Niclosamide

The final volume for each dose was 500 mL with Becker containing 10 snails. In the case of the 24-well plate, each well contained a final volume of 2 mL with one snail. In a condition, we used 10 wells with 10 mollusks for treatment (1 mollusk per well) and another 3 wells with 3 mollusks for treatment (1 mollusk per well). The observations were performed by analyzing morphological and functional criteria.
Each snail was individualized in 24-well plates, and a volume of 2 mL of the dose to be tested was added per well for the experimental group or controls and covered with a lid.
For the assays, B. glabrata were added to aqueous solutions of niclosamide, prepared in the laboratory at doses of 1 mg/L, 2 mg/L, 3 mg/L, 4 mg/L, and 5 mg/L (experimental group), distilled water (negative control group), and 1% DMSO solution (negative control group). To assess the toxic effect with a small volume of solution, the total number of animals used in triplicate tests was 21 snails and that used in decaplicate tests was 30 snails. During this period, the 24-well plates containing the doses were kept at room temperature and the animals were not fed. Each test was conducted for periods of 24 h, 48 h, and 72 h exposure. The tests were realized at least on three distinct days. The percentage of dead snails using a total number of 9 or 30 snails was calculated estimating the niclosamide molluscicidal activity as 100%. Triplicate or decaplicate assays were repeated on three distinct days. All other values were normalized in function niclosamide result.

At the screening visit, age, gender, height, weight, body mass index (BMI), diabetes duration, and blood pressure data were recorded. Urine samples were collected at baseline and after 6 months to assess UACR, MMP-7, PCX, and 8-OHdG using enzyme-linked immunosorbent assay (ELISA) kits. Blood samples were also collected at baseline and after 6 months to assess fasting blood glucose, glycosylated hemoglobin (Hemoglobin A1c), and serum creatinine using standard colorimetric methods.
The estimated glomerular filtration rate (eGFR) was calculated using the Chronic Kidney Disease Epidemiological Collaboration (CKD-EPI) equation as it is more accurate at higher levels of renal function and better to be used for clinical assessment of DKD [23 (link)]. The eGFR was calculated at baseline and the end of the study. The primary outcomes were the changes in UACR, serum creatinine, and eGFR after 6 months. Changes in other measured biomarkers were considered secondary outcomes.
Patients had regular visits every month for medication refills and to report if there are any encountered side effects. As far as we know, niclosamide can be taken with or without food and there is no evidence that food affects niclosamide effect. To prevent GIT upset, we advised patients to take niclosamide after a light meal. Every month, all administered medications by each patient were reviewed to exclude any patients taking medications that induce albuminuria or interact with niclosamide.

Statistical analysis was carried out using SPSS statistical package version 28.0, May 2021, IBM corporation software group, USA. A chi-square test was applied to compare categorical clinical variables between groups. The Shapiro–Wilk test was applied to the measured parameters before running parametric statistical analysis. Normality test results reveal the normal distribution of our data with a P-value greater than 0.05. Analysis of baseline characteristics and biomarkers was done using unpaired student t-test for parametric data. Comparing the two groups mean values and the percent change in variables were done using a student t-test with statistical significance being set at P < 0.05. Correlation analysis was done using Pearson correlation. Correlation coefficients are interpreted as weak (< 0.4); moderate (0.4–< 0.7), or strong relationship (> 0.9). [24 (link)]. A linear regression test was also conducted to evaluate the association between the measured biomarkers and UACR.
Considering that the primary objective of this trial was to compare UACR between the niclosamide arm and control arm, we calculated the minimum number of patients needed to detect the 20% change in UACR. The lower limit of 20% UACR reduction was chosen as a cut-off point representing clinical relevance which is not likely to be subjected to variance error. The assumed mean of the control group was 300 mg/g, and the expected mean difference between the control and treatment groups was 60 mg/g. Assuming 80% power, a two-sided type I error rate of 0.05, an allocation ratio (r = 1), and a standard deviation equal to 75 mg/g. After applying a 15% dropout rate, 29 participants were needed in each arm of the trial. Therefore, it was planned to enroll 30 patients per arm for 1:1 randomization.