Structural Modification Of Natural Products Based On GLUT1 And Its In Vitro Anti-brain Tumor And Blood-brain Barrier Permeability

As we all know,brain tumor is a kind of malignant tumor which is very harmful to human beings,and due to the existence of the blood-brain barrier?BBB?,many anti-tumor drugs cannot enter the brain at present,even if they could enter,ordinarily because of toxic side effects or the presence of drug resistance resulting in poor efficacy,so the drugs used to treat brain tumors are extremely limited.Therefore,the development of anti-tumor drugs that can penetrate the BBB is of milestone significance.A large number of studies have exhibited that there are highly expressed glucose transporter 1?GLUT1?on the blood-brain barrier and tumor cell membrane surface,which can facilitate the drugs to penetrate the BBB through ligand-carrier mediated transport and improve the drugs targeting to the lesions.Hereon,a series of neoteric anti-tumor natural products with monosaccharide conjugation were designed and synthesized employing GLUT1 as a drug target.In this study,we selected D-glucose,D-galactose,and D-mannose,which have inherent affinity with GLUT1,as ligands to carry tumor drugs,which can specifically bind to GLUT1 to achieve the transport function across the BBB.Then,the natural products camptothecin,podophyllotoxin,and perillyl alcohol were selected as anti-tumor effect molecules,and a series of glycoconjugates were synthesized by coupling of dicarboxylic acids such as succinic acid for glycosylation,a total of 18 compounds,which were investigated and characterized by nuclear magnetic and mass spectrometry.In vitro anti-tumor cell proliferation activity test experiments showed that the anti-tumor activity of camptothecin conjugates LQF-A4?LQF-A5?LQF-A6 to glioma cells U251 was comparable to that of camptothecin even superior to camptothecin,with IC ₅₀values of 7.60×10^-3?8.08×10^-3?1.09×10^-2?M respectively,And The IC₅₀ value of camptothecin was 8.29×10-3?m.The anti-tumor activity of LQF-B1?LQF-B2?LQF-B3 podophyllotoxin conjugates to U87 cells is comparable to that of podophyllotoxin.The podophyllotoxin was 1.44×10-3?M.The results of in vitro cytotoxicity test displayed that the toxicity of camptothecin and podophyllotoxin in brain microvascular endothelial cells was lower than that of parent drug,among which camptothecin conjugates were less than half of camptothecin,and the effect was palpable.In order to verify whether the synthetic glycoconjugate could be transported across the membrane through GLUT1,we conducted a GLUT1 substrate competition experiment using U87 cells.The experimental results showed that the glycoconjugate LQF-A7,LQF-A8,LQF-A9,LQF-B1,LQF-B2,LQF-B3 under the competition of GLUT1 substrate,the anti-tumor proliferation effect was reduced.To further verify this conclusion,brain microvascular endothelial cells in vitro model of blood-brain barrier were used for uptake experiments,in which camptothecin and its conjugates LQF-A7 were selected as uptake samples,and root endothelin was used as inhibitor to determine drug uptake concentration by LC-MS.The results showed that endothelial cells had a certain inhibitory effect on the uptake of camptothecin glycoconjugate in the presence of 100?M phlorizin,from which it can be inferred that the camptothecin glycoconjugate may be transmembrane transported into the cell via GLUT1 Within,it was speculated that the transmembrane transport of camptothecin glycoconjugate was regulated by GLUT1.For the study of the permeability of the camptothecin glycoconjugate LQF-A7 blood-brain barrier,brain microvascular endothelial cells were s elected as an model of the BBB in vitro for drug transmembrane transport experiments.Using camptothecin as the control,the concentration was 50?m,and the cellular uptake was measured at 15 min?30min?60min?120min?240min at different time nodes.As a result,the cellular uptake of camptothecin was 0.94nmol?1.00nmol?2.63nmol?5.12nmol?6.76nmol respectively.The cellular uptake of LQF-A7 was 0.32nmol?0.35nmol?0.41nmol?0.45nmol?0.64nmol respectively.According to the cellular uptake data of camptothecin sugar c onjugate LQF-A7,the uptake of it at each time node is lower than that of camptothecin.The results may be that the increased molecular weight and polarity of glycosylated drugs increase to more than 600 and increase the difficulty of blood-brain barrier.This conclusion lays a certain foundation for the selection of the maternal drug molecules targeted to GLUT1 for transportation across the BBB and the method of glycosylation modification,which has certain reference significance.In our research,the glycosylation modification of natural products was carried out for brain tumors which are formidable to treat,and the embedding of sugar molecules made the whole drug molecule reduce its toxic side effects under the condition that the anti-glioma cell proliferation activity remains unchanged,which provides a new opportunity for clinical chemotherapy.Synchronously,elucidating that natural drug sugar conjugates can trans-membrane transport through GLUT1 can contribute to construct a strategy based on GLUT1 to improve the molecular structure modification of systemic.