Identification and Clinical Evaluation of Potential Biomarkers for Breast Cancer Resistance Protein (BCRP/ABCG2)

Abstract

Clinical inhibition and genetic variation of the Breast Cancer Resistance Protein (BCRP/ABCG2) efflux transporter can significantly influence drug exposure, highlighting the need for reliable BCRP functional biomarkers. This study aimed to identify and evaluate biomarkers predictive of BCRP function in humans. A comprehensive analysis of metabolomic genome-wide association studies (mGWAS) was conducted to discover potential BCRP biomarkers, followed by evaluation in in vitro transporter assays and a clinical drug-drug interaction (DDI) study. Across multiple mGWAS datasets, plasma concentrations of three herbicide derivatives-4-hydroxychlorothalonil (4HC), 3-bromo-5-chloro-2,6-dihydroxybenzoic acid (BCDBA), and 3,5-dichloro-2,6-dihydroxybenzoic acid (DCDBA)-were significantly elevated (P < 5E-8) in individuals carrying reduced function ABCG2 polymorphisms. These compounds were confirmed as novel BCRP substrates via transporter uptake assays and selected for clinical evaluation alongside riboflavin, a known BCRP substrate and potential BCRP biomarker. In a DDI study with 11 healthy subjects, eltrombopag, a BCRP inhibitor, increased rosuvastatin concentrations by approximately twofold (P = 0.002). No significant changes in the plasma concentrations of organic anion transporting polypeptide 1B (OATP1B) biomarkers (CP-I and CP-III) or potential BCRP biomarkers (4HC, BCDBA, DCDBA, or riboflavin) were observed. Notably, two subjects were heterozygous carriers for the ABCG2 p.Q141K variant and exhibited significantly higher baseline concentrations of 4HC (P = 0.004) and BCDBA (P = 0.0003), consistent with reduced BCRP function. These findings suggest that 4HC and BCDBA are promising biomarkers for baseline BCRP function in specific populations, such as those harboring reduced function genetic polymorphisms, but do not appear suitable for detecting acute BCRP inhibition.

Figure 1

Approach for identifying and evaluating potential BCRP biomarkers. BCRP biomarker candidates were identified by mining public genomic and metabolomic databases for metabolite associations with reduced function ABCG2 genetic variants. These compounds were subsequently screened as BCRP substrates and evaluated for interactions with OATP1Bs and P‐gp. A clinical study in 11 healthy subjects assessed the proposed biomarkers' utility as indicators of BCRP modulation in both acute (clinical inhibition) and chronic (genetic variation) contexts. Created in BioRender.com.

Figure 2

Screening of proposed biomarkers as substrates of BCRP and OATP1B1/1B3 in cellular assays. Top Panel: Uptake of 4HC, BCDBA, and DCDBA was significantly reduced in HEK293 cells stably expressing the BCRP efflux transporter compared to the same cell line with inhibitor Ko143 added and in HEK293 empty vector cells. Bottom Panel: Uptake of these compounds was similar across cells expressing OATP1B1/1B3, OATP1B1 cells treated with the inhibitor CsA, and empty vector cells. All screenings were performed using 10 μM and 30 μM concentrations of each compound, with uptake quantified using normalized peak areas. 4HC, 4‐hydroxychlorothalonil; BCDBA, 3‐bromo‐5‐chloro‐2,6‐dihydroxybenzoic acid; BCRP, breast cancer resistance protein; CsA, cyclosporine A; DCDBA, 3,5‐dichloro‐2,6‐dihydroxybenzoic acid; OATP1B1/1B3, organic anion transporting polypeptide 1B1/1B3. Bars represent mean ± SD. Significance Levels: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 3

The Effect of Eltrombopag on Rosuvastatin Pharmacokinetics and OATP1B Biomarker Coproporphyrin‐I in 11 Healthy Subjects. Plasma concentrations of rosuvastatin (Top Panel) and coproporphyrin‐I (Bottom Panel) in a single‐sequence crossover study with 11 healthy subjects. In the first arm, subjects received a single 10 mg oral dose of rosuvastatin (Rosuvastatin Alone). Following a washout period of ≥7 days, subjects received a 75 mg oral dose of eltrombopag every 24 hours for 5 days, with the last dose co‐administered with a 10 mg oral dose of rosuvastatin (Rosuvastatin + Eltrombopag). Concentration‐time profiles are presented as geometric means with a 90% geometric coefficient of variation. Parameters were compared using paired t‐tests, with P values reported on the respective graphs. AUC_0‐X (Area under the concentration‐time curve for 0 to X time interval); C_max (Maximum concentration).

Figure 4

The Effect of ABCG2 p.Q141K Variant on Baseline Concentrations of Proposed BCRP Biomarkers. Baseline (pre‐treatment; T = 0 h) concentrations of 4HC, BCDBA, DCDBA, and riboflavin in nine individuals homozygous for the ABCG2 reference allele (“REF (CC)”) compared to two individuals heterozygous for the reduced function variant (“p.Q141K (CA)”). Concentrations were compared across groups using unpaired t‐tests, with P values reported on the respective graphs.

Figure 5

The Effect of Eltrombopag on Proposed BCRP Biomarkers. Plasma concentrations of 4HC, BCDBA, DCDBA, and riboflavin in 11 healthy subjects. Concentration‐time data are presented as geometric means with a 90% geometric coefficient of variation.