Study design

This trial is a single-centre, randomised, double-blinded, crossover study in patients with HNF1A-diabetes (figure 1). The study is intended to show superiority of glimepiride+linagliptin in regard to the primary end point.

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Figure 1

Trial design. Patients with hepatocyte nuclear factor 1α-diabetes will be randomly assigned to one of two double-blinded treatment sequences: (1) 16 weeks of treatment with glimepiride+linagliptin, 4 weeks of washout and 16 weeks of treatment with glimepiride+placebo (blue boxes); and (2) 16 weeks of treatment with glimepiride+placebo, 4 weeks of washout and 16 weeks of treatment with glimepiride+linagliptin (green boxes). Evaluation of the glycaemic variability will be assessed by continuous glucose monitoring (CGM) and meal and bicycle test in weeks 0, 16 and 36. For other investigations during the study, see text for more details.

Study participants

A total of 20 patients with HNF1A-diabetes will be enrolled. Patients will be recruited from the diabetes outpatient clinics at Gentofte Hospital and Steno Diabetes Center Copenhagen, Denmark or by letter to patients registered in a database at Steno Diabetes Center Copenhagen. All trial activities will be executed at Clinical Metabolic Physiology, Steno Diabetes Center Copenhagen, Gentofte Hospital, Hellerup, Denmark.

The total number of participants in the following categories will be recorded: invited, excluded before screening (including reasons for exclusion), declined to participate, not reached, screened, excluded at screening, randomised, completed intervention period, withdrew/dropped out during the study period (including reason for withdrawal/dropping out), lost to follow-up and analysed. Eligible participants will receive written and oral information about study. Sufficient time for reflection will be given before written informed consent is obtained as according to The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Good Clinical Practice (GCP) guidelines.

Eligibility of participants

Intervention

Participants will be randomly assigned to one of two treatment sequences: (1) 16 weeks of treatment with glimepiride+linagliptin, 4 weeks of washout and 16 weeks of treatment with glimepiride+placebo or (2) 16 weeks of treatment with glimepiride+placebo, 4 weeks of washout and 16 weeks of treatment with glimepiride+linagliptin. Patients treated with glimepiride at screening will continue on the same dosage after randomisation. Treatment-naïve patients will start on 0.5 mg glimepiride after randomisation. Each treatment period is divided into a drug-titration period (weeks 1–4) and a maintenance period (weeks 5–16). After initiation of linagliptin/placebo patients will continue on the initial dose of glimepiride at screening for 1 week. Patients are instructed to measure fasting plasma glucose values daily. After the first week of linagliptin/placebo, the glimepiride dose will be adjusted for the first time. Target fasting plasma glucose (average during at least 5 days) is between 4.5 and 6.0 mmol/L (both inclusive) without episodes of symptomatic or biochemical hypoglycaemia (plasma glucose≤3.9 mmol/L). In case of no hypoglycaemia and an average fasting plasma glucose>6 mmol/L, the glimepiride dose will be increased with 0.5 mg. In case of hypoglycaemia or average fasting plasma glucose <4.5 mmol/L, the dose of glimepiride will be decreased by 0.5 mg. In the remaining titration period, the glimepiride dose will be uptitrated or downtitrated once-weekly in the same treat-to-target manner with a maximum daily dose of 6 mg in both groups. A total daily dose of glimepiride≥1 mg will be divided and administered as two daily doses: one dose in the morning and one dose in the evening. If target fasting plasma glucose is not achieved during the drug-titration period, the dose of glimepiride will be adjusted after week 4 at the investigators discretion. In case of hypoglycaemia outside of the drug-titration period, glimepiride dose will be down-titrated 0.5 mg. Once-daily linagliptin 5 mg or placebo will be initiated at each treatment period and the dose kept stable throughout the experiment. After the first treatment period, the dose of glimepiride at randomisation will immediately be reintroduced for all patients and followed by a 4-week ‘linagliptin/placebo’ washout period. Participants will be encouraged not to change their lifestyle (ie, diet, exercise and smoking status) during the trial. There will be no restriction to concomitant non-antidiabetic medications. Trial medicine will comply with Good Manufacturing Practice. Drug accountability will be performed at all clinical visits to document degree of compliance.

Outcomes

Participant timeline

Participant timeline is illustrated in table 1. Study treatment will be evaluated with clinical visits and blood samplings, telephone calls, CGM and meal and bicycle test. The full study period constitutes of 36 weeks (one washout period of 4 weeks and two treatment periods of 16 weeks), preceded by a pretreatment evaluation (screening) approximately 14 days before randomisation.

Screening

Participants will be screened according to inclusion and exclusion criteria. Demographics and medical history will be obtained, including recording of current medication and previous tests verifying the diagnosis of HNF1A-diabetes. Vital signs (blood pressure and pulse), height and weight, waist and hip circumference will be measured and a physical examination will be performed. Screening blood samples will be taken including haemoglobin, creatinine, electrolytes (Na⁺, K⁺), C reactive protein (CRP), thyroid-stimulating hormone (TSH), ALT, AST, bilirubin, alkaline phosphatase, albumin, plasma glucose, HbA1c, urine examined for albumin:creatinine ratio and cardiovascular biomarkers. Fertile women will undergo a pregnancy test (urine sample). If the potential participant meets all inclusion criteria and none of the exclusion criteria, an appointment for the following visits (randomisation) will be set up. For screening failures (patients who were screened, but not randomised), a screening failure form will be completed.

Telephone contacts

During the 36 weeks, a total of six telephone calls will be conducted. At these contacts, the glimepiride treatment will be titrated as described previously according to patients’ diaries, reported hypoglycaemic episodes and adverse events (AE). Compliance will be noted. Patients will be informed to continue on 5 mg of linagliptin/placebo forward. The telephone contacts will be done in the drug-titration periods (weeks 1, 2, 3, 21, 22 and 23).

Clinical visits and blood sampling

Patients will meet for eight clinical visits. Compliance and AEs will be noted. At clinical visits dispensing of trial drugs will be done. Blood samples will be performed during clinical visits and at the end of study. The following analysis will be performed: haemoglobin, vitamin b12, creatinine, electrolytes (Na⁺, K⁺), leucocytes, thrombocytes, CRP, C-peptide, cholesterols, triglycerides, TSH, CRP, bilirubin, ALT, AST, alkaline phosphatase, albumin, coagulation factors, plasma glucose, HbA1c and fructosamine. Visits are scheduled for weeks 4, 8, 12, 16, 24, 28, 32 and 36.

Meal and bicycle test

Three meal and bicycle tests (figure 2) will be performed at baseline and in the end of each of the two treatment periods (weeks 0, 16 and 36). Patients will be subjected to a standard 4-hour liquid meal test and a concurrent 30 min bicycle test. The patients will meet in a fasting (10 hours) state. Patients will continue their therapy according to trial protocol and thus take glimepiride tablets as normal the evening before, and glimepiride and linagliptin/placebo tablets at the clinic 1 hour prior to the ingestion of the meal (time −60 min). Patients will ingest a standard liquid meal (525 kcal: 65 g carbohydrate, 20 g fat, 21 g protein) added 1.5 g of paracetamol at time 0 min. From 60 to 90 min, a 30 min bicycle test will be performed (initially 50–60 rotations per minute, with adjustment of bicycle resistance every 5 min until achieving a heart rate of 90–110 beats per min). Symptoms of hypoglycaemia will be monitored and plasma glucose measured on prespecified time points and in the event of symptoms of hypoglycaemia. Other AEs and compliance will also be noted. During the meal test, blood samples will be drawn at prespecified time points for measurement of plasma glucose, insulin, C-peptide, glucagon, GIP, GLP-1, paracetamol and counter-regulatory hormones (GH, cortisol, norepinephrine).

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Figure 2

Meal and bicycle test. Participants arrive fasting. Study medication is taken at time −60 min. At time 0 min a mixed liquid meal (525 kcal) added 1.5 g of paracetamol is served. From 60 to 90 min, a light bicycle test is performed (target heart rate 90–110 beats per min). Blood samples are taken for the analysis of plasma glucose, insulin, C-peptide, glucagon, incretins and counter-regulatory hormones. Patients will be monitored for any symptoms of hypoglycaemia. FPG, fasting plasma glucose.

Sample size

The study is a superiority study which will estimate the effect of two different treatments with the hypothesis that the effect of treatment with glimepiride+linagliptin versus glimepiride+placebo will display a significant decrease in MAGE.

For power calculation on our primary end point, the following equation has been used as recommended by Jones and Kenward22:

N = (Z_α+Z_β)²×SD²/MIREDIF²

• Z_α is a table value according to a two-sided standard normal distribution;

• Z_β is a table value according to a one-sided standard normal distribution;

• SD is the standard deviation;

• MIREDIF is the minimum relevant difference.

The power of our study (1−β) will be set at 0.8, and the risk of accepting a false hypothesis is 0.20 (β=0.20) with a significance level at 0.05 (α=0.05). Thus, Z_α=1.96 and Z_β=0.84. We have defined a minimal relevant difference (MIREDIF) of 2 mmol/L (36 mg/dL) in MAGE between the two groups. Power calculation in the current trial has been based on the SD (SD=50) in MAGE presented by Saisho et al.23

Number of participants needed:

N=(1.96+0.84)²×50²/36²=15.1≈16

In order to allow drop-outs, a total of 20 patients with HNF1A-diabetes will be randomised. With the chosen size sample a risk of type 2 error exists, thus missing an effect of glimepiride+linagliptin versus glimepiride placebo. However, we consider only an effect that can be found within a sample size of 20 patients to be clinically relevant.

Assignment of interventions

Patients will be randomised in 1:1 allocation using a computer-generated random list of numbers. The list of random numbers will be made by an employee who is otherwise uninvolved in the study. The allocation will not be known to the investigators or patients during the study. Everyone involved in the trial will be blinded until data are fully analysed. Code break of a participant’s trial medicine will occur during the trial only in case of exceptional circumstances when knowledge of treatment is absolutely necessary. Only the sponsor-investigator and an employee who is otherwise uninvolved in the study will be able to perform a code break.

Data collection, analysis and management

All data will be collected in electronic case report forms in Research Electronic Data Capture (REDCap) hosted at the Capital Region of Denmark. REDCap is a secure, web-based application designed to support data capture for research studies, providing: (1) an intuitive interface for validated data entry; (2) audit trails for tracking data manipulation and export procedures; (3) automated export procedures for seamless data downloads to common statistical packages and (4) procedures for importing data from external sources.24 The case report forms will only be available for investigators and monitors according to ICH-GCP guidelines.25 The database will be locked database after last participant’s end-of-study visit. Only investigators will have access to the final data set. To ensure adherence to protocol, a designated trial project coordinator will be responsible for reminding patients of dates for visits and clinical tests. Additionally, drug accountability will be performed to ensure compliance. CGM monitors used will be the iPro2 CGM from Medtronic, Northridge, California, USA, which has been validated elsewhere.26 To ensure data collection, only CGM-trained investigators will assemble and disassemble the CGM monitor at the study site. Investigators will receive training by Medtronic representatives before commencement of the trial. All data are stored electronically by the CGM device and uploaded to a server hosted by Medtronic. If participants have any problems with their CGM monitor, an investigator will always be accessible by phone and provide guidance. If a problem with the CGM monitor persists, additional clinical visits will be arranged to ensure data collection.

Blood and urine samples from screening visits, clinical visits and meal and bike tests will be analysed in the Department of Biochemistry at Gentofte Hospital or collaborators according to predefined local standards. Additional blood and urine will be stored in the research biobank in case of the need of re-analysis.

Data monitoring

The trial will be monitored by the GCP unit, Bispebjerg Hospital, University of Copenhagen, Copenhagen, Denmark. The GCP unit and the Danish Medicines Agency will be able to conduct an audit of the trial independent of sponsor-investigator and investigators. There will be no data monitoring committee due to (1) the relatively short duration of the study, and because (2) the drugs used in this trial are approved and considered safe. At all telephone contacts and clinical visits, patients’ adverse events, if any, will be noted in the case report forms. Additionally, patients will be encouraged to contact the investigators in case of any AE. Participants will be thoroughly informed of potential side effects when participating in this trial. All AEs will be reported to the Danish Medicines Agency and the Scientific-Ethical Committee of the Capital Region of Denmark at the end of trial. Sponsor-investigator shall be further responsible for reporting of any serious adverse event (SAE), serious adverse reaction (SAR) and suspected unexpected serious adverse reaction (SUSAR), which occur during the conduct of the study to the Scientific-Ethical Committee of the Capital Region of Denmark once yearly in accordance with the applicable laws and regulations. Sponsor-investigator will delegate the responsibility for the expedited regulatory reporting of SAE, SAR and SUSAR to Boehringer Ingelheim. The sponsor-investigator will report every SAE, SAR and SUSAR immediately to Boehringer Ingelheim within 24 hours. Boehringer Ingelheim will be responsible to report SAE, SAR and SUSAR to the Danish Medical Agency in accordance with the applicable laws and regulations.

Statistical analysis

All analyses will be performed on an intent-to-treat sample of subjects who were randomised and received at least one dose of medication or placebo. Continuous data will be summarised by descriptive statistics with the number of observations (n), mean, SD, minimum, median and maximum. Categorical data will be summarised in frequency tables using count and percentages. AUC will be calculated using the trapezoidal rule. Comparisons between treatment outcomes will be performed in linear mixed models for continuous data. Comparisons of binary or ordinal data will be performed in generalised linear models for binary or ordinal data. Missing data will implicitly be imputed by performing likelihood inference in the linear mixed models for continuous data. Interaction between treatment and treatment period will be performed in linear mixed models to test for potential carryover effect. Outcomes that are not normally distributed will be transformed for the analysis in linear mixed models. Not normally distributed outcomes will be presented as median and ranges. All tests will be carried out at a significance level of 5%. AEs will be summarised qualitatively.

Strengths and limitations

The strength of this study is the randomised, placebo-controlled, double-blinded trial design. The crossover design means the patients will be their own control. Glucose-lowering treatment will be evaluated regarding all glucose parameters: glucose variability, long-term glycaemic control (HbA1c) and by the amount of hypoglycaemic events. The duration of each treatment period (16 weeks) will provide time to asses both the efficacy of treatments and risk of hypoglycaemia. The crossover design has the advantage that it reduces the risk of confounding covariates by unbalanced randomisation. However, the disadvantage of a crossover study is if participants change during the study (eg, gain weight) or if there is a carryover effect from one treatment period to another. Given the 4-week washout period the risk of a carryover effect is minimal. The major limitation of this study is the small sample size and rather short duration of the study. Thus, the results of this study might not be generalisable to all patients with HNF1A-diabetes. Another limitation is that participants at randomisation will receive different baseline doses of glimepiride. This may cause different levels of saturation of the SUR1 and thereby potentially interindividual differences to an increment of the glimepiride dose. Third, a limitation of this study is that it might be hard to recruit patients given the criteria for participation and the relatively few diagnosed with HNF1A-diabetes in Denmark.

Patient and public involvement

This study is designed to address the well-known dysregulation and risk of hypoglycaemia in patients with HNF1A-diabetes treated with SU as monotherapy. We expect the study to provide knowledge about the combination therapy with DPP-4 inhibitors and SU, which from a mode of action point of view make a lot of sense. Currently, neither patients nor patient advisers have been involved in the design, recruitment and conduct of the study. All participants will receive a personal treatment evaluation and an evaluation of the whole population in writing after a full data analysis has been performed. The intervention burden will be assessed by the recording of all episodes of AE and/or SAE according to GCP.