Application of Microdialysis in Pharmacokinetic Studies

The objective of this review is to survey the recent literature regarding the various applications of microdialysis in pharmacokinetics. Microdialysis is a relatively new technique for sampling tissue extracellular fluid that is gaining popularity in pharmacokinetic and pharmacodynamic studies, both in experimental animals and humans. The first part of this review discusses various aspects of the technique with regard to its use in pharmacokinetic studies, such as: quantitation of the microdialysis probe relative recovery, interfacing the sampling technique with analytical instrumentation, and consideration of repeated procedures using the microdialysis probe. The remainder of the review is devoted to a survey of the recent literature concerning pharmacokinetic studies that apply the microdialysis sampling technique. While the majority of the pharmacokinetic studies that have utilized microdialysis have been done in the central nervous system, a growing number of applications are being found in a variety of peripheral tissue types, e.g. skin, muscle, adipose, eye, lung, liver, and blood, and these are considered as well. Given the rising interest in this technique, and the ongoing attempts to adapt it to pharmacokinetic studies, it is clear that microdialysis sampling will have an important place in studying drug disposition and metabolism.     

A review of membrane sampling from biological tissues with applications in pharmacokinetics, metabolism and pharmacodynamics.

This review provides an overview of membrane sampling techniques, microdialysis and ultrafiltration, and cites illustrations of their applications in pharmacokinetics, metabolism and/or pharmacodynamics. The review organizes applications by target tissue and general type of information gleaned. It focuses on recently published microdialysis studies (1999 to this writing) and offers the first review of ultrafiltration sampling studies. The advantages and limitations of using microdialysis and ultrafiltration sampling as tools for obtaining pharmacokinetic and metabolism data are discussed. Numerous examples are described including studies in which several types of data are collected simultaneously. Reports that study local metabolism of drug delivered through the probe are also presented.     

An automated blood sampler for simultaneous sampling of systemic blood and brain microdialysates for drug absorption, distribution, metabolism, and elimination studies

INTRODUCTION: A major problem in preclinical drug development where blood sampling from small animals is a routine practice is the time and labor involved in the serial sampling of small blood volumes from small animals such as rats for the duration of pharmacokinetic/pharmacodynamic (PK/PD) studies. The traditional method of manually drawing blood from the animal requires the animal to be anesthetized or restrained with some device, both of which cause stress to the animal. METHODS: An automated blood sampler (ABS) was developed to simultaneously collect blood and brain microdialysate samples at preprogrammed time points from awake and freely moving animals. The samples are delivered to fraction collectors and stored at 4 degrees C until use. The lost blood volume during collection is replaced with sterile saline to prevent fluid loss from the animal. In addition, the system is capable of collecting urine and feces for metabolism studies and monitoring the animal activity for behavioral studies. In the present study, blood samples were collected for 24 h after dosing rats orally with a 5 mg/kg dose of olanzapine (OLAN). Brain dialysates were collected for the same duration from a microdialysis probe implanted in the striatum. RESULTS: The pharmacokinetic parameters, obtained after an oral dose, are in good agreement with reported values in literature. The pharmacodynamic information obtained from brain dialysates data show that OLAN elevates the concentration of dopamine (DA) in the brain and remains in the brain even after it is cleared from the plasma. DISCUSSION: The ABS described here is a very useful tool in drug development to accelerate the pace of preclinical in vivo studies and to simultaneously provide pharmacodynamic and physiological information.     

Peritoneal microdialysis in freely moving rodents: an alternative to blood sampling for pharmacokinetic studies in the rat and the mouse.

By performing microdialysis in the peritoneal cavity, we studied the pharmacokinetics of Tramadol in awake, freely moving small laboratory animals. The systemic exposure to Tramadol was determined using both microdialysis sampling and collection of whole blood following a single intravenous injection (10 mg/kg) or a single oral dose (100 mg/kg) of Tramadol HCl. The sampling frequency of the dialysate was 10 min (mouse study) or 20 min (rat study). In rats and in mice, intraperitoneal microdialysis sampling gets reliable pharmacokinetic results without taking blood. The concentration-time curves obtained from peritoneal microdialysis were parallel to the concentration-time curves obtained from classical blood sampling. Accordingly, dose independent pharmacokinetic parameters were similar. A scaling factor, however, has to be introduced (e.g. peritoneal versus plasma AUC ratio) in order to obtain comparable pharmacokinetic results also with dose-dependent parameters. As there was no blood loss during the experiment, peritoneal microdialysis allowed the investigation of complete concentration-time curve profiles. Thus, the number of animals could be kept to a minimum. In conclusion, in vivo peritoneal microdialysis is a unique tool to obtain a complete set of free drug concentrations to determine reliable pharmacokinetic parameters from awake, freely moving rodents. Therefore, we suppose that the technique will have relevance for pharmacokinetic studies in future.     

An integrated microdialysis rat model for multiple pharmacokinetic/pharmacodynamic investigations of serotonergic agents

INTRODUCTION: Integrated in vivo models applying intracerebral microdialysis in conjunction with automated serial blood sampling in conscious, freely moving rodents are an attractive approach for pharmacokinetic (PK) and simultaneous pharmacokinetic/pharmacodynamic (PK/PD) investigations of CNS active drugs within the same animal. In this work, the ability to obtain and correlate data in this manner was evaluated for the selective serotonin (5-HT) reuptake inhibitor (SSRI) escitalopram. METHODS: An instrumented rat model equipped with an intracerebral hippocampal microdialysis probe and indwelling arterial and venous catheters was applied in the studies. Concomitant with brain microdialysis, serial blood sampling was conducted by means of an automated blood sampling device. The feasibility of the rat model for simultaneous PK/PD investigations was examined by monitoring plasma and brain extracellular concentrations of escitalopram along with SSRI-associated pharmacological activity, monitored as changes in brain 5-HT levels and plasma corticosterone levels. RESULTS: Combining intracerebral microdialysis and automated blood sampling did not cause any detectable physiological changes with respect to basal levels of plasma corticosterone or brain 5-HT levels. Furthermore, the PK of escitalopram in hippocampus following intravenous injection was not influenced by the presence of vascular catheters. Conversion of escitalopram dialysate concentrations into absolute extracellular levels by means of in vivo retrodialysis was verified by the no-net-flux method, which gave similar recovery estimates. The PK of escitalopram could be characterized simultaneously in plasma and the hippocampus of conscious, freely moving rats. Concomitantly, the modulatory and functional effects of escitalopram could be monitored as increases in brain 5-HT and plasma corticosterone levels following drug administration. DISCUSSION: The applicability of intracerebral microdialysis combined with arterial blood sampling was demonstrated for simultaneous PK/PD investigations of escitalopram in individual rats under non-stressful conditions. Together, these temporal relationships provide multiple PK/PD information in individual animals, hence minimizing inter-animal variation using a reduced number of animals.     

Determination and pharmacokinetics of ergometrine maleate in rabbit blood with on line microdialysis sampling and fluorescence detection

The study describes a flow injection on-line microdialysis system for in vivo monitoring of ergometrine maleate in rabbit blood with fluorescence detection. A flow-through microdialysis probe was used for intravenous sampling by pumping of the blood from the tested rabbit through the flow-through microdialysis probe located outside the living system at a flow rate of 15 microl min-1. The perfusion rate is 5 microl min-1. The ergometrine maleate in the dialysate was detected on-line with a flow injection fluorescence system after the ergometrine maleate administration (0.2 mg kg-1, i.v.). The dialysate sample volume was about 15 microl. The system was linearly related to the concentration of ergometrine maleate in the range 1-140 ng ml-1 (r=0.9989) with a detection limit 0.3 ng ml-1 (3sigma). The pharmacokinetic parameters of ergometrine maleate were calculated utilizing the pharmacokinetic software 'NDST-21' by a one-compartmental open model.     

Microdialysis in drug discovery

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. The development of microdialysis for the purpose of measuring drugs was initiated during the late eighties. This technique provides a means of continuous plasma sampling without repeated blood sampling and the applicability to the study of drug metabolism and pharmacokinetics in experimental animals and human. Also, the microdialysis technique allows the study of plasma protein binding and the saturation of protein binding. The implantation of the microdialysis probe in other tissues and organs, like central nervous system, adipose tissue and heart, allows the study of drug distribution. On the other hand, the measurement of endogenous substances using the microdialysis technique permits the study of the effect of drugs on neurotransmission and metabolism. Moreover, as this technique allows the simultaneous determination of different physiological parameters such as blood pressure, locomotor and convulsive activity, it is a suitable tool for pharmacokinetic-pharmacodynamic studies of drugs and pharmacokinetic-pharmacodynamic (PK-PD) modeling. Lastly, the reverse microdialysis is a powerful technique for the study of local actions of drugs in different tissues such as specific brain nuclei, myocardium, liver or skeletal muscle. So, this article reviewed the vast applications of the microdialysis technique for the study of pharmacokinetic and pharmacodynamic properties of drugs.     

Peripheral tissue microdialysis technique in unrestrained, conscious animals

The microdialysis procedure had been developed in the past 2 to 3 decades to determine levels of drug and endogenous compounds in several organs under physiological conditions. Advantages of microdialysis include: minimal stress on the experimental animals; studies may be done in unrestrained, conscious animals, and multiple determination can be made without concern for excess blood loss from small animals; and measurements can be made of drug and/or metabolites at multiple sites in the animal. By employing the microdialysis technique, I developed the method of multiple sampling for a long term period from an animal under the freely moving condition. In addition, I developed a novel microdialysis probe that was applied to several peripheral tissues and/or organs. In this paper, I will describe the fundamental procedure for peripheral tissues and/or organs such as the jugular vein and liver, using subcutaneous and ocular microdialysis sampling in unrestrained, conscious animals.     

Application of microdialysis to characterize drug disposition in tumors

Microdialysis is an in vivo sampling technique that was initially developed to measure endogenous substances in the field of neurotransmitter research. In the past decade, microdialysis has been increasingly applied to study the pharmacokinetics and drug metabolism in the blood and various tissues of both animals and humans. This paper describes the general aspects of this in vivo sampling technique followed by the survey of the recent papers regarding the application of microdialysis to characterize anticancer drug disposition in solid tumors. It can be concluded that microdialysis is a very suitable method to obtain drug concentration-time profiles in the interstitial fluid of solid tumors as well as of other variety of tissues.     

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.     

Microdialysis-perfusion sampling for the investigation of phenol metabolism

In vivo methods provide several advantages for the study of metabolism relative to the commonly used in vitro techniques. The integrity of the organism and actual physiological conditions are maintained to reflect more accurately the processes occurring on exposure to a xenobiotic compound. Experimental precision is improved because each animal serves as its own control and can be used to generate a complete pharmacokinetic experiment. This may result in the added benefit that fewer experimental animals will be needed for a metabolic investigation using in vivo techniques. The technique of microdialysis perfusion was characterized for the in vivo study of the hepatic metabolism of phenol and conjugation by glutathione. In this study, in vivo experiments were conducted by implanting a microdialysis probe into the intact, in-place liver of a killed rat. These results were compared to in vitro experiments using liver homogenate and liver-microsomal protein. Substantial differences were observed between the in situ experiments and those performed in vitro.     

Development of an intravenous microdialysis method for pharmacokinetic investigations in humans

INTRODUCTION: Limited blood volume is a major problem in pharmacokinetic investigations in specific populations, e.g. children. Intravenous microdialysis might help to obtain improved data sets as it is already successfully done in small animals. Since quantification of drugs is crucial in microdialysis, we developed an in vitro method to produce a workable intravenous microdialysis for human use. METHODS: A specifically designed microdialysis cell consisting of glass was heated to 37 degrees C. The cell was filled with Ringer's solution, plasma or whole blood. A microdialysis probe was inserted into the cell and perfused with Ringer's solution with addition of 4% dextran. The beta-receptor blocker sotalol served as a test drug. The stepwise in vitro evaluation process addressed issues of loss of dialysate, calibration by retrodialysis and relative recovery. These conditions were then applied in an in vivo pilot study to one single healthy volunteer after written informed consent. RESULTS: To address loss of perfusion fluid 4% of dextran was added and high and constant amounts of dialysate were achieved. To account for changes in the relative recovery a continuous use of retrodialysis by the calibrator atenolol was introduced. The recovery of atenolol was comparable to sotalol. The pharmacokinetic analysis revealed that sotalol concentrations from microdialysates were not different from conventional plasma samples (100+/-11%, n=33) resulting in subsequent comparable pharmacokinetic parameters. DISCUSSION: This stepwise approach using an in vitro device enabled us to demonstrate the determination of pharmacokinetic parameters of sotalol. The most important evaluation step is represented by the continuous use of retrodialysis by the calibrator atenolol because it can account for changes in the relative recovery of the drug. This approach should be a starting point to simplify pharmacokinetic studies in special populations, e.g. in small children, to improve drug treatment.     

Methodological issues in microdialysis sampling for pharmacokinetic studies

Microdialysis is an in vivo technique that permits monitoring of local concentrations of drugs and metabolites at specific sites in the body. Microdialysis has several characteristics, which makes it an attractive tool for pharmacokinetic research. About a decade ago the microdialysis technique entered the field of pharmacokinetic research, in the brain, and later also in peripheral tissues and blood. Within this period much has been learned on the proper use of this technique. Today, it has outgrown its child diseases and its potentials and limitations have become more or less well defined. As microdialysis is a delicate technique for which experimental factors appear to be critical with respect to the validity of the experimental outcomes, several factors should be considered. These include the probe; the perfusion solution; post-surgery interval in relation to surgical trauma, tissue integrity and repeated experiments; the analysis of microdialysate samples; and the quantification of microdialysate data. Provided that experimental conditions are optimized to give valid and quantitative results, microdialysis can provide numerous data points from a relatively small number of individual animals to determine detailed pharmacokinetic information. An example of one of the added values of this technique compared with other in vivo pharmacokinetic techniques, is that microdialysis reflects free concentrations in tissues and plasma. This gives the opportunity to assess information on drug transport equilibration across membranes such as the blood-brain barrier, which already has provided new insights. With the progress of analytical methodology, especially with respect to low volume/low concentration measurements and simultaneous measurement of multiple compounds, the applications and importance of the microdialysis technique in pharmacokinetic research will continue to increase.     

Microdialysis in peripheral tissues

The objective of this review is to survey the recent literature regarding the applications of microdialysis in pharmacokinetic studies and facilitating many other studies in peripheral tissues such as muscle, subcutaneous adipose tissue, heart, lung, etc. It has been reported extensively that microdialysis is a useful technique for monitoring free concentrations of compounds in extracellular fluid (ECF), and it is gaining popularity in pharmacokinetic and pharmacodynamic studies, both in experimental animals and humans. The first part of this review discusses the use of microdialysis technique for ECF sampling in peripheral tissues in animal studies. The second part of the review describes the use of microdialysis for ECF sampling in peripheral tissues in human studies. Microdialysis has been applied extensively to measure both endogenous and exogenous compounds in ECF. Of particular benefit is the fact that microdialysis measures the unbound concentrations in the peripheral tissue fluid which have been shown to be responsible for the pharmacological effects. With the increasing number of applications of microdialysis, it is obvious that this method will have an important place in studying drug pharmacokinetics and pharmacodynamics.     

The Design and Validation of a Novel Intravenous Microdialysis Probe: Application to Fluconazole Pharmacokinetics in the Freely-Moving Rat Model

Purpose. The purpose of this study was to design and validate a concentric, flexible intravenous microdialysis probe to determine drug concentrations in blood from the inferior vena cava of a freely-moving animal model. Methods. An intravenous microdialysis probe was constructed using fused-silica tubing and an acrylonitrile/sodium methallyl sulfonate copolymer hollow fiber. The probe was tested in vitro for the recovery of fluconazole and UK-54,373, a fluconazole analog used for probe calibration by retrodialysis. Subsequent in vivo validation was done in rats (n = 7) that had a microdialysis probe inserted into the inferior vena cava via the femoral vein, and the femoral artery was cannulated for simultaneous blood sampling. Comparisons of fluconazole pharmacokinetic parameters resulting from the two sampling methods were performed at 2 and 10 days after probe implantation. Results. There were no statistical differences between the microdialysis sampling and conventional blood sampling methods for the T_1/2, Cl, Vdss, and dose-normalized AUC by paired t-test (p > 0.05) for repeated dosing at day 2 and day 10 after probe placement. The probe recovery, as determined by retrodialysis, significantly decreased over the ten day period. This finding indicates the necessity for frequent recovery determinations during a long-term blood microdialysis experiment. Conclusions. These results show that microdialysis sampling in the inferior vena cava using this unique and robust probe design provides an accurate method of determining blood pharmacokinetics in the freely-moving rat for extended experimental periods. The probe design allows for a simple surgical placement into the inferior vena cava which results in a more stable animal preparation for long-term sampling and repeated-measures experimental designs.     

Evaluation of in vivo and in vitro recovery rate of anatoxin-a through the microdialysis probe

In vivo microdialysis is a versatile sampling technique commonly employed to observe changes in neurotransmitters levels that occur in response to different treatments, being these treatments administered through a microdialysis probe implanted into a specific brain region in living animals. In previous works we have used this technique to study the effects of the drug anatoxin-a, a nicotinic acetylcholine receptor agonist, on dopamine release in striatum. The aim of the present study was to assess the recovery of anatoxin-a through the microdialysis probe. This information allows knowing the exact amount of the drug crossing the microdialysis membrane, acting on extracellular tissue. High Performance Liquid Chromatography (HPLC) with Fluorescence Detection (FLD) has been used for the analysis of anatoxin-a. We observed that the recovery of anatoxin-a was about 0.5%. Under our experimental conditions, the results suggest that anatoxin-a can be used as an important tool in the study of neuronal nicotinic receptors by in vivo microdialysis technique and also show a reliable estimation of the anatoxin-a recovery through the microdialysis probe under both in vivo and in vitro conditions.     

In Vivo Microdialysis Sampling for Pharmacokinetic Investigations

In vivo microdialysis sampling coupled to liquid chromatography was used to study acetaminophen disposition in anesthetized rats. The pharmacokinetics of acetaminophen and its sulfate and glucuronide metabolites were determined using both microdialysis sampling and collection of whole blood. For microdialysis, samples were continuously collected for over 5 hr without fluid loss using a single experimental animal. Microdialysis sampling directly assesses the free drug concentration in blood. The pharmacokinetic results obtained with microdialysis sampling were the same as those obtained from blood collection. The administration of heparin, necessary when collecting blood samples, was found to double the elimination half-life of acetaminophen. Microdialysis sampling is a powerful tool for pharmacokinetic studies, providing accurate and precise pharmacokinetic data.     

Pharmacokinetic study of ketoprofen isopropyl ester-loaded lipid microspheres in rat blood using microdialysis

A blood microdialysis technique coupled with high-performance liquid chromatography was used to investigate the pharmacokinetics of unbound ketoprofen in rats after intravenous administration of a lipid-soluble ketoprofen derivate, ketoprofen isopropyl ester (KPI), loaded into lipid microspheres (LM) and ketoprofen solution. A microdialysis probe was inserted into the jugular vein of male Wistar rats. KPI-loaded LM or ketoprofen solution (24 mg/kg, i.v.) was then administrated via a femoral vein. Dialysate samples were analyzed using HPLC. The in vitro and in vivo recovery rate of the microdialysis probe was 30.42+/-0.74% (n=3) and 40.27+/-2.74% (n=3), respectively. The pharmacokinetic parameters for ketoprofen after intravenous administration of KPI-loaded LM and ketoprofen solution exhibited no statistically significant differences. The results of this pharmacokinetic study indicate that the microdialysis technique can be widely applicable to investigations of in vivo free-drug of microcarrier systems.     

The development of multiple probe microdialysis sampling in the stomach

A multiple probe approach of implanting microdialysis probes into each separate tissue layer would better represent sampling from the stomach. Presently, microdialysis sampling experiments are performed with only a single probe in the submucosa to represent sampling from the stomach tissue. The focus of this research was to develop a four-probe microdialysis sampling design to simultaneously monitor the stomach lumen, mucosa, submucosa and in the blood of the rat. Due to the small outer diameter of the microdialysis probe (350mum), implantation into each separate layer was achieved with confirmation of probe location from histological examination. To assess the significance of sampling by this approach, multiple probe microdialysis sampling was used to monitor drug absorption in the stomach. Salicylic acid, caffeine and metoprolol were individually dosed to the ligated stomach. Analysis of the dialysate samples was performed by HPLC-UV and concentration-time curves and pharmacokinetics analysis were used to determine differences between the different probe locations.     

血液微渗析技术研究酮洛芬在大鼠体内的药代动力学

目的考察酮洛芬微渗析体内外回收率及影响因素，研究酮洛芬静脉给药后非结合型药物在大鼠体内的药代动力学。方法大鼠颈静脉插入探针后，依次用不同浓度的灌注液对探针进行灌注，测定酮洛芬体内回收率及非结合型酮洛芬在大鼠体内的药代动力学。以高效液相色谱法测定微渗析液中药物浓度。体外回收率的测定采用浓差法。结果增量法及减量法测定的回收率一致。以浓差法测定的体外回收率为28.75%；反渗析法测定体内回收率为(40.3±2.7)%。酮洛芬静脉给药后非结合型药物的T_1/2，AUC和CL分别为(181±16) min，(112±27) μg·min·mL^-1和(0.22±0.05) min^-1。结论血液微渗析技术可用于研究非结合型酮洛芬在大鼠体内的药代动力学。