Design
The study is a Danish two-year, investigator-initiated, prospective, randomised, open, blinded endpoint (PROBE), multicentre, cross-over trial investigating the effect of insulin analogues versus human insulin on the frequency of severe hypoglycaemia (Figure 1). Patients are randomised to treatment with basal-bolus therapy with insulin detemir and insulin aspart (Levemir®/Novorapid®) or human NPH insulin and human regular insulin (Insulatard®/Actrapid®), in random order. Doses of insulin are adjusted according to individual patient need at the discretion of the investigator. Thus, a treat-to-target design is deliberately not used in order not to intervene with present glycaemic control and everyday clinical practice, which otherwise in itself will interfere with the risk of hypoglycaemia. The best obtainable glycaemic control for the individual patient is strived for in both treatment periods. The protocol did not specify the timing and number of insulin injections per day. In general, a four times daily basal-bolus regimen was selected, e.g. basal insulin before bedtime and prandial insulin before meals. Patients were instructed in insulin injection technique at the beginning of each treatment arm. Endpoints are assessed during the last 9 months of each treatment arm, the first 3 months being used for run-in on a new treatment regimen. Optional admissions for overnight plasma glucose measurements followed by three days of blinded continuous glucose monitoring (CGM) are performed after 6 and 12 months of treatment in each treatment arm.
Subjects
From November 2006 to June 2007 questionnaires were sent to 6112 patients with type 1 diabetes attending the outpatient clinics at Steno Diabetes Center, Hillerød Hospital, Hvidovre Hospital, Bispebjerg Hospital, Odense University Hospital, Copenhagen University Hospital (Rigshospitalet), and Aarhus University Hospital. From some centres reminders were sent once. A total of 3861 patients filled in the questionnaire (63.2%). Patients were, among other things, asked how many episodes of severe hypoglycaemia they had experienced during the last year. Patients with two or more episodes were identified (n = 720) and invited by letter to participate in the HypoAna Trial. Subjects were included in the HypoAna Trial from May 2007 to November 2009. The inclusion criteria are type 1 diabetes for more than five years, two or more episodes of severe hypoglycaemia in the previous year (defined as need for third party assistance to restore blood glucose level), age > 18 years, and a negative pregnancy test. There were no inclusion criteria regarding the pre-randomisation insulin treatment. Exclusion criteria are treated adrenal or growth hormone insufficiency or untreated hypothyroidism, myocardial infarction or coronary revascularisation, transient ischemic attack or stroke within the last three months, history of malignancy unless a disease-free period exceeding five years, history of alcohol or drug abuse, pregnancy or lactation, and women of childbearing potential who are not using chemical or mechanical contraception. Criteria for discontinuation are voluntary withdrawal of consent, pregnancy, or non-compliance with the study protocol as judged by the investigator. Discontinuing patients are not substituted, but data on discontinuing patients are included in data analyses on an intention-to-treat basis. The study was approved by The Regional Committee on Biomedical Research Ethics (# H-KA-20070008) and the Danish Medicines Agency (# 2612–3397). The study is registered at http://www.clinicaltrials.gov (# NCT00346996). The Study will be conducted in accordance with the Helsinki Declaration.
Experimental protocol
Participants attend the outpatient clinics every three months. After informed consent the participants meet fasting at the first visit in the outpatient clinic at 08:00 a.m. Information about demography, lifestyle and clinical characteristics are gathered. Weight and height are measured without shoes. Blood pressure is measured twice after 10 minutes rest in a sitting position. As a measure of peripheral and autonomic neuropathy, biothesiometry, beat-to-beat variation, and orthostatic blood pressure responses are assessed. A 12-lead electrocardiogram is recorded and visually assessed. State of hypoglycaemia awareness is classified by a validated method [27, 28]. Patients are asked: “Do you have symptoms when you have a hypo?” The subjects can answer “always” (normal awareness), “usually” (impaired awareness), and “occasionally” or “never” (unawareness). The frequencies of episodes of mild hypoglycaemia per week (defined as manageable by the patient) and severe hypoglycaemia in the previous year are determined on the basis of the aforementioned questionnaires used for screening of patients for the present trial. Recall of severe hypoglycaemia is well preserved during a one-year period [27]. Fasting blood samples are taken and samples for later analyses are centrifuged, processed and stored immediately at −80°C. HbA1c and C-peptide concentrations are measured centrally at Steno Diabetes Center. At the end of visit 1 a 30-min ACTH test (Synacthen®, 0.25 mg i.v.) is performed. After a breakfast in the outpatient clinic the patient’s insulin injection technique is tested and corrected if needed. Three questionnaires about quality of life are filled in. The EuroQol-5 dimension quality of life index (EQ-5D), the Insulin Treatment Satisfaction Questionnaire (ITSQ) and the Hypoglycemia Fear Survey (HFS). EQ-5D is a commonly used, short questionnaire assessing health-related quality of life. Patients describe their health status on five domains (mobility, self-care, usual activities, pain/discomfort, anxiety/depression) with three different answers: “I have no problem”, “some problem”, or “extreme problem”. Furthermore, patients rate health state on a visual analogue scale [29, 30]. The ITSQ measures treatment satisfaction concerning the insulin regimen and comprises 22 questions about different aspects of insulin treatment [31]. The HFS comprises 33 questions and measures to what degree patients change daily practise to avoid hypoglycaemia and measures different aspects of worries of episodes of hypoglycaemia [32]. At the end of the visit patients are centrally randomised at Steno Diabetes Center by an assistant not further involved in the trial, based on site-specific computer-generated randomisation lists in blocks of four patients. Finally, free study medication is handed out.
Endpoint registration
Severe hypoglycaemia
In case of a suspected episode of severe hypoglycaemia, patients are instructed to make a telephone call within 24 hours to a call center served by a trained diabetes nurse. The nurse performs a structured interview with the patient to validate the severity (need for assistance from others) and the plausibility of the event according to Whipple’s triad: I) characteristic hypoglycaemic symptoms; II) biochemical confirmation with plasma glucose ≤ 3.9 mmol/l; and III) adequate response to treatment with carbohydrates, glucose or glucagon. According to Whipple’s triad only episodes fulfilling all criteria can be considered definite, whereas those fulfilling two criteria can only be considered probable. Questions about time, place, blood glucose, possible cause(s) of hypoglycaemia, and types of help given to the patient are asked along with questions about health-related quality of life and questions about any consequences of the episode regarding physical or material damage and work-related absenteeism. Furthermore, at every visit to the outpatient clinic patients are asked if they have experienced any episodes of severe hypoglycaemia not recorded by telephone interview. In such cases an interview is carried out by the patient’s doctor or nurse using the same questionnaire as mentioned above. All telephone interviews are done by the same two study nurses (blinded to the treatment modality) located at Hillerød Hospital. Data from the last 9 months of each treatment arm will be used for endpoint analyses.
Mild symptomatic and asymptomatic hypoglycaemia
Patients are instructed and strongly encouraged to make a 7-point self-monitored blood glucose (SMBG) profile two days a week in the entire study period. All values are entered into a diary with study-specific guidance and at every outpatient visit the diary is evaluated by the investigator and copies of the relevant pages are saved as source data. If patients measure a SMBG value ≤ 3.9 mmol/l, they are instructed to write down if they have concomitant symptoms of hypoglycaemia. All SMBG values are measured with the Ascensia® CONTOUR® blood glucose monitoring system (Bayer Health Care, Leverkusen, Germany).
Nocturnal hypoglycaemia
Patients are instructed to measure a nocturnal blood glucose once every month at 3 a.m. Moreover, patients are invited (optional) to stay overnight at Steno Diabetes Center four times during the study (two times at month 6 and 12 in every treatment arm). In the evening before bedtime, a study-specific blinded continuous glucose monitoring (CGM) device (Guardian® REAL-time (with a “black” display), Medtronic Minimed, Northridge, USA) is mounted by a trained study nurse and the patient is instructed how to calibrate the device, using capillary glucose measurements. Thereafter, a venous line is inserted in an anticubital vein. During the night, blinded samples for subsequent plasma glucose measurements are drawn once every hour, while the patient is sleeping.
CGM
Interstitial glucose concentrations are assessed for three days in patients who accept the four optional overnight stays at Steno Diabetes Center, i.e. 12 days of CGM in total. After the three days the glucose sensor and monitor are collected and data are downloaded to a computer for further analysis. These data remain blinded until the end of the study and are not used to control glycaemic levels during the study period. Special blinded versions of the CGM device with a “black screen” were supplied by the manufacturer. The CGM data will provide a unique possibility to assess the accuracy of CGM and compare time spent at hypoglycaemia, number of unrecognized episodes of hypoglycemia in this selected patient population.
HbA1c and plasma glucose
At every visit to the outpatient clinic (totalling 9 visits per patient) blood samples are drawn to measure HbA1c levels (analysed centrally at Steno Diabetes Center) and random plasma glucose concentrations (measured locally).
Power calculation
In the power calculation, the number of events in each treatment period is assumed to be distributed according to a Poisson model. No period effect exceeding the 3 months of run-in is assumed. Based on the total number of observed events for each individual patient, the number of events for a treatment period is reduced to a binomial distribution:
Binomial (total number of events, λhuman insulin/(λhuman insulin + λinsulin analogue)), which under the null hypothesis of no difference in event rate between insulin analogues and human insulin becomes binomial (total number of events, ½). The power calculations are thus performed with the use of an exact test of number of events in a binomial distribution with the probability of p = 1/2 versus alternatively with the probability of P = λhuman insulin/(λhuman insulin + λinsulin analogue) = 1/(1+ λinsulin analogue/λhuman insulin). The sample size calculation thus indicates the total number of events needed. The study population is selected to be patients with an expected increased risk of severe hypoglycaemia. Assuming an incidence of 2.8 severe hypoglycaemic events per patient-year, corresponding to 2.09 per 9 months treatment period, and setting minimal relevant difference (MIREDIF) to a reduction in the incidence of severe hypoglycaemia of 15% (based on the conclusion of the American Diabetes Association Workgroup on Hypoglycemia ”that any significant reduction in severe hypoglycemia (that requiring the assistance of another individual), even by as little as 10–20%, would be advantageous” [26]), this will correspond to a reduction in the relation between human and analogue insulin of: λinsulin analogue/λhuman insulin = 0.85. Setting power to 80% and a test level of 5%, the total number of events needs to be 1220. With an expected number of events of 2.09 + 1.75 (1.75 = 2.09 x 15% reduction in incidence of severe hypoglycaemia) = 3.84 events per 18 months’ treatment, this corresponds to a need for 315 patients. With an expected dropout rate of 15%, 370 patients need to be included.
Statistics
The crossover design makes it possible to compare the two treatment modalities within the same person, thereby minimising between-patient variation. This design is appropriate because type 1 diabetes is a chronic, stable disease with slow progression. Therefore, the status of the patient in the first treatment period is assumed to be the same in the second period. To avoid a possible period effect and unwelcome fluctuations in blood glucose in relation to shift from one treatment modality to the next, registration of the primary endpoint is performed after three months in each arm. The long participation of two years per patient ensures a sufficient time of observation to minimise the risk of dealing with patients at particularly good or bad periods of their illness. Therefore, the analysis will be based on data from patients who complete the first treatment period and at least 6 months of the second treatment period. An analysis in a negative binomial distribution or Poisson model will be used to describe the number of events through a log link function as a linear function of treatment, treatment period, HbA1c levels and other relevant variables (state of hypoglycaemia awareness, C-peptide status, age, duration of diabetes, daily insulin dose). Individual differences in the observation period will be adjusted for in the analysis.
Timeline
“Last patient, last visit” was November 2011.
Laboratory analysis
At the first visit fasting C-peptide, haemoglobin, haematocrit, plasma creatinine, plasma sodium, and plasma potassium, thrombocyte count, white blood cell count, alanine aminotransferase, alkaline phosphatase, bilirubin, total cholesterol, high and low density lipoprotein cholesterols, triglycerides, thyroid-stimulating hormone TSH, T4 and T3 are measured. These analyses are repeated after 1 year (before changing insulin regimen) and after 2 years (final visit). Participants with C-peptide concentrations below 300 pmol/l or stimulated (venous blood glucose concentration >12 mmol/l) C-peptide concentrations below 600 pmol/l were considered C-peptide negative. At every visit HbA1c and random plasma glucose are measured. Biobank samples (serum, plasma and DNA) are gathered at baseline and six times (three times in each insulin regimen) during the study. Urinary sodium and albumin concentration are determined in urine samples collected from two separate nights at baseline and repeated after 1 year (before shifting insulin regimen) and after 2 years (final visit). HbA1c is measured centrally at Steno Diabetes Center using High Performance Liquid Chromatography method on a Tosoh Automated Glycohemoglobin Analyzer. C-peptide is measured with an AutoDELFIA C-peptide kit (detection limit 5 pmol/l). An ACTH stimulation test was undertaken at the first visit. All other analyses are performed locally by routine methods.