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CELLULAR AND MOLECULAR
Departments of Molecular Sciences (T.Z.S.), Pharmacology (M.L.W.), and Pharmacokinetics, Dynamics, and Metabolism (M.R.F.), Pfizer Global Research and Development, Ann Arbor, Michigan
Received December 14, 2004; accepted March 8, 2005.
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Abstract |
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-cylcohexanegaba; GBP), is under development by Pfizer, Inc. (Ann Arbor, MI) for the potential treatment of central nervous system disorders, including epilepsy, neuropathic pain, fibromyalgia, and generalized anxiety disorder (Feltner et al., 2003
). Both GBP and PGB were originally designed to mimic the structure of GABA. In contrast to GABA, however, both drugs are well absorbed by the intestine (Stewart et al., 1993; Bockbrader et al., 2000) and readily cross the blood-brain barrier (Luer et al., 1999; Feng et al., 2001).
Amino acids are taken up by different transport systems with overlapping substrate specificity. These functionally defined systems were originally characterized by kinetic and competition studies (Christensen, 1990). Without comprehensive kinetic analysis, it is often difficult to specify a transport system responsible for uptake of an unknown substrate (Christensen, 1990). Despite the structural similarity between GBP and PGB, GPB absorption lacks proportionality between the increasing doses and the drug levels in plasma, whereas PGB has nearly linear pharmacokinetics (Stewart et al., 1993; Randinitis et al., 2003). These early in vivo observations suggest that these two drugs may have important differences with respect to transport. GBP enters cells predominantly by the L-amino acid transport system (Su et al., 1995; Uchino et al., 2002), whereas the transport carriers for PGB uptake have not been well characterized. The mechanisms underlying the different pharmacokinetics of PGB and GBP remain unclear. One early report has shown mutual inhibition between PGB and phenylalanine in rat ileum (Jezyk et al., 1999) and suggested the involvement of type L transport system. However, the mutual inhibition alone is insufficient to conclude which specific transport system is involved in PGB transport in the ileum. Phenylalanine enters mammalian cells by as many as six different transport systems, including L, B0, B0,+, y+L, ASC, and T (Shotwell et al., 1981; Van Winkle et al., 1990), with the first four transport systems found in intestine and Caco-2 cells (Nakanishi et al., 1994; Fraga et al., 2002; Pan et al., 2002; Wasa et al., 2004). The early conclusion on system L-mediated PGB transport in the rat ileum was also complicated by lack of mutual inhibition between GBP and PGB in intestine and lack of carrier-mediated uptake of GBP and PGB in Caco-2 cells. As a follow-up study, the same research group has published a second paper revealing some interesting differences in Na+ dependence, temperature sensitivity, and regional distribution between GBP and PGB transport (Piyapolrungroj et al., 2001). In this latter report, GBP entered rat and rabbit brush-border membrane vesicle via an Na+-independent route, whereas PGB uptake was predominantly Na+-dependent. Although the authors proposed that PGB is transported by system B0 and GBP by b0,+, this proposition was mostly based on Na+ dependence, and further experimental evidence is needed. System B0 is one of the major Na+-dependent (Doyle and McGivan, 1992) and system b0,+ is one of the Na+-independent amino acid transport systems in intestine (Costa et al., 2000). System b0,+ is responsible for the uptake of both cationic and zwitterionic amino acids and was first characterized in blastocysts (Christensen, 1990). To examine the proposed interaction of PGB with system B0, we selected two system B0-enriched cell lines, Caco-2 and NBL-1 (Doyle and McGivan, 1992; Pan and Stevens, 1995), for this study. To further delineate the transport systems responsible for uptake of GBP and PGB, we also carried out comparative transport studies of these two drugs in Caco-2, NBL-1 and CHO cells, the stable cell lines overproducing GABA transporters GAT1, GAT2, and GAT3, and Xenopus laevis oocytes injected with rBAT (Bertran et al., 1992). We demonstrated that L-type amino acid transport system is the major carrier responsible for the concentrative uptake of PGB.
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). GAT1, 2, and 3 stable cell lines were made in this laboratory. In Vivo Pharmacokinetics in Rats. Male Wistar rats received oral gavage doses of PGB at 5, 25, 50, 100, and 150 mg/kg or GBP at 50, 100, and 250 mg/kg in aqueous solution. Serial plasma samples were collected. Plasma PGB concentrations in rat were determined using validated high-pressure liquid chromatography assays. PGB does not possess a useable chromophore and, therefore, is derivatized after sample collection with an UV tag, trinitrobenzenesulfonic acid, to enable quantification. The limit of quantification is 0.100 µg/ml in plasma.
Cell Culture. CHO cells were cultured in modified essential medium, and NBL-1 and Caco-2 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 100 IU/ml penicillin, 100 µg/ml streptomycin, 1% nonessential amino acids, and 10% fetal bovine serum at 37°C in 5% CO2/95% O2.
Transport Assays. The sodium-containing buffer for transport assay was Dulbecco's phosphate-buffered saline, consisting of 137 mM NaCl, 2.7 mM KCl, 10.6 mM Na2HPO4, and 1.5 mM KH2PO4. The sodium-free buffer had equimolar amounts of choline chloride and choline phosphate in place of NaCl and Na2HPO4, respectively. This buffer was referred to as PBC. Before use, both phosphate-buffered saline and PBC buffers (pH 7.4) were supplemented with 5.6 mM D-glucose, 0.49 mM MgCl2, and 0.9 mM CaCl2.
Uptake of 3H substrates was measured in cells 2 days after seeding. Except for alanine and arginine transport, the cells were depleted of intracellular pool of amino acids for 2 x 20 min in amino acid-free buffer. The cluster tray transport assay was carried out as described previously (Su et al., 1995). L-type system transport activity in CHO cells was functionally defined as 10 mM BCH-sensitive L-[3H]leucine uptake, system A as Na+-dependent [3H]MeAIB uptake, and system ASC as 10 mM MeAIB- and BCH-insensitive L-[3H]serine uptake (Shotwell et al., 1981). System B0 was defined as Na+-dependent, MeAIB-insensitive L-[3H]alanine uptake (Pan and Stevens, 1995), system y+ as Na+-independent, leucine-resistant L-[3H]arginine uptake, and system b0,+ as Na+-independent, leucine-sensitive L-[3H]arginine uptake in Caco-2 and NBL-1 cells (Doyle and McGivan, 1992; Pan and Stevens 1995; Pan et al., 2002).
The initial transport activity was determined at 37°C within the linear uptake period (0.5 min for leucine and GBP and 2 min for PGB in CHO cells; 5 min for alanine and arginine in Caco-2 and NBL-1 cells). The substrate concentrations for initial rate of transport were 50 µM. The uptake rates were referred to as saturable uptake rates, which were calculated by subtracting the nonsaturable rates measured in the presence of excess 10 mM unlabeled corresponding substrate from the total uptake rates with exception for PGB and MeAIB, where 25 and 20 mM, respectively, was used. Initial [3H]GABA uptake was carried out at 37°C for 10 min in GAT cell lines pretreated or untreated with 100 µM GBP or PGB for 1 h.
Kinetic parameters were derived from nonlinear regression to eq. 1: logV = log{[Vmax x S/(Km + S)] + P x [S]}, where V, S, Vmax, and Km have their usual meanings, and P is the first-order rate constant representing the nonsaturable uptake.
In efflux assays, the cells were preloaded with tritiated substrate for 40 min and then washed twice with prewarmed buffer. Because more than 95% of the preloaded [3H]leucine was exchangeable with extracellular leucine, the metabolism of leucine should be insignificant under these experimental conditions. PGB and GBP are known to be resistant to metabolism in vivo (M. R. Feng, unpublished data). The cells were incubated in the presence (exchange) or absence (net efflux) of substrate of interest. Exodus constant k was determined by fitting data to a first-order rate (eq. 2): y = a(1 – b x e–ck).
Transport in X. laevis Oocytes. Surgical removal and preparation of stage V or VI defoliculated X. laevis oocytes were as previously described (Su et al., 1992). The oocytes were stored in modified Barth's saline buffer containing 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.3 mM Ca(NO3)2, 1 mM CaCl2, 1 mM MgSO4, 2.5 mM sodium pyruvate, 10 mM HEPES, 1 ml/l gentamicin (Invitrogen no. 15710-064), and 10 ml/l antibiotic/antimycotic (Invitrogen no. 15240-062), pH 7.5. Each oocyte was injected using a microinjector (Nanoject II; World Precision Instruments, Inc., Sarasota, FL) with either 46 nl of nuclease-free water (control) or 3 ng of rBAT cRNA dissolved in 46 nl of nuclease-free water. Capped RNA was prepared as described previously (Bertran et al., 1992). After injection, the oocytes were incubated at 16°C in modified Barth's saline buffer with daily change of medium.
The uptake assay was run in 24-well plates with a single oocyte per well (groups of four oocytes per treatment). Three days after injection, the transport assays were started by washing oocytes twice for 15 min in sodium-free uptake buffer containing 137 mM choline chloride, 2.7 mM KCl, 10.6 mM choline phosphate, 1.5 mM KH2PO4, 5.6 mM D-glucose, 0.49 mM MgCl2, and 0.9 mM CaCl2 (0.9), pH 7.4. Five hundred microliters per well L-[3H]leucine uptake buffer consisting of Na+-free uptake buffer with 5 µCi/ml L-[3H]leucine (Amersham no. TRK 510, 1 µCi/µl, 120–190 Ci/mmol) plus or minus PGB or GBP was then added to the oocytes. Two control groups were used: one with no drug added and the other with saturating L-leucine (10 mM). Uptake was measured for 5 min, during which the uptake time course showed linearity (data not shown). After 5 min of incubation at room temperature (22°C), uptake was terminated by an immediate wash with ice-cold Na+-free buffer. Individual oocytes were then washed three more times with the same buffer, wells were aspirated dry, and 200 µl of 10% SDS was added to each well. Plates were put on a rotating mixer for 60 min to lyse and dissolve oocytes. Well contents were then transferred from the 24-well plate to individual scintillation vials and counted on a Packard Tri-Carb 2900TR (Canberra Industries, Meriden, CT) liquid scintillation counter. L-[3H]leucine uptake was calculated as picomoles per oocyte per minute and was expressed as percent untreated injected control.
Statistical Analysis. Each transport experiment was conducted in triplicate, and all experiments were confirmed by at least two independent experiments. Experimental results were reported as means ± S.D. Student's t test was used for analyzing substrate specificity, and p < 0.05 was considered to be statistically significant.
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) and humans (Stewart et al., 1993; Bockbrader 1995). In GBP-treated rats, the Cmax only increased from 16.4 to 26.8 µg/ml, when the GBP oral doses were increased from 50 to 250 mg/kg, although a nearly 5-fold increase of GBP plasma AUC was observed (79–502 µg/h/ml). Both GBP and PGB were mainly excreted in urine (>90%) in rat and human with minimum metabolism following oral administration. The less dose-dependent increase of GBP Cmax in rat may be related to a saturable absorption from the gut. The linear pharmacokinetics (linear relationship between the doses and the plasma Cmax and AUC) observed for PGB in rats are consistent with previously reported in vivo human data (Bockbrader et al., 2000).
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L-Type Amino Acid Transport System-Mediated PGB Uptake. To delineate the mechanism underlying the differences in the oral absorption between PGB and GBP, we examined interaction of these two drugs with various amino acid transport systems that are known to exist in the intestine and the kidney. One early study has suggested that PGB is absorbed in the intestine by system L (Jezyk et al., 1999). To confirm the early conclusion that PGB is a system L substrate, we first determined if transport of PGB is mutually inhibitable with leucine, a prototypical system L substrate (Shotwell et al., 1981), and GBP, a known system L substrate (Su et al., 1995). As shown in Fig. 2, uptake of PGB was predominantly an Na+-independent process in CHO and Caco-2 cells. The uptake rates for PGB were approximately 3-fold lower than that of leucine and GBP, but these three substrates were clearly mutually inhibited. In addition, uptake of all the three substrates was very sensitive to BCH, a system L-preferring substrate (Christensen, 1990), suggesting that PGB is likely to be taken up by the same route (L-type amino acid transport system) as leucine and GBP in these two cell lines.
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System L (LAT) is generally believed to be an obligatory exchanger (Verrey, 2003), although it does not seem to apply to the recently cloned LAT4 (Bodoy et al., 2005). Assuming that PGB is a substrate of L-type amino acid transport system, one may expect that PGB is exchangeable with leucine by at lease certain subtypes of LATs. To assess net efflux and exchange activity, CHO cells were preloaded with 50 µM tritiated leucine, GBP, and PGB to steady state (40 min), and then the cells were exposed to buffer (net efflux) or 50 µM unlabeled leucine (trans-stimulated exchange). As with uptake, PGB had net exodus rate (0.20 ± 0.05 min–1) 3.1- and 2.5-fold lower than that of leucine (0.61 ± 0.15 min–1) and GBP (0.49 ± 0.3 min–1) (Table 1), respectively. However, the presence of extracellular leucine clearly accelerated leucine, GBP, and PGB exodus by 2.3-, 2.9-, and 1.9-fold, respectively (trans-stimulation), although the PGB exchange rate with leucine was approximately 4-fold less than GBP. Trans-stimulation is one typical feature of L system-type transport activity, further supporting the earlier findings that PGB is taken up by the same carrier as leucine (system L; Shotwell et al., 1981).
All the data described above demonstrate that PGB uptake is characterized by slow uptake velocity, high Km, high Vmax, low kefflux, and low kexchange, implying that PGB uptake is more concentrative. To prove this assumption, concentration-dependent steady-state uptake rates were examined in both CHO and Caco-2 cells. As shown in Fig. 5B, leucine, GBP, and PGB exhibited striking differences in maximum accumulation rates (Vmax). The Vmax value for PGB (106 ± 9 nmol/mg) was 2.5- and 66-fold higher than that of GBP (43 ± 1 nmol/mg) and leucine (1.6 ± 0.2 nmol/mg), respectively. The concentrative PGB uptake was more evident in Caco-2 cells in which the accumulation rates were nearly linear from 3 to 1000 µM (Fig. 5B), reminiscent of that observed in vivo (Fig. 1).
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Effects of PGB on Other Amino Acid Transport Systems. Since PGB and GBP are GABA analogs, we first examined if PGB interacts with GABA transport by using different subtypes of GABA transporters, GAT1, GAT2 and GAT3. As shown in Table 2, neither preincubation with 100 µM nor coincubation with 10 mM of GBP and PGB altered GABA transport significantly. The IC50 values for inhibition of GABA uptake through GAT1, GAT2, and GAT3 were 19, 20, and 29 mM by GBP and 19, 16, and 24 mM by PGB, respectively. These IC50 values were approximately 3 orders of magnitude higher than the corresponding Km values of 19.7, 2.9, and 3.3 µM for GABA uptake by GAT1, GAT2, and GAT3, respectively.
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We next determined if the two other important amino acid transport systems, A and ASC, are involved in PGB uptake in CHO cells. In contrast to system L, the Na+-dependent MeAIB transport (system A) and the Na+-dependent, MeAIB-insensitive alanine transport (system ASC) were not inhibited by 25 mM PGB, thus excluding involvement of system A and ASC in the PGB uptake.
To examine effect of PGB on other major amino acid transport systems that have been found in intestine and kidney, we selected Caco-2 cells of intestinal origin and NBL-1 cells of kidney origin for further study. In addition to A and L systems, b0,+, y+, y+L, B0,+, and B0 transport systems have been reported in these two cell lines (Doyle and McGivan, 1992; Pan and Stevens 1995; Pan et al., 2002).
As illustrated in Fig. 6, L-[3H]arginine transport was mediated by both Na+-independent and -dependent systems. Inhibition of Na+-dependent arginine uptake by neutral amino acids could result from mixed inhibition of system B0,+ and y+L because y+L is inhibited by neutral amino acids only in the presence of Na+ (Christinsen, 1990). Further separation of these two transport systems was not pursued in this study. The Na+-independent arginine uptake (67 and 52% of total uptake in NBL-1 and Caco-2 cells, respectively) was significantly inhibited by leucine (36% in NBL-1, 66% in Caco-2 cells) but not inhibited by BCH, alanine, GBP, and PGB (Fig. 6; Table 2). In the presence of Na+, a similar inhibition profile was observed except for small inhibition by BCH. Inhibition of Na+-dependent uptake of arginine by BCH suggests a B0,+-like transport activity (Pan and Stevens, 1995).
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; Pan et al., 2002) and was blocked by GBP and PGB. The Na+-independent, arginine-sensitive alanine uptake (approximately 50%) was possibly through a b0,+-like system (Christensen, 1990; Costa et al., 2000). To further address the interaction with b0,+-like transport activity, we assessed effects of GBP and PGB on rBAT-induced b0,+-like transport activity in X. laevis oocytes (Bertran et al., 1992). Our studies showed that 10 mM GBP inhibited rBAT-induced leucine uptake by 87 ± 19%, whereas the same concentration of PGB only inhibited 40 ± 6% of the uptake (Table 2). The IC50 value for this inhibition by GBP was 10-fold more potent than PGB (1.9 and 19 mM, respectively).
In agreement with previous reports (Doyle and McGivan, 1992; Costa et al., 2000; Pan et al., 2002), the Na+-dependent alanine uptake in Caco-2 and NBL-1 cells was contributed by small MeAIB-sensitive (system A) and MeAIB-insensitive (system B0) components. By subtracting MeAIB-sensitive, Na+-dependent alanine uptake, GBP and PGB showed net inhibition of system B0-mediated alanine uptake by 11 and 16% in NBL-1 and 23 and 22% in Caco-2 cells, respectively, suggesting a weak interaction with system B0.
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-position of GABA. The present in vivo study has shown that PGB exhibited pharmacokinetic properties distinctive from GBP, which has saturable absorption at the therapeutic dosage. As reported in the previous brain microdialysis study in rats (Wang and Welty, 1996; Feng et al., 2001), PGB had slower brain influx (lower CLin) and particularly slower brain efflux (lower CLout) than GBP. These differences between PGB and GBP were thought to be due to the different affinities and rates of flux between PGB and GBP at L-type transport system of the blood-brain barrier. L-type amino acid transport system is an ubiquitous Na+-independent transport system for large, neutral amino acids across plasma membranes (Oxender and Christensen, 1963). In the present study, several lines of evidence supported mediation of PGB transport by L-type transport system. First, PGB uptake was predominantly a Na+-independent process, exhibiting mutual inhibition with two previously known L-type transport system substrates, leucine and GBP (Su et al., 1995). Second, as with GBP and leucine, the uptake of PGB was highly sensitive to BCH, a system L-preferring substrate (Christensen, 1990). Third, PGB competitively inhibited leucine uptake in CHO cells, with the apparent Km value identical to the Ki value for inhibition of leucine uptake. Finally, PGB was exchangeable with leucine. In contrast to a previous report (Jezyk et al., 1999), we reproducibly observed Na+-independent saturable uptake of PGB and GBP in Caco-2 cells. One interpretation for the discrepancy could be associated with the age of Caco-2 used for transport assays. The Caco-2 cells used in the present study were 2 days postseeding, whereas the early study was carried out 19 to 30 days after seeding. It has been known that the transport properties in Caco-2 cells highly depend on cell age, differentiation stage, and ambient substrate availability (Pan and Stevens, 1995; Pan et al., 2002).
It should be noted that the functionally defined L-type amino acid transport activities as described in this and many other studies may result from a mixture of functionally similar amino acid transporters that share common features such as Na+ dependence, BCH sensitivity, and neutral amino acid substrate preference. To date, at least four members of this family, LAT1, LAT2, LAT3, and LAT4, have been cloned (Bodoy et al., 2005). LAT1 and LAT2 are heterodimeric transporters that require coexpression of both LAT and 4F2hc for function and exhibit trans-stimulation (Verrey, 2003). In contrast, LAT3 and LAT4 are structurally distinct from LAT1 and LAT2 and are functional independent of 4F2hc (Babu et al., 2003; Bodoy et al., 2005). It has been shown that different LAT subtypes exhibit distinct expression profiles across tissues and species (Verrey, 2003; Bodoy et al., 2005). Moreover, different LAT subtypes show differences in substrate specificity, transport kinetics, and other properties such as pH sensitivity (Rajan et al., 2000b; Bodoy et al., 2005). LAT3 and LAT4 represent low-affinity L-type transport activities with distinct expression profiles (Babu et al., 2003; Bodoy et al., 2005). The data presented in this study did not determine exactly which subtypes of system L transporters are responsible for the observed PGB uptake. The observed differences in PGB uptake among different cell lines in this study could also be related to tissue and/or species differences. Two low-affinity L-type transporters, LAT2 and LAT4, are highly expressed in intestine and kidney (Rossier et al., 1999; Liu et al., 2003; Verrey, 2003; Dave et al., 2004). The observed low-affinity and high-capacity PGB transport profile in this study and the lack of trans-stimulation (data not shown) in Caco-2 and NBL-1 cells are consistent with some of the properties of recently cloned LAT4. Uptake of PGB into Caco-2 and NBL-1 cells, which are of intestine and kidney origin, respectively, may be mediated by a LAT4-like transport system. The high-affinity, low-capacity, and trans-stimulated transport of PGB seen in CHO cells is more likely mediated by an LAT1-like transport system. Because L-type transport system activity is ubiquitously expressed in almost all cell types, expression of specific LAT subtype genes in cells with low endogenous LAT activities, such as X. laevis oocytes, is needed to delineate which subtype of LAT is involved in PGB transport.
In the present study, we also investigated the role of other amino acid transport systems in PGB transport. Although both PGB and GBP are chemically derived from GABA, consistent with our previous GABA transport study with GBP in neuronal cells (Su et al., 1995), GBP and PGB essentially had no effect on GABA uptake by GAT1, GAT2, or GAT3 transporters (IC50 > 10 mM). In contrast to a previous study in hippocampal neurons (Whitworth and Quick, 2001), we did not observe stimulation of GABA transport following preincubation with GBP and PGB. The lack of detectable up-regulation of GAT activity in GAT-stable cell lines could result from overexpression of GAT protein in or lack of regulated translocation to the plasma membranes. The Vmax values for GAT1, GAT2, and GAT3 in the stable cell lines were 0.3, 12.7, and 22.0 nmol/min/mg protein, respectively, which are much higher than that in hippocampal neurons (331 fmol/min/mg protein).
PGB at concentrations up to 25 mM did not show noticeable inhibition of Na+-dependent MeAIB uptake (system A) or Na+-dependent and MeAIB-insensitive serine uptake (system ASC) in CHO cells. PGB did not inhibit Na+-independent (system b0,+-like) and Na+-dependent (systems B0,+- and y+L-like) arginine transport in Caco-2 and NBL-1 cells. These results exclude the importance of systems A, ASC, b0,+, y+L, and B0,+ in the uptake of PGB and GBP in these cell types. Despite the ineffectiveness of PGB and GBP as inhibitors of b0,+-like transport activity in Caco-2 and NBL-1 cells, the b0,+-like transport activity induced by rBAT in X. laevis oocytes was somewhat sensitive to GBP, with an IC50 value of 1.9 mM. The inconsistent inhibition of b0,+-like transport activity by PGB in different expression systems suggests that the b0,+-like transport system(s) in Caco-2 and NBL-1 cells may be structurally and functionally different from the rBAT-associated heterodimer in X. laevis oocytes (Rajan et al., 1999, 2000a).
Despite a small inhibition of system B0 transport activity by PGB in Caco-2 and NBL-1 cells, system B0 is unlikely to play a major role in PGB uptake because Na+-dependent GBP uptake in these two cell types was negligible. This slight inhibition of system B0 by both PGB and GBP differs from the previously reported PGB-specific and Na+-dependent transport activity (Piyapolrungroj et al., 2001). In addition, system B0 is a high-affinity transporter with Km values in the low micromolar range in Caco-2 (Pan and Stevens, 1995; Costa et al., 2000; Pan et al., 2002; Wasa et al., 2004), NBL-1 (Doyle and McGivan, 1992), and brush border membrane vesicles (Nakanishi et al., 1994), matching well with the cloned intestinal system B0 transporter ATB0 (Kekuda et al., 1997; Pollard et al., 2002). In the early report (Piyapolrungroj et al., 2001), PGB uptake was almost nonsaturable. PGB at a concentration as high as 50 mM only blocked 50% of the total [14C]PGB entry. Whether the early reported Na+-dependent PGB uptake is by yet-unidentified low-affinity transport systems as reported in dog and rabbit intestinal brush border membrane vesicles (Hatanaka et al., 2002) needs to be investigated. By examining previously established system B0 in Caco-2 and NBL-1 cells, we did not obtain evidence supporting the early hypothesis of mediation of PGB transport by system B0 (Piyapolrungroj et al., 2001). However, our results do not rule out transport of PGB by other uncharacterized Na+-dependent transport systems that may be present in brush border cells.
The second objective of the present study was to delineate the mechanism underlying the linear pharmacokinetics in PGB absorption. Our kinetic data showed several important features of PGB that differentiate it from GBP and leucine. Compared with GBP and leucine, PGB has low-affinity/high-capacity initial uptake kinetics (Km ratio, 8:2:1; Vmax ratio, 5:1.4:1, respectively), slow net efflux (exodus constant k ratio, 3.1:1.3:1, respectively) and slow exchange rate K ratio (3.7:1:1, respectively). As a result, the steady-state maximum accumulation rates for PGB were strikingly higher than that for GBP and leucine. For example, at a 300 µM extracellular concentration, the steady-state intracellular concentrations reached 1.48, 22.6, and 34.0 nmol/mg for leucine, GBP, and PGB, respectively. Based on the intracellular water volume of 4.9 µl/mg of protein in CHO cells (Su et al., 1995), the calculated intracellular concentrations for leucine, GBP, and PGB are 0.32, 8.8, and 21.6 mM, respectively. Therefore, the concentration ratios between the inside and outside of the cells are 1, 15, and 23, respectively. In Caco-2 cells, the PGB accumulation rate was almost linear from 10 µM to 3 mM, which is reminiscent of the in vivo dose/plasma response curve in animals (this study) and human (Bockbrader et al., 2000). Based on the facts that PGB and GBP are nonmetabolized in vivo (Taylor et al., 1998; T.Z. Su, M.R. Feng, and M.L. Weber, unpublished data for PGB), interaction with A and ASC systems was unnoticeable (this study), and more than 90% of the preloaded PGB and GBP were exchangeable with leucine (this study), it is unlikely that this concentrative uptake was by known concentrative transport systems such as A or ASC (Christensen 1990) or by being trapped in metabolic pool. For most natural amino acids, system L may not be a concentrative carrier. Our results for leucine transport were in agreement with this general conclusion. On the other hand, it has been shown that BCH exhibits unusually strong accumulation by system L in Ehrlich cells (Christensen 1990). Our study of PGB and GBP transport also demonstrated that system L-mediated transport of certain substrates can be more concentrative or asymmetric. Transport asymmetry is an important feature of system L (Christensen, 1990). Some substrates, such as L-alanine, are relatively good uptake substrates (Km = 167 µM) of system L but are poor efflux substrates (Km = 28 mM) at the same transporter (Meier et al., 2002). Because of the transport asymmetry, the rank order of substrate affinity for entry may not be the same as that for exodus. As a result, the steady-state levels of PGB reached much higher levels than that of GBP and L-leucine.
In summary, our present data have shown that PGB absorption from the gastrointestinal tract in rats is proportional to doses up 150 mg/kg, which contrasts to the partially saturable absorption of GBP in rats reported previously (Radulovic et al., 1995). Furthermore, our results with in vitro cellular test systems suggest that transport of PGB across membrane is largely explained by its substrate action at L-type transport system characterized as Na+-independent and BCH-sensitive neutral amino acid transporters. However, the higher affinity (Km) and lower capacity in cell accumulation at L-type system for GBP compared with PGB may help explain the saturable absorption of GBP from the gut and dose-dependent decrease in oral bioavailability (Bockbrader, 1995; Radulovic et al., 1995; Bockbrader et al., 2000). Our data further suggest that a compound such as PGB that is a substrate of L-type system, but with moderate affinity, may be pharmacokinetically superior to other higher affinity system L drugs such as GBP by virtue of having good proportionality between increasing oral dose and drug levels in plasma.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: PGB, pregabalin; GBP, gabapentin; BCH, 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid; MeAIB, 
-(methylamino)-isobutyric acid; AUC, area under the curve.
Address correspondence to: Dr. Ti-Zhi Su, Department of Molecular Science, Pfizer Global Research and Development, 2800 Plymouth Road, Ann Arbor, MI 48105. E-mail: ti-zhi.su{at}pfizer.com
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