Preparation And Pharmacodynamic And Pharmacokinatic Studies Of Microtubule Protein Inhibitor SUD Inclusion Compound Liposome

Objective: Cyclodextrins (CYD) and liposomes have been used in recentyears as drug delivery vehicles, improving the bioavailability and therapeuticindex of many poorly water soluble drugs. Liposome is a kind of ultra-fineform globular carrier preparation and encapsulates the drug in the thin filmformed by lipid bilayer. Liposome is analog to cellular structure, and has thecharacteristics and function of biological membrane. Liposomes have beenextensively used as drug carriers that can enhance the therapeutic index of anumber of drugs and reduce the toxicity. Liposomes can accumulate greateramounts of drug in some tissues such as liver, spleen and lung comparing freedrug administration.However,incorporation of lipophilic drugs into lipidbilayers of liposomes is often limited in terms of drug-to-lipid mass ration anddrug choice,whitch can interfere with bilayer formation and its stability. CYDcan accommodate water insoluble drugs in their cavities to form inclusioncomplexes. The formed inclusion complex has increased solubility, stabilityand pharmacological activity. Entrapping water-soluble drug/CYD inclusioncomplexes into the aqueous phase of liposomes combines the advantages ofcertain properties of cyclodextrins and liposomes into a single system tocircumvent problems associated with both systems. In our present study, SUDwas used as lipophilic drug model. SUD-HP-β-CD liposome was prepared toincrease the bioavailability and cut down toxicity to achieve the effect ofprolonged action. Meanwhile, some pharmacological experiments wereapplied to explore the effect of SUD-HP-β-CD liposome on A549cellproliferation and the related mechanisms, which would be expected to provideexperimental evidence in future clinical application of SUD-HP-β-CD liposome as an anti-tumor drug.Methods: Inclusion complexes of SUD with hydropropyl-β-cyclodextrin(HP-β-CD) were prepared by the saturation solution method and ultrasionmethod,respectively. However,the saturation solution method was the betterthan the ultrasion method on the basis of the load efficiency. L49(3) orthogonaldesign of the preparation technology of HP-β-CD was made to screen thedosage of adjuvant. The optimized prescription was decided according to theresult of encapsulated ratio determination by range analysis. Inclusioncomplexes were verified by differential scanning calorimetry (DSC) and X-raydiffraction (XRD).Preparation technique and prescription of liposomal of SUD-HP-β-CDwere initially determined on the basis of literature and preliminary test, andinfluential factors were determined by single factor investigation. The effect ofdifferent assay methods on encapsulated ratio was studied. Four methods suchas film dispersion method, injection method, reverse-phase method, doubleemulsion method were used to prepare liposomes, and sephadex column wasused to separate liposome with free drug. The encapsulated ratio of theliposomes was determined, which showed that the film dispersion method wasthe best preparation method. Encapsulated ratio as index, the preparationtechnology of liposomes of SUD-HP-β-CD was optimized by single factorinvestigation, Consequently, L9(34) orthogonal design was made to screen thedosage of adjuvant. The optimized prescription was decided by rangeanalysis to the encapsulated ratio determination result.The shape,appearance,viscosity,particle size distribution,and physical andchemical stability were observed. The accumulative release curve ofSUD-HP-β-CD liposomes and SUD-HP-β-CD solution was protractedrespectively and compared. Zero order, first order, Higuchi, and Weibullkinetic equations were used to fit the release process, and coefficientcorrelation were calculated.A simple HPLC method was developed for the determination of SUD inrat plasma and mice tissues. HPLC conditions: column: Diamonsil C18column (200×4.6mm,5μm); methanol: water (80:20) v/v as mobile phase; detectionwavelength was232nm; column temperature:30℃; flow rate1mL·min^-1;injection volume was20μL. The pharmacokinetic parameters were calculatedand the drug distribution in tissues of mice was investigated to evaluatetargeting in tissues.A549cells were cultured. The proliferation of A549cells was evaluatedby MTT assay. Cell-cycle distribution was determined by flow cytometryassay. The expression of PCNA and Cyclin D1was evaluated by western blotanalysis.Results: SUD-HP-β-CD prepared by saturated aqueous solution has ahigher inclusion rate, the optimal feed ratio of SUD and HP-β-CD is1:5; theinclusion temperature is50℃,the inclusion time is90min, the stirring speedis100rev/min and the average inclusion rate is75.8%(n=3). The inclusioncompound was identified by DSC method and it has been found that newendothermic peak appeared in SUD-HP-β-CD inclusion compound at105℃.Whereas, in the XRD patterns, part of the characteristic diffraction peaks ofSUD disappeared or displaced after SUD was inclused, which indicated thatthe inclusion complex had been formated indeed.The optimized prescription of SUD-HP-β-CD inclusion compoundliposomes was: phospholipid50mg·mL^-1, phospholipid to cholesterol massratio of5:1, drug to lipid ratio of1:30. Liposomes were made by filmdispersion method and the encapsulation was determined by sephadex column.The appearance of SUD-HP-β-CD inclusion compound liposomes wasuniform milky suspension, the encapsulated ratio was76.32%, the averageparticle size of about238.4nm, and the polydispersity coefficient was0.258.The release process in vitro of SUD-HP-β-CD correspond to the Weibullequation, ln[-ln(1-Q)]=0.9045lnt^-1.433, R²=0.9861. In vitro release ofliposome-encapsulated SUD-HP-β-CD inclusion compound correspond tofirst order kinetic equation, Ln (1-Q)=-0.0824t+0.0517, r²=0.9893. Thestability results showed that the liposome was unstable under heatedconditions. When stored at25℃for one month, the encapsulation efficiency decreased by33.6%, liposome deposited obviously; stored for one month at4℃, the encapsulation efficiency decreased by1.7%, indicating that theliposomes need to be stored under refrigeration.Pharmacokinetic experiments showed that the liposome elimination rateconstant k of free SUD was0.22h^-1and the k value of SUD-HP-β-CDinclusion compound liposome was0.41h^-1, indicating that the elimination ofSUD slowerd after liposome formation. AUC was517.54μg·h·mL^-1and228.72μg·h·mL^-1in liposome group and SUD group, indicating that theliposome increased the bioavailability of SUD in the body. Theconcentration of the drug in heart, spleen and lung of mice in SUD-HP-β-CDinclusion compound liposomes group was much higher than that in SUDgroup. The revalue was3.01,3.14and2.71, respectively, which indicated thatliposomes played a certain targeted purpose in heart, spleen and lung afterintravenous injection.The results of MTT assay suggested that SUD-HP-β-CD inclusioncompound liposomes significantly inhibited A549cell proliferation in aconcentration-and time-dependent manner. The results of flow cytometryassay suggested that liposome-encapsulated SUD-HP-β-CD inclusioncompound1,5and10μg·mL^-1increased percentage of cells in G0/G1phase(P<0.05or P<0.01), liposome-encapsulated SUD-HP-β-CD inclusioncompound5and10μg·mL^-1decreased the percentage of cells in S phase(P<0.01), and the percentage of cells in G₂/M phase was also decreased byliposome-encapsulated SUD-HP-β-CD inclusion compound10μg·mL^-1(P<0.01). Western blot analysis indicated that the expression of PCNA andCyclin D1was not effected by control liposomes. Whereas, in5μg·mL^-1SUDand1,5,10μg·mL^-1SUD-HP-β-CD inclusion compound liposomes groups,the level of PCNA and Cyclin D1was markedly decreased compared withcontrol group (P<0.05or P<0.01).Conclusion: HP-β-CD was used as the inclusion material, and theSUD-HP-β-CD inclusion compound was prepared by method of the saturatedaqueous solution; the SUD-HP-β-CD inclusion compound liposome, with the characteristics of simple preparation, controllable quality and goodreproducibility, was prepared by film dispersion method, and phospholipidsand cholesterol were used as the main membrane phospholipids. Theliposome-encapsulated SUD-HP-β-CD inclusion complex can improve drugtargeting and reduce toxicity, which would be expected to be developed into anew prospective dosage form. SUD-HP-β-CD inclusion compound liposomessignifacantly inhibited A549cell proliferation in vitro, and obviously arrestedthe cell-cycle progression in the G₀/G₁phase. Meanwhile, the proteinexpression of PCNA and Cyclin D1were markedly decreased bySUD-HP-β-CD inclusion compound liposomes, which would be expected toprovide experimental evidence in future clinical application of SUD as ananti-tumor drug.