Cutaneous Pharmacokinetics Of Azole Antifungal Drugs Based On Microdialysis And Ultra-fast Liquid Chromatography

Pharmacokinetics is the study of the time course of a drug within the body and incorporates the processes of absorption, distribution, metabolism and excretion, the better interpretation the effect and toxicity oh the drug, which help us to know about their mechanisms of action and their pharmacokinetic properties during the initial state of drug development and in clinical therapy. In general, the data obtained from pharmacokinetic samplings such as the collection of body fluids (blood, urine, etc.) and the homogenate method are indirect, rough and the great damage in experimental animals. In recent years, in vivo microdialysis technique is beginning to play an important role in the field of pharmacokinetics, especially in the drug distribution and metabolism. As an in vivo bio-sampling technique, microdialysis has continuous, dynamic observation, light tissue damage, better time and spatial resolution, dialysis fluid without pretreatment characteristics. Microdialysates obtained were extremely low, which must combine with high selectivity and high sensitivity, capable of micro-volume samples of quantitative analysis method. Ultra-fast liquid performance has the unique advantages in the separation and detection and become a powerful tool in the analysis of microdialysate. In our study, application of microdialysis technique combined with modern chromatographic analysis of ultra-fast liquid chromatography to analysis high-throughput pharmacokinetics of antifungal drugs (iodiconazole, miconazole nitrate and berberine hydrochloride).The establishment and evaluation of the research system of microdialysis and ultra-fast liquid chromatography1. Comparison of UFLC and HPLC system performanceThis chapter established chromatographic analysis methods by shimadzu ultra-fast liquid chromatography (UFLC) and shimadzu high-performance liquid chromatography (HPLC) to quantitate a class of insulin sensitizer drug MCC-555 respectively, and compared instrument performance and parameters of both. UFLC is a newly developed instrument offering a high power of separation and short analysis time. UFLC system supports high-speed analysis through the following features: low volume tubing and flow cell decrease extra-column band spreading; 10 sec ultra-fast injections with the Prominence SIL-20 autosampler; temperature capability up to 85°C; fast data acquisition allows for better signal tracing. These features, together with the use of a 2.2μm particle material column, can shorten analysis time 5- to 6-fold, giving a considerable saving in both instrument and analyst time; of value to those involved in high-throughput applications in the determination of antifungal drugs in microdialysate.2. Preparation of laboratory-made microdialysis probesLinear microdialysis probes and flexible microdialysis probes were self-designed, which achieve the stability and reproducibility of the level of commercial probe. Since it is laboratory-made probe, the length of dialysis membrane can be changed according to the different experimental animals and the specific requirements of experiments. Increase in the length of dialysis membrane dialysis to expand the exchange area could meet the different needs of drug analysis. It would prove to be a useful and reliable tool to study the pharmacokinetics of antifungal drugs.3. The establishment and evaluation of the research system of microdialysis and ultra-fast liquid chromatographyThe research system of microdialysis and ultra-fast liquid chromatography was evaluated by iodiconazole as a model drug. A Shimadzu Prominence UFLCTM system was used for analysis the in vitro and vivo recovery of iodiconazole. Samples were analyzed on a Shimadzu Prominence UFLCTM C18 column (2.2μm, 50 mm×2.0 mm i.d.); mobile phase consisted of acetonitrile-0.025% triethylamine solution adjusted to pH 3.6 with phosphoric acid (65:35, v/v); UV detector of 230 nm; flow rate, 0.5 ml/min; column temperature, 35°C; injection volume, 10μl; analysis time, 2 min. In vitro, the recovery determined by dialysis, retrodialysis and by the zero-net-flux (ZNF) was compared. In vivo, the recoveries were determined by retrodialysis from rat subcutaneous tissue and blood vessel. The in vitro recovery detected by dialysis method was the same as that by retrodialysis method at the flow rate (3μl/min). The recovery decreased when the perfusion rate increased. However, the recovery was independent of the drug concentration surrounding the probe. The concentration and recovery of iodiconazole in the solvent could be determined accurately by no-net-flux method. The performance of the microdialysis system was stable over an 8-h study resulting in a mean in vitro recovery of 47.64%±4.29% (linear probe), 38.05%±6.63% (flexible probe). In vivo recovery of iodiconazole was 33.57%±5.41% (linear probe), 27.86%±3.08% (flexible probe) and was stable over the 8 h period.The microdialysis sampling could be used for the pharmacokinetic study of iodiconazole.In vivo pharmacokinetics and in vitro pharmacodynamics of iodiconazole in rats based on MD-UFLC system1. Pharmacokinetic study of iodiconazole in rat dermal microdialysate and blood microdialysateIt was the first time that microdialysis combined with ultra-fast liquid chromatography were applied to pharmacokinetics of iodiconazole in rat blood microdialysate and dermal microdialysate. Microdialysis probes were implanted in the skin and blood vessels at the same time, which was of great significance for the study of the true distribution at different time points in the skin and blood. The pharmacokinetic parameters Cmax and AUC of dermal microdialysate were higher than that of blood microdialysate. The iodiconaole concentration curves of the both were roughly the same. AUCdermal and AUCblood ratio of was close to 20, which means that the free iodiconazole concentration in dermal interstitial fluid is much higher than the blood drug concentration, and has a higher distribution in target tissues. Drug AUC/Cmax of dermal microdialysate and blood microdialysate with excellent quantitative relationship shows that the drug-related in blood concentration and skin tissue concentration of transdermal formulations. The experiments show that microdialysis combined with ultra-fast liquid chromatography can be used to study the pharmacokinetics of free iodiconazole in the target organ (skin) and blood of rats.2. In vivo pharmacokinetics and in vitro pharmacodynamics of iodiconazoleThe combination of pharmacokinetics and pharmacodynamics is to clarify the three-dimensional relationship (concentration-effect-time) of drugs in the target site. This chapter studied iodiconazole in vitro antifungal activity (MIC) combined with pharmacokinetics (PK/PD parameters) and in vitro antifungal concentration-efficacy curves. Iodiconazole has a stronger antibacterial effect against clinical pathogenic fungi, and its MIC80 ranged from 0.0625μg/ml to 1μg/ml. The maximum MIC80 (0.5μg/ml) as the standard of PK/PD parameter analysis, the drug concentrations in dermal microdialysate were all higher than 1μg/ml in the 900 min sampling time. After topical administration of iodiconazole cream the duration time of dermal microdialysate (T>MIC≥15.00 h) meet the demand of antifungal activity. The AUC0～∞/MIC of dermal microdialysate is blood microdialysate 18.9 times, indicating that the skin as a target organ has the highest levels of drug concentration. At the same time, drugs have longer retention time in the skin indicating that the skin storage library formed to play a partial efficacy, to achieve local effects of local administration of drugs and to maintain a long time the role of antifungal activity. From the in vitro antifungal concentration-pharmacodynamic curve we can see iodiconazole concentration in dermal microdialysate have good inhibitory effect to 12 clinical pathogenic fungi (bacteriostasis rate≥90%) and the inhibitory rate of up to 100% in the 80~680 min. Therefore, iodiconazole is a better clinical effect of antifungal agents.Pharmacokinetics and interaction of compound ointment of miconazole nitrate and berberine hydrochloride drug based on MD-UFLC system1. Develpopment of ultra-fast liquid chromatography methods for quantification of miconazole nitrate and berberine hydrochloride in guinea pig dermal microdialysate and plasmaUFLC quantitative analysis methods of miconazole nitrate and berberine hydrochloride in dermal microdialysate and plasma were established respectively. Chromatographic separation conditions of miconazole nitrate in dermal microdialysate: the separation of miconazole was performed on a Shimadzu Prominence UFLCTM C18 column (2.2μm, 50 mm×2.0 mm i.d.). The mobile phase consisted of acetonitrile-0.033% triethylamine solution adjusted to pH 3.8 with phosphoric acid (54:46, v/v); UV detector of 230 nm; flow rate, 0.5 ml/min; column temperature, 25°C; injection volume, 10μl; analysis time, 1.5 min. Chromatographic separation conditions of miconazole nitrate in plasma: the separation of miconazole was performed on a Shimadzu Prominence UFLCTM C18 column (2.2μm, 50 mm×2.0 mm i.d.). The mobile phase consisted of acetonitrile-0.033% triethylamine solution adjusted to pH 3.8 with phosphoric acid (53:47, v/v); UV detector of 230 nm; flow rate, 0.5 ml/min; column temperature, 35°C; injection volume, 10μl; analysis time, 2 min. Chromatographic separation conditions of berberine hydrochloride in dermal microdialysate: the separation of miconazole was performed on a Shimadzu Prominence UFLCTM C18 column (2.2μm, 50 mm×2.0 mm i.d.). The mobile phase consisted of acetonitrile-0.048% triethylamine solution adjusted to pH 3.8 with phosphoric acid (32:68, v/v); UV detector of 346 nm; flow rate, 0.5 ml/min; column temperature, 25°C; injection volume, 10μl; analysis time, 1.5 min. Chromatographic separation conditions of berberine hydrochloride in plasma: the separation of miconazole was performed on a Shimadzu Prominence UFLCTM C18 column (2.2μm, 50 mm×2.0 mm i.d.). The mobile phase consisted of acetonitrile-0.048% triethylamine solution adjusted to pH 3.2 with phosphoric acid (32:68, v/v); UV detector of 346 nm; flow rate, 0.5 ml/min; column temperature, 25°C; injection volume, 10μl; analysis time, 2 min. The chromatographic assay showed good characteristics of specificity, sensitivity and precision, allowing numerous samples to be processed in clincal pharmacokinetic studies of miconazole nitrate and berberine hydrochloride.2. Assessment of in vitro and in vivo microdialysis recovery of miconazole nitrate and berberine hydrochlorideThe present investigation examined in vitro and in vivo microdialytic characteristics of miconazole nitrate and berberine hydrochloride. The performance of the microdialysis system was stable over an 8 h study resulting in a mean in vitro recovery of 78.33%±4.81% (miconazole nitrate), 61.21%±3.32% (berberine hydrochloride). In vivo recovery was 72.13%±1.77% (miconazole nitrate), 52.47%±0.53% (berberine hydrochloride) and was stable over the 8 h period.The recovery was independent of the drug concentration surrounding the probe. It was suggested that microdialysis sampling could be used for the pharmacokinetic study of miconazole nitrate and berberine hydrochloride and retrodialysis method could be used for the determination of the in vivo recovery of both.3. The pharmacokinetics and interaction research of compound recipe of miconazole nitrate and berberine hydrochloride ointmentThis chapter studied the single-dose pharmacokinetics and the drug interaction of compound recipe of miconazole nitrate and berberine hydrochloride ointment (size: each containing miconazole nitrate 1.5%, berberine hydrochloride 1.5%) in animals based on the MD-UFLC method. In this study, three male and three female guinea pigs were randomly divided into three groups using self-controlled, randomized cross-experimental methods, and three preparations×three cycles (3×3 Latin square design) dosage regimen were taken. The compound recipe of test preparation was a mixture of 1.5% miconazole nitrate and 1.5% berberine hydrochloride, the control preparations were 1.5% miconazole nitrate and 1.5% berberine hydrochloride. Through in vivo preclinical pharmacokinetic study in guinea pigs, we can estimate the relative bioavailability and other pharmacokinetic kinetic parameters (the characterization of miconazole nitrate or berberine hydrochloride in the compound berberine of miconazole nitrate and berberine hydrochloride ointment), and investigate pharmacokinetic drug interaction of both in test preparation to provide reference for clinical trials. The three-factor ANOVA analysis showed that the kinetic parameters InCmax,InAUC of the representation of miconazole nitrate and berberine hydrochloride in dermal microdialysate and plasma (compound recipe of miconazole nitrate and berberine hydrochloride ointment: total dose miconazole nitrate 15 mg, berberine hydrochloride 15 mg, the control preparations: miconazole nitrate 15 mg, berberine hydrochloride 15 mg) were no significant difference (p>0.05). The non-parametric test of tmax (Wilcoxon′s rank sum test) showed p>0.05, which means that test preparation and the corresponding control preparations had no significant difference. Further one/two-sided t-test results of InCmax,InAUC showed that the main pharmacokinetic parameters in dermal microdialysate and plasma were non-obvious change between compound recipe of miconazole nitrate and berberine hydrochloride ointment and the control preparations (miconazole nitrate ointment, berberine hydrochloride ointment), indicating that there is no pharmacokinetic drug interaction of both.