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ObjectiveTo investigate the effects of hydroxycamptothecin liposomes （LHCPT） on myocardial fibrosis in rats with heart failure by regulating the sphingosine kinase 1 （SphK1）/sphingosine-1-phosphate （S1P） signaling pathway. MethodsSD rats were divided into control group， model group， hydroxycamptothecin （HCPT） group， LHCPT group， captopril group， and LHCPT+K6PC-5 （SphK1 activator） group， with 12 rats in each group. The heart failure rat models in all groups except the control group were established by intraperitoneal injection of doxorubicin and then the corresponding drugs were given once a day. After four weeks， we applied color Doppler ultrasound to detect left ventricular end systolic diameter （LVESD）， left ventricular end diastolic diameter （LVEDD）， and left ventricular ejection fraction （LVEF） in rats； HE and Masson staining for myocardial pathological damage and myocardial fibrosis in rats， respectively； ELISA method for the levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） in rat myocardial tissues； qRT-PCR for the expression of transforming growth factor-β1 （TGF-β1）， type I collagen （Collagen I）， and type Ⅲ collagen （Collagen Ⅲ） in rat myocardial tissues； Western blot for the expression of SphK1 and S1P proteins in rat myocardial tissues. ResultsCompared with the control group， the model group showed severe myocardial pathological damage and myocardial fibrosis， increased LVESD， LVEDD， levels of TNF-α and IL-6， expression of TGF-β1， Collagen I， Collagen Ⅲ， SphK1， S1P and decreased LVEF （P<0.05）. Compared with the model group， the HCPT group， LHCPT group and captopril group showed alleviated myocardial pathological damage and myocardial fibrosis， decreased LVESD， LVEDD， levels of TNF-α and IL-6， expression of TGF-β1， Collagen I， Collagen Ⅲ， SphK1， S1P and increased LVEF （P<0.05）. Compared with the LHCPT group， the LHCPT+K6PC-5 group showed aggravated myocardial pathological damage and myocardial fibrosis， increased LVESD， LVEDD， levels of TNF-α and IL-6， expression of TGF-β1， Collagen I， Collagen Ⅲ， SphK1， S1P and decreased LVEF （P<0.05）. ConclusionLHCPT may inhibit myocardial fibrosis in heart failure rats by inhibiting the SphK1/S1P signaling pathway.
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OBJECTIVE：To study the pharmacokinetics and tissue distribution characteristics of Hydroxycamptothecin （HCPT） nanoparticles in rats ，and to investigate their targeting. METHODS ：Male SD rats were randomly divided into 2 groups，with 6 rats in each group. They were given HCPT nanoparticles and HCPT injection （4 mg/kg based on HCPT ）via tail vein respectively. 500 μL fundus venous plexus blood were sampled at 5，30，60，120，240，360，480，600 and 720 min after administration. The plasma concentration of HCPT in rats were measured by HPLC at different time points. The pharmacokinetic parameters were calculated by DAS 3.0 software. Male SD rats were randomly divided into two groups ，with 24 rats in each group. They were given HCPT nanoparticles and HCPT injection （0.6 mg/kg based on HCPT ）via tail vein ，respectively. Blood was immediately taken from the abdominal aorta ，and heart ，liver，spleen，lung，kidney and brain were removed at 30，60，120，240 min after administration. The plasma and tissue concentration of HCPT in rats were measured by HPLC. The distribution of HCPT ineach tissue and targeting were investigated. RESULTS ：HCPT nanoparticles and its injection conformed to a two-compartment model in rats. Compared with HCPT injection ，AUC0-720 min andAUC0- ∞ increased by 1.89 and 1.87 times respectively ， MRT0-720 min and MRT 0- ∞ increased by 2.74 and 3.00 times respectively， t1/2 β increased by 2.75 times，with statistical significance（P＜0.05）. At 30 min after administration ，HCPT nanoparticles and HCPT injection had the highest concentration in lung；with the passage of time ，the drug gradually accumulated in the liver and reached the highest concentration at 60 min. The relative liver uptake rate of HCPT nanoparticles was the highest （6.28）. Taking liver ad target organ ，and the targeting efficiencies of it in heart ，spleen，lung，kindey，brain and plasma were higher than those of HCPT injection. The selectivity index of HCPT nanoparticles in heart ，lung（except for 30 min after administration ），kidney，brain and plasma were significantly higher than those of HCPT injection at 30-120 min after administration. CONCLUSIONS ：HCPT nanoparticles extend the half-life of the drug ， increase its plasma concentration ，and prolong its action time in vivo ，with significant liver targeting.
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Objective: To prepare a anti-drug-resistant hybrid nanosuspension of quercetin and hydroxycamptothecin by employing the anti-drug resistance property of quercetin. Methods: The ratio of quercetin to hydroxycamptothecin was optimized by in vitro cytotoxicity test. The hybrid nanosuspension of quercetin and hydroxycamptothecin was prepared by the microprecipitation-high pressure homogenization method, with polyethylene glycol-poly(benzyl aspartateic acid)as stabilizers. The particle size, potential, morphology, crystal form and stability of nanosuspensions were studied by the dynamic light scattering, transmission electron micros-copy, differential scanning calorimetry and X-ray powder diffraction. The hemolytic effect of nanosuspension was analyzed by the UV spectrophotometry. The in vitro dissolution of nanosuspensions was investigated by the paddle method. Results: The particle size and Zeta potential of nanosuspensions were(216.3±5.93)nm and(0.197±0.035)mV, respectively. The nanosuspension showed an irregular shape in the transmission electron microscope image. Compared with the two raw drugs, the crystal forms of quercetin and hydroxycamptothecin in nanosuspension was likely transformed in the state of nanosuspension. The nanosuspension showed a good storage and dilution stability, and had good blood compatibility. The dissolution test showed that nanosuspensions could significantly increase the dissolution rate of the two drugs(both P<0.05). Conclusion: The combination of quercetin and hydroxycamptothecin in a favored ratio selected by the present study could significantly improve the antitumor effect of hydroxycamptothecin. The prepared quercetin and hydroxycamptothecin hybrid nanosuspensions showed good pharmaceutical properties, which might be used for further studies on the synergistic and detoxifying effect of the hybrid nanosuspensions.
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Chemotherapy outcomes for the treatment of glioma remains unsatisfactory due to the inefficient drug transport across the blood-brain barrier (BBB) and insufficient drug accumulation in the tumor region. Although many approaches, including various nanosystems, have been developed to promote the distribution of chemotherapeutics in the brain tumor, the delivery efficiency and the possible damage to the normal brain function still greatly restrict the clinical application of the nanocarriers. Therefore, it is urgent and necessary to discover more safe and effective BBB penetration and glioma-targeting strategies. In the present study, menthol, one of the strongest BBB penetration enhancers screened from traditional Chinese medicine, was conjugated to casein, a natural food protein with brain targeting capability. Then the conjugate self-assembled into the nanoparticles to load anti-cancer drugs. The nanoparticles were characterized to have appropriate size, spheroid shape and high loading drug capacity. Tumor spheroid penetration experiments demonstrated that penetration ability of menthol-modified casein nanoparticles (M-CA-NP) into the tumor were much deeper than that of unmodified nanoparticles. imaging further verified that M-CA-NPs exhibited higher brain tumor distribution than unmodified nanoparticles. The median survival time of glioma-bearing mice treated with HCPT-M-CA-NPs was significantly prolonged than those treated with free HCPT or HCPT-CA-NPs. HE staining of the organs indicated the safety of the nanoparticles. Therefore, the study combined the advantages of traditional Chinese medicine strategy with modern delivery technology for brain targeting, and provide a safe and effective approach for glioma therapy.
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Objective To prepare hydroxycamptothecin-phospholipid complex(HCPT-PC),characterize its physicochemi-cal properties,and evaluate the cytotoxicity. Methods The particle size and morphology of HCPT-PC were characterized by malvern particle size potentiometer,scanning electron microscopy(TEM)and transmission electron microscopy(TEM). Its composite mecha-nism was investigated by X-ray powder diffraction and infrared spectroscopy. The solubility and antitumor activity were also investigat-ed. Results The particle size of HCPT-PC was(145.08±18.37)nm. Scanning electron microscopy and transmission electron micros-copy revealed that HCPT-PC was uniformly distributed with a spherical shape. X-ray powder diffraction indicated that HCPT changed from crystalline to amorphous state in HCPT-PC. Fourier transform infrared spectroscopy showed that there was a weak interaction be-tween HCPT and PC. The solubility of HCPT-PC in water,PBS,ethanol and n-octanol was about 21.91,20.36,1.42 and 6.32 times than that of HCPT,respectively. After treated with HepG2,SMMC-7721 and H22 cells for 48 and 72 hours,IC50 of HCPT-PC was higher than that of HCPT by 3.57,11.14,2.79,37.26,21.23 and 24.49 times,respectively. Conclusion HCPT is compounded into an amorphous-state HCPT-PC by a weak interaction with the polar end of PC. Its solubility and anti-hepatocarcinoma activity are signif-icantly higher than HCPT.
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Objective To compare and analyze the Brucea javanica oil emulsion,hydroxy camptothecin bladder perfusion of recurrence after operation of superficial bladder cancer.Methods 80 cases with superficial blad-der cancer were randomly divided into group A and group B (n=40 ).Group A received intravesical instillation of Brucea javanica oil emulsion,group B received intravesical instillation of hydroxycamptothecin.The serum tumor marker levels were compared between two groups,and the recurrence rate and adverse reactions in the two groups were observed.Results At 1 day before operation,postoperative 7d,the serum Dickkopf-1 (DKK-1 ),Dickkopf-2 (DKK-2),Dickkopf-3(DKK-3),Dickkopf-4(DKK-4),vascular endothelial growth factor A(VEGFA), vascular endothelial growth factor B(VEGFB),vascular endothelial growth factor C(VEGFC)of the two groups had no statistically significant differences (P>0.05).Postoperative 6 months,the serum levels of DKK-1,DKK-2, DKK-3,DKK-4,VEGFA,VEGFB,VEGFC in group A were (20.62 ±2.84)g/L,(29.15 ±6.32)g/L,(18.66 ± 4.53)g/L,(32.34 ±6.42)g/L,(80.72 ±9.16)g/L,(60.43 ±16.73)g/L,(70.31 ±8.41)g/L,respectively, which were lower than those of group B [(27.42 ±3.76)g/L,(38.16 ±4.34)g/L,(30.37 ±5.35)g/L,(47.26 ± 7.72)g/L,(146.31 ±12.44)g/L,(83.14.14.07)g/L,(91.58 ±7.82)g/L,respectively,t=-2.92,-2.58,-2.82,-2.41,-2.80,-2.75,-2.70,all P<0.01].The recurrence rate of group A was 7.5%(3/40),which was significantly lower than 15.0%(6/40)of group B(6/40)(χ2 =6.11,P <0.05).The incidence rate of adverse reac-tions of group A was 12.5%,which was significantly lower than 27.5% of the control group(χ2 =5.46,P<0.05). Conclusion Compared with hydroxyl camptothecine vesical perfusion,the effect of intravesical instillation of Brucea javanica oil emulsion in preventing the postoperative recurrence of superficial bladder cancer is more significant,can effectively reduce the levels of serum tumor marker,with less adverse reactions,it is worth application and promotion.
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Objective:To establish an LC–MS/MS method for the determination of the active metabolite(SN-38) and secondary metabolite(SN-38G) of irinotecan in rat liver microsomes incubation system, and optimize the incubation conditions. Methods:Meth-anol was selected to precipitate protein in the samples, and then the concentrations were analyzed by LC–MS/MS. All the separation was carried out on a ZORBAX Eclipse XDB-C18 column(2. 1 mm × 50 mm, 3. 5 μm) with the mobile phase of acetonitrile – water (containing 0. 1% formic acid) (23 :77) at a flow rate of 0. 3 ml·min-1. The mass spectrometer was operated with multiple reac-tions monitoring ( MRM) using electrospray ionization ( ESI) . The incubation conditions were optimized by single factor design. Re-sults:SN-38 and SN-38G showed a good linearity ( r≥0. 9972) respectively within the range of 2. 3-920 ng·ml-1 and 2. 5-1000 ng ·ml-1. The intra-and inter-day RSD was below 14. 6%(n=6). The average recovery was within the range of 74. 1%-123. 4% with RSD below 13. 5% (n=6). The optimal incubation conditions were as follows:the concentration of liver microsomal protein was 0. 3 mg·ml-1 and the incubation time was 30 min. Conclusion:The method is rapid, sensitive and accurate in the quantification of SN-38 and SN-38G in the incubation system,which provides methodological basis for the activity determination of UGT1A1 enzyme in vitro.
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OBJECTIVE:To develop a method for simultaneous determination of irinotecan(CPT-11)and its active metabo-lite 7-ethyl-10-hydroxycamptothecin(SN-38)in human plasma.METHODS:After precipitated by acetonitrile and acidified with hy-drochloric acid,using camptothecin as internal standard,the plasma sample was determined by HPLC-FLD. The determination was performed on Waters Luna C18column with mobile phase consisted of 0.05 mol/L sodium dihydrogen phosphate-acetonitrile(70:30, V/V,adjusted pH to 4.0 by phosphoric acid)at flow rate of 1 mL/min. The excitation wavelength was set at 380 nm;the emis-sion wavelengths of CPT-11 and SN-38 were set at 480 nm and 535 nm,respectively. The column temperature was 25 ℃ and the sample size was 20 μL. RESULTS:The linear ranges were 200-1 000 ng/mL for CPT-11(r=0.999 4,n=5)and 5-45 ng/mL for SN-38(r=0.999 2,n=5). RSDs of inter-day and intra-day were 1.68%-5.57%. The relative recoveries of CPT-11 and SN-38 were 90.12%-106.93%(RSD<8%,n=5)and 92.07%-102.56%(RSD<6%,n=5);the extraction recoveries of CPT-11 and SN-38 were 72.23%-86.56%(RSD<6%,n=5)and 71.98%-83.44%(RSD<7%,n=5),respectively. The plasma concentra-tions of CPT-11 and SN-38 in 5 patients with colon cancer were 431.13-617.19,13.97-31.89 ng/mL(1 h after intravenous drip-ping)and 398.14-584.43,11.61-29.94 ng/mL(2 h after intravenous dripping). CONCLUSIONS:The method is simple,rapid, sensitive,reproducible and suitable for the determination of plasma concentration and pharmacokinetic study of CPT-11 and its metabolite SN-38.
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OBJECTIVE:To develop a method for simultaneous determination of irinotecan(CPT-11)and its active metabo-lite 7-ethyl-10-hydroxycamptothecin(SN-38)in human plasma.METHODS:After precipitated by acetonitrile and acidified with hy-drochloric acid,using camptothecin as internal standard,the plasma sample was determined by HPLC-FLD. The determination was performed on Waters Luna C18column with mobile phase consisted of 0.05 mol/L sodium dihydrogen phosphate-acetonitrile(70:30, V/V,adjusted pH to 4.0 by phosphoric acid)at flow rate of 1 mL/min. The excitation wavelength was set at 380 nm;the emis-sion wavelengths of CPT-11 and SN-38 were set at 480 nm and 535 nm,respectively. The column temperature was 25 ℃ and the sample size was 20 μL. RESULTS:The linear ranges were 200-1 000 ng/mL for CPT-11(r=0.999 4,n=5)and 5-45 ng/mL for SN-38(r=0.999 2,n=5). RSDs of inter-day and intra-day were 1.68%-5.57%. The relative recoveries of CPT-11 and SN-38 were 90.12%-106.93%(RSD<8%,n=5)and 92.07%-102.56%(RSD<6%,n=5);the extraction recoveries of CPT-11 and SN-38 were 72.23%-86.56%(RSD<6%,n=5)and 71.98%-83.44%(RSD<7%,n=5),respectively. The plasma concentra-tions of CPT-11 and SN-38 in 5 patients with colon cancer were 431.13-617.19,13.97-31.89 ng/mL(1 h after intravenous drip-ping)and 398.14-584.43,11.61-29.94 ng/mL(2 h after intravenous dripping). CONCLUSIONS:The method is simple,rapid, sensitive,reproducible and suitable for the determination of plasma concentration and pharmacokinetic study of CPT-11 and its metabolite SN-38.
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Objective To prepare a 10-hydroxycamptothecin (10-HCPT) loaded folate-receptor targeted phase-change contrast agent (FR-HCPT-PNPCA),and to study the general characteristics including drug loading,phase changing and targeting capability in vitro.Methods Using a method of two-step emulsification,the phase-change nanoparticles loading anticancer drug (10-HCPT) with lipids shell and liquid pefluorocarbon core were prepared.The entrapment efficiency and the drug-loading amounts were studied by high performance liquid chromatography,and the phase transition of the nanoparticles after heating was observed.The targeting ability was evaluated on liver cancer cell line 7721 in vitro.Results The FR-HCPT-PNPCA,with a drug encapsulation rate of about 70.42 ％ and drug loading amounts of about 20.05 ％,was prepared successfully.When being heated to 70℃,obvious phase changing and microbubbles generating could be observed under microscope.In addition,a large amount of FR-HCPT-PNPCA particles could adhere specifically around the 7721 cells.Conclusion The prepared FR-HCPT-PNPCA,which has a stable characteristic and high performance of drug loading and tumor targeting,is expected to become a promising multifunctional molecular ultrasound probe for diagnosis and treatment of tumor.
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In this study, we used Shirasu porous glass membrane (SPG) as a template and hydroxy camptothecin (HCPT) as a model drug to prepare the comet-shaped MePEG[methoxyl poly(ethylene glycol)]-PLGA[poly(lactic-co-glycolic acid)-HCPT amphiphilic block copolymer. Our method was optimized by the orthogonal design method. The partical size, zeta potential, drug-loaded content, yield, shape and status of the obtained comet-shaped MePEG-PLGA-HCPT particles were further characterized by dynamic light scattering (DLS), scanning electron microscopy (SEM)/transmission electron microscopy (TEM), X-ray diffraction (XRD) and differential scanning calorimetry (DSC) et al, respectively. In vitro release was preliminary evaluated. MTT assay to preliminary evaluate the cytotoxicity of particles against human liver BEL-7402 cells. Based on these experimental results, the optimal preparation conditions contain:weight ratio of HCPT to MePEG-PLGA was 1:1, nitrogen pressure was 100 kPa and SPG membrane pore size was 1.1 μm. The particles exhibited a comet-shaped shape, fairly uniform size and were well dispersed. The drug-loading content was 46.2%, with yield of 96.4%, and zeta -31.4 mV. The distribution of HCPT in particles was very uniform, and HCPT showed a amorphous state existed in particles. The release behavior in vitro showed sustained releasing,and with the drug loading content in proportion to the release of the drug. MTT test indicated that the HCPT-loaded comet-shaped particles had enhanced the cytotoxicity against human liver BEL-7402 cells relatively to the HCPT-loaded spherical particles in vitro. The results showed a promising potential application of the preparation in clinical treatment of tumor.
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To improve the bioavailability of 10-hydroxycamptothecin, 10-hydroxycamptothecin solid dispersion(HCPT-SD) and 10-hydroxycamptothecin-phospholipid complex-solid dispersion(HCPT-PC-SD) were prepared, and their solubility and dissolution rate were evaluated in this study. SD rates were administered intragastrically with HCPT-SD or HCPT-PC-SD respectively, then their blood samples were collected at different time intervals. The concentration of HCPT in blood was detected by HPLC method with camptothecin as internal standard, and then its pharmacokinetic parameters were calculated and obtained. The results showed that the Cmax, AUC0-t and AUC0-∞ of both kinds of solid dispersion of HCPT were significantly increased than those of crude drug. The AUC0-t of HCPT-SD was increased by 176.87%, and AUC0-t of HCPT-PC-SD was increased by 254.31% as compared with crude drug. Therefore, the two kinds of solid dispersion of HCPT could significantly enhance the bioavailability of HCPT in SD rates, and the effect of HCPT-PC-SD was more obvious.
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Objective To ch aracterize and evaluate in vitro and in vivo of the 10-hydroxycamptothecin (HCPT) loaded human serum albumin nanoparticle (HSA-NP) prepared by drug-liquid compound method. Methods The HCPT-HSA-NP was prepared with low weight polyethylene glycol drug-liquid compound and blank albumin nanoparticle. Then the in vitro evaluations were conducted with tests of entrapment efficiency, solution stability, accumulative release, morphous investigation and X-ray powder diffraction. At the same time, the primary pharmacodynamics comparison between HCPT injection and the nano preparation (8 mg/kg) was carried out on animal tumor model. Results The obtained HCPT-HSA-NP fitted to the basal features of nano preparation. The entrapment efficiency was averagely higher than 99% for each sample and the solution was stable. In vitro accumulative release study showed that the preparation had long-term release pattern over 100 hours. In vivo pharmacodynamics study showed that the HCPT-HSA-NP was significantly more effective than HCPT injection (P<0.01). Conclusion The drug-liquid compound method can be used to prepare HCPT-HSA-NP.
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Objective To characterize and evaluate in vitro and in vivo of the 10-hydroxycamptothecin (HCPT) loaded human serum albumin nanoparticle (HSA-NP) prepared by drug-liquid compound method. Methods The HCPT-HSA-NP was prepared with low weight polyethylene glycol drug-liquid compound and blank albumin nanoparticle. Then the in vitro evaluations were conducted with tests of entrapment efficiency, solution stability, accumulative release, morphous investigation and X-ray powder diffraction. At the same time, the primary pharmacodynamics comparison between HCPT injection and the nano preparation-8 mg/kg) was carried out on animal tumor model. Results The obtained HCPT-HSA-NP fitted to the basal features of nano preparation. The entrapment efficiency was averagely higher than 99% for each sample and the solution was stable. In vitro accumulative release study showed that the preparation had long-term release pattern over 100 hours. In vivo pharmacodynamics study showed that the HCPT-HSA-NP was significantly more effective than HCPT injection (P<0.01). Conclusion The drug-liquid compound method can be used to prepare HCPT-HSA-NP.
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7-Ethyl-10-hydroxycamptothecin (SN-38) is a prominent anticancer agent, but it is insoluble in water and the most pharmaceutically acceptable solvents, the lactone ring of SN-38 shows reversible pH-dependent hydrolysis and forms to the open lactone in alkaline condition. All of these factors limit its application in clinic, and a variety of chemical modification studies have been reported to improve the efficient use of SN-38 by improving its solubility in water and pharmaceutical solvents, stabilizing the lactone form, and altering the pharmacological properties. This paper reviews the pharmacological properties of commercial SN-38 derivatives in clinical trials or preclinical studies and their research progress.
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Objective To investigate the anti-tumor effects of hydroxycamptothecin ( HCPT ) with different lactone ratios on the mice models of H22 hepatoma. Methods Mice models of H22 hepatoma were established. Tumor inhibiting rates of HCPT with different lactone ratios ( 0%, 25%, 50%, 75%, 100%) and the growth status of model mice before and after chemotherapy were observed. Serum biochemical indices were determined to investigate the effects of HCPT with different lactone ratios on hepatic and renal function of the mice. Results Positive control drug and HCPT with different lactone ratios all inhibited the tumor in mice with H22 hepatoma, the inhibition rate was 65. 30%, 12. 57%, 49. 23%, 75. 47%, 90. 06% and 93. 22%, respectively. Compared with the model control group, the living conditions of the mice in HCPT groups were improved. With increasing of lactone ratios, the hepatic injury was alleviated markedly, but the renal injury was aggravated. Conclusion There is a positive correlation between lactone ratios and its anti-tumor effect, and HCPT with 75% lactone can achieve preferable anti-tumor effect with less toxicity as compared with that with 100% lactone ratio.
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Background Studies confirmed that hydroxycamptothecin cause the apoptosis of human Tenon capsule fibroblasts (HTFs) by protein kinase R-like endoplasmic reticulum stress kinase (PERK) single pathway.Autophagy and apoptosis are programmed cell death following stress reaction,so they remain a close association.However,the effect of hydroxycamptothecin on the autophagy of HTFs and its mechanism are still unclear.Objective This study was to explore the promoting effect of PERK signal pathway on hydroxycamptothecin inducing the autophagy of HTFs.Methods This study procedure was approval by Ethic Committee of Nanjing Medical University.Human Tenon capsule tissue was obtained from fresh adult donors.HTFs were cultured and passaged by explant-culture method and identified by immunofluorescence for vimentin and keratin.pLVX-PERK lentiviral packed by 293T cells was transfected into HTFs to obtain stable PERK-knockout cell line by puromycin selection.Then the HTFs were treated with 0.10 g/L of hydroxycamptothecin for 5 minutes and consecutively cultivated for 24 hours,and the untreated cells were used as the control group.Western blot assay was used to detect the expressions of autophagy specific proteins in the cells,including autophagy related gene 5 (ATG-5),Beclin-1,light chain 3 (LC-3).Cyto-ID staining was used to identify the autophagosome in the cells.The experimental results were analyzed and compared between different treating groups.Results The gray scales for the expressions of Beclin 1,ATG-5,LC-3-Ⅰ and LC-3-Ⅱ proteins in HTFs were 0.365:±0.045,0.765 ±0.055,0.120±0.030 and 0.215 ±0.035 in the control group,and those in the hydroxycamptothecin treated group were 0.980±0.070,1.495±0.095,0.585±0.025 and 0.785±0.055,showing a significant decline in the hydroxycamptothecin treated group(P=0.018,0.022,0.007,0.013).The green fluorescence of the autophagosome was stronger in the hydroxycamptothecin treated group compared with the control group.Western blot revealed that the gray scale of PERK expression in the cells was 0.130±0.030 in the PERK-knockout group,with a significant reduce in comparison with 0.765 ±0.055 of the control group (P =0.010).However,no obvious distinctions were seen in the band intensities of the expressions of Beclin-1,ATG-5 and LC-3 proteins between the two groups.Western blot indicated that the grey scale of the PERK expression in the cells was 1.790± 0.060 in the 0.10 g/L hydroxycamptothecin group,which was significantly higher than 0.880 ± 0.070 of the control group (P =0.010).Expression levels (gray scales) of Beclin-1,ATG-5,LC-3-Ⅰand LC-3-Ⅱ in the PERK-knockout+ 0.10 g/L hydroxycamptothecin group were 0.475 ± 0.045,0.390 ± 0.040,0.055 ± 0.015 and 0.075 ± 0.025,which were significantly lowed in comparison with 0.955 ± 0.065,0.765 ± 0.055,0.155 ± 0.015 and 0.280 ± 0.030 of the control+ 0.10 g/L hydroxycamptothecin group (P =0.026,0.031,0.042,0.034).In addition,the fluorescence intensity of autophagosomes was weaker in the PERK-knockout+0.10 g/L hydroxycamptothecin group compared with the control+0.10 g/L hydroxycamptothecin group.Conclusions Hydroxycamptothecin induces the autophagy of HTFs by PERK signal pathway.
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Background The fibrosis of filtering area caused by proliferation of human Tenon fibroblasts (HTFs) is one of failure causes following glaucoma surgery.Researches revealed that hydroxycamptothecin can induce the apoptosis of HTFs,but its influence on autophagy of HTFs is unclear.Objective This study attempted to investigate whether hydroxycamptothecin can cause an alteration of autophagic activity in HTFs.Methods Human Tenon capsular tissue was obtained from 3 patients during strabismus correction surgery under the informed consent of patients and their parents for the primary culture and passaged of HTFs in DMEM containing 10％ fetal bovine serum.The generation 3 to 6 cells then were incubated with 0.0,0.5,1.0,4.0,10.0 mg/L hydroxycamptothecin for 24 hours,respectively.A cell counting kit-8 (CCK-8) was used to detect the cell viability in different treated groups.The autophagic activity of HTFs was evaluated by a Cyto-ID autophagy detection kit,and then the autophagic flux was evaluated by counting the Cyto-ID positive cells under a fluorescence microscope,and the green fluorescence intensity was determined by flow cytometry.Quantitative reverse transcriptase PCR (qRT-PCR) and Western blot analysis were employed to assay the relative expressions of autophagic-associated genes and their proteins in HTFs,including Beclin-1,autophagy related gene 5 (ATG-5) and light chain 3 (LC-3).Results The cell viability of HTFs in the 0.0,0.5,1.0,4.0 and 10.0 mg/L hydroxycamptothecin groups were (100.00 ± 6.44) ％,(91.70 ± 6.36) ％,(81.47 ± 6.00) ％,(68.43 ± 6.69) ％ and (59.97 ± 6.98) ％ respectively,showing a gradually declining trend with the increase of hydroxycamptothecin doses,with a significant difference among them (F=19.040,P＜0.001),and the viability of HTFs in the 1.0,4.0 and 10.0 mg/L hydroxycamptothecin groups were significantly decreased than the control group (P＜0.05,P＜0.01,P＜0.01).qRT-PCR analysis revealed that the relative expression levels of Beclin-1 mRNA,ATG-5 mRNA and LC-3 mRNA in 4.0 mg/L hydroxycamptothecin group were (3.225 ±0.346),(2.839 ±0.418) and (3.761±0.224) folds higher than those of the control group.The expressions of Beclin-1 and ATG-5 proteins were significantly increased in the 4.0 mg/L hydroxycamptothecin group in comparison with the control group,and the expression intensity ratio of LC-3-Ⅱ/Ⅰ was 0.965±0.159 in the hydroxycamptothecin group,which was significantly higher than 0.275 ±0.860 of the control group (P =0.003).Cyto-ID staining showed that the percentage of autophagic cells increased dramatically from (11.333±4.010) ％ to (55.000±9.013) ％ upon the exposure of HTFs to 4.0 mg/L hydroxycamptothecin (P=0.002).Flow cytometry analysis showed that the green fluorescence intensity in the 4.0 mg/L hydroxycamptothecin group was (3.037 ±0.513) fold relative to that in the control group,showing a significant difference between the two groups (P =0.003).Conclusions Hydroxycamptothecin can induce autophagy in HTFs in vitro.
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7-Ethyl-10-hydroxycamptothecin (SN-38) is a prominent anticancer agent, but it is insoluble in water and the most pharmaceutically acceptable solvents, the lactone ring of SN-38 shows reversible pH-dependent hydrolysis and forms to the open lactone in alkaline condition. All of these factors limit its application in clinic, and a variety of chemical modification studies have been reported to improve the efficient use of SN-38 by improving its solubility in water and pharmaceutical solvents, stabilizing the lactone form, and altering the pharmacological properties. This paper reviews the pharmacological properties of commercial SN-38 derivatives in clinical trials or preclinical studies and their research progress.
ABSTRACT
Background The filtration surgery is the main method of treating glaucoma,which usually fails due to postoperative scarring.The study about application of anti-scarring agents in filtration surgery is a hotspot.Objective This study was to investigate whether topical administration of hydroxycamptothecin (HCPT) could be used to prevent postoperative scarring in after experimental glaucoma filtration surgery,and explore its optimal dose.Metbods Trabeculectomy was performed on the right eyes of 40 New Zealand white rabbits to establish the trabeculectomy animal models.The rabbits were then randomized into normal saline solution group,0.3 g/L mitomycin C(MMC) group,0.3 g/L HCPT group and 1.0 g/L HCPT group based on the intraoperative topical drugs using randomized number table method.The different drugs above-mentioned were put beneath the conjunctival flap and scleral flap for 5 minutes during the surgery.The intraocular pressure (IOP) was measured before and day 1,4,7,14,21 and 28 after the surgery with Icare tonometer,and the filtering area and height were measured under the slit lamp microscope to assess the efficacy of various drugs.The adverse effects were evaluated by examining the responses of the ocular anterior segment and retinal change.The specimens at operative zone were obtained in 7,14 and 28 days after surgery with the size 5 mm×5 mm for the hematoxylin-eosin staining and Masson trichrome staining to estimate the anti-fibrosis effect of various drugs,and to evaluate the survival time of functional bleb.Kaplan-Meier analysis was used to compare the survival time of functional bleb of different groups.The use and care of the animals complied with the Regulation for the Administration of Affair Concerning Experimental Animals by State Science and Technology Committee.Results The IOP was significantly different in the rabbits from different groups among various time points (Fgroup =20.79,P =0.00 ; Ftime =85.34,P =0.00 ; Fiion =2.13,P =0.01).From 1 day through 28 days after operation,the IOPs in MMC group and 1.0 g/L HCPT group were significantly lower than those before operation (all at P＜0.05).The survival time of functional bleb of different groups was (11.3 ±2.8),(19.5 ±2.4),(13.3 ±2.2) and (20.2 ± 4.5) days,respectively,showing a significant difference (log rank =11.92,P ＜ 0.01),with a considerable prolong in the 1.0 g/L HCPT group.No significant change was found in the bleb area and height among the four groups within 7 days after operation,but postoperative 7,14,28 days,the area and height values of bleb were significantly smaller in the normal saline solution group and 0.3 g/L HCPT group compared with the MMC group and 1.0 g/L HCPT group (all at P ＜ 0.05).Histopathological examination showed loosen subconjunctival tissue,less inflammatory cells and weaker collagenous fibrillary staining in the MMC group and 1.0 g/L HCPT group in comparison with the normal saline solution group and 0.3 g/L HCPT group.Conclusions The topical administration of 1.0 g/L HCPT inhibit the inflammatory response and collagen fibrosis and therefore prolong the survival time of functional bleb after glaucomatous filtering surgery.