Application of In Vivo Brain Microdialysis to the Study of Blood-brain Barrier Transport of Drugs

Drug Metab. Pharmacokin. 17 (5): 395–407 (2002).

Review

JSSX Award for Young Scientists, 2001

Application of In Vivo Brain Microdialysis to the Study of Blood-Brain Barrier Transport of Drugs Yoshiharu DEGUCHI Department of Drug Disposition & Pharmacokinetics, School of Pharmaceutical Sciences, Teikyo University, Kanagawa, Japan Summary: Recent advances in blood-brain barrier (BBB) research have led to a new understanding of drug transport processes at the BBB. The BBB acts as a dynamic regulatory interface at which nutrients necessary for neural activity are actively taken up into the brain from the blood circulation, and actively excludes metabolites that might interfere with the maintenance of brain homeostasis. Such in‰ux and eŒux transport functions at the BBB would also control the concentrations of various drugs in the brain interstitial ‰uid (ISF), which are an important determinant of the central nervous system (CNS) eŠects. Thus, direct measurement of the brain ISF concentration of drugs can provide signiˆcant information for clarifying the in‰ux and eŒux transport functions of drugs across the BBB. Although several experimental techniques have been developed to investigate transport functions across the BBB, in vivo brain microdialysis seems to be one of the most suitable techniques for characterizing the in‰ux and eŒux transport functions across the BBB under physiological and pathological conditions. This review covers studies during the past decade, in which the in‰ux and eŒux transport of drugs across the BBB was kinetically and mechanistically evaluated by means of the brain microdialysis technique. Some applications of brain microdialysis to studies on neuronal function and neurotherapeutics are also included.

Key words: brain microdialysis; blood-brain barrier; transport system gp results in an uphill concentration gradient from the brain ISF toward the blood circulation. In contrast, if a drug of interest is transported across the BBB solely by passive diŠusion, the brain ISF concentration at the steady state would be equal to the plasma unbound concentration. This suggests that simultaneous measurement of the brain ISF concentration and the plasma concentration can provide signiˆcant information for clarifying the in‰ux and eŒux transport functions of drugs across the BBB. Many experimental techniques have been developed for the measurement of concentrations of brain ISF components. These include intraventricular perfusion, cortical cup perfusion and the push-pull cannula technique.4) The prototype of the brain microdialysis technique was developed by Ungerstedt et al. in order to continuously measure the dynamic changes in neurotransmitters such as dopamine.5) Since then, the technique has been widely applied not only in the ˆeld of neurochemical research, but also in many other ˆelds, including physiology and pharmacology. However, in 1990 when we started our microdialysis studies, there

Introduction The concentration in the brain interstitial ‰uid (ISF) of centrally acting drugs, is an important determinant of the central nervous system (CNS) eŠects, and is greatly in‰uenced by the homeostatic mechanisms of the brain. In particular, in‰ux and eŒux transport processes across the blood-brain barrier (BBB), which is formed by complex tight junctions of the brain capillary endothelial cells, is considered to be the dominant factor in‰uencing the brain ISF concentration of certain drugs. The intercellular tight junctions of the brain capillaries severely restrict the paracellular transport of highly hydrophilic drugs between the brain ISF and the plasma. On the other hand, more lipophilic drugs may readily permeate through the BBB by passive diŠusion. Recently, a number of transporters and receptors for endogenous substances have been identiˆed at the BBB.1,2) It is gradually becoming clearer that they also have the ability to transport certain drugs.3) Indeed, Pglycoprotein (P-gp) acts as an eŒux pump of the anticancer drugs at the BBB. Such transport function by P-

Received; October 4, 2002, Accepted; November 5, 2002 To whom correspondence should be addressed : Yoshiharu DEGUCHI, Ph.D., Department of Drug Disposition & Pharmacokinetics, School of Pharmaceutical Sciences, Teikyo University, 1091-1 Suarashi, Sagamiko-machi, Tsukui-gun, Kanagawa 199-0195, Japan. Tel. +81-426-85-3766, Fax. +81-426-85-1345, E-mail: deguchi＠pharm.teikyo-u.ac.jp

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had been few reports of application of this technique to the study of BBB functionality for drug transport. Progress in brain microdialysis methodology during the last decade has opened up the opportunity to analyze in detail the BBB transport mechanisms of various drugs.6–8) This review deals mainly with the results of our studies to characterize the BBB transport of drugs by means of the brain microdialysis technique. Certain applications of brain microdialysis to studies on neuronal function and neurotherapeutics are also addressed.9) Basic Procedure of Brain Microdialysis Brain microdialysis usually involves implantation of a horizontal- or vertical-type microdialysis probe with a semi-permeable membrane into a speciˆc region of the brain, and the probe is then perfused with a physiological solution (dialysis ‰uid). Thus, a concentration gradient is created between the brain ISF and the dialysis ‰uid, so that diŠusion occurs from the brain ISF to the dialysis ‰uid. Therefore, virtually any compound that can pass through the semi-permeable membrane of the microdialysis probe can be examined. Other advantages of this technique are as follows: Firstly, it enables us to monitor endogenous compounds or drugs in the brain ISF of living, awake and freely moving animals, in either healthy or diseased brain sites. Secondly, it enables us to sample continuously the compounds in the brain ISF of an individual animal for hours or days. This signiˆcantly reduces of the number of animals needed for experiments, and the interindividual variability. Thirdly, since a semi-permeable membrane with low molecular weight cut-oŠ excludes large molecules, no ``clean-up'' procedure, such as extraction or deprotenization, is required, and potential degradation by enzymes is avoided. Consequently, the dialysate can be directly subjected to high resolution ES W MS10) or capillary elecanalysis, e.g., with HPLC W 11) trophoresis. On the other hand, there are some limitations to the use of this technique. One of them relates to the damage to local tissue during and after probe implantation. There is an initial period of disturbed tissue function involving increased glucose metabolism, decreased blood ‰ow, and tissue trauma.12,13) This period appears to last from 30 min to 24 hr after probe implantation. In our experience, the BBB damage is minimal by 48 hr after the probe implantation,14) but the degree of damage is greatly in‰uenced by the surgical procedure employed, and the shape of the probe. Microdialysis experiments should not usually be started until the disturbed tissue function has fully recovered. Another problem relates to the method used to estimate ``true'' concentration the brain ISF from that in the dialysate. The concentration of a compound in the

dialysate re‰ects the ``true'' concentrations in the brain ISF, but not necessarily equal. The relation between brain ISF and dialysate concentrations is not simple, but depends markedly on the geometry and characteristics of the probe (length, radius, composition and molecular cut-oŠ of the semi-permeable membrane), as well as on complex biological processes (diŠusion, metabolism and transport processes of compounds in the brain ISF). Several methods to estimate the ``true'' concentration in brain ISF have been proposed. They include the ‰ow rate method,15) the perfusion interval method,16) the zero-‰ow method17) and the retro dialysis method.18) Amberg and Lindefores,19) and Bungay et al.20) presented detailed mathematical models of microdialysis that contain descriptions of diŠusion, metabolism and cellular and capillary exchange processes. In 1991, we proposed new and practical method for estimating the ``true'' concentration in the brain ISF by using a reference compound. This method was named ``the reference method,''21) and has largely overcome the above problems. The Reference Method The classical method to estimate the brain ISF concentration of a compound is to use the ``in vitro recovery.'' The in vitro recovery is measured in buŠer solution including a known concentration of a compound, and is deˆned as the ratio of the concentration in the dialysate to that in the buŠer solution. The in vitro recovery method is based on the assumption that ``in vivo recovery'' when the probe is positioned in the brain ISF is equal to ``in vitro recovery''. However, this assumption is invalid. Therefore, the use of the in vitro recovery method leads to underestimation of the ``true'' brain ISF concentration. To solve this problem, we introduced the terms ``permeability rate constant (PA)'' and ``eŠective dialysis coe‹cient (Rd )'' into the reference method.21) The term ``PA'' represents the intrinsic permeability across the semi-permeable membrane. The reciprocal of PA is expressed as the sum of the reciprocal of the permeability rate constants of the membrane (PAm ), and of the unstirred water layers at the inner side (PAin ) and at the outer side of the probe (PAout ). 1 1 1 1 ＝ + + PAvitro PAin PAm PAvitro,out

(1)

1 1 1 1 ＝ + + PAvivo PAin PAm PAvivo,out

(2)

where PAvitro and PAvivo represent the permeability rate constants in aqueous medium and the brain ISF, respectively. It has been reported that the resistance at the outer side of the probe membrane, i.e., diŠusion in the unstirred water layer around the probe, is the rate-limiting process.19–20) Therefore, PAvitro and PAvivo are approxi-

397

Microdialysis in the BBB Transport Study Table 1.

Comparison between the reference and recovery methods for estimating the unbound concentration in the brain ISF (From ref. (14)) Unbound concentration in ISF (mg W mL)

Flow rate (mL W min) Aminopyrine 2.5 10.0 50.0 CaŠeine 2.5 10.0 50.0

Dialysate (mg W mL)

Rd

Predicteda)

Observed Reference method

Recovery method

11.9±0.4 (9) 3.68±0.07 (9) 0.698±0.015(9)

0.491±0.023(9) 0.537±0.023(9) 0.496±0.038(9)

90.8±3.3(3) 97.8±4.6(3) 90.1±4.9(3)

112±3 (9) 108±3 (9) 102±4 (9)

58.3±1.7(9) 60.9±1.4(9) 51.6±1.2(9)

5.90±0.22 (9) 2.02±0.04 (9) 0.374±0.006(9)

0.399±0.014(9) 0.435±0.010(9) 0.382±0.008(9)

36.4±1.9(3) 41.0±1.3(3) 41.3±2.3(3)

41.4±1.4(9) 47.1±1.0(9) 46.7±0.9(9)

18.1±0.9(9) 22.0±0.8(9) 17.3±0.6(9)

Values are the mean±S.E., with the number of points being indicated in parentheses. a) Values determined based on the assumption that the unbound concentration in the plasma is equal to that in ISF.

compounds in erythrocyte suspension.21) As shown in Fig. 1, a signiˆcant positive correlation was obtained between Rd and extracellular space of the erythrocyte suspension, suggesting that Rd is mainly dependent on the extracellular space. Interestingly, the Rd value was insensitive to the molecular weight and the plasma membrane permeability of compounds. Based on these ˆndings and clearance theory in pharmacokinetics, we derived the following equation to estimate brain ISF concentration (Cisf ): Cisf＝ Fig. 1. Relationship between the eŠective dialysis coe‹cient in the erythrocyte suspension (Rd,erythrocyte ) and the extracellular space (z) (From ref. (21)). Each point represents the mean of three to ˆve determinations. Compounds: 3H-water (), antipyrine (), 14C-sucrose (#), and 125Iebiratide ().

mated to PAvitro,out and PAvivo,out, respectively. The term Rd deˆned as the ratio of PAvivo and PAvitro then becomes an indicator which quantitatively explains the diŠerence between the permeability rates through the probe membrane in vitro and in vivo. R d＝

PAvivo PAvitro

(3)

Rd can also be expressed in terms of the diŠusion parameters. R d＝ a

Svivo Lvitro g Svitro l2Lvivo b

(4)

Where S, L, a and l2 are the eŠective surface area of the microdialysis probe, the diŠusion distance, the extracellular space and the tortuosity factor. g W b is the ratio of the diŠusion coe‹cients in vitro and in vivo. Based on eq. (3), we have determined the Rd values of several

Cd 1„exp („Rd PAvitro W F)

(5)

where Cd is the dialysate concentration and F is the perfusion rate. Usually, the Rd value of a compound of interest is not known. Therefore, this value is assumed to be equal to that of a reference compound, Rd,ref, because the value is insensitive to the molecular weight and plasma membrane permeability of compounds, as described above. Either antipyrine or H2 O is usually selected as a reference compound, since these compounds do not bind to the constituents of plasma and brain tissue, and readily pass through the BBB. Thus, the brain ISF concentration of these reference compounds can be predicted from the plasma concentration, and the Rd,ref value can be easily estimated. Using the Rd,ref value obtained with antipyrine, we demonstrated that the ``true'' concentrations of aminopyrine and caŠeine in the brain ISF can be successfully estimated from dialysate samples in a microdialysis study (Table 1).14) Furthermore, this method has been veriˆed to be useful and to provide reliable data for muscle tissue,21) liver22) and lung.22) Bungay et al.20) suggest that the diŠusion of a compound in the tissue is in‰uenced by metabolism in the brain ISF, uptake by the brain cells, and transport across the BBB. Therefore, the ISF concentration estimated using Rd,ref of antipyrine should be recognized as a good approximation to a real value.

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Yoshiharu DEGUCHI

Fig. 2. Schematic model for the kinetic analysis of the brain interstitial ‰uid (ISF) concentration of drug. Arrows represent (a) in‰ux rate of a drug from the blood circulation to the brain ISF across the BBB, (b) eŒux rate of a drug from the brain ISF to the blood circulation across the BBB, (c) exchange rate of a drug between the brain ISF and the nerve cell, (d) disappearance rates of a drug from the brain ISF, including metabolism, diŠusion and bulk ‰ow.

Application of the Brain Microdialysis Technique to BBB Transport Studies As shown in Fig. 2, the brain ISF concentration of drugs after systemic administration is governed by several factors, including 1) the extent of plasma protein binding, 2) the rate of in‰ux and eŒux transport processes between brain ISF and plasma across the BBB or the blood-cerebrospinal ‰uid barrier (BCSFB), and W 3) the exchange rates between brain cells and brain ISF, 4) the extent of binding to the brain cells, 5) the metabolic rate in the brain, and 6) the diŠusion rate in the brain ISF space. The brain ISF concentration of drugs for which process 5 can be ignored, can be approximated to eq. (6) from a practical viewpoint. CL dCisf CLin ＝ C „ out Cisf dt Vd p,u Vd

(6)

where CLin is the in‰ux clearance from blood to brain across the BBB, CLout is the eŒux clearance from brain to blood, Cp,u is the unbound plasma concentration, and Vd is the volume of distribution in the brain. Vd is also deˆned as the ratio of the drug amount in the brain tissue to the concentration in the brain ISF, and it includes processes 3 and 4. Although process 6 is included in CLout, the rate is normally slower than the eŒux clearance across the BBB. Under a steady state condition, the following relation is obtained. Cisf,ss CLin ＝ Cpu,ss CLout

(7)

If CLin is estimated by means of other techniques such

as the intravenous injection W integration analysis method,23) or the brain perfusion technique,24) CLout is separately evaluated. Integration of eq. (6) yields the following relationship: AUCisf CLin ＝ AUCp,u CLout

(8)

In this equation, AUCp,u and AUCisf are the areas under the time curves of plasma unbound drug concentration and brain interstitial ‰uid drug concentration, respectively. The value of Cisf,ss W Cpu,ss or AUCisf W AUCp,u can be calculated from the results of the brain microdialysis study, and has the following implications with regard to the transport mechanism at the BBB.79) (a) When Cisf,ss W Cpu,ss＝1 or AUCisf W AUCp,u＝1, CLin ＝CLout. The transport mechanism by passive diŠusion across the BBB is suggested. Also, the facilitated transport by a transporter, which does not require energy, will be suggested. Cpu,ssº1 or AUCisf W AUCp,uº1, (b) When Cisf,ss W CLinºCLout. Active or e‹cient eŒux transport from the brain ISF to the blood circulation across the BBB is the most probable explanation. However, care is needed in interpreting the results. The value of Cisf,ss W Cpu,ss or AUCisf W AUCp,u would decrease from unity when the metabolic rate and W or eŒux rate from the CSF contribute substantially to the over-all elimination rate from the brain, even if a drug is transported via passive diŠusion at the BBB. Hammarlund-Udenaes et al.79) have shown that a low AUC ratio might also be caused

399

Microdialysis in the BBB Transport Study Table 2.

Characterization of the BBB transport of drugs by the brain microdialysis technique

Drugs Passive or facilitated transport type Aminopyrine CaŠeine Carbamazepine Carbamazepine-epoxide Cocaine Codeine Phenytoin EŒux transport type Alovudine Baclofen Colchicine Cyclosporine A Gabapentin Ketoprofen 6-mercaptopurine Morphine Morphine-3-glucuronide Morphine-6-glucuronide Probenecid Quinidine Quinolone antimicrobials Fleroxacin Nor‰oxacin O‰oxacin Pe‰oxacin Spar‰oxacin Valproic acid Zidovudine Endocytosis type E-2078 Ebiratide

Cisf,ss W Cpu,ss or AUCp,u AUCisf W

Species

Calibration method

1.13 1.13 1.12±0.27 1.06±0.12 1.20±0.18 1.00±0.18 0.84¿0.87

Rat Rat Human Human Rat Rat Human

Reference method Reference method Flow rate method Flow rate method Zero-‰ow method Retrodialysis method Flow rate method

14 14 25 25 80 26 27

0.285 0.04 º0.04 — 0.13 0.15 0.0413±0.0091 0.9 0.3 0.08±0.02 0.22±0.09 0.20±0.02 —

Rat Rat Rat Rat Rat Mice Rat Mdr la knockout mide wild-type mice Rats Rat Rat Rat

Retrodialysis method Reference method Zero-‰ow method Reference method Retrodialysis method Reference method Reference method Retrodialysis method

75 23 78 39 58 61 48 37

Retrodialysis method Reference method Bungay's model

60 59 24 76

0.147±0.012 0.034±0.011 0.118±0.041 0.147±0.004 1.45 0.277 º0.03 0.20±0.05

Rat Rat Rat Rat Mdr la knockout mice wild-type mice

Reference method Reference method Reference method Reference method Dynamic-no-net-‰ux

57 57 57 57 38

Rabbit

Retrodialysis method Retrodialysis method

77 41

— —

Rat Rat

— Reference method

65 67

by active in‰ux transport across the BBB under certain circumstances. This is less probable for the majority of drugs.79) (c) When Cisf,ss W Cpu,ssÀ1 or AUCisf W AUCp,uÀ1, CLinÀCLout. Active or e‹cient in‰ux transport from the blood circulation to the brain ISF across the BBB is a probable explanation. Unfortunately, there are no reports about drugs that are classiˆed into this case. In this review, the results obtained in microdialysis experiments with various drugs have been analyzed by means of eq. (7) or eq. (8) and listed in Table 2. They are classiˆed into 1) passive transport type and 2) eŒux Cpu,ss or transport type, based on the value of Cisf,ss W AUCisf W AUCp,u. In addition, drugs which pass the BBB through the endocytosis mechanism are classiˆed into 3) endocytosis type. Passive Transport Type As shown in Table 2, examples of drugs whose concentration in the brain ISF is the same as their

Reference

plasma unbound concentration include aminopyrine,14) caŠeine,14) cocain80) and codeine26) in rats, and carbamazepine25) and phenytoin27) in human. Aminopyrine and codeine permeate the BBB, probably by a simple diŠusion mechanism. On the other hand, transportermediated system was suggested for caŠeine, carbamazepine and phenytoin.28–30) The results suggest that for this type of drug, the unbound drug concentration in the plasma is an important indicator of pharmacological e‹cacy in the brain, because the drug concentration in the brain ISF correlates with the degree of receptor binding. This is particularly useful in analyzing the antiepileptic action of phenytoin, which shows a nonlinear pharmacokinetic proˆle and has a narrow therapeutic plasma concentration range. However, there is a few reports that phenytoin and carbamazepine as well as phenobarbital might be eŒuxed via P-gp or a multidrug resistance-associated protein (MRP) in rats.29–31) Drugs that are bound avidly by plasma proteins have been suggested to be transported through the BBB by ``plasma protein-mediated transport.'' This type of

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Yoshiharu DEGUCHI

transport is based on the proposal that dissociation of a drug from the drug-protein complex would be enhanced by interaction with the surface of the brain capillaries.32,33) If the BBB transport of a drug follows this mechanism, the drug concentration in the brain ISF is equal to the plasma total drug concentration, but not the plasma unbound drug concentration. The transport of diazepam into the brain has been reported to follow this mechanism.34) In contrast, Dubey et al., using the brain microdialysis technique, compared the concentration of diazepam in the brain ISF of normal rats and that of genetically analbuminemic rats (Nagase analbuminemic rats).35) However, the concentration in brain ISF was equal to the plasma unbound concentration in either normal rats or analbuminemic rats, suggesting at least that a protein-mediated transport mechanism is not involved in the BBB transport of diazepam. EŒux Transport Type In this review, drugs for which the value of Cisf,ss W Cpu,ss or AUCisf W AUCp,u was smaller than 1 are classiˆed into this category. In particular, the drug for which the value of Cisf,ss W Cpu,ss or AUCisf W AUCp,u is smaller than 0.1 are considered to be actively eŒuxed through the BBB from the brain. Lange et al. applied brain microdialysis to knockout mice deˆcient in Mdr 1a, a type of P-gp. After the intravenous administration of rhodamine 123, a substrate of P-gp, to Mdr 1a knockout mice and wild-type mice, concentrations in the brain ISF were compared.36) The concentration of rhodamine 123 in the brain ISF was 4.4 times higher in the Mdr 1a knockout mice (15.7±7.8 nM) than in the wild-type (3.6±2.1 nM), suggesting the involvement of P-gp in eŒux transport of rhodamine 123 across the BBB. In this study, they found that in vivo recovery of rhodamine 123 in Mdr 1a knockout mice is 3.5-fold lower than that in the wild-type mice. This results suggests that loss of P-gp results in a higher resistance to tissue mass transfer of a P-gp substrate from the brain ISF to the probe, in accordance with the microdialysis theory of Bungay et al.20) This result also indicates that care is necessary when BBB transport function under a pathological condition is analyzed by the microdialysis technique. This methodology has also been applied to morphine37) and spar‰oxacin,38) a quinolone antibacterial agent, and eŒux transport by P-gp has been clearly shown to play an important role in lowering the concentrations of both drugs in the brain ISF. In contrast, there were no signiˆcant diŠerences in the brain ISF concentration of morphine-3-glucuronide (M3G) between Mdr 1a knockout mice and wild-type mice, suggesting that P-gp may not be involved in eŒux transport of M3G across the BBB.37) It has been reported that a probenecid-sensitive transport system may be involved

in the transport of M3G.60) We examined the eŠect of quinidine, an MDR-reversing agent, on the BBB eŒux transport of cyclosporin A using the microdialysis technique.39) When quinidine was infused into the brain through a microdialysis probe implanted in the rat hippocampus, the eŒux rate of cyclosporin A from the brain ISF was increased 2.5-fold over the control. This result suggests that P-gp mediates the eŒux transport of cyclosporin A at the BBB. Various eŒux transport systems at the BBB, in addition to P-gp have been demonstrated to restrict the transport of drugs into the brain. The anti-HIV drug, azidothymidine (AZT), has a low e‹cacy against intracerebral HIV virus due to its restricted distribution to the brain. The cause of this has been believed to be the low uptake rate through the BBB due to the low lipophilicity of AZT.40) Wong et al. analyzed the brain ISF concentration of AZT using brain microdialysis in rabbits, and suggested that AZT is e‹ciently eŒuxed from the brain through the BBB.41) In addition, Dykstra et al. analyzed the results of microdialysis experiments based on ``a distributed model'' that incorporated the diŠusion process within the brain, and suggested that an active eŒux transporter is involved in the e‹cient eŒux of AZT across the BBB.42) Takasawa et al. examined the mechanism of the AZT eŒux transport using the brain eŒux index method. They showed that the AZT eŒux transport was inhibited by dideoxyinosine, PAH and benzylpenicillin, but not by thymidine.43) From these results, it was suggested that a probenecid-sensitive active transporter is involved in the eŒux of AZT through the BBB. Baclofen is a GABA derivative that is used clinically as a centrally acting antispastic agent, and is characterized as a typical GABAB receptor agonist. Figure 3 shows the results of a brain microdialysis experiment in baclofen-treated rats.23) The ISF concentration of baclofen in the hippocampus region was low, being only about 1 W 25 of that in the plasma. In addition, the concentrations of baclofen in both CSF and brain tissues were also low, showing that the distribution of baclofen to the brain is restricted. In contrast, when probenecid was administered directly into the brain through the microdialysis probe 60 min after the intravenous administration of baclofen, the concentration of baclofen in the brain ISF increased with increasing concentration of probenecid. Thus, the cause of the restricted distribution of baclofen to the brain was suggested to be not a low rate of uptake through the BBB, but rather the involvement of a probenecid-sensitive eŒux transport system at the BBB. Van Bree et al. reported that a saturable neutral amino acids carrier is involved in the uptake of baclofen in the luminal side of the brain capillaries, based on a study using cultured brain capillary endothelial cells.44) This carrier has been recently identiˆed

Microdialysis in the BBB Transport Study

Fig. 3. EŠect of probenecid on the concentration of baclofen in plasma and hippocampal ISF in rats. Microdialysis probe was perfused with Krebs-Ringer phosphate buŠer (KRP) for 60 min after an i.v. administration at a dose of 50 mg W kg (period a). Thereafter, the perfusion solution was changed to KRP containing 30 mM or 120 mM probenecid, and the probe was perfused for 120 min (period b). The dotted line represents the concentration level in control rats. () plasma and () ISF for 30 mM probenecid. () plasma and () ISF for 120 mM probenecid. From ref. (23) with kind permission of Plenum Press Corporation, New York, USA.

401

at the BBB as the large neutral amino acid transporter type 1 isoform (LAT1).45) The LAT1 protein is the light chain of a heterodimer formed with the heavy chain, 4F2hc.46) The LAT1 mRNA is 100-fold more abundant at the BBB than any other tissue.45) It has not been clari4F2hc at the BBB transports ˆed whether LAT1 W baclofen in the direction from the brain ISF to the blood circulation. 6-Mercaptopurine (6-MP) has long been used as a drug for the treatment of acute lymphatic leukemia (ALL) in children. Although 6-MP is administered during the remission period when the patient's condition has stabilized, proliferation of leukemia cells that have migrated to the brain occurs in about 10z of the patients, resulting in relapse of ALL.47) A possible cause of this central relapse is restricted distribution of 6-MP to the brain, so that the brain concentration of 6-MP is not su‹ciently high to be eŠective. The concentration of 20th of 6-MP in the brain ISF was limited to about 1 W the unbound concentration in plasma.48) Moreover, the concentration of 6-MP in the brain ISF after intravenous administration was signiˆcantly increased by the intracerebral administration of several organic anions, including probenecid, PAH, benzoic acid and salicylic acid (Fig. 4). On the other hand, organic cations, such as choline and tetraethylammonium, did not aŠect the 6-MP concentration in the brain ISF. These results

Fig. 4. EŠects of several inhibitors on the concentration of 6-mercaptopurine in brain ISF at the steady state. Vertical line is shown as the ratio of brain ISF concentration and plasma unbound concentration at the steady state (Cisf,ss W Cpu,ss ). Open bars and ˆlled bars represent the results in the control rats and the rats treated with inhibitors, respectively. ***pº0.001 vs. control in Student's t-test.

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Yoshiharu DEGUCHI

Fig. 5. Disappearance curves of probenecid and methylaminoisobutyric acid (MeAIB) from the hippocampal ISF in rats (a) and an agar gel plate (b). The microdialysis probe was perfused with Krebs-Ringer phosphate (KRP) buŠer containing 120 mM probenecid, or 308.3 kBq W mL 14CMeAIB plus 100 mM unlabeled MeAIB for 30 min. Then, the perfusion buŠer was changed to KRP without probenecid or MeAIB. The dialysate was collected every 10 min for 120 min. The concentration in brain ISF (Cisf ) or the concentration in an agar plate (Cagar ), which are estimated by the reference method, are normalized with the concentration in the perfusate given initially into the brain ISF or an agar plate (Cd,inlet). () and () indicate probenecid and MeAIB, respectively. From reference (24).

suggest that 6-MP is eŒuxed from the brain ISF by the organic anion transport system at the BBB. Multidrug resistance-associated protein 5 (MRP5) is expressed in primary cultured bovine brain endothelial cells49) and 6-MP is suggested to be a substrate of the MRP5.50) In addition, PAH and salicylic acid are substrates or inhibitors of organic anion transporter (OAT) family,51) and OAT1 and OAT3 are suggested to exist in the brain and the BBB.52,53) Moreover, benzoic acid is a substrate of the monocarboxylic acid transport system that transports lactate, pyruvate and acetate at the BBB. A proton-coupled monocarboxylate transporter (MCT1) is selectively expressed at the BBB,54,55) although MCT2, MCT6, MCT7 and MCT8, as well as MCT1, are expressed over the whole brain.56) This transporter is localized at both the luminal and abluminal membranes of the brain capillary endothelial cells and is involved in bidirectional L-lactate transport at the BBB.56) It also may play a role in L-lactate eŒux transport from brain to detoxify excess L-lactate. It is therefore necessary to elucidate the molecular mechanism of BBB transport of 6-MP, although we cannot rule out the possibility that these transporters simultaneously transport 6-MP. Very recently, it was reported that 6-MP and baclofen both inhibit the eŒux transport of indoxyl sulfate, a substrate of OAT3, through the BBB.53) This result raises the possibility that 6-MP and baclofen are both eŒuxed from the brain ISF via the OAT3-mediated transport system. Future studies will be necessary to resolve this issue. Moreover, it is anticipated that concomitant use of 6-MP and a speciˆc inhibitor will increase the 6-MP concentration in the brain ISF, thereby making it possible to suppress central relapse in ALL patients. Probenecid is frequently used as an inhibitor of the BBB eŒux transport system. We have examined the

BBB eŒux transport system of probenecid using the microdialysis technique.24) One of the characteristics of brain microdialysis is that it can be used to analyze the eŒux transport process across the BBB, by measuring the concentration of a drug recovered in the dialysate after an intracerebral administration of a drug.76) The concentration of a drug in the dialysate is in‰uenced not only by the BBB eŒux transport process, but also by diŠusion in the brain, particularly under non-equilibrium conditions. Therefore, the eŠect of the diŠusion process in the brain should be analyzed by using a compound that is impermeable at the BBB, such as methylaminoisobutyric acid. As shown in Fig. 5, the apparent elimination rate constants in rat hippocampus for probenecid and 14C-methylaminoisobutyric acid were 0.0456 min„1 and 0.0144 min„1, respectively, demonstrating a 3.2-fold diŠerence between the two compounds. On the other hand, in an agar plate, in which the contribution of the eŒux process is not involved, probenecid and 14C-methylaminoisobutyric acid decreased at the same rates from the circumference of the microdialysis probe. This result suggests that the diŠusion rate in the vicinity of the probe is the same for the two compounds. Since the probenecid is not metabolized in the brain, the diŠerence in the slope of Fig. 5 is suggested to be primarily due to the eŒux rate of probenecid across the BBB. This eŒux transport of probenecid was inhibited by monocarboxylate drugs, such as salicylic acid and benzoic acid. Quinolone antibacterial such as nor‰oxacin and o‰oxacin,57) an antiepileptic drug, gabapentin,58) and ketoprofen61) have low concentrations in the brain ISF. Microdialysis and pharmacokinetic examinations have suggested that these are transported from the brain ISF across the BBB by active eŒux mechanism, although

Microdialysis in the BBB Transport Study Table 3. Concentration ratio of the brain ISF to perfusate and the CSF to perfusate of 125I-E-2078 (From ref. (65)) 125

I-E-2078

ISF W perfusate CSF W perfusate

0.292±0.050 0.00296±0.00026

14

C-Sucrose

0.00271±0.00143 0.00132±0.00043

Values are the mean±S.E. of three rats. Brain microdialysis was performed with brain perfusion for 10 min. The concentrations of 125E-2089 and 14C-sucrose in the brain interstitial ‰uid (ISF) were determined using the respective in vitro recoveries by the analysis of the brain dialysate. Cerebrospinal ‰uid (CSF) was obtained by cisternal puncture.

transporters and W or transport system have not been fully clariˆed yet. Recently, Sun et al. demonstrated by the use of the brain microdialysis that MRPs or MRP-like transport system(s) may play an important role in ‰uorescein distribution across the BBB and BCSFB.62) Moreover, endogenous quinolinic acid has been suggested to be recognized by the probenecid-sensitive organic anion transport system at the BBB,63) while aluminum citrate recognized by the monocarboxylic acid transport system.64) Endocytosis Type Entry of hydrophilic peptides and proteins into the brain is usually blocked by the BBB. Furthermore, enzymatic instability of peptides in the circulating blood also makes it di‹cult to deliver them into the brain. On the other hand, some biologically and neurologically active peptides exhibit CNS eŠects after the systemic administration, suggesting that these peptides are taken up by the brain in pharmacologically signiˆcant amounts through the BBB route or the BCSFB route or both. Measurement of peptide concentration in the brain ISF using the microdialysis technique would provide useful information in this regard. We have applied the microdialysis technique to demonstrate the transport of a dynorphin-like analgesic peptide (E-2078) across the BBB. This peptide was developed to overcome the instability of the native opioid peptide, dynorphin-(1-8), to peptidases. The brain of rats ˆtted with a microdialysis probe was perfused with 125 I-E-2078 and 14C-sucrose (an impermeable BBB marker) using the brain perfusion technique.65) The dialysate ‰owing out of the microdialysis probe was collected during the brain perfusion of 125I-E-2078. At the end of the perfusion, CSF was obtained by the cisternal puncture technique. The concentration ratio of 125I-E-2078 in brain ISF versus the perfusate was approximately 100-fold greater than that of 14C-sucrose, whereas that in CSF versus the perfusate was only 2-fold greater than that of 14C-sucrose (Table 3). These results demonstrate clearly that E-2078 is transported from the blood circulation to the brain ISF, and that the main pathway is the

403

BBB route. E-2078 is a basic peptide of isoelectric point 10.0. The results of transport experiments using isolated brain capillaries have shown that E-2078 is internalized in the brain capillary endothelial cells via the adsorptivemediated endocytosis (AME) mechanism.66) AME is triggered by an electrostatic interaction between a positively charged site of the peptide and a negatively charged region on the surface of the brain capillary endothelial cells. Thus, the results of the microdialysis experiments provided in vivo evidence of the transcytosis of E-2078 across the BBB. Similarly, the BBB transport of ebiratide, a synthetic peptide analogous to adrenocorticotropic hormone that is used to treat Alzheimer's disease, was demonstrated to occur via the AME mechanism.67) AME is system with lower a‹nity and higher capacity in comparison with the receptor-mediated endocytosis system through which transferrin, leptin, insulin.68) It also carries basic ˆbroblast growth factor (bFGF) into the brain.69) Therefore, the AME system may be useful for the delivery of therapeutic peptides and proteins to the brain. Other Important Applications of Brain Microdialysis The barrier function of the BBB can change during various diseases of the CNS e.g., in the presence of a brain tumor and during Alzheimer's disease, or during cerebral in‰ammation such as multiple sclerosis.70) The brain microdialysis technique oŠers the possibility to investigate changes in local barrier conditions in such pathophysiological states. Several investigators have demonstrated that the BBB penetration of anticancer drugs increases in the tumor bearing brain.7,71) Recently, Planas et al.72) demonstrated, using brain microdialysis, that matrix metalloproteinases, which are involved in BBB breakdown and tissue remodeling, are released and accumulated in the brain ISF after brain injury. This result suggests that microdialysis may be also a useful technique to clarify the mechanism maintaining the barrier function of the BBB. Very recently, we suggested that bFGF is internalized via HSPG, which is expressed on the abluminal membrane of the BBB, and that HSPG may play a role in maintaining the BBB function due to acceptance of the bFGF secreted from astrocyte.73) As well as the case of metalloproteinase, bFGF may be released from the foot process of astrocyte when the BBB was destroyed. The direct measurement of bFGF by brain microdialysis will provide signiˆcant information on this hypothesis. Although brain microdialysis has mainly been applied to animal studies, recent studies have demonstrated that brain microdialysis might be a valuable tool for the supervision of patients during neurointensive care.9,74) When the brain is subjected to traumatic damage or brain ischemia, the levels of endogenous substances, such as lactate, pyruvate, and glycerol in the brain ISF

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can change with the death of nerve cells and expansion of the region of brain injury. Therefore, these substances are considered to be surrogate markers to monitor the progress of disease. Ungerstedt et al. reported that drug therapy while monitoring surrogate markers by means of brain microdialysis successfully reduced neuronal cell damage in severely traumatized brain.74)

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Concluding Remarks Recent advances in studies on BBB research have led to a new understanding of drug transport processes at the BBB. The BBB acts as a dynamic regulatory interface at which compounds necessary for neural activity are e‹ciently taken up into the brain from the blood circulation. Furthermore, the BBB actively excludes the compounds that might interface with the maintenance of brain homeostasis. This review had sought to show that the brain microdialysis technique is a useful tool for characterizing in detail the transport functions of drugs at this regulatory interface, and especially for evaluating both in‰ux and eŒux transport of drugs under various physiological and pathological conditions. It has been almost 25 years since the brain microdialysis was initially developed. Further technical advances should lead to more widespread application of brain microdialysis in the ˆelds of drug metabolism and pharmacokinetics, as well as drug discovery and drug delivery. It is also expected to become available for monitoring pharmacotherapy with centrally acting drugs in severely ill patients. Acknowledgements: The author would like to thank Professor Akira Tsuji, Graduate School of Pharmaceutical Sciences, Kanazawa University, Professor Ryohei Kimura, School of Pharmaceutical Sciences, University of Shizuoka, and Professor Tetsuya Terasaki, Graduate School of Pharmaceutical Sciences, Tohoku University for their tremendous encouragement and helpful advice. Thanks are also due to Professor William M. Pardridge, UCLA School of Medicine and Associate Professor Shizuo Yamada, University of Shizuoka, for helpful discussions, as well as to Professor Kazuhiro Morimoto, Hokkaido College of Pharmacy, and many colleagues who extended their assistance. This work was supported in part by a Grant-in-Aid for Scientiˆc Research (C) provided by the Ministry of Education, Science, Sports and Culture of Japan.

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