Study On Combinative And Antitumor Effect Of Paclitaxel Inclusion Complex In Cyclodextrin

Nowadays, Cancer has become one of the main symptoms, which seriouslythreat human health. Because of the difficulties of early diagnosis, a significantnumber of cancer patients have to depend on drug therapy. Therefore, the researchand development of anti-cancer drugs is extremely imperative. Since Danjie used forthe treatment of malignant lymphoma in 1946, the drug therapy (chemical therapy)has been in great progress, and attracts much more attention gradually. Fromtreatment to cure, drug therapy is playing an important role in practice.Consequently, with the unique role in mechanisms, Paclitaxel is becoming therepresentative of the new generation of anti-cancer drugs. Since United States FDAapproved Paclitaxel in the treatment of late ovarian cancer tumor in December 1992,it has been developed extensively in the clinical application from second to first-linedrugs. So far, Paclitaxel is deemed as one of the best anti-cancer drugs. In thedevelopment, however, the medicine sources and poor dissolution become the twomain constraints on its wide use. The study uses supercritical CO₂ fluid extraction method to extract Paclitaxelfrom the leaves of northeast yew under the conditions: 27.6 Mpa, 36 degrees,methanol: water (80:20) as the agents,methanol as the absorption liquid, collectingper 20 minutes and total 4 hours. After rotary evaporation and enrichment of thecollected fluid, use LC/MS to analyze and test. Under the HPLC testing, the level ofPaclitaxel is 91%.When Paclitaxel/cyclodextrin inclusion complex are produced, firstly acomputer simulation method is applied to construct drug molecular and cyclodextrinmolecular structure documents. Optimize structure energy through molecularmechanics, molecular dynamics and quantum chemistry of the different procedures(such as MM3, PM3 and PM5 etc.). With the molecular docking operations(Docking) to construct inclusion complex structure, use the same procedures ascalculating isolated position to optimize energy structure. Through calculatingstructural parameters, deduct that inclusion complex may have multi-structure forms(combinative ratio of 1:3, 1:2 and 1:1), and calculate the lowest structure of energyas the form of 1:2 combinative ratio structure with a larger possibility. And thefluorescence spectra method certifies this result further. The stable constant is3030M-1 and inherent solubility is 4.70 × 10-7mol/L from solubility assay.Meanwhile, a variety of physical-chemical methods (NMR, DSC, XRD, IR, etc.)certify the formation of inclusion complex.We used self-made Paclitaxel/cyclodextrin inclusion complex (HP6-CD) toinvestigate the anti-tumor actively in vivo and the mechanism of anti-tumor activity.The study is looking forward to developing innovative anti-cancer drugs withpractical value.1. Anti-tumor Activity of Paclitaxel/Cyclodextrin Inclusion Complex in Vitro:The study used MTT method to detect the anti-tumor activity of Paclitaxel/cyclodextrin inclusion complex on five cancer cell lines: HT-29, Hela, K562, A1 andHO-8910 cell lines. The results showed that Paclitaxel/cyclodextrin inclusioncomplex inhibited the growth and proliferation of cancer cells with differentconcentration dependences in the concentration of 10-6, 10-5, 10-4, 10-3, 10-2, 0.1, 1,10, 100μg/ml (p<0.05). The highest rates of anti-tumor are 65.17%, 66.41%, 47.43%,23.09%, and 13.09%.2. Effect Cell Cycle and cell Apoptosis of Paclitaxel/Cyclodextrin InclusionComplex in Vitro: The study used One-Fluorescence Dying method on FlowCPPometer to detect the cell cycle and apoptosis of Paclitaxel/cyclodextrin InclusionComplex on Hela. The concentration of Paclitaxel/cyclodextrin inclusion complexstudy used was 0.1μg/ml. The results showed that experimental concentration ofPaclitaxel/cyclodextrin inclusion complex could significantly extend the G2/M cellcycle of Hela (p<0.05). At the same time, the G0 indicates thatPaclitaxel/cyclodextrin inclusion complex significantly change cell apoptosis ofHela (p<0.05).3. Anti-tumor Study of Paclitaxel/Cyclodextrin Inclusion Complex in Vivo: Thestudy set up KunMing bearing H22 and S180 mice model, which were 18-20g andmale mice. Then treated with Paclitaxel/cyclodextrin inclusion complex (2.5 mg/kg,5 mg/kg,10mg/kg) for 7 days. After that, the study measured mouse weight, tumorweight, thymus weight and spleen weight. The result of mouse weight showed thatthere was significantly difference between experimental groups with control groupincluding S180 and H22 (p<0.05). All the above indicates Paclitaxel/cyclodextrininclusion complex develop a trend on falling weights, which have a toxic side effecton the mice. The result of tumor weight showed that experimental groups and CPPgroup were obviously less than control group (p<0.05). In particular,Paclitaxel/cyclodextrin inclusion complex under concentration of 5mg/kg have ahigher rate of refraining tumors. Under this concentration, the anti-tumor ratio ofS180 and H22 tumor cells is 95.28% and 62.46% respectively (p<0.05). The results ofthymus weight and spleen weight showed that experimental groups of H22 wereobviously less than control group and CPP group (p<0.05).4. T LymphoCPPe Transformation Study of Paclitaxel/Cyclodextrin InclusionComplex in Vivo: The study used MTT method to detect the T lymphoCPPetransformation of Paclitaxel/cyclodextrin inclusion complex on KunMing bearingH22 and S180 mice. The results of T lymphoCPPe transformation showed that 2.5mg/kg and 5mg/kg groups were less than control group of H22.CPP group was lessthan control group. Experimental groups were more than control group of S180.5. SOD Vitality Study of Paclitaxel/Cyclodextrin Inclusion Complex in Vivo:The study used xanthenes oxidation enzyme method to detect SOD vitality ofPaclitaxel/cyclodextrin inclusion complex on mice bearing H22 and S180. The resultsof SOD vitality showed that the SOD of H22 less than control group but withoutstatistical significance. CPP group significantly less than control group (p<0.05).SOD of S180 is less than control group (p<0.05) of 5mg/kg Group. The remainingdoses group makes no statistical significance.6. GSH Study of Paclitaxel/Cyclodextrin Inclusion Complex in Vivo: The studyshowed that the GSH of 5mg/kg and 10mg/kg group were more than control groupof H22 (p<0.05). CPP group and 2.5mg/kg group make no statistical significance.Compared S180 and each dose group with the control group, there is no impacts andwithout statistical significance. It indicates that Paclitaxel/cyclodextrin inclusioncomplex have a certain immunity-enhanced effect on the anti-oxidation function ofH22.Conclusion: (1) The study demonstrated that CO2 supercritical fluid extractionmethod extracting Paclitaxel from the northeast yew leaves have a higher rate ofextraction and content. (2) This study showed that using different cyclodextrin tocombinative Paclitaxel and get the Paclitaxel/cyclodextrin inclusion complex with avariety of physical-chemical methods for certification. It improved the waterdissolving and stability. Furthermore, it confirmed a variety of important parameterssuch as combinative ratio and combinative constant of Paclitaxel/cyclodextrininclusion complex. (3) Using the Paclitaxel/cyclodextrin inclusion complex on thetumor cells of various mice and human, the research studyed its anti-tumor effects ofvivo and vitro. It certifyed that Paclitaxel/cyclodextrin inclusion complex caninhibited the growth and proliferation under experimental concentration onKunMing mice of HT-29, Hela, K562, A1 and HO-8910 tumor cells. Moreover, underexperimental concentration 48h-working on Hela, Paclitaxel/cyclodextrin inclusioncomplex can significantly extend the G2/M cycle of Hela cell. At the same time, theG0 showed that Paclitaxel/cyclodextrin inclusion complex significantly changes cellapoptosis of Hela.