Tetrakis(p-carboranylthio-tetrafluorophenyl)chlorin (TPFC): application for photodynamic therapy and boron neutron capture therapy

Abstract

Carboranyl-containing chlorins have emerged as promising dual sensitizers for use in both photodynamic therapy (PDT) and boron neutron capture therapy (BNCT), by virtue of their known tumor affinity, low cytotoxicity in dark conditions, and their strong absorptions in the red region of the optical spectrum. Tetrakis(p-carboranylthio-tetrafluorophenyl)chlorin (TPFC) is a new synthetic carboranyl-containing chlorin of high boron content (24% by weight). To evaluate TPFC's applicability as sensitizer for both PDT and BNCT, we performed an in vitro and in vivo study using F98 rat glioma cells and F98 rat glioma-bearing brain tumor models. For the in vivo BNCT study, we used boronophenylalanine (BPA), which is currently used in clinical BNCT studies, via intravenous administration (i.v.) and/or used TPFC via convection-enhanced delivery (CED), a method for local drug infusion directly into the brain. In the in vitro PDT study, the cell surviving fraction following laser irradiation (9 J/cm(2) ) was 0.035 whereas in the in vitro BNCT study, the cell surviving fraction following neutron irradiation (thermal neutron = 1.73 × 10(12) n/cm(2) ) was 0.04. In the in vivo BNCT study, the median survival time following concomitant administration of BPA (i.v.) and TPFC (CED) was 42 days (95% confidence interval; 37-43 days).

Keywords: BNCT; CED; CNS; PDT; TPFC; absorption; blood-brain barrier; boron-containing chlorin; cell culture; osmotic pumps.

Figure 1

TPFC’s cellular uptake of boron (ng¹⁰B/10⁶ cells) was about twice as high as BPA’s cellular uptake of boron on each time.

Figure 2

Colony-forming assay using F98 rat glioma cells exposed to 0 (control), 2.5, and 5 μg ¹⁰B/mL of TPFC and irradiated with light doses of 0, 4.5, 9, and 18 J/cm², respectively. The most efficient PDT-induced tumoricidal effect was achieved when the cells were irradiated with 9 J/cm², 18 h after exposure to 5 μg ¹⁰B/mL of TPFC (less than 0.05).

Figure 3

F98 rat glioma cells were exposed to culture medium without boron compound (= control), with 10 μg ¹⁰B/mL of BPA or TPFC for 24 h. Then they were irradiated with thermal neutron, respectively. BPA’s cell surviving fraction following thermal neutron for 30 min was 0.12. On the other hand, TPFC’s cell surviving fraction following thermal neutron for 30 min was 0.04.

Figure 4

Median survival times of untreated group, irradiated group, TPFC group, BPA group, and TPFC + BPA group were 25 days (95% CI; 22–28 days), 26 days (95% CI; 26–30 days), 31 days (95% CI; 27–42 days), 36 days (95% CI; 30–44 days), and 42 days (95% CI; 37–43 days), respectively (yellow, green, blue, brown, and red line indicated untreated, irradiated, TPFC, BPA, and TPFC and BPA group, respectively.). It was significantly between TPFC group and TPFC + BPA group (p value = 0.007 by Log-rank test and 0.004 by Wilcoxon test). However, it was no significantly between BPA group and TPFC + BPA group (p value = 0.350 by Log-rank test and 0.133 by Wilcoxon test). And it was no significantly between TPFC group and BPA group (p value = 0.110 by Log-rank test and 0.063 by Wilcoxon test).

Figure 5

(a) Fluorescence of chlorin (TPFC), (b) nuclear fluorescence by the Hoechst dye (excitation wavelength was 340–380 nm), (c) merged image (magnification of all images: ×400), and (d) bright field image.

Figure 6

In vivo fluorescence microscopy also showed fluorescence of tumor. Red fluorescence indicated the tumor and green fluorescence indicated the normal brain.