Burkhardt Laufer

Intestinal permeability of cyclic peptides: common key backbone motifs identified.

Insufficient oral bioavailability is considered as a key limitation for the widespread development of peptides as therapeutics. While the oral bioavailability of small organic compounds is often estimated from simple rules, similar rules do not apply to peptides, and even the high oral bioavailability that is described for a small number of peptides is not well understood. Here we present two highly Caco-2 permeable template structures based on a library of 54 cyclo(-D-Ala-Ala(5)-) peptides with different N-methylation patterns. The first (all-trans) template structure possesses two β-turns of type II along Ala(6)-D-Ala(1) and Ala(3)-Ala(4) and is only found for one peptide with two N-methyl groups at D-Ala(1) and Ala(6) [(NMe(1,6)]. The second (single-cis) template possesses a characteristic cis peptide bond preceding Ala(5), which results in type VI β-turn geometry along Ala(4)-Ala(5). Although the second template structure is found in seven peptides carrying N-methyl groups on Ala(5), high Caco-2 permeability is only found for a subgroup of two of them [NMe(1,5) and NMe(1,2,4,5)], suggesting that N-methylation of D-Ala(1) is a prerequisite for high permeability of the second template structure. The structural similarity of the second template structure with the orally bioavailable somatostatin analog cyclo(-Pro-Phe-NMe-D-Trp-NMe-Lys-Thr-NMe-Phe-), and the striking resemblance with both β-turns of the orally bioavailable peptide cyclosporine A, suggests that the introduction of bioactive sequences on the highly Caco-2 permeable templates may result in potent orally bioavailable drug candidates.

The Effect of Multiple N-Methylation on Intestinal Permeability of Cyclic Hexapeptides

Recent progress in peptide synthesis simplified the synthesis of multiple N-methylation of peptides. To evaluate how multiple N-methylation affects the bioavailability of peptides, a poly alanine cyclic hexapeptide library (n = 54), varying in the number of N-methyl (N-Me) groups (1-5 groups) and their position, was synthesized. The peptides were evaluated for their intestinal permeability in vitro using the Caco-2 model. Further evaluation of the transport route of chosen analogues was performed using rat excised viable intestinal tissue, a novel colorimetric liposomal model and the parallel artificial membrane permeability assay (PAMPA). While most members were found to have poor permeability (permeability coefficient, P(app) < 1 x 10⁻⁶ cm/s, lower than mannitol, the marker for paracellular permeability), 10 analogues were found to have high Caco-2 permeability, (P(app) > 1 x 10⁻⁵ cm/s, similar to testosterone, a marker of transcellular permeability). No correlation was found between the number of N-methylated groups and the enhanced permeability. However, 9/10 permeable peptides in the Caco-2 model included an N-Me placed adjacently to the D-Ala position. While the exact transport route was not fully characterized, the data suggests a facilitated diffusion. It can be concluded that multiple N-methylation of peptides may improve intestinal permeability, and therefore can be utilized in the design of orally available peptide-based therapeutics.

Synthesis of N -methylated cyclic peptides

This protocol presents a detailed description of the synthesis of N-methylated cyclic peptides. N-methylation is a powerful technique to modulate the physicochemical properties of peptides by introducing one or more methyl groups into the peptidic amide bonds. Together with peptide cyclization, this procedure confers unprecedented pharmacokinetic properties to the peptides, including metabolic stability, membrane permeability and even oral bioavailability. Here we describe two simplified methods of N-methylation of linear peptides on solid supports, which can be performed in less than 2 h and are applicable to any amino acid. Finally, we also describe two methods of peptide cyclization, which can be used to obtain the N-methylated cyclic peptide and which are not limited to specific peptide sequences. With this protocol, multiply N-methylated cyclic peptides can be synthesized in as little as 4–5 d.

Improvement of drug-like properties of peptides: the somatostatin paradigm.

Importance of the field: Peptides are promising candidates as therapeutic agents due to their wide involvement in physiological processes. However, their often non-selective activity and their poor drug-like properties, mainly their inherent low stability to enzymatic degradation and poor oral bioavailability, limit their clinical potential. Somatostatin is a peptide hormone involved in many different biological functions. The role of its five different receptor subtypes and their interplay in medicinal processes is only partially understood. In addition, it suffers from poor drug-like properties. Areas covered in this review: We review several promising chemical modifications, including head-to-tail and backbone cyclization as well as N-methylation, which were applied throughout the years in the development of various somatostatin analogs. What the reader will gain: These modifications led to enhanced metabolic stability and intestinal permeability. In addition, several analogs exhibited specific receptor subtype activation. Take home message: The results presented in this review suggest a potential use of these chemical modifications in order to achieve required characteristics for a bioactive peptide, mainly for clinical usage.

Can N‐methylated amino acids serve as substitutes for prolines in conformational design of cyclic pentapeptides?

The incorporation of proline into cyclic peptides seems to be the most promising way to induce β‐turn structures. Recently, however, it was shown that N‐methylated amino acids might be even better suited than proline for introducing turn structures. Another property of proline, the ability to effect cis‐peptide bonds, has also been reported for N‐methylated amino acids. These findings raise the question if it might be possible to replace a proline by an N‐methylated amino acid without altering the desired conformational features. The most important benefit of replacing proline by an N‐methylated residue is that one recovers the side‐chain functionalities, which could be used for enhancing binding selectivity, or to tune a cyclic peptide concerning its pharmacological properties.

Breaking the Dogma of the Metal-Coordinating Carboxylate Group in Integrin Ligands: Introducing Hydroxamic Acids to the MIDAS To Tune Potency and Selectivity†

A suitable substitute: All integrin receptors bind their ligands, which contain an aspartate residue, in the metal-ion- dependent adhesion site (MIDAS). So far all attempts to replace the carboxyl group of aspartate with other, pharmacologically favorable isosteric groups have failed. Now it has been shown that a hydroxamic acid group can replace the carboxyl group; the resulting ligand retains its high binding activity. The picture shows one such ligand in the binding site of alphavbeta3.

Polymer-free immobilization of a cyclic RGD peptide on a nitinol stent promotes integrin-dependent endothelial coverage of strut surfaces

This study examined the utility of a stabilized cyclic RGD peptide chemically modified to selectively bind to titanium-oxide for enhanced biocompatibility of self-expanding nitinol stents. Endothelial cells express integrin receptors that promote attachment to subendothelial matrix proteins. Integrin binding to arginine-glycine-aspartic acid (RGD) peptide derivatives mimic naturally occurring adherent interactions. Irreversible covalent surface coating of conventional nitinol stents with a cyclic RGD (cRGD) peptide highly specific for integrin alpha v beta 3 might foster endothelialization after stent implantation. A selective cRGD peptide was irreversibly immobilized onto titanium oxide-rich nitinol coupons or self-expanding stents. Functionality of the engrafted RGD peptide was demonstrated using in vitro endothelial bioassays. A subsequent 7-day in vivo endothelialization study was performed using cRGD-coated self-expanding nitinol stents in rabbits. cRGD peptide coating effectively promoted endothelial cell anchorage, migration, and proliferation confirmed by increased focal adhesions. Proof-of-concept studies of rabbit cRGD stent implants showed a significant increase in endothelial coverage above stent struts relative to stents coated with BSA (cRGD = 70.1 ± 21.9 vs. BSA = 49.9 ± 21.8%, p < 0.03). Immobilization of cRGD peptides on strut surfaces represents an innovative strategy to improve endothelialization, which may facilitate vascular healing after stent implantation.

N-Methylated sst2 Selective Somatostatin Cyclic Peptide Analogue as a Potent Candidate for Treating Neurogenic Inflammation.

A focused multiply N-methylated library of a cyclic hexapeptidic somatostatin analogue: MK678 cyclo(-MeAYwKVF-) was generated, which resulted in the unexpected observation of an efficacious tetra-N-methylated analogue, cyclo(-MeAYMewMeKVMeF-) with a potent inhibitory action on sensory neuropeptide release in vitro and on acute neurogenic inflammatory response in vivo. The analogue shows selectivity toward somatostatin receptor subtype 2 (sst2). Extensive 2D NMR spectroscopy and molecular dynamics simulation revealed the solution conformation of the analogue, which can be adopted as a lead for the further structure-activity relationship studies targeting neurogenic inflammation.

Efficient factor VIII affinity purification using a small synthetic ligand.

Summary.  Background: Hemophilia A is currently treated by infusions of the coagulation factor (F) VIII, of which production and purification remain a challenging task. Current purification procedures using immunoaffinity chromatography are cumbersome, expensive, and suffer from the instability of the applied antibody ligands, which elute along with the product and contaminate it. Recently, FVIII was purified using octapeptide ligands, but their use is limited due to the low resistance to proteases. Objective: Our goal was to develop and evaluate a novel ligand for FVIII purification, overcoming the drawbacks of current procedures. Methods: Peptide ligands were screened for binding of 125I‐plasma‐derived‐FVIII (pdFVIII) in a microbead assay. A selected ligand‐coated Toyopearl resin was then used for pdFVIII purification from cell‐conditioned Delbucco’s modified Eagle’s medium (DMEM) containing fetal bovine serum. The proteolytic stability of ligand was measured by incubating with human serum and proteinase K, and its cytotoxicity towards human OV‐MZ‐6 cells was assayed. Results: A high‐affinity octapeptidic FVIII ligand was modified into the small, highly stable and non‐toxic peptidomimetic ligand L4 by rational and combinatorial design without affecting its affinity for FVIII. Using ligand L4‐coated Toyopearl resin, pdFVIII was isolated from cell‐conditioned medium with high purity and 89% column retention after elution with a mild buffer containing 0.6 m NaCl at pH 6.8. Conclusions: Ligand L4 offers a valuable alternative to antibody‐based procedures for laboratory and industrial production. Its synthesis by established solid‐phase procedures is straightforward and considerably cheaper than the biotechnological production of antibodies, and safety concerns associated with the use of biological material are overcome.

The Integrin Ligand c(RGDf(NMe)Nal) Reduces Neointimal Hyperplasia in a Polymer-Free Drug-Eluting Stent System

The use of highly active and selective integrin ligands in combination with stent implantation is emerging as a promising alternative to the release of classical immunosuppressive drugs by current drug‐eluting stents (DES), which has been associated with delayed vascular healing and late stent thrombosis. Herein we present the development and biological evaluation of the integrin ligand c(RGDf(NMe)Nal) as a potent anti‐proliferative molecule that targets coronary artery smooth muscle cells (CASMCs). This peptide showed an antagonistic activity for αvβ3 and αvβ5 in the low‐nanomolar range, and selectivity against the platelet receptor αIIbβ3. In vitro, it efficiently inhibited the proliferation of CASMCs, displaying higher potency than the anti‐tumor drug candidate cilengitide. This peptide was then loaded into a polymer‐free bare metal stent (BMS), and its release studied at different time points. Up to seven days of elution, the peptide‐coated stents retained high anti‐proliferative activity toward CASMCs. Finally, the peptide was examined in vivo in a polymer‐free DES system in a rabbit iliac artery model. After 28 days of implantation, histopathological analysis revealed that the peptide clearly decreased neointimal growth and improved vessel healing and re‐endothelialization compared with the FDA‐approved Cypher DES. Our study shows that this type of lipophilic integrin ligand, when eluted from a polymer‐free stent system, has the potential to successfully decrease in‐stent restenosis in the absence of delayed vascular healing.