tri-peptide transporter hPEPT1 (SLC15A1)

J. DRUG DEL. SCI. TECH., 23 (4) 307-314 2013

Current status of rational design of prodrugs targeting the intestinal di/tri-peptide transporter hPEPT1 (SLC15A1) L. Saaby2, C.U. Nielsen1, B. Steffansen1, S.B. Larsen3, B. Brodin1* Department of Pharmacy, 2Bioneer:FARMA, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen Ø, Denmark 3 Zealand Pharma A/S, Smedeland 36, DK-2600 Copenhagen, Denmark *Correspondence: [email protected]

1

The intestinal di/tri-peptide transporter hPEPT1 has broad substrate specificity, accommodating uptake of the majority of investigated di- and tripeptides, as well as of a number of drug compounds. This transport system has a high capacity and it has been hypothesized that hPEPT1-mediated uptake of drug compounds, conjugated with promoieties so as to resemble di- or tripeptides, may be a strategy for increasing the intestinal permeability of compounds with otherwise low bioavailability. Based on the research activities of our group during the last decade, as well as on general research in the field, the present review aims at giving a brief overview of structure-activity relationships for hPEPT1, and to provide a critical evaluation of whether hPEPT1-targeted prodrugs can be rationally designed. Key words: Drug delivery – Oral bioavailability – Drug transporters – ADME – PEPT1– Substrates – Prodrug strategies.

The enterocytes of the small intestine possess a number of specialized nutrient and micronutrient transporters in their apical and basolateral membranes [1-6]. These transporters have received a lot of attention as potential routes for drug delivery for small hydrophilic drug compounds. Among these transporters, the intestinal di/tri-peptide transporter hPEPT1 (SLC15A1) remains the most thoroughly investigated [7]. This is probably due to the fact that the transporter has broad substrate specificity, a large transport capacity and has been shown to mediate intestinal absorption of several drug compounds [6]. Pept1 cDNA was initially cloned from rabbit small intestine and expressed in Xenopus laevis oocytes [8] and shortly thereafter, the cloning of hPEPT1 was reported [9]. A splice variant with putative regulatory functions, hPEPT1-RF, has been reported [10], however a recent study demonstrated that this variant is not expressed in enterocytes [11]. Another splice variant, hPEPT1-RFnt, is expressed but only as mRNA, and its function remains unclear [11]. PEPT1 is expressed in the small intestine [9], in the early parts of the proximal kidney tubules [12], the pancreas [13] and in the liver [8] and reports also point at the presence of PEPT1 in the apical membrane of human lung epithelium cell lines [14], [15]. The gene encoding hPEPT1 consists of 24 exons and is located on chromosome 13 (q33-q34). hPEPT1 is encoded by 23 exons and consists of 708 amino acids with a core molecular mass of ~79 kDA [9]. hPEPT1 has 12 putative membrane-spanning domains, two potential sites for protein kinase C phosphorylation and seven putative N-glycosylation sites [9]. The predicted structure has a large extracellular loop between the 9th and 10th membrane-spanning domain, with both the amino terminus and carboxyl terminus facing the cytosol [9]. By means of homology modeling using the bacterial lactose permease LacY as a template, it has been possible to divide the 12 membrane spanning domains into an internal group (segments 1, 4, 5, 7 and 10) and an external group (segments 2, 3, 6, 8, 9, 11 and 12) [16; 17]. The internal set of segments is believed to form a central pore and bears the key residues involved in substrate recognition [16]. hPEPT1 expression levels can be regulated by epidermal growth factor (EGF) [18], leptin [19], insulin [20], nutritional status [21] and diurnal rhythm [22]. The proposed model for intestinal transport of di/tri-peptides and peptidomimetic drug and prodrug compounds is depicted in Figure 1. hPEPT1 transports substrates across the apical membrane

of the enterocyte in an electrogenic co-transport together with protons, utilizing the proton gradient between the intestinal lumen (pH 5.56.0) and the intracellular environment of enterocytes (~ pH 7) [23]. This proton gradient is generally believed to be maintained by a Na+/ H+ exchanger in the apical membrane, which in turn is driven by the basolateral Na+/K+-ATPase. Once inside the cells, most naturally occurring peptides are hydrolyzed to their constituent amino acids and reach the blood through the activity of amino acid transporters in the basolateral membrane [24, 25]. Non-hydrolysable substrates may pass the basolateral barrier via a peptide transport mechanism, which is not fully understood. The presence of a basolateral peptide transport

Figure 1 - The transepithelial transport pathway for di/tri-peptides and peptidomimetic drug compounds. Proteins are broken down to a mixture of amino acids and small peptides in the intestinal lumen. Di/tri-peptides are taken up via hPEPT1, situated in the apical membrane of the enterocytes in the small intestine. The transport process is energized by the concentration gradient of peptides and the proton gradient across the luminal membrane of the enterocytes. The proton gradient is created by the action of the sodium/proton-exchanger (NHE3), which in turn is energized by basolateral sodium-potassium ATPase. In the cells, di- and tripeptides may be enzymatically hydrolyzed to amino acids and cross the basolateral membrane via amino acid transporters. Intact di- and tripetides, as well as peptidomimetic drug compounds, may cross the basolateral membrane via a basolateral peptide transporter system. 307

Current status of rational design of prodrugs targeting the intestinal di/tri-peptide transporter hPEPT1 (SLC15A1) L. Saaby, C.U. Nielsen, B. Steffansen, S.B. Larsen, B. Brodin

J. DRUG DEL. SCI. TECH., 23 (4) 307-314 2013

mechanism has only been shown on a functional level [25, 26]. However, it has been demonstrated that the transport is pH-dependent [26, 27] and subjected to regulation [28]. A number of drugs have been reported to be taken up via hPEPT1, including β-lactam antibiotics such as penicillins and cephalosporins [29] and the dipeptide-like antineoplastic drug bestatin [30]. Prodrugs of acyclovir, ganciclovir and L-DOPA are also recognized and transported by hPEPT1 [31-37], as well as dipeptidyl prodrugs of pamidronate and amino acid prodrugs of carbapenems [38, 39]. ACE inhibitors [40-42], renin inhibitors [43] and thrombin inhibitors [44] have also been considered classical hPEPT1 substrates. However, recent studies indicate that at least the ACE-inhibitors are not transported to a significant extent via hPEPT1 [45]. It should also be noted that some carbapenems may be absorbed via the intestinal organic anion transporters OATP1A2 and OATP2B1 [46]. Finally, substrates for hPEPT1 also include most of the dietary di- and tripeptides, while single amino acids and larger peptides are not transported [9, 47]. Adibi and Mercer have shown that in human volunteers the intestinal amino acid concentration after ingestion of a test meal containing 50 g purified bovine serum albumin result in luminal millimolar concentrations of dipeptides, and that 3 h after ingestion, luminal concentrations of peptides is still measurable [48]. Orally administered prodrugs and dipeptides from the food could thus compete for available PEPT1 transporters, thereby altering the pharmacokinetic profile of the parent drug. Depending on the dose, dipeptide concentration and the therapeutic index potential food-drug interactions could emerge. Such interactions have been demonstrated for the proton-coupled amino acid transporter hPAT1. Intestinal transport of the amino-mimetic drug candidate gaboxadol, which owes its bioavailability to uptake via PAT1, has been shown to be inhibited by amino acids proline and tryptophan in in vivo in dogs [49] and a similar inhibition may occur for hPEPT1-mediated drug uptake in the presence of di- or tri-peptides originating from the breakdown of dietary protein. Earlier reviews from the group have focused on the various classes of substrates for hPEPT1 [47], the regulation of peptide transporters [50], therapeutic applications of peptide transporters [51] and strategies for employing peptide transporters in drug development and delivery [52]. The aim of the present review is to provide a critical, retrospective evaluation of the attempts by us and others to design hPEPT1-targeted prodrugs in a "rational" manner. In this context, we define rational prodrug design as a process where knowledge of the physicochemial properties of the promoiety structure or knowledge of the allowed pharmacophore of the entire prodrug enable design of substrates, which utilize hPEPT1 as an intestinal uptake pathway. An overview of approaches used for the design of prodrugs, which may be absorbed via hPEPT1, will be provided. We will discuss whether knowledge of the binding affinities of the promoieties may be useful in terms of predicting bioavailability of prodrugs, and whether the developed quantitative structure-activity relationship (QSAR) models have a broad applicability or are restricted to the substrate class, which has been used for their generation. In the last section, we will touch upon what has been accomplished, what the present limitations are, and point to future challenges.

of PEPT1-mediated transport and regulation also include HeLa or CHO cell lines, transiently or stably transfected with hPEPT1 [54], Xenopus oocytes injected with hPEPT1 mRNA [55], transgenic yeasts [56], and perfused intestinal segments from rats [57]. By relating affinity and translocation data obtained from these model systems with physico-chemical properties (by means of molecular descriptors) of the investigated compounds, a number of QSAR models have been proposed. The aim of developing QSAR models has been to generate a set of determinants of affinity and translocation which will enable rational design of hPEPT1-targeted drugs. Andersen et al. developed a QSAR model based on a series of 25 diverse tripeptides correlating hPEPT1-affinity data with alignment-independent VolSurf descriptors. According to this model, an even distribution of hydrophobic regions rather than having the hydrophobic region concentrated in one area of the tripeptide favors binding to hPEPT1. The model also suggests that tripeptides with large hydrophobic surface areas have a higher affinity for hPEPT1 than molecules with large hydrophilic surface areas. Finally, separation between charged functional groups appears to be positively correlated with binding to the transporter. Hence, this favors tripeptides with a neutral side chain as these has a structure with maximal charge separation [58]. The overall findings of Andersen et al. generally corroborate previous QSAR models proposed by Gebauer et al. and Biegel et al. on di-/tripeptides and beta-lactam antibiotics employing CoMSIA descriptors [59, 60] as well as the conformation studies by Våbenø et al. [61]. Larsen et al. developed a QSAR model using several alignment independent descriptors (thus, assuming no specific pharmacophore model as for, e.g., CoMSIA) as well as partial least square (PLS) modeling on a data set comprising the major known chemical classes of hPEPT1 ligands compiled from the literature. An important finding from this work was that hydrophilic interactions contribute more to binding to hPEPT1 than do hydrophobic interactions; that is, H-bond donor capabilities of a ligand seem favorable for affinity, while H-bond acceptor properties are inversely correlated with affinity [62, 63]. The models described above were restricted by the methodological approach, since the affinity values originated from uptake studies of test compound in the presence of a known radiolabelled hPEPT1 substrate, and the affinity was determined as the degree of inhibition of uptake of the reference substrate (usually Gly-Sar). The models, only describe the structural features of compounds which were capable of binding to hPEPT1, thereby causing inhibition of substrate uptake. However, not all compounds which inhibit uptake of a reference substrate are substrates themselves, and several non-transported inhibitors have been identified, including compounds such as Glu(acyclovir)-Sar, LGlu(acyclothymidine)-Sar and Lys[Z(NO2)]-Pro [64-66]. Furthermore, some drug substances such as e.g. ibuprofen may apparently decrease hPEPT1-mediated transport in a non-specific manner, presumably without interacting directly with the transporter [67]. This emphasizes the need for generating correlations between structural determinants and actual translocation of substrates from the intestinal lumen into the enterocytes, as opposed to the competition studies which only predict affinity to the ‘external’ binding site of the transporter. This approach is more challenging in terms of experimental design since investigations of translocation require that the movement of test compound from the outside to the inside of a cell can be monitored. Bretschneider et al. performed a thorough comparison of affinities and bioavailability for a series of beta-lactam antibiotics and observed an overall correlation between affinity for hPEPT1 and bioavailability [29]. In this important study it was demonstrated that substrates with Ki-values above ~14 mM were unlikely to have a significant carriermediated uptake component. The first detailed study of the correlation between affinity and actual translocation was performed by Vig et al. by comparing affinity data from uptake experiments with translocation data [68]. The translocation was measured using a membrane depolarization assay as a surrogate marker for depolarization. The

I. Structure-affinity and structuretranslocation relationships for hPEPT1

Despite the great interest in hPEPT1, the crystal structure has yet to be resolved; a task which has proven difficult due to the large molecular size and the presence of membrane spanning segments [9, 53]. Therefore, existing knowledge on structural requirements for binding and translocation has been obtained from functional experimental model systems using a ligand-based approach. A large number of studies have been performed using the Caco-2 cells, a colon-derived cell line which expresses a number of small intestinal nutrient transporters. Experimental models to investigate mechanisms 308

J. DRUG DEL. SCI. TECH., 23 (4) 307-314 2013

Current status of rational design of prodrugs targeting the intestinal di/tri-peptide transporter hPEPT1 (SLC15A1) L. Saaby, C.U. Nielsen, B. Steffansen, S.B. Larsen, B. Brodin

rationale for this came from studies showing that translocation of a hPEPT1 substrate is an electrogenic process [69] where a charge is moved from the cell exterior to the cell interior during a translocation cycle, independent of the initial charge of the substrates [55, 70]. Vig et al. investigated a set of 81 compounds, with the majority of the compounds being dipeptides. In general, a good correlation between affinity and translocation was observed, however four of the tested dipeptides showed binding but no translocation. Notably, dipeptides with two positive charges at the experimental pH (Lys-Lys and ArgArg) or with bulky side chains (Trp-Trp) showed affinity to the binding site, but were not translocated by hPEPT1 [68], underlining that affinity for the transporter does not necessarily imply translocation. Using a similar experimental approach, Omkvist et al. developed a model based on a correlation between VolSurf descriptors and translocation data generated from studies on a series of 55 tripeptides and successfully predicted new inhibitors of hPEPT1, with Met-Pro-Pro being the most prominent [71]. In the model proposed by Omkvist et al. hydrogen bonding descriptors and hydrophilic region descriptors were found to have a negative effect on translocation. Omkvist et al. found that the hPEPT1 substrate Ile-Leu-Met with high affinity for the translocation process possesses a relatively smaller hydrophilic region than the poor substrate Glu-Glu-Ser and concluded that the volume of the hydrophilic region is inversely correlated to hPEPT1mediated transport [71]. A few differences between the three models discussed above can be identified. The models of Omkvist et al. and Larsen et al. found a positive influence of the integy moments, while the opposite was true for the model developed by Andersen et al. The models developed by Andersen et al. and Omkvist et al. indicated that a large hydrophilic surface area was negatively correlated to binding to hPEPT1 and hPEPT1-mediated transport [58, 71]. The differences in predicted determinants for affinity and translocation by hPEPT1 between the three models can possibly be explained by differences in measured effect parameters (binding versus translocation data) and differences between the size and composition of the training sets used to build the three models. Thus, as expected, the applicability domains of the developed QSAR models are somewhat dependent on the training sets on which they are based. Furthermore, differences in predicted determinants for binding and translocation underline the need for investigating structure-translocation relationships for broader classes of compounds.

ments in Caco-2 cells, it was shown that the affinity of ketomethylene isostere towards hPEPT1 is comparable to the native peptide Phe-Gly and that this isostere is transported into the cells in a carrier-mediated manner [73]. In contrast, the trans-hydroxy ethylidene and hydroxyl ethylene isosteres were found to be poor substrates for the transporter [73]. Thorn et al. found that three ketomethylene tripeptide analogues and their corresponding tripeptides were high affinity ligands of hPEPT1 with binding constants in the sub-millimolar range [74]. As a rule of thumb, compounds with affinities below 0.5 mM can be considered high affinity ligands, compounds between 0.5 to 5 mM can be considered moderate affinity ligands and compounds with affinities above 5 can be considered low affinity ligands [75]. As previously observed with the beta-lactam antibiotics, compounds with affinities above 14 mM are unlikely to cross the epithelium via the carrier-mediated pathway [29]. The general structure of the tested ketomethylene analogues was H-Phe-Ψ[COCH2]-Ser(Bz)-X(aa)-OH, inspired by the fact that H-Phe-Ser-Ala-OH is a translocated hPEPT1 substrate with high affinity towards hPEPT1 [76]. The results of Thorn et al. (2011) and Våbenø, thus, indicate that replacement of the amide bond with a ketomethylene group is tolerated in terms of retaining tripeptidomimetic and dipeptidomimetic affinity towards hPEPT1. However, further studies are needed to establish whether the three ketomethylene investigated tripeptidomimetics are substrates or inhibitors of hPEPT1.

2. Design of prodrugs

Dipeptides have mainly been investigated as promoieties for hPEPT1-targeted prodrugs, although suitable tripeptide promoieties have also been reported [76]. Nielsen et al. showed that model ester prodrugs, designed using the metabolically stabilized dipeptides D-Glu-Ala and D-Asp-Ala as promoieties for benzyl alcohol esterified in the carboxylic acid of the N-terminal side chain, had a high affinity to hPEPT1 [77, 78]. The release of the model drug at physiological pH values was controlled by base-catalyzed hydrolysis, indicating that the compounds could remain stable in the intestinal lumen with a pH of ~6.0, and potentially release the model drug in the blood (pH 7.4) [77]. Thomsen et al. found that ester prodrugs of acyclovir and 1-(2-hydroxyethyl)-linked thymine to the glutamic side chain of D-Glu-Ala as the promoiety had a poor affinity for hPEPT1, however the L-Glu-Sar ester prodrugs of the same two compounds had a high affinity for the transporter [79]. In concordance with the findings of Nielsen et al., in vitro metabolism studies showed that release of acyclovir and 1-(2-hydroxyethyl)-linked thymine from the respective L-Glu-Sar prodrugs primarily is controlled by specific base-catalyzed hydrolysis and can thus be expected to share a similar stability as proposed for the D-Glu-Ala and D-Asp-Ala prodrugs of benzyl alcohol [77, 79]. However, oral administration of L-Glu(acyclovir)-Sar in rats resulted in a low bioavailability of both acyclovir and the intact prodrug [65]. To support these findings, uptake and bidirectional transport studies in Caco-2 cells indicated that the L-Glu(acyclovir)-Sar prodrug is not translocated by hPEPT1 [64, 79]. A similar pattern was observed with a nucleoside and a pyrimidine Glu-Sar prodrugs, which, despite showing high affinity towards hPEPT1, were found not to be translocated by the transporter [64]. Thus, Glu-Sar seems to be a poor promoiety for hPEPT1-mediated transport. In contrast, it was demonstrated in Caco-2 cells that model prodrugs of benzyl alcohol linked to the C-terminal side chain using the ketomethylene isostere of Phe-Asp and Val-Asp as promoieties, are both stable at intestinal lumen pH and substrates of hPEPT1 with high binding affinity for the transporter [80]. Amino acids may also be used as promoieties when coupled to the parent drug compound via ester bonds. This is illustrated by the high bioavailability of the valine ester prodrugs of the antiviral drugs acyclovir and ganciclovir, as compared to the parent compounds [72]. However, since the promoiety alone has no affinity to hPEPT1,

II. Design of drug and prodrug substrates for hPEPT1

hPEPT1-mediated transport is an attractive transport route for prodrugs of hydrophilic drug compounds which themselves cannot cross the lipid barriers in the intestinal epithelium by passive means. It may, however, be both chemically and structurally challenging to modify a drug compound so as to mimic a di- or tripeptide. Moreover, it may be challenging to achieve the right balance between pre-absorption stability and post-absorption lability.

1. Design of promoieties

Promoieties are normally selected from structures already having an intrinsic high affinity for hPEPT1 and proven translocation capability via hPEPT1. Although a number of studies have shown that, promoieties not having affinity for hPEPT1, such as amino acids, may be useful promoieties [72]. Ideally, the promoiety should be stable against enzymatic and chemical degradation in the gastrointestinal tract, and at the same time, the drug should be delivered to the blood stream. In the case of peptide drugs, labile amide bonds may need to be replaced with metabolically more stable bonds while maintaining the overall structural features of the corresponding natural peptide. Våbenø et al. synthesized a series of Phe-Gly dipeptidomimetics including the ketomethylene isostere, the trans-hydroxy ethylidene and hydroxyl ethylene isosteres. Through binding and transport experi309

Current status of rational design of prodrugs targeting the intestinal di/tri-peptide transporter hPEPT1 (SLC15A1) L. Saaby, C.U. Nielsen, B. Steffansen, S.B. Larsen, B. Brodin

J. DRUG DEL. SCI. TECH., 23 (4) 307-314 2013

this approach demands that the prodrug will acquire peptidomimetic properties.

about the structural requirements for substrates of hPEPT1 can be traced back to the fact that the crystal structure of the protein has yet to be resolved. Up to now most of the known structural requirements have been obtained through SAR-models in which affinity or translocation data have been correlated to structural determinants for a range of different compounds (training sets). However, the size and diversity of these training sets are often limited due to time and cost issues, and lack of availability of a wide range of structurally diverse compounds due to synthesis-related limitations. Furthermore, the majority of the models are based on competition studies with radiolabelled hPEPT1 substrates, and the resulting molecular determinants will therefore predict binding but not necessarily translocation. It is thus limited what we can expect to learn from these models and often the developed models are not available outside the involved research group. The amount and quality of published knowledge is often limited to trends retrieved from these models, which may not be specific enough to support a truly rational design of hPEPT1 substrates. On a more positive note, the increased use of a range of experimental approaches seems promising. A number of studies combined inhibition studies with cellular permeability studies and/or bioavailability studies enabling the selection of prodrug candidates which showed, not only inhibition of hPEPT1 substrates, but actual translocation via the transporter (Table I). However, it should also be noted that the data obtained from the animal studies may not predict human bioavailability correctly. Even though the function of PEPT1 seems to be largely conserved between species, correlation of data from animal models to humans should be done with caution due to possible differences in absolute transporter expression levels, and metabolic activities.

III. Can hPEPT1 substrates be rationally designed? An overview of recent prodrug design approaches

A substantial amount of effort has been invested in identifying intrinsic physico-chemical properties common to substrates of hPEPT1 to enable rational design of prodrugs targeted at this transporter. A number of these approaches have been covered by an earlier review from our group [51]. Since then, a number of new studies describing the synthesis and evaluation of prodrugs designed with the aim of targeting hPEPT1 have been published. A number of these studies are listed and depicted schematically in Table I. The design approach for the majority of these prodrugs has been to synthesize a series of different amino acid derivatives of the parent compounds and to screen the resulting prodrugs for affinity for and cellular uptake via hPEPT1. A number of studies have also reported bioavailability data from animal models. The amino acid promoieties have either been chosen from a broad range of amino acids or through a more selective approach with the inclusion of amino acid promoieties based on hPEPT1 affinity and stability data. An example of the latter is found in the synthesis of ester prodrugs of a guanidino-group containing compound, where the amino acids L-valine, L-isoleucine and Lphenylalanine were chosen as promoieties based on previous affinity and stability data [81]. Val-ester prodrugs of the antiviral compounds oseltamivir (Tamiflu), zanamivir, didanosine and levovirin were also shown to have increased intestinal permeability, as compared to the parent compounds [82-85] and isoleucyl ester prodrugs of the anticancer compound floxuridine showed increased permeability across Caco-2 cell monolayers [86]. The metabotropic glutamate receptor agonist LY354740 was modified to an alanyl prodrug, LY5443444, with an increased oral bioavailability [87, 88]. A prodrug consisting of the iron chelator maltosine, conjugated with alanine was also observed to have an increased rate of translocation via hPEPT1 in a Xenopus Oocyte two-electrode setup [89]. A number of studies have thus demonstrated that small molecular weight compounds can increase their intestinal uptake via hPEPT1 after being chemically linked to an amino acid. Another design approach was chosen by Santos et al. [90] and Kikuchi et al. [91], who used di-peptide promoieties to synthesize prodrugs of zidovudine and rebamipide, resepctively. Santos et al. synthesized various dipeptide ester prodrugs of zidovudine [90] and the promoieties were selected with the aim of incorporating certain structural features important for molecular recognition by hPEPT1, i.e. an L,L-peptide skeleton, a free amino group and uncharged hydrophobic side-chains [90]. However, in practice a range of prodrugs covering a wide range of physico-chemical properties was synthesized and evaluated to obtain further structure-activity information. Kikuchi et al. synthesized a series of prodrugs of rebamipide, a small-molecule antiulcer agent, using both single amino acids and dipeptides as promoieties [91]. Using both in vitro and in vivo methods, the authors demonstrated that the prodrug Ser(Reb)-Gly was taken up via hPEPT1 and that the intestinal permeability of the prodrug was higher than that of the parent compound. A third approach has been to use thiodipeptides as a promoiety for hydroxyimine prodrugs of the anti-inflammatory ketone drug nabumetone, resulting in increased transport in Caco-2 cell cultures as compared to a known hPEPT1 substrate [92]. Even though the design approaches adopted in the synthesis of the prodrugs listed in Table I may be different, they generally incorporate an element of trial and error, which indicates that the basis for a truly rational design approach is not available yet. At best, the current knowledge of the structural requirements for hPEPT1 substrates has enabled researchers to adopt a semi-rational approach where a limited number of promoieties are evaluated. Then, what are the main obstacles for a truly rational design of hPEPT1-targeted prodrugs? The lack of knowledge

IV. Conclusion and future perspectives

hPEPT1 is an intestinal nutrient transporter but also an oral uptake pathway for a number of therapeutic di/tri-peptidomimetics and prodrugs. This is most likely due to the fact that hPEPT1 has broad substrate specificity and a significant transport capacity. It has been hypothesized that new prodrugs can be designed to be taken up from the intestinal lumen via hPEPT1 and subsequently release drug substance in the blood stream or site of action. A number of studies have therefore dealt with investigating the structural and physicochemical requisites for binding to- and translocation via hPEPT1. A number of prodrugs have been developed using conjugation of parent drug compounds with amino acids or dipeptides. However, knowledge from the existing structure-affinity and structure-translocation relationships has not to our knowledge been incorporated rigorously in the drug development phase, probably due to limited accessibility of the models as well as challenges related to their interpretation. There is a need for expanding the structure-affinity relationships to structure-translocation relationships, in order to be able to predict the determinants for translocation. Furthermore, it may be speculated that further comparisons of affinity and translocation requirements will yield information on structural limitations for hPEPT1 substrates. The ongoing work on resolving the transporter protein structure and thereby the structure of both the binding pocket and the transporter in the different translocation steps is also likely to yield valuable information on the types of substrates which may be translocated. Finally an often overlooked potentially rate-limiting factor in intestinal prodrug delivery is the basolateral exit step from enterocyte to the circulation. A basolateral peptide transporter with a substrate specificity profile similar to that of hPEPT1 has been observed in functional studies, but a candidate protein has not yet been identified. A prodrug must either be hydrolyzed intracellularly, followed by exit of the parent drug to the circulation, or cross the basolateral membrane in intact form. The basolateral peptide transporter should therefore be characterized in greater detail in terms of structure-translocation relationships before we can make reliable predictions on hPEPT1-mediated transcellular transport across the intestinal epithelium. 310

J. DRUG DEL. SCI. TECH., 23 (4) 307-314 2013

Current status of rational design of prodrugs targeting the intestinal di/tri-peptide transporter hPEPT1 (SLC15A1) L. Saaby, C.U. Nielsen, B. Steffansen, S.B. Larsen, B. Brodin

Table I - Recent examples of prodrugs designed for carrier-mediated transport via PEPT1. Parent drug

Promoiety (R)

hPEPT1 affinity

Comparison of prodrug versus parent drug

Ref.

[3-(hydroxymethyl)phenyl]guanidine (3-HPG)

Val Ile

0.65 (IC50, mM)a 0.63 (IC50, mM)a

Both prodrugs showed increased permeability as compared to the parent drug in an in situ rat perfusion study

[81]

Zanamivir

Val

1.19 (IC50, mM)a

The valyl ester prodrug showed increased permeability in both cellular transport studies in Caco-2 cells and in in situ rat perfusion studies as compared to the parent drug

[82]

Guanidin oseltamivir carboxylate (GOC)

Val

0.19 (IC50, mM)a

The ester prodrug showed increased cellular uptake, rat intestinal permeability and bioavailability in mice when compared to the parent drug

[83]

Didanosine

Val

0.27 (IC50, mM)a

Oral bioavailability in rats was increased from 8 % for the parent drug to 47 % for the valyl ester prodrug

[84]

Levovirin

Val

ND

Increased cellular permeability (48-fold) across Caco-2 cells and bioavailability in rats (6.9-fold, AUC) as compared to levovirin

[85]

Floxuridine

3´-Ile 5´-Ile

0.86 (IC50, mM)a 1.19 (IC50, mM)a

Both prodrugs exhibited increased cellular permeability across Caco-2 cells as compared to floxuridine

[86]

LY354740

Ala

0.3 (IC50, mM)a

Oral bioavailability of LY354740 was increased in both rats and beagle dogs when administered as the prodrug (LY544344)

[87, 88]

Maltosine

Ala-R R-Ala

0.33 (Ki, mM)b 0.16 (Ki, mM)b

Electrophysiological measurements in hPEPT1expressing oocytes showed significant increase in transport of the prodrugs as compared to maltosine

[89]

Zidovudine

Val-Gly Val-Ala

0.2 (IC50, mM)a 0.15 (IC50, mM)a

No data on cellular uptake, intestinal permeability or bioavailability was reported

[90]

Rebamipide

Ser-Gly

ND

Electrophysiological measurements in hPEPT1expressing oocytes, cellular transport studies in Caco-2 cells and intestinal in situ rat perfusion experiments showed increased transport of the dipeptide ester prodrug as compared to rebamipide

[91]

-

0.46 (Ki, mM)b

Cellular transport studies in Caco-2 cells showed increased permeability of the prodrug when compared to the known hPEPT1 substrate PheΨ[CS-NH]-Ala

[92]

Thiodipeptide conjugate of nabumetone a

Structure

In competition with Gly-Sar. bIn competition with D-Phe-Gln.

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Acknowledgements The authors wish to acknowledge the PDA consortium (a project grant under the Danish Research Council for Strategic Research), the Carlsberg Foundation and the Novo Nordisk Foundation for their support.

Manuscript Received 26 March 2013, accepted for publication 26 April 2013.

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