The Effects of Nano-Curcumin as A Nutritional Strategy on Clinical and Inammatory Factors in Children with Cystic Fibrosis: A Protocol Study and Review Article

Background: Cystic brosis(CF) is a genetic disorder, which is caused by the CFTR protein defects. Along with CFTR dysfunction, inammation plays a key role in the disease outcomes. Inammation may develop due to the internal dysfunction of the CFTR protein or external factors. Curcumin affects the CFTR protein function primarily as a corrector and potentiator and secondary as an anti-inammatory and antimicrobial agent. The present study aims to assess the impact of Nano-curcumin on clinical and inammatory markers in children with CF. Methods: This prospective, double blind control trial will be conducted at Akbar Children's Hospital in Mashhad, Iran. children with CF will be enrolled based on the eligibility criteria. Placebo and curcumin with the maximum dose of 80 milligrams considering the body surface of the patients will be administrated for three months. The primary outcome is to evaluate inammation based on serum interleukin-6, interleukin-10, and hs-CRP, stool calprotectin, and neutrophil count of nasopharyngeal swab. The secondary outcome involved clinical assessment via spirometry, anthropometrics, and quality of life. They will be assessed before and after three mounts. Discussion: Due to the multifarious effects of curcumin on CF disease, it can be used as a nutritional strategy in the treatment of cystic brosis.


Introduction Background and rationale {6a}
Cystic brosis (CF) is a common, life-shortening, autosomal recessive, genetic disorder in Caucasian populations. The disease prognosis has improved in recent decades with the advances in the treatment procedures, and the mortality rate has decreased by 2% annually. The median age of survival in the male and female patients with CF has been reported to be 65 and 56 years, respectively (1). In ammation plays a critical role in CF lung pathology and disease progression. The abnormal function of cystic brosis transmembrane conductance regulator (CFTR) alters the chloride ion transport in the epithelial cells. Hence, leading to the dehydration of the airway surface liquid. This condition causes the secreted mucin to thicken and contribute to airway obstruction, thereby predisposing the patients to infection. This is considered to be the leading cause to trigger in ammation (2). The dysregulation of the proin ammatory mediators secondary to the innate and adaptive immune dysfunction in CF patients is another cause of excessive in ammation. It remains unclear whether the intrinsic effects of mutant CFTR on the immune system are the major cause of in ammation or exposure to the CF airway infection and in ammatory environment could exert extrinsic effects toward this phenomenal (3,4). With the advent of new modi ed CFTR protein drugs such as ivacaftor, lumacaftor, tezacaftor, a signi cant impact has been observed on the treatment of patients. Ivacaftor and tezacaftor act as the potentiator through enabling the opening chloride channel, while lumacaftor acts as the corrector through increasing the tra cking of the CFTR proteins to the outer cell membrane (5,6). Although these drugs could reduce airway in ammation, they cannot eliminate it completely (7). On the other hand, these drugs affect speci c mutations and do not cover all patients with various mutations (7). Therefore, anti-in ammatory treatments are an inevitable therapy for CF patients. Curcumin is the main ingredient in turmeric with a substantial impact on the reduction of in ammatory factors (8) (9). Previous ndings have indicated that curcumin could correct the CFTR protein function (corrector) in the F508del mutation, which is associated with the CFTR protein misfolding (10). Moreover, curcumin has been reported to be acts as a potentiator and release the gating defect of CFTR protein and promoting the channel activity (11,12).According to the literature, curcumin can reduce in ammation through antimicrobial effects, which contribute to its extrinsic impact. On the other hand, curcumin is soluble in water and has a short half-life and low bioavailability. Various products have been obtained for the improvement of bioavailability through increasing the absorption rate from the gastrointestinal tract and reducing the liver metabolism (13)(14)(15).
The present study aimed to evaluate the effects of Nano-curcumin on CF patients in order to achieve the intrinsic and extrinsic effects of curcumin on the in ammatory process in these patients. This is a prospective, single center, double-blind, parallel, randomized placebo-controlled clinical trial. The study protocol was written following the standard Protocol Items Recommendation for Interventional Trials(SPIRIT) Checklist. (Additional le 1)

Objectives {7}
The present study aimed to evaluate the effects of Nano-curcumin on CF patients in order to achieve the intrinsic and extrinsic effects of curcumin on the in ammatory process in these patients.

Trial design {8}
This is a prospective, single center, double-blind, parallel, randomized placebo-controlled clinical trial. The study protocol was written following the standard Protocol Items Recommendation for Interventional Trials(SPIRIT) Checklist. (Additional le 1)

Study setting {9}
The study population will consist of the patients with known CF referring to the Cystic Fibrosis Clinic at Akbar Children's Hospital, Mashhad, Iran.

Inclusion Criteria
The inclusion criteria of the study are as follows: 1) one or more typical phenotypic features of CF and a minimum of an elevated sweat chloride concentration on two/more occasions or two mutations known to cause CF on separate alleles; 2) age of 5-18 years; 3) pulmonary and gastrointestinal involvement; 4) ability to perform spirometry maneuvers and the minimum FEV1 of ≥30% compared to the same age, gender, and height in the normal population; 5) the percentage of oxygen saturation based on pulse the oximetry of ≥90% at room temperature; 6) no cardiovascular, hepatic, and renal failure; 7) absence of celiac disease and rheumatoid arthritis; 8) no acute pulmonary exacerbation requiring hospitalization within the past four weeks; 9) absence of acute respiratory tract infection and 10) informed consent for participation.

Exclusion Criteria
The exclusion criteria of the study are the lack of adherence to the drug regimen and presence of drug intolerance like nausea or vomiting or any allergic reaction.

Who will take informed consent? {26a}
Initially, the objectives of the trial will be clari ed by the main administrator to the children's parents and legal guardians, and written informed consent will be obtained from them at the time of enrolment. The Patients with eligible criteria will be enrolled to the study.

Intervention description{11a}
The Patients with eligible criteria will be enrolled to the study. Thereafter, With the use of the strati ed randomization procedure, participants will be divided into two groups according to disease severity (severe/mild-to-moderate) using spirometry with 40% FEV1 cutoff range. Following that, simple sampling method will be used to select the patients from each group to receive either Nano-curcumin drop or Nanocurcumin-like placebo drop. (Figure 1) Nano-curcumin (Exir Nano Sina Drug Company, Iran) is prepared as Nano micelle in the form of 70 milligrams of drops in 1 cc, and the placebo with the same color, taste and odor.
To adjust the drug dose for different ages and considering that the maximum acceptable dose based with the most signi cant impact and minimum side-effects was 80 milligrams, the required amount will be obtained for each subject based on the ratio of the body surface area of the patients. The curcumin and placebo glasses will be labeled A and B by Exir Nano Sina company, respectively and made available to the patients through the double-blind design. The duration of the treatment period is three months.

Criteria for discontinuing or modifying allocated interventions {11b}
Although no special side effect has been reported until now, with any intervention related side effects we will stop the intervention and report it to the Ethics committee of Mashhad university of medical sciences (MUMS) for decision making.

Strategies to improve adherence to interventions {11c}
In order to control patients for taking curcumin and placebo, they will be followed-up by phone every month. curcumin and placebo will be given to the patients for one month and patients will be asked to bring the bottle of drug in their next visit for assessing their compliance and if anyone used less than 70% of drop, he or she will be excluded.

Relevant concomitant care permitted or prohibited during the trial {11d}
Concomitant intervention will be: 1-Pancratic Enzyme Replacement Therapy(PERT): All CF patients with pancreatic de ciency need PERT for enzyme replacement as a regard we enrolled CF patients with gastrointestinal involvement, all of our patients use Creon as a usual treatment that doses are adjusted to the level of dietary fat. 2-Antibiotic: Tobramycin inhalation is an antibiotic which is used for pseudomonas infection. 3-Anti-in ammatory: Low dose azithromycin treatment frequently prescribed chronically as an anti-in ammatory effect in CF patients six years and older. We use it in our patients. 4-Anti Acid: suggested for patient who fail to respond to maximal dose of PERT and in patient with re ux disease. 5-Inhaled hypertonic saline: nebulized hypertonic (7%)saline is recommended twice daily to all patients six years and older. We use it in our patients. 6-Inhaled Beta2 agonist: is administrated in CF patient with moderate to severe lung disease twice a week.

7-Fat soluble vitamins: for all CF patients with pancreatic de ciency.
Provisions for post-trial care {30}

Outcomes {12}
The primary outcomes of the current trial are changes from the baseline to three months of the intervention in the in ammation at three levels: systemic in ammation by assessing IL-6 as an in ammatory agent, IL-10 as an anti-in ammatory agent, and hs-CRP level in the blood samples, pulmonary in ammation with the neutrophil count, and bacterial/viral culture on the nasopharyngeal swab, and gastrointestinal in ammation with the calprotectin level in the fecal samples.
The secondary outcomes are changes in clinical assessment of the pulmonary symptoms via spirometry, anthropometric assessment stand on the BMI Z score, and evaluation of the quality of life using the CFQ from the baseline to three months of the intervention. Participant timeline {13} Nutritional recommendations will be provided to all the patients and/or their parents and legal guardians stand on the CF requirements and adjustable CREON dosing to the level of dietary fat. In addition, the patients and their parents completed the CFQ depend on age.
Anthropometric measurements (weight and height) will be performed using a digital scale (model: SECA).
Body weight will be measured without shoes, and height will be measured in the standing position without shoes with the heels stuck to the wall and the head looking frontwards with the accuracy of 0.5 centimeter.
The fecal examination will be performed before and after the intervention. Before intervention, fecal sample will be evaluated for bacterial over growth and parasite specially giardia by checking stool PH and trophozoites or cysts of giardia. Any positive result will be treated with antibiotics before stating the trials.
Blood sampling will be performed before and after the intervention. Approximately ve milliliters of blood will be collected, immediately centrifuged, the serum will be separated from the sediment, and preserved at the minimum of temperature of -20°C.
The primary and secondary consequences of the treatment will be investigated before and three months after the treatment. The clinical evaluation and follow-up of medication use and side-effects will also be carried out via phone call and paying monthly visit to the clinic, and the ndings will privately be presented to the patients. (Table 2) Sample size {14} According to the lack of complete similar clinical trials which evaluate in ammatory effect of Nanocurcumin in children with cystic brosis, we used HSCRP indices as an effect size. Based on related article, the mean ± SD of hs-CRP indices in the curcumin and placebo groups, were 5.9(2.57) and 3.6(1.58). (16). G-power analyzer was applied for calculating sample size with a con dence interval of 95% and power of 80%. By considering 10% dropout and using Deff (deign effect) for strati ed sample, a total of 30 samples for each group was calculated. In total, 60 eligible patients with CF are needed to include of randomization procedures.

Recruitment {15}
During regular monthly visit of patients at the cystic brosis clinic, treatment team specially nutritionist will explain the trial and discuss the advantages and disadvantages of therapy and answer any questions that participants may have.

Sequence generation {16a}
The Patients with eligible criteria will be enrolled to the study. Thereafter, the rst time with the use of the strati ed randomization procedure, participants will be divided into two groups according to disease severity (severe/mild-to-moderate) using spirometry with 40% FEV1 cutoff range. The second time, simple sampling method will be used to select the patients from each group to receive either Nano-curcumin drop or Nano-curcumin-like placebo drop by using table of random numbers. (Figure 1)

Concealment mechanism {16b}
Table of random number is used to select patients from each group after strati ed randomization based on disease severity. Also curcumin and placebo labelling is done by Exir Nano Sina company.

Implementation {16c}
Generate the allocation sequence, enrol participants, and assign participants to interventions will be done by the main administrative.

Assignment of interventions: Blinding
Who will be blinded {17a} The curcumin and placebo glasses will be labeled A and B by Exir Nano Sina company, respectively and made available to the patients through the double-blind design in which neither the participant nor the experimenter are aware of which group.

Procedure for unblinding if needed {17b}
Data collection and management Plans for assessment and collection of outcomes {18a} Electronic registry form of cystic brosis in Khorasan province is an instrument for collecting the outcomes result.

Plans to promote participant retention and complete follow-up {18b}
In order to control patients for taking curcumin and placebo they will be followed-up by phone every month. curcumin and placebo will be given to the patients for one month and patients will be asked to bring the bottle of drug in their next visit for assessing their compliance and if anyone used less than 70% of drop, he or she will be excluded. Data management {19}Research nurses and assistants collect the data at baseline and follow up and record it on electronic registry forms of cystic brosis in Khorasan province. Other additional date that is not available in the electronic registry form, will be recorded in the Excel le and in the paper forms at the same time. Also CFQ will be answered via electronic validated questioner by patients older than twelve years old. for patients younger than this age, clinical psychologist will ask them their questioner in the clinic.

Con dentiality {27}
Participants 'study information will be stored at the security site. All laboratory specimens, data collected, and reports will be identi ed by a coded ID number Plans for collection, laboratory evaluation and storage of biological specimens for genetic or molecular analysis in this trial/future use {33} We will try to collect a minimum two millilitre of blood sample for pharmaco-genetic and pharmacokinetic study in future use.

Statistical methods for primary and secondary outcomes {20a}
Data analysis will be performed in SPSS version 20. The differences in the quantitative dependent variables will be evaluated using paired t-test or Wilcoxon test. The difference in the quantitative independent variables between two group, Mann-Whitney test or independent t test will be considered. The analysis of variance (ANOVA) or Kruskal-Wallis test will be applied for quantitative variable more than two groups. In all the statistical analyses, the P-value of ≤0.05 will be considered signi cant.

Interim analyses {21b}
Interim analysis is not considered. In case of frequent side effects (more than previous reports), we will stop the intervention and present the results to the Ethics Committee of Mashhad University of Medical Sciences (MUMS) for further decision

Methods for additional analyses (e.g. subgroup analyses) {20b}
Any covariates will be controlled by ANCOVA or binary logistic regression.
Methods in analysis to handle protocol non-adherence and any statistical methods to handle missing data {20c} For incomplete block of date's, we will use intention to treat analysis.
Plans to give access to the full protocol, participant level-data and statistical code {31c} We will attempt to release the full study protocol and results as soon as possible, regardless of the magnitude or direction of effect. The anonymized data set and statistical code may be available from the corresponding author (Email: kianifar HR@mums.ac.ir) on reasonable request.

Oversight and monitoring
Composition of the coordinating center and trial steering committee {5d} The ethical committee and vice chancellery of Mashhad University of Medical Sciences, supervises all the study stages. It is an academic committee and has no competing interest Composition of the data monitoring committee, its role and reporting structure {21a} Any modi cation to protocol which may impact on the conduct of the study, will be approved by ethical committee of MUMS prior to implementation.

Dissemination plans {31a}
We will attempt to release the full study protocol and results as soon as possible, regardless of the magnitude or direction of effect. The anonymized data set and statistical code may be available from the corresponding author (Email: kianifar HR@mums.ac.ir) on reasonable request.

Discussion
Curcumin [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione], is a polyphenolic compound isolated from the rhizomes of Curcuma longa (turmeric). (17) Secondary metabolites of this component are phenolic acids, avonoids, alkaloids, terpenoids, tannins and saponins all of which are known to have biological effects. The antibacterial effect of curcumin has been exhibited since 1949 (18) and several studies have revealed its anti-in ammatory, anticancer, antioxidant, wound-healing, antiviral effects which are believed to have therapeutic effects in many human related diseases. (19) Curcumin is suggested as a line of treatment for cystic brosis disease. Since CF is the most common life shortening disease, Early initiation of treatments, including anti-in ammatory agent, are very important in the nal prognosis of the disease. It appears cystic brosis children are the best candidate for using this treatment.
According to previous research it is shown that Cystic brosis transmembrane conductance regulator (CFTR), is a cAMP-activated Cl ion channel in the apical membrane of epithelial cells (20), which is a wellstudied ion channel target for curcumin. (Fig. 2) Due to the limited trial that evaluated the effect of curcumin in cystic brosis patient, here we reviewed animal and cellular based studies to recognize the main mechanism of curcumin on CFTR protein. It acts as a CFTR modulatory, anti-in ammatory and anti-microbial agent.

Modi ed CFTR effect
Cystic brosis is caused by mutations, or errors, in the CFTR gene. This can result in either no CFTR protein being made or a malformed CFTR protein being created which cannot perform its key function in the cell. (21) Curcumin has several effects on CFTR protein such as increase tra cking of channel on cell membrane (corrector) and or potentiation (activity). The rst study published by Egan et al demonstrated that curcumin induced accumulation of complex-glycosylated F508 CFTR and increased cell surface density. (22) Curcumin as a sarcoplasmic/endoplasmic reticulum calcium (SERCA) pump inhibitor, could increase the appearance of functional DF508 CFTR on the plasma membrane cells. (22) On the other hand, several chaperone proteins are associated with the quality control of CFTR. Chaperons could bind to the mutant CFTR protein and carry this complex to the ER. One important chaperone is called ER chaperone calreticulin (CRT) which is one of the negative regulators of CFTR expression and function. Some studies have also demonstrated that curcumin suppressed the endogenous calreticulin mRNA transcription that regulate level of this protein in wild type and DF508 CFTR protein so that CFTR protein can be stabilized on the cell membrane. (23) In addition, curcumin enhanced tra cking of DF508 CFTR protein by phosphorylation of ser52 on keratin 18 intermediate laments on CF pancreatic epithelial cells (24) Potentiation function of curcumin has been reviewed and con rmed in a number of studies. Curcumin can react as a potentiator that repairs sub activation of CFTR protein activity, and also accelerates CFTR protein activity which has been increased by other CFTR modulation in mutations such as S1251N, G551D and F508del.(25) Considering G551D-CFTR mutation, where normal tra cking on the plasma membrane is characterized with a very short time on opening channel. Therefore, Curcumin can increase G551D-CFTR opening channel and its activity on its own, or when it is added to the channel that is further affected by genistein. Thus, these neutral components are suggested to have different mechanisms as potentiations of channels. (12) Similarly, curcumin stimulates channels which lack NBD2 due to deletion of G551D CFTR mutants, where effect is persistent and irreversible. (26) W1282X is another common CF mutations which reduces CFTR function by two mechanisms: rstly through reduction of steady state levels of CFTR mRNA, and secondly by lowering the channel activity due to the defect of the second cytosolic nucleotide binding domain (NBD2), that is a surface bind to ATP molecules (27,28). According to several studies, curcumin stimulated W1282X-CFTR after adding a saturating dose of VX-770, thus it increased the rates of channel opening. With regards to channel opening, ATP is required for binding of both nucleotide-binding domains (NBDs), which probably results in the dimerization of the two NBDs, and phosphorylation of the R domain. Therefore, both curcumin and VX-770 are acting as allosteric modulators since they stimulate activity of the CFTR channel without ATP binding, but with different mechanisms. (11) Finally, CFTR-S1251N gating mutation has been evaluated and clinical effects of the three CFTR potentiator treatments curcumin, genistein and ivacaftor have been studied. Based on previous research no clear response was seen on clinical parameters of cystic brosis patients, and the main reason was the plasma concentration of curcumin which was lower than the in vitro effective experimental range. . (29) Anti-in ammatory effect In ammation has a signi cant role in the outcome of cystic brosis patients. Studies have shown an extensive in ammation on pulmonary, gastrointestinal and systemic levels in CF patients.
Mucus plugging is suitable for microorganism growth and neutrophilic in ammation as it has many nutrients. It is believed that, delayed neutrophil apoptosis and long lifespan of neutrophils could increase the levels of neutrophil extracellular traps (NETs), thus in ammation is induced. (30) Moreover, differentiation of monocyte in to macrophage and division into M1 or M2 phenotype are unsuccessful in CF patients, and hence it does not produce IL-13Rα1 on the surface. This impaired process contributes to excessive in ammation in CF lung disease. (33). Last but not least, the highest number of T-cell subsets, which are Th17 cells, are present in the CF lung. Th17 cells produce both IL-8 and IL-17 and promote neutrophil accumulation. These T-cells are activated by IL-6, IL-23, and TGF-β, and suppressed by active T regulatory-lymphocytes (Tregs). Otherwise, a CFTR abnormality or an unknown imbalance between Th17 cells and Tregs can induce in ammation by neutrophil recruitment. (2) Antimicrobial effect Toll-like receptor-2(TLR2) is a receptor of pathogenic bacteria like Staphylococcus aureus peptidoglycan (PGN), is found in epithelial cell and has a key role in recognizing the infected pathogen, and increases in ammatory cytokines as a result, and is up regulated in CF patients.
Cystic brosis patients are prone to infection since absence of CFTR protein causes demethylation of DNA at the speci c CpG sites which overlaps a minimal region to maintain activity of TLR2 promotor. (34) Curcumin can degrade speci city protein 1 (SP1) via oxidative and proteasome degradation pathways. This factor is essential for upregulation of TLR2 expression. Consequently, curcumin decreases basal expression of TLR2. (35) Finally, curcumin has been proved to have a signi cant effect on suppressing the growth of P. aeruginosa on bio lm optical densities. Although the MIC (Minimum Bacterial concentration) which is needed for growth suppression is higher than other usual P. aeruginosa treatments such as imipenem or tobramycin. (36) Since the reduction of in ammation plays a key role in the conditions of patients with CF and considering the primary effects of curcumin on the CFTR protein, extensive investigations are required regarding the applications of this natural compound in these patients. Recently, the Nano-curcumin formulation has been extensively studied considering its impact on the increased bioavailability of curcumin. This formulation has been reported to have a great effect on the reduction of in ammatory processes.
The main limitation of the current study is the lack of assessment of CFTR protein function by nasal epithelial biopsy and cellular evaluation. However, this method is invasive and cannot be performed easily on children. To the best of our knowledge, this is the rst study which will evaluate the use of curcumin as a speci c and supportive nutritional agent in children with CF.

Trial Status
Recruitment was started on 11 July 2020 and is estimated to be completed by 2020-11-21. Recruitment was ongoing at the time of submission.  Figure 1 schematic of study design. First and second evaluation consist of: serum interleukin-6 (IL-6), IL-10, and high-sensitivity C-reactive protein (hs-CRP), fecal calprotectin, weight and body mass index (BMI), and score of quality of life of the cystic brosis questionnaire (CFQ).