Spectroscopic Studies On The Competitive Binding Of Drugs To Plasma Protein And Sonodynamic Activities Of Drugs

Fluorescence spectrophotometry is an effective spectroscopic method to study the binding properties of drugs to proteins.In recent years,it has been used in the study of the competitive binding of drugs to plasma proteins.The competitive binding of drugs to plasma proteins is a kind of pharmacokinetic interactions,which will lead to changes in the free concentration of one or more drugs,thereby affecting the therapeutic effect and the risk of side effects of the drugs.Herein,the influences of non-steroidal anti-inflammatory drugs piroxicam(PXC)and paracetamol(PCT)on the affinities of nifedipine(NDP)binding to human serum albumin(HSA)were investigated by fluorescence spectroscopy and ultrafiltration.The results are as follows:the results of the displacement experiment of binding site markers and molecular docking confirm that the binding site of NDP binding to HSA is site I,which is the same as the binding sites of non-steroidal anti-inflammatory drugs PXC and PCT.The results of spectroscopic method show that the non-steroidal anti-inflammatories drugs PXC and PCT reduce the association constant of NDP binding to HSA.The results of ultrafiltration illustrate that the non-steroidal anti-inflammatories drugs PXC and PCT reduce the amount of NDP binding to HSA.The results suggest that the non-steroidal anti-inflammatories drugs PXC and PCT can reduce the affinities of NDP binding to HSA,and will increase the free concentration of NDP in plasma,which thereby would enhance the biological effect and increase the risk of adverse drug reactions of NDP.Since the affinities of PXC binding to HSA are greater than those of PCT binding to HSA,the decreased affinities of NDP binding to HSA caused by PXC are more significant than those caused by PCT.Flavonoids are a large class of naturally bioactive polyphenols widely existing in plant foods.It is reported that flavonoids can affect the affinities of many drugs binding to plasma proteins,which will change the free concentration of drug in blood.Herein,rutin and baicalin were selected as the representatives of flavonoids in this paper.The influences of flavonoids on the association constant of theophylline(TP)and clevidipine(CVDP)binding to HSA were studied.And then the influences of flavonoids on the binding distance between the tryptophan(Trp)residues of HSA and TP and CVDP binding to HSA were analyzed.In addition,the changes of HSA conformation caused by the synergies of drugs(TP and CVDP)and flavonoids were investigated by synchronous fluorescence,three dimensional(3D)fluorescence and circular dichroism(CD)spectra.The results are as follows:The binding sites of flavonoids,rutin and baicalin,are the same to TP and CVDP,which suggests that the competitive binding to HSA between flavonoids and TP,flavonoids and CVDP,may occur.The affinities of TP and CVDP binding to HSA decrease because of the the presence of flavonoids.In addition,the decreased affinities of TP and CVDP binding to HSA induced by baicalin are more prominent than those induced by rutin.The binding distance values of TP and CVDP binding to HSA both increase due to the flavonoids.The increased effect of the binding distance induced by baicalin is larger than rutin.The results of synchronous fluorescence,3D fluorescence and CD spectra suggest that the interaction between HSA and TP,and the interaction between HSA and CVDP,can give rise to the changes of HSA conformation.The flavonoids can lead to the further changes of HSA conformation.The results of the competitive binding of drugs to plasma proteins by spectroscopic method and ultrafiltration are consistent,which indicates that it is feasible to study drugs interaction in vitro by spectroscopic method.In addition,spectroscopic method has advantages of simple equipment and quick operation,which will play an important role in the study of the competitive binding of drugs to plasma proteins.It is expected that the above results will provide a valuable research basis for the wide application of spectroscopic method in the study of the competitive binding of drugs to plasma proteins and the further study of drug interactions of NDP,TP and CVDP as well as their clinical safety application.It is one of the main contents of sonodynamic therapy(SDT)that looking for and developing sonosensitizer with good sonodynamic activity.In recent years,spectroscopic method has become an effective method to study the sonodynamic activity of compounds due to its advantages of simple,fast operation and reliable conclusion.Herein,PRP,PEP,CYP and TOP,the 3,7-diamine phenothiazide methylene blue(MB)analogues substituted by di-n-propyl,di-n-pentylamine,cyclohexane and p-toluidine were synthesized using the structure of MB as a reference.Firstly,the damage of protein caused by the synergy of ultrasound and PRP was studied by fluorescence spectroscopy and the reactive oxygen species(ROS)produced in the sonodynamic process were investigated by the oxidation-extraction photometry method.Secondly,the ROS and its kinds produced in the sonodynamic process in the presence of MB analogues were investigated by ROS scavengers and the oxidation-extraction photometry method.Finally,the effects of 3 and 7 amino substituents on the cytotoxicity and the sonodynamic activity of MB analogs were investigated by the mothod of cell damage.The results are as follows:The synergy of ultrasound and PRP can cause damage to protein,and the damage degree is stronger than that of ultrasound and sonosensitizer alone.Oxidative damage of ROS produced by the synergy of ultrasound and PRP is the main cause of protein damage.The damage degree of protein induced by the synergy of ultrasound and PRP is positively correlated with the concentration of sonosensitizer and ultrasonic irradiation time.The order of the production ability of ROS induced by the synergy of ultrasound and MB analogues is TOP>CYP>PEP>PRP>MB,indicating that the extension of amine carbon chain of 3,7-diamine phenothiazine compounds and the introduction of cyclohexane and p-toluidine in the amine group can improve the ability of the compound to produce ROS.In addition,the kinds of ROS produced by the synergy of ultrasound and MB analogues are the same,which are mainly hydroxyl radical(·OH),singlet oxygen('02)and superoxide anion radical(·O2-).Different concentrations of MB analogues all have cytotoxicity to K562 cells.The extension of amine carbon chain of 3,7-diamine phenothiazine compounds will help to strengthen the inhibition effects of tumor cell proliferation.The introduction of cyclohexane and p-toluidine in the amine group of 3,7-diamine phenothiazine compounds reduce the inhibition effects of tumor cell proliferation.The cytotoxicity of the synergistic effect of ultrasound and MB analogue to K562 cells is greater than that of ultrasound and drug alone,and the difference is significant.The extension of amine carbon chain of 3,7-diamine phenothiazine compounds will increase the inhibition effects of K562 cells proliferation induced by the synergistic effect of ultrasound and MB analogue.There was no significant difference of the inhibition effects of K562 cells proliferation between ultrasound combined with CYP and ultrasound combined with MB,and the result of TOP and MB was the same as that of CYP and MB.In order to confirm the sonodynamic activity of eosin B(EB)and brilliant cresyl blue(BCB),the sonodynamic damage to protein in the presence of EB and BCB were studied by fluorescence spectra with serum albumin as a model of protein.The ROS and its kinds produced in the sonodynamic process were investigated by the oxidation-extraction photometry method.In addition,the quantity and kinds of ROS produced by the synergistic effects of ultrasound and EB were compared with those in the presence of fluorescein sodium(Na-FS).The results are as follows:The synergistic effects of ultrasound and sonosensitizers(EB and BCB)can efficiently induce the damage of protein.The damage degree of protein induced by the synergistic effects of ultrasound and sonosensitizer is more serious than that induced by ultrasound or sonosensitizer alone,which indicates that both EB and BCB possess sonodynamic activity.In the certain concentration range of a sonosensitizer,the damage degree of protein induced by the synergistic effects of ultrasound and sonosensitizer increases with the increasing concentration of sonosensitizer.The protein damage induced by the synergistic effects of ultrasound and sonosensitizer increases with the extension of ultrasonic irradiation time.The sonosensitizers can be activated by ultrasound,and then induce the generation of ROS.The damage degree of protein is closely related to the ROS produced by the synergistic effects of ultrasound and sonosensitizer,indicating that the oxidative damage of ROS plays a major role in the sonodynamic damage of protein.The quantities of ROS generation induced by the synergistic effects of ultrasound and EB are higher than those of Na-FS.The major kinds of ROS generated by the synergistic effects of ultrasound and EB,and the synergistic effects of ultrasound and Na-FS,are both ·OH and·O2-.The major kinds of ROS generated by the synergistic effects of ultrasound and BCB are 1O2,·OH and ·O2-.It can be seen from the above results that both spectroscopic method and cell damage method can be used to confirm whether the compounds have sonodynamic activity.The advantage of spectroscopic method is simple and suitable for the preliminary screening of sonosensitive compounds in various laboratories.It is expected that the above results will provide valuable research basis for the wide application of spectroscopic method in the screening and the study of the structure-activity relationship of sonosensitizers,and the development of sonosensitizers with good sonodynamic activity for SDT.