| Background and Objetive:Diabetes mellitus is one of the most common chronic diseases in nearly all countries, and continues to increase in numbers and significance, as changing lifestyles lead to reduced physical activity and increased obesity. In our country, DM has become the third common disease followed cardiovascular diseases and cancer. The World Health Organization(WHO) published estimates for the years2000and2030, using data from40countries but extrapolated to the191WHO member states. These estimates suggest that in2010there will be285million people worldwide with diabetes.For developed countries, the majority with diabetes are aged over60years, whereas for developing countries most people with diabetes are of working age, between40and60years.Population growth, ageing of populations, and urbanization with associated lifestyle change is likely to lead to a54%increase in worldwide numbers with diabetes by2030. Diabetic complication is the material cause inducing death and mutilation to diabetics. And among the various diabetic complications, dermatosis (skin disease) posses a very high incidence. According to the relevant information,25%～30%diabetic suffering from skin diseases. Ketoconazole is one of the most widely used drug for fungous infection.But the toxic and side-effect for oral use is very severe。A great many diabetics should use ketoconazole frequently because of the high incidence rate of fungous infection which is one of the various complications of diabetes. According to investigation statistics,25%～30%diabetics would suffer from dermatoses。 So they usually need reasonable application of ketoconazole to match the treatment in the symptomatic treatment of skin disease so as to relieve symptoms.Therefore, the question about how to make the logical application of ketoconazole more reasonably to diabetics was elicited from above-mentioned situation.Microdialysis (MD) is a minimal invasive technique where it is possible to measure various metabolites in almost any animal bodily compartment. The MD probe mimics a capillary blood vessel,by perfusing a thin dialysis catheter implanted into the tissue of interest, it is possible to measure the concentration of a variety of metabolites in interstitial fluid or a cavity. The biggest advantage of microdialysis is it can comply in-vivo, real-time and on-line sampling in the body during normal course of life with no interference.In our study, we chose ketoconazole gelatin,cream and ointment as model drug preparations, applied microdialysis and Franz technology to measure the concentrations of KTZ permeating through the nomal and diabetic rats’skin, made a compare between the two groups, then analysed the subcutaneous tissue/plasma drug concentration-time profiles, and finally got the various kinetic parameters.The fingdings was aimed to provided evidence for the clinical pharmacist consultation to give reasonable guidence of topical use of KTZ preparations to patients with diabetes, and also provide experimental reference for clinicians and nurses in taking care of diabetics when the patients topically use KTZ preparation.Method and Result: In order to study the influence of diabetic skin’s change on drug’s transdermal activity, we need to establish a diabetes animal model. In our study, DM rat model was prepared with an easy and simple,highly applicable method of streptozocin (STZ,40mg·kg-1) intraperitoneal injection. After the injection of STZ, the blood glucose of STZ-treated rats was sharply increased(>16.7mmol·L-1) and subsequently remained in the high level indicating that the modeling method was feasible.In vitro study, male wistar rats were randomly divided into six groups:KTZ gelatin-control group and KTZ gelatin-DM group, KTZ cream-control group and KTZ cream-DM group, KTZ ointment-control group and KTZ ointment-DM group, while each group contained6rats. Abdominal skin was cut to carry out an in-vitro penetration experiment on an improved Franz diffusion cells, and phosphate buffer contained10%alcohol (PBS, pH7.4) was used as receptor solution. The solution was analyzed by HPLC, and then the penetrating rate can be calculated. The HPLC conditions were as follows:the mobile phase was methyl alcohol-pH6.8phosphate buffer (70:30, v/v), and the flow rate was1.0mL·min-1with the detection wavelength at235nm, and the injection volume was20μL. The penetrating rate of KTZ gelatin-control group and KTZ gelatin-DM group were3.38±0.36and4.56±0.67μg·h-1`cm-2, respectively. There was significant difference between the two groups(P<0.05).The penetrating rate of KTZ cream-control group and KTZ cream-DM group were1.33±0.41and1.87±0.11μg·h-1·cm-2, respectively. There was significant difference between the two groups(P<0.05). The penetrating rate of KTZ ointment-control group and KTZ ointment-DM group were0.77±0.42and1.72±0.86μg·-1·cm-2, respectively. There was significant difference between the two groups(P<0.05).Ringer’s solution,a constant perfusion rate of lμL·min-1was maintained during the strdy. The dialysate was collected in45min interals for12samples. KTZ concerntrations in dialysate were quantitatively determined by LC-MS/MS using positive ion electrospray ionisation(ESI) with the multiple reaction monitoring transitions performed at531.2â†'82.1. The separation was achieved on an Agilent C18colum and the mobile phase was acetonitrile-water with0.1%formic acid(70:30,v/v) at a flow rate of0.3ml-min-1with a10μL injection volume. Nitrogen was used as nebulizer gas and nebulizer pressure was set at30psi. Cesolvation gas (nitrogen) was heated to350℃and delivered at a flow rate of11L-min-1. Fregmentor and collision energry was205eV and46eV respectively.After KTZ Gelatin/cream/ointment administrationed on rat’s skin, the distribuion and concentration of KTZ in blood and abdominal subcutaneous were moitored by microdiaylsis. The results of in-vivo microdialysis showed that subcutaneous drug concentrations in diabetic rats was significantly higher than normal rats, the largest plasma concentration (Cmax) and area under curve (AUC0-540) compared with the normal group was with significant difference(P<0.05), indicating that there surely be changes in the skin of diabetic rats and increase the topical drug absorption. However, in the consequence of blood microdialysis, the two groups were not significant difference.Conclusion and discussionIn this study, a feasible diabetic rat modelling methods was established, and a HPLC and a highly selective, sesitive LC-MS/MS method were developed for the quantification of KTZ.We also improved the methods of KTZ microdialysis experiments. We applied in vitro(Franz) and in vivo(Microdialysis) tecnology to examine the the influence of diabetic skin’s change on drug’s transdermal activity. Based on the results from the above in vitro and in vivo experiments, it can conclude that diabetes induce some pathological changes on skin, which then result in the increasing of the permeation of KTZ. Therefore, it is necessary to adjust the dose while KTZ or some other topical preparations were applied on the skin in case of diabetes mellitus, in order to reduce drug side effects and improve the diabetics’ quality of life.In the research, for the reasons of limited time, energy and other factors, there are still many deficiencies. For instance, the sample size of diabetic rats in microdialysis experiment is still a little insufficient; the calculation of recovery of probe also needs a futher improvement.These disappintments hoped to be performed in the future research. |
PDF Full Text Request