In vitro evaluation of N-methyl amide tripeptidomimetics as substrates for the human intestinal di-/tri-peptide transporter hPEPT1.

Oral absorption of tripeptides is generally mediated by the human intestinal di-/tri-peptide transporter, hPEPT1. However, the bioavailability of tripeptides is often limited due to degradation in the GI-tract by various peptidases. The aim of the present study was to evaluate the general application of N-methyl amide bioisosteres as peptide bond replacements in tripeptides in order to decrease degradation by peptidases and yet retain affinity for and transport via hPEPT1. Seven structurally diverse N-methyl amide tripeptidomimetics were selected based on a principal component analysis of structural properties of 6859 N-methyl amide tripeptidomimetics. In vitro extracellular degradation of the selected tripeptidomimetics as well as affinity for and transepithelial transport via hPEPT1 were investigated in Caco-2 cells. Decreased apparent degradation was observed for all tripeptidomimetics compared to the corresponding natural tripeptides. However, affinity for and transepithelial transport via hPEPT1 were only seen for Gly-Sar-Sar, AsnPsi[CONCH(3)]PhePsi[CONCH(3)]Trp, and Gly-Sar-Leu. This implies that tripeptidomimetics originating from tripeptides with neutral side chains are more likely to be substrates for hPEPT1 than tripeptidomimetics with charged side chains. The results of the present study indicate that the N-methyl amide peptide bond replacement approach for increasing bioavailability of tripeptidomimetic drug candidates is not generally applicable to all tripeptides. Nevertheless, retained affinity for and transport via hPEPT1 were shown for three of the evaluated N-methyl amide tripeptidomimetics.     

CHO/hPEPT1 cells overexpressing the human peptide transporter (hPEPT1) as an alternative in vitro model for peptidomimetic drugs

The present study characterized Chinese hamster ovary cells overexpressing a human intestinal peptide transporter, CHO/hPEPT1 cells, as an in vitro model for peptidomimetic drugs. The kinetic parameters of Gly-Sar uptake were determined in three different cell culture systems such as untransfected CHO cells (CHO-K1), transfected CHO cells (CHO/hPEPT1) and Caco-2 cells. Vmax in CHO/hPEPT1 cells was approximately 3-fold higher than those in Caco-2 cells and CHO-K1 cells, while Km values were similar in all cases. The uptake of beta-lactam antibiotics in CHO/hPEPT1 cells was three to twelve fold higher than that in CHO-K1 cells, indicating that CHO/hPEPT1 cells significantly enhanced the peptide transport activity. However, amino acid drugs also exhibited high cellular uptake in both CHO-K1 and CHO/hPEPT1 cells due to the high background level of amino acid transporters. Thus, cellular uptake study in CHO/hPEPT1 cells is not sensitive enough to distinguish the peptidyl drugs from amino acid drugs. The potential of CHO/hPEPT1 cells as an in vitro model for peptidomimetic drugs was also examined through the inhibition study on Gly-Sar uptake. Peptidomimetic drugs such as beta-lactam antibiotics and enalapril significantly inhibited Gly-Sar uptake whereas the nonpeptidyl compounds, L-dopa and alpha-methyldopa, did not compete with Gly-Sar for cellular uptake within the therapeutic concentrations. In conclusion, the present study demonstrates the further characterization of CHO/hPEPT1 cells as an uptake model as well as inhibition study and suggests their utility as an alternative in vitro model for drug candidates targeting the hPEPT1 transporter.     

Affinity and translocation relationships via hPEPT1 of H-Xaa-Ser-OH dipeptides: Evaluation of H-Phe-Ser-OH as a pro-moiety for ibuprofen and benzoic acid prodrugs

The intestinal di/tri-peptide transporter 1 (hPEPT1) has been suggested as a drug delivery target for peptide-based prodrugs. The aim of the study was to synthesize a series of 11 serine-containing dipeptides (H-X_aa-Ser-OH) and to investigate the relationship between binding to and transport via hPEPT1. An additional aim was to design a dipeptide which could serve as a pro-moiety for prodrugs targeted to hPEPT1. X_aa was chosen from the 20 proteogenic amino acids. The dipeptides were synthesized using solid phase peptide synthesis. The K_i-values of H-X_aa-Ser-OH dipeptides for hPEPT1 in MDCK/hPEPT1 cells ranged from 0.14 mM (log IC₅₀ = −0.85 ± 0.06) for H-Tyr-Ser-OH to 0.89 mM (log IC₅₀ = −0.09 ± 0.02) for H-Gly-Ser-OH, as measured in a competition assay with [¹⁴C]Gly-Sar. The dipeptides were translocated via hPEPT1 with K_m-values in the range of 0.20 (log IC₅₀ = −0.69 ± 0.04) for H-Met-Ser-OH to 1.04 (log IC₅₀ = 0.02 ± 0.04) mM for H-Gly-Ser-OH. The relationship between ligand and transportate properties indicated that the initial binding of the ligand to hPEPT1 is the major determinant for translocation of the investigated dipeptides. H-Phe-Ser-OH was selected as a pro-moiety, and two prodrugs were synthesized, i.e. H-Phe-Ser(Ibuprofyl)-OH and H-Phe-Ser(Bz)-OH. Both H-Phe-Ser(Ibuprofyl)-OH and H-Phe-Ser(Bz)-OH had high affinity for hPEPT1 with K_i-values of 0.07 mM (log IC₅₀ = −0.92 ± 0.12) and 0.12 mM (log IC₅₀ = −1.17 ± 0.40), respectively. However, none of the prodrugs were translocated via hPEPT1. This indicated that the coupling of the drug compounds to the peptide backbone did not decrease transporter binding, but abolished translocation, and that high affinity of prodrugs does not necessarily translate into favourable permeation properties.     

Synthesis and intestinal transport of the iron chelator maltosine in free and dipeptide form

Maltosine, a 3-hydroxy-4-pyridinone derivative of lysine formed in the course of the advanced Maillard reaction, is an effective metal chelating agent. It therefore represents an interesting compound for the treatment of metal ion storage diseases. We synthesized 6-(3-hydroxy-4-oxo-2-methyl-4(1H)-pyridin-1-yl)-l-norleucine (free maltosine) and its dipeptide derivatives alanylmaltosine (Ala-Mal) and maltosinylalanine (Mal-Ala) and examined the transepithelial flux of these compounds across Caco-2 cells and their interaction with membrane transporters. Transepithelial flux of maltosine was significantly higher when added as Ala-Mal and Mal-Ala than in free form. Assays at Caco-2 cells and at HeLa cells expressing the human peptide transporter (hPEPT)1 revealed that Ala-Mal and Mal-Ala show medium to high affinity to the system. Only free but not peptide-bound maltosine inhibited the uptake of l-[³H]lysine in Caco-2 and OK cells. Maltosine dipeptides were transported by hPEPT1 across cell membranes and accumulated in hPEPT1-transfected HeLa cells. In electrophysiological measurements at hPEPT1-expressing Xenopus laevis oocytes, Ala-Mal and Mal-Ala induced significant inward directed currents. We conclude that Ala-Mal and Mal-Ala are transported by hPEPT1 into intestinal cells and then hydrolyzed to free maltosine and alanine. The results suggest that the oral bioavailability of maltosine can be increased significantly by applying this drug candidate in peptide-bound form.     

Enhanced Transdermal Peptide Delivery and Stability by Lipid Conjugation: Epidermal Permeation,Stereoselectivity and Mechanistic Insights

Purpose

Efficient delivery of therapeutic peptides to the skin will facilitate better outcomes in dermatology. The tetrapeptide AAPV, an elastase inhibitor with potential utility in the management of psoriasis was coupled to short chain lipoamino acids (Laa: C6-C10) to enhance the peptide permeation into and through human epidermis.

Methods

AAPV was conjugated to Laas by solid phase synthesis. Peptide stability, skin distribution and permeation, elastase activity and surface activity were determined.

Results

Laas increased peptide permeation into the skin. The permeation lag time and amount of peptide remaining in the skin increased with the carbon chain length of the Laa conjugate. We also demonstrated stereoselective permeation enhancement in favour of the D-diastereomer. Importantly, the elastase inhibition activity of the peptide was largely retained after coupling to the Laa conjugates, showing potential therapeutic utility. The Laa-peptide structures were shown to be surface active, suggesting that this surfactant-like activity coupled with enhanced lipophilicity may contribute to their interaction with and permeation through the lipid domains of the stratum corneum.

Conclusions

This study suggests that the Laa conjugation approach may be useful for enhancing the permeation of moderately sized peptide drugs with potential application in the treatment of skin disorders.     

hPEPT1 affinity and translocation of selected Gln-Sar and Glu-Sar dipeptide derivatives

The intestinal di- and tripeptide transporter hPEPT1 is considered responsible for the absorption of di- and tripeptides arising from digestion, along with several drugs and prodrugs. In order to gather information on the binding site of the protein, several structure-affinity relationships have been suggested. However, these are not necessarily predictive of compounds that are actually translocated by hPEPT1. More information on affinity to and translocation via hPEPT1 of side-chain-modified dipeptides may be gained by conducting a study of selected dipeptide derivatives with variety in size, hydrophobicity, and bond type. The aim of the present study was to synthesize new esters and amides based on L-Glu-Sar and investigate the effects that bond type and size of modification of the N-terminal side chain of sarcosine-containing dipeptides have on the affinity to and translocation via hPEPT1. The esters L-Glu(O-i-Bu)-Sar and L-Glu(OCH(2)Ada)-Sar and the amides L-Gln(N,N-dimethyl)-Sar and L-Gln(N-piperidinyl)-Sar were synthesized, and affinity to and translocation via hPEPT1 were investigated in mature Caco-2 cell monolayers, grown on permeable supports. Affinity was estimated in a competition assay using (14)C-labeled Gly-Sar. Translocation was measured as fluorescence ratios induced by the substrates using the fluorescent probe BCECF and an epifluorescence microscope setup. All compounds showed high affinity to hPEPT1, but only the amides L-Gln(N,N-dimethyl)-Sar and L-Gln(N-piperidinyl)-Sar were translocated by hPEPT1. hPEPT1 is very susceptible to modifications of the N-terminal amino acid side chain of dipeptidomimetic substrates, in terms of achieving compounds with high affinity for the transporter. However, as affinity is not predictive of translocation, derivatization in this position must be performed with great caution since some of the compounds investigated turn out not to be translocated by the transporter.     

Ibuprofen is a non-competitive inhibitor of the peptide transporter hPEPT1 (SLC15A1): possible interactions between hPEPT1 substrates and ibuprofen

BACKGROUND AND PURPOSE

Recently, we identified etodolac as a possible ligand for the human intestinal proton-couple peptide transporter (hPEPT1). This raised the possibility that other non-steroidal anti-inflammatory drugs, and especially ibuprofen, could also interact with hPEPT1. Here, we have assessed the interactions of ibuprofen with hPEPT1.

EXPERIMENTAL APPROACH

The uptake of [¹⁴C]Gly-Sar, [³H]Ibuprofen and other radio-labelled compounds were investigated in Madin–Darby canine kidney cells (MDCK)/hPEPT1, MDCK/Mock, LLC-PK₁ or Caco-2 cells. The transepithelial transport of ibuprofen and hPEPT1 substrates was investigated in Caco-2 cell monolayers.

KEY RESULTS

Ibuprofen concentration dependently inhibited hPEPT1-mediated uptake of Gly-Sar in MDCK/hPEPT1 cells (K_i^app= 0.4 mM) but uptake of ibuprofen in Caco-2 cells and MDCK/hPEPT1 cells was not inhibited by hPEPT1 substrates. The maximum uptake rate for Gly-Sar uptake was reduced from 522 pmol·min⁻¹·cm⁻² to 181 pmol·min⁻¹·cm⁻² and 78 pmol·min⁻¹·cm⁻² in the presence of 0.5 mM and 1 mM ibuprofen, respectively. The interaction between ibuprofen and hPEPT1 was thus non-competitive. In LLC-PK1 cells, ibuprofen (1 mM) did not influence the transporter-mediated uptake of glycine or α-methyl-D-glycopyranoside. In Caco-2 cell monolayers the absorptive transport of δ-aminolevulinic acid was reduced by 23% and 48% by ibuprofen (1 and 10 mM), respectively. Likewise the transport of Gly-Sar was reduced by 23% in the presence of ibuprofen (1 mM).

CONCLUSIONS AND IMPLICATIONS

Ibuprofen is a non-competitive inhibitor of hPEPT1. As ibuprofen reduced the transepithelial transport of δ-aminolevulinic acid, drug–drug interactions between ibuprofen and hPEPT1 drug substrates at their site of absorption are possible if administered together.     

<Emphasis Type="Italic">In Vitro</Emphasis> and Pharmacophore-Based Discovery of Novel hPEPT1 Inhibitors

No HeadingPurpose. The human proton-coupled small peptide carrier (hPEPT1) is a low-affinity, high-capacity transporter with broad substrate specificity. We have taken an iterative in vitro and in silico approach to the discovery of molecules with hPEPT1 affinity.Methods. A pharmacophore-based approach was taken to identifying hPEPT1 inhibitors. The well-characterized and relatively high affinity ligands Gly-Sar, bestatin, and enalapril were used to generate a common features (HIPHOP) pharmacophore. This consisted of two hydrophobic features, a hydrogen bond donor, acceptor, and a negative ionizable feature.Results. The pharmacophore was used to search the Comprehensive Medicinal Chemistry (CMC) database of more than 8000 drug-like molecules and retrieved 145 virtual hits mapping to the pharmacophore features. The highest scoring compounds within this set were selected and tested in a stably transfected CHO-hPepT1 cell model. The antidiabetic repaglinide and HMG CoA reductase inhibitor fluvastatin were found to inhibit hPEPT1 with sub-millimolar potency (IC₅₀ 178 ± 1.0 and 337 ± 4 M, respectively). The pharmacophore was also able to identify known hPEPT1 substrates and inhibitors in further database mining of more than 500 commonly prescribed drugs.Conclusions. This study demonstrates the potential of combining computational and in vitro approaches to determine the affinity of compounds for hPEPT1 and, in turn, provides insights into key molecular interactions with this transporter.     

Role of the intestinal peptide transporter PEPT1 in oseltamivir absorption: in vitro and in vivo studies

It was reported that oseltamivir (Tamiflu) absorption was mediated by human peptide transporter (hPEPT) 1. Understanding the exact mechanism(s) of absorption is important in the context of drug-drug and diet-drug interactions. Hence, we investigated the mechanism governing the intestinal absorption of oseltamivir and its active metabolite (oseltamivir carboxylate) in wild-type [Chinese hamster ovary (CHO)-K1] and hPEPT1-transfected cells (CHO-PEPT1), in pharmacokinetic studies in juvenile and adult rats, and in healthy volunteers. In vitro cell culture studies showed that the intracellular accumulation of oseltamivir and its carboxylate into CHO-PEPT1 and CHO-K1 was always similar under a variety of experimental conditions, demonstrating that these compounds are not substrates of hPEPT1. Furthermore, neither oseltamivir nor its active metabolite was capable of inhibiting Gly-Sar uptake in CHO-PEPT1 cells. In vivo pharmacokinetic studies in juvenile and adult rats showed that the disposition of oseltamivir and oseltamivir carboxylate, after oral administration of oseltamivir, was sensitive to the feed status but insensitive to the presence of milk and Gly-Sar. Moreover, oseltamivir and oseltamivir carboxylate exhibited significantly higher exposure in rats under fasted conditions than under fed conditions. In humans, oral dosing after a high-fat meal resulted in a statistically significant but moderate lower exposure than after an overnight fasting. This change has no clinical implications. Taken together, the results do not implicate either rat Pept1 or hPEPT1 in the oral absorption of oseltamivir.     

A peptide prodrug approach for improving bisphosphonate oral absorption

This work was aimed at improving the absorption of bisphosphonates by targeting carrier systems in the intestine and the intestinal peptide carrier system (hPEPT1), in particular. (14)C-Labeled pamidronate and alendronate as well as radiolabeled and "cold" peptidyl-bisphosphonates, Pro-[(3)H]Phe-[(14)C]pamidronate, and Pro-[(3)H]Phe-[(14)C]alendronate were synthesized. In situ single-pass perfusion studies revealed competitive inhibition of transport by Pro-Phe, suggesting peptide carrier-mediated transport. Prodrug transport in the Caco-2 cell line was significantly better than that of the parent drugs, and the prodrugs exhibited high affinity to the intestinal tissue. Oral administration of the dipeptidyl prodrugs resulted in a 3-fold increase in drug absorption following oral administration in rats, and the bioavailability of Pro-Phe-alendronate was 3.3 (F(TIBIA)) and 1.9 (F(URINE)) times higher than that of the parent drug. The results indicate that the oral absorption of bisphosphonates can be improved by peptidyl prodrugs via the hPEPT1; however, other transporters may also be involved.     

Enhancement of drug absorption through the blood-brain barrier and inhibition of intercellular tight junction resealing by E-cadherin peptides

E-cadherin-mediated cell-cell interactions in the zonula adherens play an important role in the formation of the intercellular tight junctions found in the blood-brain barrier. However, it is also responsible for the low permeation of drugs into the brain. In this study, HAV6 peptide derived from the EC1 domain of E-cadherin was found to enhance the permeation of 1?C-mannitol and [3H(G)]-daunomycin through the blood-brain barrier of the in situ rat brain perfusion model. In addition, HAV6 peptide and verapamil have a synergistic effect in enhancing the BBB permeation of daunomycin. A new intercellular-junction resealing assay was also developed using Caco-2 monolayers to evaluate new peptides (BLG2, BLG3, and BLG4) derived from the bulge regions of the EC2, EC3, and EC4 domains of E-cadherin. BLG2 and BLG4 peptides but not BLG3 peptides were found to be effective in blocking the resealing of the intercellular junctions. The positive control peptides (ADT10, ADT6, and HAV10) block the resealing of the intercellular junctions in a concentration-dependent manner. All these findings suggest that E-cadherin-derived peptides can block E-cadherin-mediated cell-cell interactions. These findings demonstrate that cadherin peptides may offer a useful targeted permeation enhancement of therapeutic agents such as anticancer drugs into the brain.     

In-depth evaluation of Gly-Sar transport parameters as a function of culture time in the Caco-2 cell model.

The aim of the present study was to investigate the influence of culture time on hPEPT1-mediated transport in Caco-2 cell monolayers. Peptide transport activity in Caco-2 cells grown in standard media and in a "rapid" 4-day model was first compared. The rapid 4-day Caco-2 cell model, cultured using a cocktail of growth factors and agonists, displayed lower peptide uptake capacity than Caco-2 cells grown for 4 days in conventional media, and was judged to be unsuitable for peptide transport studies. Peptide transport activity as well as monolayer integrity and tissue morphology were evaluated in the standard >21 days model as a function of the culture time. Peptide transport activity was studied using [14C]-glycylsarcosine ([14C]-Gly-Sar). Monolayer integrity was evaluated by transepithelial electrical resistance (TEER) measurements and [3H]-mannitol permeabilities. Tissue morphology and hPEPT1 expression were studied using confocal laser scanning microscopy (CLSM) and conventional staining/immunostaining. Caco-2 cells grown in conventional media became confluent after 3-4 days. Mannitol permeability decreased from day 5 to 21 and TEER increased steadily until approximately day 21. Apical hPEPT1 uptake activity appeared to be maximal in cells cultured for >21 days, whereas basolateral uptake reached a maximum already after 12 days in culture. In some of the passages studied, a secondary increase in hPEPT1 transport activity was observed in cells grown for >25 days. A large carrier-mediated transepithelial peptide flux component was evident from day 14.     

Pore induction in human epidermal membrane during low to moderate voltage iontophoresis: A study using AC iontophoresis

The present study aimed to investigate new pore induction as a flux-enhancing mechanism in human epidermal membrane (HEM) with low to moderate voltage electric fields. The extent of pore induction and the effective pore sizes of these induced pores were to be assessed using a low frequency (12.5 Hz) low to moderate voltage (2. 0 to 4.0 V) square-wave alternating current (ac) "passive" permeation method (ac iontophoresis). This ac approach was to allow for inducing and sustaining a state of pore induction in HEM while permitting no significant transport enhancement via electroosmosis; thus, transport enhancement entirely due to new pore induction (enhanced passive permeation) was to be assessed without any contributions from electroosmosis. Good proportionality between the increase in HEM permeability and its electrical conductance was found with the "passive" transport data obtained during square-wave ac iontophoresis using urea as the model permeant. Typically, at 3.0 to 4.0 V, HEM conductance increases (and permeability increases) ranged from around 3- to 30-fold. These results appear to be the first direct evidence that new pore induction in HEM is a significant flux enhancing mechanism under moderate voltage conditions. The extents of pore induction in HEM under low frequency moderate voltage (2.0 to 3.0 V) ac, pulsed direct current (dc), and continuous dc were also compared. The extents of pore induction from square-wave ac and pulsed dc were generally of the same order of magnitude but somewhat less than that observed during continuous dc iontophoresis at the same applied voltage and duration, suggesting less extent of pore induction with reversing polarity or when a brief delay is provided between pulses to allow for membrane depolarization. The average effective pore sizes calculated for the induced pores from the experimental data with urea and mannitol as probe permeants and the hindered transport theory were 12 +/- 2 A, which are of the same order of magnitude as those of preexisting pores determined from conventional passive diffusion experiments.     

Transport and metabolism of opioid peptides across BeWo cells, an in vitro model of the placental barrier

In keeping with the advance of biotechnology, cell culture becomes an important tool for investigating the transport and the metabolism phenomena. A cell line of human origin, the BeWo choriocarcinoma cell line, was used for the study of the transport and metabolism of opioid peptides across the in vitro model of the placental barrier. Opioid peptides, both naturally occurring and their synthetic analogs, are of interest to be developed as potent analgesics and were included in this study. The apparent permeability coefficients (Pe)s of the peptides containing 4-11 amino acid or analog residues were in the range of 0.23-14.6 x 10(-5) cm/s. The (Pe)s of these peptides were comparable to those of sucrose or dextrans, hydrophilic markers. The (Pe)s of low molecular weight (MW) peptides was not dependent on their MW or molecular size, whereas an inversely linear correlation between (Pe)s and molecular size was observed with the larger peptides. Molecular sieving of the BeWo monolayer restricted the transport of the peptides with MW> or =1033 Da or molecular size > or =6.6 A. Membrane partitioning ability and charge of the peptides were also investigated and found to be the minor factors regulating the extent of peptide permeation. Contrasting to the transport of Tyr-[D-pen-Gly-Phe-D-Pen] (DPDPE) peptide analog across the blood-brain barrier, the transport of DPDPE across the BeWo monolayers were not indicated to be via carrier-mediated transport. The major transport pathway of the opioid peptides across the BeWo monolayers was found to be via paracellular route. In metabolism studies, aminopeptidase was found to be a major enzyme type responsible for the degradation of naturally occurring peptides but not for the synthetic analogs. The finding obtained from the present study reveals the applicability of the BeWo cell line as an in vitro model for investigating placental transport and metabolism of opioid peptides.     

High-affinity interaction of sartans with H+/peptide transporters

Sartans are very effective drugs for treatment of hypertension, heart failure, and other cardiovascular disorders. They antagonize the effects of angiotensin II at the AT(1) receptor and display p.o. bioavailability rates of 13 to 80%. Because some sartans sterically resemble dipeptide derivatives, we investigated whether they are transported by peptide transporters. We first assessed the effects of sartans on [(14)C]glycylsarcosine uptake into Caco-2 cells expressing H(+)/peptide transporter (PEPT) 1 and into SKPT cells expressing PEPT2. Losartan, irbesartan, valsartan, and eprosartan inhibited [glycine-1-(14)C]glycylsarcosine ([(14)C]Gly-Sar) uptake into Caco-2 cells in a competitive manner with K(i) values of 24, 230, 390, and >1000 microM. Losartan and valsartan also strongly inhibited the total transepithelial flux of [(14)C]Gly-Sar across Caco-2 cell monolayers. In SKPT cells, [(14)C]Gly-Sar uptake was inhibited with K(i) values of 2.2 microM (losartan), 65 microM (irbesartan), 260 microM (valsartan), and 490 microM (eprosartan). We determined by the two-electrode voltage-clamp technique whether the compounds elicited transport currents by PEPT1 or PEPT2 when expressed in Xenopus laevis oocytes. No currents were observed for any of the sartans, but the compounds strongly and reversibly inhibited peptide-induced currents. Uptake of valsartan, losartan, and cefadroxil was quantified in HeLa cells after heterologous expression of human PEPT1 (hPEPT1). In contrast to cefadroxil, no PEPT1-specific uptake of valsartan and losartan was found. We conclude that the sartans tested in this study display high-affinity interaction with PEPTs but are not transported themselves. However, they strongly inhibit hPEPT1-mediated uptake of dipeptides and cefadroxil.     

Synthesis and evaluation of the affinity toward mu-opioid receptors of atypical, lipophilic ligands based on the sequence c[-Tyr-Pro-Trp-Phe-Gly-

An ultimate and general model describing the interaction between opioid ligands and mu-opioid receptors is not available yet, so the mode of action of atypical peptide analogues or peptidomimetics is worthy of investigation. In this context, the peptide c[-Tyr-d-Pro-d-Trp-Phe-Gly-] was observed to act as an agonist toward mu-opioid receptors with appreciable potency, albeit deprived of a protonable nitrogen. This compound was synthesized as a member of a library of diastereo- or enantiomeric cyclic peptides based on the sequence of endomorphin-1, aiming to obtain lipophilic peptide ligands active at the mu-opioid receptors, having good performances in terms of resistance to enzymatic degradation and permeation of biological barriers.     

Pyrimidine and nucleoside gamma-esters of L-Glu-Sar: synthesis, stability and interaction with hPEPT1.

The aim of the present study was to improve the synthetic pathway of bioreversible dipeptide derivatives as well as evaluate the potential of using l-Glu-Sar as a pro-moiety for delivering three newly synthesised nucleoside and pyrimidine l-Glu-Sar derivatives. l-Glu(trans-2-thymine-1-yl-tetrahydrofuran-3-yl ester)-Sar (I), l-Glu(thymine-1-yl-methyl ester)-Sar (II) and l-Glu(acyclothymidine)-Sar (III) were synthesised and in vitro stability was studied in various aqueous and biological media. Affinity to and translocation via hPEPT1 was investigated in mature Caco-2 cell monolayers, grown on permeable supports. Affinity was estimated in a competition assay, using [14C] labelled Gly-Sar (glycylsarcosine). Translocation was measured as pHi-changes induced by the substrates using the fluorescent probe BCECF and an epifluorescence microscope setup. All dipeptide derivatives released the model drugs quantitatively by specific base-catalysed hydrolysis at pH>6.0. II was labile in aqueous buffer solution, whereas I and III showed appropriate stability for oral administration. In 10% porcine intestinal homogenate, the half-lives of the dipeptide derivatives indicated limited enzyme catalyzed degradation. All compounds showed good affinity to hPEPT1, but the Compounds I and III showed not to be translocated by hPEPT1. The translocation of the l-Glu-Sar derivative of acyclovir, l-Glu(acyclovir)-Sar was also investigated and showed not to take place. Consequently, l-Glu-Sar seems to be a poor pro-moiety for hPEPT1-mediated transport.     

Synthesis and evaluation of the physicochemical properties of esterase-sensitive cyclic prodrugs of opioid peptides using coumarinic acid and phenylpropionic acid linkers

Abstract: In an attempt to improve the membrane permeabilities of opioid peptides, we have synthesized cyclic prodrugs of [Leu⁵]-enkephalin and DADLE using a coumarinic acid or a phenylpropionic acid linker. The synthesis of the coumarinic acid- and phenylpropionic acid-based cyclic prodrugs followed similar strategies. Key intermediates were the compounds with the C-terminal amino acids of opioid peptides (L-Leu, [Leu⁵]-enkephalin; D-Leu, DADLE) attached to the phenol hydroxyl group and the remaining amino acids of the peptide linked via the N-terminal amino acid (L-Tyr) attached to the carboxylic acid groups of the prodrug moieties (coumarinic acid or propionic acid). Cyclization of these linear precursors gave the cyclic prodrugs in 30–50% yields. These cyclic prodrugs exhibited excellent transcellular permeation characteristics across Caco-2 cell monolayers, an in vitro model of the intestinal mucosa. To correlate the cellular permeabilities of these cyclic prodrugs with their physicochemical properties, we calculated their Stokes–Einstein molecular radii from their diffusion coefficients which were determined by NMR and we determined their membrane interaction potentials using immobilized artificial membrane (IAM) column chromatography. The cyclic prodrugs exhibited molecular radii similar to those of the parent compounds, [Leu⁵]-enkephalin and DADLE. However, these cyclic prodrugs were shown to have much higher membrane interaction potentials than their corresponding opioid peptides. Therefore, the enhanced cellular permeation of the cyclic prodrugs is apparently due to the alteration of their lipophilicity and hydrogen bonding potential, but not their molecular sizes.