Predicting in vivo performance of fenofibrate amorphous solid dispersions using in vitro non-sink dissolution and dissolution permeation setup.

Amorphous solid dispersion (ASD) is emerging as a useful formulation strategy to increase the bioavailability of active pharmaceutical ingredients with poor solubility. In vitro dissolution testing under non-sink conditions has often been used to evaluate the ability of ASDs to generate and maintain supersaturation to predict the in vivo performance. However, such a single compartment dissolution setup can fail to predict the oral bioavailability, due to an interdependence between precipitation and permeation. Hence, the use of two compartment dissolution-permeation setups is emerging. In this study, three ASDs containing fenofibrate as model drug substance were developed using Soluplus®, and Hypromellose Acetate Succinate in two different grades (high and low), respectively. The aim was to compare the use of a small-scale in vitro non-sink dissolution setup and a small-scale in vitro dissolution-permeation setup to predict the in vivo oral exposure of the ASDs in rats. The maximum concentration (Cmax) and area under curve (AUC) obtained in the in vitro studies were used to predict the in vivo rank order of the formulations. The results showed that the two in vitro studies resulted in the same rank order based on both Cmax and AUC. Interestingly, Cmax resulted in a better in vitro/in vivo correlation than the in vitro AUC, and based on the in vitro Cmax, the in vivo rank order was predicted.

Affinity and translocation relationships via hPEPT1 of H-X aa-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 (logIC(50)=-0.85 ± 0.06) for H-Tyr-Ser-OH to 0.89 mM (logIC(50)=-0.09 ± 0.02) for H-Gly-Ser-OH, as measured in a competition assay with [(14)C]Gly-Sar. The dipeptides were translocated via hPEPT1 with K(m)-values in the range of 0.20 (logIC(50)=-0.69 ± 0.04) for H-Met-Ser-OH to 1.04 (logIC(50)=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 (logIC(50)=-0.92 ± 0.12) and 0.12 mM (logIC(50)=-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.

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 [(14)C]Gly-Sar, [(3)H]Ibuprofen and other radio-labelled compounds were investigated in Madin-Darby canine kidney cells (MDCK)/hPEPT1, MDCK/Mock, LLC-PK(1) 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.

A quantitative structure-activity relationship for translocation of tripeptides via the human proton-coupled peptide transporter, hPEPT1 (SLC15A1).

The human intestinal proton-coupled peptide transporter, hPEPT1 (SLC15A1), has been identified as an absorptive transporter for both drug substances and prodrugs. An understanding of the prerequisites for transport has so far been obtained from models based on competition experiments. These models have limited value for predicting substrate translocation via hPEPT1. The aim of the present study was to investigate the requirements for translocation via hPEPT1. A set of 55 tripeptides was selected from a principal component analysis based on VolSurf descriptors using a statistical design. The majority of theses tripeptides have not previously been investigated. Translocation of the tripeptides via hPEPT1 was determined in a MDCK/hPEPT1 cell-based translocation assay measuring substrate-induced changes in fluorescence of a membrane potential-sensitive probe. Affinities for hPEPT1 of relevant tripeptides were determined by competition studies with [14C]Gly-Sar in MDCK/hPEPT1 cells. Forty tripeptides were found to be substrates for hPEPT1, having K(m)(app) values in the range 0.4-28 mM. Eight tripeptides were not able to cause a substrate-induced change in fluorescence in the translocation assay and seven tripeptides interacted with the probe itself. The conformationally restricted tripeptide Met-Pro-Pro was identified as a novel high-affinity inhibitor of hPEPT1. We also discovered the first tripeptide (Asp-Ile-Arg) that was neither a substrate nor an inhibitor of hPEPT1. To rationalise the requirements for transport, a quantitative structure-activity relationship model correlating K(m)(app) values with VolSurf descriptors was constructed. This is, to our knowledge, the first predictive model for the translocation of tripeptides via hPEPT1.

Discovery of ligands for the human intestinal Di-/Tripeptide transporter (hPEPT1) using a QSAR-assisted virtual screening strategy.

The discovery of novel ligands for the hPEPT1 transporter is reported. By exploiting a fast and rigorously validated QSAR model in combination with the distance in activity-centered chemical space (DACCS) approach, a database of commercially available compounds (Sigma-Aldrich) was screened for virtual hits. Twelve compounds were then purchased and characterized in an apical [14C]Gly-Sar uptake competition assay. Four compounds displayed affinity in the medium-to-high range. A simple benzophenone derivative displayed high affinity with a sub-millimolar binding constant (Ki=0.24 mM). The results of this study will serve as starting points for future projects, including the design and synthesis of compound libraries that seek to systematically explore the fundamental requirements for binding and transport by hPEPT1.

The existence of multiple conformers of interleukin-21 directs engineering of a superpotent analogue.

The high resolution three-dimensional structure of human interleukin (hIL)-21 has been resolved by heteronuclear NMR spectroscopy. Overall, the hIL-21 structure is dominated by a well defined central four-helical bundle, arranged in an up-up-down-down topology, as observed for other cytokines. A segment of the hIL-21 molecule that includes the third helical segment, helix C, is observed to exist in two distinct and interchangeable states. In one conformer, the helix C segment is presented in a regular, alpha-helical conformation, whereas in the other conformer, this segment is largely disordered. A structure-based sequence alignment of hIL-21 with receptor complexes of the related cytokines, interleukin-2 and -4, implied that this particular segment is involved in receptor binding. An hIL-21 analog was designed to stabilize the region around helix C through the introduction of a segment grafted from hIL-4. This novel hIL-21 analog was demonstrated to exhibit a 10-fold increase in potency in a cellular assay.