The effect of melatonin on cisplatin-induced nephrotoxicity: A pilot, randomized, double-blinded, placebo-controlled clinical trial

European Journal of Integrative Medicine 34 (2020) 101065

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The effect of melatonin on cisplatin-induced nephrotoxicity: A pilot, randomized, double-blinded, placebo-controlled clinical trial

T

Elliyeh Ghadrdana, Sanambar Sadighib, Sholeh Ebrahimpourc, Alireza Abdollahid, Molouk Hadjibabaeia, Kheirollah Gholamia, Zahra Jahangard-Rafsanjania,e,* a

Department of Clinical Pharmacy. Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran Cancer Institute, Tehran University of Medical Sciences, Tehran, Iran c Department of Clinical Pharmacy, Virtual University of Medical Sciences, Tehran, Iran d Department of Pathology, Faculty of Medicine, Imam Khomeini Hospital Complex, Tehran University of Medical Sciences, Tehran, Iran e Research Center for Rational Use of Drugs, Tehran University of Medical Sciences, Tehran, Iran b

A R T I C LE I N FO

A B S T R A C T

Keywords: Acute kidney injury Cisplatin Kidney injury molecule-1 Neutrophil gelatinase-associated lipocalin Melatonin Randomized controlled trial

Introduction: Cisplatin-induced nephrotoxicity (CIN) is a major dose-limiting factor in cisplatin use for the management of human cancers. This study was designed to evaluate the effect of melatonin on CIN as the first clinical trial in humans. Methods: This pilot, randomized, double-blinded, placebo-controlled clinical trial was conducted with cancer patients. Participants were randomly assigned to receive either 20 mg/day oral melatonin or placebo from 24 to 48 hours before cisplatin therapy. Urine samples were collected at 0, 6 and 24 h following cisplatin infusion to evaluate urinary neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), and creatinine. Serum creatinine (SCr) levels were assessed at baseline and 72 h after cisplatin administration. Results: Fifty-five patients were randomly assigned to receive either melatonin (n = 25) or placebo (n = 30), where 3 (7.5 %) versus 9 (21.4 %) acute kidney injury (AKI) episodes were reported in each group respectively. Although urinary biomarkers increased in both groups, the magnitude of the increase was smaller in the melatonin group compared to placebo. In addition, differences of urine KIM-1/creatinine ratio at 6 h and 24 h from baseline were significantly lower in the melatonin group (2.37(−71.16 to 63.70) ng/mg and −1.45(−80.28 to 48.76) ng/mg) compared to placebo (3.83(−11.19 to 37.05) ng/mg and 0.38(−5.42 to 70.00) ng/mg). Conclusions: This study showed that the number of AKI episodes in patients who received melatonin was lower compared to the placebo group, although it was not found to be statistically significant due to the small sample size. Moreover, melatonin administration in cisplatin recipients reduced tubular kidney injury, evidenced by less increase in KIM-1/creatinine and NGAL/creatinine ratios.

1. Introduction Cisplatin is an alkylating agent which is administered for the management of solid tumors such as gastrointestinal, urogenital, lung, head and neck, sarcoma and breast cancers [1,2]. Cisplatin-induced nephrotoxicity (CIN) is a dose-limiting adverse effect (AE) of this agent [2]. CIN mainly occurs in the renal proximal tubules, particularly in the S3 segment of the tubular epithelial cells which is due to the high intensity of cisplatin accumulation compared to other cells. In addition to the renal proximal tubules, the glomeruli and distal tubules are affected by cisplatin [3]. The apoptosis of tubular cells, oxidative stress,

inflammatory response, mitochondrial dysfunction, and renal vasoconstriction are principal causes of CIN [2,3]. The renal toxicity manifestations in cisplatin-treated patients include acute kidney injury (AKI), the decline in glomerular filtration rate (GFR), salt wasting, hyperuricemia, hypocalcemia and hypomagnesemia [4]. Transient polyuria occurs concomitant with normal GFR over the first 24−48 hours post cisplatin administration which is then followed by persistent polyuria accompanied by reduced GFR after 72−96 hours [5]. AKI increases the risk of progression to chronic kidney disease (CKD) [6]. The risk of developing CKD and end-stage renal disease is

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Corresponding author at: Department of Clinical Pharmacy, Faculty of Pharmacy, Tehran University of Medical Sciences, 16th Azar St., Enqelab Sq., Tehran, Iran. E-mail addresses: [email protected] (E. Ghadrdan), [email protected] (S. Sadighi), [email protected] (S. Ebrahimpour), [email protected] (A. Abdollahi), [email protected] (M. Hadjibabaei), [email protected] (K. Gholami), [email protected] (Z. Jahangard-Rafsanjani). https://doi.org/10.1016/j.eujim.2020.101065 Received 1 October 2019; Received in revised form 19 January 2020; Accepted 7 February 2020 1876-3820/ © 2020 Elsevier GmbH. All rights reserved.

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were recruited for this study. Adult patients (18 years and older) with a Karnofsky performance status greater than 70 % and GFR of greater than 50 ml/min/1.73m2 (based on the modification of diet in renal disease (MDRD) equation) were included in this study. Exclusion criteria comprised of anyone having an active infection, chronic obstructive pulmonary disease, pancreatitis, heart failure, liver impairment and history of nephrotoxic drug administration (aminoglycoside, amphotericin B, vancomycin and contrast media) within a month prior to study enrollment, as well as concomitant use of methotrexate and pemetrexed with cisplatin. According to the Cancer Institute’s Protocol, all patients received 2 L of intravenous normal saline one day prior to cisplatin infusion, 1 L of normal saline with 10 mL of potassium chloride 15 % w/v and 2 mL of magnesium sulfate 50 % w/v just before cisplatin administration, followed by 500 mL of normal saline after completion of cisplatin infusion. Patients were assigned to receive either melatonin (Best Naturals, USA) or placebo (pharmaceutical incubator of Tehran University of Medical Sciences, Iran) using block randomization method. Randomization was done using a computer-generated randomization schedule (a block size of 4) created by supervisor of the research. Each tablet box containing melatonin or placebo tablets with similar shapes was numbered based on randomization schedule and assigned to participants sequentially as they entered into the study. Patients were studied within the two consecutive cycles of chemotherapy. In each chemotherapy cycle, melatonin (20 mg/day orally) or placebo was started 24−48 hours before cisplatin infusion and continued for 5 days. To avoid daytime sedation, melatonin was given in the early night hours. During the study, patients were followed for adherence to drug therapy and adverse drug reactions. Blood samples were obtained at baseline, before cisplatin infusion for the assessment of SCr, urea, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, sodium, potassium, calcium, albumin, bilirubin, and complete blood cell (CBC) profile. Also, a second blood specimen was collected 72 h after cisplatin administration to measure SCr concentration and follow patients for AKI occurrence. Urine NGAL, KIM-1, and creatinine were measured at three timepoints: baseline, 6 and 24 h post cisplatin infusion. Urine samples were centrifuged at 10,000 rpm for 5 min and then frozen at −70 °C until the time of analysis. Urine NGAL and KIM-1 concentrations were assayed using the enzyme-linked immunosorbent assay (ELISA) method (Bioassay Technology Laboratory, Shanghai Crystal Day Biotech Co., Ltd., Shanghai, China). NGAL and KIM-1 concentrations were adjusted based on urine creatinine to eliminate being affected by hydration therapy and/or cisplatin-related polyuria. Of note, urinary NGAL and KIM-1 were studied because urine sample results have shown more reliable data compared to serum samples [34]. Serum and urine creatinine levels were measured by the Jaffé method. CIN was assessed based on the Acute Kidney Injury Network (AKIN) classification. AKI was defined as an increase in SCr by ≥0.3 mg/dL or at least a 50 % rise from baseline [35]. Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 was used to assess adverse events [36]. As there were no previous human studies on the protective effects of melatonin against CIN, by considering a type-I error of 5 % (α = 0.05), study power of 80 % (β = 0.20), and assuming a 30 % decrease in the incidence rate of CIN with a dropout rate of 10 %, a sample size of 30 patients was calculated for each group. All participants were verbally informed about the study protocol and filled out the consent form. The study protocol was approved by the institute of pharmaceutical sciences of Tehran University of Medical Sciences (IR.TUMS.TIPS.REC.1397.016). All data were analyzed using Statistical Package for the Social Sciences (SPSS) version 23. Shapiro-Wilk’s test was utilized to assess the normality distributions of variables. Continuous variables were expressed as mean ± standard deviation (SD) or median (range). Independent sample t-test and Mann–Whitney U test were performed to compare normal and non-normal quantitative variables between the two groups. Categorical variables were presented as frequency and

two-fold in those who experience AKI episodes [7]. Therefore, attempts to prevent AKI provide renal protection from subsequent CKD. Despite hydration and electrolyte management, CIN remains a major clinical concern. In an effort to prevent this AE, several protective agents such as metformin [8], ascorbic acid, tocopherol [9], theophylline [10], amifostine [11], and N-acetyl cysteine [12] have been investigated. However, serious AEs, insufficient clinical data and the unknown impact on chemotherapy efficacy have prevented identification of a safe and effective nephroprotective strategy to prevent CIN. Melatonin is an endogenous hormone synthesized from the amino acid tryptophan by the pineal gland which regulates the body’s circadian rhythm [13]. Melatonin is a well-known antioxidant agent with immunomodulatory effects [14]. Several animal studies have addressed the promising protective effects of melatonin against CIN. Melatonin treatment decreases CIN through the regulation of nuclear factor erythroid 2-related factor 2 (Nrf2), induction of antioxidant enzyme heme oxygenase-1 (HO-1), and suppression of nuclear factor kappa B (NF-κB), as well as caspase 3 [15–17]. Moreover, the effects of melatonin on the nephrotoxicity induced by antineoplastic agents [18], antibiotics [19,20], cyclosporine [21] and radiotherapy [22] have previously been evaluated. These studies concluded that the protective effects of melatonin against renal toxicity were due to its free radical scavenging properties, antioxidant effects, anti-inflammatory and anti-apoptotic characteristics [18–23]. Some studies have demonstrated that the combination of melatonin with chemotherapeutic agents does not reduce their anticancer effects [24–26], whereas recent studies have indicated that melatonin can act as an oncostatic agent through different mechanisms such as antioxidant activity, the regulation of apoptosis, inhibition of angiogenesis and suppression of metastasis [16,26]. Surprisingly, melatonin can reduce the apoptosis of healthy renal cells while enhancing the apoptosis of malignant cells [27]. Moreover, in some cancer patients, melatonin given as an adjuvant therapy has not only proven to be effective in enhancing the therapeutic effects of anticancer therapy, but also in reducing chemotherapy-related AEs [16]. To date, an increase in serum creatinine (SCr) and decrease in GFR have been the hallmark for defining AKI, but a delayed increase in SCr following AKI and the need for early AKI detection have led to the introduction of novel AKI biomarkers such as neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) [28]. KIM-1 is produced in very low quantities in a healthy kidney, but has an increased expression subsequent to proximal tubular cell injury. NGAL is produced in other tissues besides the kidney, such as the prostate, gastrointestinal system, lung and liver. NGAL dramatically increases as soon as 3 h [29] and KIM-1 begins to increase at approximately 3−12 hours following ischemic or nephrotoxic kidney injury [30,31]. Renal vasoconstriction, proteinuria due to tubular injury and glomerular atrophy may be possible causes of hypofiltration and GFR reduction following cisplatin administration [5]. Thus, due to tubular damage caused by cisplatin, the evaluation of urinary KIM-1 and NGAL as tubular markers concomitant with SCr were considered for AKI assessment in this study. Given the efficacy of melatonin in the prevention of CIN in animal models [15,17,32,33] this study was designed to evaluate its protective effects for the first time in humans. Additionally, this study investigated the effects of melatonin on NGAL and KIM-1 as biomarkers of tubular injury. 2. Methods This pilot, randomized, double-blind, placebo-controlled clinical trial was carried out from July 2018 to March 2019 at the Cancer Institute of Imam Khomeini Hospital Complex affiliated to Tehran University of Medical Sciences (ID number: IRCT20171225038073N1). Cancer patients who received a 2 h infusion of cisplatin with an unfractionated dose of 50 mg/m2 or greater and met inclusion criteria 2

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Fig. 1. CONSORT flow diagram.

group (Standardized β = 2.32, P = 0.020), while the elevation in these urinary biomarkers in the melatonin group did not reach statistical significance at this timepoint (Standardized β = 0.75, P = 0.452 for KIM-1/creatinine ratio and Standardized β = 1.25, P = 0.211 for NGAL/creatinine ratio). Table 3 shows that the urine KIM-1/creatinine and NGAL/creatinine ratios at 24 h following cisplatin treatment fell below their baseline values in the melatonin group, whereas they remained above baseline values in the placebo group. The comparison between melatonin and placebo groups using the GEE model are presented in Table 4. The differences at 6 h from baseline for the KIM-1/creatinine ratio were significantly lower in the melatonin versus placebo group (2.37(−71.16 to 63.70) versus 3.83(−11.19 to 37.05), P = 0.040). KIM-1/creatinine ratio at 24 h demonstrated a higher difference from baseline in placebo compared to the melatonin group (0.38(−5.42 to 70.00) versus -1.45(−80.28 to 48.76), P = 0.004). The differences at each timepoint from baseline for NGAL/creatinine ratio did not reach statistical significance in either group. Melatonin and placebo tablets were well-tolerated and the only reported AE of melatonin was daytime drowsiness which significantly showed a higher incidence in the melatonin group compared to placebo. The causality assessment of this AE using the Naranjo scale was probable (score 5–8) [37]. Other reported AEs were related to the chemotherapy regimens and there was no significant difference between the two groups in nausea, vomiting, and cytopenia based on CTCAE Version 5.0 [36] (Table 5).

percentages. Chi-square or Fisher’s exact test was used to compare categorical variables between the two groups. Generalized estimating equation (GEE) model was applied during the intragroup and intergroup comparisons of the repeated data. Standardized β was reported to facilitate comparison of variables. In all comparisons, P-values less than 0.05 were considered as significant. 3. Results Of the 121 patients screened, 59 met the study inclusion criteria. Following randomization, 30 and 29 subjects were assigned to the placebo and melatonin groups, respectively. During the study, 4 patients dropped out of the melatonin group. In total, 25 patients in the melatonin group (undergoing 40 chemotherapy cycles) and 30 patients in the placebo group (undergoing 42 chemotherapy cycles) completed the study (Fig. 1). The mean age of participants in this study was 53.88 ± 13.19 years and 63.4 % were men. Although not statistically significant, the cumulative dose of cisplatin was higher in the melatonin group compared to placebo (180 (80–680) mg versus 150 (70–480) mg). Baseline demographic characteristics and laboratory data are presented in Table 1. Gastrointestinal cancer was the most frequent (43 %) among our patients. Both study groups were similar regarding chemotherapy regimens (Table 2). There were 3 (7.5 %) AKI episodes in the melatonin group and 9 (21.4 %) AKI episodes in the placebo group (P = 0.096). All AKI patients experienced stage 1 AKI based on AKIN criteria [35]. The SCr levels of patients who experienced AKI returned to their baseline values before the next chemotherapy cycle in both groups. In both melatonin and placebo groups, comparisons between urinary markers at various study timepoints compared to baseline were performed using the GEE model (Table 3). In comparison to baseline values, urine KIM-1/creatinine and NGAL/creatinine ratios increased at 6 h after cisplatin administration in both groups. The rise of urinary KIM-1/creatinine ratio at 6 h after cisplatin administration compared to baseline was significant in the placebo group (Standardized β = 2.79, P = 0.005). The NGAL/creatinine ratio at 6 h compared to baseline also showed a significant increase compared to baseline in the placebo

4. Discussion Cisplatin is a potent chemotherapeutic agent used to treat a broad spectrum of malignancies. The administration of the full dose of cisplatin for cancer treatment has been limited by major AEs such as nephrotoxicity. The manifestations of CIN include salt wasting, loss of urine concentrating ability and AKI [2]. After cisplatin administration, cisplatin or its metabolites may be absorbed by the renal tubular cells through the organic cation transporter-2. Several mechanisms have been suggested for the 3

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Table 1 Baseline demographic characteristics and laboratory data in melatonin and placebo groups at the time of trial initiation. Parameters

Melatonin group (n = 25)

Placebo group (n = 30)

P-value

Gender, male, n (%) Age (years)a Body surface area (m2)a Cisplatin dose (mg)a Cisplatin cumulative dose (mg)b Serum creatinine (mg/dL)a GFR (mL/min/1.73 m2)a Urea (mg/dL)b Serum sodium (mEq/L)a Serum potassium (mEq/L)a Serum calcium (mg/dL)a Serum albumin (g/dL)a Leukocyte count (109/L)b Hemoglobin (g/dL)a Platelet count (109/L)a Serum AST (U/L)b Serum ALT (U/L)b Serum alkaline phosphatase (U/L)b Total bilirubin (mg/dL)a Uric Acid (mg/dL)a Urine KIM-1/creatinine (ng/mg)b Urine NGAL/creatinine (ng/mg)b

14 (56) 54.44 ± 13.63 1.72 ± 0.16 98.88 ± 30.04 180 (80–680) 0.96 ± 0.26 84.41 ± 27.76 28.50 (12–70) 137.28 ± 4.72 4.45 ± 0.49 8.92 ± 0.64 4.76 ± 0.96 7.05 (5.20–14) 11.32 ± 1.44 297.60 ± 110.33 21 (12–69) 15 (10–77) 236.5 (115–1000) 0.68 ± 0.28 5.09 ± 1.17 13.48 (0.41–89.40) 219.64 (81.69–748.52)

22 (73) 52.00 ± 13.76 1.73 ± 0.18 105.77 ± 30.57 150 (70–480) 0.92 ± 0.29 93.77 ± 35.29 26.50 (16–51) 137.30 ± 4.43 4.32 ± 0.43 8.81 ± 0.75 4.45 ± 0.69 7.20 (3.70–15.30) 12.24 ± 1.93 318.97 ± 101.35 20 (11–59) 15 (10–48) 239.5 (136–1397) 0.64 ± 0.24 4.84 ± 1.36 1.43 (0.4–10.43) 74.88 (22.96–306.72)

0.178 0.514 0.670 0.406 0.456 0.558 0.286 0.619 0.986 0.326 0.567 0.175 0.598 0.094 0.462 0.648 0.667 0.866 0.555 0.476 0.0001 0.0001

Abbreviations: GFR: glomerular filtration rate; AST: alanine aminotransferase; ALT: aspartate aminotransferase; KIM-1: kidney injury molecule-1; NGAL: neutrophil gelatinase-associated lipocalin. a Data are presented as mean ± SD. b Data shown as median (range).

pathophysiology of CIN. Following cisplatin accumulation in the renal tubular cells, initiation of the apoptotic and necroptotic cell death process, inflammation, and oxidative stress cause CIN [7]. The programmed necrosis or necroptosis is established by receptor-interacting serine/threonine-protein kinase (RIPK) 1 and RIPK3. Furthermore, induction of the apoptotic pathway resulting in activation of caspase-3 plays an important role in cisplatin associated tubular cell apoptosis [17]. Cisplatin treatment increases reactive oxygen species (ROS) and decreases antioxidant activity through the reduction of superoxide dismutase, glutathione, HO-1 and catalase activity [7]. Additionally, cisplatin dysregulates the Nrf2 signaling pathway which contributes to oxidative stress [15]. Moreover, the expression of NF-κB and activator protein 1 (AP-1) have the main roles in the inflammatory responses and production of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF- α) [18] (Fig. 2). Given the pathophysiology of CIN, the possible nephroprotective effects of various pharmacological agents have been studied, but none of them showed a promising effect [38]. Currently, the preventive modalities against CIN are limited to hydration and electrolyte management [2]. To the best of our knowledge, this is the first human study that has evaluated the possible nephroprotective effect of melatonin

Table 2 Chemotherapy regimens and malignancy types in both groups. Treatment regimen, n (%)

Melatonin group (n = 25)

Placebo group (n = 30)

P-value

Cisplatin, docetaxel, 5-FU (TCF) Cisplatin, epirubicin, 5-FU (ECF) Cisplatin, gemcitabine Cisplatin, paclitaxel Cisplatin,5FU Cisplatin, etoposide Cisplatin, doxorubicin Cisplatin Malignancy types, n (%) Gastrointestinal cancer Breast cancer Sarcoma Urogenital cancer Lung cancer Head and neck

10 (40)

8 (26.7)

0.553

2 (8)

1(3.3)

4 1 3 3 2 0

5 0 5 9 1 1

(16) (4) (12) (12) (8)

12 (48) 1 (4) 1 (4) 3 (12) 2 (8) 6 (24)

(16.7) (16.7) (30) (3.3) (3.3)

12 (40) 1 (3.3) 2 (6.7) 6 (20) 4 (13) 5 (17)

0.917

Abbreviations: 5-FU: 5-fluorouracil.

Table 3 Evaluation of urinary biomarkers at 6 and 24 h compared to baseline in the melatonin and placebo groups. Group

Melatonin

Variable

Median (Range)

KIM-1/creatinine at baseline KIM-1/creatinine at 6 hours KIM-1/creatinine at 24 hours NGAL/creatinine at baseline NGAL/creatinine at 6 hours NGAL/creatinine at 24 hours

13.97 (0.41–89.40) 20.18 (0.24–76.70) 12.63 (1.39–62.62) 232.89 (8.72–1508.49) 298.29 (51.26–1343.39) 200.79 (5.34–1487.26)

Placebo Statistics

Median (Range)

Standardized β

P-value

0.75 0.86

0.452 0.385

1.25 0.86

0.211 0.389

1.72 (0.24–23.57) 3.86 (0.46–47.43) 1.86 (0.41–76.31) 81.42 (13.40–883.11) 222.38 (34.23–825.34) 71.79 (23.49–1738.15)

Standardized β is found by dividing the unstandardized β coefficient by the standard error of the mean (SEM). β is the change in response as a result of one unit change in the independent variable. Abbreviations: KIM-1: kidney injury molecule-1; NGAL: neutrophil gelatinase-associated lipocalin. 4

Statistics Standardized β

P-value

2.79 1.64

0.005 0.099

2.32 1.12

0.020 0.260

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Table 4 Comparison of changes of biomarkers in the melatonin group compared to the placebo group. Variable

KIM-1/creatinine difference 6 h from baseline KIM-1/creatinine difference 24 h from baseline NGAL/creatinine difference 6 h from baseline NGAL/creatinine difference 24 h from baseline

Melatonin

Placebo

Statistics

Median (Range)

Median (Range)

β

SEM

P-value

2.37 (−71.16 to 63.70) −1.45 (−80.28 to 48.76) 39.52 (−1384.58 to 805.29) −37.02 (−1347.28 to 1278.15)

3.83 (−11.19 to 37.05) 0.38 (−5.42 to 70.00) 98.55 (−726.16 to 728.71) 6.16 (−718.57 to 1504.57)

−12.50 −20.08 −127.87 −267.20

6.08 6.99 135.28 154.78

0.040 0.004 0.345 0.084

β is the change in response as a result of one unit change in the independent variable. Abbreviations: KIM-1: kidney injury molecule-1; NGAL: neutrophil gelatinase-associated lipocalin; SEM: standard error of the mean.

group. The incidence of CIN has been reported 8–40 % in other studies [39,40]. Differences in AKI definition, baseline hydration, renal function and performance status of patients receiving cisplatin therapy are several factors which contribute to the variability in the incidence of AKI post cisplatin administration. So far, there have been no human studies that have investigated the impact of melatonin on a nephrotoxic agent such as cisplatin. In several animal studies, melatonin has been administered for the prevention of CIN using a dosing range of 4–20 mg/kg within a specific timeframe. Melatonin ameliorated CIN through suppression of oxidative stress, inflammatory processes and apoptotic and necroptotic pathways in renal cells [15,17,32]. In more recent animal studies, melatonin has been reported to attenuate CIN through cell protection against oxidative stress by free radicals scavenging, modulating the Nrf2, increasing HO-1 signaling, reducing oxidative agents and antioxidant enzymes activation [15]. The anti-inflammatory effects of melatonin caused by decreasing AP-1 and NF-κB activation as well as pro-inflammatory mediators such as TNFα and IL-6 also contribute to its nephroprotective effects [41]. Furthermore, cisplatin-induced apoptotic and necroptotic pathways have also been reported to be significantly attenuated by melatonin through suppression of caspase 3, RIPK1 and RIPK3 [17] (Fig. 2). Based on the findings of this study, the number of AKI episodes was lower in the melatonin group, although it did not reach statistical significance. Some reasons for not observing a statistically significant decrease in AKI episodes are as follows: First, it may be due to the low

Table 5 Adverse drug reactions in the study population based on common terminology criteria for adverse events (CTCAE) version 5.0. Adverse drug reactions, n (%)

Melatonin (25)

Placebo (30)

P-value

Nausea Grade 1 Grade 2 Grade 3 Vomiting Grade 1 Grade 2 Grade 3 Anemia Grade 1 Grade 2 Grade 3 White blood cell count decreased Grade 1 Platelet count decreased Daytime drowsiness Grade 1 Grade 2

12 (48) 7 4 1 7 (28) 4 2 1 20 (80) 14 5 1 2 (8) 2 0 4 (16) 2 2

14 (46.67) 11 2 1 8 (26.67) 5 2 1 23 (76) 14 9 0 1 (3.33) 1 0 0 0 0

0.840

0.981

0.622

0.579 N/A 0.037

Grades 4 and 5 reactions were not seen during the study.

against CIN. In this study, a total of 12 AKI episodes (14.6 %) occurred (based on the AKIN criteria), including 3 episodes (report 7.5 % here) in the melatonin group and 9 episodes (report 21.4 % here) in the placebo

Fig. 2. The mechanisms of the protective effect of melatonin on cisplatin-induced nephrotoxicity. Abbreviations: ↑: Increase; ↓: Decrease; SOD: Superoxide dismutase; CAT: Catalase; GSH: Glutathione; ROS: Reactive oxygen species; Nrf2: Nuclear factor erythroid 2-related factor 2; HO-1: Heme2 oxygenase-1; NF-κB: Nuclear factor kappa B; AP-1: Activator protein 1; IL-6: Interleukin-6; TNFα: Tumor necrosis factor-α; RIPK1: Receptor-interacting serine/threonine-protein kinase 1; RIPK3: Receptor-interacting serine/ threonine-protein kinase 3; OCT2: Organic cation transporter2.

5

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regulation of proximal tubular function [27]. This study had several limitations: the small sample size and short follow-up time of study participants did not allow detecting a statistically significant finding regarding AKI occurrence in the comparison of the two groups. Given the low bioavailability of melatonin, increasing its dose and duration of treatment is likely to be effective in detecting melatonin’s full effects, however, it may increase the risk of melatonin complications. Hence, further studies with larger sample sizes and greater doses over longer durations are suggested.

dose and short duration of melatonin treatment. Due to the lack of human studies investigating melatonin effects on CIN, the administered dose was chosen based on the proposed melatonin dose used as adjuvant therapy in cancer treatment [14,26]. In such studies, melatonin was prescribed in a range of 10−50 mg/day with most having chosen a dose of 20 mg/day. On the other hand, increasing the melatonin dose causes a rise in complications such as daytime drowsiness [42]. Considering a balance between the AE and threshold for efficacy, a 20 mg single daily dose was chosen in this study. Second, the reported absolute bioavailability of melatonin is low, ranging from 3 %–37 % with large inter-individual variations in plasma levels [43,44]. It is speculated that the low bioavailability of oral melatonin can reduce its effectiveness. Lastly, the small sample size of this study limits the trial’s results and findings. However, determining the optimal sample size for first clinical trials is limited due to lack of data availability from similar trials. The baseline renal functions of participants in this study were measured using estimated GFR with the MDRD equation. It has been documented that GFR assessment by directly measuring the clearance of radioisotope technetium-99 m diethyl-triamine-penta-acetic acid was the most accurate method for GFR measurement [45]. Between the two usual mathematical formulae used for GFR estimation (Cockcroft-Gault and MDRD), MDRD correlates better with direct GFR measurement [46,47]. Also, direct GFR measurement is not routinely available in all centers due to the high cost and complexity of measurement protocols. Furthermore, in the current study CIN was defined by an increase in SCr according to the AKIN criteria. Khurana and colleagues assessed SCr level, direct measurements of GFR and estimated GFR for CIN detection. They showed that after cisplatin therapy, abnormal SCr was found in 12.5 % of patients while direct GFR was found to be abnormal in 34.38 % of the patients [47]. Additionally, the increase in SCr may not reflect CIN in all patients; since tubular damage is the main consequence of CIN, tubular markers might more accurately detect nephrotoxicity compared to SCr in cisplatin-treated patients. Thus, it seems that the assessment of renal tubular indicators such as KIM-1, NGAL, N-acetyl-bD-glucosaminidase, and alpha glutathione S-transferase concomitant with filtration markers is more reasonable for CIN detection [48]. The high sensitivity and specificity of KIM-1 and NGAL in the detection of CIN had also been reported in recent studies [48–51]. Therefore, in this study, urine NGAL and KIM-1 were assessed as early tubular injury biomarkers in order to evaluate the effect of melatonin on cisplatinrelated tubular damage. In this study, the trend of urinary biomarkers showed that urine NGAL-creatinine and KIM-1/creatinine ratios increased and peaked 6 h after cisplatin treatment, then decreased at 24 h in both groups. But the KIM-1/creatinine and NGAL/creatinine ratios at 24 h reached a magnitude lower than baseline values in the melatonin group. However, the level of these biomarkers still remained above baseline in the placebo group. Based on the results of this study, the differences at 6 and 24 h compared to baseline for KIM-1/creatinine ratio in the melatonin group were significantly lower than the placebo group. These differences for NGAL/creatinine ratio were lower in melatonin versus placebo group but did not reach statistical significance. These findings may be associated with tubular damage reduction by melatonin administration evidenced by a significant increase in KIM-1/creatinine ratio in placebo compared to the melatonin group. In line with our findings, the immunohistochemical analysis of Woo Kim et al. illustrated that the administration of melatonin significantly ameliorated the increase in KIM1 and NGAL levels after cisplatin therapy. Interestingly, melatonin induced a significant reduction in the cisplatin-induced elevation of Kim1 level [17]. Both KIM-1 and NGAL are elevated in tubular damage, but urinary KIM-1 had higher sensitivity in detecting the damage of S3 segment of proximal tubule compared to NGAL [44]. On the other hand, melatonin receptors are predominantly localized in the proximal tubule. This localization might suggest how melatonin influences the

5. Conclusion In this study, a decrease in AKI episodes was observed in the melatonin group compared to placebo, although it was not statistically significant. While urinary KIM-1/creatinine and NGAL/creatinine ratios increased in both groups, the magnitude of the increase in the melatonin group was smaller than placebo. Finally, differences of urine KIM1/creatinine ratio at 6 h and 24 h from baseline were significantly lower in the melatonin group compared to placebo, which denote the possible nephroprotective effect of melatonin through reducing cisplatin-associated tubular cell injury. Authors’ contribution Elliyeh Ghadrdan contributed to the literature search, acquisition of data, statistical analysis, and writing the original and revised manuscript. Sholeh Ebrahimpour, Sanambar Sadighi, Alireza Abdollahi, Molouk hadjibabaei, Kheirollah Gholami and Zahra Jahangard-Rafsanjani were responsible for the study design and writing the original and revised manuscript. Alireza Abdollahi was responsible for measurement of laboratory data. Elliyeh Ghadrdan, Sholeh Ebrahimpour and Zahra JahangardRafsanjani were responsible for data analysis and interpretation. Zahra Jahangard-Rafsanjani supervised the study. All authors approved the final paper. Financial support This study was supported by Tehran University of Medical Sciences. Declaration of Competing Interest None. References [1] S. Dasari, P.B. Tchounwou, Cisplatin in cancer therapy: molecular mechanisms of action, Eur. J. Pharmacol. (2014) 364–378. [2] V. Volarevic, B. Djokovic, M.G. Jankovic, et al., Molecular mechanisms of cisplatininduced nephrotoxicity: a balance on the knife edge between renoprotection and tumor toxicity, J. Biomed. Sci. 26 (1) (2019) 25. [3] L.A.B. Peres, A.D.D. unha Júnior, Nefrotoxicidade aguda da cisplatina: mecanismos moleculares, J. Bras. Nefrol. 35 (2013) 332–340. [4] R.P. Miller, R.K. Tadagavadi, G. Ramesh, W.B. Reeves, Mechanisms of cisplatin nephrotoxicity, Toxins (Basel). 2 (11) (2010) 2490–2518. [5] V. Launay-Vacher, J.B. Rey, C. Isnard-Bagnis, G. Deray, M. Daouphars, Prevention of cisplatin nephrotoxicity: state of the art and recommendations from the European Society of Clinical Pharmacy Special Interest Group on Cancer care, Cancer Chemother. Pharmacol. 61 (6) (2008) 903–909. [6] S.G. Coca, S. Singanamala, C.R. Parikh, Chronic kidney disease after acute kidney injury: a systematic review and meta-analysis, Kidney Int. 81 (5) (2012) 442–448. [7] S.J. Holditch, C.N. Brown, A.M. Lombardi, K.N. Nguyen, C.L. Edelstein, Recent advances in models, mechanisms, biomarkers, and interventions in cisplatin-induced acute kidney injury, Int. J. Mol. Sci. 20 (12) (2019). [8] J. Li, Y. Gui, J. Ren, et al., Metformin protects against cisplatin-induced tubular cell apoptosis and acute kidney injury via AMPKα-regulated autophagy induction, Sci. Rep. 6 (2016) 23975. [9] D.M. Maliakel, T.V. Kagiya, C.K. Nair, Prevention of cisplatin-induced nephrotoxicity by glucosides of ascorbic acid and alpha-tocopherol, Exp. Toxicol.

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