The Study On Transdermal Pharmacokinetics Of Podophyllotoxin-loaded Solid Lipid Nanoparticles Gel Using Microdialysis

BackgroundPodophyllotoxin (PPT) extracted from podophylltom resin is a kind of natural and live material with significant cytotoxic activity.0.5%PPT tincture is one of the first-line drugs to control Condyloma Acuminatum (CA) recommend by WHO in 1990. While the existence of some side effect,such as strong skin irritation, short function time, poor skin targeting, easy system absorbing toxicity, has limited its further application in the clinic.Solid lipid nanoparticles(SLN) has becoming more and more hot as drug carrier to skin during the recent years, which is considered to be a new generation preparation of liposome to carry medicine. Most studies showing that SLN has good physiology compatibility, has good skin targeting, can control drug release,can avoid chemical degradation of unsteady medicine, and can reduce side effect such as skin irritation etc. Therefore, SLN has the good development prospects as drug carrier in local application on skin. In order to increase the amount of PPT in the skin, to enhance its target effect on pathological lesions, to prolong its function time on the pathological cells, to attain the treatment result of CA,to reduce the CA to relapse, and to attain the purpose of minimizing the local and systemic side effect at the same time, we have used the solid lipid material-stearic acid and stearamide with low toxicity and good biocompatibility as drug carrier to adsorb or wrap up PPT, taking Brij78 and soybean lecithin as composite surfactant,by the method of modified emulsion evaporation in high temperature and solidification in low temperature to prepare PPT-SLN. Because the gel has a full three dimensional network structure which can disperse solid lipid nanoparticles droplets to further stability,good biological adhesion, we prepared PPT-SLN gel. It is necessary to compare with podophyllotoxin tincture to prove the superiority of PPT-SLN gel.Skin is the target organ of some diseases.In the treatment of local skin lesions_drugs play a role in the target tissue, therefore it is very important to detect the drug concentration in the target tissue. The drug concentration in the skin lesions is not only an important quantitative indicators of local pharmacodynamic effect, but also the key parameters in skin pharmacokinetic study. However, topical formulations development appears to be very delayed because of the limitations on the research methods and analysis equipment. For many years, the commonly used method for estimation of cutaneous drug delivery is still diffusion through excised animal/human skin or artificial membranes in the classical two-compartment Franz-type diffusion cells. Even though this in vitro system has proven to be a robust screening system for early qualitative prediction of bioequivalence/bioavailability, the method obviously have several limitations. Among the most critical is the lack of elimination routes in terms of the vascular system and viable metabolising enzymes, alterations in the stratum corneum structure due to water uptake, so transdermal research results can not accurately reflect the absorption, distribution, metabolism, excretion of the drug in the skin.While in vivo data are commonly obtained via tape stripping,indirect radiochemical methods,skin biopsies or suction blisters,which are nonspecific or too traumatic to be used and can not meet the minimally invasive, real-time and precise positioning.Microdialysis(MD), a valuable technique for continously sampling,is used to measure the in vivo concentration of the unbound fraction of drugs and/or endogenous substances in the extracellular fluid of many tissues. It is applicable to both animal and human studies. The basic principle is to mimic the function of a capillary blood vessel by perfusing a dialysis probe with physiological solution after it is inserted into the tissue of interest. MD sampling provides several advantages, such as direct in vivo sampling in an aesthetized or freely moving animals and continuous sampling from specific target tissues with minimally invasive and capable of producing concentration-time profiles in the target tissue, so it is widely used. Microdialysis has been shown to be a very promising technique for assessent of cutaneous drug delivery.Among several applications, cutaneous microdialysis has gained popularity to study drug pharmacokinetics from topical formulations during the last decade. The cutaneous drug concentration of animal or human can be continuously detected in vivo by Cutaneous microdialysis,which makes it is possible to study pharmacokinetic and bioequivalence of topical agents. A better understanding of the pharmacokinetic of drug in dermis will promote the development of therapy schedules, drug formulations. Dermis is the target of several diseases and the site from which drugs can be absorbed into blood from transdermal delivery systems,so we can further evaluate the systemic side effect using blood microdialysis. However, A major limitation with the use of MD is the production of extremely small volumes of dialysate coupled with the extremely low drug concentrations (ng/ml or even pg/ml).Hence, an analytical method that either can make use of small sample volumes collected during the MD process, or having the necessary sensitivity to measure the drug concentration in the dialysate is essential. The recent introduction of liquid chromatography tandem mass spectrometer (LC-MS/MS) seeks to address the limitations associated with sensitivity,rapid and low volume samples,thus LC-MS/MS combined with microdialysis has become one of the important methods for studying the biochemistry and pharmacokinetics.In the study we developed and validate a LC-MS/MS assay for the quantification of podophyllotoxin concentrations in microdialysate samples and investigate and optimize the in vitro and in vivo recovery and influencing factors of podophyolloxin in microdialysis probe.In order to study the bioequivalence we studied the transdermal pharmacokinetics of podophyllotoxin-loaded solid lipid nanoparticles gel and PPT tincture using cutaneous microdialysis.Meanwhile the blood microdialysis was used to study the absorbing toxicity.Objective1,To develop and validate a selective and sensitive method for the quantitative analysis of podophyllotoxin in blood and dermal microdialysis samples of rats by LC-MS/MS;2,To investigate the in vitro and in vivo recovery and influencing factors of podophyolloxin in microdialysis probe;3,To study the pharmacokinetics of podophyllotoxin-loaded solid lipid nanoparticles gel and podophyllotoxin tincture in cutaneous and blood using microdialysis and evaluate the bioequivalence of the two kinds of topical formulations.Methods1,Microdialysis samples were prepared by liquid-liquid extraction using ethyl acetate with etoposide as internal standard(IS). Podophyllotoxin was separated with a Agilent Zorbax XDB-C18 column (2.1mm×50mm,3.5μm).The mobile phase consisted of acetonitrile:10mmol/L ammonium acetate (40:60,v/v) at a flow rate of 0.3mL/min and analysis was performed at ambient temperature. LC-MS/MS system was operatated under the multiple-reaction monitoring(MRM)using the electrospray ionization technique in positive mode.2,Severals factors such as the perfusion rate, temperature, concentration were studied in vitro. The zero-net flux method was used to investigate if podophyolloxin in microdialysis probe was dispersion dependence. After microdialysis probe was inserted into the jugular vein of male SD rats, the probe was infused with Ringer's.The retrodialysis method was used to study the in vivo recovery of podophyolloxin from rat subcutaneous tissue and blood. Dialysate samples were determined by LC-MS/MS.3,Eight male SD rats are randomly divided into two groups on average. SD rats were anesthetized with urethane (1.25 g/kg, i.p.) during the surgical procedure。Microdialysis probes were inserted into the jugular vein/right atrium and dermis of SD rats. Both probes were connected to a microinjection pump and perfused with Ringer's solution at a flow rate of 1μL/min, After 1 h of stabilization,0.5% PPT-SLN gel or PPT tincture was administered to the skin directly overlying the subcuteous probe and samples were collected at 30 min intervals for 10 h.Podophyllotoxin concentration in dialysates was determined by LC-MS/MS.4,Statistical and analysis:The pharmacokinetic parameters were analyzed with DAS 2.1.1 pharmacokinetic program.Statistic software SPSS 13.0 was used for stastistic analysis.Measurement data were expressed with X±S and indepent-sample T test was used for significant test with two groups. One-way Annova analysis and Repeated measure was used for significant test with 3 groups or more than 3 groups. Differences were significant if P<0.05.Results1,Podophyllotoxin and etoposide responses were optimised at the transitions m/z 432.7→397.3 and 589.5→229.5,respectively. Calibration curves were linear over the range 2.0-1000ng/mL with correlation coefficients>0.99.The lowest limit of quantification(LLOQ) and the lowest limit of detection(LOD) values were found to be 2.0ng/mL and 0.7ng/mL,respectively.The inter-and intra-day precision(RSD%) and accuracy(RE%) were both less than 10%.2,In microdialysis in vitro,the recovery decreased as the perfusion rate was increased and increased as the temperature was increased.However the recovery was independent of the drug concentration surrounding the probe. The recovery and delivery were coincident by zero-net flux method, the in vitro recovery was 47.89%.The in vivo recovery of subcutaneous tissue and blood by retrodialysis method was 28.46±2.42%,15.61±1.92%,respectively, and was stable over the 10 h study period.3,After administration of PPT-SLN gel the concentrations of podophyllotoxin in blood can not be detected in the microdialysis samples. Variance analysis of Repeated measure shows that main effect of group of concentrations of podophyllotoxin under skin has statistical significance (F=255.822, P=0.000), main effect of time has statistical significance (F=85.98, P=0.000), interactive effect has significant difference (F=14.362, P=0.000). Indepent-sample T test shows the cutaneous concentrations of podophyllotoxin between the podophyllotoxin gel and podophyllotoxin tincture at different time points has statistical significance (P<0.05),except for 0.5 hour (t=1.794, P=0.123).The cutaneous concentrations of podophyllotoxin of PPT tincture is higher than that of PPT-SLN gel. Indepent-sample T test shows the main pharmacokinetic parameters between the two groups,such as Tmax (t=2.777, P=0.032), Cmax (t=-7.806, P=0.000), AUC0-10h (t=-16.361, P=0.000), AUC0-∞(t=-4.841, P=0.003),have statistical significance.The Cmax,AUC0-10h and AUC0-∞of PPT-SLN gel are lower than these of PPT tincture,while the Tmax is higher.Conclusion 1,This selective and sensitive method was successfully applied to quantity podophyllotoxin in blood and dermal microdialysates of rats.2,The retrodialysis method can be used to investigate the in vivo recovery of podophyolloxin in microdialysis probe.Microdialysis would prove to be a useful and reliable tool to study the pharmacokinetics of podophyolloxin.3,PPT-SLN gel has the feature of epidermal targeting and low systemic toxicity and can release slowly.