Doluisio’s (Closed-Loop) Rat Intestinal Perfusion

All the experimental protocols were approved by the Jiangsu University Animal Ethics and Experimentation Committee and followed the principles of laboratory and animal care of the university (UJS-LAER-AP-2018030809).

Male Sprague-Dawley rats (180–220 g) were obtained from the Jiangsu University Animal Center (Zhenjiang, People’s Republic of China). The absorption rate coefficients of nintedanib and its SMEDDS were determined in four intestinal segments (the duodenum, jejunum, ileum and colon) using the in situ “closed-loop” perfusion method based on the Doluisio’s Technique26,27 with minor modification. Briefly, rats were fasted for 4 hrs (free access to water) and randomly divided into 2 groups (nintedanib solution and NDNB-SMEDDS groups). After anesthesia by using sodium pentobarbital (40 mg∙kg⁻¹), the rat was fixed on a heated surface maintained at 37°C and a midline abdominal incision was made.28 According to the position shown in Table 1, four individual 10 cm of intestinal segments (the duodenum, jejunum, ileum and colon) or entire small intestine (from the duodenum to ileum) were cut and cannulated at both ends. The bile duct was tied up to avoid drug enterohepatic circulation and the presence of bile salts in the lumen. In order to remove the intestinal contents, the four intestinal segments were flushed with a 37°C physiological solution. Then, the intestinal segments were connected with three-way stopcock valves. Throughout the experiment, the abdomen was covered with a cotton wool pad to avoid peritoneal liquid evaporation and heat loss. The nintedanib solution and NDNB-SMEDDS were diluted with a K-R solution to 20 μg∙mL⁻¹, which contained phenol red which was then introduced into each segment and circulated at 2.5 mL∙min⁻¹ and 37°C. Samples were taken at 0.5, 1, 1.5, 2, 2.5 and 3 hrs, while the same volume of blank medium was added. Next, samples were centrifuged at 10,000 r∙min⁻¹ for 10 mins, and the drug concentration was determined by an HPLC assay.

The Specific Location Of The Intestinal Segments

X₀ was the drug concentration in the intestinal circulating fluid at 0 hr, LnX is the ln value of the remaining nintedanib dose in the enteric circulation fluid at each time point.

According to Equation 4, linear regression was carried through on LnX and t, the slope of the line was the absorption rate coefficient (K_a).

K_a was transformed into a permeability value (P_app) with the following equation:

where R is the effective radius of the intestinal segment, while R was calculated from the area/volume relationship considering a 2 mL and 5 mL volume of each intestinal segments and colon, respectively.

The predicted oral fraction absorbed (F_abs) obtained with the perfusion approach was calculated from the following equation:

R represents the effective radius of the small intestine (fixed at a value of 0.1630 cm) or colon (fixed at a value of 0.3718 cm), and T is the effective absorption time.