Methodological issues in microdialysis sampling for pharmacokinetic studies

Advanced Drug Delivery Reviews 45 (2000) 125–148

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Methodological issues in microdialysis sampling for pharmacokinetic studies Elizabeth C.M. de Lange*, A.G. de Boer, Douwe D. Breimer Leiden /Amsterdam Center for Drug Research, Division of Pharmacology, Sylvius Laboratory, University of Leiden, P.O. Box 9503, 2300 RA Leiden, The Netherlands Received 28 January 2000; accepted 10 August 2000

Abstract 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.  2000 Elsevier Science B.V. All rights reserved. Keywords: Review; Microdialysis; Methodology; Pharmacokinetics; Drugs; Blood–brain barrier

Abbreviations: A, Membrane area or surface; Cdial , Concentration in the dialysate; CECF ,Bulk extracellular fluid concentration outside the microdialysis probe; Cin , Concentration in the perfusate; De , Diffusion coefficient in extracellular; Deff , Effective diffusion coefficient in the brain; Dp , Diffusion coefficient in the perfusate; ECF, Extracellular fluid; EEG, Electro-encephalogram; F, Flow rate of the perfusate; k ep , Rate constant for extracellular-microvascular exchange; k em , Rate constant for irreversible extracellular metabolism; k e:im , Composite rate constant for irreversible intracellular metabolism and intracellular-extracellular exchange; K0 , Mass transfer coefficient over the dialysis membrane; r, Radius of the dialysis probe; R, In vivo recovery; R vitro , In vitro recovery; R e , Resistance of external medium to mass transport; R m , Resistance of membrane to mass transport; R p , Resistance of perfusate to mass transport; t, Time; t*, Time, normalized with respect to T; T, Time, expressed as r 2 /Deff . *Corresponding author. Tel.: 131-71-527-6330 / 6211; fax: 131-71-527-6292. E-mail address: [email protected] (E.C.M. de Lange). 0169-409X / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0169-409X( 00 )00107-1

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Contents 1. Introduction ............................................................................................................................................................................ 1.1. The principle of microdialysis ........................................................................................................................................... 1.2. The microdialysis experimental setup ................................................................................................................................ 1.3. Recovery / extraction efficiency ......................................................................................................................................... 1.4. Advantages and limitations ............................................................................................................................................... 2. Methodological aspects of microdialysis ................................................................................................................................... 2.1. Experimental variables ..................................................................................................................................................... 2.1.1. Probe geometry and materials ................................................................................................................................. 2.1.2. Composition and temperature of the perfusate .......................................................................................................... 2.1.3. Tubing connections ................................................................................................................................................ 2.1.4. Flow rate of perfusion and semipermeable membrane surface.................................................................................... 2.2. Tissue integrity after implantation of the probe................................................................................................................... 2.2.1. Factors that influence tissue integrity ....................................................................................................................... 2.2.2. BBB integrity measured with intracerebral microdialysis .......................................................................................... 2.2.3. Continuous sampling / repeated experiments ............................................................................................................. 2.3. Analytical considerations .................................................................................................................................................. 2.3.1. In general .............................................................................................................................................................. 2.3.2. The sample ............................................................................................................................................................ 2.3.3. Detection............................................................................................................................................................... 2.4. Factors that influence recovery .......................................................................................................................................... 2.4.1. Absolute and relative (concentration) recovery ......................................................................................................... 2.4.2. In vitro recovery .................................................................................................................................................... 2.4.3. In vivo recovery..................................................................................................................................................... 2.4.4. Some methods to determine in vivo recovery; quantification of microdialysis data ...................................................... 2.4.4.1. The no-net-flux methodologies ................................................................................................................... 2.4.4.2. Internal standard / retrodialysis / reverse dialysis methods............................................................................... 2.4.4.3. Importance of quantification, an example using genetically modified animals................................................. 2.5. Considerations in data analysis.......................................................................................................................................... 3. The added value of microdialysis in pharmacokinetic studies...................................................................................................... 3.1. Membrane transport processes .......................................................................................................................................... 3.2. In vivo synthesis rates, ECF concentrations, distribution ..................................................................................................... 3.3. Combined measurement of concentration and effect ........................................................................................................... 4. Conclusions ............................................................................................................................................................................ References ..................................................................................................................................................................................

1. Introduction

1.1. The principle of microdialysis Microdialysis involves the insertion of a microdialysis probe into a selected tissue or (body) fluid. The probe consists of a small semipermeable hollow fiber membrane, connected to an inlet and outlet tubing with a small diameter. The probe is continuously perfused with a physiological solution, the perfusate. The perfusate is an aqueous solution that must closely match the (ionic) composition of the (extracellular) fluid surrounding the probe in order to prevent unwanted changes in composition of periprobe fluid due to drainage or introduction of molecules. Molecules able to pass the semipermeable

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membrane will diffuse over the membrane down their concentration gradient into or out of perfusate. The solution that exits the probe, the dialysate, can be collected for analysis (Fig. 1). Any analytical technique can be used for microdialysate samples as long as it is able to deal with the typical small sample volumes and often low concentrations. The concentrations of the drug in the dialysate reflect the concentrations in the (extracellular) fluid around the semipermeable part of the probe. However, as the dialysis procedure is not performed under equilibrium conditions, the concentration in the dialysate will be different from that in the periprobe fluid. The term recovery is used to describe this relationship and should be determined by a suitable method for quantification of microdialysis data.

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Fig. 1. The basic principle of microdialysis. A microdialysis probe is inserted into a selected tissue. The probe is perfused with a physiological medium. By means of diffusion down their concentration-gradient, molecules small enough to cross the semipermeable membrane of the probe will enter the probe lumen, and will be taken with the perfusion flow. The resulting dialysate can be analyzed ex-vivo by an appropriate analytical technique.

1.2. The microdialysis experimental setup A basic microdialysis set-up consists of a microdialysis probe, a subject (an animal or human), a perfusion pump, inlet and outlet tubing, and a (refridgerated) microfraction collector. The microdialysis probe can be ‘‘home-made’’ or purchased commercially. The perfusion pump should be able to provide an exact and pulse-free flow rate in the nl / min and ml / min range, while the microfraction collector should be able to collect volumes exactly according to pre-set volumes or pre-set time. Perfusate (inlet) tubing, the microdialysis probe, and dialysate (outlet) tubing should not interact with the drug. The length and inner diameter of the outlet

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tubing should be considered to minimize mixing of the dialysate and to prevent hydrostatic pressure build-up across the probe membrane. A syringe selector, an in vitro stand for the probes, swivels for inlet and outlet connection tubing, and an on-line analysis system can extend the equipment. A syringe selector accomplishes a change of perfusate syringes without interrupting the flow. An in vitro stand is useful for the safe storage of reusable probes and for testing in vitro recovery and may be ‘‘homemade’’ as well as commercially purchased. Swivels can be used to prevent tangling and twisting of the inlet and outlet tubing by the freely moving animal. An on-line injector enables direct collection and injection of the microdialysate when the analysis can be performed directly, for example by high-pressure liquid chromatography (HPLC) and an appropriate detection method. An example of an on-line experimental set-up is given in (Fig. 2).

1.3. Recovery /extraction efficiency The term recovery describes the relation between concentrations of the drug in the periprobe fluid and those in the dialysate. These concentrations will differ from each other in case of a constant flow of the perfusate by which concentration equilibrium will never be reached. In vitro, a number of parameters influencing recovery can be investigated. These

Fig. 2. This is an example of an on-line microdialysis experimental setup. An infusion pump pumps the physiological solution via inlet tubing through the probe. The probe is present in the brain of the animal The outlet tubing guides the dialysate into a loop of an HPLC system in which the sample can be collected during the sample interval. At the end of each sample interval the loop sample is loaded onto an HPLC column. The sample is separated, and subsequently the detector measures the content of the drug in each sample.

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parameters include temperature, perfusate composition, perfusate flow rate, characteristics of the semipermeable membrane, probe geometry, surface of the semipermeable membrane, and the characteristics of the drug. Also the diffusion of the drug through the periprobe fluid may influence recovery. In vivo, however, effective diffusion of the drug through the extracellular fluid of a tissue, will also be affected by uptake into cells, metabolic conversion rate, active transport across membranes, extent of tissue vascularization and blood flow. Special quantification methods are needed to determine the actual relation between dialysate concentrations and those in the extracellular fluid.

ly, lipophilic drugs sticking to tubing and probe components, thereby complicating the relation between dialysate and extracellular concentrations. At last, most importantly, a disadvantage of the technique is the need to determine in vivo recovery of the drug to calculate true concentrations in the extracellular fluid of the surrounding tissue. This may be time-consuming and partly counteract the advantage of the decrease of the number of subjects needed.

2. Methodological aspects of microdialysis

2.1. Experimental variables 1.4. Advantages and limitations Microdialysis has a number of advantages. With this technique, sampling can be performed continuously without fluid loss. Thereby, one can obtain high-resolution concentration profiles of drugs and metabolites from (freely moving) individual subjects. This reduces the number of subjects needed for pharmacokinetic investigations. Then, the probe is present at a certain location within the selected tissue whereby dialysate concentrations will reflect extracellular concentrations in a distinct region. With the dialysis principle providing protein free samples, which may be of special value from a pharmacological point of view, potential ex vivo enzymatic degradation is eliminated and clean up procedures for analysis will not be needed. Moreover, ex vivo analysis of the dialysate samples permits the measurement of drug concentrations by virtually every analytical technique able to deal with the small dialysate volumes, which contributes to the selectivity and sensitivity. Also, disadvantages in the use of the technique exists. Implantation of the probe will elicit tissue reactions that may interfere with the system under investigation. Therefore, the valid use of the technique should be investigated for each application. Then, the diluting effect of the dialysis procedure leads to lower concentration samples, which requires sensitive analytical methods. Increase of sensitivity of analytical methods in microdialysis will therefore lead to an increase of the possible applications of the technique. Another problem is associated with, most-

2.1.1. Probe geometry and materials Microdialysis probes are available or can be made with much different geometry, to be chosen on the basis of its possible use in virtually any tissue or fluid of the body and its surgical accessibility. In general, probes will either have a longitudinal, a semicircular or an I-shaped design. For soft peripheral tissues like muscle, skin, liver, tumor, and fluids like blood and bile, flexible probes can be used. Intracerebral probes can be rigid, as these can be fixed onto the surface of the skull. In the latter case, also guide cannulas can be implanted which opens up the possibility to insert the probe itself after surgical recovery, thereby decreasing effects of anesthesia. Obviously, probes that can be removed and re-implanted have advantages for chronic studies. Examples of probe designs for different applications are shown in Fig. 3. Probes can be ‘‘homemade’’ to suit specific research needs [1–16]. Probes can also be purchased commercially such as I-shaped probes and guides (CMA microdialysis AB, Stockholm Sweden; Europhor, Toulouse, France; Bio Analytical Systems, West Lafayette, IN, USA). For example, the procedure of making a homemade probe suited for horizontal introduction in the brain is given in Fig. 4. Dialysis probes are made of various materials which differ with respect to inner and outer diameters (150–500 mm, composition (for example celluloses and copolymers like polyacetonitrile / sodium methallyl sulfonate and polycarbonate / ether), molecular mass cut off (5–50 kDa), inertness and per-

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Fig. 3. Different types of probes as used in intracerebral microdialysis experiments.(a) longitudinal; or horizontal probe, (b) I-shaped probe, with the possibility to be removed if a guide cannula has been introduced is present and (c) a semicircular probe.

Fig. 4. Fabrication procedure for a longitudinal probe, suitable for horizontal introduction into the cortical brain of the rat or mouse [16,88].

meability to solutes [2,7,17–23]. The choice of the membrane type is an essential element in searching the optimal probe for a particular application. A wide range of kidney dialysis fibers has been employed for

this purpose (Table 1). It is of importance that the membrane (but also other material like tubing connections etc) do not interact with the drug, as this may clearly affect the dialysis concentration in

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Table 1 Microdialysis membranes (from Levine and Powell [18] with kind permission from Academic Press Inc.) Membrane materials

O.D. (mm)

M.W. cutoff

Cellulose Cellulose Cellulose Copolymer (polyacetronitrile / sodium methallyl sulfonate) Copolymer (polycarbonate / polyether) Acrylic copolymer Polysulfone

0.20 0.25 0.32 0.31 0.50 0.30 0.30

6000 5000 5000 15 000 20 000 50 000 100 000

response to true periprobe fluid concentrations (Fig. 5); [24,25].

2.1.2. Composition and temperature of the perfusate Perfusion media used in microdialysis experiments vary widely in composition and pH [19]; Table 2). Ideally the composition, ion strength, osmotic value and pH of the perfusion solution should be as close as possible to those of the extracellular fluid of the dialyzed tissue. Extracellular fluids mostly contain only very small concentrations of proteins. But, in some cases, proteins have been added to the perfusion medium to prevent sticking of the drug to the microdialysis probe and tubing connections [18,21,26]. Also, the composition of the perfusate can be changed with the intention to study the effects on the system under investigation. Deviations in ion composition have been shown to affect brain

dialysate levels of neurotransmitters [27–29]. and drugs [16].The latter investigators have shown that a non-physiological (hypotonic) perfusion medium used in daily repeated experiments to measure blood–brain barrier permeability resulted in a substantial increase of the dialysate levels of the hydrophilic drug atenolol with days, presumably reflecting increased blood–brain barrier permeability. Most investigators use a perfusion medium at room temperature before entering the probe. As a result a temperature gradient exists between the probe and its environment. This may have an effect on tissue processes and consequently on the results. De Lange et al. [16] used a subcutaneous cannula by which the perfusion fluid could equilibrate to body temperature before entering the cortical brain probe. The results on the area under the concentration–time curve values for acetaminophen following intravenous administration obtained with ‘‘prewarmed’’ iso-

Fig. 5. Differences in dialysate serotonin (5-HT) concentration with the use of different probe membranes. The GF membrane has a rapid response to changes in perfusion medium, as shown by the almost instantaneous equilibrium in dialysate outcomes, while the HOSPAL membrane gives results that take much more time to reach equilibrium. By the use of the HOSPAL membrane quick changes in serotonin concentrations cannot be measured [25].

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Table 2 Different compositions of perfusion medium, in mM unless stated otherwise (From Benveniste and Huttemeier [19]. With kind permission by Pergamon Press Ltd., Oxford, UK) Distilled water Saline: 0.9% NaCl Saline: 0.9% NaCl; 1.85% CaCl 2 (pH 7.2) Saline: 0.9% NaCl; 0.5% bovine serum albumin Ringers solution 134 NaCl; 5.9 KCl; 1.3 CaCl 2 ; 1.2 MgCl 2 (O 2 -sat.) 147 NaCl; 2.4 CaCl 2 ; 4 KCl (pH 6.0) 147 NaCl; 3.4 CaCl 2 ; 4 KCl (pH 6.1) 147 NaCl; 1.3 CaCl 2 ; 4 KCl (pH 7.2) 155 NaCl; 2.3 CaCl 2 ; 5.5 KCl 189 NaCl; 3.4 CaCl 2 ; 3.9 KCl Modified Ringers solution 145 NaCl; 1.2 CaCl 2 ; 2.7 KCl, 1 MgCl 2 ; 0.2 ascorbate (pH 7.4) Buffered Ringers solution 147 NaCl; 3.4 CaCl 2 ; 2.8 KCl, 1.2 MgCl 2 ; 0.6 K 2 HPO 4 ; 114 mM ascorbate (pH 6.9) Krebs Ringer solution 147 NaCl; 3.4 CaCl 2 ; 4.0 KCl (pH 6.0) 138 NaCl; 1 CaCl 2 ; 5 KCl; 1 MgCl 2 ; 11 NaHCO 3 ; 1 NaHPO 4 , 11 glucose (pH 7.5) Krebs Ringer bicarbonate 122 NaCl; 1.2 CaCl 2 ; 3 KCl, 1.2 MgSO 4 ; 25 NaHCO 3 ; 0.4 KH 2 PO 4 , (pH 7.4) Krebs-Henseleit bicarbonate buffer 118 NaCl; 2.5 CaCl 2 ; 4.7 KCl, 0.6 MgSO 4 ; 25 NaHCO 3 ; 1.2 NaH 2 PO 4 , 11 glucose Mock-cerebrospinal fluids 120 NaCl; 1.5 CaCl 2 ; 5 KCl; 15 NaHCO 3 ; 1.0 MgSO 4 ; 6 glucose (pH 7.4) 127 NaCl; 1.1 CaCl 2 ; 2.4 KCl; 0.85 MgCl 2 ; 28 NaHCO 3 ; 0.5 KH 2 PO 4 , 0.5 Na 2 SO 4 ; 5.9 glucose (pH 7.5) 127 NaCl; 1.3 CaCl 2 ; 2.5 KCl; 0.9 MgCl 2 ; (pH 6.0) 127 NaCl; 1.3 CaCl 2 ; 2.5 KCl; 2 133 NaCl; 3.0 KCl; 24.6 NaHCO 3 ; 6.7 urea; 3.7 glucose 135 NaCl; 1.2 CaCl 2 ; 3.0 KCl; 1.0 MgCl 2 ; 2.0 phosphate (pH 7.4) 140 NaCl; 2.5 CaCl 2 ; 3.0 KCl; 1 MgCl 2 ; 1.2 NaHPO 4 , 0.27 NaH 2 PO 4 ; 7.2 glucose (pH 7.4) 150 NaCl; 1.7 CaCl 2 ; 3 KCl; 0.9 MgCl 2 ; 120 NaCl; 1.3 CaCl 2 ; 4.8 KCl; 1.2 MgSO 4 ; 1.6 NaHPO 4 , 1.2 KH 2 PO 4 , (pH 7.2)

tonic and hypotonic perfusate were compared to those obtained with perfusate at room temperature. A temperature effect was observed only for the use of the hypotonic perfusate, with a two-fold higher dialysate area under the concentration-time curve value obtained with the room temperature perfusion medium (Fig. 6). It was hypothesized that the periprobe tissue, already ‘‘stressed’’ by the hypotonic condition, loses its capability to compensate temperature effects. This indicates that perfusate tem-

perature may be especially important in pathological circumstances. However, it is recommended to perform all microdialysis experiments with perfusion fluids at body temperature.

2.1.3. Tubing connections Tubing connections should ideally have no interaction with the drug as this may have a profound effect on the relation between the concentration of the drug found in the dialysate and the periprobe

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Fig. 6. Influence of the perfusate temperature (248 versus 388C) on AUCbrain ECF values of acetaminophen as obtained from the rat cortex with the isotonic (iso) and hypotonic (hypo) perfusate at 24 h after implantation of the probe. (From De Lange et al. [16] with kind permission of Elsevier Science-NL, Sara Burghartstraat 25, 1055 KV, Amsterdam, The Netherlands).

fluid concentrations, similar to effects of interaction with the probe membrane (Fig. 3). The inner diameters of the tubing connections may be of importance with respect to build-up of fluid pressure and thereby fluid loss over the semipermeable membrane. This may be prevented by using inlet tubing (from perfusion pump to the probe) with an inner diameter smaller than that of the probe itself, and an outlet tubing (from probe to collection site) with an inner diameter being larger than that of the probe.

2.1.4. Flow rate of perfusion and semipermeable membrane surface Flow rate of perfusion and sample interval will both determine sample size, temporal resolution, but will also affect recovery (Fig. 7). Typical perfusion flow-rates applied in microdialysis experiments today range between 0.1 and 5 ml / min. The tendency is to use lower flow-rates as this may increase recovery, provided that an analytical technique is available to deal with the smaller sized samples. The semipermeable membrane is generally chosen as long as possible, typically between 1 and 10 mm, as this will increase recovery, provided that the target tissue is homogeneous with respect to the system under investigation.

Fig. 7. Relationship between flow-rate and relative or absolute recovery.

2.2. Tissue integrity after implantation of the probe 2.2.1. Factors that influence tissue integrity Although the small size of the probe, mostly 300 mm O.D., causes minimal disturbances within the tissue, implantation of a probe is invasive and effects hereof on the system under investigation should be determined in order to prevent the possibility of generating erroneous results. Peripheral tissues including dermis, muscle, tumor, and liver have been examined on their response to probe implantation [30–32]. Initial infiltration of neutrophils was found, followed by macrophages, for dermis, muscle and liver. For tumors, little or no inflammatory response was found up to 72 h after probe implantation. In the evaluation of tissue trauma following intracerebral implantation of a microdialysis probe, initial formation of eicosanoids, local disturbances in cerebral blood flow and glucose metabolism were present [33,34]. These changes were more or less normalized 1 day after surgery. Histological evaluation revealed that glial reactions (gliosis) usually started 2 or 3 days after implantation of the probe. Reactions were confined mainly to a very small region around the probe [35–40]. Perfusion as such also had an effect on the

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Fig. 8. Mean tissue scores of individual parameters, as the sum of 3 zones around the probe, for daily repeated perfusion periods of 2.5 h at post surgery intervals of: 1(24 h); 1 1 (48 h); 1 1 1 (72 h); and 1 1 1 1 (96 h) all with perfusion of the probe, as well as the unperfused control with the probe present for 96 h (22 2 2). (From De Lange et al. [16] with kind permission of Elsevier Science-NL, Sara Burghartstraat 25, 1055 KV, Amsterdam, The Netherlands).

course of tissue reactions (Fig. 8, [40]). These results indicate that the optimal period to perform (repeated) intracerebral microdialysis experiments in a particular experimental design must be determined [16,40,41]. In general, the optimal post-surgery interval lies between 1 and 2 days after implantation of the probe; after recovery from early tissue reactions, and before the start of long-term reactions. With respect to anesthesia the most relevant studies are obtained when experiments are performed in the conscious state because the use of anesthetics may interfer with physiological processes [42]. To reduce the effects of anesthesia, a guide cannula can be used through which the probe can be inserted after recovery from surgery [43,44].

2.2.2. BBB integrity measured with intracerebral microdialysis The majority of applications of microdialysis thusfar include the brain. In pharmacokinetic studies on the distribution of drugs into the brain, transport

across the blood–brain barrier is an essential factor. With the introduction of a microdialysis probe it is important that the blood–brain barrier is intact at time of measurement. A good relationship between blood–brain barrier permeability and log P/(MW ) CHR.4.17; (the logarithm of the partition coefficient over octanol and physiological buffer (pH 5 7.4) divided by the square root of molecular mass), exists for many drugs that cross the blood–brain barrier by passive diffusion [45]. Such transport across the blood–brain barrier occurs via the paracellular and / or the transcellular route. Hydrophilic drugs can only diffuse paracellularly and are therefore restricted in transport across the blood–brain barrier by the presence of the tight junctions between the brain endothelial cells. More lipophilic drugs can diffuse transcellularly as well, and may for that reason exhibit a more pronounced distribution into the brain [45–47]. De Lange et al. [16,48,49] found that this relation between lipophilicity and extent of blood–brain

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barrier transport was also reflected by the microdialysis technique, indicating blood–brain barrier integrity. First, blood–brain barrier transport was considerably higher for intravenously administered acetaminophen (18%) then for atenolol (3.4%), being a moderately lipophilic and hydrophilic drug respectively [16]. Second, for intravenously administered atenolol, blood–brain barrier transport was considerably increased after blood–brain barrier opening by intracarotid injection of hypertonic mannitol. This was as expected on the basis of increased paracellular diffusion through the enlarged spaces between the tight junctions of the blood–brain barrier upon hyperosmolar opening [48]. Third, when the above-mentioned drugs were administered by constant local ‘‘infusion’’ microdialysis probe and resulting concentrations at different interprobe distances were measured by a ‘‘detection’’ probe, spatial concentration gradients at steady-state could be determined. Thereby, the ratio of the transfer coefficient to blood / the effective diffusion coefficient in extracellular fluid (k bp /Deff ) could be estimated. This value was substantially higher for acetaminophen compared with atenolol. As the drugs have comparable values of Deff , the higher value found for acetaminophen was indicative of higher blood–brain barrier transport of this moderately lipophilic drug [49].

2.2.3. Continuous sampling /repeated experiments Microdialysis allows continuous sampling, whereby data can be obtained during the entire course of drug administration and elimination within individual subjects. This gives the possibility to reduce the number of subjects needed, compared with methods that involve postmortem analysis at several single time points after drug administration, in which each animal provides only a single time point. Moreover, since no fluid is removed during sampling, continuous sampling with high temporal resolution makes microdialysis a powerful tool for the determination of pharmacokinetic parameters of drugs. Furthermore, since protein-bound molecules cannot pass through the membrane, only the extracellular ‘‘free’’ drug is measured. This may provide important pharmacokinetic information for drugs that exert their action via interaction of the free drug with receptors in the extracellular space.

Continuous sampling as well as repetitive experiments for longitudinal or cross-over studies will only be of value if tissue characteristics will not change significantly upon probe presence and / or perfusion, as has been discussed above. Histological and functional studies on the repeatability of intracerebral microdialysis experiments with respect to blood– brain barrier transport has been performed by De Lange et al. [16,40] Perfusion was performed on 1, 2, 3 or 4 consecutive days and compared with no perfusion for 4 days. Histological findings indicated no important histological changes in periprobe tissue, up till 2 days after implantation of the probe in combination with two consecutive experiments performed, or with the ‘‘silent’’ presence of the probe for 4 days. At 3 and 4 days of consecutive experiments tissue reactions became significant [40]. In terms of blood–brain barrier transport, microdialysis results remained reproducible at 2 consecutive days for acetaminophen and 3 consecutive days for atenolol, and changed thereafter. This indicates that repeated blood–brain barrier transport measurements are feasible within a certain time frame [16] It should be realized, however, that this was only the case when microdialysis experiments were performed under carefully controlled conditions. Another part of this study focussed on the effects of a nonphysiological perfusion medium, which resulted in a significant increase of dialysate concentrations of atenolol within the course of three consecutive days, indicating changes in blood–brain barrier permeability upon ‘‘bad’’ microdialysis experimental conditions [16].

2.3. Analytical considerations 2.3.1. In general The reliable use of intracerebral microdialysis greatly depends on and is limited by the sensitivity of the assay method to analyze the drug in microdialysis samples. Such methods should be able to handle small volume samples (nano- to microliter range) which often contain very low concentrations (pico- to nanomolar range). So progress in the area of microanalysis will shift further the frontiers of microdialysis applications. The measurement of hydrophilic drugs is in general more straightforward than that of lipophilic drugs. With the latter there is a

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higher chance to encounter problems like the drug sticking to tubing and / or probe and / or parts of the analytical device. For studies on such drugs a steadystate approach will be the most appropriate. Moreover, lipophilic drugs will probably prefer the intracellular compartment, which leads to very low concentrations in the extracellular fluid and even lower concentrations in the dialysate. But, on the other hand, the concentrations of lipophilic drugs will often change less rapidly than will hydrophilic drugs, and in such cases longer sample intervals can be used in which higher amounts of the lipophilic drug can be collected.

2.3.2. The sample When microdialysis sampling is applied in vivo, the subject, the microdialysis sampling method and the analytical system must be considered in relation to each other. Microdialysis samples are typically aqueous and its constituents are usually small molecular weight molecules that are moderately-to-highly soluble in water. The dialysate samples are usually free of endogenous compounds like lipids and proteins that might interfere with analysis procedures and hence no additional sample preparation is needed. Perfusion flow-rates usually range between 0.1 and 5 ml / min. With a 10-min collection interval, sample volumes of 1–50 ml are obtained. By on-line analysis, sample loss can be minimized, which is important when dealing with very small sample volumes as evaporation will become more important [50,51]. For on-line sample analysis the analysis time should be shorter than the sample collection time. Sample volumes can be increased by using a higher flow and / or extended collection interval. However, it should be noted that a higher flow reduces recovery (Fig. 7). But, as previously mentioned, recovery can be increased by enlargement of the dialysis surface provided that the dialyzed tissue is still homogeneous under those conditions. On the other hand, extension of the collection interval will decrease the timeresolution of the measurements. A decrease in the number of time points may be unfavorable for drugs that are subjected to relative fast changes in their concentrations. The dialysate collected from the brain by in vivo microdialysis can be analyzed by any available analytical procedure, provided that it is

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able to deal with small sample volumes. With the use of an appropriate analytical method, parent drugs and metabolites can be monitored together [52–58]. It is also possible to measure a drug together with possibly affected endogenous compounds [59,60].

2.3.3. Detection Mostly, reversed phase high liquid chromatography combined with ultraviolet, fluorescence, electrochemical detection, or mass spectroscopy, are used because these may provide sensitive analysis of small volumes. If drugs are present in the dialysate at very low concentrations, as is often the case, more sensitive analytical techniques are needed. Then, microbore liquid chromatography, capillary liquid chromatography combined with on-column focussing, and capillary electrophoreses become more appropriate [61–64]. These techniques can handle extremely low sample volumes (nanoliters) which means that a lower perfusate flow-rate can be used (increasing the recovery) and / or shorter collection intervals can be used (increasing the time-resolution). 2.4. Factors that influence recovery 2.4.1. Absolute and relative (concentration) recovery One of the most important questions in microdialysis is how to relate the concentrations in the dialysate to the true concentrations outside the probe. In other words: to which extent is the drug recovered by the dialysate (or lost if a certain drug is added to the perfusate). The concentrations of the drug in the dialysate reflect the concentrations in the (extracellular) fluid around the semipermeable part of the probe. However, as the dialysis procedure is not performed under equilibrium conditions, the concentration of a drug in the dialysate will be different from that in the periprobe fluid and the term recovery is used to describe this relationship. Absolute recovery (or mass recovery) is defined as the amount of drug that is extracted (or delivered) by the dialysate as a function of time. Relative recovery or, better, concentration recovery of a substance is defined as the concentration of the drug in the dialysate (Cdial ) divided by its uniform concentration in the periprobe

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fluid (or bulk concentration, Cbulk ). In general the term recovery refers to the concentration recovery.

2.4.2. In vitro recovery In vitro, effects of a number of parameters on recovery can be investigated, knowing the concentration of the drug in the bulk solution. These parameters include perfusate flow-rate, temperature, perfusate composition, characteristics of the drug, characteristics of the semipermeable membrane, the surface of the semipermeable membrane, and the characteristics of the semipermeable membrane. Also the diffusion of the drug through the periprobe fluid may influence recovery. Various in vitro experiments have been performed to examine the relationship between flow-rate and recovery. Most investigators now use a flow-rate that ranges from 0.1 to 5 ml / min. The in vitro recovery is inversely dependent on the flow-rate and often linearly dependent on the concentration in the periprobe fluid [2,7,18,65–68]. A higher flow-rate will increase the fluid pressure inside the probe, which may result in net transport of fluid across the dialysis membrane and thereby counteract the diffusion of the drug into the dialysis fluid. Fluid pressure gradients will be greatly affected by the sequence of dimensions of the perfusate (inlet) tubing and the dialysis (outlet) tubing [38]. Also, the length of the outlet tubing should be considered [69]. It is safe to use a sequence of diameters of tubing that slightly increases from the perfusion pump ultimately to the collection site and to check flow-rate with and without the probe interconnected. As diffusion increases with higher temperature, a higher temperature will increase recovery. Also perfusate composition will have an effect on recovery. Especially a difference between the osmotic value of the perfusate and that in the periprobe fluid will affect recovery as water movement due to this difference will either add on or counteract diffusion of the drug across the semipermeable membrane [70]. Then, an increase of the dialysis surface (length) will increase the recovery [1,68]. This will be a linear increase with small surfaces, while at larger surfaces the increase in recovery will start to lag behind the increase in surface because the concentration difference between periprobe fluid and dialysate traversing along the semipermeable mem-

brane will gradually diminish with the longer length of the membrane. Furthermore, the characteristics of the drug and the characteristics of the semipermeable membrane will affect recovery.

2.4.3. In vivo recovery All parameters that influence in vitro recovery will also influence in vivo recovery. However, in vivo, tissue characteristics will play an important role and may ultimately determine the recovery. In vivo recovery depends on diffusion in three regions; probe lumen, dialysis membrane and the periprobe environment [71–73]. The first two regions can be characterized in vitro. Diffusion in probe lumen is limiting only with the use of very low flow rates. Diffusion through the dialysis membrane is limiting only when transport through the periprobe environment is rapid. Rapid diffusion through the periprobe environment occurs in most flowing systems (like blood). In tissues, effective diffusion through the extracellular fluid determines the recovery of the microdialysis probes [71,72]. Under these conditions, calibration performed in vitro may not be valid in vivo. Such a difference between in vivo and in vitro recovery has been shown by Hsiao et al. [20]. Effective diffusion of a drug through the extracellular space of a tissue will be affected by uptake into cells, metabolic conversion rate, active transport across membranes, extent of tissue vascularization and blood flow through the tissue. Bungay et al. [71] have developed a mathematical framework for in vivo concentration recovery in tissue. It was assumed that: the probe is inserted in tissues with a normal state, with intimate contact between tissue and outer surface of the probe, for which diffusion through tissues for hydrophilic drugs takes place through the extracellular fluid, and for which transport processes are linear in the concentration-range studied. Other assumptions may lead to other expressions, which according to the authors are nevertheless likely to fit within the general mass transport laws. For in vivo concentration recovery the following equation can be used, viewing the transport of a molecule from the tissue to the dialysate being determined by a series of mass transfer resistances: Ed 5 (Cout 2 Cin ) /(brain ECF 2 Cin ) 5 1 2 exp h 2 1 / f F (R d 1 R m 1 R e ) g j

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Here Ed 5in vivo recovery (or dialysate extraction fraction); Cout 5dialysate concentration; Cin 5 perfusate concentration; F 5flow rate of the perfusate; R5mass transfer resistance with subscripts d5 dialysate, m5membrane and e5external medium. For a probe in tissue it generally holds that R e . .R m . .R d , leaving only the R e to be further considered: R e 5 h(K0 /K1 ) /(2.ro. L.De .we ) jG Here K0 and K1 are modified Bessel functions, r 0 is the radius of the probe, L is the length of the semipermeable part of the membrane, De is the diffusion coefficient for the extracellular phase, we is the accessible volume fraction of the extracellular phase, and G is the profile depth parameter. The profile depth parameter is defined as:

G 5 œ hDe /(k ep 1 k em 1 k e→im ) j in which k ep , k em and k e→im represent first-order rate constants for efflux to the microvasculature, irreversible extracellular metabolism, and the composite of irreversible intracellular metabolism and extracellular-intracellular exchange respectively. Thus, it can be understood that under steady-state conditions all processes that contribute to elimination of a drug will affect in vivo concentration recovery. Influx of the drug does not affect the in vivo concentration recovery, but plays a role in determining the actual concentrations of the drug in the extracellular fluid. In more popular terms, the presence of additional elimination of a drug out of the tissue at steady-state conditions will result in a higher turnover of that drug, as a certain concentration will be maintained. This leads to a better ‘‘delivery’’ of the drug to the probe and therefore to a higher recovery by the dialysate. In vivo, the implications of some parameters will be different from in vitro. In vivo, net transport of fluid across the probe by differences in fluid pressure inside and outside the probe or by differences in (ionic) composition of perfusate relative to that in the extracellular fluid should be avoided as it may affect normal physiology. Then, higher flow rates will remove more endogenous drugs and thereby more easily distort normal physiology [74]. Also perfusate temperature should be considered with respect to

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periprobe tissue physiology, as shown in Section 2.1.2. For flowing fluid systems like blood, an in vitro calibration can provide valuable in vivo recovery values if perfusion flow-rate and temperature are well controlled, provided that the characteristics of the membrane do not change upon implantation and / or during the experiment. However, here also physiological responses to probe implantation may result in changes to the membrane environment, for example, clot formation around an intravenously implanted probe may occur.

2.4.4. Some methods to determine in vivo recovery; quantification of microdialysis data 2.4.4.1. The no-net-flux methodologies The ‘‘no-net-flux’’ (NNF) method has been developed by Lonnroth et al. [75]. The method involves consecutive perfusion of the microdialysis probe with different concentrations (Cin ), if steadystate conditions of the drug are present in the tissue (Fig. 9a). The resulting dialysate concentrations (Cdialysate , or Cout ) are measured and the difference between Cin and Cout is calculated and plotted as a function of Cin . This function will cross the x-axis at the value where Cin equals the periprobe concentration in the extracellular fluid, CECF , which represents the no-net-flux condition. If a linear relationship and true steady-state conditions exist, the slope of the line gives the in vivo concentration recovery of the drug (Fig. 9b). With this method no assumptions on periprobe behavior of the drug have to be made, due to the fact that at no-net-flux conditions no mass-transfer of the drug from further positions to the probe is taking place. However, this method is inadequate for monitoring CECF and probe recovery as a function of time. On the basis of an extended version of the NNF approach this difficulty can be overcome. To estimate in vivo recovery as a function of time, Olson and Justice [76] presented an extended or modified version of the NNF method, which is called here the ‘‘dynamic-no-net-flux’’ (DNNF) method. Instead of serial perfusion of individual animals with different concentrations via the probe, a group of animals are continuously perfused with one selected perfusion concentration (Fig. 10a). Different groups receive

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¨ Fig. 9. The principle of the no-net-flux (NNF) method of Lonnroth et al. [75]. (a) With the tissue at steady-state concentration for the drug of interest, the probe is subsequently perfused with different concentrations of the drug in the perfusion medium (Cin ). The resulting dialysate concentration, Cout , is measured. (b) Graphically, the difference between Cout and Cin can be depicted as a function of the Cin . The condition of no net flux (Cin 5Cout ) is met at the cross-section of the line with the x-axis, when Cin 5CECF in (a).

Fig. 10. The principle of the dynamic-no-net-flux (DNNF) method of Olson and Justtice [76]. (a) Different groups of animals are treated identically with respect to body drug administration. However, at the level of probe the different groups are perfused with different perfusion concentrations (Cin ) for the whole experiment. The concentration of the drug in the tissue may vary with time. The resulting dialysate concentrations, Cout , are measured. (b) Graphically, the difference between Cout and Cin for each group can be depicted as a function of the Cin and as a function of time. The condition of no net flux (Cin 5Cout ) is met at the cross-section of the lines with the xy-plane, when Cin 5CECF for a certain time. In (a) the no-net-flux conditions are indicated by the dotted rectangles.

different concentrations and the results are combined at each time point. Regression of the mean data points of the different groups at a particular point in time will give the actual CECF with the associated in vivo concentration recovery value at that time (Fig. 10b). These investigators showed that in vivo recovery might change during the course of an experiment, with or without concomitant changes in CECF ,

as shown for changes in extracellular dopamine concentrations when cocaine or amphetamine are co-administered. This has important implications for the interpretation of results obtained in earlier microdialysis studies, which might have been erroneous. Although this is a powerful experimental setup, more experimental animals are needed, which in part reduces the advantage of minimizing the use

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of living experimental animals by the microdialysis technique.

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2.4.4.2. Internal standard /retrodialysis /reverse dialysis methods Another approach to determine in vivo recovery for every dialysate sample during the experiment is the use of an internal standard, which is added to the perfusate during the course of the experiment. The internal standard should match the characteristics of the drug as close as possible so that the concentration loss of the internal standard will predict the concentration recovery, or gain, of the drug (Fig. 11). In vitro, the recoveries of both drugs can be measured, and the ratio between the values for internal standard and drug can be determined. With the assumption that the obtained ratio in vitro would remain the same in vivo, it can be used to calculate in vivo recovery of the drug as a function of time. This method would be suited to determine changes in recovery if brought about by factors that decrease probe efficiency, such as the formation of air bubbles on the inside of the semipermeable membrane or occlusion of membrane pores by cells or sticky drugs [77–79]. However, in vivo, effective diffusion of the internal standard and drug is assumed to be equal.

This may not be realistic as illustrated by Stahle [80] for theophylline and caffeine, for which a difference in in vivo recovery was found using the NNF method. Moreover, the difference found was dependent on the tissue, with the highest deviation in the brain. Also interaction of the internal standard with the drug should be ruled out. This stresses prudence to be exercised in the use of internal standards. Other investigators also used and validated this method against the no net flux method [81–83]. In reverse dialysis or retrodialysis, the drug itself may be added to the perfusate and its in vivo loss may be used as a measure for in vivo recovery (Fig. 12). It is a one-point simplification of the NNF method. In principle this measurement can be conducted before the start of the real experiment at the time when there is no drug present in the body. This may be a relatively easy and useful approach, but should be validated, as has been shown by a tissue specific asymptotic profile in recovery that results under increasing concentration gradient conditions [84]. A stronger approach, a combination of internal standard and retrodialysis, has been developed by Bouw et al. (Fig. 13) [85]. This method has been compared with the DNNF method in a study on

Fig. 11. The internal standard method [77–79] assumes that the relative loss of the internal standard into the tissue (compound B) is representative for the recovery or relative gain of the drug (compound A) from the tissue during the experiment, if compound A and B behave similarly in vitro.

Fig. 12. The retrodialysis method [81,82] assumes that the relative loss of the drug into the tissue, before the experiment, is representative for its recovery or relative gain from the tissue during the experiment.

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Fig. 13. In the improved retrodialysis method [85] is a combination of the internal standard and retrodialysis method. In the first phase of the study, where no drug has been administered yet to the body of the subject, the probe is perfused with the internal standard (compound B) and the drug of interest (compound A). The relative loss for both compounds is determined and related to each other by calculation of their ratio. The second phase involves the washout period for the drug, by perfusion of the probe with the internal standard only. The third phase of the study involves administration of the drug to the body of the subject, and measuring the content of the drug in the dialysate samples. During this phase, internal standard is still present in the perfusion solution and its relative loss is taken as being representative for the recovery or relative gain of the drug for each sample interval, using the loss ratio as determined in the first phase of the study.

microdialysis has been applied in mdr1a (2 / 2) and wild-type mice and the importance of quantification of microdialysate data will be exemplified beneath for two drugs for which in vivo recovery values were influenced by differences in active elimination of these drugs by absence versus presence of mdr1aencoded P-glycoprotein. The first example includes rhodamine-123, used as a model substrate to detect P-glycoprotein functionality [88]. Total brain and blood levels of rhodamine123 were determined after a constant intravenous infusion. The about four-fold difference in brain levels between mdr1a (2 / 2) and wild-type mice at steady state, however, was not observed in associated end-of-experiment dialysate concentrations of these mice. A possible difference in in vivo recovery of rhodamine-123 in mdr1a (2 / 2) and wild-type mice was investigated using the NNF method. Brain ECF values were estimated and on average a five-fold difference evolved in rhodamine-123 levels between mdr1a (2 / 2) and wild-type mice, as was found between the corresponding brain homogenate levels (Fig. 14). Different values for in vivo recovery of rhodamine-123 of the two types of mice might seem surprising at first sight, but is, however, in line with the theoretical mathematical framework on in vivo concentration recovery developed by Bungay et al. [71]. All microdialysis experimental parameters were identical, but between the mdr1a (2 / 2) and wildtype mice the difference is the absence of P-glycoprotein-dependent elimination of the substrate (efflux to the microvasculature) in the mdr1a (2 / 2) mice. This will have an effect on the profile depth parameter G, which is defined as:

blood–brain barrier transport of morphine [89], as presented below.

G 5 œ hDe /(k 1 1 k 2 1 k 3 ) j

2.4.4.3. Importance of quantification, an example using genetically modified animals The mdr1a-encoded P-glycoprotein is an active pump being expressed on the luminal face of the cells constituting the blood–brain barrier. Thereby P-glycoprotein will counteract brain penetration of its widely diverse substrates as implicated by large differences in brain distribution of a number of P-glycoprotein substrates observed between mdr1a (2 / 2) and wild-type mice [86,87]. Intracerebral

with k 1 , k 2 and k 3 representing the first-order rate constants for efflux to the microvasculature, irreversible extracellular metabolism, and the composite of irreversible intracellular metabolism and extracellular–intracellular exchange respectively. The term k 1 can be subdivided into k 1a and k 1b , representing the rate constants for elimination to microvasculature that is not P-glycoprotein dependent and the Pglycoprotein dependent one, respectively. Then after some rearrangements one will obtain that the ratio of

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Fig. 14. (a) Concentration-time profiles of rhodamine-123 (R123) in dialysate of mdr1a (2 / 2) and wild-type mice. (b) Concentration-time profiles of R123 in cortical brain extracellular fluid of mdr1a (2 / 2) and wild-type mice, after correction of the dialysate concentrations for in vivo recovery values. (c) Total brain concentrations of R123 at the end of the experiment for of mdr1a (2 / 2) and wild-type mice. It can be seen that the end-of-experiment dialysate concentration ratio is not, but end-of-experiment cortical brain extracellular fluid concentration ratio is comparable to the corresponding total brain concentration ratio [88]. It stresses the importance of correction for in vivo concentration recovery. Furthermore, extracellular concentrations are much lower due to accumulation of R123 in (brain) tissue cells.

mass transfer resistance in mdr1a (2 / 2) over wildtype mice, R e, (2 / 2) /R e, wild-type , equals

h(k 1a 1 k 1b 1 k 2 1 k 3 ) /(k 1a 1 k 2 1 k 3 ) j which shows that R e, (2 / 2) /R e, wild-type .1. Therefore, R e, (2 / 2) is higher than R e, wild-type . A larger value for the mass transfer resistance, R e , will lead to a lower value of in vivo recovery. Thus, it can be understood that for a P-glycoprotein substrate, the absence of P-glycoprotein results in a higher resistance to tissue mass transfer and therefore to a lower in vivo recovery as was found in these mdr1a (2 / 2) mice compared with the wild-types. Also for morphine in vivo recovery values were influenced by differences in active elimination by mdr1a-encoded P-glycoprotein [89]. Blood–brain barrier transport of morphine was investigated by intracerebral microdialysis in mdr1a (2 / 2) and wild-type mice. Morphine concentrations in brain ECF were estimated during constant intravenous infusion. Two methods to estimate in vivo recovery were used: extended retrodialysis with nalorphine as an internal standard, and the DNNF method. Retrodialysis loss of morphine and nalorphine, determined before intravenous administration of mor-

phine, was similar in vivo. Also here a difference in in vivo recovery of morphine between mdr1a (2 / 2) and wild-type mice was found, and similarly for the two quantification methods used. The recovery ratios of mdr1a (2 / 2) over wild-type mice was 0.63 and 0.53 for the extended retrodialysis and DNNF method, respectively. For both methods comparable results were obtained. Morphine brain ECF profiles for mdr1a (2 / 2) mice were significantly higher than those found for mdr1a (1 / 1) mice. This indicates a role of P-glycoprotein in the elimination of morphine from the brain.

2.5. Considerations in data analysis Concentration versus time profiles of drugs can be obtained by the microdialysis method, and pharmacokinetic parameters can be derived. Microdialysate data represent ‘‘average’’ concentrations obtained in the sampling interval, mostly ranging between 5 and 20 min. This should be considered in the description of the concentration–time profiles [90]. For pharmacokinetic data, in most cases, the use of the interval midpoint to relate the dialysate concentrations to is valid. Adjustments are required when the half-life of a process, such as absorption or

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elimination, is short in comparison with the sampling interval. Calculations of half-life and slopes are similar to standard methods for equal sample intervals. Calculation of area-under-the-curve and clearance values may be even more accurate for microdialysis data than for normal sampling at discrete time-points. This is because of the time-integral character of the microdialysis technique as well as the typical more frequent data points. Stahle used a general method to deal with multi-compartment models. Under such conditions the adjusted timepoints are not equidistant, although sample intervals are equal. This is because the slope will be different in different portions of the ln(conc) versus time curve. Then, the time point T, measured from the initial time-point t i of the sampling interval a] t at which the extracellular concentration coincides with the dialysate concentration should be found. With the assumption that the data can be locally approximated by a mono-exponential function the following relation can be derived: T / a] t 5 f ln(q) 2 ln(1 2 e 2q) g /q Here q 5 k a] t with k being the slope of the log(conc) versus time profile. When a ]

t , , t2

T / a] t will be about 0.5, the interval midpoint. For longer sampling intervals T / a] t will shift to values lower than 0.5. Thus, a drug with a first phase elimination half-life of 10 min, a second phase elimination half-life of 30 min and a sampling interval of 10 min the values of the sampling midpoint T / ht will be 0.42 and 0.47 in the first and second elimination phase respectively

3. The added value of microdialysis in pharmacokinetic studies

3.1. Membrane transport processes Quantitative microdialysis is now possible with the use of the in vivo recovery methods that have

been developed over the past years. Therefore, experiments to determine pharmacokinetic parameters can be performed. Microdialysis is a technique that is perfectly suited to specifically determine membrane transport processes in vivo, because it enables measurement of free concentrations at both sides of a membrane. Especially transport across the blood–brain barrier may be of interest as this is a very special and important membrane. By a microdialysis-pharmacokinetic approach first applied to zidovudin [91], Wang and Welty [92] studied the influx and efflux blood–brain barrier permeability of the anticonvulsant gabapentin, and validated the model by using the conventional brain tissue homogenate-pharmacokinetic model [93]. In rats, steady-state levels of gabapentin in plasma were achieved by intravenous infusion, and microdialysis was used to measure gabapentin concentrations in brain ECF during infusion. At termination of the experiment, brain tissue concentrations were measured. In the model used, it was assumed that exchange of gabapentin between intra- and extracellular space was more rapid than transport across the blood–brain barrier. Simulations of the concentration–time course of gabapentin in brain tissue based on this model correlated well with the time-course of brain concentrations determined after intravenous bolus administration and validated the microdialysis– pharmacokinetic approach for estimations of influxand efflux blood–brain barrier permeability. The values found indicated that brain efflux was more efficient, 8-fold higher, than was brain influx. The total brain concentrations of gabapentin were significantly higher than were brain ECF concentrations, which points at intracellular accumulation and tissue binding. An important conclusion was that because the concentration of gabapentin in brain homogenate was much higher than in brain ECF , the brain homogenate method underestimated gabapentin efflux permeability. This shows the big advantage of using microdialysis although the effect of brain metabolism was not considered here, not being necessary for gabapentin. In the model in vivo metabolism and efflux cannot be distinguished, unless very rich data on metabolites in both plasma and brain ECF are available, which in principle can be obtained within one sample using an appropriate analytical procedure.

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The fact that microdialysis enables investigations on membrane transport processes also opened up the possibility to have a closer look at drug equilibration across the blood–brain barrier, by examining microdialysis profiles that have been obtained for drugs in brain ECF in relation to their profiles in plasma (Hammarlund-Udenaes et al.) [94]. The influence of different rates of transport into and out of the brain, either active and / or passive, on free brain concentration–time profiles given a certain blood profile was simulated. In the model, one body compartment and one brain compartment were defined with linear (diffusion) or saturable (active) transport into and out of the brain and the results were compared with experimental results from microdialysis studies. Special emphasis was put on hydrophilic drugs for which diffusion across the blood–brain barrier is impeded by the presence of the tight junctions between the brain endothelial cells. Simulations combined with microdialysis data from literature show that active transport components across the blood–brain barrier are more likely present for more drugs than expected. For most drugs studied, the equilibration of drug concentrations between brain and blood is very rapid. Thereby the blood profile appeared to be a strong predictor for the concentration profile in the brain also for hydrophilic drugs. This contradicts earlier assumptions on hydrophilic drugs having a slow (passive) equilibration across blood–brain barrier due to low permeability [95], and shows the added value of microdialysis in pharmacokinetic studies.

3.2. In vivo synthesis rates, ECF concentrations, distribution The special potential of the microdialysis technique is also shown in a study that employed microdialysis to quantify the dynamics of quinolinic acid levels in brain extracellular fluid [96]. Subcutaneous infusion of quinolinic acid was used to determine the influence of blood concentrations to extracellular fluid concentrations and tissue levels. Local quinolinic acid production rates and influx and efflux rates across the blood–brain barrier were calculated from in vivo recovery, probe geometry, tissue diffusion coefficients, the extracellular volume

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fraction, and the ratio of quinolinic acid levels in brain dialysate over the ones in blood. For this purpose different isotopes of quinolinic acid were used. Experiments were performed under normal conditions but also during central nervous system inflammation by local infusion of endotoxin, which appeared to have an enormous effect on the pharmacokinetic parameters studied for this endogenous drug. The multiple isotope approach can also provide a means to study brain metabolism of drugs or more in general its pharmacokinetic fate.

3.3. Combined measurement of concentration and effect In principle the monitoring of brain extracellular fluid concentrations by microdialysis provides the opportunity to study the relationship between brain drug concentrations and effects. By means of an assay that simultaneously measures the opiate morphine as well as serotonin and its metabolites, Matos et al. [59] studied the effect of morphine on brain extracellular fluid concentrations of serotonin and its metabolites in cerebrospinal fluid, spinal cord, and several regions of the brain. These data were related to observations on behavioral analgesia in order to identify the site of action of morphine. Stain et al. [60] studied the pharmacokinetics of morphine and its metabolite morphine-6-b-D-glucuronide together with the analgesic response, and could deduce that the metabolite had a higher analgesic potency than morphine. Nicolaysen et al. [97] have combined the monitoring of extracellular cocaine and dopamine concentrations, following intraperitoneal cocaine injection. The maximum cocaine concentration was found at 30 min after administration after which its concentration rapidly declined. A non-linear fit of a kinetic model to the experimental cocaine data gave a first order rate constant for appearance as well as disappearance of cocaine in the extracellular space of the striatum. Comparison of the cocaine and dopamine data showed a high correlation between their concentrations for the same point in time. The microdialysis technique has also been used to elucidate the delayed anticonvulsant action of gabapentin, for which anticonvulsant activity appeared to be maximal at much later times as com-

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pared to other drugs with anticonvulsant action like phenotoin, lidocaine, GABA a receptor benzodiazepines, valproic acid, and glutamate antagonists. It appeared that the anticonvulsant effect of gabapentin was delayed by time-dependent events other than distribution from blood to brain [98]. Ludvig et al. [99] combined the microdialysis technique with EEG recordings. An electrode-microdialysis unit was used in which a guide cannula is present, which permits the introduction of the measuring probe after postsurgical recovery. In this study various drugs were delivered locally into the brain by means of a probe and simultaneously the EEG activity of the dialyzed area was monitored. Infusion of the drugs resulted in consistent, clearly defined, and dose-dependent EEG effects. Although this setup is obviously also applicable for measuring drugs which have been administered via another route, the local infusion makes it possible that drugs are delivered and washed out in specific brain sites. However, care should be taken within such experiments because microdialysis can influence the experimental or pathological conditions under study, by buffering transient changes in extracellular fluid composition. For example, Obrenovitch et al. [100] have shown that microdialysis markedly inhibited the propagation of spreading depression.

sion medium can be used to investigate the response of the system as result of such changes. Flow-rates up to 10 ml / min can be used safely if the largest pressure drop takes place before the perfusion fluid enters the semipermeable part of the probe in order to prevent fluid loss that may affect normal physiology. Tissue trauma may be related to the shape, diameter and material of the probe as well as by the surgical and experimental procedure. Such tissue trauma may limit the usefulness of microdialysis and the validity of the results and therefore optimal experimental conditions always have to be searched for. And at last, effects induced by anesthesia may in part be reduced by the use of a guide cannula implanted some time before the start of the microdialysis experiment. Quantification of microdialysis data has been shown to be crucial in microdialysis experiments and is now possible by several practical methods. Thus, pharmacokinetic parameters can be determined. Especially detailed information on drug equilibration across membranes like the blood–brain barrier, and drug distribution and elimination into and out of different compartments within tissues represent important applications of the technique, which is of special value compared with existing in vivo techniques.

4. Conclusions

References

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