Acute administration of cyclosporine A does not

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Acute administration of cyclosporine A does not impair attention or memory performance in healthy men Anna L. Kahla·*, Julia Kirchhofa•*, Anna Fütinga, Bernd-Otto Hütterb, Benjamin Wildec, Oliver Witzkec,d, Sven Bensona, Martin Hadamitzkya and Manfred Schedlowskia There is clinical and experimental evidence that treatment with immunesuppressive and antiproliferative drugs such as the calcineurin inhibitor cyclosporine A (CsA) is associated with mental health problems and neuropsychological disturbances in patients. However, it remains unclear whether and to what extent cognitive functions such as memory and attention processes are affected by the pharmacological treatment This is partly because of the fact that it is difficult to refer the observed neuropsychological disturbances in patients to the drug itself, to drug-induced immune suppression, or to interaction with other medication or comorbidities. Thus, in a double-blind study with healthy male participants (n = 30), we investigated whether short-term intake of therapeutic doses of CsA (4 x 2.5 mg/kg) affects attention, working memory performance, and anxiety Ieveis, measured with the Tests of Attentional Performance and the State-Trait Anxiety lnventory. The data indicate that short-term CsAadministration and subsequent suppression in interleukin-2 production are accompanied neither by a decrease in attention or memory performance nor by increased anxiety

lntroduction Sixty years after the first renal transplantation, the calcineurin inhibitor cyclosporine A (CsA) has become a widely used immunosuppressive dmg in clinical practice, especially for preventing graft rejection (Kahan, 2009), as weil as for the treatment of autoimmune diseases, for example, rheumatoid arthritis or psoriasis (Maza et al., 2011; Casrrejon et al., 2013). However, chronic treatment with CsA induces unwanted neurotoxic side effects such as tremor, seizures, neuralgia, peripheral neuropathy, impaired cognitive performance, psychotic disorders, or depression (Wijdicks, 2001; Tombazzi et al., 2006; Saner et al., 2007; Lang et al., 2009; Anghel et al., 2013) in 10--28% of patients (Bechstein, 2000). Increasing semm Ievel of CsA in transplanred patients was inversely associated with aspects of attentional performance, that is, divided attention, visual attention, and rask switching (Griva et al., 2004). Furthermore, several case reports show that CsA treatment affects memory performance and the acquisition of new information in patients (Craven, 1991). In heart transplant patients, CsA neurotoxicity is strongly suggested to be related to a relative long-term decline in cognitive brain functioning (Grimm et al., 1996), potentially affecting the patients' quality of life (Bechstein, 2000). 0955-8810 Copyright © 2017 Wolters Kluwer Health, lnc. All rights reserved.

Ieveis in healthy male volunteers, suggesting that the shortterm intake of CsA does not impair cognitive functioning. Further studies in healthy humans are needed to determine neurocognitive functions and mood states after short-term or subehrenie treatment with different immunesuppressive and antiproliferative drugs. Behavioura/ Pharmaco/ogy 28:255-261 Copyright © 2017 Wolters Kluwer Health, lnc. All rights reserved. Behavioural Pharmacology 2017, 28:255-261 Keywords: anxiety Ieveis, attention, cognitive function, cyclosporine A, working memory, human "Institute of Medical Psychology and Behavioral lmmunobiology, Departments of bNeurosurgery, cNephrology and dlnfectious Diseases, University Hospital Essen, University of Duisburg-Essen, Essen, Germany Correspondence to Manfred Schedlowski, PhD, Institute of Medical Psychology and Behavioral lmmunobiology, University Hospital Essen, Hufelandstrasse 55, D-45122 Essen, Germany e-mail: [email protected] • Anna L. Kahl and Julia Kirchhof contributed equally to the writing of this article. Received 4 July 2016 Accepted as revised 16 November 2016

Anima( experiments document neurotoxic and neuropsychological consequences for acute as weil as for chronic treatment with CsA (Bosche et al., 2015). For example, CsA treatment augmented the intensity of intracerebroventricularinduced convulsions in mice (Fujisaki et al., 2002), enhanced anxiety-like behavior in rodents (Sato et al., 2007; Mineur et al., 2014), and induced alterations of neuronal activity in the amygdala (Pacheco-Lopez et al., 2013). Moreover, a bilateral intracranial CsA administration inhibited memory formation in chicks (Bennen et al., 1996). In patients, it remains problematic, however, to refer the observed side effects to the drug itself, to drug-induced immune Suppression, or to interaction with other medication or to comorbidity in general (Bosche et al., 2015). Experimental data in rodents indicate that even shortterm peripheral treatment of CsA induces acute changes in neural activity and behavior (von Horsten et al., 1998; Pacheco-Lopez et al., 2013; Bosche et al., 2015). Thus, we investigated whether short-term intake of relatively high therapeutic dosages of CsA (a total of 4 X 2.5 mg/kg on 3 consecutive days with intervals between doses of 14, 8, and 16 h, equiva lent to 5 mg/kg per day), as used in DOI: 10.1 097/FBP.0000000000000281

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routine clinical treatment in patients with ulcerative colitis, atopic dermatitis, or ocular diseases (Jabs et al., 2000; van Assehe et al., 2003; Brandt et al., 2009), affects attention and working memory performance, as measures of cognitive functioning, in healthy male volunteers. As we previously observed that learning processes during short-term CsA-treatment in healthy male volunteers were associated with state anxiety (Ober et al., 2012), we additionally assessed state anxiety Ievels as a potential confounder.

Table 1 Demographie characteristics of the experimental and the control group Demographie parameters Age BMI School education (years) < 12 ~ 12

CsA

Placebo

P·value

25.5 ± 3.5 22.9 ± 2.5

25.1 ± 3.5 24.4 ± 1.7

0.796 0.054 0.543

2 13

14

Mean difference (95%· Cl) -0.33 (-2.95-2.29) 1.56 (- 0.03-3.1 5)

All data are expressed as mean ± SO. Cl, confidence interval; CsA, CsA-treated experimental group; placebo, placebotreated control group.

Methods Participants

A total of 30 healthy male volunteers with a mean age of 25.3 ± 0.6 years (age range: 20-32 years) and a BMI of 23.6±0.4 (range: 18.6-27.3) were recruited by public announcement in the surrounding community. Overall, 90% of the participants had 12 years or more offormal education, which is equivalent to high school education. All volunteers underwent a substantial physical and psychiatric estimation through self-reported questionnaires and an interview of their medical history. In addition, the physicians of the Department of Nephrology performed and evaluated an ECG and an ultrasonography of the kidneys. Furthermore, a urinalysis as weil as a full blood exam including assessment of renal, liver, and thyroid parameters was performed. Participants were excluded if one of the following criteria was identified: blood donations of more than 200 ml wirhin the last 2 months, intolerance for the test medication or other substances used in the study (e.g. Iactose intolerance), known history or indication of cardiovascular, hematological, liver, kidney, or respiratory diseases, alcohol or drug addiction, clinical signs of malignoma, hepatomegaly, hepatitis, or diabetes mellitus, treatment with an immunosuppressive drug or a drug affecting the immune system as weil as previous or persistent psychiatric or neurologic disorders. After inclusion, participants were allocated randomly to the experimental group (i.e. CsA-treated, 11 = 15) or the placebo group (control, 11 = 15). Participants randomized to the experimental and control groups did not differ in age, BMI, or education (Table 1). The study was approved by the local ethics committee of the University of Duisburg-Essen and follows the rules stated in the Declaration of Helsinki. All participants provided written informed consent and were paid for participation. Study design

In this double-blind study, on 3 sequential days, the participants of the experimental group received a total of four oral doses of 2.5 mglkg body weight of CsA (Sand immun Optoral; Novartis, Nürnberg, Gerrnany) in capsule form as established in previous studies of our group (Aibring et a!, 2012, 2014; Ober et a!, 2012). On day 1, they took the medication at 18:00 h, on day 2 at 08:00 and 16:00 h, and on day 3 at 08:00 h again. Blood was drawn on day 1 at 10:00 h for the baseline

measurements and on day 3 at 10:00 h again, which was 2 h after the last intake of CsA, to analyze the pharmacological effect of CsA. The control group was treated in the same rnanner, but received placebo capsules during the study. To document a putative effect of short-term CsA-treatment on attention, working rnemory performance, or anxiety Ievels, participants completed six subtests (i.e. working rnemory, alertness, flexibility, divided attention, Go/Nogo, incompatibility) of the Tests of Attentional Performance (TAP, Testbatterie zur Aufmerksamkeitsprüfung') (Zimmermann and Fimm, 2009) along with the state-version of the StateTrait Anxiety Inventory (Laux et a!, 1981). All tests were carried out on study days 1 and 3 at 10:00 h.

Attentional performance

Different subcomponents of attention were assessed using the German TAP, which is a standardized and wellestablished test battery consisting of a total of 13 subtests enabling assessment of different subcomponents of attentional performance. The TAP is used widely in clinical and experimental setrings and contains normative data for both healthy and patient populations. Here, participants completed the following six subtests, all presented on the highest Ievel of difficulty: (l) Alertness (duration: 4 min, 30 s): Tonic and phasic

alertness was assessed using the 'alertness' subtest. Briefly, participants were instructed to fixate the middle of the black computer screen and to immediately press a button when an 'X' was presented. In half of the trials, an acoustic warning signal preceded the appearance of the 'X'. (2) Flexibi lity (duration: not less than 3 min): This 'set shifting' task was used to assess the performance in shifting the attentional focus. Two figures (one rounded, one angular) were simultaneously presented on the black screen. Participants were instructed to respond as fast as possible either to the rounded or to the angular figure by pressing one of two buttons corresponding to the position of the target (i.e. the left button if the target was presented on the left side of the screen). The target (i.e. the rounded or the angular figure) changed from trial to trial.

CsA does not impair cognitive performance Kahl et a/.

(3) Go/Nogo (duration: 2 min, 45 s): Selective attention was resred using the TAP 'Go/Nogo' subtest. Five different figures were presented in a randomized order. Participanrs had to press the button as fast as possible only if rwo previously defined figures were shown. (4) lncompatibility (duration: 3 min): This 'stroop-like ' paradigm measured interference. An arrow was shown either at a left or a right position on the black screen, pointing either to the left or to the right. Participants were instructed to immediately p ress the left button if the arrow pointed to the left (and to press the right button if the arrow pointed to the right), irrespective of the position of the arrow. (5) Divided attention (duration: 3 min, 25 s): A 'dual-task' test was used to measure divided attention. Participants were instructed to respond to rwo different signals that were presented simultaneously. Two alternating tones (a high-pitched and a deep tone) were presented, and participants had to respond as fast as possible by pressing a button if the same tone occurred twice in succession. At the same time, 16 quadratic arranged dots (4 X 4) were presented in the middle of a black screen. Simultaneously with the acoustic signals, eight dots changed to 'X'es in a randomized manner. Participants had to press a button as soon as possible if four of the 'X' es formed a square. (6) Working memory (duration: 5 min): In the TAP 'working memory' subtest, a sequence of digits was presented on a black screen. Participants had to immed iately press a button if the presented digitwas similar to the penultimate digit. The tests were performed in a quiet and dark room. Participants sat in a comfortable chair with a fixed distance to the computer screen. Standardized instruction tests were presented on the computer screen and participants completed test trials to become familiarized with the task before the each subtest was started. Subtests were presented in the same order for all participants on both srudy days. Depending on the subtest and according to the TAP manual, different parameters (i.e. reaction time and/or number of mistakes/omissions) were analyzed. Cyclosporine A blood concentrations

CsA-concentration in whole blood was measured using an affinity chromatography-mediated immunoassay performed with Siemens DIMENSION Xpand (Siemens Ag, München, Germany) according to the manufacturer's recommendations. Measurement of immune parameters Ce// isolation

Blood was drawn using Li-Heparin Monoverres (Sarstedt, Nümbrecht, Germany). To isolate peripheral blood mononuclear cells from whole blood, a density gradient

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centrifugation (Ficoll Paque Plus; GE Healthcare, Munich, Germany) was applied according to the manufacturer's protocol. After isolation, cells were washed twice with Hanks' Balanced Salt Solution (Gibco Life Technologies, Darmstadt, Germany). For further analyses, cells were counted using an automated hematology analyzer (Sysmex XP-300; Sysmex Europe GmbH, Norderstedt, Germany) and adjusted to 5 x 106 cells/ml in cell culture medium (RPMI 1640 supplemented wirh GlutaMAX I, 25 mmol/1 Hepes, 10% fetal bovine serum, 50 g gentamicin; Life Technologies, Darmstadt, Germany). lnterleukin-2 expression

For interleukin (IL)-2 mRNA expression analysis, peripheral blood mononuclear cells were incubated with a 40 ng/ml mouse anti-CD3 monoconal antibody Solution (Clone: Hit 3A; BD Pharmingen, San Diego, California, USA) for 4 hat 37°C, 5% C0 2 • Cells were lysated and total R A was extracted using the R easy Micro Kir (QIAGE , Hilden, Germany) according to the manufacturer 's recommendations. With a high-capacity Reverse Transcription Kir (Applied Biosystems, Darmstadt, Germany), single-stranded cDNA was synthesized. For the real-time quantitative PCR, a 7500 Fast Real-Time PCR System (Applied Biosciences) was used in combination with Takyon Low Rox Probe MasterMix (Eurogentec, Cologne, Germany). The following cycle conditions were chosen: 5 min at 95°C, followed by 40 cycles of 3 s ar 95°C, 20 s at 60°C, and 26 s at 72°C. Primers (forward: 5'-CCAGGATGCTCACATTTAAGT TTTAC-3'; reverse: 5'-GAGGTTTGAGTTCTTCTT CTAGACACTGA-3') and probe (5'-6-FAM-TGCCCAA GAAGGCCACAGAACTGAA-BHQl-3') were designed using Primer Express 3.0 software (Applied Biosystems) and were purchased from Eurogentec. With serially diluted cDNA samples, created from purified specific PCR producrs (High Pure PCR Product Purification Kir; Roche Diagnostics, Mannheim, Germany), as an external Standard in each run, the total amount of RNA in fg/,.d was measured. Statistical analysis

Data were analyzed using PASW srausncs (version 22, SPSS Inc., Chicago, Illinois, USA). The Kolmogorov-Smimov test was applied to assess whether rhe dara fulfilled the assumption of normality. Variables (i.e. IL-2 mRNA expression, reaction time in the Alerrness and Go/Nogo rask) were log-transforrned before data analysis where appropriate. Sociodemographic characteristics of the experimental and control group were compared using independent-samples Itests or /-tests, respectively. Immunological parameters as weil as psychological parameters were analyzed using repeated-measures analysis of variance (ANOVA), followed by post-hoc computed Bonferroni-corrected I-tests. Only ANOVA interaction effects are reported if not otherwise indicated. For the six subtests of the TAP, reaction time and/

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or the number of mistakes/omissions were analyzed. For the TAP 'divided attention' task, data were analyzed separately for visual and auditory signals. For the TAP 'alertness' task, data were analyzed separately for trials wirh and without warning signals. 'Phasic alertness' was calculated as the difference berween reaction times for trials with and wirhaut warning signals. For all TAP subtests, reaction times were analyzed using repeated-measures ANOVA. Nonparametrie Wilcoxon and Mann-Whitney U-tests were used to compare the number of mistakes and omissions within and berween srudy groups. The Ievel of significance was set ar P < 0.05 (P < 0.025 for Bonferroni-corrected post-hoc tests). In addition, we calculated the effect sizes as IJ; for ANOVA interactions and as Cohen's d for differences berween means. All data are expressed as mean ±SO.

Results The pharmacological effects of short-term trearment with CsA were reflected by a significant increase in blood CsA-Ievels (F =4 16.6; P