β-mercaptoethanol (Qiagen, Valencia, CA). Total RNA was
      extracted using the RNeasy® Mini Kit according to the manufacturer-recommended protocol
      (Qiagen, Valencia, CA) and analyzed as described above.

      Control groups Because subjects in the anakinra study were not randomized, we used two
      different pre-existing control groups. Subjects in control group A were previously enrolled
      at our institution under a separate, IRB-approved protocol in a randomized, controlled trial
      examining the effect of the initial choice of longer-acting insulin on the rate of loss of
      beta cell function. Subjects were enrolled during their hospitalization for diabetes
      diagnosis and randomized to receive either NPH or basal insulin (glargine or detemir).
      Diabetes care and routine clinic visits, schedule and procedures for mixed meal tolerance
      testing and microarray analysis were the same as for our anakinra-treated subjects.. Control
      group A includes all patients in that study randomized to receive basal insulin, except those
      with negative autoantibody results.

      Subjects in control group B were identified in a chart review of all new diabetes diagnoses
      from April 2008 through November 2009 in patients aged 9-18 years (to match date range of
      recruitment and age of anakinra-treated subjects). Subjects were included in this control
      group if they had at least one positive autoantibody and were not enrolled in the anakinra
      study. We collected age, gender, race/ethnicity, weight, height, and beta-hydroxybutyrate at
      diagnosis. We also collected HbA1c and total daily insulin dose (expressed as units/kg/day)
      at diagnosis and for clinic visits at 1 month, 4 months, and 7 months after diagnosis.

      A subset of 10 of these subjects consented to enroll in an IRB-approved study in which blood
      was drawn for microarray analysis at diagnosis and again 1 month after diagnosis. Microarray
      procedures were the same as described above for the anakinra-treated subjects.

      Statistical analysis Descriptive statistics were compiled and all comparative analyses
      performed using SAS version 9.2 (Cary, NC). C-peptide area under the curve (AUC) was
      calculated for each MMTT using the trapezoidal method.. Insulin-dose adjusted A1c (IDAA1c)
      was calculated as outlined by Mortensen, et. al. (9), IDAA1c = hemoglobin A1C (percent) + [4
      × insulin dose (units per kilogram per 24 h)]. Hemoglobin A1c and IDAA1c between groups were
      compared using Wilcoxon rank-sum tests. Since total daily insulin dose was normally
      distributed, between groups comparisons were performed by standard t-test.
    

Intervention name(s): Anakinra
Intervention type(s): Drug
Intervention description(s): Patients will receive daily anakinra therapy for 28 days
Other intervention name(s): Kineret
Intervention MeSH terms: Interleukin 1 Receptor Antagonist Protein
Intervention model: Single Group Assignment
Masking: None (Open Label)
Primary purpose: Treatment
Keywords: Type 1 diabetes mellitus; IL1 beta; Anakinra