Pregnancy related hormones increase CYP3A mediated buprenorphine metabolism in human hepatocytes: a comparison to CYP3A substrates nifedipine and midazolam

Abstract

Introduction: Pregnancy increases the clearance of CYP3A4 substrate drugs and pregnancy-related hormones (PRHs) induce hepatic CYP3A4 expression and metabolism. However, it remains unclear to what extent the magnitude of PRH-evoked changes in hepatic CYP3A metabolism varies across multiple substrates. This study quantified the impact of PRHs on CYP3A protein concentrations and buprenorphine metabolism in human hepatocytes, and compared the magnitude of these effects to nifedipine and midazolam metabolism. Methods: Sandwich-cultured human hepatocytes (SCHH) from female donors were exposed to PRHs, administered in combination across a range of physiologically relevant concentrations, for 72 h. Absolute protein concentrations of CYP3A4, CYP3A5, and CYP3A7 in SCHH membrane fractions were quantified by nanoLC-MS/MS, and norbuprenorphine (nor-BUP), dehydro-nifedipine (dehydro-NIF), and 1-hydroxy-midazolam (1-OH-MDZ) formation was evaluated. Results: Compared to control, PRH exposure increased CYP3A4, CYP3A7, and total CYP3A protein concentrations, but not CYP3A5 concentrations, and increased nor-BUP, dehydro-NIF, and 1-OH-MDZ formation in a concentration-dependent manner. The formation of nor-BUP, dehydro-NIF, and 1-OH-MDZ each positively correlated with PRH-mediated changes in total CYP3A protein concentrations. The PRH-evoked increase in nor-BUP formation was evident in all donors; however, the PRH induction of dehydro-NIF and 1-OH-MDZ formation was diminished in a hepatocyte donor with high basal CYP3A5 expression. Discussion: These findings demonstrate that PRHs increase buprenorphine, nifedipine, and midazolam metabolism in SCHH via induction of CYP3A4 and total CYP3A protein concentrations, and the magnitude of these effects vary across hepatocyte donors in a substrate-specific manner. These data provide insight into the contribution of PRH induction of CYP3A4 metabolism to increased buprenorphine clearance during pregnancy.

Keywords: CYP3A4; CYP3A5; buprenorphine; hepatic metabolism; nifedipine; pregnancy; pregnancy related hormones; targeted proteomics.

FIGURE 1

Protein expression of CYP3A isoforms in sandwich-cultured human hepatocytes (SCHH). SCHH (n = 5 donors) were exposed to vehicle control or rifampin (RIF) for 72 h (n = 3–4 replicates per group). (A) Basal absolute protein concentrations of total CYP3A, CYP3A4, CYP3A5, and CYP3A7 in SCHH exposed to vehicle control (0.1% DMSO) by hepatocyte donor. (B) Fold-induction of CYP3A isoforms in SCHH exposed to 10 µM RIF relative to vehicle control across all donors (n = 5 per group). ***p < 0.001 versus vehicle control.

FIGURE 2

PRHs increased CYP3A4 and total CYP3A protein concentration in sandwich-cultured human hepatocytes (SCHH). SCHH (n = 5 donors) were exposed to vehicle control or PRH cocktails targeting average trimester 2 (T2), average trimester 3 (T3), or upper range of T3 (T3-90%) for 72 h. The line graphs depict mean ± SD absolute protein concentration of (A) total CYP3A, (B) CYP3A4, (C) CYP3A5, and (D) CYP3A7 within each hepatocyte donor (n = 3–4 replicates per group). The dotted line at y = 0.1 pmol/mg represents lower limits of quantitation. The corresponding bar graphs exhibit the net mean ± SD fold-difference for each protein relative to the vehicle control across all donors (n = 5 per group). *p < 0.05, **p < 0.01, ***p < 0.001 versus vehicle control. #Represents the concentration-dependent effect across PRH groups (#< 0.05, ##p < 0.01).

FIGURE 3

PRHs increased buprenorphine (BUP), nifedipine (NIF), and midazolam (MDZ) metabolism in sandwich-cultured human hepatocytes (SCHH). SCHH (n = 4 donors) were exposed to vehicle control or PRH cocktails targeting average trimester 2 (T2), average trimester 3 (T3), and upper range of T3 (T3-90%) for 72 h prior to incubation with CYP3A substrates BUP, NIF, or MDZ. The line graphs depict the mean ± SD formation of nor-BUP (A), dehydro-NIF (B), and 1-OH-MDZ (C) within each donor (n = 3–4 replicates per group; n = 2 replicates in the MDZ experiment T3 group in donor Hu8375 due to a concentration measurement analytical failure). The corresponding bar graphs exhibit the net mean ± SD fold-difference for each metabolite relative to vehicle control across all donors (n = 4 per group). *p < 0.05, **p < 0.01, ***p < 0.001 versus vehicle control. # Represents the concentration-dependent effect across PRH groups (#< 0.05).

FIGURE 4

Correlation between total CYP3A or CYP3A4 protein concentration and metabolite formation in sandwich-cultured human hepatocytes (SCHH). Correlation between total CYP3A (CYP3A4 + 3A5 + 3A7) (A–C) and CYP3A4 (D–F) absolute protein concentrations with norbuprenorphine (nor-BUP) (A,D), dehydro nifedipine (dehydro-NIF) (B,E), and 1-OH-midazolam (1-OH-MDZ) concentration (C,F) in SCHH exposed to vehicle control or PRH cocktails (n = 4 donors). Each data point represents the mean concentration for each treatment group within each donor. The Pearson correlation coefficient (r) and p-values are provided.