Received: 26 October 2020
-
Revised: 31 December 2020-
Accepted: 11 January 2021 DOI: 10.1002/dmrr.3440R E S E A R C H A R T I C L E
Combined Etanercept, GAD‐alum and vitamin D treatment:
an open pilot trial to preserve beta cell function in recent
onset type 1 diabetes
Johnny Ludvigsson
1| Indusmita Routray
2| Tore Vigård
3,4| Ragnar Hanås
5,6|
Björn Rathsman
7| Annelie Carlsson
8,9| Stefan Särnblad
10| Anna‐Karin Albin
11|
Carl‐Göran Arvidsson
12| Ulf Samuelsson
1| Rosaura Casas
21Department of Biomedical and Clinical Sciences, Crown Princess Victoria Children´s Hospital and Division of Pediatrics, Linköping University, Linköping, Sweden 2Department of Biomedical and Clinical Sciences, Division of Pediatrics, Linköping University, Linköping, Sweden
3Department of Clinical Sciences, Pediatric Endocrinology, Lund University, Malmö, Sweden 4Department of Pediatrics, Skåne University Hospital, Malmö, Sweden
5Department of Pediatrics, NU Hospital Group, Uddevalla, Sweden
6Institute of Clinical Sciences, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden 7Sachska Pediatric Hospital, Södersjukhuset, Stockholm, Sweden
8Department of Clinical Sciences, Pediatric Autoimmunity, Lund University, Lund, Sweden 9Skåne University Hospital, Lund, Sweden
10Department of Pediatrics, University Hospital, Örebro, Sweden
11Department of Pediatrics, Helsingborg Central Hospital, Helsingborg, Sweden 12Department of Pediatrics, Västerås Central Hospital, Västerås, Sweden
Correspondence
Johnny Ludvigsson, Division of Pediatrics, Department of Biomedical and Clinical Sciences, Linköping University, Linköping, Sweden.
Email:Johnny.Ludvigsson@liu.se
Funding information
Barndiabetesfonden (Swedish Child Diabetes Foundation); Diabetesfonden (Swedish Diabetes Association); FORSS (the Research Council of Southeast Sweden); ALF (Region Östergötland); Diamyd Medical
Abstract
Aim: We aimed to study the feasibility and tolerability of a combination therapy
consisting of glutamic acid decarboxylase (GAD‐alum), Etanercept and vitamin D in
children and adolescents with newly diagnosed with type 1 diabetes (T1D), and
evaluate preservation of beta cell function.
Material and Methods: Etanercept Diamyd Combination Regimen is an open‐
labelled multi‐centre study pilot trial which enrolled 20 GAD antibodies positive
T1D patients (7 girls and 13 boys), aged (mean ±SD): 12.4 ± 2.3 (8.3–16.1) years,
with a diabetes duration of 81.4 ± 22.1 days. Baseline fasting C‐peptide was
0.24 ± 0.1 (0.10–0.35) nmol/l. The patients received Day 1‐450 Vitamin D
(Calciferol) 2000 U/d per os, Etanercept sc Day 1‐90 0.8 mg/kg once a week and
GAD‐alum sc injections (20 μg, Diamyd™) Day 30 and 60. They were followed for
30 months.
Results: No treatment related serious adverse events were observed. After
6 months 90‐min stimulated C‐peptide had improved in 8/20 patients and C‐peptide
This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.
© 2021 The Authors. Diabetes/Metabolism Research and Reviews published by John Wiley & Sons Ltd.
Diabetes Metab Res Rev. 2021;e3440. wileyonlinelibrary.com/journal/dmrr
-
1 of 10area under the curve (AUC) after Mixed Meal Tolerance Test in 5 patients, but
declined thereafter, while HbA1c and insulin requirement remained close to
base-line. Administration of Etanercept did not reduce tumour necrosis factor (TNF)
spontaneous secretion from peripheral blood mononuclear cells, but rather GAD65‐
induced TNF‐α increased. Spontaneous interleukin‐17a secretion increased after
the administration of Etanercept, and GAD65‐induced cytokines and chemokines
were also enhanced following 1 month of Etanercept administration.
Conclusions: Combination therapy with parallel treatment with GAD‐alum,
Eta-nercept and vitamin D in children and adolescents with type 1 diabetes was feasible
and tolerable but had no beneficial effects on the autoimmune process or beta cell
function.
K E Y W O R D S
C‐peptide, Etanercept, GAD‐alum, immune intervention, type 1 diabetes, vitamin D
1
|
INTRODUCTION
In spite of a modern devices and drugs, the treatment of type 1 diabetes is heavy and the disease cause complications and increased mortality.1,2Residual insulin secretion facilitates treatment, improves
metabolic control, decreases the risk of both acute and long‐term complications.3,4 Some immune interventions have shown limited
efficacy to preserve residual beta cell function,5‐13but the effect has been transient and some treatments have caused risks and adverse events. Autoantigen treatment could be a way to get a more specific and long‐lasting effect with less adverse events. Glutamic acid decarboxylase (GAD‐alum) treatment has been easy for both patients and staff, tolerable and without treatment‐related adverse events. Efficacy was found in a Phase II trial using two GAD‐alum sc in-jections,14but the treatment failed in another Phase II trial with a
different age range of patients and three sc injections.15In a
Euro-pean Phase III trial, GAD‐alum treatment failed to reach primary endpoint but efficacy was seen in pre‐specified subgroups.16Swine
influenza epidemic and accompanying vaccination parallel to the GAD‐alum treatment seemed to contribute to lack of efficacy,17and patients in the European Phase III trial, who had already been followed for 30 months when the trial was interrupted, and got no swine flu vaccination in connection with the GAD‐alum treatment, showed significant efficacy.18A meta‐analyses showed >97%
prob-ability that GAD‐alum does preserve residual beta cell function.19
We decided to conduct a series of pilot experiments to see if the GAD‐alum efficacy could be improved. We have earlier reported a combination therapy using GAD‐alum sc, combined with vitamin D and Ibuprofen.20 In that study, we found support for the use of
vitamin D, while addition of a short period with Ibuprofen did not improve efficacy. Here we report results from the Etanercept Diamyd Combination Regimen (EDCR) study where we used the same GAD‐ alum treatment and oral vitamin D, but instead of Ibuprofen we tried combination with the tumour necrosis factor‐α (TNF‐α)—inhibitor Etanercept. Vitamin D is said to cause Th2 deviation, improve
dendritic cell function, increase T‐regulatory cells, and perhaps also protect beta cells and improve insulin sensitivity.21,22TNF‐α‐
inhibition should be justified as TNF‐α is involved in the autoimmune process leading to beta cell destruction.23,24
Etanercept is a recombinant soluble TNF‐α receptor fusion protein that binds to TNF‐α. It acts by clearing TNF‐α from the circulation, thereby blocking the biological activity of this inflamma-tory cytokine. Although Etanercept is used in the treatment of many autoimmune diseases including ankylosing spondylitis, juvenile rheu-matoid arthritis, psoriasis, psoriatic arthritis and rheurheu-matoid arthritis,25,26it has to our knowledge only been tested once earlier in
young people with type 1 diabetes (T1D).27In that study, the beta cell
function was preserved for 6 months and the percent change in C‐peptide area under the curve from baseline to Week 24 showed a 39% increase in the Etanercept group compared to a 20% decrease in the placebo group (p < 0.05), while insulin dose decreased significantly more, as well as HbA1c, in the treatment group compared to placebo. With this background, we found it reasonable to try a combi-nation therapy with GAD‐alum sc, vitamin D per os and Etanercept given sc. with the aim to see if such a therapy was feasible, safe and tolerable, and efficacious enough to justify possible risks.
2
|
PATIENTS AND DESIGN
The study was an one‐arm, open‐label multi‐centre, clinical trial. No sample size calculation was done for this pilot study where we aimed to get only 20 patients included. The following inclusion criteria had to be fulfilled: T1D according to the American Diabetes Association (ADA) classification with <100 days diabetes duration at time of screening, age 8.00–17.99 years at time of screening, fasting serum C‐peptide at time of screening ≥ 0.12 nmol/L and positivity for glutamic acid decarboxylase GAD65‐autoantibodies (GADA) but < 50,000 Random Units. At the screening visit, all patients were screened for tuberculosis according to established Swedish and
T A B L E 1 Baseline characteristics
Variable Response category Group (n = 20)
Gender Female 7
Male 13
Age (years) Mean (SD) 12.36 (2.3)
Median 12.4
Min 8.3
Max 16.1
T1D duration (days) Mean (SD) 81.3 (22.1)
Median 83 Min 44 Max 118 Weight (kg) Mean (SD) 45.69 (12.1) Median 43.05 Min 27.5 Max 72.3 Height (cm) Mean (SD) 156.1 (14.5) Median 155.7 Min 133.8 Max 182.7 BMI (kg/m2) Mean (SD) 18.4 (2.1) Median 18.2 Min 15.36 Max 21.9
Fasting glucose (mmol/L) Mean (SD) 6.29 (1.17)
Median 5.8
Min 4.4
Max 8.6
HbA1c unadjusted (mmol/mol) Mean (SD) 45.7 (7)
Median 48.5
Min 35
Max 55
HbA1c adjusted Mean (SD) 8.45 (0.7)
Median 8.7
Min 6.6
Max 9.3
Average insulin dose/kg/24 h (IU) Mean (SD) 0.53 (0.15)
Median 0.51
Min 0.23
Max 0.87
international guidelines. Patients with active or inactive (latent) tuberculosis should be excluded. The evaluation included a detailed medical history regarding previous potential exposure for tubercu-losis, recent travel to countries with possible high exposure to tuberculosis and previous and/or current immunosuppressive treat-ments. Pulmonary X‐ray as well as QuantiFeron®test was performed.
All patients were also tested for hepatitis B and C. Patients with prior or active hepatitis B and C were excluded from the study.
Out of 47 patients screened at eight Swedish paediatric clinics 20 were eligible (FigureS1). Clinical characteristics at baseline are shown in Table 1. The patients got from Day 1 to 450 Vitamin D (Calciferol) 2000 U/d per os; Day 1‐90 Etanercept (TNF‐α inhibitor) 0.8 mg/kg body weight (max 50 mg) given sc once a week; and 20 μg Diamyd™ sc at Day 30 and 60. During the Etanercept treatment the patients were carefully observed.
All patients and their parent(s)/legal guardian(s) were informed by the investigator and study team that they should seek medical help immediately in case the patient treated with Etanercept devel-oped signs and/or symptoms of blood dyscrasia or infections (long‐ lasting low‐grade fever, sore throat, bruises, bleedings, weight loss, unexpected productive cough lasting 2–3 weeks or more and/or paleness (TableS1).
The patients were followed for a total of 30 months. The primary endpoint was to evaluate the tolerability of this combination therapy with Diamyd, Vitamin D and Etanercept. Secondary endpoints were to evaluate how the above‐mentioned treatment influences the im-mune system and endogenous insulin secretion: C‐peptide (90 min value and AUCmean 0–120 min) during an Mixed Meal Tolerance Test
(MMTT), proportion of patients with a stimulated maximum C‐peptide level above 0.2 nmol/L, and fasting C‐peptide.
Haemoglobin A1c (HbA1c) and exogenous insulin dose per kg body weight and 24 h was also evaluated. Insulin‐adjusted HbA1c was used as a measure of partial remission28
3
|
LABORATORY TESTS
Laboratory analyses were performed at Linköping University, Sweden. Blood samples were drawn after fasting overnight. Pe-ripheral blood mononuclear cells (PBMCs) were isolated within
24 h using Leucosep (Greiner Bio One) according to the manu-facturer's instructions. Serum gel tubes were used for analysis of autoantibodies, and C‐peptide and EDTA‐tubes for measurement of HbA1c.
Serum C‐peptide was determined using a solid phase‐two side enzyme immunoassay (Mercodia), and results were validated with the inclusion of a Diabetes Antigen Control Human (Low/High) (Mercodia). Inter and intra assay variation were 7% and 4%, respectively.
Serum levels of GAD65 (GADA) and IA‐2A autoantibodies were estimated in duplicate with a radio‐binding assay using35S‐labelled
recombinant human GAD65 and IA‐2A as previously described.29
Sepharose protein A was used to separate free from antibody‐bound labelled GAD65 and IA‐2A. Levels were expressed as U/ml. Evalua-tion of the test performance at a diabetes autoantibody standardi-zation program (IASP) has shown that GADA assay has a sensitivity and specificity of 70 % and 100%, respectively, and for IA‐2A was 99% sensitivity and 100% specificity.29
Lymphocyte proliferation, and cytokines and chemokines secre-tion analysis were performed in samples from baseline and 1, 2, 3, 6, 9, 15 and 30 months.
Cytokines were quantified in PBMC supernatants, after cell culture for 7 days in the presence of 5 μg/ml recombinant human GAD65 (Diamyd Medical), CD3/CD28 beads (Gibco; Life Technolo-gies AS) or in medium (AIM‐V) alone at 37°C in 5% CO2. The cyto-kines interleukin IL‐1, IL‐2, IL‐4, IL‐5, IL‐6, IL‐7, IL‐10, IL‐13, IL‐17, IL‐12(P70), TNF‐α and interferon (IFN)‐γ were using Bio‐Plex Pro Cytokine Panel (Bio‐Rad) according to the manufacturer's in-structions. The chemokines G‐CSF, GM‐CSF, MCP‐1, MIP‐1 b and IL‐8 were quantified PBMC supernatants. Data were collected using the Luminex 200 ™ (Luminex xMAP™ Corporation). The antigen‐ induced cytokine secretion level was calculated by subtracting the spontaneous secretion (i.e., secretion from PBMC cultured in medium alone) from the one following stimulation with GAD65 or CD3/CD28 beads.
PBMC proliferative response was analysed in the presence of 5 μg/ml rhGAD65 (Diamyd Medical), CD3/CD28 beads (Gibco; Life Technologies AS) and in medium alone. Data were expressed as stimulation index, calculated as the mean of triplicates in presence of stimulus divided by the mean of triplicates with medium alone.
T A B L E 1 (Continued)
Variable Response category Group (n = 20)
Fasting C‐peptide (nmol/L) Mean (SD) 0.24(0.08)
Median 0.24
Min 0.1
Max 0.4
GADA (U/ml) Mean (SD) 957.2 (1810.8)
Median 157.6
4
|
STATISTICS
Demographics and baseline characteristics are presented using descriptive statistics. Efficacy data regarding C‐peptide and immune system as well as serious(S) adverse events (AE) and other safety data are also summarized descriptively. The AE/SAE are presented using a standardized tabulation of the frequency and incidence rate of all observed AEs/SAEs. The frequencies and incidence rates are calculated on a per patient basis. p‐values <0.05 were considered statistically significant.
5
|
RESULTS
The treatment was feasible, well tolerated, with no serious adverse events related to the treatment. AEs are shown in Table S2. No patient dropped out from the study.
Compared to baseline 90‐min stimulated C‐peptide improved in 8/20 patients after 6 months (Figure 1A). Of these patients, seven were ≥12 years old. One female patient aged ≥12 years had a mild disease with only 6% C‐peptide decrease from Baseline to Month 30. Otherwise the decreases were ≥43%.
C‐peptide AUC after MMTT increased up to 6 months in five patients, 4/5 > 12 years old (Figure 1B), but thereafter an age‐ independent consistent decline in C‐peptide values was seen. The female patient aged ≥12 years with a mild disease had only 2% C‐peptide decrease from baseline to Month 30); otherwise the decreases were ≥46%. The proportion of patients with a MMTT stimulated C‐peptide maximum level >0.2 nmol/L decreased consis-tently during the study: 100.0% at Baseline, 94.4% at Month 6, 70.0% at Month 15 and 42.1% at Month 30. The mean fasting C‐peptide values peaked at Month 1, declined thereafter and were above the Baseline level up to Month 6. The mean decreases from baseline were 0.02 nmol/L at Month 6, 0.10 nmol/L at Month 15 and 0.15 mmol/L at Month 30.
HbA1c reached lowest levels at Month 1, increased thereafter. The mean increases from baseline were 0.80 mmol/mol at Month 6, 6.15 mmol/mol at Month 15 and 7.55 mmol/mol at Month 30. The external insulin requirements (per kg body weight and 24 h) were lowest at Month 1, increased thereafter. The mean increases in in-sulin requirements were 0.01 IU at Month 6, 0.25 IU at Month 15 and 0.42 IU at Month 30. The insulin dose‐adjusted HbA1c was lowest Month 1, increased thereafter. The mean increases from baseline were 0.09 units at Month 6, 1.56 units at Month 15 and 2.37 units at Month 30. (TableS3). Vitamin D in serum is shown in TableS4.
F I G U R E 1 (A and B). C‐peptide after
Mixed Meal Tolerance tests from baseline during the 30 months follow‐up
5.1
|
Autoantibodies in serum
GADA autoantibody levels started to rise after the first injection of GAD‐alum, and were significantly higher at 3 months, after the sec-ond GAD‐alum dose. The levels started to wane thereafter, with a statistically significant reduction over the study (Figure2). Levels of IA‐2 were not affected by the treatment (data not shown).
5.2
|
Cytokines and chemokines secretion
Levels of cytokine and chemokine secretion in PBMC supernatants cultured in medium alone did not change along the study, with the exception of IL‐17 that was significantly increased between baseline and 3 months (Figure3A).
Following 1 month administration of Etanercept in‐vitro stimu-lation with GAD65 induced higher levels of IL‐4 (p = 0.03; Figure3B). IL‐4 further increased at 2 months, after the first GAD‐alum dose, but dropped following the second injection (Figure3B). Higher IL‐13 levels were also detected at 2 months (p = 0.03), but decreased after the second GAD‐alum dose (p = 0.03; Figure3C). Levels of GAD‐ induced IFN‐gamma and also TNF‐α were enhanced following administration of Etanercept and were higher at 1 month compared to baseline samples (p = 0.002 and p = 0.02, respectively) (Figure3D, E). Subcutaneous injections of GAD‐alum did not produce any further effect, and both cytokines decreased at 6 months compared to 1 month levels (p = 0.005 and p = 0.003, respectively; Figure3D,E). GAD65‐induced levels of the chemokines GM‐CSF and MIP were enhanced after 1 month administration of Etanercept (p = 0.02; Figure3F,G), but the first injection of GAD‐alum did not lead to a further increase. MCP‐1 started to increase at 1 month, and became higher than baseline at 3 months (p = 0.02; Figure3H). The chemo-kines started to wane after the second injection of GAD‐alum and were significantly lower at 6 months compared to 2 months levels (Figure3F–H).
Stimulation of samples with CD3/CD28 beads did not induce any change in cytokines and chemokines secretion along the study.
F I G U R E 2 Levels of glutamic acid decarboxylase (GAD65) from
baseline to 30 months. GAD antibodies (GADA) were measured in serum samples (n = 20) using 35S‐labelled recombinant human GAD65, and levels were expressed as U/ml. The significance differences were indicated as p values
F I G U R E 3 Cytokine and chemokine secretion in peripheral blood mononuclear cells (PBMCs). PBMC samples from the patients (n = 20)
collected at baseline (Day 1) and after 1, 2, 3, 6, 15 and 30 months were cultured for 7 days in in medium (AIM‐V) alone (spontaneous secretion) or in the presence of 5 μg/m rhGAD65 (Diamyd Medical) at 37°C in 5% CO2. Cytokines and chemokines were measured using Bio‐ Plex Pro Cytokine Panel (Bio‐Rad). GAD65‐induced levels were calculated by subtracting the spontaneous secretion, and levels were expressed as pg/ml. The significance differences are indicated as p values
5.3
|
Proliferative response to GAD65
GAD65‐induced proliferation was not affected by GAD‐alum in-jections. The low proliferation observed at baseline decreased along the study, and it was lower at 30 months than baseline (Figure4A). No changes were observed in CD3/CD28‐induced proliferation (data not shown)
6
|
GADA IN RELATION TO C‐PEPTIDE
PRESERVATION
We stratified patients according to the preservation of c‐peptide at 15 months: (i) good responders (GR): patient with loss of C‐peptide AUC≤ 30% (n = 5, Figure 4B, blue colour) (GR) and (ii) poor responders (PR): patient with loss in c‐peptide AUC> 30% (n = 15, Figure 4B, red colour) GADA titres increased after GAD‐alum treatment both in GR and PR, but GADA was significantly higher in the GR individuals at 3 months. No other association between C‐peptide and immune responses was observed.
7
|
DISCUSSION
The combination treatment including Etanercept, an effective TNF‐α‐ inhibitor, was well tolerated and gave no treatment‐related SAEs. However, even though we have no controls, we dare to conclude that a 3‐month treatment period with adequate doses of Etanercept, a TNF‐ α‐inhibitor, given parallel to sc GAD‐alum plus vitamin D did not lead to any substantial preservation of C‐peptide, as had been seen in a pre-vious 6‐month trial,27and this type of combination therapy did not
improve GAD‐alum therapy. Somewhat surprisingly we got no decrease of TNF‐α concentrations and instead of the usual Th2‐devi-ation we are used to see in connection with GAD‐alum therapy,18,20,29
the increase of IL4 and IL‐3 was very transient, but instead we noticed a stable increase of IL‐17. Thus, we observed that spontaneous secretion of IL‐17a started to increase after the administration of Etanercept, and it remained higher throughout the study. It has been shown that treatment with Etanercept inhibited Th‐17 cells and IL‐17a levels in psoriasis patients.30In contrast, anti‐TNF therapy
(adalimu-mab) of patients with rheumatoid arthritis induced both Th17‐cells and IL‐17 secretion, irrespectively of disease activity,31suggesting a
different disease‐related outcome. The pathogenic contribution of Th17 cells with T1D progression of T1D has been suggested by several studies,32,33and circulation of Th17 lymphocytes was observed in
peripheral blood of long‐standing T1D patients.34 Enhanced IL‐17
expression in the pancreas of newly diagnosed T1D patients35and
increased expression of IL‐17 genes on circulating lymphocytes of T1D patients have been also reported.36It was interesting that
adminis-tration of Etanercept in our study did not reduce TNF‐α spontaneous secretion from PBMC in T1D, but on the contrary GAD65‐induced TNF‐α increased 1 month after Etanercept treatment. Interleukin‐17, which increased, initiates inflammation by enhancing production of pro‐inflammatory cytokines such as TNF‐α, IL‐1β and IL‐6.37It has
been shown that circulating CD4+T cells from T1D patients produced
IL‐17 when activated by β‐cell autoantigens including proinsulin, insulinoma‐associated protein and GAD65 peptides.35,38
We have previously shown that GAD65‐recall response of PBMC from T1D patients who received subcutaneous administra-tion of GAD‐alum was characterized by the secreadministra-tion of several cytokines, including IL‐5, IL‐10, IL‐13, IL‐17, IFN‐g and TNF‐a.14In
the present study, increase of the levels of GAD65‐induced
F I G U R E 4 (A) Glutamic acid decarboxylase (GAD65)‐induced peripheral blood mononuclear cells (PBMCs) proliferation. PBMC samples
from the patients (n = 20) collected at baseline (Day 1) and after 1, 2, 3, 6, 15 and 30 months were cultured in in medium (AIM‐V) alone (spontaneous secretion) or in the presence of 5 μg/m rhGAD65 (Diamyd Medical) at 37°C in 5% CO2. GAD65‐induced proliferation was expressed as stimulation index, calculated from the mean of triplicates in the presence of stimulus divided by the mean of triplicates with medium alone. Significant differences are indicated as p‐values. (B) Levels of glutamic acid decarboxylase GAD65 and c‐peptide preservation. GADA levels from baseline to 30 months were compared in patients stratified patients according to the preservation of c‐peptide at 15 months: good responders: patient with loss of c‐peptide AUC ≤ 30% (n = 5, blue colour) and poor responders: patient with loss in c‐peptide AUC > 30% (n = 15, red colour). Significant differences are indicated as p‐values
cytokines and chemokines was observed already after 1‐month treatment with Etanercept, before the first injection of GAD‐alum. Interestingly, PBMC proliferation was not enhanced after treatment, but instead it waned over time, and was lower after 30 months compared to baseline. This is in strong contrast with previous studies showing enhanced GAD65‐induced proliferation as part of the effect of subcutaneous administration of GAD‐alum injections.39 Administration of Etanercept seems to induce activation of existing GAD‐specific circulating CD4+ T cells, as has been shown in a
previous study.35 Parallel administration of Etanercept and GAD‐
alum seems to prevent any further induction of GAD‐specific T cell response.
Induction of GADA titres in our study suggests that combination of Etanercept and GAD‐alum acts in different ways on the T and B cells specific responses. The few individuals with better preservation of c‐peptide after 15 months had higher levels of GADA, while no association between C‐peptide preservation and any other analysed marker was observed.
7.1
|
Limitations of the study
Although studies in experimental animals can give valuable infor-mation, such studies cannot replace clinical studies in humans when we want to know how to preserve residual beta cell function in humans. Neither can studies in adults replace studies in children and adolescents as the T1D disease differ depend on age.40 However,
when using new combination therapies, with possible risks the number of included children has to be kept as low as possible. Treatment with Etanercept in children means addition of more in-jections and could increase the risk for serious infections. Thus, for ethical reasons, we chose to do a pilot trial in a small group of pa-tients, with deliberately no power to show clear efficacy unless this would be pronounced. For safety reasons, we chose not to do a double‐blind placebo‐controlled trial, but an open pilot trial without controls, which may be justified in early phase pilot trials.41,42Our
small pilot trial did give results clear enough regarding the lack of efficacy of a short treatment period with Etanercept given parallel to a GAD‐alum/auto‐antigen treatment. Thus, we are able to add a piece of the puzzle regarding the design of future trials. The regimen we used can be avoided. However, whether another design, with Etanercept and GAD‐alum/autoantigen given at different time‐ points, separated from each other, would give another result cannot be answered from our trial. As said in another paper43‘progress in
medicine is, at best, two steps forward and one step back, and progress towards halting or preventing T1D has been no different’. We agree. Our results are just one small piece of the puzzle.
In summary, the combination of GAD‐alum, vitamin D and Etanercept was safe and tolerable. However, patients in the study did not go into pronounced or long remission and the beta cell function improved just in a minority of patients during the first 6 months but thereafter declined. The immune response did not suggest mitigation
of the immune process. We can conclude that 3 months addition of sc Etanercept parallel to sc treatment with GAD‐alum and oral vitamin D can be ruled out as a promising therapy.
A CKNOWLEDGEM ENTS
We are grateful for the important work done by diabetes research nurses at the participating clinics, to paediatricians who have helped with seeing some patients at certain visits and laboratory staff such as Ingela Johansson and Gosia Smolinska. This trial was generously funded by Barndiabetesfonden (Swedish Child Diabetes Foundation), Diabetesfonden (Swedish Diabetes Association), FORSS (the Research Council of Southeast Sweden), ALF (Region Östergötland) and unrestricted grants from Diamyd Medical.
C O N F L I C T O F I N T ER E ST
None of the authors has anything to disclose or any financial interest in the products studied.
E T H IC A L A P P R O V A L
The study (EudraCT: 2014‐001323‐76; Clin Gov NCT 02464033) was approved by Medical Product Agency in Sweden and by the Research Ethics Committee, Linköping (Dnr 2014/148‐31). All patients and their parents/caregivers gave their consent after oral and written information.
AUT HOR CONTRIB UTION S
Johnny Ludvigsson had the idea, designed the EDCR study, was coordinating investigator and sponsor, conceived the study, and wrote the first draft of the manuscript. Indusmita Routray and Rosaura Casas performed experiments and analysed data. Rosaura Casas conceived the study, designed data set and data analysis. Tore Vigård, Ragnar Hanås, Björn Rathsman, Annelie Carlsson, Stefan Särnblad, Anna‐Karin Albin, Carl‐Göran Arvidsson, Ulf Samuelsson and Johnny Ludvigsson recruited and followed patients. All the authors read and approved the final version of the manuscript. Johnny Ludvigsson and Rosaura Casas are guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data, and the accuracy of the data analysis.
DA TA AVA ILA BLITY STA TEM ENT
The data sets are available from the corresponding author on reasonable request.
ORCID
Johnny Ludvigsson https://orcid.org/0000-0003-1695-5234
R EFERENCES
1. Bojestig M, Arnqvist HJ, Hermansson G, Karlberg BE, Ludvigsson J. Declining incidence of nephropathy in insulin‐dependent diabetes mellitus. N Engl J Med. 1994;330(1):15‐18. https://doi.org/10.1056/ nejm.199401063300103.
2. Lind M, Svensson AM, Kosiborod M, et al. Glycemic control and excess mortality in type 1 diabetes. N Engl J Med. 2014;371:1972‐ 1982. https://doi.org/10.1056/NEJMoa1408214.
3. Madsbad S, Alberti KG, Binder C, et al. Role of residual insulin secretion in protecting against ketoacidosis in insulin‐dependent diabetes. Br Med J. 1979;2:1257‐1259. https://doi.org/10.1136/ bmj.2.6200.1257.
4. Steffes MW, Sibley S, Jackson M, Thomas W. Beta‐cell function and the development of diabetes‐related complications in the diabetes control and complications trial. Diabetes Care. 2003;26(3):832‐836. https://doi.org/10.2337/diacare.26.3.832.
5. Stiller CR, Laupacis A, Dupre J, et al. Cyclosporine for treatment of early type I diabetes: preliminary results. N Engl J Med. 1983; 308(20):1226‐1227.
6. Coutant R, Landais P, Rosilio M, et al. Low dose linomide in type I juvenile diabetes of recent onset: a randomized placebo‐controlled double‐blind trial. Diabetologia. 1998;41(9):1040‐1046. https://doi. org/10.1007/s001250051028.
7. Herold KC, Hagopian W, Auger JA, et al. Anti‐CD3 monoclonal antibody in new‐onset type 1 diabetes mellitus. N Engl J Med. 2002;346(22):1692‐1698. https://doi.org/10.1056/NEJMoa012864. 8. Keymeulen B, Vandemeulebroucke E, Ziegler AG, et al. Insulin needs after CD3‐antibody therapy in new‐onset type 1 diabetes. N Engl J
Med. 2005;352(25):2598‐2608. https://doi.org/10.1056/NEJMoa 043980.
9. Sherry N, Hagopian W, Ludvigsson J, et al. Teplizumab for treatment of type 1 diabetes (Protege study): 1‐year results from a random-ized, placebo‐controlled trial. Lancet. 2011;378(9790):487‐497. https://doi.org/10.1016/S0140-6736(11)60931-8.
10. Hagopian W, Ferry RJ, Jr, Sherry N, et al. Teplizumab preserves C‐peptide in recent‐onset type 1 diabetes: two‐year results from the randomized, placebo‐controlled. Protégé Trial. Diabetes.
2013;62(11):3901‐3908. https://doi.org/10.2337/db13-0236. 11. Pescovitz MD, Greenbaum CJ, Krause‐Steinrauf H, et al. Type 1
diabetes trial Net anti‐CD20 study group. Rituximab, B‐lymphocyte depletion, and preservation of beta‐cell function. N Engl J
Med. 2009;361(22):2143‐2152. https://doi.org/10.1056/NEJMoa0
904452.
12. Rigby MR, Harris KM, Pinckney A, et al. Alefacept provides sus-tained clinical and immunological effects in new‐onset type 1 dia-betes patients. J Clin Invest. 2015;125(8):3285‐3296. https://doi.org/ 10.1172/JCI81722.
13. Haller MJ, Gitelman SE, Gottlieb PA, et al. Anti‐thymocyte globulin/ G‐CSF treatment preserves β cell function in patients with estab-lished type 1 diabetes. J Clin Invest. 2015;125(1):448‐455. https:// doi.org/10.1172/JCI78492.
14. Ludvigsson J, Faresjo M, Hjorth M, et al. GAD treatment and insulin secretion in recent‐onset type 1 diabetes. N Engl J Med. 2008; 359(18):1909‐1920. https://doi.org/10.1056/NEJMoa0804328. 15. Wherrett DK, Bundy B, Becker DJ, et al. Antigen‐based therapy with
glutamic acid decarboxylase (GAD) vaccine in patients with recent‐ onset type 1 diabetes: a randomised double‐blind trial. Lancet. 2011;378(9788):319‐327. https://doi.org/10.1016/S0140-6736(11) 60895-7.
16. Ludvigsson J, Krisky D, Casas R, et al. GAD65 antigen therapy in recently diagnosed type 1 diabetes mellitus. N Engl J Med. 2012;366(5):433‐442. https://doi.org/10.1056/nejm.oa1107096. 17. Tavira B, Cheramy M, Axelsson S, Åkerman L, Ludvigsson J, Casas R.
Effect of simultaneous vaccination with H1N1 and GAD‐alum on GAD65‐induced immune response. Diabetologia. 2017;60:
1276‐1283. https://doi.org/10.1007/s00125-017-4263-x.
18. Ludvigsson J, Chéramy M, Axelsson S, Phil M, Åkerman L, Casas R. GAD‐treatment of children and adolescents with recent‐onset type 1 diabetes preserves residual insulin secretion after 30 months.
Diabetes Metab Res Rev. 2013;30(5):405‐414. https://doi.org/
10.1002/dmrr.2503.
19. Beam CA, MacCallum C, Herold KC, Wherrett DK, Palmer J, Lud-vigsson J. Type 1 Diabetes TrialNet Study Group. GAD vaccine re-duces insulin loss in recently diagnosed type 1 diabetes: findings from a Bayesian meta‐analysis. Diabetologia. 2017;60(1):43‐49. https://doi.org/10.1007/s00125-016-4122-1.
20. Ludvigsson J, Routray I, Elluru S, et al. Combined vitamin D, ibuprofen and glutamic acid decarboxylase‐alum treatment in recent onset Type I diabetes: lessons from the DIABGAD randomized pilot trial. Future Sce OA. 2020;6(7):FSO604. https://doi.org/10.2144/ fsoa-2020-0078.
21. Piemonti L, Monti P, Sironi M, et al. Vitamin D3affects
differentia-tion, maturadifferentia-tion, and function of human monocyte‐derived dendritic cells. J Immunol. 2000;164(9):4443‐4451. https://doi.org/10.4049/ jimmunol.164.9.4443.
22. Mathieu C, Gysemans C, Giulietti A, Bouillon R. Vitamin D and diabetes. Diabetologia. 2005;48:1247‐1257. https://doi.org/10.1007/ s00125-005-1802-7.
23. Mandrup‐Poulsen T, Zumsteg U, Reimers J, et al. Involvement of interleukin 1 and interleukin 1 antagonist in pancreatic β‐ cell destruction in insulin‐dependent diabetes mellitus. Cytokine. 1993; 5(3):185‐191. https://doi.org/10.1016/1043-4666(93)90003-N. 24. Argiles JM, Lopez‐Soriano J, Lopez‐Soriano FJ. Cytokines and
dia-betes: the final step? Involvement of TNF‐α in both type I and II diabetes mellitus. Horm Metab Res. 1994;26(10):447‐449. https:// doi.org/10.1055/s-2007-1001730.
25. Weisman MH, Paulus HE, Burch FX, et al. A placebo‐controlled, randomized, double‐blinded study evaluating the safety of eta-nercept in patients with rheumatoid arthritis and concomitant comorbid diseases. Rheumatology. 2007;46(7):1122‐1125. https:// doi.org/10.1093/rheumatology/kem033.
26. Braun J, McHugh N, Singh A, Wajdula JS, Sato R. Improvement in patient‐reported outcomes for patients with ankylosing spondylitis treated with etanercept 50 mg once‐ weekly and 25 mg twice‐ weekly. Rheumatology. 2007;46(6):999‐1004. https://doi.org/ 10.1093/rheumatology/kem069.
27. Mastrandrea L, Yu J, Behrens T, et al. Etanercept treatment in children with new‐onset type 1 diabetes: pilot randomized, placebo‐ controlled, double‐blind study. Diabetes Care. 2009;32(7): 1244‐1249. https://doi.org/10.2337/dc09-0054.
28. Mortensen HB, Hougaard P, Swift P, et al. New definition for the partial remission in children and adolescents with type 1 diabetes.
Diabetes Care. 2009;31(8):1384‐1390. https://doi.org/10.2337/ dc08-1987.
29. Tavira B, Barcenilla H, Wahlberg J, Achenbach P, Ludvigsson J, Casas R. Intralymphatic glutamic acid decarboxylase‐alum administration induced Th2‐like‐spe cific immunomodulation in responder patients: a pilot clinical trial in type 1 diabetes. J Diabetes Res. 2018;2018: 9391845. https://doi.org/10.1155/2018/9391845.
30. Zaba LC, Cardinale I, Gilleaudeau P, et al. Amelioration of epidermal hyperplasia by TNF inhibition is associated with reduced Th17 responses. J Exp Med. 2007;204(13):3183‐3194. https://doi.org/ 10.1084/jem.20071094.
31. Aerts NE, De Knop KJ, Leysen J, et al. Increased IL‐17 production by peripheral T helper cells after tumour necrosis factor blockade in rheumatoid arthritis is accompanied by inhibition of migration associated chemokine receptor expression. Rheumatology.
2010;49(12):2264‐2272. https://doi.org/10.1093/rheumatology/ keq224.
32. Bellemore SM, Nikoopour E, Krougly O, Lee‐Chan E, Fouser LA, Singh B. Pathogenic T helper type 17 cells contribute to type 1 diabetes independently of interleukin‐22. Clin Exp Immunol. 2015;183(3):380‐388. https://doi.org/10.1111/cei.12735.
33. Abdel‐Moneim A, Bakery HH, Allam G. The potential pathogenic role of IL‐17/Th17 cells in both type 1 and type 2 diabetes mellitus.
Biomed Pharmacother. 2018;101:287‐292. https://doi.org/10.1016/j.
biopha.2018.02.103.
34. Bradshaw EM, Raddassi K, Elyaman W, et al. Monocytes from pa-tients with type 1 diabetes spontaneously secrete proinflammatory cytokines inducing Th17 cells. J Immunol. 2009;183(7):4432‐4439. https://doi.org/10.4049/jimmunol.0900576.
35. Arif S, Moore F, Marks K, et al. Peripheral and islet interleukin‐17 pathway activation characterizes human autoimmune diabetes and promotes cytokine‐mediated beta‐cell death. Diabetes. 2011;60(8): 2112‐2119. https://doi.org/10.2337/db10-1643.
36. Kumar P, Natarajan K, Shanmugam N. High glucose driven expres-sion of pro‐inflammatory cytokine and chemokine genes in lym-phocytes: molecular mechanisms of IL‐17 family gene expression.
Cell Signal. 2014;26(3):528‐539. https://doi.org/10.1016/j. cellsig.2013.11.031.
37. Katz Y, Nadiv O, Beer Y. Interleukin‐17 enhances tumor necrosis factor α‐induced synthesis of interleukins 1, 6, and 8 in skin and synovial fibroblasts : a possible role as a “fine‐tuning cytokine” in inflammation processes. Arthritis Rheum 2001;44(9):2176‐2184. https://doi.org/10.1002/1529-0131(200109)44:9<2176::AID-ART 371>3.0.CO;2-4.
38. Li Y, Liu Y, Chu CQ. Th17 cells in Type 1 diabetes: role in the pathogenesis and regulation by gut microbiome. Mediat Inflamm. 2015;2015:638470. https://doi.org/10.1155/2015/638470. 39. Axelsson S, Cheramy M, Åkerman L, Pihl M, Ludvigsson J, Casas R.
Cellular and humoral immune responses in type 1 diabetic patients participating in a phase III GAD‐alum intervention trial. Diabetes
Care. 2013;36(11):3418‐3424. https://doi.org/10.2337/dc12-2251.
40. Ludvigsson J, Carlsson A, Deli A, et al. Decline of C‐peptide during the first year after diagnosis of Type 1 diabetes in children and adolescents. Diabetes Res Clin Pract. 2013;100(2):203‐209.https:// doi.org10.1016/j.diabres.2013.03.003.
41. Haller MJ, Wasserfall CH, McGrail KM, et al. Autologous umbilical cord blood transfusion in very young children with type 1 diabetes.
Diabetes Care. 2009;32(11):2041‐2046. https://doi.org/10.2337/
dc09-0967.
42. Ludvigsson J, Tavira B, Casas R. More on intralymphatic injection of autoantigen in type 1 diabetes. N Engl J Med. 2017;377(4):403‐405. https://doi.org/10.1056/nejm.c1703468.
43. Leslie RD, Jerram ST. Looking back and looking forward: a brief history of research into type 1 diabetes. Diabetes Metab Res Rev. 2019 Sep;35(6):e3196. https://doi.org/10.1002/dmrr.3196.
S U P P O R T I N G I N F O R M A TI O N
Additional supporting information may be found online in the Sup-porting Information section at the end of this article.
How to cite this article: Ludvigsson J, Routray I, Vigård T,
et al. Combined Etanercept, GAD‐alum and vitamin D treatment: An open pilot trial to preserve beta cell function in recent onset type 1 diabetes. Diabetes Metab Res Rev. 2021; e3440.https://doi.org/10.1002/dmrr.3440
