Experimental animals
Male Sprague Dawley rats were purchased from The SLAC Laboratory Animal Center (Shanghai, China). The rats were maintained under specific pathogen-free conditions, which were housed in a local facility for laboratory animal care and fed a standard diet and water. Rats weighing 180 to 230 g were used for the experiments. Animals were held in plastic cages with hardwood chips. They were provided with food and water ad libitum. The experimental protocol was approved by the Committee of Animal Care of Fudan University.
Dosing regimens and sample collection
MMF was purchased from Roche Pharmaceuticals Co. (Shanghai, China), and made into powder. Mofetil was purchased from Sinopharm Chemical Reagent Co. (Shanghai, China). EC-MPS (Novartis Pharma Stein AG) was made into enteric-coat microcapsule of mycophenolate sodium by School of Pharmacy Fudan University. Fourty rats were randomly assigned into five groups (each group, n = 8): (1) the control group, which was given normal saline (NS); (2) the MMF group, which was given 100 mg/kg·d MMF; (3) the EC-MPS group, which was given 72 mg/kg·d EC-MPS; (4) the mofetil group, which was given 30 mg/kg·d mofetil; and (5) EC-MPS + mofetil group, which was given 72 mg/kg·d EC-MPS and 30 mg/kg·d mofetil. This is designed to evaluate the contribution of mofetil to the effect of MMF and EC-MPS on MPA GI complication. The drugs were diluted in NS and used to treat rats through oral gavage for 14 consecutive days. 100 mg MMF, 72 mg EC-MPS were equivalent to 50 mg MPA.
Following the last treatment, blood (100 μl) was collected at 0, 15, 30, 60, 120, and 240 min by tail nick for determination of MPA and MPAG pharmacokinetic profiles. The rats were sacrificed with an intraperitoneal injection of urethane (1.5 g/kg), GI tissues including stomach, duodenum, jejunum, ileum, colon, and rectum were collected and each was divided into two parts. One part was homogenized and the other part was fixed in 10% formalin for histological analysis. In each animal (n = 8 for each group), one sample of every GI site was collected for histologic analysis.
Gastrointestinal segments were defined as follows: stomach, the part close to pyloric sphincter; duodenum, at the site 1 cm below pyloric sphincter to ligament of Trietz; upper jejunum, at the site 5 cm below stomach (upper half of remaining small intestine); lower jejunum-ileum, at the site 5 cm above cecum (lower half of small intestine); colon, at the site 5 cm below ileum-cecum (cecum to rectum); and rectum, at the site 3 cm above anus [28, 29].
Determination of MPA and MPAG in plasma and GI tissue
Plasma and tissue MPA and MPAG were determined using a high-performance liquid chromatography (HPLC) with ultraviolet detection [30]. Briefly, plasma and tissue homogenates were precipitated with acetonitrile, spiked with propafenone hydrochloride as an internal standard (50 μg/ml in sample). MPA and MPAG were determined using liquid chromatography/mass spectrometry (LC/MS). The HPLC conditions included a C18 column (150*4.6 mm; Kromasil, AkzoNobel, Sweden), isocratic mobile phase [46% methanol: 54% aqueous trifluoroacetic acid (0.1%; pH 2.5)]. Analysis was performed under a 20 μl injection, solvent flow of 1.5 ml/min, total run time of 15 min per injection, and UV detection at 295 nm. The appropriate standard curves for MPA and MPAG were linear over the range of 0.5-100 μg/ml and 2.5-100 μg/ml, respectively.
Non-compartmental model (linear trapezoidal model) was used to calculate pre-dose concentration (C0), maximum concentration (Cmax), and area under the plasma concentration-time curve from 0 to 240 min.
Assessment of diarrhea grade and body weight
Body weight and stools were monitored daily. Food intake was not assessed. Stools were graded for degree of diarrhea by the following scale (0, firm stool; 1, malformed stool; 2, watery stool with perianal staining; 3, severe perianal staining).
Histological analysis
Tissue samples fixed in 10% formalin were processed for histological examination following the standard procedure. The tissues in formalin were embedded in paraffin and cut 5-6 μm sections using rotary microtome. The sections were stained with hematoxylin and eosin and analyzed under light microscope [31].
The histological changes in the GI tract was scored using a semi-quantitative scale. The gastric injury were graded from 0 to 5 (0, no lesions; 1, lesions seen in mucosal surface, no damaged gastric pit cell; 2, damages with gastric pits, no damaged gastric gland cell; 3, lesions of gastric gland cells; 4, partial mucosal necrosis, multiple linear ulcer and hemorrhage; 5, total mucosal necrosis). The intestinal injury were graded from 0 to 5 according to Criteria of Chiu grading (0, normal mucosa villi; 1, development of subepithelial Gruenhagen’s space, usually at the apex of the villus and often with capillary congestion; 2, extension of the space with moderate lifting of epithelial layer from the lamina propria; 3, massive epithelial lifting with a few denuded villi; 4, denuded villi with exposed capillaries; 5, disintegration of the lamina propria, ulceration and hemorrhage) [32]. The histopathological studies were conducted by a pathologist who was blinded to the study.
Statistical analysis
The data are presented as the mean ± standard deviation (SD). Statistical differences between groups were analyzed using one way analysis of variance test. Histological data were analyzed using χ2 test. Differences were considered statistically significant if the p value was less than 0.05.