OATP1A/1B, CYP3A, ABCB1, and ABCG2 limit oral availability of the NTRK inhibitor larotrectinib, while ABCB1 and ABCG2 also restrict its brain accumulation

Abstract

Background and purpose: Larotrectinib is a FDA-approved oral small-molecule inhibitor for treatment of neurotrophic tropomyosin receptor kinase fusion-positive cancer. We here investigated the functions of the multidrug efflux transporters ABCB1 and ABCG2, the SLCO1A/1B (OATP1A/1B) uptake transporters, and the multispecific drug-metabolizing enzyme CYP3A in larotrectinib pharmacokinetic behaviour.

Experimental approach: In vitro, transepithelial drug transport and uptake assays were performed. In vivo, larotrectinib (10 mg·kg^-1 ) was administered orally to relevant genetically modified mouse models. Cell medium, plasma samples, and organ homogenates were measured by a sensitive and specific LC-MS/MS larotrectinib assay.

Key results: In vitro, larotrectinib was avidly transported by human (h) ABCB1 and mouse (m) Abcg2 efficiently by hABCG2 and modestly by hOATP1A2. In vivo, both mAbcb1a/1b and mAbcg2 markedly limited larotrectinib oral availability and brain and testis accumulation (by 2.1-fold, 10.4-fold, and 2.7-fold, respectively), with mAbcb1a/1b playing a more prominent role. mOatp1a/1b also restricted larotrectinib oral availability (by 3.8-fold) and overall tissue exposure, apparently by mediating substantial uptake into the liver, thus likely facilitating hepatobiliary excretion. Additionally, larotrectinib is an excellent substrate of CYP3A, which restricts the oral availability of larotrectinib and hence its tissue exposure.

Conclusions and implications: ABCG2 and especially ABCB1 limit the oral availability and brain and testis penetration of larotrectinib, while OATP1A/1B transporters restrict its systemic exposure by mediating hepatic uptake, thus allowing hepatobiliary excretion. CYP3A-mediated metabolism can strongly limit larotrectinib oral availability and hence its tissue concentrations. These insights may be useful in the further clinical development of larotrectinib.

FIGURE 1

Transepithelial transport of larotrectinib (5 μM) assessed in MDCK‐II cells either non‐transduced (a, b) or transduced with hABCB1 (c, d), hABCG2 (e, f) or mAbcg2 (g, h) cDNA. At t = 0 hr, drug was applied in the donor compartment and the concentrations in the acceptor compartment at t = 1, 2, 4, and 8 hr were measured and plotted as larotrectinib transport (pmol) in the graph (n = 3). (b, d–h) Zosuquidar (Zos, 5 μM) was applied to inhibit human and/or endogenous canine ABCB1. (f, h) The ABCG2 inhibitor Ko143 (5 μM) was applied to inhibit ABCG2/Abcg2‐mediated transport. AB, translocation from the apical to the basolateral compartment; BA, translocation from the basolateral to the apical compartment. Data shown are means ± SD; r, relative transport ratio

FIGURE 2

Plasma concentration–time curves of larotrectinib in male wild‐type, Abcb1a/1b ^−/−, Abcg2 ^−/−, and Abcb1a/1b;Abcg2 ^−/− mice over 1 hr after oral administration of 10 mg·kg⁻¹ of larotrectinib. Data are given as mean ± SD (wild‐type and Abcb1a/1b ^−/−, n = 7; Abcg2 ^−/− and Abcb1a/1b;Abcg2 ^−/−, n = 6)

FIGURE 3

Organ concentration (a, c) and organ‐to‐plasma ratio (b, d) of larotrectinib in male wild‐type, Abcb1a/1b ^−/−, Abcg2 ^−/−, and Abcb1a/1b;Abcg2 ^−/− mice 1 hr after oral administration of 10 mg·kg⁻¹ of larotrectinib (wild‐type and Abcb1a/1b ^−/−, n = 7; Abcg2 ^−/− and Abcb1a/1b;Abcg2 ^−/−, n = 6). ^* P < .01, significantly different from wild‐type mice. ^# P < .01, significantly different from knockout mouse strains. Statistical analysis was applied after log transformation of linear data

FIGURE 4

Plasma concentration–time curves of larotrectinib in male wild‐type and Slco1a/1b ^−/− mice over 1 hr after oral administration of 10 mg·kg⁻¹ of larotrectinib. Data shown means ± SD (n = 7)

FIGURE 5

Tissue concentrations (a, c, e, g) and tissue‐to‐plasma ratios (b, d, f, h) of larotrectinib in male wild‐type and Slco1a/1b ^−/− mice 1 hr after oral administration of 10 mg·kg⁻¹ of larotrectinib (n = 7). SI, small intestinal tissue. ^* P < .01, significantly different from wild‐type mice. Statistical analysis was applied after log transformation of linear data

FIGURE 6

Plasma concentration–time curves (a) and semi‐log plot of plasma concentration–time curves (b) of larotrectinib in male wild‐type, Cyp3a ^−/−, and Cyp3aXAV mice 4 hr after oral administration of 10 mg·kg⁻¹ of larotrectinib. Data shown are means ± SD (n = 6)

FIGURE 7

Tissue concentrations for the brain (a), liver (b), kidney (c), small intestine (d), lung (e), and testis (f) of larotrectinib in male wild‐type, Cyp3a ^−/−, and Cyp3aXAV mice 4 hr after oral administration of 10 mg·kg⁻¹ of larotrectinib (n = 6). ^* P < .01, compared with wild‐type mice. ^# P < .01 significantly different as indicated. Statistical analysis was applied after log transformation of linear data