Patients with recent-onset type 1 diabetes were referred for participation in the study from hospitals or outpatient clinics in Bavaria, Germany, between November 2000 and 2006. They were selected according to the following criteria: they were 18–39 years of age, had been treated with insulin for less than 2 months (62 days), had a positive result on testing for islet autoantibodies (anti-GAD antibodies or anti-IA-2 antibodies), had plasma levels of calcium, phosphate, alkaline phosphatase, and creatinine within the normal ranges, and were compliant with insulin treatment. Exclusion criteria were disorders in calcium metabolism, kidney diseases, malignancy, and arterial hypertension. Pregnant or lactating women were excluded, and female patients with child-bearing potential had to practice an acceptable contraceptive technique from enrollment until 30 days after the last dose of study drug. Written informed consent was obtained from each patient. The study was conducted at the Diabetes Research Institute, Munich, Germany, and was approved by the ethics committee of the Medical Faculty at the Ludwig-Maximilians University, Munich, Germany.
The study had two phases. The first was designed to assess safety at the dose selected for investigation. It was an open study that included 25 patients (median age ± SD, 31.2 ± 7.3 years; 14 men) who received treatment with 0.25 μg 1,25(OH)2D3 as Rocaltrol (F. Hoffmann-La Roche, Basel, Switzerland) daily at breakfast for 9 months and were followed for a total of 18 months (9 additional months after treatment). Safety parameters (plasma levels of calcium, phosphate, alkaline phosphatase, and creatinine and urinary calcium excretion) were measured at study entry and at 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, and 18 months. Kidney sonography was performed at study entry and at 9 and 18 months.
The second phase was subsequently performed to assess the effect of the study drug on residual β-cell function via a phase II, monocenter, randomized, double-blind, placebo-controlled study. Forty-one patients were screened, and 40 of these were randomly assigned to oral 0.25 μg 1,25(OH)2D3 (n = 22; median age ± SD 31.4 ± 6.8 years; 16 men; median days after diagnosis 35.0) or oral placebo (n = 18; age 24.0 ± 6.0 years; 13 men; median days after diagnosis 40.0) daily at breakfast over a 9-month period. Randomization was performed by an independent pharmacy (Bastei-Apotheke, Munich, Germany), and the study medication was sent coded to the Diabetes Research Institute. Patients returned to the study center to receive their study drug supply and have efficacy and safety assessments performed at 0, 1, 2, 3, 4, 6, 7, 9, 12, and 18 months for safety, A1C, and insulin dose, and at 0, 9, and 18 months for a mixed-meal tolerance test (MMTT) and kidney ultrasound.
In both phases of the study, patients continued their normal insulin regimen as intensive insulin therapy, unless changes were clinically indicated. To avoid possible confounding through differences in glycemic control among the groups, diabetes management and glycemic targets were standardized as much as possible in all patients. Patients with A1C levels >8% had additional contacts with the study investigator to improve their metabolic control.
Safety parameters included laboratory tests (plasma calcium, phosphate, creatinine, and alkaline phosphatase and urinary calcium excretion in 24 h), ultrasound of the kidneys at baseline, month 9 and month 18, and documentation of adverse events and concomitant medication. Laboratory tests were performed centrally at the Institute for Clinical Chemistry of the Klinikum Schwabing using accredited methods. 1,25(OH)2D3 concentration was measured in an accredited laboratory by radioimmunoassay (MVZ Prof. Seelig, Karlsruhe, Germany).
End point assessment during second phase
The primary efficacy variable was the area under the curve (AUC0–120 min
) C-peptide (AUC C-peptide) of a MMTT at 18 months. The MMTT (Boost High Protein from Novartis in an amount of 360 ml) was administered to collect stimulated C-peptide samples (0, 30, 60, 90, and 120 min) at baseline, at month 9 immediately after the end of treatment, and at the final visit at month 18. Secondary efficacy variables were the peak C-peptide after MMTT at 18 months, daily insulin intake, and glycemic control, assessed by A1C levels. C-peptide concentrations were measured in Trasylol-stabilized EDTA plasma samples using an automated immunoassay analyzer (AIA 360; Tosoh, San Francisco, CA). The interassay coefficient of variation of the C-peptide assay is 4.2% at a concentration of 0.71 nmol/l and the lower limit of detection is 0.07 nmol/l. A1C was measured centrally by high-pressure liquid chromatography. Antibodies to insulin, GAD65, IA-2, and ZnT8 were measured by radiobinding assay as described previously (18
). All measurements were performed with the operator blinded to group assignment.
For the analysis of safety, data from the first (safety) and second (efficacy) component of the study were combined, and all subjects who had at least one baseline and postbaseline measurement of safety parameters were included. For the analysis of efficacy, all randomly assigned patients who had taken at least one dose of study drug and had some postbaseline data for the primary efficacy parameter (AUC C-peptide) were included. Comparisons between groups and between baseline and follow-up time points were made using parametric tests (t test). With the assumption that the AUC C-peptide values would decrease by 40% over 18 months in the placebo-treated group, the efficacy phase of the study had 50% power to detect 50% preservation of AUC C-peptide after 18 months. All hypotheses testing was two-sided and performed at the 5% significance level.