Preliminary Study Of Photosensitizing Properties And Pharmacokinetic Of The Novel Chlorin E₄ Derivatives

ObjectiveTo study the spectrum properties and the photodynamic effect of six kinds of novel Chlorin e₄ derivatives in human lung cancer A549 cells. Then to choose the most excellent derivative among them to investigate its tissue distribution in the S₁₈₀ tumor bearing mice. In order to investigate its clinical application perspective in tumor, the pharmacokinetic rule of it in rabbit was studied after intravenous injection. Methods and results(1) The absorption spectra, fluorescence emission spectra of the six kinds of novel Chlorin e₄ derivatives (1401, 1402, 1403, 1404, 1405, 1601) in Phosphate Buffered Saline (PBS) and physiological saline with 10 percent serum were measured using UV spectrophotometer and fluorospectrophotometer, respectively. Futhermore, the characterization of the absorption and fluorescence spectra were analyzed and compared with that of the traditional second photosensitizer HMME. Experimental results showed that there were three absorption peaks between 300⁷00nm in PBS of the six kinds of derivatives, and the maximum absorption peaks appeared at 402nm, the second absorption peaks at 654nm, the minimum absorption peaks at 500nm. The maximum peaks of HMME appeared at 394nm, besides this, there are four absorption peaks between 500⁶50nm (500, 530, 560, 613nm, respectively), and the absorption intensity will decrease with the wavelength increasing. The absorption intensity(absorbance value) at 654nm of the six derivatives were in order 1404(0.051) >1402(0.048)>1401(0.033) >1403(0.031)>1601(0.025)>1405(0.013) , The absorption intensity of Chlorin e₄ derivates was 6²5-floder higher than that of HMME at 613nm.The peaks had a red shift with 5⁹nm in the physiological saline with 10 % serum. The fluorescence emission spectra results showed that there was one emission peak between 650⁶70nmfor six kinds of Chlorin q* derivatives, respectively. The fluorescence emission intensity of 1402 is the highest among them, and that of 1405 is the lowest. The two emission peaks of HMME were at 613nm and 671nm, respectively. Except 1405, the others' maximum fluorescence intensity was stronger than HMME. In the physiological saline with 10 % serum, the emission peaks of Chlorin e4 derivatives had a red shift with 4nm and HMME lOnm, respectively.(2) To compare the dark cytotoxicity of the six kinds of Chlorin e4 derivatives in human lung cancer A549 cell line. A549 cells were incubated 4h in vitro with the six derivatives dissolved in DMEM at different concentrations, and the cell survival rate at each concentration point of each derivative was measured by MTT assay after 24h incubation. And to compare the phototoxicity of the six derivatives, A549 cells incubated with the above photosensitizers were irradiated with 630nm laser ( fluence rate= 20mW/cm2 ) for 1000 s. Cell survival rate was measured by MTT assay with same methods. Accordingly, cell survival curve of different photosensitizer was described. The equation of each cell survival curve was analyzed. Finally, IC50 (50% inhibition concentration) of each photosensitizer was caculated. The results showed that the dark cytotoxicity was in order 1403 >1401 >1402 >1404 >1601 >HMME >1405;the photoinduced cell-killing activity (phototoxicity) was in order 1403 > 1404 > 1401 > 1402 > 1601 > HMME> 1405. In this study we designed the ratio between the dark cytotoxicity IC50 values and phototoxicity IC50 values of a photosensitizer as its safety coefficient. The higher safety coefficient indicated a lower dark cytotoxicity and higher phototoxicity. The result showed that the safety coefficient was in order HMME > 1405 > 1601 > 1404 > 1401 > 1403 > 1402. Considering the standard of safety and high-performance, we selected Chlorin ^ derivatives 1404 as the promising photosensitizer for the further study in vivo.(3) After the Si go tumor-bearing mice model being established, thirty mice and five rabbits were administered intravenously with the Chlorin e* derivatives 1404 (lOmg/kg, 15mg/kg respectively). Fluorospectrophotometry was applied to measure the tissue distribution of 1404 in Si go tumor-bearing mice and plasma concentration of 1404 inrabbits. Pharmacokinetics parameters were calculated according to the experimental data. The results showed that 1404 distributed in mice immediately after intravenous administration. The maximal levels of photosensitizer concentration in each tissue were achieved at l³h post-injection, and the photosensitizer concentration of the liver is the highest among them. At 24⁴8h post-injection, the photosensitizer in other tissues had all been cleared except liver, skin and tumor remained only lower concentration of photosensitizer. And the clearance speed of photosensitizer in tumor and skin is the slowest. At 3h post-injection, the photosensitizer concentrations ratio of tumor and skin was maximum(tumor/skin 3.63). At the same time, the photosensitizer concentrations ratio of tumor and muscle was 20. The photosensitizer didn't diffuse through the blood-brain barrier. The results of pharmacyokinetics of 1404 in rabbits after intravenous injection indicated that the drug were absorpt strongly and cleared slowly in animal body.ConclusionThe absorption coefficient of Chlorin e4 derivatives is higher than that of HMME in red light area, which indicate that Chlorin e4 derivatives can be applied to clinical PDT of tumor. The fluorescence intensity of other Chlorin e4 derivatives is higher than that of HMME, except 1405. It suggests that Chlorin e4 derivatives can be used in the fluorescence diagnosis. The dark cytotoxicity of Chlorin e4 derivatives 1404 is lower and the phototoxicity is stronger for human lung cancer A549 cell among the six kinds of derivatives, it is a promising photosensitizer in PDT of tumor. The clearance speed of the normal tissue of 1404 in Si go tumor-bearing mice is fast, but that of skin is slow, so the risk of skin's photosensitivity would be high. The derivatives 1404 can't diffuse through the blood-brain barrier. The results of pharmacy okinetics of 1404 in rabbits after intravenous injection indicate that the drug distributes widely in many organs and is cleared slowly in animal body.