Design And Synthesis And Machanism Of Chlorin-type Photosensitizers &HDACi Dual Mode System For Antitumor Therapies

In recent years,photodynamic therapy(PDT)has been widely applied in clinic,and plays a pivatal role in treating with superfacial tumor such as dermology,gynecology and urinary system cancers.Due to low toxicity,fast therapeutic effect,none drug resistance and no damage to adjacent tissues,PDT has become one of the most active field in anti-tumor field,and occupys large portions in Pyrormaceutical and medical research.Though great advantages and potentials,there are still some drawbacks of PDT:(1)photosensitizers are non-specific targeted drugs with poor solubility and cellular uptake,large doses should be given in clinic to guarantee the therapeutic effect of drugs,correspondingly,patients suffer from drug allergy,heat reaction,dermal phototoxicity and cutaneous pigmentationn;(2)recent research demonstrates that acetylation level of tumor cells were greatly decreased after PDT,while acitivity of Histone deacetyases is significantly increased,which may be the main cause of the fast proliferation of tumor cells.This problem is the main reason of tumor relapse and metastasis,also a harsh challenge in clinical practice.Therefore,based on the evidence from literatures that combination of photosensitizers and HDACi potentiate sernegetic antitumor effect,attempts of combination of PDT and HDACi are put forward to achieve dual-mode anti-tumor effect to inhibit tumor relapse and metastasis.Considering this conception,three routes are designed to achieve the dual mode antitumor effect of HDACi and PDT.1.Pharmacophore integration strategy of HDACi and photosentitizersBy the traditional Pharmacophore integration strategy,introduction of Pyrormacophores of HDACi into the molecule structures of photosensitizer chlorin e4 and e6 to explore wide spectrum,high effeciency dual mode antitumor lead molecules.Based on earlier works,clorin rings of second generation photosensitizers porphin e4 and e6 are designed as CAP group of aromatic zone of HDACi molecule to provide 17 novel porphin-HDACi dual mode anti-tumor photosensitizer with different linker groups.Photophysicochemical properties of all the compounds have been determined,and their dark cytotoxicity and light cytotoxicity against A549 and HCT116 have been detected to screen a compoond 5d with outstanding antitumor activity in vitro.Its IC₅₀ values of dark cytotoxicity and light cytotoxicity against A549 are 1.5 ?M and 0.026 ?M respectively,which are comparable to the clinical use HDACi SAHA(vorinostat,1.67?M),which demonstrated lead molecule of photosensitizer combining HDACi pharmacophoresuccessfully achieved the function of HDACi;additionally,its antitumor activity(light cytotoxicity)is much better than the dark phototoxicity of itself and SAHA,also much better than the clinical second generation photosensitizer Talaporfin(4.71?M),which demonstrates that the lead molecule of photosensitizer combining HDACi pharmacophore maintained the anti-tumor activity of PDT.Hence,compound 5d with high antitumor effectwas chosen to probe into the antitumor mechanismvia tumor cellular uptake,apoptosis,yield of reactive oxygen species(ROS),subcellular localization and cell cycle arrest to investigate its antitumor effect,and the results indicate,cellular uptake of compound 5d is greatly higher than that of Talaporfin;With PDT,5d induces the increasing of reactive oxygen species(ROS)in tumor cells with concentration dependent characteristic;Without PDT,5d at a concentration of 2.5 ?M could induce cell apoptosis at a rate of 91.41%,demonstrating that compound 5d functioning as a HDACi to induce cell apoptosis;With PDT,5d plays the role as a photosensitizer to induce tumor cell apoptosis;subcellular localization indicates that 5d mainly locates in mitochondria and endoplasmic reticulum,and cell cycle arrest test indicates the cell cycle presents the characteristics of both PDT and HDACi in cell cycle arrest.Animal experiment in vivo demonstrate,with PDT,5d(2.0 mg/kg)could significantlyinhibit the growth of tumor volume,indicating that 5d exert comparable antitumor effect to Talaporfin;and without PDT,both 5d and SAHA showed no significant tumor inhibition effect,hinting further work may focus on adjusting tumor bearing animal model to verified the therapeutic effect of the 5d.2.Prodrug design strategyConsidering the steric hindrance of pharmacophores,prodrug design stratagy was used to couple HDACi and photosensitizer together to provide compound which could play therapeutic roles in the tumor site respectively.Potential linkers includesacyl hydrazone,disulfide and ester bond were considered as the linker candidate,and finally ester bond was chosen as the linker to achieve the goal that HDACi and photosensitizer were seperated in the targeting cells.Our results indicated,although the four compounds demonstrated outstanding photodynamic activity,but they didn't show any HDACi activity through their dark cytotoxicity.Among those compounds,compound 6 was chose to explore its antitumor effect in cell cycle arrest and animal experiment in vivo due to its high activity of PDT.Experimental results indicate: 6 could exert cell cycle arrest characteristic of HDACi,however,6 only could demonstrated therapeutic effect as a photosensitizer,reminding us further work should concentrate on optimizing the animal experiment model to investigate the therapeutic effect of compound 6.3.Molecule self-assembly strategyIn this part,a noval p H-responsive and lysosome triggered release micelleplex,polyethylene glycol-b-poly(asparaginyl-vorinostat),simplified as FPPS.This strategy not only guaranteed the synergetic therapeutic effect of HDACi and PDT.also the drug delivery system reduced the systematic toxicity and increased the effective therapeutic concentration of drugs in tumor site.In a buffer solution at p H7.4,as a strong hydrophobic interaction between Pyro and FPPS,Pyro could be encapsulated to form micelleplex.Under TEM,Pyro@FPPS showed regular sphere and good dispersity with a diameter about 110 nm.In the simulation of drug release in vitro,the results indicated that Pyro could be well released at p H5.2 responding to the p H changes;also,with the generation of lipase in the lysosome,SAHA could be released due to the rupture of ester bond between SAHA and the polymer chains.Responsive release of SAHA and Pyro guaranteed the highly synergetic therapeutic effect in machnism.Cycle arrest performance of tumor cells treated with Pyro@FPPS were just like the combination of the performances of those tumor cells blocked with HDACi and PDT respectively,which indicated an ideal synergistic effect of these two drugs.In animal experiment level,Pyro@FPPS showed excellent tumor inhibition effect and significantly prolonged the average survival period of tumor-bearing mice.Importantly,Pyro@FPPS could significantly inhibit the metastasis of tumor in tumor-bearing mice modals.In a word,acetylation level of tumor cells were greatly decreased after PDT,while acitivity of Histone deacetyases is significantly increased,which may be the main cause of the fast proliferation of tumor cells,therefore,three strategies including pyrormacophore integration,coupling photosensitizer and HDACi with enviromental responsive linkers,and self-assembly photosensitizer and HDACi with tumor-targeting micelleplex were adopted to achieve the synergistic effect of HDACi and PDT.Relatively,p H responsive and drugs lysosome triggered released tumor-targeting micelleplex was the ideal administration of both HDACi and photosensitizer in vivo.With the knowledge of Medicinal Chemistry,Pyrormaceutics and Polymers,administration of HDACi and photosensitizers synchronously was deeply investigated,and the result provided potential application value in clinic to overcome the relapse and metastasis of cancer in tumor photodynamic therapy.