Pharmacokinetic, biodistribution, safety and efficacy studies of borophenylalanine (BPA) in BNCT in hepatocellular carcinoma cells and tumor-bearing mouse model

Abstract

Boron neutron capture therapy (BNCT), using boronophenylalanine (BPA) as the main boron carrier, is a dual-targeted particle radiotherapy at the cellular level. Although BPA shows clinical promise in liver cancer, there is relatively little basic research on its effect on hepatocellular carcinoma. Therefore, we systematically evaluated the uptake, safety, pharmacokinetics, and therapeutic efficacy of BPA. Boron uptake in hepatocellular carcinoma cells (Hepa1-6, HepG2) was quantified by ICP-AES, revealing concentration- and time-dependent accumulation (plateau at 6 h), while CCK-8 assays indicated significant cytotoxicity at 24 h. Pharmacokinetic studies in Sprague-Dawley (SD) rats showed rapid boron distribution (peak at 25 ± 5.8 min) with a blood clearance half-life of 74.71 ± 52.22 min. In tumor-bearing mouse models, BPA achieved tumor-specific targeting, with tumor-to-normal tissue (T/N) and tumor-to-blood (T/B) ratios exceeding 2 and 4, respectively, at 2 h post-injection, followed by rapid systemic clearance. Cell viability significantly decreased after BPA-BNCT irradiation, and the tumor growth inhibition rate in mice reached 77%. BPA did not produce tissue damage in vivo, and there were no abnormalities in blood counts or liver or kidney function in vivo after irradiation. These findings suggest that BPA can be selectively enriched in hepatocellular tumors with good pharmacokinetics and therapeutic efficacy, supporting its clinical application in BNCT of hepatocellular carcinoma.

Keywords: BPA; Boron neutron capture therapy; Distribution of drugs; Pharmacokinetic.

Fig. 1

Neutrons combine with boron-bearing tumor cells to undergo nuclear fission.

Fig. 2

Standard curves of ICP-AES. (A) Structure of BPA. (B) Linear correlation between ICP-AES values and cell count. (C) Linear correlation between ICP-AES values measured in normal saline containing different concentrations of boron and boron concentrations.

Fig. 3

(A,B) The comparison of cell viability after co-culturing BPA with Hepa1-6 and HepG2 cells; the figure displays the cell viability values measured after 3 h, 6 h, 12 h, and 24 h of co-culture. Data were expressed as the means ± SD. N = 6. (C,D) Drug uptake of BPA at different concentrations and different times in Hepa1-6 and HepG2 cells. Data were expressed as the means ± SD. N = 6.

Fig. 4

(A) Time curve of blood boron concentration—BPA in rats. The dose of BPA was 500 mg/kg. Female rats were 8 weeks old. Blood was collected at 10, 20, 30, 60, 90, and 150 min after BPA injection for boron determination. Data are expressed as mean ± SD. N = 4. (B) The boron concentration-time curves of BPA in the heart(a), liver(b), spleen(c), lung(d), kidney(e), brain(f), and blood(g) of rats. The dose of BPA was 500 mg/kg. Female rats were 8 weeks old. Blood and tissues were collected at 10, 20, 30, 60, 90, and 150 min after BPA injection for boron determination. Data are expressed as mean ± SD. N = 4.

Fig. 5

(A) Tumor-bearing mouse model. We selected female 6-week-old BALB/c nu mice. N = 4. The injection method used was intravenous push injection. The mice were divided into 3 groups, namely the 0.5-hour post-BPA injection group, 1-hour post-BPA injection group, and 2-hour post-BPA injection group. Boron concentrations in tumors and tissues of subcutaneously tumor-bearing mouse models. (B) Boron concentrations in tumors, blood, and tissues of heart, liver, spleen, kidney, lung and brain (0.5, 1, 2 h). Female mice were 6 weeks old. The dose of BPA was 500 mg/kg. Data are expressed as mean ± SD. N = 4. (C) Ratios of boron concentrations in tumor/blood or tumor/tissue. Female mice were 6 weeks old. The dose of BPA was 500 mg/kg. Blood and tissues were collected at 0.5, 1 and 2 h after BPA injection for boron measurement. Data are expressed as mean ± SD. N = 4.

Fig. 6

Antitumor effects of BNCT combined with BPA in hepatocellular carcinoma models in vitro and in vivo. CK: Control; 10: 10 min neutron irradiation alone (BNCT); 20: 20 min neutron irradiation alone (BNCT); N10: BPA + 10 min BNCT irradiation; N20: BPA + 20 min BNCT irradiation. (A) In vitro cytotoxicity assay: Hepa1-6 cells were divided into five groups. BPA-treated groups were incubated with 500 µg/mL BPA for 6 h prior to irradiation. Cell viability was assessed at 24, 48, 72, and 96 h post-irradiation using CCK-8. **p < 0.01, ****p < 0.0001 compared to CK group. Data were expressed as the means ± SD. N = 6. (B) Colony formation assay: Post-irradiation cells were seeded into 60 mm dishes and cultured for 14 days. Colonies were stained with crystal violet and quantified. (C) In vivo tumor growth kinetics: Subcutaneous Hepa1-6 xenografts were established in the right dorsum of Balb/c nude mice. Female mice were 6 weeks old. Data are expressed as mean ± SD. N = 4. Tumor volume was recorded starting 7 days post-inoculation. At the third day of recording, BPA (500 mg/kg) was intravenously administered, followed by neutron irradiation 2 h later. Tumor volume was monitored and calculated as (length × width²)/2. (D) Representative tumors from control and irradiated groups at day 17 post-treatment.

Fig. 7

(A) Paraffin sections of normal tissues from mice in the non-injection group and the intravenous injection BPA group. N = 3 (B) degree of hemolysis at different concentrations of BPA; The BPA concentration (0.1−1 mg/mL); Data were expressed as the means ± SD. N = 4. (C) Changes in body weight of BPA-injected and control groups of tumor-bearing mice. On the third day of recording body weight, the experimental group was injected with BPA 500 mg/kg, and the change in body weight was recorded continuously for 1 week. Data were expressed as the means ± SD. N = 4.