Pharmacokinetic Effects of Different Models of Nonalcoholic Fatty Liver Disease in Transgenic Humanized OATP1B Mice

Abstract

Organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 (collectively, OATP1B) transporters encoded by the solute carrier organic anion transporter (SLCO) genes mediate uptake of multiple pharmaceutical compounds. Nonalcoholic steatohepatitis (NASH), a severe form of nonalcoholic fatty liver disease (NAFLD), decreases OATP1B abundance. This research characterized the pathologic and pharmacokinetics effects of three diet- and one chemical-induced NAFLD model in male and female humanized OATP1B mice, which comprises knock-out of rodent Oatp orthologs and insertion of human SLCO1B1 and SLCO1B3. Histopathology scoring demonstrated elevated steatosis and inflammation scores for all NAFLD-treatment groups. Female mice had minor changes in SLCO1B1 expression in two of the four NAFLD treatment groups, and pitavastatin (PIT) area under the concentration-time curve (AUC) increased in female mice in only one of the diet-induced models. OATP1B3 expression decreased in male and female mice in the chemical-induced NAFLD model, with a coinciding increase in PIT AUC, indicating the chemical-induced model may better replicate changes in OATP1B3 expression and OATP substrate disposition observed in NASH patients. This research also tested a reported multifactorial pharmacokinetic interaction between NAFLD and silymarin, an extract from milk thistle seeds with notable OATP-inhibitory effects. Males showed no change in PIT AUC, whereas female PIT AUC increased 1.55-fold from the diet alone and the 1.88-fold from the combination of diet with silymarin, suggesting that female mice are more sensitive to pharmacokinetic changes than male mice. Overall, the humanized OATP1B model should be used with caution for modeling NAFLD and multifactorial pharmacokinetic interactions. SIGNIFICANCE STATEMENT: Advanced stages of NAFLD cause decreased hepatic OATP1B abundance and increase systemic exposure to OATP substrates in human patients. The humanized OATP1B mouse strain may provide a clinically relevant model to recapitulate these observations and predict pharmacokinetic interactions in NAFLD. This research characterized three diet-induced and one drug-induced NAFLD model in a humanized OATP1B mouse model. Additionally, a multifactorial pharmacokinetic interaction was observed between silymarin and NAFLD.

Fig. 1.

Representative H&E liver histology for SC (A), HFHF (B), HFHC (C), and MCD (D) mice. Inflammation scores in OATP1B mice fed a SC, HFHF, HFHC, or MCD diet in both sexes (E, n = 12), males (F, n = 6), and females (G, n = 6). Steatosis scores in OATP1B mice fed a SC, HFHF, HFHC, or MCD diet in both sexes (H), males (I), and females (J).

Fig. 2.

Representative H&E liver histology for saline (A) and tunicamycin (B) treated mice. Inflammation scores in OATP1B mice administered saline or tunicamycin in both sexes (C, n = 12), males (D, n = 6), and females (E, n = 6). Steatosis scores in OATP1B mice administered saline or tunicamycin in both sexes (F), males (G), and females (H).

Fig. 3.

SLCO1B1 mRNA qRT-PCR analysis for SC, HFHF, HFHC, or MCD mice in both sexes (A, n = 12), males (B, n = 6), and females (C,n = 6). SLCO1B3 mRNA qRT-PCR analysis for SC, HFHF, HFHC, or MCD mice in both sexes (D, n = 12), males (E, n = 6), and females (F, n = 6). Bar height and error bars represent arithmetic mean and standard deviation, respectively. One-way ANOVA with Dunnet’s post-test for multiple comparisons: * P < 0.05 or ** P < 0.01. qRT-PCR, real time quantitative polymerase chain reaction.

Fig. 4.

SLCO1B1 mRNA qRT-PCR analysis for mice administered saline or tunicamycin in both sexes (A, n = 12), males (B, n = 6), and females (C, n = 6). SLCO1B3 mRNA qRT-PCR analysis for mice administered saline or tunicamycin in both sexes (D, n = 12), males (E, n = 6), and females (F, n = 6). Bar height and error bars represent arithmetic mean and standard deviation, respectively. Student’s t test: * P < 0.05. qRT-PCR, real time quantitative polymerase chain reaction.

Fig. 5.

Relative protein expression of OATP1B3 in SC, HFHF, HFHC, or MCD mice in both sexes (A, n = 12), males (B, n = 6), and females (C, n = 6). Protein expression was normalized to amido black total protein staining for each sample and represented relative to SC. A representative Western blot for OATP1B3 and amido black staining in a male mouse (D). Bar height and error bars represent arithmetic mean and standard deviation, respectively.

Fig. 6.

Relative protein expression of OATP1B3 in mice administered saline or tunicamycin in both sexes (A, n = 12), males (B, n = 6), and females (C, n = 6). Protein expression was normalized to amido black total protein staining for each sample and represented relative to saline. A representative Western blot for OATP1B3 and amido black staining in male saline and tunicamycin mice (D). Bar height and error bars represent arithmetic mean and standard deviation, respectively. Student’s t test: * P < 0.05 or ** P < 0.01.

Fig. 7.

PIT plasma concentration-time profiles for SC, HFHF, HFHC, MCD mice in both sexes (A, n = 12) with corresponding PIT AUC data (B). PIT plasma concentration-time profiles and corresponding PIT AUCs for male (C and D, n = 6) and female (E and F, n = 6) mice. For box and whisker plots, the box extends to the 25th and 75th percentiles with the median represented as the horizontal line, and the whiskers represent the 90% confidence interval. Kruskall–Wallis test with Dunn’s post-test for multiple comparisons: * P ≤ 0.05.

Fig. 8.

Male (n = 6) and female (n = 6) PIT plasma concentration-time profiles and corresponding PIT AUC data for SC (A and B), HFHF (C and D), HFHC (E and F), or MCD (G and H) mice. For box and whisker plots, the box extends to the 25th and 75th percentiles with the median represented as the horizontal line, and the whiskers represent the 90% confidence interval. Unpaired Mann–Whitney t test: * P ≤ 0.05 or ** P ≤ 0.01.

Fig. 9.

PIT plasma concentration-time profiles for mice administered saline or tunicamycin in both sexes (A, n = 12) and corresponding PIT AUC data (B). PIT plasma concentration-time profiles and corresponding PIT AUCs for male (C and D, n = 6) and female (E and F, n = 6) mice. For box and whisker plots, the box extends to the 25th and 75th percentiles with the median represented as the horizontal line, and the whiskers represent the 90% confidence interval. Unpaired Mann–Whitney t test: ** P ≤ 0.01 or **** P ≤ 0.0001.

Fig. 10.

PIT plasma concentration-time profiles for SC and HFHF mice orally administered vehicle or silymarin (A, n = 12) with corresponding PIT AUC data (B). PIT plasma concentration-time profiles and corresponding PIT AUCs for male (C and D, n = 6) and female (E and F, n = 6) SC and HFHF mice orally administered vehicle or silymarin with corresponding PIT AUC data. For box and whisker plots, the box extends to the 25th and 75th percentiles with the median represented as the horizontal line, and the whiskers represent the 90% confidence interval. Statistical test for AUCs of all diet groups used the nonparametric unpaired two-way ANOVA. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001.