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CELLULAR AND MOLECULAR
Molecular Nutrition Unit, Institute of Nutritional Sciences, Technical University of Munich, Freising-Weihenstephan, Germany (M.F., S.T., H.D.); and Probiodrug AG, Halle, Germany (A.M., H.-U.D.)
Received for publication
February 3, 2004
Accepted
March 29, 2004.
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Abstract |
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; Leibach and Ganapathy, 1996; Adibi, 1997; Daniel and Herget, 1997; Nussberger et al., 1997; Covitz et al., 1998; Fei et al., 1998). It is known that in addition to di- and tripeptides, both mammalian peptide transporters have the capability to accept a large number of peptidomimetic drugs such as 
-lactam antibiotics, angiotensin-converting enzyme inhibitors, selected peptidase inhibitors, and prodrugs (Rubio-Aliaga and Daniel, 2002). PEPT1 possesses a high transport capacity, is expressed along the entire small intestine epithelium, and is central for the known high oral availability of peptidomimetic drug substrates. The function of the high-affinity type transporter PEPT2 in drug transport is mainly reabsorption of the compounds after glomerular filtration in the kidney. PEPT2 expression also has been shown in other tissues such as epithelium of the choroid plexus, lung, mammary gland, and glia cells of the central nervous system. Recent studies have identified major differences in the structural requirements for binding and transport in substrates of PEPT1 and PEPT2. Whereas 
-amino fatty acids and amino acid-arylamides are recognized and transported by PEPT1 as high-affinity substrates, PEPT2 does not accept -amino fatty acids and displays a wide range of affinities and different transport characteristics when studied with amino acid-arylamides (Borner et al., 1998; Doring et al., 1998a). However, modifications, such as the introduction of a carbonyl group into the backbone of omega-amino fatty acids, can transform a substrate with very low affinity into a high-affinity compound that is transported by PEPT2 electrogenically (Theis et al., 2002), suggesting that PEPT2 has more specific requirements for substrate recognition. This also could be important for a specific delivery of drugs such as dipeptidyl peptidase IV (DPIV) or prolyl endopeptidase (PEP) inhibitors.
The therapeutic potential of inhibitors of postproline cleaving enzymes has been the focus of recent pharmaceutical research (Rosenblum and Kozarich, 2003). DPIV (EC 3.4.14.5
[EC]
), also known as CD26, is a membrane-associated peptidase found in numerous tissues. Significant DPIV-like activity also is detectable in plasma from humans and rodents (De Meester et al., 1999). Special attention has recently been given to DPIV inhibitors, which where found to act as efficient antidiabetic drug candidates in first human trials (Ahren et al., 2002; Hoffmann et al., 2001). Similar, inhibitors of the related enzyme PEP (EC 3.4.21.26
[EC]
) have potential as drugs to treat neurodegenerative disorders (Rosenblum and Kozarich, 2003).
DPIV inhibitors with structures of or similar to di- and tripeptides offer the opportunity to target the mammalian peptide transporter PEPT1 for oral delivery, which would result in an efficient intestinal permeability. The specific DPIV inhibitor isoleucine-thiazolidide (di-[2S,3S]-2-amino-3-methyl-pentanoic-1,3-thiazolidine fumarate; L-threo-isoleucine-thiazolidide; Ile-Thia), when given orally in humans, resulted in a dose-dependent decrease of plasma DPIV and a concomitant increase of active glucagon-like peptide 1 (Hoffmann et al., 2001), suggesting its efficient intestinal absorption possibly by PEPT1. Because DPIV/CD26 possesses multiple functions, a central question associated with the emerging use of DPIV inhibitors is that of potential side effects such as immunosuppression or inhibition of DPIV-like enzymes, which are partially intracellularly located enzymes (Kubota et al., 1992; Sedo and Malik, 2001). Therefore, peptide transporters in peripheral tissues could be of importance in this respect by allowing DPIV or PEP inhibitors to be taken up into specific cells.
Here, we have analyzed whether 1) Ile-Thia based on its peptide-like structure is a substrate of the mammalian peptide transporters PEPT1 and PEPT2, and 2) whether structurally related compounds can be identified for a targeted delivery and high oral availability. Because proline-containing tri- and tetrapeptide analogs are capable to inhibit DPIV and PEP, we also included such structures into the study.
We first determined the apparent affinity of Ile-Thia and the other test compounds for interaction with peptide transporters based on competition with the uptake of the radiolabeled dipeptide D-Phe-Ala in Pichia pastoris yeast cells expressing heterologously either human PEPT1 or PEPT2. To be able to differentiate between compounds that only interact with the substrate-binding site of the transporter and those that are electrogenically transported, we performed electrophysiological studies by recording inward currents induced by the compounds in Xenopus laevis oocytes expressing either of the peptide transporter forms. This procedure also permitted a comparison between the two transporters with respect to differences in substrate specificity for binding and transport.
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Materials and Methods |
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) and Demuth et al. (1993).
P. pastoris Strains and Transport Assays in Yeast. Cultures of P. pastoris strains expressing human PEPT1 or PEPT2 were prepared as described previously (Doring et al., 1997, 1998b). Cells were centrifuged at 3000g for 10 min and formed into a pellet, washed twice with 100 mM potassium phosphate buffer (PPB; pH 6.5), and resuspended to 5 x 107 cells/20 µl of PPB. Uptake measurements were performed at 22-24°C using a rapid filtration technique on 96-well filter plates (filter material HATF type, 0.45-µM pore size; Millipore Corporation, Eschborn, Germany). In brief, uptake was initiated by mixing 20 µl of the cell suspension with 30 µl of PPB containing 0.1 µCi corresponding to 50 nM (PEPT1) or 0.05 µCi corresponding to 25 nM (PEPT2) of the radiolabeled dipeptide D-Phe-Ala either with or without competitors (final concentration 0-20 mM). After 15 min of incubation, the uptake was terminated by the addition of 200 µl of ice-cold PPB followed by filtration. The filters were washed four more times with 200 µl of PPB. The radioactivity associated with the filter was directly measured by liquid scintillation counting.
X. laevis Oocytes Handling and cRNA Injection. Female X. laevis frogs were purchased from Nasco (Fort Atkinson, WI). Oocyte handling and transporter cRNA injection has been described previously (Boll et al., 2002). Oocytes were injected with 15 nl of sterile water (control), 15 nl of rabbit PEPT1-cRNA (15 ng), or 15 nl of rabbit PEPT2-cRNA (30 ng), respectively. The oocytes were kept in modified Barth's solution [88 mM NaCl, 1 mM KCl, 0.8 mM MgSO4, 0.4 mM CaCl2, 0.3 mM Ca(NO3)2, 2.4 mM NaHCO3, and 10 mM HEPES, pH 7.5] at 18°C until further use (3-5 days after injection).
Two-Electrode Voltage Clamp. Two-electrode voltage-clamp experiments were performed as described previously (Kottra and Daniel, 2001). Briefly, the oocyte was placed in an open chamber and continuously superfused with modified Barth's solution or with solutions of Gly-Gln and/or substrates to be tested. Electrodes with a resistance between 1 and 10 M
were connected to a TEC-05 amplifier (NPI Electronic, Tamm, Germany). Oocytes were voltage-clamped at -60 mV, and current-voltage relations were measured using short (100-ms) pulses separated by 200-ms pauses in the potential range -160 to +80 mV. Current-voltage measurements were made immediately before and 20 to 30 s after substrate application when current flow reached steady state. The current evoked by PEPT1 or PEPT2 at a given Vm was calculated as the difference between the currents measured in the presence and the absence of substrate. Each substrate was tested against the maximal inward current elicited by 5 mM Gly-Gln, allowing for a comparison of current recordings that are independent of the level of functional expression of various oocyte batches. Current traces were recorded during voltage clamping to -60 mV, and substrates were perfused at concentrations of 5 mM for 20 s. The electrophysiological measurement data are representative of at least two independent experiments with different oocyte batches.
Statistics. All calculations (linear as well as nonlinear regression analyses) were performed using Prism software (GraphPad Software Inc., San Diego, CA). At least two independent experiments with three replicates were carried out for each variable in the yeast competition assays. Transport measurements in Xenopus oocytes were performed at least two times with oocytes from different oocyte batches. Data are presented as mean ± S.E.M.
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) and therefore have chosen an incubation time of 15 min for all uptake experiments. We screened a wide range of different dipeptides and structurally related compounds. As shown in Fig. 1, the compounds when provided at a concentration of 10 mM displayed very different potencies for reducing the influx of the tracer [3H]D-Phe-Ala (50 nM) via PEPT1 into the yeast cells. Dipeptides and tripeptides, inter alia Lys-Phe (Fig. 2, 1), and Lys-Phe-Pro (Fig. 2, 2) were able to reduce the uptake of [3H]D-Phe-Ala to 8.2 ± 1.8 and 10.3 ± 0.7% of control levels, but also some of the DPIV-inhibitors such as Ile-Thia (Fig. 2, 11) and His-Thia (Fig. 2, 9) reduced tracer uptake to 19.9 ± 2.6 and 34.8 ± 1.4% of that in controls.
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Kinetics of Transport of Ile-Thia- and Thia-Derivatives. We next determined inhibition constants of D-Phe-Ala uptake via PEPT1 by the DPIV inhibitor Ile-Thia. D-Phe-Ala uptake was assessed at a concentration of 10 µM in the presence of increasing Ile-Thia concentrations (0-20 mM) at an external pH of 6.5. Inhibition of D-Phe-Ala uptake by Ile-Thia followed typical first order competition kinetics with an apparent EC50 value of 510 ± 94 µM (Fig. 3A). This apparent affinity is in the similar range as the EC50 value of 164 ± 18 µM of the dipeptide Gly-Gln (Fig. 3A). This demonstration that an amino acid coupled via its carboxy group to a thiazolidide function is recognized by PEPT1 led to the question whether di- or tripeptides with a thiazolidide group replacing the free carboxy group are also potential PEPT1 substrates. The dipeptide derivative Glu-Gly-Thia competed dose dependently with D-Phe-Ala for uptake by PEPT1 but with a considerably lower affinity than Ile-Thia (EC50 = 3.23 ± 0.05 mM) (Fig. 3B). Pro-Ile-Thia exhibited almost no affinity for the PEPT1 transporters as indicated by extrapolated EC50 values greater than 30 mM (Fig. 3B). In the case of the tripeptide derivatives Gly-Pro-Ile-Thia and Pro-Pro-Ile-Thia, affinity for PEPT1 was abolished (Fig. 3B). Because concentration-dependent competition with a dipeptide for binding at the substrate binding site of PEPT1 does not establish that the thiazolidide-derivatives are indeed transported, we used the two-electrode voltage-clamp technique in X. laevis oocytes expressing PEPT1. As shown in Fig. 3C, perfusion of oocytes with 5 mM Gly-Gln induced a PEPT1-mediated inward current of 700 nA at a Vm of -60 mV and in the same oocyte, 5 mM Ile-Thia induced a current of around 200 nA. In water-injected controls neither Gly-Gln nor Ile-Thia induced any inward currents (Fig. 3C). In contrast to Ile-Thia, all dipeptide and tripeptide derivatives (Glu-Gly-Thia, Pro-Ile-Thia, Gly-Pro-Ile-Thia, and Pro-Pro-Ile-Thia) failed to induce any inward currents at substrate concentrations of 5 mM (data not shown).
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This establishes that although Glu-Gly-Thia possesses considerable affinity for interaction with the substrate binding domain of PEPT1, it is an inhibitor, whereas Ile-Thia is an electrogenically transported substrate.
Transport Properties of Radiolabeled Ile-Thia. To demonstrate by independent methods that amino acid thiazolidide-derivatives such as Ile-Thia are in fact taken up into the cell by PEPT1, we used radiotracer flux studies in P. pastoris cells with [14C]L-Ile-Thia as a representative substrate to assess its transport characteristics by PEPT1. As shown in Fig. 4A, labeled Ile-Thia was taken up into the yeast cells expressing PEPT1 in a concentration-dependent manner after Michaelis-Menten kinetics. An apparent Kt value of 684 ± 80 µM was determined using nonlinear regression (Prism; GraphPad Software Inc.). Moreover, uptake of [14C]Ile-Thia was inhibited by classical substrates of PEPT1 such as Gly-Gln, D-Phe-Ala, and cefadroxil (Fig. 4B). The free amino acid isoleucine failed to inhibit uptake of Ile-Thia. These results confirm that Ile-Thia is a PEPT1-substrate with a substrate affinity similar to normal dipeptides.
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Because PEPT2 has a similar but not identical substrate specificity as PEPT1, we also determined the interaction of the various DPIV inhibitors with the high-affinity peptide transporter expressed in P. pastoris cells and in X. laevis oocytes. Ile-Thia displayed in the competition assay with D-Phe-Ala as a substrate a fairly low apparent EC50 value of 530 ± 40 µM. The dipeptide derivative Pro-Ile-Thia possessed an EC50 value of 1.50 ± 0.01 mM, whereas Val-Thia showed with a EC50 value of 60 ± 17 µM strong inhibitory potency. However, none of the tested compounds was able to induce substrate-evoked inward currents in oocytes expressing PEPT2 compared with the dipeptide glycyl-glutamine, even at hyperpolarized membrane potentials of -120 mV, demonstrating that PEPT2 does not transport these compounds, although they are inhibitors with lower affinities.
Role of the Amino Acid Residue in Amino Acid-Thiazolidide-Derivatives on Transport by PEPT1. Comparative analysis of transport of a series of compounds with a C-terminal thiazolidide-function and various N-terminal amino acid residues established that all of the tested aminoacyl-thiazolidides interacted with the peptide transporter PEPT1 in P. pastoris cells. The apparent EC50 values obtained for the different substrates are summarized in Table 1. Similar to normal dipeptides, hydrophobic N-terminal amino acid residue such as isoleucine, leucine, or valine increase the substrate affinity to PEPT1 compared with compounds with more hydrophilic properties, such as glutamate. This effect also was observed for their interaction with PEPT2. Val-Thia exhibited with an apparent EC50 value of 60 ± 17 µM a very high affinity for inhibition of D-Phe-Ala uptake, whereas Glu-Thia displayed a very low affinity, represented by an EC50 value of 4.36 ± 1.51 mM. Despite the high affinity of Val-Thia, we did not observe electrogenic transport of this compound in oocytes expressing PEPT2. Perfusion of the other Thia-derivatives also did not lead to a depolarization of the oocyte membrane potential (Table 1), whereas oocytes expressing PEPT1 responded to perfusion of Val-Thia with an inward current as high as that of the reference dipeptide Gly-Gln. Additionally, Ile-Thia, Leu-Thia, and Glu-Thia also displayed similar PEPT1-mediated transport currents (Table 1).
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Selective Recognition of Isoleucine-Thiazolidide Stereoisomers by PEPT1 and PEPT2. Because of the second asymmetric carbon center at carbon position 3 along the side chain of isoleucine, there are four possible configurations of isoleucine-thiazolidide. We therefore examined whether the various Ile-Thia stereoisomers display different transport characteristics for PEPT1 and PEPT2. In the case of PEPT1, L-allo-Ile-Thia possessed an up to 40-fold higher affinity than the corresponding D-enantiomer (Fig. 5; Table 2). However, L-allo-Ile-Thia displayed, with an EC50 value of 130 ± 31 µM, a 4-fold higher affinity than its diastereomer L-threo-Ile-Thia (510 ± 60 µM). D-allo-Ile-Thia exhibited almost 5-fold lower affinity than its diastereomer D-threo-Ile-Thia (Fig. 5; Table 2). Similarly, all L-isoleucine-containing enantiomers displayed 3- to 4-fold higher affinities compared with the D-isomers for binding to PEPT2 and substrates with allo-configuration also showed the highest affinities. Interestingly, D-allo-Ile-Thia was found to be selective for PEPT2. With this compound, we observed a 30-fold higher affinity to PEPT2 compared with PEPT1.
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, 1998b). The transgenic yeast cells can be grown in large quantities with high expression levels of the transporters. We have used this system before in combination with the X. laevis system for defining the template for substrate recognition by the peptide transporters (Theis et al., 2002; Knutter et al., 2004). Here, we describe that the combination of these two peptide transporter expression systems also may be useful for predicting the oral availability of peptide-derived peptidase inhibitors as a new class of potential drug candidates.
The peptide transporter PEPT1 and PEPT2 have proven to be relevant targets for drug delivery due to their uniquely broad substrate specificity and predominant expression in brush-border membranes of epithelial cells of the small intestine, lung, choroid plexus, and kidney (Rubio-Aliaga and Daniel, 2002). It was shown in different cell systems, expressing the peptide transporter either heterologously or endogenously, that -lactam antibiotics, angiotensin-converting enzyme inhibitors, and selected peptidase inhibitors are high-affinity substrates of both transporters. In the case of PEPT1, these substrates also show good oral availability (Rubio-Aliaga and Daniel, 2002). Moreover, poorly absorbed drugs can be turned into rapidly and efficiently transported compounds by rendering them into PEPT substrates, as shown, for example, for the antiviral nucleoside acyclovir by esterification with L-valine (Ganapathy et al., 1998; Lycke et al., 2003).
Based on this knowledge, we have screened a variety of DPIV and PEP inhibitors for their transport by mammalian peptide transporters to assess whether they could be orally available by efficient uptake via PEPT1 or delivered via PEPT2 to the lung epithelium or for renal reabsorption that could alter the compounds' pharmacokinetics. By uptake inhibition experiments with D-Phe-Ala as a substrate, we demonstrate that Ile-Thia and other amino acids coupled via the carboxy group to a thiazolidide function do interfere with the substrate binding domains of PEPT1 and PEPT2 with high affinities. That the thiazolidide-derivatives are recognized by the transporters is not necessarily surprising because they match at least partially the template for structural requirements in substrates which consist of 1) a free N terminus or instead a weakly basic group in 
-position at the N terminus; 2) an extended planar backbone, not longer than four methylene groups, with a correctly positioned backbone carbonyl group; and 3) an ionic function at the C terminus (Doring et al., 1998a; Bailey et al., 2000; Brandsch et al., 2004; Daniel and Kottra, 2004). Although a carboxyl or C-terminal ionic group is missing in the amino acid thiazolidide-derivatives, other compounds lacking this function such as alanyl aryl amides also were shown to be transported by PEPT1 (Borner et al., 1998). Very recently, Gebauer et al. (2003) reasoned by means of three-dimensional quantitative structure-activity relationship analysis based on a series of dipeptide substrates that an ionized C-terminal group can be replaced by a ring system with a high electronic density without a loss in substrate affinity. This may explain the good affinity of the aminoacyl-thiazolidides and is obviously also sufficient to allow transport of the compounds, at least in the case of PEPT1.
However, when di- and tripeptides were coupled with a thiazolidide function no transport was observed, although some of the compounds retained reasonable affinities for binding. That the tripeptide derivatives are not transported is also in accordance with other findings on substrate specificity and template requirements (Terada et al., 2000; Chen et al., 2002). However, the dipeptide derivatives that by coupling to the thiazolidide moiety mimic a tripeptide structure also lacked electrogenic transport while displaying high affinities. This can currently not be explained. This finding addresses the important question of whether transport predictions made on basis of competition studies are valid. Template predictions available are so far based on substrate affinities as derived from competition experiments in different expression systems. They thereby do allow a fair description of substrate requirements for binding to PEPT1 or PEPT2 but not necessarily for transport. We here demonstrate by combining competition assays and electrophysiological measurements that most of the DPIV inhibitors, despite of their good substrate affinity, cannot be transported. However, the aminoacyl-thiazolidides are transported substrates of PEPT1. Using radiolabeled Ile-Thia, we also demonstrate that hPEPT1 in P. pastoris cells allows efficient Ile-Thia uptake and that this influx is inhibited by dipeptides and aminocephalosporines. That Ile-Thia may be a PEPT1 substrate was suggested by uptake inhibition experiments in Caco-2 cells with glycyl-sarcosine as a substrate of the endogenous PEPT1 protein but with lower affinity than found here (Brandsch et al., 1999). This difference, however, may be explained in part by the different experimental conditions such as cell system (yeast and oocyte versus Caco-2), the tracer used (D-Phe-Ala versus Gly-Sar), and membrane potential. In particular, the latter is known to affect substrate affinity significantly; therefore, studies carried out under voltage-clamp conditions as provided here in oocytes allow a more defined determination of substrate affinity.
Electrogenic transport was in our studies also observed for Leu-Thia, Val-Thia, and Glu-Thia. Val-Thia exhibited highest transport currents and is also an effective DPIV inhibitor. The amino acid-thiazolidide-derivatives tested do show the known stereospecificity of the peptide transporters (Li et al., 1998) with all L-isomers displaying higher affinities than the corresponding D-isomers. This was most pronounced when comparing L-allo-Ile-Thia with D-allo-Ile-Thia, with a difference in affinity of more than 40-fold.
The inhibitory effects of various di- and tripeptides as well as aminoacyl-thiazolidides and -pyrrolidides on DPIV in vitro is well known (Rahfeld et al., 1991; Demuth and Heins, 1995). However, recently the inhibition of DPIV also was observed in vivo after oral administration of Ile-Thia in diabetic animals and patients (Hoffmann et al., 2001; Pospisilik et al., 2002a,b). In vivo studies in rats, dogs, and monkeys using the DPIV inhibitors Ile-Thia and its allo-stereoisomer have recently shown that these compounds are well absorbed, because more than 80% of the administered dose was found in plasma (Beconi et al., 2003). Our findings suggest that this high oral availability of Ile-Thia in vivo and that of the similar derivatives is based on their efficient uptake via PEPT1 across the apical membrane into the intestinal epithelial cell. A recent study using rat jejunum has shown a close correlation between the intestinal permeability of identified PEPT1 substrates and the PEPT1 protein expression level (Naruhashi et al., 2002). Although a paracellular permeation of the DPIV inhibitors also may contribute to intestinal absorption, their good affinity and the carrier-mediated transport shown here argue for a significant contribution of PEPT1 to overall absorption of the compounds.
Regarding PEPT2, we demonstrate that this protein does not allow Ile-Thia- and Thia-derivatives to be transported, which may be important in view of possible unwanted side effects by uptake of the inhibitors into extraintestinal tissues. Finally, there is a clear selectivity of aminoacyl-thiazolidine-derivatives for PEPT1 over related tri- and tetrapeptide derivatives, which further helps discriminating DPIV and PEP activity inhibition by such compounds.
In conclusion, we provide direct evidence that the transport of the DPIV inhibitor isoleucine-thiazolidide occurs by the intestinal peptide transporters PEPT1 with all the characteristics known such as membrane potential dependence, affinity pattern, and stereospecificity. Efficient handling of Ile-Thia by PEPT1 does have pharmacological implications for treatment of diabetes based on the Ile-Thia-induced specific inhibition of the dipeptidyl peptidase DPIV/CD26 that in turn increases the circulating levels of the insulinotropic gut hormones GLP-1 and glucose-dependent insulinotropic polypeptide. Moreover, our expression systems for PEPT1 and PEPT2 in P. pastoris cells in combination with electrophysiological recordings in X. laevis oocytes proved to be valid in rapidly screening a large numbers of potential drug candidates and to easily distinguish that solely bind to the transporters from those that also are transported.
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ABBREVIATIONS: PEPT, peptide transporter; Vm, membrane potential; DPIV, dipeptidyl peptidase IV; PEP, prolyl endopeptidase; Thia, thiazolidide; Ac, acetate; Pyrr, pyrrolidide; Boc, tert-butyloxycarbonyl; Bz, benzoyl; Z, benzyloxycarbonyl; PPB, potassium phosphate buffer.
Address correspondence to: Prof. Dr. Hannelore Daniel, Institute of Nutritional Sciences, Center of Life and Food Sciences, Technical University of Munich, Hochfeldweg 2, D-85350 Freising-Weihenstephan, Germany. E-mail: daniel{at}wzw.tum.de
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