The present trial has a double-blind, placebo-controlled, parallel-group design in which participants are randomised to receive weekly infusions of either Epo (Eprex; 40,000 IU; Janssen-Cilag) or saline over 8 weeks. Patients complete a broad neurocognitive test battery assessing memory, executive function and attention at baseline, after completion of treatment (week 9) and at follow-up (week 14). Mood, symptoms and quality of life are measured at baseline and weeks 5, 9 and 14 using a variety of rating scales including the Hamilton Depression Rating Scale 17 items (HDRS-17) [1
], Young Mania Rating Scale (YMRS) [40
], Beck Depression Inventory (BDI) [41
], Massachusetts General Hospital Cognitive and Physical Functioning Questionnaire (CPFQ) [42
], and the EuroQol visual analogue scale (VAS) for quality of life (EQ-5D) [43
]. Patients undergo an fMRI scan at baseline and week 14. Further, plasma and whole blood BDNF and inflammatory markers will be investigated with blood tests at baseline and weeks 5, 9 and 14. Finally, we will assess metabolic parameters with blood tests at baseline and weeks 5, 9 and 14, and body fat mass by Dual energy X-ray absorptiometry (DXA) scanning at baseline and week 9. For an overview of investigations and their timing see table .
Participants and screening
Patients with treatment-resistant major depression (study 1) and patients with bipolar disorder in full or partial remission but with cognitive problems (study 2) will be recruited through Clinic for Affective Disorders, Department of Psychiatry, Copenhagen University Hospital, Rigshospitalet. The centre receives patients with treatment-resistant major depression or bipolar disorder from the whole Capital Region. Patients will be screened with Schedules for Clinical Assessment in Neuropsychiatry (SCAN) [44
] to confirm diagnosis.
Inclusion Criteria are a diagnosis of either treatment-resistant major depression (defined as failure to respond to at least two different types of antidepressants given in sufficient doses over sufficient time) in moderate to severe degree (HDRS score ≥ 17), or bipolar disorder in full or partial remission (HDRS ≤ 14 and YMRS ≤ 14) and subjective experience of moderate to severe cognitive problems on the CPFQ (score ≥ 4 on ≥ 2 domains), age between 18-65 years and unchanged antidepressant or mood stabilising treatment for minimum 2 weeks prior to inclusion in the study.
Exclusion criteria are any significant medical conditions (incl. diabetes, renal failure, epilepsy, untreated hypertension, present or past malignancies and thrombosis), smoking, hypertension, overweight (BMI > 30) or a body weight < 45 or > 95 kg, schizophrenia, present alcohol or substance misuse, acute suicidal risk, present or previous suicide attempts in the past 2 years, pregnancy or breast feeding, contraceptive medication, or a first-degree family history of thromboembolic events or seizure disorders.
A pregnancy test will be performed on female patients in their fertile age before and every second week during the study. Blood screening and physical examinations will be performed at baseline and every week during the treatment period as well as 1, 2 and 6 weeks after treatment cessation to monitor patients' level of red blood cells and ensure patient safety. Written informed consent will be obtained from all patients before their inclusion in the study and letters sent to their general practitioners to avoid any potential medical complications.
Block randomisation for the two studies has been performed by Pharma Consulting Group AB http://www.pharmaconsultinggroup.com
. Pharma Consulting Group has created randomisation lists for each study and emergency envelopes for each patient ID number. Treatment groups are stratified for age (< or ≥ 35 years) and gender. Upon inclusion of participants, the date of birth, gender, diagnosis, and to which of the above strata the participant belongs are recorded. Numbering within each stratum is consecutive and study identification numbers are recorded in the patients' individual case report forms (CRFs).
Study personnel involved in the evaluation of outcomes and Epo/placebo infusions and study participants are blinded to study medication allocation. Blinding is maintained throughout the study, data management, outcome assessment and data analysis. This is possible because Epo is a clear colorless liquid similar to saline and because safety monitoring and bloodlettings are performed by a medic who is not involved in the evaluation of outcome measures or drug administration.
The primary outcome for treatment-resistant depressed patients (study 1) is depression severity as measured with the HDRS-17 [1
] at week 9. Global Assessment of Function (GAF) scores at week 9 will be reported in addition to the primary outcome. The secondary outcome is remission defined as HDRS score ≤ 8 at week 9. Confidence intervals will be reported in relation to the proportions in remission. The tertiary outcomes are memory and executive functions, measured with the Rey Auditory Verbal Learning Test (RAVLT) [2
] and Rapid Visual Processing (RVIP) test from the Cambridge Neuropsychological Test Automated Battery (CANTAB), respectively, facial expression recognition measured with a facial expression recognition test, as well as self-ratings of cognitive difficulties on the CPFQ, depressive symptoms and life quality at week 9.
For bipolar patients in remission (study 2) the primary outcome is memory performance measured with the RAVLT at week 9. The secondary outcome is sustained attention measured with the RVIP test and recognition of emotional facial expressions assessed with a facial expression recognition test at week 9. Finally, the tertiary outcomes are ratings of mood, symptoms, quality of life and cognitive function at week 9.
Clinical mood ratings and patients' self-ratings will be performed at baseline, and weeks 5, 9 and 14 to assess the speed and duration of potential effects on these measures. Neuropsychological testing is performed at baseline, and weeks 9 and 14 to assess effects of Epo vs. placebo treatment and their duration. For overview see table .
For study 1, it is hypothesised that Epo vs. placebo treatment will: 1) reduce HDRS-17 scores 2) increase remission rates measured with the HDRS-17 3) improve memory and executive function, reduce the perception of negative facial expressions, and improve symptoms and quality of life.
Study 2 is designed to test the hypothesis that Epo vs. placebo treatment will: 1) improve memory 2) facilitate sustained attention and reduce the perception of negative facial expressions measured, and 3) improve mood, symptoms and life quality.
Further outcome assessments: biomarkers of potential clinical effects
fMRI is performed before and after treatment to investigate the neurophysiological underpinnings for effects of repeated Epo administration on cognitive function and mood. The second fMRI scan is scheduled 6 weeks after treatment cessation (week 14) because hematocrit levels in the two groups should be comparable at this time point [20
] and thus not confound data interpretation. We will investigate whether potential memory improvement after Epo vs. placebo administration is associated with increased hippocampal engagement during picture encoding consistent with increased hippocampal BDNF and plasticity [34
]. The neural correlates of potential improvements in executive function with Epo vs. placebo treatment will be investigated with a spatial N-back working memory test. Finally, we will investigate whether potential antidepressant effects of Epo are accompanied by reduced cognitive and neural response to fearful facial expressions in a face-processing test similar to effects seen with existing antidepressants [32
]. This would point to the modulation of emotional bias as a common mechanistically important effect of compounds with diverse neurochemical signalling mechanisms. Structural T1-weighted MRI and diffusion weighted imaging (DWI) at these time points will investigate whether Epo-treatment induces structural changes in the hippocampus and influences functional brain connectivity, respectively. Arterial spin labelling (ASL) will investigate whether Epo introduces enduring changes in global and regional cerebral blood flow (after the return of haematocrit levels to baseline).
It is known that Epo increase hippocampal BDNF and nerve growth factor (NGF) and has anti-inflammatory actions, but it is unclear whether these actions are mechanistically important for potential antidepressant effects of Epo. We will therefore investigate the effect of Epo on plasma and whole blood BDNF, NGF and inflammatory markers incl. interleukin-6 (IL-6) and C-reactive protein (CRP). Given diurnal variation in peripheral BDNF, blood samples are collected at approximately the same time for all patients. For overview of timing see table .
Preclinical research suggests that Epo protects against diet-induced obesity and has beneficial effects on metabolic parameters [45
], which may play a role in the treatment of affective disorder [46
]. The study therefore investigates the effects of Epo on metabolic parameters (fasting glucose, fasting insulin, HgbA1c, HDL-cholesterol, LDL-cholesterol and TAG) in these patients through blood sampling before and during Epo vs. placebo treatment. DXA-scanning (Lunar Prodigy, GE Medical Systems Wisconsin, USA, version 8.8) is performed to assess whether Epo affects the total lipid- and lipid-free lean mass in the body. For overview of timing see table .
Sample size and power calculation
Sample size and statistical power for the two studies were calculated using nQuery Advisor 5.0 software. The primary outcome in study 1 is depression severity reflected by HDRS-17 after Epo vs. placebo treatment. The clinically relevant difference is defined as minimum 3 scores on this scale. The mean standard deviation in the two treatment groups is estimated to be 3 HDRS-17 scores and the mean scores to be 18 and 21 after Epo and placebo treatment, respectively. With these assumptions a sample of n = 40 patients (n = 20 per drug group) has a statistical power of 86% to detect a clinically relevant difference in HDRS-17 scores between the two treatment groups at an alpha level of 5% (2-sided test).
The primary outcome in study 2 is verbal memory, reflected by the RAVLT total score after Epo vs. placebo treatment. A recent study has shown that the mean RAVLT total score for patients with bipolar disorder in remission is 52.0 (while the mean score for healthy age-matched controls was 60.7) out of a maximum of 75 [47
]. We estimate that a clinically relevant difference between drug groups in the change in RAVLT scores is at least 4 points (half-way to normal function). The mean standard deviation in RAVLT scores in the two treatment groups is estimated to be 4 points and the mean scores to be 56 and 52 after Epo and placebo treatment, respectively. With these assumptions a sample of n = 40 patients (n = 20 per drug group) has a statistical power of 86% to detect a clinically relevant difference in RAVLT total scores between the two treatment groups at an alpha level of 5% (2-sided test).
The key aspects of cognitive function affected in mood disorder, executive function, attention, memory and emotional processing, are assessed using an extensive battery of neurocognitive tests. The test battery includes following manual tests: RAVLT, Verbal and Semantic Fluency Tests, WAIS-III Letter Number Sequencing, Trail Making A and B, the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) digit span and coding tests, and the following computerised tests: Simple Reaction Time, Rapid Visual Information Processing, Delayed Match to Sample and Spatial Working Memory. Tests used for fMRI scanning are an N-back working memory task, a picture encoding task modified from Hariri et al [38
], an emotional face processing task using fearful and happy Nimstim picture stimuli http://www.macbrain.org/resources.htm
and a facial expression recognition task using Ekman and Friesen morphed stimuli sets [48
The computerised neuropsychological test battery CANTAB (Cambridge Cognition Ltd.) are employed for a precise estimation of drug effects on accuracy and reaction times during the above mentioned tasks probing executive function, attention and memory. The effects of Epo on the neural basis of executive function, memory and emotion processing as well as potential structural changes are investigated using the in vivo non-invasive techniques of fMRI, structural T1 weighted MRI, DWI, and ASL-based measurements of cerebral blood flow. Imaging data will be collected using a Siemens Trio scanner operating at 3.0 T, at the Danish Research Centre for Magnetic Resonance (DRCMR), Copenhagen University Hospital, Hvidovre. fMRI data will pre-processed and analysed using FEAT (FMRIB Expert Analysis Tool) version 5.43, part of FSL (FMRIB Software Library version 3.3) http://www.fmrib.ox.ac.uk/fsl
. Structural T1-weighted MRI data is analysed with FAST (FMRIB's Automated Segmentation Tool), FIRST (FMRIB's Integrated Registration and Segmentation Tool) http://www.fmrib.ox.ac.uk/fsl
and FreeSurfer http://surfer.nmr.mgh.harvard.edu/
. DWI data is analysed with FDT (FMRIB's Diffusion Toolbox; http://www.fmrib.ox.ac.uk/fsl/fdt/
). Finally analysis of ASL data is performed with SPM software http://www.fil.ion.ucl.ac.uk/spm/software/spm8/
and FABBER http://www.fmrib.ox.ac.uk/fsl/fabber
Data will be collected until dropout from all randomised patients and analysed using repeated measures analysis of variance (ANOVA). Significant interactions were analysed further with simple main effect analyses. In the case of missing or invalid data, we will perform Per Protocol (PP) analyses for complete data sets and Intention to Treat (ITT) analysis. In the case of disparity between the results of these analyses, the outcome of the ITT analysis will be regarded as the result of the study. The statistical threshold for which results are considered significant if p ≤ 0.05 (2-tailed). Statistical analyses will be performed using the Statistical Package for Social Sciences (SPSS).
Data for each participant is kept in a Case Record File (CRF), which fulfil the Danish law for medical doctors' obligation to keep patients records. The study personnel involved in the evaluation of clinical outcomes are blinded to the treatment until the data analysis is completed. Blood test results and lists of potential adverse effects are therefore kept separately in a locked safe to which only medical doctors responsible for patient safety and the person involved in the blinding of the study medication have access.
Safety profile of Epo
Epo is widely used in the treatment of anaemia and generally has a good safety profile. Some potential acute side effects are transient flu-like symptoms, rash at the injection site and headache, which are normally reported as mild and disappear within a few hours. However, long-term Epo-treatment can induce epileptic seizures in medically ill patients with chronic renal failure and past history of seizures [49
]. A very rare adverse effect of Epo treatment is a condition where the patient develops neutralising antibodies against Epo resulting in pure red cell aplasia (PRCA). This problem has only been observed in patients treated with subcutaneous injections and was solved in 2003 when the manufacturer changed the preparation, packaging and transport of Epo [50
]. Today, Epo-antibody formation is thus an extremely rare condition observed with long-term subcutaneous Epo-treatment of patients with chronic renal failure [51
Meta-analysis of 5243 renal failure patients in long-term Epo treatment showed a slight increase in mortality (17%; 95% C.I. 1-35%) potentially because of thrombosis at patients' arterial dialysis access site and insufficient treatment of hypertension [52
]. Recently, a study of 522 stroke patients treated with Epo vs. placebo also revealed greater mortality in the Epo group, possibly because of interaction with recombinant tissue plasminogen activator (rtPA) used for thrombolysis as well as an unusual low death rate in the placebo group [53
Weekly high-dose (40,000/48,000 IE) Epo administration to patients with neuropsychiatric disorder increases haematocrit after 5 weeks [20
] calling for bloodlettings in about 40% of patients when values exceed 50% and 48% for men and women, respectively. However, continued Epo-treatment in the absence of any iron substitution was well tolerated and not associated with further haematocrit increase after 8 weeks of treatment [20
Given the above safety concerns and increased mortality in certain patient groups, the study inclusion and exclusion criteria (see 'Patients and screening') are strictly adhered to and patients' safety monitored closely throughout the study. Given the very low incidence of PRCA, our use of intravenous (vs. subcutaneous) administration and exclusion of patients with renal failure, we believe that the risk of PRCA in the current study is negligible. Nevertheless, reticulocyte counts as first indicator of PRCA will be carefully monitored (see below).
Patients are informed of all potential adverse effects before their inclusion in the study, instructed that iron supplements are strictly prohibited (since they would boost haematocrit levels) and advised to immediately contact their local emergency department if (1) they experience any potential symptoms of thrombosis or (2) their physician suspects PRCA. They are given a pocketsize plastic card with instruction about whom to contact in the event of these symptoms, their contact details, and important information to the medical doctor at the local emergency department.
It is ensured the temperature of the Epo is kept at 2-8°C at all times during transport and storage to avoid damage of the compound and potential associated adverse effects. To minimise the risk of acute allergic reactions, Epo/placebo is dissolved in 100 ml saline and administered as intravenous infusions over 15 minutes. After this, patients' well-being is monitored for at least 30 minutes by a nurse at the clinic in the case of potential transient side effects of Epo like flu symptoms, rash at the infusion site or head ache.
Safety parameters and monitoring
Patient safety is monitored with medical examinations, blood pressure measurements, blood tests, ECG and other safety measures at baseline and throughout the study until 2 weeks after cessation of Epo/placebo treatment. For an overview of the safety tests, their frequency and timing see table .
If blood tests show significantly increased haematocrit (>50% for men and >48% for women) at two consecutive measurements within a week, bloodletting(s) (450 ml each) will be performed weakly with no cessation of treatment until haematocrit values are normalised. In the case of significant increase in thrombocytes (> 400 billion/L) or drop in reticulocytes (< 1‰) 2 repeated controls will be performed in the following week. If these values remain abnormal, the patient is taken out of the study and monitored with weekly medical examinations and blood tests until the values are normalised or is hospitalised for observation.
Procedures for acting on serious adverse effects
Procedures for breaking the randomisation code are established for the case of severe adverse reactions potentially related to the drug intervention. These will be followed when knowledge of the patient's drug treatment will have implications of the treatment of the observed adverse effects. It is the decision of the principal investigator or the medic responsible for safety monitoring to break the emergency envelope for the affected participant.