Chemicals and Reagents
Dulbecco’s Modified Eagle Medium (DMEM) with 4500 mg/ml glucose, 110 mg/l sodium pyruvate and 0.584 mg/l L-glutamine; Bovine Fetal Serum (BFS); Penicillin/ streptomycin/neomycin (P/S/N) solution with 5000 units penicillin, 5 mg streptomycin and 10 mg neomycin/ml; phosphate buffered saline; 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), ibuprofen, dimethylsulfoxide (DMSO), acetic acid, tween 80, k-carrageenan. All materials and reagents were purchased from Sigma-Aldrich. The thiazolidin-4-one derivatives of ibuprofen (4a-n, Fig. 1) were previously synthesized and characterized by our research group [7].
Animals
Swiss albino mice and Wistar rats provided from the Biobase of “Grigore T. Popa” University of Medicine and Pharmacy from Iasi, were used. The animals were housed in polyethylene cages with access to water and food ad libitum for 7 days before starting the experiments. The environmental conditions during the study were maintained relatively constant at a 23±2°C temperature, 40-60% relative humidity and a 12 h light/dark cycle. The food and the water were withdrawn 18 h before starting the experiments. The animals (mice and rats respectively) were randomly divided into several groups (n = 8), depending of the method applied. The inclusion criteria used in the design of the experiment refer to the weight of animal (20-30 g for mice, 150-200 g for rats) and healthy state and the exclusion criteria applied were any pathological conditions observed to the animals. All the experiments were designed to cause minimum harm to animals. At the end of the experiments, the animals were anesthetized with ethyl ether and then the cervical dislocation procedure was used to euthanasia the animals. Prior to the disposal, the animal death was confirmed by observing the movement, heartbeat, respiration and eye reflex. All procedures were strictly conducted by the expert personnel and were in agreement with the guideline of laboratory animal studies.
Ethics approval and consent to participate
The study was conducted in agreement with actual deontology and ethics guidelines about laboratory animal studies (Law no. 206/27 May 2004, EU/2010/63 - CE86/609/EEC) and was approved by Research Ethics Committee of “Grigore T. Popa” University of Medicine and Pharmacy from Iasi (resolution no. 292).
Cell viability assay using the MTT method is based on the capacity of cell to reduce the slightly yellow tetrazolium salts to intense purple formazan by intracellular reduction system mostly located in the mitochondria. The amount of formazan, which is correlated with number of viable cells, is spectrophotometrically measured at 570 nm [8]. Primary cells, mesenchymal type with stem cell potential, isolated by collagenization from adipose tissue, were used. The cell line was maintained in DMEM supplemented with BFS (10%) and P/S/N in a humidified incubator with 5% CO2 at 37°C. The cells were seeded in 24-well plates (104 cells/ml) and treated after 24 h with different concentrations of ibuprofen derivatives (4a-n) (50 µg/ml, 10 µg/ml, 2 µg/ml) for 24 h, 48 h and 72 h. DMSO was used as solvent and a negative control (blank, DMSO 0.2 %) and a positive control (DMSO 5%) were used in similar conditions. The culture media was removed and MTT (500 μl) was next added to each well and the cells were further incubated for 3 h at 37°C. The medium was then removed and isopropanol was added. After 15 min from each well were transferred 100 µl in a 96-well plate and the absorbance was recorded at 570 nm with a microplate reader [9, 10]. The cell viability rate was calculated using the following formula [9]:
Cell viability (%) = (As/Ac) ´ 100
in which,
As = the absorbance of culture cells incubated with the sample (ibuprofen derivatives);
Ac = the absorbance of culture cells incubated with DMSO (0.2%).
The experiments were performed in triplicate and the results are presented as mean ± standard deviation (SD).
Acute toxicity assay was performed on mice and the tested compounds (4a-n) were suspended in tween 80 and administrated orally, in volume of 0.1 ml/10 g animal. The different doses (1000-3000 mg/kg body weight) of the tested compounds were used [11] and the survival rate was noted at the different timelines: 24 h, 48 h, 72 h, 7 days and 14 days. The LD50 was calculated on base of Kärber arithmetic method [12] using the following formula:
LD50 = LD100 - (∑(a + b)/n),
in which:
a = the difference between two consecutive doses of administered the tested compound;
b = the mean number of dead animals from two consecutive groups;
n = the number of animals for each group;
LD100 = the lethal dose 100 (the dose of the tested compounds that leads to death of all animals within the treated group).
Carrageenan-induced paw edema assay was used to evaluate the anti-inflammatory effect, according to the protocols describes in the literature [13, 14] with slightly modifications. The edema was induced by intra-plantar administration of 0.2 ml of 1% suspension of k-carrageenan in physiological saline into the left hind paw of rat and the volume of paw was measured using the digital pletismometer LE. After induction of edema, the ibuprofen derivatives (4a-n) were administered orally in a daily dose representing 1/20 of LD50 as suspension in tween 80 (0.5 mL/100 g b.w.). Ibuprofen as reference drug and tween 80 (0.5 mL/100 g b.w.) as control, were used in similar conditions (Table 1).
Table 1 The doses (mg/kg b.w.) of ibuprofen derivatives (4a-n) used for anti-inflammatory and analgesic assays
Group
|
Compound
|
Dose (mg/kg b.w.)
|
Group
|
Compound
|
Dose (mg/kg b.w.)
|
1
|
4a
|
81.25
|
9
|
4i
|
86.00
|
2
|
4b
|
81.25
|
10
|
4j
|
92.00
|
3
|
4c
|
86.40
|
11
|
4k
|
78.25
|
4
|
4d
|
81.70
|
12
|
4l
|
79.50
|
5
|
4e
|
91.00
|
13
|
4m
|
82.50
|
6
|
4f
|
89.50
|
14
|
4n
|
85.00
|
7
|
4g
|
91.00
|
15
|
Ibuprofen
|
68.75
|
8
|
4h
|
89.50
|
16
|
Tween 80
|
0.5 ml/100 g
|
The volume of the left hind paw was measured at different timelines (2 h, 4h, 6 h and 24 h).The edema inhibition (%) was calculated, using the following formula:
Edema inhibition (%) = (ΔVc-ΔVt) x 100/ΔVc
in which:
ΔVc= the rat paw’s volume recorded for control group;
ΔVt= the rat paw’s volume recorded for groups treated with ibuprofen derivatives.
Analgesic effect
Tail-flick assay, used as model of thermal nociception is based on measuring the sensibility of rats when a thermal stimulus is applied on tail. According to the experimental protocol, the animals were initially tested by applying a radiant beam at the distal part of the tail, measuring the latency at time 0 (T0) [15, 16]. The response to pain was quantified using a Tail-flick algesimeter (Harvard Apparatus, United States of America). The ibuprofen derivatives (4a-n) were administered by oral gavage in a daily dose representing 1/20 of LD50, as suspension in tween 80 (0.5 mL/100 g b.w.) (Table 1). Ibuprofen, as reference drug, was used in similar conditions. In order to assess the analgesic effect, the initial response to pain and at 4 h after administration of ibuprofen derivatives (4a-n) it was determined. The maximum allowed time (cut-off time), for not causing tissue lesions was established to be 10 sec (Tm).
The pain inhibition (%) was calculated for each tested compound, using the following formula:
Pain inhibition (%) = (Tt-T0)/(Tm-T0) x 100
in which:
Tt = the nociceptive response measured at 4 h after of ibuprofen derivatives administration;
T0 = the nociceptive response measured before any treatment (initially);
Tm = the maximum allowed time (cut-off time).
The presence of analgesic effect (anti-nociceptive potential) is highlighted by an increased latency response after administration of the ibuprofen derivatives in reference with the initially value.
Writhing assay is a model of visceral pain, induced to mice by intraperitoneal administration of acetic acid. It is characterized through abdominal contractions, body movements (especially of the posterior members), writhing of the dorsal-abdominal muscles with reduced locomotor activity. The applied experimental protocol was in agreement with the literature data with slightly modifications [17, 18]. The ibuprofen derivatives (4a-n) were administered by oral gavage in a daily dose representing 1/20 of LD50 as suspension in tween 80 (0.1 mL/10 g b.w.) (Table 1). Ibuprofen, as reference drug and tween 80 (0.1 ml/10 g b.w.), as control, were used in similar conditions. One hour after administration of tested compounds, the acetic acid (0.6% water solution), as irritating agent, in volume of 0.1 ml/10 g b.w. was intraperitoneally injected. After other 5 min the number of writhings for each mouse was noted, every 5 min, during 30 min. The analgesic effect, expressed as inhibition (%) of writhings was calculated for each tested compounds, using the following formula [14]:
Inhibition (%) = (Nc-Nt) x 100 / Nc
in which:
Nc = the writhings number recorded for mice from control group
Nt = the writhings number recorded for mice of group treated with ibuprofen derivatives
It is estimated that the analgesic activity is higher if the writhings number is decreased in comparison with control group.
Statistical analysis
Data are presented as mean ± standard deviation. The one-way analysis of variance (ANOVA) and Tukey post hoc test were used to determine whether there are any statistically significant differences between tested compounds and control. The p value <0.05 was considered to be statistically significant.