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. 2020 Nov 4;25(21):5123.
doi: 10.3390/molecules25215123.

Synthesis and Evaluation of PEG-PR for Water Flux Correction in an In Situ Rat Perfusion Model

Guo Chen  1 , Xingqi Min  1 , Qunqun Zhang  1 , Zhiqiang Zhang  1 , Meiqiang Wen  1 , Jun Yang  1 , Meijuan Zou  1 , Wei Sun  2 , Gang Cheng  1
Affiliations

Synthesis and Evaluation of PEG-PR for Water Flux Correction in an In Situ Rat Perfusion Model

Guo Chen et al. Molecules. .

Abstract

Phenol red (PR) is a widely used marker for water flux correction in studies of in situ perfusion, in which intestinal absorption usually leads to the underestimation of results. In this paper, we propose a novel marker polyethylene glycol (PEG)-PR (i.e., PR modified by PEGylation) with less permeability and evaluate its application in an in situ perfusion model in rats. PEG-PR was synthesized by the chemical conjunction of polyethylene glycol-4k/5k (PEG-4k/5k) and PR. The synthesized PEG-PR was then characterized using 1H-NMR, 13C-NMR, ultraviolet (UV), X-ray diffraction (XRD), and differential scanning calorimetry (DSC) analyses. The low permeability of PEG-PR was assessed using everted gut sac (EGS) methods. The apparent permeability coefficients (Papp, 3-8 × 10-7 cm/s) of PEG4k/5k-PR exhibited a nearly 15-fold reduction compared to that of PR. The different concentrations of PEG4k/5k-PR did not contribute to the Papp value or cumulative permeable percentage (about 0.02-0.06%). Furthermore, the larger molecular weight due to PEGylation (PEG5k-PR) enhanced the nonabsorbable effect. To evaluate the potential application of the novel marker, atenolol, ketoprofen, and metoprolol, which represent various biopharmaceutics classification system (BCS) classes, were selected as model drugs for the recirculation perfusion method. The water flux corrected by PEG4k/5k-PR reflected the accuracy due to the nonabsorbable effect, while the effective intestinal membrane permeability (Peff) of atenolol corrected by PEG4k/5k-PR showed a statistically significant increase (p < 0.05) in different intestinal segments. In conclusion, PEG-PR is a promising marker for the permeability estimation when using the in situ perfusion model in rats.

Keywords: PEGylation; everted gut sac (EGS); in situ perfusion model; permeability; phenol red; water flux correction.

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Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
Recirculation method in a rat model.
Scheme 1
Scheme 1
Synthetic route of polyethylene glycol (PEG)-PR.
Figure 2
Figure 2
Structure and appearance of PEG4k-PR (A) and PEG5k-PR (B).
Figure 3
Figure 3
UV spectra of PR, PEG4k-PR, and PEG5k-PR.
Figure 4
Figure 4
X-ray diffraction (XRD) spectra of PR, PEG4k/5k, PEG4k/5k-PR, and the physical mixtures of PEG4/5k and PR.
Figure 5
Figure 5
Differential scanning calorimetry (DSC) data of PR, PEG4k/5k, and PEG4k/5k-PR.
Figure 6
Figure 6
The apparent permeability (Papp) of PR, PEG4k-PR, and PEG5k-PR at 56 μM in different intestinal segments (everted gut sac). Data are shown as mean ± SD, n = 4–6; * p < 0.05, ** p < 0.01 and *** p < 0.001.
Figure 7
Figure 7
Cumulative intestinal permeable amount versus time of PR (A), PEG4k-PR (B), and PEG5k-PR (C) at 56 μM in different intestinal segments. Data are shown as mean ± SD, n = 3.
Figure 8
Figure 8
Cumulative intestinal permeable amount versus time of PR (A), PEG4k-PR (B), and PEG5k-PR (C) in the jejunum (a), ileum (b), and colon (c) at different concentrations (28–112 μM). Data are shown as mean ± SD, n = 3.
Figure 9
Figure 9
Water flux (Jwater) corrected by PR, PEG4k-PR, and PEG5k-PR at 56 μM during intestinal perfusion. Data are shown as mean ± SD, n = 6; * p < 0.05 and ** p < 0.01.
Figure 10
Figure 10
Effective intestinal membrane permeability (Peff) of atenolol, ketoprofen, and metoprolol corrected by PR, PEG4k-PR, and PEG5k-PR in different intestinal segments. Data are shown as mean ± SD, n = 3; * p < 0.05 and ** p < 0.01.
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