| BackgroundPodophyllotoxin(POD)is a natural active lignan containing a significant antiviral and antitumor effects which is extracted from roots of A.littoralis and A.Quadrangularis.In 1990,the WHO recommended podophyllotoxin tincture(POD-T)as a first-line treatment of genital warts.However,application of POD-T on the reproductive tract mucous membrane accompany with a serve irritation,heavy damage,increased risk of local tissue infection and easy to be absorbed poisoning,so it is not suitable for the treatment of female cervix CA.In order to overcome these shortcomings and ensure POD as the first-line drug administration of cervix CA,in the preliminary work,we had successfully prepared 0.5%podophyllotoxin nanostructured lipid carriers(POD-NLC)with characteristics of strong stability,small entrapment rate,high drug loaded,and it can effectively promote drug delivery,improve bioavailability,reduce irritation,and reduce the absorption toxicity.In this study,we prepared 0.5%POD-NLC,and evaluated the physicochemical properties and metabolic characteristics of the drug.ObjectiveTo preparate the POD-NLC and tested The physical and chemical properties.We established a tibet mini-pigs model for drug application on cervix to compare the irritation and systemic toxicity with POD tincture and to evaluate the safety further.Materials and Method1.We prepared 0.5%POD-NLC and 0.5%POD-NLC gel by modified rotary-evaporative emulsification method The morphology of 0.5%POD-NLC appearance was observed,and the particle size was detected by Malvern particle size analyzer,and the POD concentration was detected by HPLC and microscopic morphology was obtained by scanning electron microscopy.2.Different dose of POD-NLC was used as the experimental group,normal saline(NS)as the negative control,0.5%POD-T as the positive control,and after 3 days of continuous drug administration,we observed the morphology and pathological changes of the cervical mucosa.The concentrations of IL-1?,IL-6 and IL-8 in the cervical secretions of different experimental groups were measured by LUMINEX.3.The POD concentrations of cervical secretions and cervical mucosal tissue were detected after 2h,4h,6h and 8h of 0.5%POD-NLC and different concentration of 0.5%POD-T(0.5%,1%,2%)were applied,and the metabolism of 0.5%POD-NLC was evaluated in vivo.4.Different dose of 0.5%POD-NLC and 0.5%POD-T were administrated to cervix mucosa of pigs.Blood drug concentration were measured after the administration of 2h?4h?6h?8h and 10h.The changes of POD concentration in the blood were analyzed to assess system safety.5.0.5%POD-NLC was used as the experimental groupand 0.5%POD-T as the control group,and the drug was administered to the cervix mucosa,and blood collection was took from the anterior vena cava of the miniature pig.The biochemical parameters were measured by the automatic biochemical analyzer,and the blood cell count and hemoglobin content were measured by automatic blood cell counter and the changes of blood physiological and biochemical indexes of the pigs were evaluated after POD-NLC administration to cervix mucosa.StatisticsSPSS20.0(PASW)was used for data statistics,description and analysis.The data were expressed as X±S.There was statistically significant between each groups when P<0.05.Results and analysis1.0.5%POD-NLC was preparated successfully,and the appearance perform as a uniform texture translucent colloidal emulsion.The particle size of POD-NLC was 178.5±20nm and the encapsulation efficiency was(75.7±0.44)%.The POD-NLC was regular round or quasi-circular circular with uniform distribution in microscopic.2.The irritation score results show that the POD-T grouphad the highest score of 29.3±1.5,which was significantly different from the POD-NLC group(POD-NLC small dose group)(p=0.001).The total score of POD-NLC high dose groupwas 27.2±1.0,which was significantly higher than that of POD-NLC low dose group(4.3±0.8)(p=0.001).The total score of NS group(3.3±1.0)was slightly lower than that of POD-NLC low dose group(p=0.092).The results of inflammatory cytokine concentration showed that the levels of IL-1?,IL-6 and IL-8 in POD-NLC low dose groupwere significantly lower than those in POD-NLC high dose group(p<0.01),but there was no difference with NS group(P>0.01).The concentration in POD-T groupwas higher than that of POD-NLC high dose group(p<0.05),the difference was statistically significant(p<0.05).3.During the same time of drug applied,the POD concentrations in 0.5%POD-NLC groupwere higher than those in 0.5%POD-T groups(p<0.05).0.5%POD-T treatment group,the highest peak concentration of drug in cervical mucous tissue appeared at 4 hours,then the concentration decreased rapidly.The drug concentration in the mucosal tissue of 0.5%POD-NLC groupincreased slowly and continuously,and no obvious drug concentration peak was observed.The drug concentration in mucosal tissue of 0.5%POD-NLC groupbegan to be higher than 2%POD-T groupat 10h of drug applied.4.When 1ml 0.5%POD-NLC was applied to cervix,it was failed to measure POD blood concentration although monitoring 10h continuously.The highest peak blood drug concentration(17.4±1.0ng/ml)of 0.5%POD-T groupappeared at 4h after and appeared at low concentration within 10h.When 2ml applied,the blood drug concentration of 0.5%POD-T groupwas significantly higher than that of 0.5%POD-NLC group(P<0.05),and gently changed in 0.5%POD-NLC group,and the highest drug concentration peak(9.1±0.7 ng/ml)appeared after 6h of drug administration,while failed to measure after 10 h.In 0.5%POD-T group,the highest peak drug concentration was(24.9±3.1 ng/ml),and appear at low concentration within 10h after administration.5.Physiological and biochemical indicators of liver and kidney,and blood routine examination indicated that the average values of ALT and AST,white blood cell count were significantly higher than those of POD-NLC and POD-T before the drug administration to cervical mucosa and with no significant difference(p>0.05).It showed no obvious systemic toxicity after administration 2ml of 0.5%POD-NLC to cervix mucosa.ConclusionIt was confirmed that the self-made POD-NLC had the characteristics of small particle size,good stability and high entrapment efficiency.Amild local irritation in the POD-NLC group,and it will stay longly in cervical secretions,releasing slowly in cervical mucosal tissue.So the irritation is mild and with less absorption by the mucosa when the conventional treatment dose of POD-NLC applied to cervical mucosa,and it has a better system security. |
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