Optimization of intestinal microsomal preparation in the rat: A systematic approach to assess the influence of various methodologies on metabolic activity and scaling factors

Abstract

The metabolic capacity of the intestine and its importance as the initial barrier to systemic exposure can lead to underestimation of first-pass, and thus overestimation of oral bioavailability. However, the in vitro tools informing estimates of in vivo intestinal metabolism are limited by the complexity of the in vitro matrix preparation and uncertainty with the scaling factors for in vitro to in vivo extrapolation. A number of methods currently exist in the literature for the preparation of intestinal microsomes; however, the impact of key steps in the preparation procedure has not been critically assessed. In the current study, changes in enterocyte isolation, the impact of buffer constituents heparin and glycerol, as well as sonication as a direct method of homogenization were assessed systematically. Furthermore, fresh vs. frozen tissue samples and the impact of microsome freeze thawing was assessed. The rat intestinal microsomes were characterized for CYP content as well as metabolic activity using testosterone and 4-nitropheonol as probes for CYP and UGT activity, respectively. Comparisons in metabolic activity and scaled unbound intestinal intrinsic clearance (CL_intu,gut ) were made to commercially available microsomes using 25 drugs with a diverse range of metabolic pathways and intestinal metabolic stabilities. An optimal, robust and reproducible microsomal preparation method for investigation of intestinal metabolism is proposed. The importance of characterization of the in vitro matrix and the potential impact of intestinal scaling factors on the in vitro-in vivo extrapolation of F_G needs to be investigated further. © 2017 The Authors Biopharmaceutics & Drug Disposition Published by John Wiley & Sons Ltd.

Keywords: in vitro-in vivo extrapolation; intestinal metabolism; scaling factors.

Figure 1

Summary of method and optimization steps for optimizing preparations of rat intestinal microsomes. E1, ETDA 5 mm; E2, EDTA 1.5 mm; T1. 60 min elution; T2, 20 min elution; H1, 6 W sonication; H2, 18 W sonication; H3, 30 W sonication; B1, no heparin or glycerol; B2, glycerol 20% v/v; B3, heparin 3 U/ml, glycerol 20% v/v; B4, heparin 9 U/ml. S1, no glycerol; S2, glycerol 20% v/v. Solid lines represent progression of optimized method, dashed lines represent sub‐optimal method combinations. The applicable preparation method number in relation to Table 1 is shown within each circle. Each preparation method represents three independent preparations of three pooled intestinal samples

Figure 2

Comparison of microsomal CYP content (A), total microsomal content (B), microsomal recovery (C) and MPPGM (D). *Significantly different to condition (3) (p < 0.05), ×Significantly different to condition (5) (p < 0.05). Preparation numbers correspond to those in Table 1 and represent the mean ± SD for pooled observations of three rats on three separate occasions. MPPGM, microsomal protein per gram of mucosa

Figure 3

(A) Correlation between CL _int,u in pool 1 and pool 2 rat intestinal microsomes using combined CYP and UGT cofactors. n = 22 compounds. Data represent mean ± SD of n = 3 of duplicate incubations. (B) Correlation between CL _int,u for in‐house pools (1 and 2) and Han Wistar commercial rat intestinal microsomes using combined CYP and UGT cofactors. n = 11 compounds. Data represent mean ± SD of n = 3 of duplicate incubations. Solid line represents line of unity, dashed lines 2‐fold

Figure 4

Comparison of extrapolated measures of CL _int,u,gut (l/h) for individual pools and commercial microsomes for a range of study compounds. (A) Comparison of pool 1 vs. pool 2 extrapolated CL _int,u,gut. Line of unity (solid line). Extrapolated CL _int,u,gut (l/h) for pool 1, pool 2 were scaled using pool specific scaling factors of intestinal weight and microsomal protein per gram intestine. (B) Comparison of in‐house and commercial microsomes extrapolated CL _int,u,gut. Line of unity (solid line). In‐house pooled and Han Wistar commercial microsomes were scaled using mean weights and scalars were used from the two in‐house prepared pools. Compound abbreviations in Table 5

Figure 5

Ratios of extrapolated measures of CL _int,u,gut (l/h) for individual pools and comparison with commercial microsomes for a range of study compounds. (A) Ratio of pool 1 and pool 2 extrapolated CL _int,u,gut. Line of unity (solid line). Extrapolated CL _int,u,gut (l/h) for pool 1, pool 2 were scaled using pool specific scaling factors of intestinal weight and microsomal protein per gram intestine. (B) Ratio of in‐house and commercial microsomes extrapolated CL _int,u,gut. Line of unity (solid line). In‐house pooled and Han Wistar commercial microsomes were scaled using mean weights and scalars were used from the two in‐house prepared pools. Compound abbreviations in Table 5