| Objectives:Multidrug resistance,the principal mechanism by which many cancers develop resistance to chemotherapy drugs,is a major factor in the failure of many forms of chemotherapy.It affects patients with a variety of blood cancers and solid tumors,including breast,ovarian,lung,and lower gastrointestinal tract cancers.Tumors usually consist of mixed populations of malignant cells,some of which are drug-sensitive while others are drug-resistant.Chemotherapy kills drug-sensitive cells,but leaves behind a higher proportion of drug-resistant cells.As the tumor begins to grow again,chemotherapy may fail because the remaining tumor cells are now resistant.To address the problem of resistance,researchers have developed nanoparticles that simultaneously deliver chemotherapy drugs to tumors and inhibit the MDR proteins that pump the therapeutic drugs out of the cell.Taxanes are complexes of diterpenoid natural products and semisynthetic analogs.Presently,these drugs belong to prominent anticancer agents used for combined chemotherapy.PTX was found to be effective in treating a broad spectrum of advanced human cancer including breast and ovarian cancer as well as non-small cell lung carcinama,melanoma and head and neck cancer.However,the present-day chemotherapy employing Taxol is accompanied by serious problems.One of the major problems associated with this formulation is the fact,that the diluted Cremophor EL/ethanol vehicle is toxic.Liposomes represent versatile and advanced nano delivery systems for a wide range of biologically active compounds.These relatively non-toxic systems have a considerable potential to entrap both hydrophobic and hydrophilic drugs.The entrapment of the drug into the lipsomes is used to bypass the frequent generic toxicity associated with the drug.Thus,it represents a very effective route that enhances the drug therapeutic effect.It was demonstrated that liposomal carriers are superior to Taxol as regards the delivery of PTX.This work aimed to develop hyaluronic acid(HA)decorated pluronic 85(P85)coated solid lipid nanoparticles(SLN)loaded with paclitaxel(HA-PTX-P85-SLN)and to evaluate its potential to overcome drug resistance and to increase antitumor efficacy in mice bearing cervical and breast tumor.Methods:1.Synthesis and characterization of P85-DSPE The P85-DSPE conjugate was synthesized from P85 and DSPE by coupling in the presence of 1,10-carbonyldiimidazole(CDI)as a catalyst.P85-DSPE was purified and obtained by dialyzing and lyophilizing.2.Preparation of SLNThe SLN were prepared by the hot homogenization technique and electrostatic interaction.GMS,P85-DSPE(5:1,w/w)and PTX were melted and dissolved in warm ethanol(70?)to form the lipid phase.The aqueous phase was prepared by dissolving DOTAP(0.2%,w/v)and Poloxamer 188(0.1%,w/v)in Milli-Q water and heated to the same temperature as that of the lipid phase.The lipid phase was then added to the aqueous phase drop by drop.Thereafter,the mixture was continuously stirred for 10 min and then passed through a high pressure homogenizer for several cycles and kept on a stirring until solvent completely evaporated.For preparation of HA-PTX-P85-SLN,1 mL of PTX-P85-SLN dispersion was added into HA solution(0.03%,w/v)dropwise under gentle stirring.After 30 min,electrostatic interactions between HA and cationic SLN led to the formation HA-PTX-P85-SLN.3.The identification of physicochemistry of the SLNPTX-loaded SLN was characterized for mean diameter,zeta potential,morphology,entrapment efficiency(EE),drug loading capacity(LC)and in vitro drug release.The particle size and zeta potential of SLN were determined by dynamic light scattering using Zetasizer Nano ZS90.The shape and morphology of SLN were studied by transmission electron microscopy.The drug entrapment efficiency(EE)and drug loading capacity(LC)of SLN was determined by HPLC.Release of PTX from SLN was evaluated using the dialysis method.4.The effect of H A-PTX-P85-SLN on breast cancer cells and cervical cancer cellsThe cellular uptake efficiency of HA-PTX-P85-SLN was determined by flow cytometer.MTT assay was performed to evaluate the cytotoxicity of different PTX loaded SLN and free PTX against HeLa cells,HeLa/PTX cells,MCF-7 cells and MCF/PTX cells respectively.Female BALB/c mice bearing HeLa/PTX tumor(90?100 mm3)were randomly divided into five groups(n = 6),including control(0.9%saline),free PTX.PTX-SLN,PTX-P85-SLN and HA-PTX-P85-SLN at a dose of 10 mg/kg.Mice of each group were given the above formulations by tail vein injection on every other day.15 days later,all the mice were sacrificed and the tumor tissue samples were taken out.In vivo animal evaluation containing antitumor effect,pharmacokinetics and biodistribution were conducted in mice bearing cervical and breast tumor.Results:The HA-PTX-P85-SLN showed a mean diameter of 160.3nm,negative zeta potential(-31.6mv),EE of 88.2%,and LC of 4.9%.PTX from HA-PTX-P85-SLN exhibited greater sustained drug release profiles compared free PTX.As showed in the results,cellular uptake efficiency of HA-PTX-P85-SLN was significantly higher than other SLNs(P<0.05).And HA-PTX-P85-SLN showed the significantly higher cytotoxicity toward cervical and breast cancer cells than other drug formulations respectively.The group treated with HA-PTX-P85-SLN maintained stable tumor growth control up to 14 days,and had the greatest inhibition of tumor growth when compared to the other treatment groups.Also it showed no weight loss during the treatment period,while those treated with free PTX or saline showed weight loss.The pharmacokinetic test showed that the drug was rapidly cleared from the plasma within 3 hours for free PTX,whereas PTX-loaded SLN showed the sustained release in vivo(over 24 h).The terminal half life(t1/2?),mean residence time(MRT),body clearance(CL)and the area under the plasma concentration-time curve(AUCO??)were 2.23±0.09 h,1.26±0.52 h,0.71 ±0.06 L/h/kg,12.89±2.37?g*h/mL and 13.12±0.82 h,8.17±0.85 h,0.17±0.01 L/h/kg,59.33±6.39?g*h/mL for free PTX and HA-PTX-P85-SLN respectively.Biodistriution results revealed that HA-PTX-P85-SLN exhibited higher tumor drug concentration compared with free PTX.HA-PTX-P85-SLN exhibited maximum drug concentration in the tumor tissue.HA-PTX-P85-SLN and PTX-P85-SLN exhibited 5.5-fold increase and 4.1-fold increase in comparison to free PTX in tumor drug concentration.The concentration of PTX for PTX-loaded SLN was higher in the liver and lung compared with free PTX,whereas lower in the heart and kidney(p<0.05).Conclusions:In the present study,a novel HA-P85-SLN delivery system was designed and compared with free PTX.In vitro cell viability studies using cervical and breast cancer cells revealed that HA-PTX-P85-SLN had highest cytotoxicity than other formulations.In vivo animal evaluation(pharmacokinetics and tissue distribution,and anti-tumor study)demonstrated that HA-P85-SLN prolonged MRT in circulation,and increased PTX accumulation in HeLa/PTX tumor tissue.Therefore,HA-P85-SLN could be a potential drug delivery vehicle of PTX for the effective treatment of the cervical and breast tumor that overcomes MDR.Objectives:With the increasing incidence of malignant tumor,the incidence of gynecological cancers in premenopausal or perimenopausal stage becomes more and more common.At the same time tumor onset age tends to be younger.It seriously affects the physical and mental health so that the patients may lose the confidence of the treatment.These patients underwent resection of ovary or chemoradiation,leading to a sharp drop in serum estrogen levels.Then the menopause symptoms present ahead of time.It has been confirmed that postmenopausal hormone therapy can effectively alleviate symptoms of menopause.Other medicine cannot replace HT.However,whether the menopausal hormone treatment(MHT)can be safely used in patients with malignant tumor is still controversial.Kuntai capsule,a traditional Chinese medicine,is widely used in the treatment of menopausal syndrome in our country.It has been confirmed that Kuntai capsule can effectively alleviate symptoms of menopause.The current study discussed the efficacy and safety of Kuntai capsule in the treatment of patients who have accepted bilateral ovaries removed and/or radiation and chemotherapy for malignant tumor,This work aimed to explore the effect of Kuntai capsule on the menopause syndrome in patients with gynecological malignant tumor after treatment FSH and E2 in patients treated with Kuntai capsule orally.Methods:1.General informations120 patients with gynecology malignant tumor(mean age:38)were recruited in the study in the Department of Gynecology,Shandong provincial hospital affiliated to Shandong University from July 2014 to January 2016.Informed consent was obtained from all participants and the study was approved by the institutional review board of Shandong Provincial Hospital affiliated to Shandong University.Jinan,China.The patients were randomly assigned to the experimental group(n=60),and control group(n=60).Diagnosis was confirmed or excluded by histological examination..All patients underwent radioactive therapy,chemotherapy,or surgery.They all suffered from menopausal syndrome after treatment.2.Main CriteriaThe main criteria were as follows:(a)Vasomotor symptoms,including hot flashes and night sweats;(b)Autonomic nerve disorder,such as palpitations,dizziness,headache,insomnia,tinnitus,etc.3.Exclusion CriteriaThe main exclusion criteria were as follows:(a)current contraindication to Kuntai;(b)severe or current disease which could interfere with the climacteric complaints,or the actual or expected treatment which could interfere with the study objectives;(c)liver damage,severe diabetes,hypertension,cardiovascular or cerebrovascular disease;(d)drug abuse or alcohol addiction;(e)participation in another clinical trial.All patients were followed-up for 12 weeks;and the patients who had treatment interruption less than 12 weeks would be ruled out from the study.No significant difference was noted in the age,body mass index(BMI)serum E2 and FSH,KMI between the two groups before Kuntai capsules treatment(P>0.05,table 1).4.TreatmentsThe patients in experimental group(n=60)were treated with Kuntai capsules orally(Guiyang Xintian Pharmaceutical Co.Ltd.,State Medical Approval number:Z20000083;4 capsules,3 times/day.po,half an hour after meal for 12 weeks).The patients in control group were treated without any drug during 12 week.We took the date of 1 week after the first radioactive therapy or Chemotherapy or surgery as 0 week.The follow-up of the patients was carried out every 4 weeks for 12 weeks.The first follow-up began before therapy.The next two follow-up began at 4 weeks and 8weeks after the first radioactive therapy,Chemotherapy,or surgery.The patients who discontinued therapy within 12 weeks,used other hormonal drugs,or took non-hormonal drugs(including nutrition),were removed from the statistical analysis.5.Evaluation and observation5.1 Safety assessmentLiver and renal function were examined initially in the 4th and 12th week after taking Kuntai capsule.Routine blood and urieme tests and electrocardiographic examination(ECG)of each patient were also performed.All data collected were analyzed statistically.Personnel records adverse reactions in every follow-up.5.2 Curative effect evaluation5.2.1 Modified Kupperman Menopause Index(KMI)score was used to evaluate the symptoms.The modified KMI is an internationally recognized and validated scale for the quantitative determination of menopausal symptoms.The modified KMI consists of 11 items,including hot flash/sweating,paresthesia,insomnia,nervousness,melancholia,vertigo,fatigue,arthralgia,headache,palpitation,and formication.15-20,21-35,and>35 were used to rate the degree of severity as mild,moderate,and severe,respectively.Single points = weighted score × degree score.The total score is the sum of each single score.5.2.2 Serum estradiol and follicle stimulating hormone level2 ml venous blood of each patient was collected before and after treatment.Then the serum was isolated in 2h from venous blood and saved at-70?.The serum FSH level of all the patients enrolled in this study was determined by radioimmunoassay(Diagnostic ProductsCorporation,Co.,Tianjin,China):and serum estradiol level was determined by chemiluminescence immunoassay(Diagnostic ProductsCorporation,Co.,Tianjin,China).6.Statistical analysisStatistical analysis was performed using SPSS 13.0 software(SPSS Inc.,Chicago,IL,USA).The data were presented as mean ± standard deviation(SD),using analysis of variance for different groups.Count data were analyzed by?2-test..A two-sided value of P<0.05 was considered to be statistically significant.Conclusions:1.When Kuntai capsule treating the patients with gynecological malignant tumor accompanied with menopausal symptoms,the duration of drug use and the clinical efficacy of the capsule were correlated.The serum sex hormone FSH decreased significantly after 20 weeks,while the E2 increased significantly.2.ymptoms of abnormal feeling,insomnia,palpitation,and dizziness improved significantly after 4-week treatment of Kun Tai capsule,while Symptoms of the hot flashes and sweating improved significantly after 12 weeks.3.Kun Thai capsule can be an alternative hormone therapy for malignant tumors,which can improve symptoms and enhance the quality of life.Discussion:After removal of bilateral ovaries,radiation or chemical therapy,the menopausal symptoms appear earlier and heavier in menopausal or premenopausal patients with malignant gynecologic tumors.Hormone replacement therapy(HRT)can effectively relieve symptoms of menopause.However,for patients with malignant tumors,the safety of HRT is controversial.More and more patients are inclined to Chinese medicine 5-8.From the Chinese point of view,Kuntai capsule,consisting of Rehmannia glutinosa,Rhizoma Coptidis,Radix Paeoniae Rubra,Radix Scutellariae,Ejiao,Fuling,Ziyin Reduce Pathogenic Fire,Qingxin Chufan,Yijing Tiansui,can soothe the nerves and regulate the balance of yin and yang.In the current study,we found that Kuntai capsules can improve serum E2 levels in menopausal/perimenopausal patients and may shift the vaginal cell maturation index.Studies by Zhang Shaofen showed that Kuntai capsule can increase ovarian volume and the number of corpus luteum in menopausal rats,suggesting that Kuntai capsule plays an important role in improving ovarian function.In the current study,all patients suffered from removal of bilateral ovaries due to gynecological malignancies.We found that kuntai capsules treatment significantly increased serum E2 level and decreased serum FSH level.The KMI score was also obviously improved after 12 weeks treatment.However,the study by Fu changling found that no difference was noted between treatment for 4 weeks and 8 weeks.During Kuntai capsules treatment.no toxic side effect were observed in the 4 weeks and 12 weeks of treatment,which indicated that Kuntai capsule was not only convenient to take,but also had fewer adverse reactions.In summary,for patients undergo removal of bilateral ovarian for gynecological malignancies,Kuntai capsule can be used as an alternative treatment of HRT in order to improve postoperative menopausal symptoms. |
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