Title page Number of words: 4773 Title: Effects of Erythropoietin on Depressive Symptoms and Neurocognitive Deficits in Depression and Bipolar Disorder

Background: Depression and bipolar disorder are associated with reduced neural plasticity and deficits in memory, attention and executive function. Drug treatments for these affective disorders have insufficient clinical effects in a large group and fail to reverse cognitive deficits. There is thus a need for more effective treatments which aid cognitive function. Erythropoietin (Epo) is involved in neuroplasticity and is a candidate for future treatment of affective disorders. The investigators have demonstrated that a single dose of Epo improves cognitive function and reduces neurocognitive processing of negative emotional information in healthy and depressed individuals similar to effects seen with conventional antidepressants. The current study adds to the previous findings by investigating whether repeated Epo administration has antidepressant effects in patients with treatment resistant depression and reverses cognitive impairments in these patients and in patients with bipolar disorder in remission. Methods/design: The trial has a double-blind, placebo-controlled, parallel-group design. 40 patients with treatment-resistant major depression and 40 patients with bipolar disorder in remission are recruited and randomized to receive weekly infusions of Epo (Eprex; 40,000 IU) or saline (NaCl 0.9%) for 8 weeks. Randomization is stratified for age and gender. The primary outcome parameters for the two studies are: depression severity measured with the Hamilton Depression Rating Scale 17 items (HDRS-17) [1] in study 1 and, in study 2, verbal memory measured with the Rey Auditory Verbal Learning Test (RAVLT) [2,3]. With inclusion of 40 patients in each study we obtain 86% power to detect clinically relevant differences between intervention and placebo groups on these primary outcomes. Trial registration: The trial is approved by the Local Ethics Committee: H-C-2008-092, Danish Medicines Agency: 2612-4020, EudraCT: 2008-04857-14, Danish Data Agency: 200841-2711 and ClinicalTrials.gov: NCT 00916552. Background and objective Background Depression and bipolar disorder are associated with neurodegenerative processes, reduced neuroplasticity and neuropsychological dysfunction, which often persist even after clinical remission. Current pharmacological treatment strategies have several limitations including a significant treatment-onset-response delay, only partial or no response in a large group of patients and limited effects on their cognitive deficits, which often persist into periods of remission [4-9]. These enduring cognitive impairments affect patients’ psychosocial and occupational function, quality of life and prognosis [7,8,10,11]. Better treatment options are thus required to improve the onset of efficacy, address patients who are treatment resistant and remediate neurocognitive deficits. Converging evidence suggests that neural atrophy and reduced neuroplasticity are involved in depression and bipolar disorder, while restoration of synaptic plasticity may be an important mechanism of chronic antidepressant and mood stabilizing drug treatment [12-14]. This hypothesis is supported by evidence that increased hippocampal brain-derived-neurotrophic-factor (BDNF) is necessary for the behavioural effects of antidepressant treatment [15] and that infusion of BDNF into the hippocampus mimics the behavioural effects of antidepressants in rodents [16]. These effects and the role of BDNF in learning and memory suggest that compounds, which induce rapid and prolonged upregulation of neuroplasticity, may reduce the latency to antidepressant effects and alleviate neurocognitive dysfunction. Erythropoietin (Epo) has interesting properties, which make it a candidate for investigation as a novel therapeutic agent in neuropsychiatric diseases. It is used for the treatment of anaemia but has received renewed attention because of its direct neurobiological actions mediated through a non-haematopoietic Epo receptor system in the brain [17]. Systemically administered Epo crosses the blood–brain barrier (BBB) and has neuroprotective and neurotrophic effects in animal models of acute brain damage and chronic neurodegenerative conditions [18,19]. In humans, repeated Epo administration improves neuropsychological function in patients with schizophrenia [20] and multiple sclerosis [21]. Several complex biochemical pathways mediate these actions, including rapid up-regulation of neuroplasticity mechanisms including BDNF and neurogenesis [22,23], and anti-inflammatory actions [24,25]. Functional magnetic resonance imaging (fMRI) evidence highlights the functional neuroanatomy of emotional processing and working memory as potential neurobiological markers for depression [26,27]. Antidepressant drugs with different neurochemical actions show similar effects in such human biomarker models relevant to depression and antidepressant effects. In particular, serotonergic and noradrenergic antidepressants increase the neural and cognitive processing of positive emotional information and down-regulate the processing of negative threat-relevant information after acute and 7 days administration and in the absence of changes in mood in healthy volunteers [28,29] and in depressed patients [30]. Such actions contrast with heightened recognition and neural responses to threatening information in depression [31,32] and may be an important mechanism of antidepressant drug treatment. We have recently reported cognitive and neural effects of a single dose of Epo (40,000 IU; i.v.) in human models of antidepressant drug action independent of any haematological effects in healthy volunteers [33-36]. In particular, Epo enhanced memoryrelevant hippocampal engagement during picture retrieval 1 week post administration [34] consistent with increased hippocampal BDNF and plasticity [37,38]. Epo also modulated frontoparietal activation during spatial working memory (WM) and improved verbal fluency performance at this time after administration [35]. Finally, Epo reduced parietal-occipital activation during presentation of fearful facial expressions and the behavioural recognition of this emotion [33] consistent with decreased attentional processing and perception of threatrelevant stimuli 7 days post administration similar to effects seen with conventional antidepressant drug administration to healthy volunteers [28,29]. Notably, we demonstrated a similar reduction in the neural and cognitive response to negative threat-relevant picture stimuli following a single dose of Epo vs. placebo to acutely depressed patients [39]. Together with its known effects on neuroplasticity in animal models, these findings highlight Epo as a new candidate treatment for affective disorders. Objective The aim of the study is to investigate whether these early effects of Epo translate into improvement of mood and neurocognitive function with repeated administration in patients with treatment-resistant depression or bipolar disorder in remission and to elucidate the underlying biomarkers of potential treatment effects. Beneficial effects on these measures would highlight Epo as a candidate for adjunct treatment for affective disorders to attenuate mood symptoms and aid cognitive function in these patients groups.