Human subcutaneous tissue distribution of fluconazole: comparison of microdialysis and suction blister techniques

AIMS: To investigate uptake of fluconazole into the interstitial fluid of human subcutaneous tissue using the microdialysis and suction blister techniques. METHODS: A sterile microdialysis probe (CMA/60) was inserted subcutaneously into the upper arm of five healthy volunteers following an overnight fast. Blisters were induced on the lower arm using gentle suction prior to ingestion of a single oral dose of fluconazole (200 mg). Microdialysate, blister fluid and blood were sampled over 8 h. Fluconazole concentrations were determined in each sample using a validated HPLC assay. In vivo recovery of fluconazole from the microdialysis probe was determined in each subject by perfusing the probe with fluconazole solution at the end of the 8 h sampling period. Individual in vivo recovery was used to calculate fluconazole concentrations in subcutaneous interstitial fluid. A physiologically based pharmacokinetic (PBPK) model was used to predict fluconazole concentrations in human subcutaneous interstitial fluid. RESULTS: There was a lag-time (approximately 0.5 h) between detection of fluconazole in microdialysate compared with plasma in each subject. The in vivo recovery of fluconazole from the microdialysis probe ranged from 57.0 to 67.2%. The subcutaneous interstitial fluid concentrations obtained by microdialysis were very similar to the unbound concentrations of fluconazole in plasma with maximum concentration of 4.29 +/- 1.19 microg ml(-1) in subcutaneous interstitial fluid and 3.58 +/- 0.14 microg ml(-1) in plasma. Subcutaneous interstitial fluid-to-plasma partition coefficient (Kp) of fluconazole was 1.16 +/- 0.22 (95% CI 0.96, 1.35). By contrast, fluconazole concentrations in blister fluid were significantly lower (P < 0.05, paired t-test) than unbound plasma concentrations over the first 3 h and maximum concentrations in blister fluid had not been achieved at the end of the sampling period. There was good agreement between fluconazole concentrations derived from microdialysis sampling and those estimated using a blood flow-limited PBPK model. CONCLUSIONS: Microdialysis and suction blister techniques did not yield comparable results. It appears that microdialysis is a more appropriate technique for studying the rate of uptake of fluconazole into subcutaneous tissue. PBPK model simulation suggested that the distribution of fluconazole into subcutaneous interstitial fluid is dependent on tissue blood flow.     

提高微透析探针回收率方法的研究进展

微透析是将灌流取样和透析技术结合起来的研究物质动态变化的一种新型采样技术,适合于深部组织和重要器官的活体研究。微透析探针的回收率是影响微透析结果准确性的重要因素之一。微透析探针对物质的回收率通常较低,需结合灵敏的检测方法才能实现物质的定量分析,因此限制了微透析技术在科研中的应用及发展。如何提高微透析探针的回收率是亟待解决的问题,本文综述了国内外研究中提高微透析探针对物质回收率的方法,为探针回收率的提高提供一些参考。     

Analytical considerations for microdialysis sampling

Adaptations in microdialysis probe designs have made it possible to obtain samples from the extracellular fluid of a variety of tissues with high temporal resolution. The resulting small volume samples, often with low concentration of the analyte(s) of interest, present a particular challenge to the analytical system. Rapid separations can be coupled on-line with microdialysis to provide near real-time data. By combining microdialysis sampling with a liquid chromatographic or capillary electrophoretic separation and a highly sensitive detection method, a separation-based sensor can be developed. Such sensors have been applied to the investigation of drug entities as well as to study endogenous analytes.     

Pharmacokinetic estimations from microdialysis data

Summary Microdialysis has recently been adapted for sampling the extracellular fluid of various organs in order to measure drug concentrations, and the first clinical application has been published. My aim here is to provide simple rules about how to analyse pharmacokinetic data from such studies. The plotting of data on a time scale and the estimation of C (0) and slopes is not a trivial problem when multicompartmental models are assumed or sampling intervals are unequal. I have developed formulae and algorithms to solve the problem. A simple rule of thumb is given, suggesting when these formulae need to be applied. It is shown that the calculations of half-life and slopes is similar to standard methods for equal sample intervals and that calculation of AUC and clearance may be even more accurate for microdialysis data than for ordinary blood sampling, because of the time-integral character of the dialysis method. I have dealt with both zero-order and first-order kinetics.     

Application of microdialysis in clinical pharmacology

Microdialysis has been developed during the last 25 years by several authors primarily to study brain function and changes in levels of endogenous compounds such as neurotransmitters or metabolites in different laboratory animals. However, in the last ten years microdialysis sampling has been introduced as a versatile technique in the clinical setting. Although, microdialysis sampling has been extensively used for metabolic monitoring in patients, it was also employed for the study of distribution of different therapeutic agents especially anti-infective and antineoplasic drugs. In addition, clinical effect of drugs in patients could be also determined by means of microdialysis. So, this article reviewed the vast applications of the microdialysis technique for the study of pharmacokinetic and pharmacodynamic properties of drugs in the clinical setting.     

青藤碱微透析体外回收率研究

目的考察浓度、流速对回收率和损失率的影响,为体内青藤碱微透析实验提供参考依据。方法利用增量法和减量法测定回收率,采用超高效液相测定青藤碱含量。结果流速相同时,探针的回收率和损失率与周围药物浓度无关;探针的回收率随着流速的增加呈下降趋势,增量法和减量法测定的回收率与损失率在各流速下相近。结论微透析法可用于青藤碱体内药动学研究,反向透析法可用于青藤碱微透析体内回收率的测定。     

In vitro microdialysis sampling of docetaxel

Microdialysis is a technique that allows sampling compounds from the extracellular fluid in different tissues, such as muscle, lung, and brain. However, the feasibility of using this technique with lipopohilic and high molecular weight compounds has been questioned, since these compounds are less likely to diffuse through the dialysis membrane. Therefore, it was the objective of this study to investigate the feasibility of doing microdialysis of docetaxel by determining its recovery by the microdialysis probe. Three different methods were investigated: extraction efficiency, retrodialysis, and no-net-flux. For the first two methods, three different concentrations were tested: 2.5, 5, and 9 mg/l. The recovery obtained for each concentration was 49.3 +/- 6.7 (n = 4), 44.6 +/- 5.4 (n = 3), and 34.7 +/- 2.1 (n = 4) by extraction efficiency, and 53.4 +/- 7.9 (n = 3), 61.4 +/- 7.6 (n = 3), and 64.2 +/- 1.9 (n = 3) by retrodialysis, respectively. The average recovery obtained by no-net-flux was 68.7 +/- 9.6 (n = 5). Although it has been reported that microdialysis cannot be applied to lipophilic compounds, the results here show the opposite. The high recoveries obtained for docetaxel in all methods applied show that the compound can diffuse through the probe membrane. Overall, docetaxel seems to be very suitable for microdialysis despite its lipophilicity and high molecular weight.     

An equivalent length model of microdialysis sampling

One of the critical issues in microdialysis sampling is how to predict the extraction fraction (E(d)), based on transport properties of analytes in both tissues and probes. A one-dimensional (1-D) model has been used widely in previous studies to predict E(d) at the steady state. However, this model is valid only for long probes. To this end, an equivalent length (EL) model was developed for probes with any length used in experiments. The key idea in the model was to replace the probe length (L) in the 1-D model with an equivalent length (L(E)) when calculating transport resistance in surrounding tissues. The length difference, (L(E)-L), was assumed to be proportional to the penetration depth of analytes (Gamma). The proportionality constant (lambda) was determined through minimizing the errors in predicted E(d). We found that, the EL model could accurately predict E(d) when lambda=0.369. The maximum error in EL model predictions was <6%, for model constants varying in the same ranges as those in microdialysis experiments. This error was one order of magnitude smaller than that in 1-D model predictions.     

Improving microdialysis extraction efficiency of lipophilic eicosanoids

Microdialysis recovery of the lipophilic analytes prostaglandin B₂, leukotriene B₄ and C₄ was studied in vitro. Relative recovery (RR) through different commercially-available microdialysis probes for prostaglandin B₂ and leukotrienes was examined using different flow rates. The enhancing effect at different concentrations of binding agents such as , β, γ-cyclodextrins (, β, γ-CD) on the microdialysis RR for different eicosanoids was evaluated. Small organic molecules such as ethanol, propylene glycol and dimethyl sulfoxide (DMSO) were studied in terms of their effect on enhancing RR. Inclusion of arachidonic acid in either the perfusion fluid or the sample medium caused the microdialysis RR for these hydrophobic analytes to be increased.     

脑微透析技术在脑内研究中的应用

脑微透析技术可用于采样脑内神经递质、神经蛋白和激素,并结合高灵敏度的微量化学分析技术对其进行定量分析,从而研究与中枢神经递质相关的疾病、病理及治疗方法等。本文介绍了脑微透析技术的基本原理和过程,并对其在中枢神经递质如兴奋性氨基酸、乙酰胆碱、多巴胺、五羟色胺等及相关药物如选择性五羟色胺再摄取抑制剂、自由基清除剂研究中的应用进行综述。     

Assessment of cutaneous drug delivery using microdialysis

During the last decade microdialysis has been successfully applied to assess cutaneous drug delivery of numerous substances, indicating the large potential for bioequivalence/bioavailability evaluation of topical formulations. The technique has been shown to be minimally invasive and supply pharmacokinetic information directly in the target organ for cutaneous drug delivery with high temporal resolution without further intervention with the tissue after implantation. However, there are a few challenges that need to be addressed before microdialysis can be regarded as a generally applicable routine technique for cutaneous drug delivery assessments. Firstly, the technique is currently not suitable for sampling of highly lipophilic compounds and, secondly, more studies are desirable for elucidation of the variables associated with the technique to increase reproducibility. The present literature indicates that the condition of the skin at the individual assessment sites is the main variable, but also variables associated with relative recovery, differentiation between the pharmacokinetic parameters (i.e., lag time, distribution, absorption and elimination rate) can influences the reproducibility of the technique. Furthermore, it has been indicated that cutaneous microdialysis in rats may be useful for prediction of dermal pharmacokinetic properties of novel drugs/topical formulations in man.     

Measurement of drug release from microcarriers by microdialysis

The purpose of this study was to examine the feasibility of the microdialysis sampling technique as a method to precisely and conveniently measure drug release from microcarrier systems such as liposomes and microspheres. Release of 5-fluorouracil (5-FU) from liposomes and microspheres was evaluated in vitro using microdialysis. Retrodialysis calibration using 5-chlorouracil (5-CU) was performed in conjunction with on-line HPLC analysis. At a microdialysis perfusate flow rate of 0.5 muL/min, concurrent 5-FU gain and 5-CU loss ranged from 72% to 75%, while concurrent 5-FU loss and 5-CU ranged from 69% to 71%. After calibration, simultaneous 5-FU release profiles were obtained by continuous microdialysis and discrete equilibrium dialysis sampling using a side-by-side diffusion apparatus. Release rates were characterized by a first-order release model. The release rate constants for a representative liposomal formulation were 0.30 and 1.85/h by microdialysis in the acceptor and donor compartments, respectively, and 0.39/h by equilibrium dialysis in the acceptor compartment. The calculated release rate constant determined by equilibrium dialysis in the donor compartment (1.98/h) agrees with that determined by microdialysis (1.85/h) when the resistance of the equilibrium dialysis membrane with associated first-order rate constant of transfer of 0.42/h is taken into account. Release profiles of 5-FU from a number of different liposome and microsphere formulations were determined. The results indicate that a convenient and reproducible characterization of drug release from various liposome and microsphere formulations is readily obtainable by microdialysis.     

Application of long-term microdialysis in circadian rhythm research

Our laboratory has pioneered long-term microdialysis to monitor pineal melatonin secretion in living animals across multiple circadian cycles. There are numerous advantages of this approach for rhythm analysis: (1) we can precisely define melatonin onset and offset phases; (2) melatonin is a reliable and stable neuroendocrine output of the circadian clock (versus behavioral output which is sensitive to stress or other factors); (3) melatonin measurements can be performed extremely frequently, permitting high temporal resolution (10 min sampling intervals), which allows detection of slight changes in phase; (4) the measurements can be performed for more than four weeks, allowing perturbations of the circadian clock to be followed long-term in the same animals; (5) this is an automated process (microdialysis coupled with on-line HPLC analysis), which increases accuracy and bypasses the labor-intensive and error-prone manual handling of dialysis samples; and (6) our approach allows real-time investigation of circadian rhythm function and permits appropriate timely adjustments of experimental conditions. The longevity of microdialysis probes, the key to the success of this approach, depends at least in part on the methods of the construction and implantation of dialysis probes. In this article, we have detailed the procedures of construction and surgical implantation of microdialysis probes used currently in our laboratory, which are significantly improved from our previous methods.     

In vitro recovery of triamcinolone acetonide in microdialysis

The purpose of this study was to assess the factors affecting the calibration of the microdialysis probe for the in vitro recovery of triamcinolone acetonide (TA). Recoveries of TA were determined in microdialysis, retrodialysis, and no-net flux methods. Experiments were performed at room temperature or 37 degrees C while the reservoir medium was either stirred or unstirred. The effect of the viscosity of the medium on the recovery was studied using methylcellulose gel spiked with TA. Recovery was also calculated by the no-net-flux method in Ringer's solution and in plasma. Stirring the medium increased the recovery of TA by 30%. The recovery was higher at 37 degrees C under stirred or unstirred conditions and was same in either direction of dialysis. Increasing viscosity of the reservoir medium decreased the recovery (55% in Ringer's solution to 14% in 20% methylcellulose gel). Recovery from spiked plasma under stirred conditions was only 15% and this shift which was also seen in no-net-flux method was accounted for by the protein binding. Binding of TA, determined by ultrafiltration, was 20% in 5% gel and 81% in plasma. The recovery determined by the no-net-flux method was similar to the retrodialysis result. Stirring, temperature, viscosity and protein binding in the reservoir medium affected the in vitro recovery of TA.     

Brain microdialysis of isolation-reared mice under freely-moving conditions

Brain microdialysis has become a well established and a widely-used technique for in vivo measurement of extracellular levels of neurotransmitters. However, a single neurotransmitter in the perfusates has been measured in most studies. The present paper describes a simultaneous measurement of serotonin and dopamine release in the prefrontal cortex of mice. Using this technique, we examined the modulation by serotonin(1A) receptors of serotonin, dopamine, and noradrenaline release in the cortex of isolation-reared mice. The isolation-reared mouse is a useful model of complicated mood disorders including phenotypes of anxiety, depression, and aggression. The study shows that isolation rearing selectively decreases the sensitivity of serotonin(1A) receptors to increase dopamine release.     

Gentamicin enzyme-linked immunosorbent assay for microdialysis samples.

The authors investigated the use of a commercially available gentamicin enzyme-linked immunosorbent assay (ELISA) for the quantitative analysis of gentamicin concentrations in microdialysis samples. The assay demonstrated good accuracy and precision in the concentration range of 100 to 967 pg/mL. The developed quantitative ELISA is a highly sensitive and valid method for measuring gentamicin concentrations in microdialysis samples. This assay may be a useful alternative to other assay methodologies where analysis may be restricted by sample volume requirements and limited sensitivity.