Lapatinib distribution in HER2 overexpressing experimental brain metastases of breast cancer

Abstract

Purpose: Lapatinib, a small molecule EGFR/HER2 inhibitor, partially inhibits the outgrowth of HER2+ brain metastases in preclinical models and in a subset of CNS lesions in clinical trials of HER2+ breast cancer. We investigated the ability of lapatinib to reach therapeutic concentrations in the CNS following (14)C-lapatinib administration (100 mg/kg p.o. or 10 mg/kg, i.v.) to mice with MDA-MD-231-BR-HER2 brain metastases of breast cancer.

Methods: Drug concentrations were determined at differing times after administration by quantitative autoradiography and chromatography.

Results: (14)C-Lapatinib concentration varied among brain metastases and correlated with altered blood-tumor barrier permeability. On average, brain metastasis concentration was 7-9-fold greater than surrounding brain tissue at 2 and 12 h after oral administration. However, average lapatinib concentration in brain metastases was still only 10-20% of those in peripheral metastases. Only in a subset of brain lesions (17%) did lapatinib concentration approach that of systemic metastases. No evidence was found of lapatinib resistance in tumor cells cultured ex vivo from treated brains.

Conclusions: Results show that lapatinib distribution to brain metastases of breast cancer is partially restricted and blood-tumor barrier permeability is a key component of lapatinib therapeutic efficacy which varies between tumors.

Figure 1

¹⁴C-Lapatinib in brain and brain metastases at 2 (a–c) and 12 (d–f) hours following ¹⁴C-lapatinib administration (100 mg/kg, p.o.) in mice with 231-BR-HER2 tumors. Representative coronal brain sections showing green EGFP metastasis fluorescence (a,d), Texas Red 3kD dextran fluorescence (b,e), and ¹⁴C-lapatinib radioactivity (c,f) from an animal with 231-BR-HER2 brain metastases that received ¹⁴C-lapatinib for 2 (a–c) or 12 (d–f) hours. Met = metastasis.

Figure 2

Distribution of ¹⁴C-lapatinib between (a) and within (b) brain metastases, as well as relation of ¹⁴C-lapatinib concentration to metastasis size (c) and barrier permeability (d). Data are for animals (n=6-8) at 2 hours after oral administration of 100 mg/kg ¹⁴C-lapatinib. Similar results were found at 12 h. (a) Brain metastasis ¹⁴C-lapatinib concentrations were divided into three mutually exclusive groups: comparable to brain (<mean brain concentration + 3 × SD), <10-fold higher relative to brain, and >10-fold higher relative to brain. Bars represent group average concentration ± SEM. The percentage of metastases within each group is listed above the bar. (b) Distribution of ¹⁴C-lapatinib concentration within a single representative brain metastasis (Met-1 in Figure 1f). Each bar represents the relative frequency of concentration within small regions (pixel = 25 ×25 μm) of the tumor. Although the average concentration for the metastasis was 604 ng/g, individual regions within the metastasis varied from 30 to >4300 ng/g. (c,d) Points represent data for individual brain metastases. Color dotted lines denote average metastasis diameter (d) in mm. The Pearson correlation is shown as a solid line and was statistically significant (P<0.05).

Figure 3

¹⁴C-Lapatinib in lung (a–d) metastases at 12 hour after ¹⁴C-lapatinib administration (100 mg/kg, oral). Representative sections showing lung metastasis EGFP fluorescence (a), Texas Red 3kDa dextran fluorescence (b), cresyl violet staining (c) and ¹⁴C-autoradiography (d) from an animal with 231-BR-HER2 metastases that received ¹⁴C-lapatinib at 12 hours prior to euthanasia. Matching metastases were also measured in brain from the same animal.

Figure 4

¹⁴C-Lapatinib concentrations in brain and peripheral metastases, as well as blood and other tissues, at 12 hours after ¹⁴C-lapatinib administration (100 mg/kg, oral) in immunocompromised mice with 231-BR-HER2 metastases. Bars represent mean ± SD for n=5 animals. Lapatinib concentration (y-axis) is shown in log scale. Points represent concentrations in individual metastases; mean concentration is shown by a line.

Figure 5

Radiochromatography of ¹⁴C-radioactivity in plasma at 30 min after intravenous administration of 10 mg/kg ¹⁴C-lapatinib. Radiolabeled lapatinib was found to be >98% pure and co-eluted with cold lapatinib as confirmed with HPLC.

Figure 6

¹⁴C-Lapatinib distribution at 30 min after i.v ¹⁴C-lapatinib administration (10 mg/kg) in immunocompromised mice with 231-BR-HER2 metastases. Bars represent mean ± SD for n=3 animals. Lapatinib concentration (y-axis) is shown in log scale. Points represent concentrations in individual metastases; mean concentration is shown by a line.

Figure 7

HER2 expression is maintained upon ex vivo culture of lapatinib-treated brain metastatic tumor cells. 231-BR-HER2 cells were injected into immunocompromised mice, treated with either 100 mg/kg lapatinib twice daily or vehicle. At necropsy, tumor cells were cultured ex vivo from brains of lapatinib-treated (L) or vehicle-treated (V) mice. The HER2 expression of lysates of each culture was determined by western blot, using α-tubulin as a loading control.

Figure 8

Ex vivo cultures of lapatinib-treated 231-BR-HER2 cells remain sensitive to lapatinib in vitro. (a-b) Cultures described in Fig. 7 were assayed for viability after 72 hours of lapatinib treatment (8 μM lapatinib, a; 10 μM lapatinib, b) by MTT assay. Data represent the mean ± SD of triplicate cultures. (c) Clonogenic assays of the ex vivo cultures in 4 μM lapatinib.