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Physiological pharmacokinetics and membrane transport for drug delivery research
International Congress Series 1284 (2005) 266 – 273
www.ics-elsevier.com
Physiological pharmacokinetics and membrane transport for drug delivery research Tetsuya Terasaki*, Sumio Ohtsuki Graduate School of Pharmaceutical Sciences, and New Industry Creation Hatchery Center (NICHe), Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan Solution-Oriented Research for Science and Technology (SORST), Japan Science and Technology Agency (JST), Japan
Abstract. In the case of patients undergoing dentistry and oral surgery, several drugs, including anaesthetics, antibiotics, analgesics, muscle relaxants, steroids, antidepressants and anticancer drugs are administered. Physiological pharmacokinetics is the best way to integrate the processes of drug absorption, distribution, metabolism and excretion (ADME). We have developed physiological pharmacokinetic models for h-lactam antibiotics, quinolone antibiotics, a muscle relaxant and anticancer drugs. These models enable us to compare the characteristics of drug ADME among different derivatives and to predict drug concentrations in target organs. As far as the factors affecting drug ADME are concerned, plasma membrane transport is one of the most important. The molecular mechanism of drug transport is very important as far as the bioavailability and targeting efficiency are concerned. Members of the ATP binding cassette (ABC) transporter superfamily, such as ABCB1, ABCC4 and ABCG2 have been shown to play an important role in drug efflux as a part of detoxifying system in the body. The expression and function of the transporters is not static, but can be up- or downregulated by several factors including oxidative stress, osmotic pressure and substrates of the transporter as well as by cell-to-cell interactions. Thus, plasma membrane transporters can act as a part of the dynamic interface in the body. For safe, efficient and rational drug development and treatment, it is important to understand the physiological role of these transporters. D 2005 Elsevier B.V. All rights reserved. Keywords: Physiological pharmacokinetics; Drug delivery; Transport; ABC transporter
* Corresponding author. Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan. Tel.: +81 22 795 6831; fax: +81 22 795 6886. E-mail address: [email protected] (T. Terasaki). 0531-5131/ D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2005.06.070
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1. Introduction A number of drugs are routinely used in dentistry and oral surgery, such as anaesthetics, antibiotics, analgesics, muscle relaxants, steroids, antidepressants, and anticancer drugs. For safe and effective drug therapy, it is important to understand the molecular mechanisms of drug distribution in the body and the pharmacokinetics integrating all the process in the body after administration, such as absorption, distribution, metabolism and excretion (ADME). Table 1 shows a list of the possible factors that affect drug distribution. For the rational development of new drugs, it is important to understand their major elimination pathways and the rate-limiting process for candidate compounds as well as factors affecting distribution to the target organ(s). In this review, we shall highlight the importance of physiological pharmacokinetics in analyzing this drug distribution mechanism. As plasma membrane transport is one of the most important factors involved in drug delivery to the target organ, we will also discuss the importance of the molecular mechanism and physiological role of plasma membrane transport. 2. Physiological pharmacokinetics and tissue distribution mechanism The drug concentration is the most important factor governing the therapeutic effect. If the plasma membrane receptors or the ecto-enzyme of the plasma membrane are a site of drug action, the interstitial fluid drug concentration may be directly related to the therapeutic effect. If the intracellular enzyme, protein and nucleic acid are a site of drug action, the intracellular drug concentration is directly related to this. Therefore, it is very important to identify the molecular mechanism governing drug distribution to the target organ. h-Lactam antibiotics are substrates of the oligopeptide transporters, PEPT1 and PEPT2, and the organic anion transporter, OAT3. Small intestinal epithelial cells express PEPT1, while renal epithelial cell express PEPT2 and OAT3, and h-lactam antibiotics can be transported to these organs by such transporters. In contrast, as the passive diffusion rate of h-lactam antibiotics in the plasma membrane is low, the drug cannot cross the plasma membrane in the muscle, heart, lung, skin and fat. Fig. 1A illustrates an extracellular localization model used as a physiological pharmacokinetic model to predict the distrubution of h-lactam antibiotics in the body [1,2]. The volume of extracellular fluid and plasma is the determinant factors for the distribution volume of h-lactam antibiotics. Depending on the age, the extracellular fluid volume of muscle changes significantly. Physiological pharmacokinetics based on the model shown in Fig. 1A can accurately Table 1 Possible factors affecting drug distribution Process
Factors
Absorption Distribution Metabolism Excretion
Membrane permeability, first-pass metabolism, solubility, gastric emptying time, food, lumen pH Membrane permeability, plasma protein binding, tissue binding Enzyme affinity and amount, membrane permeability, tissue binding Membrane permeability, plasma binding, bile flow rate, urine pH
Blood flow rate and tissue volume are the factors that can be involved in all the processes.
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Fig. 1. Schematic diagram of drug distribution in the organ. A: Extracellular localization model; B: Intracellular distribution model.
predict the organ distribution in rats that are 1, 7, 50 and 100 weeks old [3]. The unbound h-lactam antibiotic concentration in the interstitial fluid of an infected organ will be very similar to that in the plasma after systemic administration. If the permeability rate across the capillary is not significantly low, Fig. 1A may be used for the prediction of the pharmacokinetics of physiological peptides such as growth hormone. If the drug can cross the plasma membrane, an intracellular distribution model, shown in Fig. 1B, can be used for this analysis [4]. In this case, the plasma unbound fraction, tissue unbound fraction and plasma membrane permeability are the determinant factors for the organ distribution. A concentration gradient of unbound drug between the intracellular and extracellular fluid will be generated by the ratio of the influx permeability rate and the efflux permeability rate across the plasma membrane. If a drug is able to permeate the plasma membrane by passive diffusion, there will be no concentration gradient in usual cases. If a drug is taken up into the cells by an active influx transporter, the intracellular unbound drug will be significantly higher than that in the extracellular fluid and plasma. If a drug can be pumped out of the cell by the active efflux transporter, the intracellular unbound drug concentration will be significantly lower than that in the extracellular fluid and plasma. Therefore, if intracellular proteins or nucleic acids are drug target molecules, the plasma membrane transport mechanism of the target organ will play a very important role in determining the pharmacological effect. The intracellular distribution model in Fig. 1B was validated by examining the differential distribution of doxorubicin among the organs [4] in rats, guinea pigs and rabbits. The differential distribution of quinolone antibacterials, among the derivatives, was also analyzed by a similar model. The results showed that there was no significant difference in tissue binding among the quinolone derivatives and the lower plasma protein binding of drug resulted in a higher concentration in the tissues, except in the brain [5]. If the plasma membrane permeability is not saturable and very low, the plasma unbound drug concentration is proportional to the unbound drug concentration in the cell of target organ, and this acts as an appropriate marker of the drug effect. The organ blood flow rate may be a determinant factor of the time-dependence of drug distribution. Inaperison, a muscle-relaxant, distributes in the brain and causes a significant increase in the blood flow rate in muscle and fat. The rapid distribution of inaperison in the muscle and fat is attributed to the enhanced blood flow rate in these tissues [6].
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In dentistry and oral surgery, most drugs are administered locally. The drug diffusion in tissues is also a determinant of the retention and elimination of the drug in oral tissues. Macromolecules may be significantly retained in these tissues where the drug is injected [7], if the capillary permeability is significantly restricted. 3. Mechanism of plasma membrane transport and its importance for drug distribution The modes of plasma membrane transport can be classified as follows: passive diffusion, facilitated transport, primary active transport and secondary active transport (synport and antiport). The drug transport rate of passive diffusion increases in parallel with drug lipophilicity and inversely with the square root of molecular size. When the partition coefficient between n-octanol and water is greater than 1, the drug can easily cross the plasma membrane by passive diffusion. Nevertheless, low solubility in water becomes problem, if the partition coefficient is greater than 4. At the plasma membrane, various transporters (solute carrier protein; SLC) operate to transport amino acids, dipeptides, glucose and lactate. Therefore, drugs recognized by these transporters can cross the plasma membrane. GLUT1/SLC2A1 is one subtype of glucose transporter. The K m (half saturation concentration) of GLUT1 is higher than the plasma glucose concentration, and GLUT1 is not saturated by plasma glucose under normal conditions. On the other hand, using saturated transporters is not an efficient method of drug delivery to target organs. l-DOPA is transported to the brain from the blood by LAT1/SLC7A5 [8], a transporter for large neutral amino acids, which is expressed at the blood–brain barrier [9]. However, LAT1 is saturated by plasma amino acids, since its K m is lower than the plasma concentration of substrate amino acids. On the brain side of the plasma membrane of brain capillary endothelial cells, organic anion transporter 3 (OAT3/SLC22A8) is expressed to excrete neurotransmitter metabolites from the brain to the blood [10] (Fig. 2). Homovanillic acid, a dopamine metabolite, is an OAT3 substrate, and, during its elimination process, OAT3 plays a major (N 90%) role in the brain side of the membrane transport. Indoxyl sulfate is a uremic toxin, which accumulates in the body of patients with renal failure, and it is excreted from the brain to the blood by OAT3 [11]. It has been suggested that OAT3 transports other uremic toxins (Fig. 2). OAT3 also accepts anionic drugs. The distribution of 6-mercaptopurine (6-MP) into the brain is low, and this low distribution has been suggested to be due to the predominant brain-to-blood efflux transport [12]. Recently, we have reported that OAT3 is involved in this efflux transport [13] (Fig. 2). In the kidney, the renal epithelial cells forming the uriniferous tubules express OAT3 and OAT1. Therefore, drugs which are OAT3 substrates tend to be excreted from the brain and kidney. P-glycoprotein (P-gp/MDR1/ABCB1) is a subtype of the ATP binding cassette (ABC) transporter superfamily, and is classified as a primary active transporter directly consuming ATP hydrolysis energy. P-gp was initially identified in cancer cells, and subsequently reported to be expressed in normal cells including brain capillary endothelial cells, small intestinal epithelial cells and hepatocytes. P-gp accepts relatively lipophilic
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Fig. 2. Physiological roles of the brain-to-blood efflux transport systems at the blood–brain barrier (BBB). The BBB expresses various brain-to-blood efflux transport systems, which excrete neurotransmitters, neurotransmitter metabolites, neuromodulators and uremic toxins. The BBB efflux transporters also act to limit drug distribution into the brain. Furthermore, the BBB possesses efflux transport system for peptides and proteins, such as apotransferrin, immunoglobulin and h-amyloid. These BBB efflux transporters function cooperatively as the CNS supporting and protecting system. Cited from Ref. [15].
drugs as substrates. If drugs are recognized by P-gp, they are excreted from the inside of cells to the outside even although the drugs can cross the plasma membrane by diffusion. This results in a very low brain distribution and low intestinal absorption of P-gp substrate drugs. In humans, 48 subtypes of ABC transporters have been identified [14]. Among them, the ABCC (MRP) family and ABCG2 have been reported to limit the tissue distribution of drugs as well as P-gp [15]. The expression of these efflux transporters in target organs is important for the purposes of drug development. In addition, even for commercially available drugs, substrate recognition of these efflux transporters is an important factor as far as drug efficacy is concerned. To date, the limiting factor in percutaneous absorption is thought to be the horny layer of the epidermis. Recently, claudin 1, a tight junction protein, has been reported to play an important role in this epidermal barrier [16]. Furthermore, it has been reported that ABCC1/MRP1 is acting as a significant efflux pump in the epithelial cells in the epidermis [17]. Therefore, the strategy for developing drug for percutaneous absorption needs to be fundamentally changed. 4. Regulation of membrane transport Membrane transport involves a number of functions and is not constant. The expression of P-gp by the brain capillary endothelial cells increase with development after birth [18]. The expression and function of TAUT and ATA2, which transport taurine and proline, respectively, is induced under hypertonic conditions in brain capillary endothelial cells
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Fig. 3. Principle of establishment of cell line. A: Traditional method using primary cultured cell and gene transfection. B: Transgenic animal method for conditionally immortalized cell line from temperature-sensitive SV40 large T-antigen gene transgenic rats or mice.
[19,20]. This induction is likely to contribute to osmoregulation in the brain. TAUT is also induced by cytokines and suppressed by excess of taurine [19]. xCT, an l-cystine and lGlu exchange transporter, is induced by oxidative stress [21]. ABCG2 expressed in the brain capillary endothelial cells is induced by soluble factors secreted from astrocytes, but not those from pericytes [22], while occludin, a tight junction protein, is induced by angiopoietin1 secreted from pericytes [23]. The changes in transporter functions and identification of the regulating factors will provide us with important information for a better understanding of drug distribution to target organs. 5. Conditionally immortalized cell lines To analyze membrane transport, in vitro cell culture systems, such as freshly prepared cells, primary cultured cells, immortalized cells and conditionally immortalized cells, are useful. Fig. 3 shows the establishment of conditionally immortalized cell lines.
Fig. 4. Comparison of the blood–brain barrier (BBB) permeability clearance between in vivo and in vitro experiments. Closed symbols and open symbols indicate the blood-to-brain influx and the brain-to-blood efflux in vivo BBB permeability clearance, respectively. The BBB permeability clearance was estimated from transport activity measured by conditionally immortalized rat and mouse brain capillary endothelial cells (TR-BBB (square) and TM-BBB (circle), respectively). The estimations were performed with the following surface area parameters: TM-BBB, 18 cm2/mg protein; TM-BBB, 22 cm2/mg protein; in vivo brain capillary, 100 cm2/g brain. 3-OMG, 3-O-methyl-glucose; DHEAS, dehydroepiandrosterone sulfate; GABA, g-aminobutylic acid; l-Pro, lproline; TAPA, H-Tyr-d-Arg-Phe-h-Ala-OH (Opioid Peptide). Cited from Ref. [24].
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Conditionally immortalized cell lines involve cell lines isolated from transgenic animals harbouring temperature sensitive SV40 large T antigen and cultured at 33 8C [24]. Using this method, it is possible to establish immortalized cell lines from very small tissues, such as the rat retina [25]. Compared with the classical method of introducing an immortalized gene into primary cultured cells, this method using transgenic animals can establish cell lines under milder conditions, and the established cells fairly retained their in vivo function. Indeed, the transport activity of a variety of compounds in conditionally immortalized brain capillary endothelial cells has been found to match that of the in vivo blood–brain barrier [24] (Fig. 4). We have established conditionally immortalized cell lines from many tissues, such as brain capillary endothelial cells, choroid plexus epithelial cells, brain pericytes and retinal capillary endothelial cells [24]. In dental research, establishing immortalized cell lines using transgenic animals will be a useful strategy for analyzing transport function, cell–cell interactions and signal transduction. 6. Conclusion Physiological pharmacokinetic models enable us to compare the characteristics of drug ADME among different derivatives and to predict drug concentrations in the target organ. The molecular mechanism of drug transport plays a very important role in determining the drug bioavailability and targeting efficiency. The plasma membrane transporters can act as a part of the dynamic interface in the body. In order to ensure safe, efficient and rational drug development and treatment, it is important to fully understand the physiological role of transporters. References [1] A. Tsuji, et al., Physiologically based pharmacokinetic model for cefazolin in rabbits and its preliminary extrapolation to man, Drug Metab. Dispos. 13 (6) (1985) 729 – 739. [2] A. Tsuji, et al., Physiologically based pharmacokinetic model for h-lactam antibiotics I: tissue distribution and elimination in rats, J. Pharm. Sci. 72 (11) (1983) 1239 – 1252. [3] A. Tsuji, et al., Age-related change in tissue-to-plasma partition coefficient of cefazolin for noneliminating organs in the rat, J. Pharm. Sci. 78 (7) (1989) 535 – 540. [4] T. Terasaki, et al., Pharmacokinetic study on the mechanism of tissue distribution of doxorubicin: interorgan and interspecies variation of tissue-to-plasma partition coefficients in rats, rabbits, and guinea pigs, J. Pharm. Sci. 73 (10) (1984) 1359 – 1363. [5] E. Okezaki, et al., Structure-tissue distribution relationship based on physiological pharmacokinetics for NY198, a new antimicrobial agent, and the related pyridonecarboxylic acids, Drug Metab. Dispos. 16 (6) (1988) 865 – 874. [6] O. Nagata, et al., Physiological pharmacokinetics of a new muscle-relaxant, inaperisone, combined with its pharmacological effect on blood flow rate, Drug Metab. Dispos. 18 (6) (1990) 902 – 910. [7] D. Hovgaard, et al., Clinical pharmacokinetic studies of a human haemopoietic growth factor, GM-CSF, Eur. J. Clin. Invest. 22 (1) (1992) 45 – 49. [8] T. Kageyama, et al., The 4F2hc/LAT1 complex transports l-DOPA across the blood–brain barrier, Brain Res. 879 (1–2) (2000) 115 – 121. [9] R.J. Boado, et al., Selective expression of the large neutral amino acid transporter at the blood–brain barrier, Proc. Natl. Acad. Sci. U. S. A. 96 (21) (1999) 12079 – 12084. [10] S. Mori, et al., Rat organic anion transporter 3 (rOAT3) is responsible for brain-to-blood efflux of homovanillic acid at the abluminal membrane of brain capillary endothelial cells, J. Cereb. Blood Flow Metab. 23 (4) (2003) 432 – 440.
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[11] S. Ohtsuki, et al., Role of blood–brain barrier organic anion transporter 3 (OAT3) in the efflux of indoxyl sulfate, a uremic toxin: its involvement in neurotransmitter metabolite clearance from the brain, J. Neurochem. 83 (1) (2002) 57 – 66. [12] Y. Deguchi, et al., Brain distribution of 6-mercaptopurine is regulated by the efflux transport system in the blood–brain barrier, Life Sci. 66 (7) (2000) 649 – 662. [13] S. Mori, et al., Organic anion transporter 3 is involved in the brain-to-blood efflux transport of thiopurine nucleobase analogs, J. Neurochem. 90 (4) (2004) 931 – 941. [14] M. Dean, T. Annilo, Evolution of the ATP binding cassette (ABC) transporter superfamily in vertebrates, Annu. Rev. Genomics Hum. Genet. (Epub ahead of print February 15, 2005). [15] T. Terasaki, S. Ohtsuki, Brain-to-blood transporters for endogenous substrates and xenobiotics at the blood– brain barrier: an overview of biology and methodology, NeuroRx 2 (1) (2005) 63 – 72. [16] M. Furuse, et al., Claudin-based tight junctions are crucial for the mammalian epidermal barrier: a lesson from claudin-1-deficient mice, J. Cell Biol. 156 (6) (2002) 1099 – 1111. [17] Q. Li, Y. Kato, Y. Sai, T. Imai, A. Tsuji, Multidrug resistance-associated protein 1 functions as an efflux pump of xenobiotics in the skin, Pharm. Res. 22 (6) (2005) 842 – 846. [18] Y. Matsuoka, et al., Developmental expression of P-glycoprotein (multidrug resistance gene product) in the rat brain, J. Neurobiol. 39 (3) (1999) 383 – 392. [19] Y.S. Kang, et al., Regulation of taurine transport at the blood–brain barrier by tumor necrosis factor-a, taurine and hypertonicity, J. Neurochem. 83 (5) (2002) 1188 – 1195. [20] H. Takanaga, et al., ATA2 is predominantly expressed as system A at the blood–brain barrier and acts as brain-to-blood efflux transport for l-proline, Mol. Pharmacol. 61 (6) (2002) 1289 – 1296. [21] K. Hosoya, et al., Enhancement of l-cystine transport activity and its relation to xCT gene induction at the blood–brain barrier by diethyl maleate treatment, J. Pharmacol. Exp. Ther. 302 (1) (2002) 225 – 231. [22] S. Hori, et al., Functional expression of rat ABCG2 on the luminal side of brain capillaries and its enhancement by astrocyte-derived soluble factor(s), J. Neurochem. 90 (3) (2004) 526 – 536. [23] S. Hori, et al., A pericyte-derived angiopoietin-1 multimeric complex induces occludin gene expression in brain capillary endothelial cells through Tie-2 activation in vitro, J. Neurochem. 89 (2) (2004) 503 – 513. [24] T. Terasaki, et al., New approaches to in vitro models of blood–brain barrier drug transport, Drug Discov. Today 8 (20) (2003) 944 – 954. [25] K. Hosoya, et al., Conditionally immortalized retinal capillary endothelial cell lines (TR-iBRB) expressing differentiated endothelial cell functions derived from a transgenic rat, Exp. Eye Res. 72 (2) (2001) 163 – 172.
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Physiological pharmacokinetics and membrane transport for drug delivery research Tetsuya Terasaki*, Sumio Ohtsuki Graduate School of Pharmaceutical Sciences, and New Industry Creation Hatchery Center (NICHe), Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan Solution-Oriented Research for Science and Technology (SORST), Japan Science and Technology Agency (JST), Japan
Abstract. In the case of patients undergoing dentistry and oral surgery, several drugs, including anaesthetics, antibiotics, analgesics, muscle relaxants, steroids, antidepressants and anticancer drugs are administered. Physiological pharmacokinetics is the best way to integrate the processes of drug absorption, distribution, metabolism and excretion (ADME). We have developed physiological pharmacokinetic models for h-lactam antibiotics, quinolone antibiotics, a muscle relaxant and anticancer drugs. These models enable us to compare the characteristics of drug ADME among different derivatives and to predict drug concentrations in target organs. As far as the factors affecting drug ADME are concerned, plasma membrane transport is one of the most important. The molecular mechanism of drug transport is very important as far as the bioavailability and targeting efficiency are concerned. Members of the ATP binding cassette (ABC) transporter superfamily, such as ABCB1, ABCC4 and ABCG2 have been shown to play an important role in drug efflux as a part of detoxifying system in the body. The expression and function of the transporters is not static, but can be up- or downregulated by several factors including oxidative stress, osmotic pressure and substrates of the transporter as well as by cell-to-cell interactions. Thus, plasma membrane transporters can act as a part of the dynamic interface in the body. For safe, efficient and rational drug development and treatment, it is important to understand the physiological role of these transporters. D 2005 Elsevier B.V. All rights reserved. Keywords: Physiological pharmacokinetics; Drug delivery; Transport; ABC transporter
* Corresponding author. Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan. Tel.: +81 22 795 6831; fax: +81 22 795 6886. E-mail address: [email protected] (T. Terasaki). 0531-5131/ D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2005.06.070
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1. Introduction A number of drugs are routinely used in dentistry and oral surgery, such as anaesthetics, antibiotics, analgesics, muscle relaxants, steroids, antidepressants, and anticancer drugs. For safe and effective drug therapy, it is important to understand the molecular mechanisms of drug distribution in the body and the pharmacokinetics integrating all the process in the body after administration, such as absorption, distribution, metabolism and excretion (ADME). Table 1 shows a list of the possible factors that affect drug distribution. For the rational development of new drugs, it is important to understand their major elimination pathways and the rate-limiting process for candidate compounds as well as factors affecting distribution to the target organ(s). In this review, we shall highlight the importance of physiological pharmacokinetics in analyzing this drug distribution mechanism. As plasma membrane transport is one of the most important factors involved in drug delivery to the target organ, we will also discuss the importance of the molecular mechanism and physiological role of plasma membrane transport. 2. Physiological pharmacokinetics and tissue distribution mechanism The drug concentration is the most important factor governing the therapeutic effect. If the plasma membrane receptors or the ecto-enzyme of the plasma membrane are a site of drug action, the interstitial fluid drug concentration may be directly related to the therapeutic effect. If the intracellular enzyme, protein and nucleic acid are a site of drug action, the intracellular drug concentration is directly related to this. Therefore, it is very important to identify the molecular mechanism governing drug distribution to the target organ. h-Lactam antibiotics are substrates of the oligopeptide transporters, PEPT1 and PEPT2, and the organic anion transporter, OAT3. Small intestinal epithelial cells express PEPT1, while renal epithelial cell express PEPT2 and OAT3, and h-lactam antibiotics can be transported to these organs by such transporters. In contrast, as the passive diffusion rate of h-lactam antibiotics in the plasma membrane is low, the drug cannot cross the plasma membrane in the muscle, heart, lung, skin and fat. Fig. 1A illustrates an extracellular localization model used as a physiological pharmacokinetic model to predict the distrubution of h-lactam antibiotics in the body [1,2]. The volume of extracellular fluid and plasma is the determinant factors for the distribution volume of h-lactam antibiotics. Depending on the age, the extracellular fluid volume of muscle changes significantly. Physiological pharmacokinetics based on the model shown in Fig. 1A can accurately Table 1 Possible factors affecting drug distribution Process
Factors
Absorption Distribution Metabolism Excretion
Membrane permeability, first-pass metabolism, solubility, gastric emptying time, food, lumen pH Membrane permeability, plasma protein binding, tissue binding Enzyme affinity and amount, membrane permeability, tissue binding Membrane permeability, plasma binding, bile flow rate, urine pH
Blood flow rate and tissue volume are the factors that can be involved in all the processes.
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Fig. 1. Schematic diagram of drug distribution in the organ. A: Extracellular localization model; B: Intracellular distribution model.
predict the organ distribution in rats that are 1, 7, 50 and 100 weeks old [3]. The unbound h-lactam antibiotic concentration in the interstitial fluid of an infected organ will be very similar to that in the plasma after systemic administration. If the permeability rate across the capillary is not significantly low, Fig. 1A may be used for the prediction of the pharmacokinetics of physiological peptides such as growth hormone. If the drug can cross the plasma membrane, an intracellular distribution model, shown in Fig. 1B, can be used for this analysis [4]. In this case, the plasma unbound fraction, tissue unbound fraction and plasma membrane permeability are the determinant factors for the organ distribution. A concentration gradient of unbound drug between the intracellular and extracellular fluid will be generated by the ratio of the influx permeability rate and the efflux permeability rate across the plasma membrane. If a drug is able to permeate the plasma membrane by passive diffusion, there will be no concentration gradient in usual cases. If a drug is taken up into the cells by an active influx transporter, the intracellular unbound drug will be significantly higher than that in the extracellular fluid and plasma. If a drug can be pumped out of the cell by the active efflux transporter, the intracellular unbound drug concentration will be significantly lower than that in the extracellular fluid and plasma. Therefore, if intracellular proteins or nucleic acids are drug target molecules, the plasma membrane transport mechanism of the target organ will play a very important role in determining the pharmacological effect. The intracellular distribution model in Fig. 1B was validated by examining the differential distribution of doxorubicin among the organs [4] in rats, guinea pigs and rabbits. The differential distribution of quinolone antibacterials, among the derivatives, was also analyzed by a similar model. The results showed that there was no significant difference in tissue binding among the quinolone derivatives and the lower plasma protein binding of drug resulted in a higher concentration in the tissues, except in the brain [5]. If the plasma membrane permeability is not saturable and very low, the plasma unbound drug concentration is proportional to the unbound drug concentration in the cell of target organ, and this acts as an appropriate marker of the drug effect. The organ blood flow rate may be a determinant factor of the time-dependence of drug distribution. Inaperison, a muscle-relaxant, distributes in the brain and causes a significant increase in the blood flow rate in muscle and fat. The rapid distribution of inaperison in the muscle and fat is attributed to the enhanced blood flow rate in these tissues [6].
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In dentistry and oral surgery, most drugs are administered locally. The drug diffusion in tissues is also a determinant of the retention and elimination of the drug in oral tissues. Macromolecules may be significantly retained in these tissues where the drug is injected [7], if the capillary permeability is significantly restricted. 3. Mechanism of plasma membrane transport and its importance for drug distribution The modes of plasma membrane transport can be classified as follows: passive diffusion, facilitated transport, primary active transport and secondary active transport (synport and antiport). The drug transport rate of passive diffusion increases in parallel with drug lipophilicity and inversely with the square root of molecular size. When the partition coefficient between n-octanol and water is greater than 1, the drug can easily cross the plasma membrane by passive diffusion. Nevertheless, low solubility in water becomes problem, if the partition coefficient is greater than 4. At the plasma membrane, various transporters (solute carrier protein; SLC) operate to transport amino acids, dipeptides, glucose and lactate. Therefore, drugs recognized by these transporters can cross the plasma membrane. GLUT1/SLC2A1 is one subtype of glucose transporter. The K m (half saturation concentration) of GLUT1 is higher than the plasma glucose concentration, and GLUT1 is not saturated by plasma glucose under normal conditions. On the other hand, using saturated transporters is not an efficient method of drug delivery to target organs. l-DOPA is transported to the brain from the blood by LAT1/SLC7A5 [8], a transporter for large neutral amino acids, which is expressed at the blood–brain barrier [9]. However, LAT1 is saturated by plasma amino acids, since its K m is lower than the plasma concentration of substrate amino acids. On the brain side of the plasma membrane of brain capillary endothelial cells, organic anion transporter 3 (OAT3/SLC22A8) is expressed to excrete neurotransmitter metabolites from the brain to the blood [10] (Fig. 2). Homovanillic acid, a dopamine metabolite, is an OAT3 substrate, and, during its elimination process, OAT3 plays a major (N 90%) role in the brain side of the membrane transport. Indoxyl sulfate is a uremic toxin, which accumulates in the body of patients with renal failure, and it is excreted from the brain to the blood by OAT3 [11]. It has been suggested that OAT3 transports other uremic toxins (Fig. 2). OAT3 also accepts anionic drugs. The distribution of 6-mercaptopurine (6-MP) into the brain is low, and this low distribution has been suggested to be due to the predominant brain-to-blood efflux transport [12]. Recently, we have reported that OAT3 is involved in this efflux transport [13] (Fig. 2). In the kidney, the renal epithelial cells forming the uriniferous tubules express OAT3 and OAT1. Therefore, drugs which are OAT3 substrates tend to be excreted from the brain and kidney. P-glycoprotein (P-gp/MDR1/ABCB1) is a subtype of the ATP binding cassette (ABC) transporter superfamily, and is classified as a primary active transporter directly consuming ATP hydrolysis energy. P-gp was initially identified in cancer cells, and subsequently reported to be expressed in normal cells including brain capillary endothelial cells, small intestinal epithelial cells and hepatocytes. P-gp accepts relatively lipophilic
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Fig. 2. Physiological roles of the brain-to-blood efflux transport systems at the blood–brain barrier (BBB). The BBB expresses various brain-to-blood efflux transport systems, which excrete neurotransmitters, neurotransmitter metabolites, neuromodulators and uremic toxins. The BBB efflux transporters also act to limit drug distribution into the brain. Furthermore, the BBB possesses efflux transport system for peptides and proteins, such as apotransferrin, immunoglobulin and h-amyloid. These BBB efflux transporters function cooperatively as the CNS supporting and protecting system. Cited from Ref. [15].
drugs as substrates. If drugs are recognized by P-gp, they are excreted from the inside of cells to the outside even although the drugs can cross the plasma membrane by diffusion. This results in a very low brain distribution and low intestinal absorption of P-gp substrate drugs. In humans, 48 subtypes of ABC transporters have been identified [14]. Among them, the ABCC (MRP) family and ABCG2 have been reported to limit the tissue distribution of drugs as well as P-gp [15]. The expression of these efflux transporters in target organs is important for the purposes of drug development. In addition, even for commercially available drugs, substrate recognition of these efflux transporters is an important factor as far as drug efficacy is concerned. To date, the limiting factor in percutaneous absorption is thought to be the horny layer of the epidermis. Recently, claudin 1, a tight junction protein, has been reported to play an important role in this epidermal barrier [16]. Furthermore, it has been reported that ABCC1/MRP1 is acting as a significant efflux pump in the epithelial cells in the epidermis [17]. Therefore, the strategy for developing drug for percutaneous absorption needs to be fundamentally changed. 4. Regulation of membrane transport Membrane transport involves a number of functions and is not constant. The expression of P-gp by the brain capillary endothelial cells increase with development after birth [18]. The expression and function of TAUT and ATA2, which transport taurine and proline, respectively, is induced under hypertonic conditions in brain capillary endothelial cells
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Fig. 3. Principle of establishment of cell line. A: Traditional method using primary cultured cell and gene transfection. B: Transgenic animal method for conditionally immortalized cell line from temperature-sensitive SV40 large T-antigen gene transgenic rats or mice.
[19,20]. This induction is likely to contribute to osmoregulation in the brain. TAUT is also induced by cytokines and suppressed by excess of taurine [19]. xCT, an l-cystine and lGlu exchange transporter, is induced by oxidative stress [21]. ABCG2 expressed in the brain capillary endothelial cells is induced by soluble factors secreted from astrocytes, but not those from pericytes [22], while occludin, a tight junction protein, is induced by angiopoietin1 secreted from pericytes [23]. The changes in transporter functions and identification of the regulating factors will provide us with important information for a better understanding of drug distribution to target organs. 5. Conditionally immortalized cell lines To analyze membrane transport, in vitro cell culture systems, such as freshly prepared cells, primary cultured cells, immortalized cells and conditionally immortalized cells, are useful. Fig. 3 shows the establishment of conditionally immortalized cell lines.
Fig. 4. Comparison of the blood–brain barrier (BBB) permeability clearance between in vivo and in vitro experiments. Closed symbols and open symbols indicate the blood-to-brain influx and the brain-to-blood efflux in vivo BBB permeability clearance, respectively. The BBB permeability clearance was estimated from transport activity measured by conditionally immortalized rat and mouse brain capillary endothelial cells (TR-BBB (square) and TM-BBB (circle), respectively). The estimations were performed with the following surface area parameters: TM-BBB, 18 cm2/mg protein; TM-BBB, 22 cm2/mg protein; in vivo brain capillary, 100 cm2/g brain. 3-OMG, 3-O-methyl-glucose; DHEAS, dehydroepiandrosterone sulfate; GABA, g-aminobutylic acid; l-Pro, lproline; TAPA, H-Tyr-d-Arg-Phe-h-Ala-OH (Opioid Peptide). Cited from Ref. [24].
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Conditionally immortalized cell lines involve cell lines isolated from transgenic animals harbouring temperature sensitive SV40 large T antigen and cultured at 33 8C [24]. Using this method, it is possible to establish immortalized cell lines from very small tissues, such as the rat retina [25]. Compared with the classical method of introducing an immortalized gene into primary cultured cells, this method using transgenic animals can establish cell lines under milder conditions, and the established cells fairly retained their in vivo function. Indeed, the transport activity of a variety of compounds in conditionally immortalized brain capillary endothelial cells has been found to match that of the in vivo blood–brain barrier [24] (Fig. 4). We have established conditionally immortalized cell lines from many tissues, such as brain capillary endothelial cells, choroid plexus epithelial cells, brain pericytes and retinal capillary endothelial cells [24]. In dental research, establishing immortalized cell lines using transgenic animals will be a useful strategy for analyzing transport function, cell–cell interactions and signal transduction. 6. Conclusion Physiological pharmacokinetic models enable us to compare the characteristics of drug ADME among different derivatives and to predict drug concentrations in the target organ. The molecular mechanism of drug transport plays a very important role in determining the drug bioavailability and targeting efficiency. The plasma membrane transporters can act as a part of the dynamic interface in the body. In order to ensure safe, efficient and rational drug development and treatment, it is important to fully understand the physiological role of transporters. References [1] A. Tsuji, et al., Physiologically based pharmacokinetic model for cefazolin in rabbits and its preliminary extrapolation to man, Drug Metab. Dispos. 13 (6) (1985) 729 – 739. [2] A. Tsuji, et al., Physiologically based pharmacokinetic model for h-lactam antibiotics I: tissue distribution and elimination in rats, J. Pharm. Sci. 72 (11) (1983) 1239 – 1252. [3] A. Tsuji, et al., Age-related change in tissue-to-plasma partition coefficient of cefazolin for noneliminating organs in the rat, J. Pharm. Sci. 78 (7) (1989) 535 – 540. [4] T. Terasaki, et al., Pharmacokinetic study on the mechanism of tissue distribution of doxorubicin: interorgan and interspecies variation of tissue-to-plasma partition coefficients in rats, rabbits, and guinea pigs, J. Pharm. Sci. 73 (10) (1984) 1359 – 1363. [5] E. Okezaki, et al., Structure-tissue distribution relationship based on physiological pharmacokinetics for NY198, a new antimicrobial agent, and the related pyridonecarboxylic acids, Drug Metab. Dispos. 16 (6) (1988) 865 – 874. [6] O. Nagata, et al., Physiological pharmacokinetics of a new muscle-relaxant, inaperisone, combined with its pharmacological effect on blood flow rate, Drug Metab. Dispos. 18 (6) (1990) 902 – 910. [7] D. Hovgaard, et al., Clinical pharmacokinetic studies of a human haemopoietic growth factor, GM-CSF, Eur. J. Clin. Invest. 22 (1) (1992) 45 – 49. [8] T. Kageyama, et al., The 4F2hc/LAT1 complex transports l-DOPA across the blood–brain barrier, Brain Res. 879 (1–2) (2000) 115 – 121. [9] R.J. Boado, et al., Selective expression of the large neutral amino acid transporter at the blood–brain barrier, Proc. Natl. Acad. Sci. U. S. A. 96 (21) (1999) 12079 – 12084. [10] S. Mori, et al., Rat organic anion transporter 3 (rOAT3) is responsible for brain-to-blood efflux of homovanillic acid at the abluminal membrane of brain capillary endothelial cells, J. Cereb. Blood Flow Metab. 23 (4) (2003) 432 – 440.
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