WO2007068983A1 - New treatment - Google Patents

Abstract

A method for the treatment or prophylaxis of depression and/or anxiet y, in particular major depression, involving administration of erythropoietin or a derivative or analogue thereof.

Description

New Treatment

This invention relates to a new treatment of depression and/or anxiety, in particular major depression.

According to the DSM-IV-TR Diagnostic and Statistical Manual of Mental Disorders (4^th Edition), American Psychiatric Association, Washington 2000, mood disorders are divided into depressive disorders ("unipolar depression"), bipolar disorders, and two disorders based on etiology - mood disorder due to general medical conditions and substance-induced mood disorder.

Depressive disorders (i.e. major depressive disorder, dysthymic disorder, and depressive disorder not otherwise specified) are distinguished from bipolar disorders by the fact that there is no history of ever having had a manic, mixed, or hypomanic episode.

Mood disorder due to a general medical condition is characterized by a prominent and persistent disturbance in mood that is judged to be a direct physiological consequence of a general medical condition.

Major depressive disorder is characterized by one or more major depressive episodes (i.e. at least 2 weeks of depressed mood or loss of interest accompanied by at least four additional symptoms of depression). Dysthymic disorder is characterized by at least 2 years of depressed mood for more days than not, accompanied by additional depressive symptoms that do not meet criteria for a Major Depressive Episode. Depressive disorder not otherwise specified is included for coding disorders with depressive features that do not meet criteria for major depressive disorder, dysthymic disorder, adjustment disorder with depressed mood, or adjustment disorder with mixed anxiety and depressed mood"

For the avoidance of doubt, references to depression in this description do not include bipolar disorder or mood disorder due to a general medical condition.

Major depression is characterized by feelings of intense sadness and despair, mental slowing and loss of concentration, pessimistic worry, agitation, and self- deprecation. Physical changes also occur, especially in severe or "melancholic" depression. These include insomnia or hypersomnia, anorexia and weight loss (or sometimes overeating), decreased energy and libido, and disruption of normal circadian rhythms of activity, body temperature, and many endocrine functions.

Anxiety is a group of disorders in which symptoms of anxiety are prominent. They include panic disorder, phobias (such as agoraphobia, specific phobia, social phobia), obsessive-compulsive disorder, posttraumatic stress disorder, generalised anxiety disorder, anxiety disorder due to a general medical condition, substance-induced anxiety disorder and anxiety disorder not otherwise specified. According to the DSM-IV-TR Diagnostic and Statistical Manual of Mental Disorders (4^th Edition), American Psychiatric Association, Washington 2000, there is a close relation between depression and anxiety in that individuals with a Major Depressive Episode frequently present symptoms including obsessive rumination, anxiety, phobias and excessive worries over physical health. During a Major Depressive Episode, some individuals have panic attacks that occur in a pattern that meets criteria for panic disorder. Depression and anxiety have a high rate of co-morbidity and drug treatments for these disorders are, to a large extent, overlapping. A number of therapeutic agents have been used in the treatment of depression and anxiety, including tricyclic antidepressants (TCAs, monoamine oxidase inhibitors (MAOIs), selective serotonin re-uptake inhibitors (SSRIs) and selective norepinephrine re-uptake inhibitors (SNRIs). In general, these agents are believed to act by modulating the action and effects of neurotransmitters. Although these treatments have considerable clinical success, a major limitation of conventional antidepressants is their significant time-lag to a treatment effect which prolongs the suffering of patients, reduces patient compliance and increases risk of suicide. Novel treatment strategies with more rapid onset of antidepressant effects would therefore have immense impact on public health. Further, new agents for the treatment or prophylaxis of depression and anxiety are needed, particularly those which are recursive, or depressions that are difficult to treat, i.e. where the patient does not respond to traditional antidepressant drugs and need an adjunct treatment. If primary pathology in these disorders results from maladaptive neuronal structural change and regional neural atrophy, Epo is a potential candidate for future treatment strategies of depression and anxiety.

Erythropoietin (Epo) is a glycoprotein occurring naturally in the body with a molecular weight of about 34,000. It is now known to protect the brain and spinal cord from ischaemic and traumatic injury, the peripheral nerve from diabetic damage, the kidney from ischaemic or toxic insults and the heart from acute ischemia, either permanent or with reperfusion. Epo is used clinically in the treatment of anaemia associated with chronic renal failure in paediatric and adult patients on haemodialysis and adult patients on peritoneal dialysis, as well as in the treatment of severe anaemia of renal origin accompanied by clinical symptoms in adult patients with renal insufficiency not yet undergoing dialysis. Epo is also used to treat anaemia in cancer patients undergoing chemotherapy.

A clinically used formulation is sold under the registered trademark Eprex and is used at a concentration of from 2000 to 40,000 IU (16.8 - 336 μg/mL) in water for injection containing sodium dihydrogen phosphate dehydrate, disodium phosphate dehydrate, sodium chloride, polysorbate 80, glycine.

We have now found that Epo and its derivatives and analogues may be used in the treatment of depression and anxiety, in particular major depression.

Thus according to the invention there is provided a method for the treatment or prophylaxis of depression or anxiety which comprises administration of a therapeutically effective amount of Epo or a derivative or an analogue thereof to a patient suffering from such a condition.

The Epo may be administered parenterally/systemically, e.g. by injection. Systemic administration may be effected vascularly, intranasally and/or by inhalation.

The administration may be effected intravenously, subcutaneously or intramuscularly. Preferably, Epo is administered intravenously. Generally the patient will be a mammal, in particular a human being.

Where the depression is acute, it may be treated with a single dose from 2000 to 40,000 IU. However, generally, Epo is indicated in the treatment of chronic depression.

Typical daily doses range from 2,000 to 200,000 IU, which may be given from 1 to 4 times over the course of the day. More typically a daily dose is from 20,000 to 80,000 IU, for example 40,000 IU.

Epo derivatives include asialoerythropoietin (asialoEPO) and carbamylated Epo (CEPO).

We prefer to use human Epo, which is made by recombinant techniques from cloned hamster ovaries which have received a gene for human erythropoietin.

The Epo or a derivative or analogue thereof is generally formulated as an aqueous solution.

According to the invention we further provide the use of Epo or a derivative or an analogue thereof in the manufacture of a medicament for the treatment of depression or anxiety, particularly major depression.

The invention will now be described in more detail with reference to the following tables and figures in which:

Table 1 shows haematological parameters for subjects treated with Epo and placebo. Table 2 shows brain regions that were significantly activated during picture encoding and recognition and effects of Epo.

Table 3 shows peak cluster activation in brain regions of significantly increased BOLD response during facial expression processing in placebo-treated volunteers. Table 4 shows peak cluster activation in brain regions of significantly increased BOLD response during 2-back spatial working memory vs. control task performance in placebo- treated volunteers (main effect of task) and effects of Epo.

Figure 1 shows neuronal response during picture encoding and effects of Epo.

Figure 2 shows hippocampus response during picture recognition under Epo and placebo. Figure 3 shows neural response to overt presentations of feariul vs. neutral faces in volunteers given placebo and plot of mean percent BOLD signal change in this region under Epo and placebo.

Figure 4 shows neural response to overtly presented fearful, happy and neutral faces, effects of Epo and plot of mean percent signal change in this cluster under Epo (dark bars) and placebo

Figure 5 shows recognition of fearful facial expressions across different emotion intensity levels.

Figure 6 shows neural response during N-back working memory and control task performance, effects of Epo (areas of increased activation) and plot of mean percent signal change in this cluster under Epo and placebo.

Figure 7 shows neural response during N-back WM and control task performance, effects of Epo (areas of reduced activation) and plot of mean percent signal change in these areas under Epo and placebo.

Figure 8 shows verbal fluency performance of subjects given Epo and placebo. The effects of Erythropoietin on neural and cognitive function in human models of antidepressant drug action

Aims and Objectives

The present study explored whether Epo affects emotional, cognitive and neural function in healthy volunteers using models relevant to anxiety and depression and the effects of conventional antidepressant medication. The actions of Epo on neural responses during these processes were assessed using functional magnetic resonance imaging (fMRI), allowing the key sites of action to be identified.

Method

Subjects

Ethical approval of the study's methods was obtained from the Oxfordshire Research Ethics Committee. Healthy subjects between 18 and 41 years were screened through a medical examination and psychiatric interview using the Structured Clinical Interview for DSM-Clinical Version (SCID-IV). Exclusion criteria were: current or past history of psychiatric disorder, any significant medical conditions (including diabetes, epilepsy, hypertension, and thrombosis), previous exposure to recombinant human erythropoietin (rh-EPO), or current medication (including the contraceptive pill). Subjects who have first-degree relatives with a history of blood clotting disorders or seizure disorders were excluded. fMRI scanning also required the following exclusion criteria: spectacles, heart pacemaker, mechanical heart valve or any mechanical implants, potential pregnancy, and claustrophobia. IQ was assessed with the National Adult Reading Test (NART). A blood test was taken to check that haemoglobin, renal function, liver function and ferritin were normal. A pregnancy test was performed on female volunteers as pregnancy was an exclusion criterion. After complete description of the study to the subjects, written informed consent was obtained. Letters were sent to volunteers' general practitioners two weeks before the study began to ensure that the GP was not aware of any contraindications to the person participating.

Procedure

Study subjects received an intravenous injection of Epoetin-alfa (Eprex) (40,000 IU/ml formulation) or saline administered by a medical doctor in a double-blind randomised design. A randomisation code was drawn up by a researcher who was not directly involved in the study and the information was kept in a sealed envelope. Subjects' blood pressure, well-being and mood were monitored for three hours following the injection. Subjects were given sets of mood questionnaires to take home. Daily ratings on these allowed for an assessment of any changes in mood and well-being in the week following the drug administration.

7 or 8 days after the injection, subjects attended a test session at the John Radcliffe Hospital of approximately 4hrs. Performance on neuropsychological measures was assessed using a battery of tasks previously found to be sensitive to depression as well as antidepressant drug administration, including emotional processing and memory. Neural function was measured using fMRI during tasks designed to activate the hippocampus, amygdala, and prefrontal cortex (duration approx. lhr). A blood test was taken to control for any rise in levels of hematocrit, haemoglobin (Hb), and red cell count in the experimental group.

Psychological tests

A battery of tasks tapping into different aspects of emotional processing, executive function and memory were used in this study. These tasks have been extensively validated and refined and are sensitive to both acute and repeated administration of established antidepressants with different mechanisms of action. Hence, sub-chronic (7 days) administration of either a selective serotonin reuptake inhibitor (SSRI) or a selective noradrenergic reuptake inhibitor (SNRI) decreased the recognition of fearful facial expressions (Harmer et al. 2004). Acute administration of the SSRI citalopram has also been found to increase memory consolidation (Harmer et al. 2002). We have hypothesised that these changes in psychological function play a key role in the clinical efficacy of these antidepressant drugs; drug treatments may work in depression by altering the neural processing of emotional information which is typically negatively biased in emotional disorders and which is believed to play a critical causal role in the maintenance of symptoms. We believe that these tasks could therefore provide an early indicator of therapeutic potential of novel antidepressant drug treatments; an approach which has been endorsed by a consortium of pharmaceutical companies. In this study we used a combination of behavioural measures to assess responses to emotional and neural stimuli as well as a battery of tasks used to probe neural function in combination with fMRI, targeting neural areas playing a key role in emotion, executive function and depression.

Memory

Picture encoding and retrieval: These picture tasks were modified from Hariri et al. 2003 and were presented during scanning. Stimuli pictures were presented in a blocked paradigm in order to maximize power and sensitivity for BOLD signal change (Birn et al. 2002). In both tasks, eight blocks of pictures of complex visual scenes (24 sees) were presented interleaved with passive rest conditions (20 sees) all preceded by brief (2 sees) instructions, resulting in a task durance of 6 mins. All pictures were matched in terms of emotional valence, arousal and visual complexity. During encoding and retrieval blocks, subjects viewed six serially presented pictures. In the encoding task, they determined whether the picture represented an "indoor" or "outdoor" scene, and in the subsequent recognition task, whether the picture was "old" or "new", i.e. appeared in the previous task or not. During the interleaved rest blocks, subjects viewed a centrally presented cross. They responded by button pressing according to the instruction. Thereby, accuracy and reaction time could be assessed.

Emotional processing

Faces processing: During fMRI, the facial expression processing task was projected from a computer using e-prime software (version 1.0; Psychology Software Tools Inc., Pittsburgh, PA) onto an opaque screen at the foot of the scanner bore. Subjects viewed the screen through angled mirrors and responded by pressing the keys of a response pad with their thumbs. Facial stimuli taken from the Pictures of Affect Series (Ekman and Friesen 1976) were presented in a blocked paradigm with covert and overt conditions. In the covert condition, 4 blocks each of fearful, happy and neutral faces were presented for 17ms followed by a neutral mask face for 183ms. Mask images were faces different from target faces but of the same gender. In the overt condition, 4 blocks each of fearful, happy and neutral faces were presented for 200ms. There were 24 stimulus blocks in total, each 20s long and consisting of 10 faces/face-mask pairs. Stimulus blocks were presented in random order interspersed with 20s of fixation cross leading to a total task time of 16min. During this time, subjects performed a simple gender discrimination task.

Facial expression recognition: Following the fMRI scan, subjects were given a facial expression recognition task using a different set of facial stimuli. Pictures of faces from Ekman and Friesen (1976) were presented sequentially on a computer screen (randomized order) for 500msec. Faces expressed one of six basic emotions, happiness, surprise, sadness, fear, anger and disgust, and subjects were instructed to determine the particular emotions by pressing the corresponding keys on the keyboard as quickly and accurately as possible. Emotional expressions had been morphed between two standard images, each prototype (full emotion) and neutral, by taking a variable percentage of the shape and texture differences between the two in 10% steps (for details, see Harmer et al 2004). Four examples of every emotion at each intensity level were presented, and every face was also given in a neutral expression, making a total of 250 stimuli presentations. Accuracy, reaction time (RT) for correct choices, and misclassifications were recorded. It was hypothesized that Epo would induce similar changes to those reported following established antidepressant drug administration i.e. decrease the recognition of negative vs. positive facial expressions (Harmer et al 2003b, 2004).

Executive function

N-back working memory: The N-back task was presented in a blocked paradigm to maximise sensitivity for hippocampal BOLD signal change (Birn et al 2002). During

WM blocks, subjects viewed pseudo-random sequences of letters in a grid and indicated when a letter (target) was presented in the same square as "2 trials back" (cue). For accurate performance, volunteers were thus required to continuously update their memory, holding in their memory the square, in which a letter was displayed, as well as the location of the two preceding squares. For control blocks (0-back), they pressed a button every time they saw any letter in the centre square of the grid. Except from the

WM load, the control task was similar in every aspect to the active 2-back condition.

The N-back task consisted of three 2-back WM blocks presented interleaved with three control blocks. Each block comprised 20 stimuli presentations and lasted 1 minute incl. 500 ms stimulus display and 2500 ms inter-stimulus delay. In addition, a blank screen was presented for 15 seconds in between each block to allow for normalisation of blood flow, leading to task duration of 7.5min. During this time, subjects responded by pressing the left button if the target was identical to the cue.

All subjects received training on the 2-back task before scanning to allow for assessment of neuronal responses underlying successful memory retrieval during scanning.

Verbal fluency: Following the IMRI scan, executive function was measured with a verbal fluency task using letters previously been described as difficult (Fu et al 2002) to avoid ceiling effects. Subjects were given the letters A, G, O, N, E sequentially and instructed to generate as many words beginning with these letters as possible. Any words were allowed except for proper names or grammatical variations of a previously said word.

fMRI control experiment: Visual stimulation

A control visual stimulation paradigm was used to assess whether drug-related effects observed during facial expression processing may reflect global effects of Epo on baseline cerebral blood flow. A flashing checkerboard (frequency = 8 Hz) was presented in blocks of 15 seconds alternating with 15 seconds of a fixation cross for a total of 20 cycles. Subjects were instructed to lie with their eyes open during this control task

Subjective ratings

Mood and subjective state were assessed using the Beck Depression Inventory (BDI), Eysenck Personality Questionnaire (EPQ), Befindlichkeits Scale (BFS), State-Trait Anxiety Inventory (STAI), Visual Analogue Scales (VAS), and the positive and negative affective mood scales (PANAS) (Watson et al. 1988).

Data analysis

fMRI data acquisition

Imaging data were collected using a Siemens Sonata scanner operating at 1.5 T, at the Oxford Centre for Clinical Magnetic Resonance Research. The functional scan parameters were: Voxel Size = 3 mm³ isotropic, TR = 3000 ms, TE = 50 ms, Flip angle = 90 deg. In each EPI sequence the scanner collected 150 whole brain volumes. To facilitate later co-registration of the fMRI data into standard space, we also acquired a Turbo FLASH sequence [TR=12ms, TE= 5.65ms] voxel size=lmm³. The first two EPI images in each session were discarded to avoid Tl equilibration effects.

fMRI data analysis

One subject was excluded from the fMRI analysis because of left-handedness. The analysis was subsequently run on 23 subjects of whom 11 had received Epo. fMRI data were pre- processed and analysed using FEAT (FMRIB Expert Analysis Tool) version 5.43, part of FSL (FMRIB Software Library) (www.fmrib.ox.ac.uk/fsl). This included within- subject image realignment, spatial normalisation to a standard template using an affine procedure and spatial smoothing using a Gaussian kernel (5mm full- width-half-maximum). The time series in each session was high pass-filtered to remove large-scale non-stationary components and low frequency noise. FSL was used to compute individual subject analyses in which the time series were pre-whitened to remove temporal autocorrelation (Jezzard et al. 2001). A mixed- effects group cluster analysis (a second-level analysis) was carried to establish group effects for these same contrasts (higher-level GLM analysis; corrected for multiple comparisons, Z = 2.0, P = 0.05). Brain areas with significant activation were localised using Talairach co- ordinates (Stereotaxic Atlas of the Human Brain) (Talairach & Tournoux, 1988).

To investigate responses specific to the hippocampus during picture encoding and recognition, a region of interest (ROI) analysis was performed. ROIs for the left and right hippocampus in standard space were obtained using mri3dX (http://www.aston.ac.uk/lhs/staff/singhkd/mri3dX/mri3dX.isp), which utilises a stored representation of the Talairach Daemon Database (Lancaster et al. 2000). Mean percent hippocampal BOLD signal change during encoding and recognition was computed and compared between Epo and placebo groups.

Because of the involvement of amygdala in emotion processing (Breiter et al 1996) and modulation of amygdala response to emotional faces by conventional antidepressants (Harmer et al 2006), a ROI analysis of amygdala was performed. ROIs for the left and right amygdala in standard space were obtained with mri3dX rhttp://www.aston.ac.uk/lhs/staff/singhkd/mri3dX/mri3dX.isp^>) (Lancaster et al 2000). BOLD signal change during conscious and non-conscious processing of feariul, happy and neutral expressions was computed in left and right amygdala and compared between groups.

For the control stimulation paradigm, we compared neuronal response in subjects given Epo vs. placebo within a region of the occipital (calcarine) cortex activated by photic stimuli. The mask was based on standardized neuroanatomic divisions (Maldjian et al 2003). Any effect of Epo in this region would suggest a global drug effect on baseline cerebral haemodynamics or neural coupling.

Statistics

Behavioural data and mood ratings were analysed using repeated measures analysis of variance with group as the between-subjects factor and time as the within-subjects factor. Significant interactions were analysed further using simple main effect analyses. To obtain a further measure of memory accuracy corrected for the subject's response tendency, signal detection theory was applied. The proportion of correctly recognised pictures (cr) and of falsely recognised pictures (fr) constitute the parametric sensitivity measure: d'= 0.5 ((cr-fr) (1+cr-fr) / 4 cr (1-fr)), with a higher d- value reflecting greater accuracy of memory (Grier 1971). All statistical analyses were performed using the Statistical Package for Social Sciences (SPSS).

Results

Biological tests

Analysis of blood samples before and after the injection demonstrated no significant differences between the groups in the levels of haemoglobin, hematocrit or the red cell count (all P > 0.31; Table 1).

Mood and subjective state

The two groups were well matched in terms of general mood, personality and subjective state, indicated by the absence of significant baseline differences in BDI, EPQ, STAI, and VAS scores (all P > 0.05).

Daily mood ratings on the BFS, VAS and PANAS revealed no differences between groups over the week after drug administration (all p > 0.07). However, the PANAS demonstrated some mood improvement in the first 3 days, as reflected by reduced scores on negative affect in volunteers given Epo compared to placebo (main effect of group: F(l,21) = 4.95, P = 0.04). No adverse effects of Epo were reported and there was no effect on self-rated sleep (P > 0.2). Despite the transient mood effect of Epo, there were no differences between groups on the day of testing in baseline mood (BDI and BFS) or transient subjective state (VAS and STAI) (all P > 0.2). Absence of mood effects is also seen following short term administration of antidepressants to healthy volunteers and allows us to examine changes in the neural processing of emotional information unconfounded by changes in subjective state.

BOLD response during picture encoding and memory Main effect of task

ROI analysis revealed that left and right hippocampus were significantly activated across all subjects during picture encoding (ANOVA; main effect of encoding: F(l,22) = 35.5, P < 0.001) and recognition (ANOVA; main effect of recognition: F(l,22) = 21.6, P < 0.001) as expected. In agreement with previous findings, picture encoding and recognition additionally produced activation in a distributed neural network involved in visuospatial processing, including bilateral parahippocampal gyri, middle and inferior frontal gyri, cingulate gyri and middle temporal, parietal and occipital cortices (Hariri et al 2003) (Fig. La and l.b; areas marked with mid-grey).

Epo x task interactions

Analysis of signal change within the hippocampi revealed that Epo significantly enhanced bilateral hippocampus response during picture recognition compared with placebo (main effect of group: F(l,21) = 4.7, P = .04). This is shown in Figure 2. Epo (N = 11) enhanced bilateral hippocampal BOLD signal change during picture recognition compared to placebo (N = 12) (ANOVA; main effect of group: F(l,21) = 4.7, P = 0.04). Values represent mean percent signal change. Error bars represent the standard error (s.e.m.).

To explore whether Epo influenced neural response in other brain regions, we performed whole brain mixed-effects group cluster analyses (thresholded at Z = 2.0, P = .05, corrected). This revealed that Epo influenced a broad neuronal network engaged in encoding and recognition of complex visual scenes. During encoding, Epo-treated volunteers (N=I l) displayed increased activation in a left-lateralized frontoparietal network including the middle frontal gyrus, caudate, precuneus, and cuneus compared to placebo (N = 12) (Fig. l.a; for cluster maxima see Table 2). During recognition Epo (N = 11) up-regulated activation in the bilateral superior frontal cortex and left middle and inferior temporal cortices compared to placebo (N = 12) (Fig. l.b; for cluster maxima see Table 2). In Figures Ia and Ib areas of increased response under Epo vs. placebo are marked with dark grey; areas activated by task under placebo are marked with mid-grey. Images are thresholded at Z = 2.0, P = .05, corrected. For cluster maxima, see Table 2.

Memory performance

Recognition accuracy during the encoding and recognition tasks were high (mean encoding accuracy: 98%; mean recognition accuracy corrected for response bias: d' = 0.85). Epo did not affect accuracy or response times in either of the tasks (all P > 0.05).

BOLD response during facial expression processing

Main effect of task

Consistent with previous reports (Morris et al 1996; Vuilleumier et al 2001, 2004), conscious processing of fearful, happy and neutral facial expressions activated a largely overlapping neural network including bilateral amygdale, inferior frontal cortex, fusiform gyrus and occipital cortex and right-lateralized medial and superior temporal gyrus and inferior parietal lobule in placebo-treated volunteers. For peak cluster activation in regions activated by overt fearful, happy and neutral faces, see Table 3. Compared to overt neutral faces, presentations of overt fearful faces produced significantly greater activation in the fusiform gyrus; see Table 2. Non-conscious processing of fearful and happy expressions elicited neural response within a similar network including the right amygdala and medial temporal gyrus, left inferior and medial frontal gyrus and bilateral inferior parietal (BA 40) and occipital regions (data not shown).

Epo x task interactions

Fusiform gyrus ROI: Mean percent BOLD signal change was extracted from the area specifically engaged in processing of overt fearful vs. neutral faces in placebo-treated volunteers and compared between groups. This revealed that Epo significantly reduced fusiform response to overt fear vs. neutral faces compared to placebo (F(l,21) = 23.9, P < 0.001; Fig. 3). Figure 3.a shows neural response to overt presentations of fearful vs. neutral feces in volunteers given placebo. Overt fearful expressions produced increased fusiform gyrus activation compared with neutral faces. Images are thresholded at Z = 2.0, P = 0.05, corrected. Figure 3.b. shows a plot of mean percent BOLD signal change in this region under Epo (dark bars) and placebo (light bars). Bars show the mean; error bars show the standard error (σM). Epo reduced response to overt fearful vs. neutral faces compared to placebo (F(l,21) = 23.9, P < 0.001).

Amygdala ROI: We found no effects of Epo on amygdala BOLD signal change during conscious and non-conscious processing of happy and fearful expressions (all P > 0.07). Whole brain: During overt presentations of fearful vs. neutral expressions, there was a significant interaction between drug group and emotion within a region encompassing the right precuneus and inferior parietal and middle occipital cortex with peak cluster activation in the right precuneus (Table 3; Fig. 4.a). Figure 4.a shows neural response to overtly presented fearful, happy and neutral faces and effects of Epo. Shaded areas are the regions significantly activated by overt fearful, happy and neutral faces across control subjects. Epo reduced activation during conscious processing of fearful vs. neutral faces in a cluster encompassing the right precuneus and middle occipital cortex (area marked with blue) (cluster maximum: x = 38, y = -92, z = 12, Z-score = 3.16) compared to placebo. Images are thresholded at Z = 2.0, P = 0.05, corrected. Analysis of BOLD signal change in this region revealed that Epo reduced neural response to overt fearful vs. neutral faces compared with placebo (ANOVA; F(l,21) = 26.15, P < 0.001). Figure 4.b shows a plot of mean percent signal change in this cluster under Epo (dark bars) and placebo (light bars). Bars show the mean; error bars show the standard error (σM). There were no effects on response to covert presentation of emotional facial expressions.

Facial expression recognition

Epo reduced the behavioral recognition of fear at high intensity levels (interaction group x emotion intensity: F(9,198) = 2.10, P = 0.03; Fig. 5). Referring to figure 5, volunteers given Epo (dashed line) demonstrated reduced recognition of fearful facial expressions at high emotion intensity levels (80% and 90% intensity of emotion) compared to placebo (solid line). 1 star (*) represents p<0.05 and 2 stars (**) represents p<0.01. There were no effects on the recognition of other emotions (all P > 0.06). These results are similar to the pattern seen after both SSRI and NRI administration in healthy volunteers (Harmer et al. 2004).

Bold response during N-back working memory

Main effect of task

In agreement with previous findings (Fletcher & Henson 2001), spatial working memory activated a broad neural network including bilateral middle and inferior frontal cortex, anterior cingulate, insula, thalamus, middle temporal cortex and precuneus in placebo- treated volunteers (Fig. 6; see cluster maxima in Table 4). Region of interest analysis of the hippocampus demonstrated no consistent engagement of this region in spatial working memory (all P > 0.39), which is consistent with hippocampal disengagement in healthy human volunteers during working memory in contrast with schizophrenic patients (Meyer-Lindenberg et al 2001).

Epo x task interactions

ROI demonstrated no effect on hippocampal activation during N-back WM (P > 0.16). Exploratory whole brain mixed-effects group cluster analysis revealed a complex up- and down regulation of neural response by Epo during working memory vs. control task; Epo increased activation in the right-hemisphere middle and superior frontal gyri, precuneus and cuneus with peak cluster activation in the right precuneus and inferior frontal gyrus (Fig 6.a; for cluster maxima see Table 4). Referring to figure 6.a, which shows neural response during N-back WM and control task performance and effects of Epo, areas marked with light grey are the regions significantly activated by N-back WM vs. control task performance across control subjects. Epo increased activation during N-back WM vs. control task performance in a right-lateralised fronto-parietal network (dark areas which are circled) compared to placebo. Images are thresholded at Z = 2.0, P = 0.05, corrected. Analysis of mean percent BOLD signal change in this network demonstrated a significant interaction between task and drug group (ANOVA; task x group interaction: F(l,21) = 47.0, P < 0.001) with Epo enhancing BOLD signal during spatial working memory in Epo-treated subjects ( 1= 3.24, df = 21, P = 0.004). Figure 6.b shows a plot of mean percent signal change in these sites under Epo (dark bars) and placebo (light bars). Bars show the mean; error bars show the standard error (σM). Epo additionally down-regulated activation in network encompassing left medial frontal gyrus and bilateral precentral, cingulate, superior temporal and parietal cortices and insula (Fig 7. a; for cluster maxima, see Table 4). Figure 7.a shows neural response during N-back WM and control task performance and effects of Epo. Areas marked with light grey are the regions significantly activated by N-back WM vs. control task performance across control subjects. Epo reduced activation during N-back WM vs. control task performance in the left medial frontal gyrus and bilateral precentral, superior temporal and parietal cortices, cingulate and insula (areas marked with mid-grey) compared to placebo. Images are thresholded at Z = 2.0, P = 0.05, corrected. Analysis of mean percent BOLD signal change in these regions demonstrated reduced neural response during spatial working memory vs. control task performance (ANOVA; task x group interaction: F(l,21) = 24.23, P < 0.001; t = -1.75, df = 21, P = 0.09). Figure 7.b. shows a plot of mean percent signal change in these areas under Epo (dark bars) and placebo (light bars). Bars show the mean; error bars show the standard error (σM). Accuracy of spatial working memory was high for all volunteers (accuracy corrected for response bias: d' = 0.96). Epo did not affect accuracy or speed of working memory or response times in the control task (P > 0.05).

Verbal fluency Speed of verbal fluency in volunteers given placebo revealed that the letters O, N, and E were significantly more difficult than A and G (paired samples t-test: t= -5.18, df = 11, P < 0.001). In support of our hypothesis, Epo increased the number of words generated to these most difficult letters (O, N & E: main effect of group: F(1, 22) = 5.02, P = 0.04). This is illustrated in Figure 8 which shows the verbal fluency performance of subjects given Epo (dark) and placebo (light). Visual stimulation control experiment

Epo had no significant effect on neuronal response to photic stimulation in the region of occipital (calcarine) cortex normally activated by photic stimulation (thresholded at Z = 2.0, P = 0.05, corrected), indicating that the observed effects during faces processing reflected emotion-specific rather than global haemodynamic changes.

Discussion

The present study has addressed the question whether Epo influences cognitive and neurobio logical function in man and, if so, whether these effects could indicate a potential clinical value of Epo in the treatment of depression and anxiety. Collectively, the data demonstrate that one dose of Epo (40,000 IU) applied peripherally to healthy volunteers (1) modified neurobio logical function in a manner that suggested increased neurogenesis in the hippocampus and improved memory processes (2) influenced cognitive function and emotional processing in ways similar to serotonergic and noradrenergic antidepressant drugs and opposite to the negative biases reported in depression, (3) improved mood in the days after drug administration and after one week

(4) had no effect on haematological parameters suggesting that the observed effects of

Epo on cognitive and neurobiological function were therefore due to its direct neurobiological actions.

Epo modulates neural response during picture encoding and retrieval

In the present study, Epo modified the neural underpinnings of picture encoding and recognition in healthy volunteers. In particular, Epo enhanced bilateral hippocampal response during picture recognition. This is comparable to effects of increased BDNF expression found a similar paradigm (Hariri et al. 2003) and consistent with in vitro and in vivo evidence that Epo directly upregulates biologically active BDNF in the hippocampus (Viviani et al. 2005). Increased hippocampal activation has been reported during correct vs. incorrect picture recognition (Cansino et al 2002) which is consistent with a beneficial effect of Epo on recognition memory.

The DG of the hippocampus plays a role in spatial memory acquisition which suggests that neurogenesis in this region is important for spatial learning and memory. Newborn cells take weeks to mature but may influence learning processes in early stages because of unique properties such as increased capacity for long term potentiation (LTP) - a main neurobiological mechanism underlying early memory formation (Aimone et al. 2006). Computational simulation shows that neurogenesis improves network capacity for new information storage and forgetting of old irrelevant information (Chambers et al. 2004). Increased hippocampal neurogenesis may hence improve learning and adaptive cognitive and emotional responses to novel challenging contexts, whereas reduced neurogenesis might impair ability to cope with stress and be a basis for psychiatric disorders like depression (Chambers et al. 2004). If upregulation of BDNF and neurogenesis in the hippocampus and increased response in hippocampus-dependent tasks are important factors in the treatment of depression as hypothesized, Epo seems a promising candidate for novel treatment strategies.

Whole-brain exploratory analysis revealed that Epo increased activation in a left- lateralized frontoparietal network during picture encoding. Enhanced fronto-parietal activation during picture encoding predicts retrieval success (Cansino et al. 2002; Iidaka et al. 2006) in agreement with our hypothesis of augmented memory processes following Epo administration. During recognition, Epo additionally up-regulated activation in the bilateral superior frontal cortex and temporal cortices; again consistent with enhanced recognition memory (Cansino et al. 2002).

Despite the beneficial effects of Epo on neural response during retrieval memory, we found no effects on recognition performance. This could be a result of ceiling effects due to high accuracy in all participants in this task which was optimized for the detection of hippocampal response rather than behavioral differences. Additional studies using more difficult tasks outside a scanning environment are needed to clarify whether the increased hippocampus engagement in picture retrieval reflects improved memory.

Epo reduces neuronal response to fear

The processing of facial expressions is crucial for social interaction. Abnormalities in the processing of emotional facial expressions in depression (Bouhuys et al 1999) are thought to contribute to the difficulties which these patients experience in interpersonal relationships (Lawrence et al 2004). A bias toward perceiving others' facial emotions as negative has been shown to be a long-term vulnerability factor for depression relapse (Bouhuys et al 1999).

In the current study, placebo-treated volunteers displayed greater fusiform gyrus activation during conscious processing of fearful vs. neutral faces. This is consistent with reports of increased fusiform response to emotional, particularly fearful, compared to neutral faces (Vuilleumier and Pourtois 2006) and is thought to be mediated by anatomical feedback connections from the amygdala towards early visual areas. Epo reduced neural response to fearful vs. neutral faces in this area responding specifically to fearful facial stimuli, which suggests direct down-regulation in threat-relevant processing. Additional exploratory whole brain analysis revealed that Epo reduced neuronal response to overt fearful vs. neutral faces in a region encompassing the right precuneus and inferior parietal and occipital cortex. Increased occipito-parietal response to fearful versus happy faces in healthy subjects (Pourtois et al 2006) has been suggested as a mechanism by which attentional resources are captured by biologically important and threatening stimuli. The reduced neural response in this region is therefore consistent with the hypothesis of reduced threat-relevant processing following Epo administration.

Although attention to threat stimuli is an evolutionary adaptive defense mechanism, hypervigilance to threat stimuli is believed to play a key role in the cause and maintenance of depression and anxiety (Williams et al 1997). It has been suggested that reduced processing of fear relevant stimuli seen following SSRI administration may be important in the mechanism of conventional antidepressant drug treatment (Harmer et al 2004, 2006) therefore making Epo an intriguing candidate for treatment of emotional disorders. The amygdala plays a central role in directing attention toward emotionally salient information (Morris et al 1996). Increased amygdala response to negative faces in depressed and anxious compared to healthy subjects is thought to contribute to the hypervigilance toward negative information in these disorders (Rauch et al 2003). We have previously reported decreased amygdala responses to fearful vs. happy facial expressions following one week of the serotonergic antidepressant citalopram in healthy volunteers (Harmer et al 2006). The absence of an effect of Epo on amygdala response could therefore indicate that Epo influences fear processing through mechanisms that are different to those of serotonergic antidepressants.

Epo reduces recognition of fear The effects of Epo on neuronal responses during facial expressions processing were accompanied by modest reduction in recognition of fear in a facial expression recognition task after the scan. This is similar to the effects of repeated doses of SSRIs and SNRIs in healthy volunteers (Harmer et al 2004). Behavioral effects may be more difficult to demonstrate in a scanning environment and after repeated exposure to a similar set of images during scanning, so this finding lends important support to the primary neuroimaging outcome measures. Taken together, these results suggest that Epo reduces threat-relevant processing and is a promising agent for the treatment of anxiety and depression. Clinical application of Epo in depressed and anxious patients in combination with conventional antidepressants may represent a multi-mechanism pharmacological treatment approach.

Epo modulates neural response during spatial N-back working memory

A clinically interesting discovery was that Epo modulated executive function, as reflected by a complex up- and down- regulation of neural responses during 2-back spatial working memory. In particular, Epo increased response in a right-lateralised fronto-parietal network and reduced activation in the left medial frontal gyrus and bilateral precentral, superior temporal and parietal cortices, cingulate and insula.

Compiling neuroimaging evidence suggests a degree of hemisphere domain dominance of spatial and non-spatial working memory. While verbal working memory activates a predominantly left-lateralised fronto-parietal network, spatial working memory recruits more right-hemisphere homologous regions (Prabhakaran et al 2000). Support for such domain dominance in the PFC comes from comparison of two fMRI studies in which verbal and spatial working memory were assessed using physically identical stimuli (Walter et al 2003a). The currently employed n-back task contained manipulation of both letters and spatial locations, of which only the latter were task- relevant. The up-regulation of right-lateralised and down-regulation of left-lateralised fronto-parietal activation by Epo is therefore consistent with enhanced task-relevant strategies. This is noteworthy because schizophrenic patients fail to show such prefrontal domain dominance during verbal versus spatial n-back working memory tasks in contrast with healthy controls (Walter et al 2003b).

Indeed, the down-regulation of left medial frontal and bilateral precentral, cingulate, temporal and parietal activation could suggest a relative ease of performance in Epo-treated subjects. This notion receives support from the finding that depressed patients display increased activation in the left inferior and medial frontal, precentral and cingulate cortices during working memory compared to healthy controls (Harvey et al 2005). Reduced insula response could further indicate that Epo-treated subjects found the task less aversive than to volunteers given placebo (e.g. Davidson 2003), which would be consistent with reduced task demand.

Epo improves verbal fluency

Remarkably, Epo-treated subjects generated more words to the most difficult letters compared to subjects given placebo, suggesting a facilitating effect of Epo on some aspects of executive function in healthy volunteers. Such effects may again be relevant to drug treatment of psychiatric disorders like schizophrenia and depression since executive dysfunction is believed to be a core deficit in these disorders (Henry and Crawford 2005).

Epo influences mood

Unexpectedly, there was some improvement in mood following Epo which lasted for three days post administration, as indicated by a reduction in negative affect scores on the PANAS. This effect is similar to decreased negative affect scores on the PANAS following one week and four weeks of daily SSRI vs. placebo administration to healthy volunteers (Knutson et al 1998). This mild but rapid effect of Epo may be clinically significant in light of the significant time-lag to antidepressant actions of conventional drug treatments. A single intravenous dose of the N-methyl-D-aspartate antagonist ketamine was recently found to have rapid and robust antidepressant effects (Zarate et al 2006). However, ketamine also introduced severe adverse effects including hallucinations, dizziness and confusion, which may preclude clinical use. In contrast, acute administration of Epo was well tolerated by our subjects and its wide clinical use and high safety could make Epo an attractive clinical compound.

Limitations

A limitation of pharmacological fJvIRI studies is that the characterization of drug effects upon task-specific brain activity may be confounded by non-specific effects of the drug on neural coupling and cerebral haemodynamics (Bonne et al 1999). There is some evidence of a positive correlation between blood pressure and BOLD response (Wang et al 2006) which may confound data interpretation. Repeated administration of Epo increases red cell mass and thereby blood pressure. However in the present study, Epo did not increase red cell mass or affect neural BOLD response in the primary visual cortex normally activated by photic stimulation. It is therefore likely that the observed effect of Epo on occipitoparietal response was specific to facial expression processing rather than a result of global haemodynamic changes.

The present findings suggest that Epo alters emotional processing in ways compatible with an antidepressant action in healthy volunteers and indicate that Epo may have clinical effects in patients suffering from anxiety and depression. If a reduction in the processing of negative threat-related information is important in the therapeutic actions of antidepressant drugs as hypothesized (Harmer et al 2004), then Epo may have some potential benefit in these patients.

In conclusion, the present study provides novel insights to the effects of Epo on the memory, processing of emotional information, executive function and mood in healthy humans. One week after administration, a single dose of Epo (40,000 IU) enhanced hippocampal response during hippocampus-dependent memory retrieval, reduced the psychological and neural processing of fearful facial expressions in ways consistent with effects of antidepressant drugs, and had beneficial effects on executive function in healthy volunteers. Further, Epo also improved self-reported mood for three days post administration. Together, the present results provide evidence of the suitability of Epo as a neuroprotective and neurotrophic adjunct treatment of depression and anxiety.

Table 1 Red cell mass. Hemoglobin, hematocrit, and red cell count in the Epo (n=12) placebo groups (n=12) before and after drug administration.

Table 2 Brain regions showing significant activation during picture encoding and recognition and effects of Epo (N = 11) vs. placebo (N = 12).

Task and Region Z- Coordinates value X Y Z

Picture encoding

Main Effect of Task

Right calcarine sulcus (BA 17) 7.18 8 -86 -4

Left anterior cingulate gyrus (BA 32) 4.45 -4 2 54

Right inferior frontal gyrus (BA 44) 5.64 48 6 32

Epo > Placebo

Left precuneus (BA 19) 3.38 -14 -86 40

Left caudate body 3.53 -10 12 18

Picture recognition

Main Effect of Task

Left fusiform gyrus (BA 37) 8.43 -36 -56 -14

Epo > Placebo

Left medial frontal gyrus (BA 9) 3.21 -8 46 46

Right superior frontal gyrus (BA 9) 3.26 14 48 40

Left inferior temporal gyrus (BA 21) 4.18 -60 -8 -18

MNI coordinates (x, y, z) refer to peak activation within each cluster identified thresholded at Z=2.0 and p<0.05 corrected. BA, Brodmann area.

Table 3. Peak cluster activation in brain regions of significantly increased BOLD response during facial expression processing in placebo-treated volunteers (main effect of task).

Task and Region Z- Coordinates value X Y Z

Main Effect of Task Overt fearful faces

Right fusiform gyrus (BA 37) 7.37 44 -54 -12 Left precental gyrus (BA 6) 3.76 -50 0 40 Overt happy faces Right fusiform gyrus (BA 18) 5.53 40 -70 -14 Right medial frontal gyrus (BA 6) 5.21 38 -12 52 Left medial frontal gyrus (BA 32) 4.49 -4 4 56 Left inferior frontal gyrus (BA 45) 4.11 -30 24 4 Left medial frontal gyrus (BA 6) 4.54 -48 0 44 Left postcentral gyrus (BA 43) 4.06 -60 -16 24 Overt neutral faces Left middle occipital gyrus (BA 19) 5.6 -44 -76 -12 Right inferior frontal gyrus (BA 44) 5.15 36 0 32 Left precentral gyrus (BA 4) 3.98 -52 -4 50 Left medial frontal gyrus (BA 6) 4.91 -6 4 56 Right inferior parietal lobule (BA 40) 4.33 48 -56 46 Overt fearful vs. neutral faces Left fusiform gyrus (BA 37) 4.35 -42 -52 -14

MNI coordinates (x, y, z) refer to peak activation within each cluster identified thresholded at Z=2.0 and p<0.05 corrected. BA, Brodmann area. Table 4 Peak cluster activation in brain regions of significantly increased BOLD response during 2-back spatial working memory vs. control task performance in placebo-treated volunteers (main effect of task) and effects of Epo.

Task and Region Z- Coordinates ^Value X Y Z

Main effect of task

Right medial frontal gyrus (BA 6) 6.38 20 0 54

Epo > placebo

Right parietal lobe, precuneus (BA 7) 4.44 20 -66 50

Right frontal lobe, inferior frontal gyrus (BA 47) 3.61 50 50 -10

Placebo > Epo

Left frontal lobe, precentral gyrus (BA 4) 4.31 -48 -16 42

Right parietal lobe, inferior parietal lobule (BA 40) 3.72 48 -32 26 Left frontal lobe, medial frontal gyrus (BA 6) 4.31 0 -24 56

MNI coordinates (x, y, z) refer to peak activation within each cluster identified thresholded at Z=2.0 and p<0.05 corrected. BA, Brodmann area.

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Claims

Claims

1. Method for the treatment or prophylaxis of depression or anxiety which comprises administration of a therapeutically effective amount of Epo or a derivative or analogue thereof to a patient suffering from such a condition.

2. Method according to the preceding claim, characterised in that the administration is effected parenterally/systemically.

3. Method according to either of the preceding claims, characterised in that the administration is effected vascularly, intranasally and/or per inhalation.

4. Method according to any one of the preceding claims, characterised in that the administration is effected intravenously, subcutaneously and/or intramuscularly.

5. Method according to any one of the preceding claims, characterised in that the patient is a human being.

6. Method according to any one of the preceding claims, characterised in depression is acute depression.

7. Method according to any one of the claims 1 to 5, characterised in that the depression is chronic depression.

8. Method according to any one of the preceding claims where the daily dose is from 2000 to 200,000 IU.

9. Method according to any one of the preceding claims, wherein the daily dose is 40,000 IU.

10. Use of Epo or a derivative or an analogue thereof in the manufacture of a medicament for the treatment or prophylaxis of depression.

11. Use of Epo or a derivative or an analogue thereof in the manufacture of a medicament for the treatment or prophylaxis of anxiety.