Effects of Standardized Brazilian Green Propolis Extract (EPP-AF®) on Inflammation in Haemodialysis Patients: A Clinical Trial

Background Patients on haemodialysis (HD) present a significant inflammatory status, which has a pronounced negative impact on their outcomes. Propolis is a natural resin with anti-inflammatory and immunomodulatory properties. We assessed the safety and impact of a standardized Brazilian green propolis extract (EPP-AF®) on the inflammatory status in patients under conventional HD. Methods Patients were assigned to receive 200 mg/day of EPP-AF® for 4 weeks followed by 4 weeks without the drug, and changes in plasma levels of interleukins (ILs), interferon gamma (IFN-γ), tumour necrosis factor-alpha (TNF-α), and high-sensitivityc-reactive protein (HsCRP) were measured. A heatmap was used to illustrate trends in data variation. Results In total, 37 patients were included in the final analysis. Patients presented an exacerbated inflammatory state at baseline. During EPP-AF® use, there was a significant reduction in IFN-γ (p=0.005), IL-13 (p=0.04 2), IL-17 (p=0.039), IL-1ra (p=0.008), IL-8 (p=0.009), and TNF-α (p  <  0.001) levels compared to baseline, and significant changes were found in Hs-CRP levels. The heatmap demonstrated a pattern of pronounced proinflammatory status at baseline, especially in patients with primary glomerulopathies, and a clear reduction in this pattern during the use of EPP-AF®. There was a tendency to maintain this reduction after suspension of EPP-AF®. No significant side effects were observed. Conclusion Patients under haemodialysis presented a pronounced inflammatory status, and EPP-AF® was demonstrated to be safe and associated with a significant and maintained reduction in proinflammatory cytokines in this population. This trial is registered with Clinicaltrials.gov NCT04072341.


Introduction
In recent decades, chronic kidney disease (CKD) has gained a pandemic status [1]. Despite advances in its management, the mortality of CKD patients, especially on haemodialysis, remains high, mainly due to cardiovascular (CV) events [2,3]. In addition to traditional CV risk factors (dyslipidaemia, sedentary lifestyle, obesity, and smoking), haemodialysis patients are subject to nontraditional factors (uraemic toxins, hypervolemia, bone disease, oxidative stress, anaemia, and malnutrition/infammation syndrome), all of which are associated with an exacerbated and chronic infammatory state, which induces unfavourable outcomes [2][3][4][5].
Due to the multifactorial nature of infammation in CKD, it is unlikely that a single strategy could halt the entire process. In this sense, the following two therapeutic rationales are important: strategies aimed at the removal of cytokines, such as the use of dialysis techniques using diffusive and convective clearance (haemodiafltration, HDF); new pharmacological therapies that act in the reduction or modulation of proinfammatory molecules [2]. HDF potentially removes proinfammatory cytokines from the circulation [2,4,7]. However, despite convective methods, the infammatory status persists, and new therapies that modulate the production of infammatory mediators are needed.
However, it remains unknown whether propolis modulates the systemic infammatory process, which cytokines are modulated by propolis and its safety in haemodialysis patients. Terefore, we designed the present study to assess safety and impact of a standardized Brazilian green propolis extract (EPP-AF ® ) on systemic infammation in adults on haemodialysis. A standardized Brazilian green propolis extract, which is composed mainly of a green propolis produced in southeast Brazil and processed with a specifc extraction and drying process, was selected for use in this study due to its batch-tobatch reproducibility [10]. Te dosage of 200 mg/day ofered 10.6 mg of total favonoids, such as quercetin (measured according to procedures previously described) [15], as well as 27 mg of total phenolics, such as gallic acid [16].

Study Design and
Te dose of propolis was selected based on previously completed clinical studies with EPP-AF ® and with the evaluation of safety of the product [8,13,14]. Patients were instructed to take the propolis capsules after the dialysis sessions on the days of treatment so that there would be no interference with their action because removal of the compound by dialysis is unknown. Blood collections were performed at baseline and after each period. Each patient was his or her own control. All authors guarantee data integrity and fdelity to the study protocol. Tis study was the initiative of the principal investigator and correspondent.

Patient Selection and Haemodialysis
Modality. Adults on classic haemodialysis treatment for at least 1 month were included. Te following patients were excluded from this analysis: patients who missed dialysis sessions, pregnant women, patients with propolis allergy, patients using shortterm catheters, patients with recent fstula thrombosis (less than 30 days), patients undergoing kidney transplantation or some surgical treatment, patients using immunosuppressants, patients diagnosed with cancer or rheumatic diseases, and patients with active infections or need for hospitalization in the period.
All patients were under the same haemodialysis treatment throughout the study as follows: classic haemodialysis mode; 03 (three) times a week; blood fow ranging from 300 to 350 ml/min; average dialysate fow of 500 ml/min; capillary FX 100 classix or FX 80 classix, Fresenius 4008S machine (Fresenius Medical Care AG, Bad Homburg, Germany); and adjusted time sufcient for single pool KT/V greater than or equal to 1.2.

Participant Monitoring.
Patients were monitored to ensure safety, which is the major premise of the entire study. To assess the potential toxicity of the compound, an adverse efect questionnaire (time to onset, duration, type, and severity) with questions directed by systems was established, and the researchers alerted patients to be informed of the frst sign of new clinical changes as well as about the suspension of the use of the product.
Participants were evaluated during haemodialysis sessions and were followed up by telephone in the interdialytic period. Laboratory data for safety analysis were also collected. Data analysis was performed by an external and impartial statistician with no patient involvement. Adverse events that could compromise patient safety or considered serious were reported to the local research Ethics Committee, and the study was discontinued in this situation.

Laboratory Tests and Cytokine
Analysis. All biochemical tests were analysed at the Hospital São Rafael Central Laboratory, a certifed laboratory that follows international standards. Blood for analysis in each study period was always collected before the dialysis procedure. Hs-CRP analysis was performed by immunoturbidimetric assay.
For the evaluation of ILs, the blood of the patients was centrifuged, and the plasma was frozen and stored at −80°C for further analysis. Plasma levels of IFN-c, IL-1β, IL-1α, IL-1RA, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17, and TNF-α were measured in cryopreserved EDTA plasma samples using a commercially available MILLIPLEX kit based on Luminex xMAP technology (Merck, Darmstadt, Germany) according to the manufacturer's instructions. Te heatmap was used to illustrate trends in data variation over the studied periods. Tis approach was used to summarize the large numbers of comparisons.
To evaluate the potential for liver, pancreatic, or muscle toxicity, analyses were performed before and after the use of EPP-AF with aspartate aminotransferase (AST), alanine aminotransferase (ALT), amylase, and creatine phosphokinase (CPK) techniques according to conventional laboratory techniques.

Outcomes.
Te primary endpoint was the assessment of infammatory parameters using the interleukin and hs-CRP panel. Te secondary outcome was the safety assessment of EPP-AF through an adverse efect questionnaire and laboratory tests.

Statistical Analysis.
Continuous variables are expressed as the mean and standard deviation (SD) or as the mean and 95% confdence interval (95% CI). Categorical variables are expressed as absolute and relative frequencies. Student's t test or χ 2 test were used for parametric data and nonparametric variables, respectively. We used intention-to-treat analyses for the primary and secondary endpoints.
Wilcoxon signed-rank exact tests were performed to compare the IL; in the case of paired ranks, Wilcoxon signed-rank tests were performed with continuity correction. Efect sizes were calculated using the following formula: Z/(√N) [17].
Te McNemar-Bowker test was used to evaluate the proportions in paired samples for the analysis of hs-CRP and stratifed by the cut-of points of <1 mg/L, between 1 and 3 mg/L and greater than 3 mg/L, which were based on a previous study that correlated these ranges with low, moderate, and high cardiovascular risk, respectively [18].
Data for each biomarker were log-transformed, and the z score was normalized to build heatmaps to illustrate the overall trends of data variation in the studied periods. Hierarchical cluster analyses (Ward's method) were used to group the biomarkers with similar distributions between clinical groups and time points. In such analyses, dendrograms represent Euclidean distance. Te data processing and analyses were performed in JMP Pro (version 13.0.0), Graph Pad Prism (version 8), or R (version 4.1.2).

Study Population.
Te baseline characteristics are shown in Table 1. We initially evaluated 61 patients and identifed 50 eligible patients. Of these eligible patients, 13 patients were excluded due to infection, transplantation, or other surgical procedures, which could interfere with the assessment of infammatory parameters. Te fow diagram is shown in Figure 1.

International Journal of Nephrology
Heatmap analysis demonstrated a pronounced degree of infammation at baseline and a clear reduction in infammation after EPP-AF ® treatment with a tendency to maintain this reduction in the period after discontinuation of medication (posttreatment) (Figure 3(a)). In addition, we also calculated fold diferences in the concentration values of the cytokines between each time point as shown in Figure 3 (Table 2).
Heatmap analysis identifed a cluster of more infamed individuals at baseline according to the aetiology of CKD. Patients with primary glomerulopathies (GPs) showed a pronounced infammatory cytokine expression profle in plasma compared to patients with other aetiologies of CKD (diabetes or arterial hypertension) (Figure 4).

Secondary Outcomes.
Treatment with EPP-AF at the dose studied was found to be safe in this population with no description of adverse reactions or the need to discontinue the treatment. Te biochemical data for the evaluation of liver, pancreatic, and muscle enzymes are described in Table 4. Tere was no signifcant variation in these markers with EPP-AF ® treatment.

Discussion
To the best of our knowledge, this is the frst randomized study using propolis in patients under haemodialysis. In this study, we observed a high baseline infammatory state in this population composed of patients on conventional haemodialysis and a signifcant reduction in some of these infammatory molecules after the use of EPP-AF. Interestingly, by comparing the clinical groups, we observed that patients with glomerulopathies exhibited a unique biosignature characterized by a distinct profle of proinfammatory interleukins before treatment. Tere are no data in the literature comparing the degree of systemic infammation in dialysis patients due to primary glomerulopathies with other aetiologies (diabetes mellitus or hypertensive nephrosclerosis), and further studies may give strength to our fndings.
Proinfammatory cytokines are higher in haemodialysis patients than in healthy people [19], and this increase is multifactorial and caused by a greater production and reduction of their clearance with reduced renal function [2,7]. Some cytokines and other proinfammatory mediators are considered medium molecules, being part of the "uraemic toxins," representing approximately 20% of these compounds, and they are not signifcantly removed  by conventional haemodialysis [20]. Recently, important advances have been achieved in the haemodialysis technology, especially in new capillaries with a greater capacity for medium molecule clearance [21]. Removing proinfammatory molecules is an important target, but it may not be enough; reducing or modulating their production in a safe way may be an essential complementary therapy, especially in patients who do not have access to haemodiafltration or are not yet on dialysis [2]. Previous studies testing the clearance of beta-2 microglobulin in haemodialysis have shown a rebound of this molecule with a peak plasma level after dialysis, which can also occur with other medium molecules, such as interleukins [22,23]. In the present study, the use of EPP-AF reduced proinfammatory cytokines, and there was an important tendency to maintain them at lower levels after a 4-week period without treatment, which indicated a lasting immunological efect.
In haemodialysis patients, infammation is related to monocyte activation and immune dysregulation with the production of proinfammatory cytokines, resulting in acute or long-term adverse events [5]. Some infammatory mediators, such as tumour necrosis factor-alpha (TNF-α) and IL-6, have been identifed as the cause of the proinfammatory state as well as the vascular calcifcation process in haemodialysis patients [5,24]. In haemodialysis patients, it was already suggested an increase in T17 cells and decrease in Treg cells, which denotes a functional imbalance and a greater predisposition to the release of proinfammatory molecules among this population [25]. Despite its reduced use, it has also been demonstrated a signifcant increase in IL-8 in haemodialysis patients when compared with healthy patients in a control group, which may indicate a greater monocyte activation [26]. In the present study, there was a signifcant reduction in IL-6 in the posttreatment period and in TNF-α and other cytokines, such as INF-c, IL-1Ra, IL17, IL-13, and IL-8, after the use of EPP-AF. Previous studies on macrophage cell cultures have also demonstrated that propolis inhibits the production of IL-1β, an important component of the infammasome infammatory pathway [12]. Our fndings supported experimental evidence that indicates the immunomodulatory capacity of Brazilian green propolis extract through innate and adaptive immunity [27][28][29].
Cardiovascular mortality in haemodialysis patients is still high despite the advances over the last few decades [3]. It has been suggested that infammation plays an important role in atherosclerosis and cardiovascular disease [2,4,21,30]. Hs-CRP is an infammatory marker that has also been shown to be a predictor of cardiovascular outcome [18]. Interestingly, analysis of hs-CRP showed that there was an increase in the number of patients in the lower cardiovascular risk group (hs-CRP <1) during the EPP-AF treatment compared to the baseline period, but this increase was not statistically signifcant. Te short treatment period may explain the modifcation of the cytokine pattern without a signifcant change in hs-CRP. In the present study, the impact on cardiovascular outcomes was not measured by the short period of treatment, and long-term studies will be needed to clarify this hypothesis.  International Journal of Nephrology Te use of convective clearance (HDF), unlike conventional HD, is associated with the removal of medium molecules with proinfammatory potential [31]. TNF-alpha (17 kDa) and IL-6 (26 kDa) are considered medium molecules, and they have clearance in continuous modalities of the renal replacement therapy using high cut-of capillaries, but the same does not occur in conventional haemodialysis [32]. A recent study in a paediatric population has shown that the use of haemodiafltration attenuates the infammation status more than conventional haemodialysis [33]. Another study has suggested the potential beneft in endothelial protection when using convective clearance [34]. However, HDF is still poorly available due to its high cost, and most haemodialysis patients use classical therapy, which does not efectively remove medium molecules. In the present study, EPP-AF reduced these proinfammatory markers independent of dialysis as all patients were on conventional haemodialysis.
Te present study had several limitations. Tis was a single-centre study with a relatively small sample size, thus requiring additional studies in other and larger populations. Although the patients kept control of their use of medication, the study was open and there was no use of placebo. However, the present study demonstrated the safety of EPP-AF at a 200 mg/day in haemodialysis patients and assessed data on infammation that bring information that can be used in the clinical practice.
Propolis has several properties of interest in medicine and nephrology, and some studies have suggested good prospects of propolis. Due to its variable composition and dependence on geographic region, fora, and climate, the use of standardized and validated extracts of propolis, such as EPP-AF ® , is essential. An experimental study in a rat sepsis model has reported the renal protection capacity of EPP-AF ® as an antioxidant and as an antiinfammatory molecule due to its ability to reduce the Te heatmap (a) shows data for the mean plasma concentration of each indicated marker per time point, and the data were log-transformed and Z score normalized. Tis approach was used to illustrate trends in data variation. A hierarchical cluster analysis (Ward's method with 100X bootstrap) was used to group the biomarkers with similar distributions between the time points. Dendrograms represent Euclidean distance. Te red and blue colours demonstrate a higher and lower infammatory pattern, respectively. Te fold diferences between the indicated means were calculated, and log10 values were plotted (b). Diferences between each time point were examined using the Wilcoxon matched paired test. Red bars indicate mediators that were signifcantly diferent between the groups. expression of toll-like receptor 4 (TLR-4) and nuclear factor-kappa B (NF-kB) as well as cytokines in the renal tissue [11].

Conclusions
Te present study demonstrated that in this population of CKD patients under conventional haemodialysis, there was a signifcant increase in proinfammatory cytokines, especially in patients with primary glomerulopathies. Te Brazilian standardized green propolis extract (EPP-AF