Isotretinoin and its Metabolites Alter mRNA of Multiple Enzyme and Transporter Genes In Vitro, but Downregulation of Organic Anion Transporting Polypeptide Does Not Translate to the Clinic

Abstract

Isotretinoin [13-cis-retinoic acid (13cisRA)] is widely used for the treatment of neuroblastoma and acne. It acts via regulating gene transcription through binding to retinoic acid receptors. Yet, the potential for isotretinoin to cause transcriptionally mediated drug-drug interactions (DDIs) has not been fully explored. We hypothesized that isotretinoin and its active metabolites all-trans-retinoic acid (atRA) and 4-oxo-13cisRA would alter the transcription of enzymes and transporters in the human liver via binding to nuclear receptors. The goal of this study was to define the DDI potential of isotretinoin and its metabolites resulting from transcriptional regulation of cytochrome P450 and transporter mRNAs. In human hepatocytes (n = 3), 13cisRA, atRA, and 4-oxo-13cisRA decreased OATP1B1, CYP1A2, CYP2C9, and CYP2D6 mRNA and increased CYP2B6 and CYP3A4 mRNA in a concentration-dependent manner. The EC₅₀ values for OATP1B1 mRNA downregulation ranged from 2 to 110 nM, with maximum effect (E_max ) ranging from 0.17- to 0.54-fold. Based on the EC₅₀ and E_max values and the known circulating concentrations of 13cisRA and its metabolites after isotretinoin dosing, a 55% decrease in OATP1B1 activity was predicted in vivo. In vivo DDI potential was evaluated clinically in participants dosed with isotretinoin for up to 32 weeks using coproporphyrin-I (CP-I) as an OATP1B1 biomarker. CP-I steady-state serum concentrations were unaltered following 2, 8, or 16 weeks of isotretinoin treatment. These data show that isotretinoin and its metabolites alter transcription of multiple enzymes and transporters in vitro, but translation of these changes to in vivo drug-drug interactions requires clinical evaluation for each enzyme. SIGNIFICANCE STATEMENT: Isotretinoin and its metabolites alter the mRNA expression of multiple cytochrome P450s (CYPs) and transporters in human hepatocytes, suggesting that isotretinoin may cause clinically significant drug-drug interactions (DDIs). Despite the observed changes in organic anion transporting polypeptide 1B1 (OATP1B1) mRNA in human hepatocytes, no clinical DDI was observed when measuring a biomarker, coproporphyrin-I. Further work is needed to determine whether these findings can be extrapolated to a lack of a DDI with CYP1A2, CYP2B6, and CYP2C9 substrates.

Graphical abstract

Fig. 1.

Metabolic scheme of isotretinoin.

Fig. 2.

Clinical sample analysis workflow to test if CP-I serum C_ss, as a biomarker of OATP1B1 function, is affected by varying lengths of retinoid treatment in vivo. Matched retinoid treatment time points were selected to allow for verification of a potential DDI from two independent studies.

Fig. 3.

Impact of retinoid treatment on enzyme and transporter mRNA in human hepatocytes. Fold change of mRNA for each enzyme and transporter is shown following 30 μM treatment with (1) 13cisRA, (2) atRA, and (3) 4-oxo-13cisRA. The bar graphs show average mRNA fold change compared with solvent control (DMSO). Data from each donor is shown as a separate data point (triangle, circle, or square). All experiments were performed as technical triplicates.

Fig. 4.

Concentration-dependent downregulation of OATP1B1 mRNA by 13cisRA, atRA, and 4-oxo-13cisRA in human hepatocytes from three donors. Results from treatment with 13cisRA are shown in panels A, B, and C; treatment with atRA in panels D, E, and F; and treatment with 4-oxo-13cisRA in panels G, H, and I. For donor 3, two independent experiments on separate days were performed (A and B). Technical replicates (n = 3) were performed for each datapoint. The data are presented as fraction of vehicle control (DMSO). EC₅₀ (nM) and E_max (fold change) are calculated as described in eq. 1. Each panel shows the EC₅₀ and E_max values and the 90% CI. In panels C, F, and I, the values are means from two independent experiments, and no CI is shown.

Fig. 5.

Concentration-dependent effect of 13cisRA, atRA, and 4-oxo-13cisRA treatment on OATP1B3 mRNA in human hepatocytes from three donors. Results from treatment with 13cisRA are shown in panels A, B, and C; treatment with atRA in panels D, E, and F; and treatment with 4-oxo-13cisRA in panels G, H, and I. For donor 3 (panels C, F, and I), two independent experiments on separate days were performed (A and B). Technical replicates (n = 3) were performed for each data point. The data are normalized to a housekeeping gene and presented as fraction of vehicle control (DMSO). In panels C, F, and I, EC₅₀ (nM) and E_max (fold change) are calculated as described in eq. 1, and the values are means from two independent experiments.

Fig. 6.

Inter- and intraindividual variability in CP-I serum concentrations and power calculations for the use of the biomarker CP-I to assess OATP1B1 activity. (A) Baseline CP-I serum concentrations averaged from the screening and baseline samples for each individual in the three studies. The intraindividual variability in CP-I concentrations in samples from (B) RA2 and (C) RA4 study participants is shown from a baseline screen and at the start of treatment. The P value for paired t test comparing baseline values is shown in each panel. Time points from a given individual were at least 1 week apart and up to 7 weeks apart. The average S.D. between each participants screening and week 0 sample was 0.228 nM, which was used in the power calculation. Power curves presented in panel D show the calculation of the number of subjects required to detect a CP-I C_ss ratio of 1.1, 1.2, 1.3, and 1.5, with different levels of power with the dotted black line showing 80% power, based on a paired t test with α = 0.05, using the intraindividual variability and population means of baseline samples from RA2 and RA4.

Fig. 7.

Comparison of baseline CP-I serum concentrations to CP-I concentrations following (A) 2 weeks of treatment with isotretinoin 40 mg twice a day (RA3, n = 8) (baseline mean CP-I concentration is 1.35 ± 0.30 nM; week 2 is 1.34 ± 0.22 nM), (B) 16 weeks of treatment with isotretinoin 20 mg twice a day (RA2, n = 19, baseline mean CP-I concentration calculated from screening visit, and week 0 is 1.62 ± 0.48 nM; week 16 is 1.61 ± 0.56 nM), and (C) 8 weeks of treatment with isotretinoin 20 mg twice a day (RA4, n = 8, baseline mean CP-I concentration calculated from screening visit, and week 0 is 1.75 ± 0.30 nM; week 8 is 1.66 ± 0.49 nM). The P value for paired t test comparing baseline and treatment day values is shown in each panel.