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Clinical microdialysis: Current applications and potential use in drug development
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Trends in Analytical Chemistry, Vol. 25, No. 7, 2006
Clinical microdialysis: Current applications and potential use in drug development Martin Brunner, Hartmut Derendorf Biochemical and pharmacological events usually take place in the tissue and not in the bloodstream. However, diagnostic and therapeutic decisions in medical practice are still generally based on blood levels of drugs and/or endogenous molecules. Microdialysis is a catheter-based sampling method that enables in vivo measurement of tissue chemistry in humans. The technique is semi-invasive and is feasible in virtually every human organ. It is currently being used to monitor brain ischemia and metabolic control, transdermal drug distribution and tissue pharmacokinetics, and might become an important tool in drug monitoring and drug development in the future. ª 2006 Elsevier Ltd. All rights reserved. Keywords: Drug development; Drug distribution; In vivo; Microdialysis; Tissue pharmacokinetics
1. Introduction Martin Brunner* Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria and College of Pharmacy, Department of Pharmacy Practice, University of Florida, Gainesville, Florida, USA Hartmut Derendorf College of Pharmacy, Department of Pharmaceutics, University of Florida, Gainesville, Florida, USA
*
Corresponding author. Tel.: +43 1 40400 2981; Fax: +43 1 40400 2998; E-mail: martin.brunner@ meduniwien.ac.at
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Although it is generally acknowledged that tissue concentrations are usually more predictive of clinical outcome than plasma concentrations [1], diagnostic and therapeutic decisions in medical practice are still based on monitoring blood concentrations of drugs and/or endogenous molecules. However, the assessment of drug distribution and tissue pharmacokinetics (PK) has long been treated as a ‘‘forgotten relative’’ [2]. One of the main reasons for this neglect has been the lack of appropriate experimental methodology providing in vivo access to target sites within tissues and organs. Consequently, PK research was restricted to drug-concentration measurements from biological specimens that are relatively easy to obtain, such as tissue biopsies, urine, saliva or skin blister fluid, or to indirect modeling of tissue concentrations from plasmaconcentration curves. Recent years have seen the introduction of several new techniques and approaches for the assessment of drug distribution and
target-tissue PK in humans, including in vivo microdialysis (MD) [3] and imaging techniques, such as magnetic resonance spectroscopy (MRS), and positron emission tomography (PET) [4]. Results from studies using these techniques have underlined the importance of the previously neglected drug-distribution process to the target site as a crucial determinant for clinical outcome. Furthermore, regulatory guidance documents issued by the Food and Drug Administration (FDA) in the USA and the Committee for Proprietary Medicinal Products (CPMP) in the European Union (EU) emphasize the value and the importance of human-tissue drug-concentration data and support the use of clinical MD to obtain this information [5,6]. The roots of MD date back to the early 1960s, when push-pull cannulas, dialysis sacs and dialytrodes were inserted into animal tissues to study tissue biochemistry directly. In the following years, these sampling devices were steadily improved and finally resulted in the type of MD probes currently in use (Fig. 1). Over the years, MD has become a standard technique in preclinical neurosciences. However, the first application in human studies dates back only about 20 years [7]. Since then, cornerstones of a constant development towards MD as a widely used clinical technique were the first publication of MD in humans characterizing interstitial glucose concentrations in healthy volunteers in 1987 [7], first reports on MD in clinical drug studies in 1991, clinical applications for in vivo studies in brain [8] or lung tissue [9] and the availability of MD probes approved for the use in peripheral human
0165-9936/$ - see front matter ª 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2006.05.004
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Figure 1. Panel a) shows a commercially available microdialysis needle probe with a semi-permeable membrane at the tip. The inlet tubing is connected to a precision pump and the probe is constantly perfused with a physiological solution at a flow rate of 1–10 lL/min. As shown in the scheme in panel b), the dialysate leaves the probe and can be continuously collected from the outflow tubing. (Panel a) is reproduced from [39] with permission of the BMJ Publishing Group).
tissues and the brain by the European Union. To date, the PubMed database [http://www.ncbi.nlm.nih.gov/entrez] has records of more than 1500 original publications on MD in humans, a number that is constantly increasing. This is also driven by the availability of MD equipment for use in humans, which is marketed by several companies. Furthermore, special probes are available for insertion into different human tissues, such as soft tissues, the brain, the liver and the peritoneal cavity. The cost of MD probes is in the range €100–250 per probe. The price for a MD pump for the use in human experiments is around €2500.
2. Principles of microdialysis MD is a semi-invasive, focal sampling method, based on the use of probes with a semi-permeable membrane at the probe tip (Fig. 1). Probe insertion into the tissue of interest is only slightly different for standard subcutaneous or intramuscular needle injections. The pain from this procedure is minimal and many subjects do not report any discomfort at all. After insertion, the membrane is located in the interstitial space, which directly surrounds cells and might be considered the anatomically defined target compartment for many drugs (e.g., most antimicrobial agents). The probe is connected to a precision pump via the probe inlet and then constantly perfused with a physiological solution at a flow-rate of 1–10 ll/min. The theoretical fundamentals of MD and important aspects to be considered in human studies have been
reviewed [10]. Briefly, after probe insertion, substances present in the interstitial space fluid pass the membrane by passive diffusion. According to FickÕs law of diffusion, the concentration gradient between perfusate and the interstitial space fluid is the driving force for solute movement. Exchange of molecules over the membrane can take place in both directions. Depending on the molecule, its charge and sterical configuration, only substances with a molar mass of approximately one quarter of the membrane cut-off will be recovered in the fluid leaving the probe via the probe outlet, the so-called dialysate (Fig. 1). The dialysate is therefore usually devoid of large molecules, such as proteins, as they are restricted from entering the probe. Currently, for most commercially available probes for use in humans, the molecular cut-off is in the range 20–100 kD. The ratio between the concentration of a substance in the dialysate and the fluid surrounding the probe is called relative recovery. Besides the pharmacological and chemical properties of the analyte and membrane cutoff, relative recovery depends on to the area of the membrane, temperature, flow rate and perfusate composition. High flow rates decrease relative recovery but increase sample volume, which is in the microliter range in human studies, whereas the opposite is true for low flow rates. In general, the choice of flow rate is influenced by the analytical capabilities of the assay method used, which in turn also influences the duration of the sampling interval and consequently the temporal resolution of an experiment. One advantage of MD compared with other sampling techniques is that no substantial
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fluid loss occurs, as there is no net exchange of fluid between tissue and the probe as long as the perfusion medium is isotonic to the body fluid. Changing the composition of the perfusate by, for example, adding osmotic agents has been employed to increase relative recovery [11], although this approach complicates estimation of tissue concentrations. Location of the probe in the interstitial space and membrane properties enable selective access to the unbound (i.e. the pharmacologically active) drug fraction. As samples are protein-free, a further preparatory step is unnecessary before analysis and samples can be stored without immediate fear of enzymatic degradation. However, due to small sample volumes (e.g., a sampling interval of 20 min with a flow rate of 1.5 lL/min yields a total sample volume of 30 lL) and low substrate concentrations, sample analysis requires highly sensitive analytical methods, such as liquid chromatographytandem mass spectrometry (LC-MS2). So far, diverse methods have been employed for dialysate analysis, which can be performed either in off-line or on-line, and they have been reviewed in detail [10,12]. 2.1. Tissue damage Tissue trauma after probe insertion could potentially influence the experiment, and several compounds, such as thromboxane B2, adenosine triphosphate, glucose, lactate, and the lactate/pyruvate ratio, have been studied as markers for tissue trauma [3]. As baseline values are usually reached within about 1 hour after probe insertion, probes are routinely perfused with a physiological solution for more than half-an-hour to allow ‘‘tissue equilibration’’ before initiating calibration. When response to probe implantation was investigated, there was no immediate evidence of edema or tissue disruption in the skin, although lymphocyte infiltration started 6 hours after the implantation procedure [13] and fibrosis or gliosis were observed after several days in brain tissue [14]. However, lymphocyte infiltration was considered not to affect probe performance [13]. Gliosis, however, could interfere with long-term brain MD. 2.2. Calibration As MD probes are constantly perfused, MD is performed under non-equilibrium conditions and dialysate concentrations represent only a fraction of actual concentrations in the medium surrounding the MD probe. To obtain and quantify interstitial concentrations from dialysate concentrations, MD probes need to be calibrated. Provided there are proper in vivo calibration procedures, intra-individual variation for tissue concentration measurements was shown to be in the range 10–20% depending on the analyte [15]. Different techniques have been developed for in vivo calibration of MD probes. Traditionally, the ‘‘equilibrium method’’ or ‘‘no-net flux method’’, developed by Lo¨nn676
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roth et al. for in vivo calibration during steady state is considered the method of choice [7]. During calibration, different known concentrations of the compound of interest are added to the perfusate and the net loss or gain of the substance in the dialysate is measured and plotted in relation to its concentration in the perfusate. The point of no-net flux corresponds to the tissue concentration. The slope of the regression line gives the relative recovery of the substance. However, the nonet flux method is time consuming and requires steadystate conditions, which are usually not attainable in a clinical setting. Alternatively, it has been suggested to use endogenous substances, such as urea, as reference for recovery determination, which would provide a less timeconsuming calibration approach without the need for steady-state conditions. So far, however, results of studies employing this approach have been inconsistent [10,16]. For use in human studies, a highly reproducible, less time-consuming calibration technique, called ‘‘retrodialysis’’ or ‘‘reverse dialysis’’ has proved to be the most practical calibration technique [15]. The principle of this method relies on the fact that the diffusion process is quantitatively equal in both directions across the semipermeable membrane. The study drug or an internal standard closely matched to the study drug can therefore be added to the perfusion medium at a known concentration (Cperfusate) and its disappearance rate across the membrane is determined. The in vivo recovery value can then be calculated by the following formula: Recovery ð%Þ ¼ 100 ð100 Cdialysate =Cperfusate Þ: In order not to affect the concentration gradient across the membrane, probe calibration by retrodialysis should be performed before an experiment, when the tissue surrounding the probe is devoid of the analyte. For the same reasons, a sufficiently long wash-out period has to be considered after calibration to remove the drug previously delivered to the tissue during retrodialysis. For this purpose, the probe is perfused with a physiological solution. Thereafter, the MD measurements are initiated by administering the study drug. 2.3. Limitations To date, MD has usually been limited to sampling watersoluble substances, in part because an aqueous perfusate (RingerÕs solution or physiological saline solution) is used [3]. Attempts to measure tissue PK of lipophilic substances in vivo have so far only partly been successful [17,18]. The use of lipid emulsions as perfusate, has proved suitable in vitro, although in vivo studies so far have failed to replicate these findings [19]. Furthermore, MD is limited to the measurement of low molecular weight substances due to its membrane cut-off. However, there have been efforts to introduce new membranes with a cut-off up to 3000 kD to allow for sampling of macromolecules and proteins [20].
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3. Current applications of clinical microdialysis In clinical research, MD is currently employed in different clinical fields, such as monitoring of secondary ischemia in neurointensive care [21] or glucose monitoring for long-term metabolic control in patients with diabetes mellitus [22]. Further areas of research comprise studies on the local physiology and metabolism of peripheral tissues [23] or local drug administration by means of MD with the aim of achieving high target-site concentrations without inducing systemic side effects. This approach also allows the simultaneous measurement of the corresponding tissue response in one experiment [24]. In clinical pharmacology, research focuses on the use of MD to measure target-site concentrations of antibiotics [25] or anti-cancer drugs [26] in different tissues and organs and subsequently to relate target-site PK to pharmacodynamics (PD) [27]. Equally challenging is the characterization of skin penetration of the active drug fraction from transdermal therapeutic systems [28]. In contrast to other, often technically demanding and expensive methods, such as imaging techniques, MD can be readily employed for clinical studies in almost any research centre at a reasonable price. Drawbacks stem from the semi-invasive nature of the technique. Consequently, most human studies have so far been performed in easily accessible tissues, such as skeletal muscle and subcutaneous adipose tissue [29], skin [30], tendons [31], superficially located tumors [26] or blood [32]. However, combined with surgical procedures, almost every human tissue is within reach for MD-probe implantation, as demonstrated by studies in brain [33], lung [9], bone [34], heart [35], liver [36] or peritoneal cavity [37]. For the latter two applications, special probes for use in humans have become available. In the following sections, we give a short, up-to-date overview of the main areas of application of clinical MD. More detailed information on methodological aspects and clinical applications of MD is available from various recently published review articles [1,3,10,25,26,38,39]. 3.1. Anti-infective drugs Inadequate tissue penetration of antibiotics can lead to therapeutic failure and bacterial resistance. Pharmacokinetic evaluation of antibiotics should therefore be based on tissue, rather than serum concentrations as in most cases, only the concentration of free unbound antibiotic in the interstitial space fluid at the infection site promotes the antibacterial effect [1]. Currently, however, anti-infective drugs are still mainly selected on the basis of their presumed ability to reach the infection site rather than a rational scientific approach. This discrepancy is well known to regulatory authorities, who are encouraging pharmaceutical companies to
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submit PK data of new anti-infective drugs at the site of action during the drug-application process [40]. MD provides exclusive and continuous access to the interstitial space, the target compartment for most bacterial pathogens, in a minimally invasive way. Thus, this technique appears particularly attractive for clinical distribution studies of anti-infective substances. This has also been acknowledged by FDA advisory committees [5]. To date, MD has been employed for the measurement of tissue PKs of various antimicrobial agents, including quinolones, b-lactams, fusidic acid, aminoglycosides, glycopeptides, ketolides, oxazolidinones, fosfomycin and rifampicin, in diverse tissues and organs, such as soft tissues, brain, lung, heart, bone and blood in human volunteers and patients [3,25]. In addition, MD has been employed to study the in vivo tissue distribution of nucleoside antiviral agents [41] and the antifungal agents fluconazole and metronidazole [42,43]. In healthy volunteers, tissue PK and the effect of protein binding or obesity on tissue penetration have been examined [44]. In patients, it has been investigated how sepsis, intensive care management, diabetes mellitus, angioplasty, head injury, blood flow and local or systemic inflammation affect plasma-to-tissue distribution [3,25]. Results from these in vivo MD studies have provided new understanding of antibiotic tissue distribution in general and therapeutic failure of anti-infective therapy in particular by demonstrating that local inflammation only had minor effects on target-site concentrations, whereas the loss of capillary integrity during systemic inflammation in septic patients markedly impaired tissue distribution [45]. Other studies provided evidence that tissue concentrations might be sub-inhibitory, although plasma concentrations were effective against relevant bacteria [46]. However, sub-inhibitory concentrations were shown to promote the selection of resistant strains in surviving bacteria. Consistent for all classes of antibiotics was the finding that drug penetration in patients was far more variable than in healthy subjects. This appears to be influenced by the disease itself and/or the severity of the disease, although other factors, such as co-medication, also have to be taken into account. Beyond its application in easily accessible soft tissues, such as muscle and adipose tissue, MD has also been adopted for measurements in the human lung and brain. Several recent studies have provided evidence for the feasibility of clinical lung MD to study penetration of different antibiotics into inflamed and healthy lung tissue in patients undergoing lung surgery [9]. These studies corroborated the use of the studied antibiotics in treatment of lung infections caused by extracellular bacteria. Only a limited number of studies have so far addressed the issue of in vivo brain penetration of antibiotics [47], although there clearly is a lack of detailed knowledge on to what extent and with which consequences disease and/or transporter expression at the http://www.elsevier.com/locate/trac
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blood-brain-barrier influence brain penetration of antibiotics. MD data might also be employed to link time-versusdrug-concentration profiles with the antimicrobial effect by means of a dynamic in vivo PK/in vitro PD simulation [27]. This approach is based on the in vivo measurement of interstitial drug PKs by MD and a subsequent PD simulation of the time-versus-drug-concentration profile in an in vitro setting on bacterial cultures. In a third step, data are analysed by an integrated PK/PD model to link unbound antibiotic concentrations to bacterial kill rates. Using these experiments, different dosing scenarios can be simulated without the need for large clinical trials. PK/PD data can be used to support dose optimization and re-evaluate current concepts for establishing dosing guidelines for select tissue infections. This approach is also in accordance with the current reasoning of regulatory authorities, which not only require investigators to measure the antibiotic distribution to unaffected and infected target sites but also to relate unbound drug concentrations at the site of action to the in vitro susceptibility of the infecting microorganism [6,40]. Based on the results from clinical MD studies, it seems justified to re-evaluate previous concepts of plasma-totissue penetration of antimicrobial substances. Furthermore, these results favor the idea of individualized drug dosing in special patient populations, such as intensivecare patients, in order to reach sufficiently high effective tissue concentrations. Depending on their class, MD may also be used to address the question of whether antibiotics are available at the infection site either for a sufficiently long time or at a sufficiently high target-site concentration to be effective against bacterial pathogens without promoting the emergence of bacterial resistance. This knowledge could help to modify appropriately dosing regimens in different disease conditions. Integrating target-site concentration measurements into drug development of anti-infectives at an early stage potentially provides a means for a more rational, effective drug-development process. 3.2. Topical drug application Topical drug application aims at delivering therapeutic drug concentrations to an affected region beneath the area of drug application, while keeping systemic drug concentration as low as possible to avoid systemic adverse drug effects. However, it is often unclear whether effective tissue-drug concentrations are attained. Techniques such as tape stripping, currently used for the measurement of dermal drug PK and bioequivalence [48], usually fail to yield information on drug distribution into deeper skin layers, as only the uppermost layer of the skin, the Stratum corneum, is removed and analysed. MD has been suggested as a promising alternative approach for the assessment of cutaneous drug delivery 678
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and is recognized by regulatory authorities as a potential tool for bioequivalence evaluation of topical dermatological dosage forms [49]. In a recently published study, dermal concentrations of a newly developed diclofenac formulation were compared with concentrations attained after standard oral treatment [50]. By means of MD, it could be demonstrated that topical drug application led to substantial dermal penetration of diclofenac with 250-fold lower plasma concentrations as compared with the oral treatment scheme. Dermal MD studies have furthermore corroborated the notion that the Stratum corneum is the main barrier for the penetration of hydrophilic compounds into deeper skin layers and that disruption of this barrier dramatically increases skin penetration [51]. A consistent finding of in vivo transdermal studies was the considerable inter-individual variability in the skin penetration of drugs, which is might be explained by factors such as the nature of the drug and vehicle, as well as skin integrity and hydration status. During development of novel transdermally applied substances, MD could not only be used for addressing bioequivalence issues, but in vivo MD data on dermal drug PK might also be employed to identify formulations and doses of topically applied drugs that lead to effective local concentrations. 3.3. Anti-cancer drugs Drug penetration into the interstitial space of solid tumors represents a rate-limiting step in clinical response to chemotherapy. MD provides the opportunity for direct study of tumor-drug exposure and metabolism in a minimally invasive way [26]. However, relatively few studies have so far been performed in a clinical setting due to both ethical and methodological considerations. Since the first study in cancer patients in 1996, where carboplatin PK was measured in melanoma metastases [52], clinical MD has been used to evaluate the tumor disposition of commonly used anti-cancer drugs [26], including 5-Fluorouracil (5-FU), capecitabine, cisplatin, dacarbazine, and methotrexate, in tumor lesions accessible to MD-probe insertion, such as breast cancer, melanoma, and oral cancer. In addition to its use in PK studies, a different application was proposed by Ronquist et al. [53], who employed MD for therapeutic site-specific drug delivery to treat inoperable brain tumors. As for anti-infectives, combined in vivo PK/in vitro PD simulations might also be employed to link intratumoral target-site concentration measurements with in vitro inhibition of tumor-cell growth [54]. This approach might assist the identification of new anti-cancer compounds with favorable tumor penetration. To date, MD has almost exclusively been confined to the study of low molecular weight substances. However, the recent introduction of largepore MD membranes offers the possibility of measuring
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tumor exposure of new high molecular anti-tumor medications, such as molecular targeted therapies. 3.4. Neurosciences and combined use with imaging techniques MD was introduced as an intracerebral sampling method for clinical neurosurgery in 1990 [33] and has since proved to be a safe, effective monitoring technique for measuring the neurochemistry of acute brain injury and epilepsy. Although data from brain-MD studies strongly suggest that changes in local markers of brain metabolism might precede the onset of secondary neurological deterioration [33], cerebral MD is still mainly used as a clinical research tool in neurosurgery and its use to influence clinical therapeutic decision-making has been restricted to only a few institutions worldwide. Still, brain MD is one of few methods for neurochemical measurements in the interstitial compartment of the human brain and has become a valuable translational research tool, providing new and important insights into the neurochemistry of acute human brain injury. However, isolated interpretation of biomarkers derived from brain MD experiments should be done cautiously and might require additional validation, particularly in clinical studies, in which experimental conditions cannot be easily standardized. The simultaneous use of complementary imaging techniques, such as PET or MRS, might be crucial for biomarker interpretation [33]. In the past 20 years, imaging techniques have evolved as tools for the non-invasive study of drug distribution in vivo as well as for studying drug effects at their target sites. So far, however, only a few clinical studies have been published combining MD and imaging, most of them in the neurosurgical field to study alterations of brain metabolism as a consequence of brain trauma or surgery [55]. Comparative characteristics of MD and imaging techniques have been published [56]. Whereas MD is comparatively inexpensive, as it is not bound to a specialized center and can be performed at any research institution, it still is a semi-invasive technique that needs to be combined with surgical procedures when studying certain organs. However, imaging techniques are totally non-invasive and can measure drug concentrations in all human organs. PET offers excellent spatial resolution of the order of few millimeters, allowing the study of concentration differences within one organ, whereas the spatial resolution of MRS imaging is low. MRS also has the lowest sensitivity of the three methods mentioned. Furthermore, PET and MRS are not able to discern concentrations of drugs in different compartments, whereas MD provides selective access to the interstitial space of tissue. Recently, a study by Langer et al. combined MD and PET, not in the brain but in peripheral tissues, to assess
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intracellular drug PK in vivo [57], concluding that the MD/PET combination might be useful in research and development of new drugs, for which knowledge of intracellular concentrations is of interest.
4. Summary Quantification of target-site PK of drugs plays an important role in drug discovery and development. However, for many years, PK research was limited to blood and plasma concentration measurements. The introduction of in vivo MD into clinical PK studies now enables the study of drug distribution and receptor phase PK of a large variety of drug molecules in different clinical settings and diverse human tissues and organs. Compared with other techniques, the costs of MD experiments are reasonable and, if adequate analytes are available, target-tissue PK of drug molecules can be quantified. Because MD offers the selective measurement of unbound target-site PKs of drugs, it can be regarded as a suitable scientific tool to satisfy regulatory requirements for PK distribution and bioequivalence studies. Furthermore, MD serves as a research tool helping to define meaningful surrogate markers for drug efficiency along the critical path of drug development and might potentially serve as translational tool for clinical decision-making [58]. Acknowledgement Martin BrunnerÕs work was supported by the ‘‘Erwin Schro¨dinger Fellowship’’ Program of the Austrian Science Fund (Project-Number J 2403-B11). References [1] M. Mu¨ller, A. de la Pena, H. Derendorf, Antimicrob. Agents Chemother. 48 (2004) 1441. [2] H.G. Eichler, M. Mu¨ller, Clin. Pharmacokinet. 34 (1998) 95. [3] C. Joukhadar, M. Mu¨ller, Clin. Pharmacokinet. 44 (2005) 895. [4] R.E. Port, W. Wolf, Invest. New Drugs 21 (2003) 157. [5] http://www.fda.gov/cder/present/anti-infective798/073198.pdf. [6] http://www.emea.eu.int/pdfs/human/ewp/265599en.pdf. [7] P. Lonnroth, P.A. Jansson, U. Smith, Am. J. Physiol. 253 (1987) E228. [8] H. Benveniste, J. Neurochem. 52 (1989) 1667. [9] H. Herkner, M.R. Mu¨ller, N. Kreischitz, B.X. Mayer, M. Frossard, C. Joukhadar, N. Klein, E. Lackner, M. Mu¨ller, Am. J. Respir. Crit. Care Med. 165 (2002) 273. [10] N. Plock, C. Kloft, Eur. J. Pharm. Sci. 25 (2005) 1. [11] W.J. Trickler, D.W. Miller, J. Pharm. Sci. 92 (2003) 1419. [12] T.P. Obrenovitch, E. Zilkha, Methods 23 (2001) 63. [13] J.M. Ault, C.M. Riley, N.M. Meltzer, C.E. Lunte, Pharm. Res. 11 (1994) 1631. [14] Y. Qu, E. van der Gucht, A. Massie, E. Vandenbussche, F. Vandesande, L. Arckens, Brain Res. Brain Res. Protoc. 7 (2001) 52.
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M. Kreilgaard, Adv. Drug Deliv. Rev. 54 (Suppl 1) (2002) S99. M. Mu¨ller, BMJ 324 (2002) 588. http://www.fda.gov/cder/guidance/2580dft.pdf. N. Borg, E. Gotharson, E. Benfeldt, L. Groth, L. Sta˚hle, Acta Derm. Venereol. 79 (1999) 274. L. Sasongko, K.M. Williams, R.O. Day, A.J. McLachlan, Br. J. Clin. Pharmacol. 56 (2003) 551. J. Karjagin, R. Pahkla, T. Karki, J. Starkopf, J. Antimicrob. Chemother. 55 (2005) 341. U. Hollenstein, M. Brunner, B.X. Mayer, S. Delacher, B. Erovic, H.G. Eichler, M. Mu¨ller, Clin. Pharmacol. Ther. 67 (2000) 229. C. Joukhadar, M. Frossard, B.X. Mayer, M. Brunner, N. Klein, P. Siostrzonek, H.G. Eichler, M. Mu¨ller, Crit. Care. Med. 29 (2001) 385. M.A. Zeitlinger, P. Dehghanyar, B.X. Mayer, B.S. Schenk, U. Neckel, G. Heinz, A. Georgopoulos, M. Mu¨ller, C. Joukhadar, Antimicrob. Agents Chemother. 47 (2003) 3548. M. Brunner, A. Reinprecht, U. Illievich, C.K. Spiss, P. Dittrich, M. van Houte, M. Mu¨ller, Br. J. Clin. Pharmacol. 54 (2002) 548. V.P. Shah, Am. J. Clin. Dermatol. 2 (2001) 275. V.P. Shah, Int. J. Clin. Pharmacol. Ther. 42 (2004) 379. M. Brunner, P. Dehghanyar, B. Seigfried, W. Martin, G. Menke, M. Mu¨ller, Br. J. Clin. Pharmacol. 60 (2005) 573. E. Benfeldt, J. Serup, Arch. Dermatol. Res. 291 (1999) 517. B. Blo¨chl-Daum, M. Mu¨ller, V. Meisinger, H.G. Eichler, A. Fassolt, H. Pehamberger, Br. J. Cancer 73 (1996) 920. G. Ronquist, R. Hugosson, U. Sjolander, U. Ungerstedt, Acta Neurochir. (Vienna) 114 (1992) 8. M. Mu¨ller, J. Bockenheimer, U. Zellenberg, N. Klein, G.G. Steger, H.G. Eichler, R.M. Mader, Breast Cancer Res. Treat. 60 (2000) 211. P.J. Hutchinson, A.K. Gupta, T.F. Fryer, P.G. Al-Rawi, D.A. Chatfield, J.P. Coles, M.T. OÕConnell, R. Kett-White, P.S. Minhas, F.I. Aigbirhio, J.C. Clark, P.J. Kirkpatrick, D.K. Menon, J.D. Pickard, J. Cereb. Blood Flow. Metab. 22 (2002) 735. O. Langer, M. Mu¨ller, Curr. Drug Metab. 5 (2004) 463. O. Langer, R. Karch, U. Mu¨ller, G. Dobrozemsky, A. Abrahim, M. Zeitlinger, E. Lackner, C. Joukhadar, R. Dudczak, K. Kletter, M. Mu¨ller, M. Brunner, J. Nucl. Med. 46 (2005) 1835. http://www.fda.gov/oc/initiatives/criticalpath/whitepaper.html.
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Clinical microdialysis: Current applications and potential use in drug development Martin Brunner, Hartmut Derendorf Biochemical and pharmacological events usually take place in the tissue and not in the bloodstream. However, diagnostic and therapeutic decisions in medical practice are still generally based on blood levels of drugs and/or endogenous molecules. Microdialysis is a catheter-based sampling method that enables in vivo measurement of tissue chemistry in humans. The technique is semi-invasive and is feasible in virtually every human organ. It is currently being used to monitor brain ischemia and metabolic control, transdermal drug distribution and tissue pharmacokinetics, and might become an important tool in drug monitoring and drug development in the future. ª 2006 Elsevier Ltd. All rights reserved. Keywords: Drug development; Drug distribution; In vivo; Microdialysis; Tissue pharmacokinetics
1. Introduction Martin Brunner* Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria and College of Pharmacy, Department of Pharmacy Practice, University of Florida, Gainesville, Florida, USA Hartmut Derendorf College of Pharmacy, Department of Pharmaceutics, University of Florida, Gainesville, Florida, USA
*
Corresponding author. Tel.: +43 1 40400 2981; Fax: +43 1 40400 2998; E-mail: martin.brunner@ meduniwien.ac.at
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Although it is generally acknowledged that tissue concentrations are usually more predictive of clinical outcome than plasma concentrations [1], diagnostic and therapeutic decisions in medical practice are still based on monitoring blood concentrations of drugs and/or endogenous molecules. However, the assessment of drug distribution and tissue pharmacokinetics (PK) has long been treated as a ‘‘forgotten relative’’ [2]. One of the main reasons for this neglect has been the lack of appropriate experimental methodology providing in vivo access to target sites within tissues and organs. Consequently, PK research was restricted to drug-concentration measurements from biological specimens that are relatively easy to obtain, such as tissue biopsies, urine, saliva or skin blister fluid, or to indirect modeling of tissue concentrations from plasmaconcentration curves. Recent years have seen the introduction of several new techniques and approaches for the assessment of drug distribution and
target-tissue PK in humans, including in vivo microdialysis (MD) [3] and imaging techniques, such as magnetic resonance spectroscopy (MRS), and positron emission tomography (PET) [4]. Results from studies using these techniques have underlined the importance of the previously neglected drug-distribution process to the target site as a crucial determinant for clinical outcome. Furthermore, regulatory guidance documents issued by the Food and Drug Administration (FDA) in the USA and the Committee for Proprietary Medicinal Products (CPMP) in the European Union (EU) emphasize the value and the importance of human-tissue drug-concentration data and support the use of clinical MD to obtain this information [5,6]. The roots of MD date back to the early 1960s, when push-pull cannulas, dialysis sacs and dialytrodes were inserted into animal tissues to study tissue biochemistry directly. In the following years, these sampling devices were steadily improved and finally resulted in the type of MD probes currently in use (Fig. 1). Over the years, MD has become a standard technique in preclinical neurosciences. However, the first application in human studies dates back only about 20 years [7]. Since then, cornerstones of a constant development towards MD as a widely used clinical technique were the first publication of MD in humans characterizing interstitial glucose concentrations in healthy volunteers in 1987 [7], first reports on MD in clinical drug studies in 1991, clinical applications for in vivo studies in brain [8] or lung tissue [9] and the availability of MD probes approved for the use in peripheral human
0165-9936/$ - see front matter ª 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2006.05.004
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Figure 1. Panel a) shows a commercially available microdialysis needle probe with a semi-permeable membrane at the tip. The inlet tubing is connected to a precision pump and the probe is constantly perfused with a physiological solution at a flow rate of 1–10 lL/min. As shown in the scheme in panel b), the dialysate leaves the probe and can be continuously collected from the outflow tubing. (Panel a) is reproduced from [39] with permission of the BMJ Publishing Group).
tissues and the brain by the European Union. To date, the PubMed database [http://www.ncbi.nlm.nih.gov/entrez] has records of more than 1500 original publications on MD in humans, a number that is constantly increasing. This is also driven by the availability of MD equipment for use in humans, which is marketed by several companies. Furthermore, special probes are available for insertion into different human tissues, such as soft tissues, the brain, the liver and the peritoneal cavity. The cost of MD probes is in the range €100–250 per probe. The price for a MD pump for the use in human experiments is around €2500.
2. Principles of microdialysis MD is a semi-invasive, focal sampling method, based on the use of probes with a semi-permeable membrane at the probe tip (Fig. 1). Probe insertion into the tissue of interest is only slightly different for standard subcutaneous or intramuscular needle injections. The pain from this procedure is minimal and many subjects do not report any discomfort at all. After insertion, the membrane is located in the interstitial space, which directly surrounds cells and might be considered the anatomically defined target compartment for many drugs (e.g., most antimicrobial agents). The probe is connected to a precision pump via the probe inlet and then constantly perfused with a physiological solution at a flow-rate of 1–10 ll/min. The theoretical fundamentals of MD and important aspects to be considered in human studies have been
reviewed [10]. Briefly, after probe insertion, substances present in the interstitial space fluid pass the membrane by passive diffusion. According to FickÕs law of diffusion, the concentration gradient between perfusate and the interstitial space fluid is the driving force for solute movement. Exchange of molecules over the membrane can take place in both directions. Depending on the molecule, its charge and sterical configuration, only substances with a molar mass of approximately one quarter of the membrane cut-off will be recovered in the fluid leaving the probe via the probe outlet, the so-called dialysate (Fig. 1). The dialysate is therefore usually devoid of large molecules, such as proteins, as they are restricted from entering the probe. Currently, for most commercially available probes for use in humans, the molecular cut-off is in the range 20–100 kD. The ratio between the concentration of a substance in the dialysate and the fluid surrounding the probe is called relative recovery. Besides the pharmacological and chemical properties of the analyte and membrane cutoff, relative recovery depends on to the area of the membrane, temperature, flow rate and perfusate composition. High flow rates decrease relative recovery but increase sample volume, which is in the microliter range in human studies, whereas the opposite is true for low flow rates. In general, the choice of flow rate is influenced by the analytical capabilities of the assay method used, which in turn also influences the duration of the sampling interval and consequently the temporal resolution of an experiment. One advantage of MD compared with other sampling techniques is that no substantial
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fluid loss occurs, as there is no net exchange of fluid between tissue and the probe as long as the perfusion medium is isotonic to the body fluid. Changing the composition of the perfusate by, for example, adding osmotic agents has been employed to increase relative recovery [11], although this approach complicates estimation of tissue concentrations. Location of the probe in the interstitial space and membrane properties enable selective access to the unbound (i.e. the pharmacologically active) drug fraction. As samples are protein-free, a further preparatory step is unnecessary before analysis and samples can be stored without immediate fear of enzymatic degradation. However, due to small sample volumes (e.g., a sampling interval of 20 min with a flow rate of 1.5 lL/min yields a total sample volume of 30 lL) and low substrate concentrations, sample analysis requires highly sensitive analytical methods, such as liquid chromatographytandem mass spectrometry (LC-MS2). So far, diverse methods have been employed for dialysate analysis, which can be performed either in off-line or on-line, and they have been reviewed in detail [10,12]. 2.1. Tissue damage Tissue trauma after probe insertion could potentially influence the experiment, and several compounds, such as thromboxane B2, adenosine triphosphate, glucose, lactate, and the lactate/pyruvate ratio, have been studied as markers for tissue trauma [3]. As baseline values are usually reached within about 1 hour after probe insertion, probes are routinely perfused with a physiological solution for more than half-an-hour to allow ‘‘tissue equilibration’’ before initiating calibration. When response to probe implantation was investigated, there was no immediate evidence of edema or tissue disruption in the skin, although lymphocyte infiltration started 6 hours after the implantation procedure [13] and fibrosis or gliosis were observed after several days in brain tissue [14]. However, lymphocyte infiltration was considered not to affect probe performance [13]. Gliosis, however, could interfere with long-term brain MD. 2.2. Calibration As MD probes are constantly perfused, MD is performed under non-equilibrium conditions and dialysate concentrations represent only a fraction of actual concentrations in the medium surrounding the MD probe. To obtain and quantify interstitial concentrations from dialysate concentrations, MD probes need to be calibrated. Provided there are proper in vivo calibration procedures, intra-individual variation for tissue concentration measurements was shown to be in the range 10–20% depending on the analyte [15]. Different techniques have been developed for in vivo calibration of MD probes. Traditionally, the ‘‘equilibrium method’’ or ‘‘no-net flux method’’, developed by Lo¨nn676
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roth et al. for in vivo calibration during steady state is considered the method of choice [7]. During calibration, different known concentrations of the compound of interest are added to the perfusate and the net loss or gain of the substance in the dialysate is measured and plotted in relation to its concentration in the perfusate. The point of no-net flux corresponds to the tissue concentration. The slope of the regression line gives the relative recovery of the substance. However, the nonet flux method is time consuming and requires steadystate conditions, which are usually not attainable in a clinical setting. Alternatively, it has been suggested to use endogenous substances, such as urea, as reference for recovery determination, which would provide a less timeconsuming calibration approach without the need for steady-state conditions. So far, however, results of studies employing this approach have been inconsistent [10,16]. For use in human studies, a highly reproducible, less time-consuming calibration technique, called ‘‘retrodialysis’’ or ‘‘reverse dialysis’’ has proved to be the most practical calibration technique [15]. The principle of this method relies on the fact that the diffusion process is quantitatively equal in both directions across the semipermeable membrane. The study drug or an internal standard closely matched to the study drug can therefore be added to the perfusion medium at a known concentration (Cperfusate) and its disappearance rate across the membrane is determined. The in vivo recovery value can then be calculated by the following formula: Recovery ð%Þ ¼ 100 ð100 Cdialysate =Cperfusate Þ: In order not to affect the concentration gradient across the membrane, probe calibration by retrodialysis should be performed before an experiment, when the tissue surrounding the probe is devoid of the analyte. For the same reasons, a sufficiently long wash-out period has to be considered after calibration to remove the drug previously delivered to the tissue during retrodialysis. For this purpose, the probe is perfused with a physiological solution. Thereafter, the MD measurements are initiated by administering the study drug. 2.3. Limitations To date, MD has usually been limited to sampling watersoluble substances, in part because an aqueous perfusate (RingerÕs solution or physiological saline solution) is used [3]. Attempts to measure tissue PK of lipophilic substances in vivo have so far only partly been successful [17,18]. The use of lipid emulsions as perfusate, has proved suitable in vitro, although in vivo studies so far have failed to replicate these findings [19]. Furthermore, MD is limited to the measurement of low molecular weight substances due to its membrane cut-off. However, there have been efforts to introduce new membranes with a cut-off up to 3000 kD to allow for sampling of macromolecules and proteins [20].
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3. Current applications of clinical microdialysis In clinical research, MD is currently employed in different clinical fields, such as monitoring of secondary ischemia in neurointensive care [21] or glucose monitoring for long-term metabolic control in patients with diabetes mellitus [22]. Further areas of research comprise studies on the local physiology and metabolism of peripheral tissues [23] or local drug administration by means of MD with the aim of achieving high target-site concentrations without inducing systemic side effects. This approach also allows the simultaneous measurement of the corresponding tissue response in one experiment [24]. In clinical pharmacology, research focuses on the use of MD to measure target-site concentrations of antibiotics [25] or anti-cancer drugs [26] in different tissues and organs and subsequently to relate target-site PK to pharmacodynamics (PD) [27]. Equally challenging is the characterization of skin penetration of the active drug fraction from transdermal therapeutic systems [28]. In contrast to other, often technically demanding and expensive methods, such as imaging techniques, MD can be readily employed for clinical studies in almost any research centre at a reasonable price. Drawbacks stem from the semi-invasive nature of the technique. Consequently, most human studies have so far been performed in easily accessible tissues, such as skeletal muscle and subcutaneous adipose tissue [29], skin [30], tendons [31], superficially located tumors [26] or blood [32]. However, combined with surgical procedures, almost every human tissue is within reach for MD-probe implantation, as demonstrated by studies in brain [33], lung [9], bone [34], heart [35], liver [36] or peritoneal cavity [37]. For the latter two applications, special probes for use in humans have become available. In the following sections, we give a short, up-to-date overview of the main areas of application of clinical MD. More detailed information on methodological aspects and clinical applications of MD is available from various recently published review articles [1,3,10,25,26,38,39]. 3.1. Anti-infective drugs Inadequate tissue penetration of antibiotics can lead to therapeutic failure and bacterial resistance. Pharmacokinetic evaluation of antibiotics should therefore be based on tissue, rather than serum concentrations as in most cases, only the concentration of free unbound antibiotic in the interstitial space fluid at the infection site promotes the antibacterial effect [1]. Currently, however, anti-infective drugs are still mainly selected on the basis of their presumed ability to reach the infection site rather than a rational scientific approach. This discrepancy is well known to regulatory authorities, who are encouraging pharmaceutical companies to
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submit PK data of new anti-infective drugs at the site of action during the drug-application process [40]. MD provides exclusive and continuous access to the interstitial space, the target compartment for most bacterial pathogens, in a minimally invasive way. Thus, this technique appears particularly attractive for clinical distribution studies of anti-infective substances. This has also been acknowledged by FDA advisory committees [5]. To date, MD has been employed for the measurement of tissue PKs of various antimicrobial agents, including quinolones, b-lactams, fusidic acid, aminoglycosides, glycopeptides, ketolides, oxazolidinones, fosfomycin and rifampicin, in diverse tissues and organs, such as soft tissues, brain, lung, heart, bone and blood in human volunteers and patients [3,25]. In addition, MD has been employed to study the in vivo tissue distribution of nucleoside antiviral agents [41] and the antifungal agents fluconazole and metronidazole [42,43]. In healthy volunteers, tissue PK and the effect of protein binding or obesity on tissue penetration have been examined [44]. In patients, it has been investigated how sepsis, intensive care management, diabetes mellitus, angioplasty, head injury, blood flow and local or systemic inflammation affect plasma-to-tissue distribution [3,25]. Results from these in vivo MD studies have provided new understanding of antibiotic tissue distribution in general and therapeutic failure of anti-infective therapy in particular by demonstrating that local inflammation only had minor effects on target-site concentrations, whereas the loss of capillary integrity during systemic inflammation in septic patients markedly impaired tissue distribution [45]. Other studies provided evidence that tissue concentrations might be sub-inhibitory, although plasma concentrations were effective against relevant bacteria [46]. However, sub-inhibitory concentrations were shown to promote the selection of resistant strains in surviving bacteria. Consistent for all classes of antibiotics was the finding that drug penetration in patients was far more variable than in healthy subjects. This appears to be influenced by the disease itself and/or the severity of the disease, although other factors, such as co-medication, also have to be taken into account. Beyond its application in easily accessible soft tissues, such as muscle and adipose tissue, MD has also been adopted for measurements in the human lung and brain. Several recent studies have provided evidence for the feasibility of clinical lung MD to study penetration of different antibiotics into inflamed and healthy lung tissue in patients undergoing lung surgery [9]. These studies corroborated the use of the studied antibiotics in treatment of lung infections caused by extracellular bacteria. Only a limited number of studies have so far addressed the issue of in vivo brain penetration of antibiotics [47], although there clearly is a lack of detailed knowledge on to what extent and with which consequences disease and/or transporter expression at the http://www.elsevier.com/locate/trac
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blood-brain-barrier influence brain penetration of antibiotics. MD data might also be employed to link time-versusdrug-concentration profiles with the antimicrobial effect by means of a dynamic in vivo PK/in vitro PD simulation [27]. This approach is based on the in vivo measurement of interstitial drug PKs by MD and a subsequent PD simulation of the time-versus-drug-concentration profile in an in vitro setting on bacterial cultures. In a third step, data are analysed by an integrated PK/PD model to link unbound antibiotic concentrations to bacterial kill rates. Using these experiments, different dosing scenarios can be simulated without the need for large clinical trials. PK/PD data can be used to support dose optimization and re-evaluate current concepts for establishing dosing guidelines for select tissue infections. This approach is also in accordance with the current reasoning of regulatory authorities, which not only require investigators to measure the antibiotic distribution to unaffected and infected target sites but also to relate unbound drug concentrations at the site of action to the in vitro susceptibility of the infecting microorganism [6,40]. Based on the results from clinical MD studies, it seems justified to re-evaluate previous concepts of plasma-totissue penetration of antimicrobial substances. Furthermore, these results favor the idea of individualized drug dosing in special patient populations, such as intensivecare patients, in order to reach sufficiently high effective tissue concentrations. Depending on their class, MD may also be used to address the question of whether antibiotics are available at the infection site either for a sufficiently long time or at a sufficiently high target-site concentration to be effective against bacterial pathogens without promoting the emergence of bacterial resistance. This knowledge could help to modify appropriately dosing regimens in different disease conditions. Integrating target-site concentration measurements into drug development of anti-infectives at an early stage potentially provides a means for a more rational, effective drug-development process. 3.2. Topical drug application Topical drug application aims at delivering therapeutic drug concentrations to an affected region beneath the area of drug application, while keeping systemic drug concentration as low as possible to avoid systemic adverse drug effects. However, it is often unclear whether effective tissue-drug concentrations are attained. Techniques such as tape stripping, currently used for the measurement of dermal drug PK and bioequivalence [48], usually fail to yield information on drug distribution into deeper skin layers, as only the uppermost layer of the skin, the Stratum corneum, is removed and analysed. MD has been suggested as a promising alternative approach for the assessment of cutaneous drug delivery 678
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and is recognized by regulatory authorities as a potential tool for bioequivalence evaluation of topical dermatological dosage forms [49]. In a recently published study, dermal concentrations of a newly developed diclofenac formulation were compared with concentrations attained after standard oral treatment [50]. By means of MD, it could be demonstrated that topical drug application led to substantial dermal penetration of diclofenac with 250-fold lower plasma concentrations as compared with the oral treatment scheme. Dermal MD studies have furthermore corroborated the notion that the Stratum corneum is the main barrier for the penetration of hydrophilic compounds into deeper skin layers and that disruption of this barrier dramatically increases skin penetration [51]. A consistent finding of in vivo transdermal studies was the considerable inter-individual variability in the skin penetration of drugs, which is might be explained by factors such as the nature of the drug and vehicle, as well as skin integrity and hydration status. During development of novel transdermally applied substances, MD could not only be used for addressing bioequivalence issues, but in vivo MD data on dermal drug PK might also be employed to identify formulations and doses of topically applied drugs that lead to effective local concentrations. 3.3. Anti-cancer drugs Drug penetration into the interstitial space of solid tumors represents a rate-limiting step in clinical response to chemotherapy. MD provides the opportunity for direct study of tumor-drug exposure and metabolism in a minimally invasive way [26]. However, relatively few studies have so far been performed in a clinical setting due to both ethical and methodological considerations. Since the first study in cancer patients in 1996, where carboplatin PK was measured in melanoma metastases [52], clinical MD has been used to evaluate the tumor disposition of commonly used anti-cancer drugs [26], including 5-Fluorouracil (5-FU), capecitabine, cisplatin, dacarbazine, and methotrexate, in tumor lesions accessible to MD-probe insertion, such as breast cancer, melanoma, and oral cancer. In addition to its use in PK studies, a different application was proposed by Ronquist et al. [53], who employed MD for therapeutic site-specific drug delivery to treat inoperable brain tumors. As for anti-infectives, combined in vivo PK/in vitro PD simulations might also be employed to link intratumoral target-site concentration measurements with in vitro inhibition of tumor-cell growth [54]. This approach might assist the identification of new anti-cancer compounds with favorable tumor penetration. To date, MD has almost exclusively been confined to the study of low molecular weight substances. However, the recent introduction of largepore MD membranes offers the possibility of measuring
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tumor exposure of new high molecular anti-tumor medications, such as molecular targeted therapies. 3.4. Neurosciences and combined use with imaging techniques MD was introduced as an intracerebral sampling method for clinical neurosurgery in 1990 [33] and has since proved to be a safe, effective monitoring technique for measuring the neurochemistry of acute brain injury and epilepsy. Although data from brain-MD studies strongly suggest that changes in local markers of brain metabolism might precede the onset of secondary neurological deterioration [33], cerebral MD is still mainly used as a clinical research tool in neurosurgery and its use to influence clinical therapeutic decision-making has been restricted to only a few institutions worldwide. Still, brain MD is one of few methods for neurochemical measurements in the interstitial compartment of the human brain and has become a valuable translational research tool, providing new and important insights into the neurochemistry of acute human brain injury. However, isolated interpretation of biomarkers derived from brain MD experiments should be done cautiously and might require additional validation, particularly in clinical studies, in which experimental conditions cannot be easily standardized. The simultaneous use of complementary imaging techniques, such as PET or MRS, might be crucial for biomarker interpretation [33]. In the past 20 years, imaging techniques have evolved as tools for the non-invasive study of drug distribution in vivo as well as for studying drug effects at their target sites. So far, however, only a few clinical studies have been published combining MD and imaging, most of them in the neurosurgical field to study alterations of brain metabolism as a consequence of brain trauma or surgery [55]. Comparative characteristics of MD and imaging techniques have been published [56]. Whereas MD is comparatively inexpensive, as it is not bound to a specialized center and can be performed at any research institution, it still is a semi-invasive technique that needs to be combined with surgical procedures when studying certain organs. However, imaging techniques are totally non-invasive and can measure drug concentrations in all human organs. PET offers excellent spatial resolution of the order of few millimeters, allowing the study of concentration differences within one organ, whereas the spatial resolution of MRS imaging is low. MRS also has the lowest sensitivity of the three methods mentioned. Furthermore, PET and MRS are not able to discern concentrations of drugs in different compartments, whereas MD provides selective access to the interstitial space of tissue. Recently, a study by Langer et al. combined MD and PET, not in the brain but in peripheral tissues, to assess
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intracellular drug PK in vivo [57], concluding that the MD/PET combination might be useful in research and development of new drugs, for which knowledge of intracellular concentrations is of interest.
4. Summary Quantification of target-site PK of drugs plays an important role in drug discovery and development. However, for many years, PK research was limited to blood and plasma concentration measurements. The introduction of in vivo MD into clinical PK studies now enables the study of drug distribution and receptor phase PK of a large variety of drug molecules in different clinical settings and diverse human tissues and organs. Compared with other techniques, the costs of MD experiments are reasonable and, if adequate analytes are available, target-tissue PK of drug molecules can be quantified. Because MD offers the selective measurement of unbound target-site PKs of drugs, it can be regarded as a suitable scientific tool to satisfy regulatory requirements for PK distribution and bioequivalence studies. Furthermore, MD serves as a research tool helping to define meaningful surrogate markers for drug efficiency along the critical path of drug development and might potentially serve as translational tool for clinical decision-making [58]. Acknowledgement Martin BrunnerÕs work was supported by the ‘‘Erwin Schro¨dinger Fellowship’’ Program of the Austrian Science Fund (Project-Number J 2403-B11). References [1] M. Mu¨ller, A. de la Pena, H. Derendorf, Antimicrob. Agents Chemother. 48 (2004) 1441. [2] H.G. Eichler, M. Mu¨ller, Clin. Pharmacokinet. 34 (1998) 95. [3] C. Joukhadar, M. Mu¨ller, Clin. Pharmacokinet. 44 (2005) 895. [4] R.E. Port, W. Wolf, Invest. New Drugs 21 (2003) 157. [5] http://www.fda.gov/cder/present/anti-infective798/073198.pdf. [6] http://www.emea.eu.int/pdfs/human/ewp/265599en.pdf. [7] P. Lonnroth, P.A. Jansson, U. Smith, Am. J. Physiol. 253 (1987) E228. [8] H. Benveniste, J. Neurochem. 52 (1989) 1667. [9] H. Herkner, M.R. Mu¨ller, N. Kreischitz, B.X. Mayer, M. Frossard, C. Joukhadar, N. Klein, E. Lackner, M. Mu¨ller, Am. J. Respir. Crit. Care Med. 165 (2002) 273. [10] N. Plock, C. Kloft, Eur. J. Pharm. Sci. 25 (2005) 1. [11] W.J. Trickler, D.W. Miller, J. Pharm. Sci. 92 (2003) 1419. [12] T.P. Obrenovitch, E. Zilkha, Methods 23 (2001) 63. [13] J.M. Ault, C.M. Riley, N.M. Meltzer, C.E. Lunte, Pharm. Res. 11 (1994) 1631. [14] Y. Qu, E. van der Gucht, A. Massie, E. Vandenbussche, F. Vandesande, L. Arckens, Brain Res. Brain Res. Protoc. 7 (2001) 52.
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