Effect of recombinant erythropoietin on inflammatory markers in patients with affective disorders: A randomised controlled study

Accepted Manuscript Effect of recombinant erythropoietin on inflammatory markers in patients with affective disorders: a randomised controlled study Maj Vinberg, Pia Weikop, Niels Vidiendal Olsen, Lars Vedel Kessing, Kamilla Miskowiak PII: DOI: Reference:

S0889-1591(16)30114-3 http://dx.doi.org/10.1016/j.bbi.2016.05.006 YBRBI 2871

To appear in:

Brain, Behavior, and Immunity

Received Date: Revised Date: Accepted Date:

25 January 2016 30 April 2016 10 May 2016

Please cite this article as: Vinberg, M., Weikop, P., Olsen, N.V., Kessing, L.V., Miskowiak, K., Effect of recombinant erythropoietin on inflammatory markers in patients with affective disorders: a randomised controlled study, Brain, Behavior, and Immunity (2016), doi: http://dx.doi.org/10.1016/j.bbi.2016.05.006

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Title page Effect of recombinant erythropoietin on inflammatory markers in patients with affective disorders: a randomised controlled study.

Maj Vinberg* MD, Ph.d. [email protected] **Pia Weikop, MSc., Ph.d [email protected] ***Niels Vidiendal Olsen, MD, DMSc, [email protected] *Lars Vedel Kessing, Professor, MD, DMSc, [email protected] *Kamilla Miskowiak, MSc, Ph.d. [email protected]

•

Psychiatric Centre Copenhagen, Rigshospitalet, University Hospital of Copenhagen Blegdamsvej 9, DK-2100 Copenhagen, Denmark.

**Laboratory of Neuropsychiatry, Psychiatric Centre Copenhagen, Rigshospitalet, University Hospital of Copenhagen. *** Department of Neuroanaesthesia. The Neuroscience Centre, University Hospital of Copenhagen (Rigshospitalet) and Department of Neuroscience and Pharmacology, The Health Faculty, University of Copenhagen.

Corresponding author: *Maj Vinberg, Psychiatric Centre Copenhagen, Rigshospitalet, University Hospital of Copenhagen, Blegdamsvej 9, DK-2100 Copenhagen, Denmark Tel +45 3864 7026, e-mail: [email protected], Fax +45 3864 7022

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1. Introduction Erythropoietin (EPO) activates distinct cellular pathways in different cells, has pleiotropic cellular protective effects and may decrease inflammation, oxidative stress and apoptosis (Toba et al., 2012). EPO is a potent growth factor that may protect central nervous system (CNS) cells against apoptosis and promote proliferation of neuronal cells (Chong et al., 2013). Evidence from preclinical studies, human neuroimaging studies, and recent clinical trials provide some indication for beneficial effects of EPO on cognition that might be mediated by action on EPO receptors located in the CNS (Ehrenreich et al., 2008; Mengozz, et al.,2012). EPO may further prevent cellular inflammation by inhibiting several proinflammatory cytokines including interleukin 6 (IL6), interleukin 18 (IL-18) (Wiedenmann et al., 2015) and C-reactive protein (CRP) involved in chronic inflammatory disorders including atherosclerosis and cardiovascular disease perhaps indicating an underlying cellular stress phenomenon (Rietzschel et al., 2012).

However, the effect of EPO on the inflammatory response in humans is still unresolved. Some studies have demonstrated pro-inflammatory effects of EPO (Hojman et al., 2009; Poulsen et al., 2009) while findings from other studies point to no effect (Joyeux-Faure et al., 2012) or an antiinflammatory effect (Avasarala et al., 2005). There is accumulating evidence that affective disorders are associated with immune activation and neuro-inflammation which may be a key mediator of patients’ deficits in memory and other cognitive functions (Ryan et al., 2015). We have previously shown that treatment with EPO improves cognition (Miskowiak et al., 2014a; Miskowiak et al.,2014b). Nevertheless, the association between peripheral inflammatory markers and EPO administration has not been studied in patients with affective disorders and it is an intriguing question whether the observed pro-cognitive effects of EPO across unipolar disorder (UD) and BD could be mediated by reduction in inflammation states. In the present study, a cohort

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of patients with treatment resistant depression (TRD) and patients with BD were randomized to weekly EPO versus saline infusions over 8 weeks. Plasma IL-6, Il-18 and high sensitive CRP (hsCRP) were measured at baseline (week one), at weeks 5 (half-way through treatment), 9 (one week after treatment cessation) and 14 (six weeks follow-up). Our primary hypothesis was that EPO would influence levels of inflammatory biomarkers in TRD and BD. Secondary, to explore whether there was an association between changes in inflammatory markers and verbal memory.

2. Methods and materials 2.1. Study design and participants The two original EPO trials from which the present data is derived had a double-blind, placebocontrolled, parallel-group design and their primary outcomes have been published elsewhere (Miskowiak et al., 2014a; Miskowiak et al.,2014b). Patients were recruited through the Copenhagen Clinic for Affective Disorders, Psychiatric Centre Copenhagen. Eligible patients had an ICD-10 diagnosis of TRD, defined as failure to respond to adequate treatment with at least two different types of antidepressants given in adequate time and doses (Posternak et al., 2004) with moderate depression (HDRS-17 score >17) (sub-study 1) or a ICD-10 diagnosis of BD in full or partial remission (HDRS-17 and YMRS scores <14) with moderate cognitive difficulties according to the Cognitive and Physical Functioning Questionnaire (Fava et al., 2009) (score >4 on >2 domains) (sub-study 2).

For an extensive description of the screening procedure, randomisation, and

masking, exclusion criteria, and safety precautions see (Miskowiak et al., 2014a; Miskowiak et al., 2014b). Written informed consent was obtained from all patients. The study was approved by The Capital Regions Local Ethics Committee: H-C-2008-092, Danish Medicines Agency 2612-4020, EudraCT: 2008-04857-14, Danish Data Agency: 2008-41-2711 and ClinicalTrials.gov: NCT 00916552.

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2.2. Procedures Patients were randomised to receive weekly infusions of EPO (Eprex; 40,000 IU/ml; Janssen-Cilag) or saline (NaCl 0.9%) for eight weeks in addition to their current medication. Blood tests were taken at a weekly basis for safety monitoring. Mood symptoms were assessed at weeks 1, 5, 9, and 14 with the HDRS-17. Cognition was assessed at weeks 1, 9 and 14 with a comprehensive neuropsychological test battery including the Rey Auditory Verbal Learning Test (RAVLT).

2.3.Sampling and biochemical analyses Blood samples for inflammatory measurements were obtained at weeks 1, 5, 9, and 14 by venepuncture in the fasting state between 8.00-10.00 A.M. Five millilitres of blood was drawn into a vacuum tube containing EDTA (Vacuette®), and processing according to standard procedures and kept frozen at -80ºC until assayed. Plasma concentrations of IL-6, Il-18 were measured using a commercially available ELISA kit (Quantikine®ELISA, R&D Systems, Minneapolis, USA). Samples were analysed in duplicates and mean concentrations were calculated from a doublelogarithmic fitted model standard curve. Investigators performing the analyses were blinded to treatment group. The detection limits were 0.039 pg/ml (IL-6) and 12.5 pg/ml (IL-18), respectively. Intra and inter-assay Coefficient of Variability were 4.56 % and 12.0% for IL-6, 3.78 % and 12.8% for IL-18, respectively. Plasma hsCRP concentration was determined using a particle-enhanced immunoturbidimetry assay (Roche/Hitachi) range 0.1–20 mg/L and lowest detection limit 0.03 mg/L on an Cobas 8000, c502 modul (Roche, Basel, Schweiz).

2.4.

Statistical analysis

The primary outcome in the present study were changes in IL-6, IL-18 and hsCRP levels from baseline to post-treatment (weeks 1 to 9) as defined a priori. In additional exploratory analyses, we

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also included inflammation levels at week 5 in the analysis and explored any potential long-term effects of EPO by including the inflammation levels obtained at the six weeks follow-up assessment. Independent t-tests were used to test differences in clinical variables between the two groups at baseline, and the chi-squared test was used to examine differences in categorical demographic and clinical variables. All comparative analyses between the treatment groups (EPO, saline) were intention-to-treat (ITT) using last observation carried forward (LOCF) for missing values. Data was analysed for each diagnostic group separately (TRD, BD) using repeated measures analyses of covariance (ANCOVA) with treatment group (EPO, saline) as the between-group factor, time (weeks 1-9 / weeks 1-14) as the within-subject factor and with adjustment for stratification variables (age and gender) to minimize effects of any baseline imbalances. In additional post-hoc analyses IL-6, IL-18 and hsCRP levels at week 5 were added. Finally, the measurements from the week 14 follow-up was added to the analyses to determine whether there was a sustained effect of EPO versus saline on IL-6, IL-18 and hsCRP levels. Spearman’s correlations were used to analyse bivariate associations between change over time (from weeks 1-9 and weeks 1-14) and the three inflammatory markers and HDRS-17 and RAVLT scores, respectively. A logarithmic transformation was performed on IL-6, IL-18 and hsCRP data because of the more stable standard deviations.

3. Results 3.1. Study characteristics Patients were included and randomised for the two trials between September 2009 and October 2012. Of the 84 randomised patients, one patient withdrew on the inclusion day, leaving 83 patients for analyses (EPO: N=41; saline: N=42), of whom 74 completed per protocol (PP) (EPO: N=34, saline: N=40). Of the nine patients who did not complete PP, six patients (EPO) discontinued

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medication after 5-6 infusions because of increased platelet count (>4x109/l) but completed all assessments and three patients (one EPO week 6, one EPO week 10, one placebo, week 10) were admitted to the hospital because of acute suicide risk. As seen from Table 1, the two groups were well matched in baseline characteristics (p-values ≥ 0.12). All patients continued their medication as usual and did not change their medication in the study period.

3.2. Effects of EPO on inflammatory markers in TRD IL-6, IL-18 and hsCRP levels at baseline, and weeks 5, 9, and 14 are presented in Table 2, upper part. Independent t-test comparing IL-6, IL-18 and hsCRP levels at baseline revealed no significant differences between groups (p=0.65, p=0.35, p=0.07, respectively). In repeated measure ANCOVA, there were no general changes over time (weeks 1-9) in IL-6, IL-8 or hsCRP (p ≥0.25). There were also no differential changes over time in IL-6 or IL-18 levels between the EPO versus saline groups (p≥0.51). In contrast, hsCRP levels increased significantly in the EPO versus saline treated patients from weeks 1 to 9 (mean±SD change in ng/L: EPO: 0.43±1.64; Saline:-0.90±2.43; F (1, 39) =4.78, p=0.04). When repeating the ANCOVA including hsCRP levels at week 5 the association rendered non-significant (F (1, 39)=1.98, p=0.16). Finally, when adding the follow-up visit in week 14, there was no significant effect on hsCRP levels in the EPO-treated TRD patients (p≥0.35) (Table 2).

3.3. Effects of EPO on inflammatory markers in BD IL-6, IL-18 and hsCRP levels at baseline, weeks 5, 9, and at follow-up week 14 in patients with BD are presented in Table 2, lower part. Independent t-test comparing IL-6, Il-18 and hsCRP levels at baseline revealed no significant differences between the EPO and saline groups (p=0.06, p=0.66, p=0.24, respectively). Repeated measures ANCOVA revealed no general changes over time in IL-6, and hsCRP levels. There was a borderline significant effect of time on IL-18 levels between weeks

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1 to 9 (F (1, 42)=4.25, p=0.05) indicating a general increase, but this effect rendered non-significant after adding week 5 in the analysis (p=0.23). Group comparisons revealed no differential change in IL-6, IL-18 and hsCRP levels between the two treatment groups over time (p≥0.25 (Table 2)).

3.4. Associations between inflammatory markers, depression, BMI and cognition. In further analyses adding our primary outcome parameters,(changes in depression severity (HDRS17 scores) and verbal memory (total RAVLT scores)) between weeks 1-9, and BMI at baseline as covariates, respectively revealed no significant associations in either the patients with TRD nor in the patients with BD (p-levels≥0.14). There were no significant correlations between changes weeks 1-9 in any of the three inflammatory markers and in HDRS-17 (p≥0.10) or RAVLT scores (p≥ 0.31).

3.5. Exploratory analyses in patients with increased or normal inflammation levels Given the negative findings in the majority of the primary analyses, post-hoc exploratory analyses within the EPO and saline groups (all participants) separately were conducted to examine (i) if there was any preliminary evidence for an influence of EPO on the three inflammatory markers, , (ii) whether this would be specific for the EPO group and (iii) whether would depend on baseline inflammation levels, thus hsCRP levels were dichotomised based on the cut-off for increased inflammation (the 2.0 ng/l split is based on previous published literature including patients with bipolar disorder (Dickerson et al., 2013, Goldstein et al., 2011).

In the saline group (n=42), Spearman´s correlation analyses between changes in IL-6, IL-18, hsCRP, haemoglobin, RAVLT total and HDRS-17 scores (weeks 1-9) revealed no significant correlations (p≥0.15). Further multiple regression analyses revealed that using change in hsCRP

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levels from weeks 1-9 as the dependent variable including age, gender and hsCRP group according to hsCRP level at baseline (hsCRP≥2,0 ng/l: n=12, hsCRP<2,0 ng/l: n=30) no significant associations were found.

In the EPO group (n=41), HRDS-17 scores (weeks 1-9) showed significant positive correlations between increase in hsCRP and in IL-18 levels (r=0.48, P=0.002) and between changes in haemoglobin and IL-18-levels (r=0.35, p=0.03). Further multiple regression analyses revealed that using changes in hsCRP levels from weeks 1-9 as the dependent variable including age, gender and hsCRP group according to baseline hsCRP level (hsCRP≥2.0 ng/l; n=8, hsCRP<2.0 ng/l; n=33) , EPO-treated patients with high baseline hsCRP levels displayed a significant reduction in hsCRP levels from baseline to week 9 (F (8, 33)=-2.51, p=0.02).

4. Discussion The present study explored the effect of EPO on peripheral levels of IL-6, IL-18 and hsCRP in patients with affective disorders. First, no effects of EPO on IL-6 and IL-18 were observed in TRD or BD. Age, gender, baseline depression severity or changes from baseline to week 9 in depression severity and in verbal memory did not influence the results. EPO was associated with an increase in hsCRP levels in patients with TRD but produced no change in hsCPR levels in patients with BD. Second, no correlations were seen between verbal memory (primary cognition outcomes in the original BD trial) or depression and the three inflammatory markers; therefore, the observed effects of EPO on these outcomes did not seem to be mediated through the inflammatory pathway.

The absence of effects of EPO on IL-6 and IL-18 is consistent with the lack of clear evidence for either anti-inflammatory or pro-inflammatory actions of EPO as reflected by discrepancies between

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the positive outcomes regarding protection towards hypoxia in a wide range of cells, derived from animal studies, and the conducted clinical studies where EPO has been investigated for brain, heart and kidney protection and overall has failed to demonstrate benefits (Pearl, 2014; Nichol et al., 2015). This could be a result of the complexity of the relationship between vascular regulation and inflammatory mediators and the present observed positive correlations on changes in weeks 1-9 between haemoglobin, IL-18 and hsCRP levels in the EPO group only may indicate that the increase in hsCRP levels could be due to general cellular proliferation effects of EPO. Anyhow, the mechanism behind the possible inflammatory effects of EPO is still unclear and this study was not designed in a way so we were able to capture the acute effects of EPO on inflammation, as the present analyses were based on four sampling points (weeks 1, 5, 9 and 14) in relation to an eight week regimen of once weekly treatment. A potential more effective use of EPO for treatment of neuropsychiatric disorders necessitates thus better insight in EPO´s effects on the relevant processes at the cellular level (Freddolino et al., 2012). Further, increasing knowledge on how to target the individual patient for relevant factors that may affect outcome seems also crucial. Interestingly in this study, a subgroup of eight EPO-treated patients with elevated inflammation levels at baseline (hsCRP ≥ 2.0 ng/l) displayed a significant decrease in hsCRP levels from week 1 to 9. This suggests that the direction of an EPO-associated change in inflammatory markers depends on the individual patient´s baseline inflammation level. EPO´s potential anti-inflammatory actions may thus only be present in patients with inflammatory disturbances although this would need to be verified in a larger population stratified according on the basis of inflammatory status.

4.1. Implications

Although some studies have shown an association between immune dysfunction and cognitive impairment (Rosenblat et al., 2015) there was no correlation between inflammatory markers and

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cognition in the present study, possibly because of the strict study exclusion criteria. In particular, we excluded patients with BMI above 30, smokers or comorbid somatic disorders. Nevertheless, diabetes, obesity and cardiovascular disease have similar reports on cognitive impairment as affective disorders and there seems to be a substantial overlap between the involved brain regions and brain circuits across the aforementioned disorders (Rincon et al., 2013; Mansur et al.,2015). It would therefore be of interest to investigate agents that can safely be administered to patients with comorbid somatic disease such as glucagon-like peptide-1 (GLP-1) receptor agonist with similar pro-cognitive and anti-inflammatory potential and a less restrictive safety profile (Jespersen et al., 2013).

4.2. Conclusions Repeated EPO infusions had no cumulative effect on IL-6 and IL-18 levels in TRD or BD but produced a modest increase in hsCRP levels in patients with TRD. Changes over time in hsCRP and the other inflammatory markers were not correlated with changes in cognition or depressive symptoms, suggesting that the inflammatory system is not involved in the previously observed procognitive effects of EPO.

Financial disclosures The study was funded by the Danish Ministry of Science, Innovation and Higher Education, Novo Nordisk Foundation, Beckett Fonden, Savværksejer Juhl’s Mindefond, and the Augustinus Foundation.

The funding source had no role in the study design, in the collection, analyses, and interpretation of data, in writing the manuscript, or in the decision to submit the paper for publication.

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Table 1. Patients´ characteristics.

Age, Years Gender, no. female (%) Years of education BMI HDRS-17 score at baseline HDRS-17 score week 9 RAVLT total score baseline RAVLT total score week 9

Unipolar patients sub-study 1 EPO group N = 18 41 (9) 13 (72) 15 (3) 25 (3) 20 (4) 15 (6) 44 (11) 50 (9)

Saline group N = 21 45 (14) 14 (66) 15 (3) 26 (3) 20 (4) 16 (6) 43 (9)

Bipolar patients sub-study 2 EPO group N = 23 41 (13) 14 (61) 15 (4) 24 (2) 10 (4) 3 (2) 46 (9))

Saline group N =21 40 (11) 13 (62) 14 (4) 25 (3) 9 (2) 3 (2) 50 (9)

45 (11)

50 (9)

50 (11)

10(44)

8(38)

7(7) 6(8) 4(1)

5(4) 3(4) 4(1)

Bipolar I, diagnosis, no. (%) No. of previous depressive hypomanic manic episodes

7(5)

5 (3)

In brackets: Mean Standard Deviation Abbreviations: Epo: erythropoietin; BMI: body mass index; HDRS-17: Hamilton Depression Rating Scale 17 items; RAVLT Rey Auditory Verbal Learning Test.

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Table 2. Results for unipolar patients with treatment resistant depression and for patients with bipolar disorder. IL 6, IL 18 and hsCRP levels levels ng/l (in brackets, Mean Standard Deviation). At baseline (week 1), half-way through treatment (week 5), and upon treatment completion (week 9) and follow-up week 14. Factor time, P# and factor time by treatment group, P interactions including baseline and week 9 levels (weeks 1,9) and baseline, week 5, and week 9 (weeks 1-5-9). Unipolar patients sub-study 1 EPO group N=18 Saline group N=21 df (1, 39) IL-6 EPO

Week 1 Week 5 baseline

Week 9

Week 14

Time (weeks 1,9) P# Time (weeks 1,9) by treatment group P-values

1.31 (0.85)

1.26 (0.67)

1.28 (0.82)

1.32 (0.60)

P#=0.82, F=0.06

Time (weeks 1-59)P# Time (weeks 1, 5, 9) by treatment group P-values P#=0.95, F=0.03

IL-6 Saline

1.44 (0.99)

1.58 (1.02)

1.19 (0.59)

1.48 (1.32)

P=0.51, F=0.45

P=0.39, F=0.92

IL-18 EPO

39.17 (5.12)

41.42 (5.45)

41.74 (5.65)

38.02 (5.20)

P#=0.25 F=1.37

P#=0.56, F=0.56

IL-18 Saline

46.00 (5.11)

48.63 (5.67)

44.60 (5.77)

49.58 (5.84)

P=0.08, F=3.24*

P=0.17, F=1.84

hsCRP EPO

1.12 (0.75)

1.30 (1.10)

1.55 (1.74)

1.42 (1.20)

P#=0.84, F=0.04

P#=0.61, F=0.39

hsCRP Saline

2.36 (2.68)

2.30 (4.54)

1.45 (1.08)

1.94 (1.42)

P=0.04, F=4.78

P=0.16, F=1.98

Bipolar patients sub-study 2 Epo group N=23 Saline group N=21 df (1, 44) IL-6 1.69 EPO (1.79)

1.00 (0.55)

1.67 (1.80)

1.00 (0.58)

P#=0.67, F=0.19

P#=0.55, F=0.49

IL-6 Saline

0.91 (0.49)

0.87 (0.52)

1.15 (1.61)

0.79 (0.43)

P=0.72, F=0.13

P=0.43, F=0.78

IL-18

41.73

47.17

43.44

41.56

P#=0.05, F=4.25

P#=0.23, F=1.51

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EPO

(5.02)

(8.68)

(5.33)

(5.50)

IL-18 Saline

39.03 (2.99)

36.19 (3.49)

36.23 (3.16)

39.40 (3.87)

P=0.25, F=1.38

P=0.22, F=1.57

hsCRP EPO

2.58 (4.76)

1.02 (1.21)

2.51 (4.33)

1.35 (1.67)

P#=0.63, F=0.24

P#=0.47, F= 0.24

hsCRP Saline

1.28 (1.63)

1.66 (2.12)

1.56 (2.23)

1.30 (1.95)

P=0.84, F=0.04

P= 0.17, F=1.88

Covariates for repeated-measures ANCOVA in all analyses: age and gender. *Age significantly associated with change in IL-18 between baseline and week 9, P= 0.02, F=5.54

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•

Repeated intravenous infusions of erythropoietin (EPO) had no effect on IL-6 and IL-8 in patients with affective disorder

•

EPO produced a modest increase in hsCRP levels in patients with treatment resistant depression

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In patients with higher baseline inflammation (hsCRP ≥ 2.0 ng/l), EPO produced a reduction in hsCRP which was not replicated in the saline group

•

Changes over time in inflammatory markers were not correlated with changes in cognition suggesting that the inflammatory system is not involved in previously observed procognitive effects of EPO

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