Tadalafil Treatment Improves Inflammation, Cognitive Function, And Mismatch Negativity Of Patients With Low Urinary Tract Symptoms And Erectile Dysfunction

Patients with Benign prostatic hyperplasia, low urinary tract symptoms, and erectile dysfunction (BPH/LUTS-ED) present chronic inflammation. We studied in patients with BPH/LUTS-ED the effect of tadalafil treatment (5 mg/day) on changes in peripheral inflammation, cognitive function, and the auditory evoked potential, “mismatch negativity” (MMN). Nine patients with BPH/LUTS-ED and 12 controls performed psychometric tests, MMN. IL-6, IL-17, IL-18, cGMP and CD4+CD28− autoreactive T-cells were measured in blood. Patients with BPH/LUTS-ED performed psychometric tests, MMN, and blood extraction at baseline and after tadalafil treatment. Patients with BPH/LUTS-ED showed increased CD4+CD28− autoreactive T-cells (p < 0.05), and higher levels of pro-inflammatory interleukins IL-6 (p < 0.001), IL-17 and IL-18 (p < 0.05), compared to controls. Patients got lower scores than controls in psychometric tests assessing mental processing speed and attention (p < 0.05), and showed lower amplitude (p < 0.01) and area (p < 0.05) of MMN wave than controls. Inflammatory, psychometric and electrophysiological parameters were normalized after tadalafil treatment. In conclusion, there is a pro-inflammatory environment in blood in patients with BPH/LUTS-ED which would induce cognitive impairment and alter MMN. Phosphodiesterase-5 inhibition with tadalafil exerts anti-inflammatory effects and ameliorates cognitive function and MMN parameters. Tadalafil could be a promising candidate for chronic treatment in other inflammatory pathologies associated with mild cognitive impairment.

Benign prostatic hyperplasia (BPH) is one of the most common benign diseases in aging man which becomes a clinical entity when associated with lower urinary tract symptoms (LUTS). BPH/LUTS, or benign prostatic hyperplasia associated with lower urinary tract symptoms, can lead to benign prostatic hypertrophy, and is usually associated with erectile dysfunction (ED) 1 .
The etiology of BPH/LUTS is multifactorial and age and volume of the prostate are associated with its development. These factors can identify patients with increased risk of progression and it is advisable to initiate early treatment.
Selective attention was assessed with the Stroop test. Controls read 118 ± 2 words in 45 seconds, in the congruent task, whereas patients read fewer words (105 ± 6; p < 0.05) (Fig. 1B). After tadalafil treatment there is a trend to increase this score in the patients, reaching the control values.
In the neutral task, the controls named 82 ± 2 colors; the patients named 74 ± 2 colors, which was significantly lower than controls (p < 0.05), and they recovered to levels similar to controls after 3 months of tadalafil treatment (83 ± 3; p < 0.01).
In the incongruent task patients with BPH/LUTS-ED got lower mean values than controls (45 ± 1 and 49 ± 1, respectively; p < 0.05) (Fig. 1B). There are no changes after tadalafil treatment. Figure 2 shows that serum levels of pro-inflammatory interleukins IL-6, IL-18, and IL-17 were significantly increased in patients compared to controls (p < 0.001, p < 0.05, and p < 0.05, respectively). Tadalafil treatment reduced significantly (p < 0.05) these levels after 6 months of treatment, getting values similar to controls.
Mean values for MMN area were lower in patients (120 ± 20 µV.ms) than controls (164 ± 17 µV.ms) (p < 0.05), and was significantly increased after six months of tadalafil treatment (231 ± 30 µV.ms) compared to values before treatment (p < 0.01) (Fig. 4C). Figure 5 shows an example of MMN wave obtained before and after treatment with Tadalafil in one of the patients with BPH/LUTS-ED.
Effects of tadalafil treatment on the IPSS and IIEF5 scores. Correlations with neuropsychological and neurophysiological parameters. There was an improvement of IPSS and IIEF5 scores after 6 months of tadalafil treatment. The mean IPSS score before treatment was of 11 ± 1, that was decreased after treatment (7 ± 1; p = 0.013). The mean score for IIEF5 was 14 ± 1 before treatment, and increased to 21 ± 1 (p = 0.0004) after 6 months of tadalafil treatment. Moreover, there was a good correlation between these scores (IPSS vs. IEEF5: r = −0.618; p = 0.01).

Discussion
By using an approach combining neuropsychological tests, electrophysiological measures, and biochemical analysis, we show that tadalafil improves the psychometric and attention tests in patients with BPH/LUTS-ED, and that parameters of the "mismatch negativity" event-related brain potential were also normalized after tadalafil treatment. Moreover, tadalafil reduces the peripheral pro-inflammatory interleukins and normalize the subset of autoreactive T cells in patients with BPH/LUTS-ED.
Patients with BPH/LUTS-ED in this study showed some alterations in mental processing speed and attention compared to controls, and after tadalafil treatment the scores in psychometric tests improved. MMN area improved in parallel with performance in mental processing speed and attention, as measured by DST test, given the good correlation found (r = 0.665; p = 0.018).
To the best of our knowledge this is the first study on effects of tadalafil on event-related brain potentials. MMN is an auditory event-related cognitive potential that reflects an attentional trigger. Multiple neuronal elements generate the MMN wave. MMN latency depends on the response speed of the neurons, whereas the amplitude is related to the maximum response of the sum of all the neurons that respond synchronously. The area is related to the accumulated response of all neurons during the entire MMN wave until returning to basal levels. In patients with BPH/LUTS-ED there are no alterations in latency, but the amplitude and the area are reduced, which is indicative of the activation of a lower number of neurons and during shorter periods. Tadalafil treatment normalizes these parameters, indicating an improvement of cortical activity.
This study agrees with those from Shim et al. 14 , who showed that a daily dosing with udenafil for 2 months improved cognitive function, in addition to erectile function, in patients with ED. Tadalafil has been reported to improve cognitive function in a mouse model of Alzheimer's disease 17 .
A study assessing the central side-effects of sildenafil on attention and memory functions in healthy young men 18 reported higher amplitude of P3 component of event-related potential (ERP) suggesting a more availability of processing resources in the sildenafil condition, and an increased capacity to focus attention on streams of auditory stimuli. Our results indicate that tadalafil would exert similar effects in patients with BPH/LUTS-ED.  www.nature.com/scientificreports www.nature.com/scientificreports/ The mechanisms by which tadalafil improves cognition in patients with BPH/LUTS-ED could be related with the function of tadalafil as PDE5 inhibitor. PDE5 is specific for cGMP hydrolysis, and enhances cGMP signaling via reducing the degradation of this cyclic nucleotide 19 . Patients in this study were treated with a daily low-dose of tadalafil (5 mg/day), which did not increase the amount of plasma cGMP, but had positive effects on cognitive functions in patients, given the improvement in psychometric tests and MMN parameters. As tadalafil has been reported to cross the blood-brain barrier 17 , it may affect attention-related processes by promoting increases of cGMP in cortical neurons. Compounds that restore pathway function and cGMP levels restore learning capacity in rats 7 .
BPH is associated with inflammation 8,9 and with chronic activation of prostate-infiltrating lymphocytes 20 . In this study we show that patients with BPH/LUTS-ED present higher levels of pro-inflammatory interleukins in peripheral blood. Moreover, a subset of autoreactive T lymphocytes (CD4 + CD28 − ) are increased in blood from these patients (about two-fold compared to controls), indicating a pro-inflammatory environment in their blood which would be affecting in some degree to cognitive functions such as attention. CD4 + CD28 − T cells expand in several diseases associated with chronic inflammation (e.g. autoimmunity, atherosclerosis, etc) 16 .The increased amount of CD4 + CD28 − T cells in patients with BPH/LUTS-ED, which can secrete inflammatory cytokines such as interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), could be promoting the differentiation of CD4 + T cells to IL-17 producers, process in which TGF-β and IL-6 are involved, and that is amplified by TNF-α and IL-1β 21 . Moreover, dysregulation of the immune response in patients with BPH/LUTS-ED can be through the high expression of IL-17 that stimulates the production of IL-6, which contributes to stromal growth in BPH 9 . In our study, patients with BPH/LUTS-ED showed increased levels of pro-inflammatory cytokines IL-6, and IL-17. IL-17 is an important player in the establishment of prostatic inflammation, and can be considered a modulator of BPH immune responses, by maintaining the local inflammatory microenvironment and amplifying the damage to prostatic tissue 8 .
In patients with BPH/LUTS-ED, PDE5 inhibition by tadalafil treatment exerts an anti-inflammatory effect by normalizing the levels of these proinflammatory interleukins and the levels of autoreactive T lymphocytes. This result agrees with Vignozzi et al. 22 , who demonstrated that tadalafil possesses antioxidant as well as antinflammatory action in addition to its vasodilatory property, and that PDE5 inhibitors suppressed other TNFα-induced genes related to inflammation (IL-6, MCP1, IL-12, COX2). The changes observed in peripheral blood could reflect those in prostatic tissue.
Moreover, this anti-inflammatory effect of tadalafil is associated to an improvement in attention and coordination functions, given the correlations found between the psychometric tests and proinflammatory interleukins, and with levels of autoreactive T-cells, as well as with IPSS scores.
We also found higher serum IL-18 levels in patients with BPH/LUTS-ED than controls. This interleukin was found to be expressed at significantly higher levels in BPH tissues, both rat and human, than in normal prostate tissues, and may act directly in BPH pathogenesis by inducing Thrombospondin-1 production in prostatic smooth muscle cells via Akt phosphorylation 23 .

Conclusion
In conclusion, there is a pro-inflammatory environment in blood from patients with BPH/LUTS-ED which would be affecting in some degree to cognitive functions and auditory evoked potential. PDE5 inhibition by tadalafil exerts an anti-inflammatory activity, and ameliorates psychometric tests, and MMN parameters. As tadalafil requires less frequent administration than other PDE5 inhibitors and it is safe in chronic treatments it may be a promising candidate for chronic treatment in other inflammatory pathologies associated with mild cognitive impairment.

Patients and Methods
Participants. Nine patients with BPH/LUTS-ED (50-70 years old) were enrolled after written informed consent. Patients were diagnosed for BPH/LUTS by the International Prostate Symptom Score (IPSS), getting a score from low to moderate (mean IPSS score: 11 ± 1; ranging from 9 to 18). Patients included presented a mild to moderate erectile dysfunction, with a mean score for IIEF5 (International Index of Erectile Function) of 14 ± 1, ranging from 12 to 17. At time of entering the study, the patients did not take any medication. Mean values for analytical parameters were: mean PSA levels: 1.26 ± 0.3 ng/mL (normal range 0-4 ng/mL); mean Testosterone levels: 495 ± 51 ng/dL (normal range 241-827 ng/dL). Exclusion criteria were: high PSA levels (>4 ng/mL); a rectal examination suspicious for prostate cancer, other diagnosed pathology; taking any other medication. Other urological pathologies (vesical tumours, litiasis…) were discarded by abdominal echography.
Twelve age-matched healthy controls (mean age: 62; range: 50-70) without BPH/LUTS-ED were also included, after written informed consent. All subjects were volunteers.
Psychometric tests, attention Stroop test and blood extraction were performed on the same day. The auditory evoked EEG potential, "mismatch negativity" (MMN), was performed in the following week after psychometric tests.
Patients with BPH/LUTS-ED were treated by the urologist with 5 mg/day of tadalafil (Cialis ® , Eli Lilly Nederland B.V.). Neuropsychological assessment and blood extraction were performed at 3 and 6 months of tadalafil treatment; MMN study was repeated after 6 months of treatment. Study protocols were approved by Scientific and Ethical Committee of Arnau de Vilanova hospital, Valencia, Spain, and conform to ethical guidelines of Helsinki Declaration. Neuropsychological assessment. We used a battery of five psychometric tests (PHES, Psychometric Hepatic Encephalopathy Score) which is usually used for the diagnosis in other pathologies with mild neurological impairment such as in minimal hepatic encephalopathy 24 .This battery is composed of five psychometric Mismatch negativity study. Controls and patients performed the mismatch negativity (MMN) analysis as previously described 15 using an 8-channels system for evoked potentials (NeuropackM1, Nihon-Kohden) and software adapted for MMN. The stimulation protocol used a stimulus train consisting of a sequence of standard tones of one frequency and duration (10 ms) which were followed by an inter-train interval of 300 ms. The first tone of the next train (differing in frequency) correspond to the "deviant". Twelve frequencies ranging in 50 Hz steps from 750 to 1250 Hz were used. The number of tones in each stimulus train varied randomly and can be 2, 4, 8, 16 or 36. A total of 4500 stimuli and 400 deviants were delivered. During the 45 min EEG recording, subjects watched a silent self-selected video film. EEG was recorded continuously from electrodes Fz, F3, F4, Cz, left and right mastoids placed according to the international 10-20 system. The vertical electrooculogram was recorded from electrodes placed above the right eye and the right outer canthus. System band pass was 0-70 Hz, with a digital sampling rate of 500 Hz. The ground electrode was placed on the central forehead and reference on the bridge of the nose. Data were analyzed as in Felipo et al. 15 The latency (ms), amplitude (μV) and area (μV. ms) of the resulting MMN wave were calculated. In patients treated with tadalafil, the MMN was analyzed twice, before and 6 months after treatment with tadalafil to assess the evolution. Statistical analysis. All data are expressed as mean values ± standard error (SEM). The Kolmogorov-Smirnov test was used for testing whether the variables had a normal distribution.
Differences between controls and patients before treatment were analyzed by t-student test or Mann Whitney test (for non-parametric variables). Differences between before and after 3 and 6 months of tadalafil treatment were analyzed by repeated measures ANOVA followed by post-hoc Tukey's multiple comparison test, except for non-parametric variables in which Friedman test followed by Dunn's test was used. As mismatch negativity was carried out before and after 6 months of treatment, results were analyzed using a Paired t-test, except for non-parametric variables, in which Wilcoxon matched-pairs signed rank test was used. The significance level was set at P < 0.05. The statistical analyses were performed using the Graphpad Prism 6.0. Pearson correlation analyses were performed with SPSS (Version 22.0, Chicago, IL).

Data availability
All data generated or analysed during this study are included in this published article.