5.1 Parasite
Plasmodium berghei ANKA was obtained from Addis Ababa University, Ethiopia. The parasite was maintained by serial intraperitoneal injection into mice. The injection volume for all mice was 106 parasitized erythrocytes in citrate saline.
5.2 Mice
Male and female Balb/c mice (weight 20–30 g) were obtained from the Animal House of the College of Pharmacy, King Saud University, Riyadh. The experiments were approved by the unit of Biomedical Ethics Research Committee, King Abdulaziz University, Saudi Arabia (Reference No 168 − 15; may 24, 2015). This study followed the ARRIVE guidelines.
5.3 Drugs
Two drugs were used in this study: silver sulfadiazine (non-soluble in water) and chloroquine (soluble in water). Both drugs were received from Riyadh Pharma, Riyadh, Saudi Arabia.
5.4 Preparation of free drugs
The silver sulfadiazine free suspension was prepared by adding 50 mg of the drug to 50 mL water, followed by sonication for 5–10 min and incubation overnight in a water bath shaker. Chloroquine (50 mg) was dissolved in 1 mL of water with vertexing. The final volume was made to be 50 mL by adding water.
5.5 Preparation of in-situ gel
Silver sulfadiazine and chloroquine were dispersed in 30 % poly (lactide-co-glycolide) 50:50 (CAS: 26780-50-7, molecular weight: 24–38 kDa) in N-methylpyrrolidone (NMP), as reported previously by Yu and Jagdish (Yu and Jagdish, 2008). The suspensions were then vortexed for 5 min, placed in a 25°C water bath, and incubated with shaking for 24–72 h.
5.6 Mouse groups
All mice received intramuscular prophylactic treatment 4 days before infection (Table 1, n = 15 in each group). The first two groups were treated with free silver sulfadiazine (1.05 mg/kg/d for 5 days) and free chloroquine phosphate (10 mg/kg/day). In-situ gel groups received one inoculum containing the total dose of the drug, 5.25 mg/kg silver sulfadiazine, and 40 mg/kg chloroquine phosphate. The control group received vehicle in-situ gel for five days. Four days after treatment, all mice were infected with 106 parasitized erythrocytes. Parasitemia was assessed via collection of mouse tail blood on days 3, 5, 7, and 9 post-infection, and life span was monitored for each mouse.
Table 1
the experimental design of mouse experiments
| Group 1 | Group 2 | Group 3 | Group 4 | Group 5 | No. of mice |
---|
Treatment | Free silver sulphadiazine | Free chloroquine phosphate | In-situ gel silver sulphadiazine | 40 mg/kg chloroquine phosphate | Vehicle in-situ gel | 15 |
Dose | 0.05 mg/kg/d | 10 mg/kg/d | 5.25 mg/kg | 40 mg/kg | 0.2 mL | |
Repetition | 5 days | 4 days | 1 day | 1 day | 5 days | |
Treatment route | i.m. | i.m. | i.m. | i.m. | i.m. | |
Infection | 106 parasitized erythrocytes | 106 parasitized erythrocytes | 106 parasitized erythrocytes | 106 parasitized erythrocytes | 106 parasitized erythrocytes | |
Parasitemia | Counted on days 3, 5, 7, 9 post-infection | Counted on days 3, 5, 7, 9 post-infection | Counted on days 3, 5, 7, 9 post-infection | Counted on days 3, 5, 7, 9 post-infection | Counted on days 3, 5, 7, 9 post-infection | |
Life span | Monitored | Monitored | Monitored | Monitored | Monitored | 15 |
Liver and kidneys enzyme | tested | Tested in a previous work* | tested | Tested in a previous work* | Tested | 3 |
Pharmacokinetic | tested | Tested in a previous work* | tested | Tested in a previous work* | tested | 3 |
* Accepted but not yet published. No, number; i.m., intramuscular; |
5.7 Liver and kidney enzyme analysis
Urea, creatinine, and uric acid, as well as aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) levels in blood plasma were measured using Vitros 350 (Johnson & Johnson, USA).
5.8 Pharmacokinetic analysis of drugs and their primary active metabolites
Plasma samples (250 µL) were spiked with serial silver sulfadiazine dilutions and its metabolite sulphamerazine which were used as standards for in vivo pharmacokinetic analysis. Plasma samples containing silver sulfadiazine were pre-treated to extract the drug from plasma after the addition of the internal standard (62.5 µg/mL).
Dilutions of 62.50, 31.25, 15.63, 7.81, 3.90, and 1.95 µg/mL silver sulfadiazine and N-acetyl sulfadiazine were prepared in mobile phase (1000:99:9, water:acetonitrile:phosphoric acid). In a separate tube, 62.5 µg/mL of sulfamerazine was prepared in mobile phase. Blank plasma (250 µL) was spiked with different dilutions of 1:1 silver sulfadiazine and N-acetyl sulfadiazine.
Nine plasma samples were collected from mice individually injected intramuscularly with a single dose of 50 mg/kg silver sulfadiazine suspension or silver sulfadiazine nanocapsules. Samples were taken after 6, 12, 24, and 48 h. In addition, plasma was extracted from mice administered silver sulfadiazine suspensions or nanocapsules (5.25 mg/kg/5 d) or silver sulfadiazine in-situ gel (26.25 mg/kg) by intramuscular injection. The time points were 6 h after the first dose, 6 h after the second dose, 6 h after the third dose, 6 h after the fifth dose, and on the seventh, ninth, and fifteenth day after injection.
All standards and samples were the processed and analyzed in the same manner. Briefly, perchloric acid (20 µL, 65 %) was added to the plasma-drug mixture, vortexed for 30 s, and centrifuged for 5 min at 11.300 × g. Supernatants were extracted and placed into new tubes. To each tube, 50 µL of 2 M dipotassium hydrogen phosphate was added; tubes were then mixed and centrifuged at 11.300 × g for 1 min. The resulting supernatants were supplied with 200 µL sulfamerazine and injected into the HPLC system (Shimadzu, Tokyo, Japan) under the same conditions used for in vitro dissolution. The standard curve and standard equation were derived using Microsoft Excel (Amini and Ahmadiani, 2007).
5.9 Data analysis
Parasitemia, pharmacokinetics, as well as liver and kidney enzyme data were expressed as means ± standard error of the mean (SEM). Life span was calculated as a percentage of the mean. All data was tested for Gaussian distribution using the D’Agostino-Pearson omnibus noramlity test and Shapiro-Wailk normality test. Experiments were designed with no matching or pairing. One-way analysis of variance (ANOVA) and Tukey’s multiple comparisons test was used to anlayze the data for significance. P-value < 0.05 were considered statistically significant.