An alternative to insulin-based therapy treatments of type 2 diabetes mellitus (T2DM) is based on the intriguing peptide hormone glucagon-like-peptide-1 (GLP-1). Several decades passed from the initial discovery of GLP-1 in the 1980s to the launch of the first GLP-1 receptor agonist-based therapy. However, in the past 10 years several new products have been launched, and we continue to learn more and more about this fascinating hormone class both from a biological and structural perspective. The field is continuously growing, and just recently an oral tablet of a GLP-1 receptor agonist was approved by the American Food and Drug Administration for treatment of T2DM. This chapter will provide an overview of the pharmaceutical developments and future directions within GLP-1 receptor agonist-based therapies.

Peptide agonists acting on the glucagon-like peptide 1 receptor (GLP-1R) promote glucose-dependent insulin release and therefore represent important therapeutic agents for type 2 diabetes (T2D). Previous data indicated that an N-terminal type II β-turn motif might be an important feature for agonists acting on the GLP-1R. In contrast, recent publications reporting the structure of the full-length GLP-1R have shown the N-terminus of receptor-bound agonists in an α-helical conformation. To reconcile these conflicting results, we prepared N-terminally constrained analogues of glucagon-like peptide 1 (GLP-1) and exendin-4 and evaluated their receptor affinity and functionality in vitro; we then examined their crystal structures in complex with the extracellular domain of the GLP-1R and used molecular modeling and molecular dynamics simulations for further investigations. We report that the peptides' N-termini in all determined crystal structures adopted a type II β-turn conformation, but in vitro potency varied several thousand-fold across the series. Potency correlated better with α-helicity in our computational model, although we have found that the energy barrier between the two mentioned conformations is low in our most potent analogues and the flexibility of the N-terminus is highlighted by the dynamics simulations.

Acylation of peptide drugs with fatty acid chains has proven beneficial for prolonging systemic circulation, as well as increasing enzymatic stability and interactions with lipid cell membranes. Thus, acylation offers several potential benefits for oral delivery of therapeutic peptides, and we hypothesize that tailoring the acylation may be used to optimize intestinal translocation. This work aims to characterize acylated analogues of the therapeutic peptide salmon calcitonin (sCT), which lowers blood calcium, by systematically increasing acyl chain length at two positions, in order to elucidate its influence on intestinal cell translocation and membrane interaction. We find that acylation drastically increases in vitro intestinal peptide flux and confers a transient permeability enhancing effect on the cell layer. The analogues permeabilize model lipid membranes, indicating that the effect is due to a solubilization of the cell membrane, similar to transcellular oral permeation enhancers. The effect is dependent on pH, with larger effect at lower pH, and is impacted by acylation chain length and position. Compared to the unacylated peptide backbone, N-terminal acylation with a short chain provides 6- or 9-fold increase in peptide translocation at pH 7.4 and 5.5, respectively. Prolonging the chain length appears to hamper translocation, possibly due to self-association or aggregation, although the long chain acylated analogues remain superior to the unacylated peptide. For K(18)-acylation a short chain provides a moderate improvement, whereas medium and long chain analogues are highly efficient, with a 12-fold increase in permeability compared to the unacylated peptide backbone, on par with currently employed oral permeation enhancers. For K(18)-acylation the medium chain acylation appears to be optimal, as elongating the chain causes greater binding to the cell membrane but similar permeability, and we speculate that increasing the chain length further may decrease the permeability. In conclusion, acylated sCT acts as its own in vitro intestinal permeation enhancer, with reversible effects on Caco-2 cells, indicating that acylation of sCT may represent a promising tool to increase intestinal permeability without adding oral permeation enhancers.

L'invention concerne un derive d'un peptide GLP-1, lequel peptide comprend un premier residu Lys au niveau d'une position correspondant a la position 36 de GLP-1(7-37) (SEQ ID NO : 1), un second residu Lys a une position correspondant a la position 37 de GLP-1(7-37) (SEQ ID NO : 1), et au maximum sept changements d'acides amines compare a GLP-1(7-37) (SEQ ID NO :1) ; ledit derive comportant deux extracteurs fixe auxdits premier et second residus Lys, respectivement, chacun par l'intermediaire d'un lieur ; dans lequel l'extracteur est choisi parmi : Chem. 1 : HOOC-C 6 H 4 -0-(CH 2 ) y -CO-* et Chem. 2 : HOOC-(CH 2 ) x -CO-*, ou y est un nombre entier compris entre 8 et 11 et x est 12 ; et ou le lieur comprend au moins l'un de ceux-ci : Chem. 3 : *-NH-CH(COOH)-(CH 2 ) 2 -CO-*, Chem. 4 : *-NH-CH((CH 2 ) 2 -COOH)-CO-* et/ou Chem. 5 : *-NH-(CH 2 ) 2 -[0-(CH 2 ) 2 ] k -0-[CH 2 ] n -CO-*, ou k est un nombre entier compris entre 1 et 5 et n est un nombre entier compris entre 1 et 5 ; ou un sel, amide ou ester pharmaceutiquement acceptable de celui-ci. L'invention concerne egalement les utilisations pharmaceutiques de ceux-ci, telles que pour le traitement du diabete et de l'obesite, ainsi que les peptides GLP-1 faisant partie des derives qui ont des residus Lys aux positions 36 et 37 et pas d'autres residus Lys, et les fragments GLP-1(9-37) de ceux-ci. L'invention concerne en outre un produit intermediaire comprenant un acide 3-carboxyphenoxy-nonanoique porteur d'un groupe de protection au niveau du groupe carboxy de l'acide nonanoique, eventuellement par l'intermediaire d'un lieur. Les derives ont une tres bonne puissance d'action et une longue demi-vie, ce qui les rend potentiellement utiles pour, par exemple, une administration par voie orale.

Background: Acylation of peptide drugs with fatty acid chains has proven beneficial for prolonging systemic circulation as well as increasing enzymatic stability without disrupting biological potency. Acylation has furthermore been shown to increase interactions with the lipid membranes of mammalian cells. The extent to which such interactions hinder or benefit delivery of acylated peptide drugs across cellular barriers such as the intestinal epithelia is currently unknown. The present study investigates the effect of acylating peptide drugs from a drug delivery perspective.Purpose: We hypothesize that the membrane interaction is an important parameter for intestinal translocation, which may be used to optimize the acylation chain length for intestinal permeation. This work aims to characterize acylated analogues of the intestinotrophic Glucagon-like peptide-2 by systematically increasing acyl chain length, in order to elucidate its influence on membrane interaction and intestinal cell translocation in vitro.Results: Peptide self-association and binding to both model lipid and cell membranes was found to increase gradually with acyl chain length, whereas translocation across Caco-2 cells depended non-linearly on chain length. Short and medium acyl chains increased translocation compared to the native peptide, but long chain acylation displayed no improvement in translocation. Co-administration of a paracellular absorption enhancer was found to increase translocation irrespective of acyl chain length, whereas a transcellular enhancer displayed increased synergy with the long chain acylation.Conclusions: These results show that membrane interactions play a prominent role during intestinal translocation of an acylated peptide. Acylation benefits permeation for shorter and medium chains due to increased membrane interactions, however, for longer chains insertion in the membrane becomes dominant and hinders translocation, i.e. the peptides get 'stuck' in the cell membrane. Applying a transcellular absorption enhancer increases the dynamics of membrane insertion and detachment by fluidizing the membrane, thus facilitating its effects primarily on membrane associated peptides.

Diamino acids are commonly found in bioactive compounds, yet only few are commercially available as building blocks for solid-phase peptide synthesis. In the present work a convenient, inexpensive route to multiple-charged amino acid building blocks with varying degree of hydrophobicity was developed. A versatile solid-phase protocol leading to selectively protected amino alcohol intermediates was followed by oxidation to yield the desired di- or polycationic amino acid building blocks in gram-scale amounts. The synthetic sequence comprises loading of (S)-1-(p-nosyl)aziridine-2-methanol onto a freshly prepared trityl bromide resin, followed by ring opening with an appropriate primary amine, on-resin N(β)-Boc protection of the resulting secondary amine, exchange of the N(α)-protecting group, cleavage from the resin, and finally oxidation in solution to yield the target γ-aza substituted building blocks having an Fmoc/Boc protection scheme. This strategy facilitates incorporation of multiple positive charges into the building blocks provided that the corresponding partially protected di- or polyamines are available. An array of compounds covering a wide variety of γ-aza substituted analogs of simple neutral amino acids as well as analogs displaying high bulkiness or polycationic side chains was prepared. Two building blocks were incorporated into peptide sequences using microwave-assisted solid-phase peptide synthesis confirming their general utility.

We have studied secretory phospholipase A(2)-IIA (sPLA(2)) activity toward different phospholipid analogues by performing biophysical characterizations and molecular dynamics simulations. The phospholipids were natural substrates, triple alkyl phospholipids, a prodrug anticancer etherlipid, and an inverted ester. The latter were included to study head group-enzyme interactions. Our simulation results show that the lipids are optimally placed into the binding cleft and that water molecules can freely reach the active site through a well-defined pathway; both are indicative that these substrates are efficiently hydrolyzed, which is in good agreement with our experimental data. The phospholipid analogue with three alkyl side chains forms aggregates of different shapes with no well-defined sizes due to its cone-shape structure. Phosphatidylglycerol and phosphatidylcholine head groups interact with specific charged residues, but relatively large fluctuations are observed, suggesting that these interactions are not necessarily important for stabilizing substrate binding to the enzyme.

Cette invention concerne un derive d'un analogue de GLP-1, ou son sel, amide, ou ester de qualite pharmaceutique, ledit analogue comprenant un premier residu K a une position correspondant a la position 18 de GLP-1 (7-37) (SEQ ID No: 1), un second residu K a une autre position, et un maximum de douze modifications d'acides amines, comparativement a GLP-1 (7-37); ledit derive comprenant deux fragments etendus lies audit premier et second residu K, respectivement, par l'intermediaire d'un lieur, le fragment etendu etant choisi parmi Chem. 1 : HOOC-(CH2)x-CO-*, et Chem. 2 : HOOC-CeH4-0-(CH2)y- CO-*, ou x est un nombre entier dans la plage de 6 a 18, et y est un nombre entier dans la plage de 3 a 17; et le lieur comprenant Chem. 3 : *-NH-(CH2)q-CH[(CH2)w-NH2]-CO-*, ou q est un nombre entier dans la plage de 0 a 5, et w est un nombre entier dans la plage de 0 a 5. L'invention concerne egalement son utilisation pharmaceutique, par exemple, pour traiter et/ou prevenir toutes les formes du diabete et autres maladies apparentees, ainsi que les nouveaux peptides et les intermediaires a chaine laterale correspondants. Les derives sont puissants, etendus, et appropries pour l'administration par voie orale.

L'invention concerne un derive d'un analogue de GPL-1, ledit analogue comprenant un premier residu K a une position correspondant a la position 18 de GLP-1 (7-37) (SEQ ID NO : 1), un second residu K a une autre position, et un maximum de douze modifications d'acides amines en comparaison a GLP-1 (7-37) ; le derive comprenant deux fractions etendues et fixees auxdits premier et second residus K, respectivement, par l'intermediaire d'un liant, la fraction etendue etant choisie parmi Chem. 1, Chem. 2 et Chem.3 : Chem. : HOOC-(CH 2 ) x -CO-* Chem. 2: HOOC-C 6 H 4 -0-(CH 2 ) y -CO-* Chem. 3: R 2 -C 6 H 4 -(CH 2 ) z -CO-*, ou x est un entier se situant dans la plage de 6-18, y est un entier se situant dans la plage de 3-17, z est un entier se situant dans la plage de 1-5, et R 2 est un groupe ayant une masse molaire non superieure a 150 Da ; et le liant comprenant Chem. 4: *-NH-(CH 2 ) 2 -(0-(CH 2 ) 2 ) k -0-(CH 2 ) n -CO-*, ou k est un entier se situant dans la plage de 1-5, et n est un entier se situant dans la plage de 1-5 ; ou un sel, un amide ou un ester pharmaceutiquement acceptable de celui-ci. L'invention concerne egalement son utilisation pharmaceutique, par exemple dans le traitement et/ou la prevention de toutes formes de diabete et de maladie associees, ainsi que des nouveaux peptides et intermediaires de chaine laterale correspondants. Les derives sont adaptes a une administration par voie orale.

Secretory phospholipase A(2) (sPLA(2)) is an interesting enzyme for triggered liposomal drug delivery to tumor tissue due the overexpression of sPLA(2) in cancerous tissue. A drug delivery system based on the triggered release of therapeutics from sPLA(2)-sensitive liposomes constituted of pro anticancer ether lipids, which become cytotoxic upon sPLA(2)-catalyzed hydrolysis has previously been established. To optimize the hydrolysis rate of the lipids and thereby optimizing the release profile of the drugs from the liposomes, we have synthesized a thio-ester pro anticancer ether lipid. Liposomes constituted of this lipid showed an altered rate of hydrolysis by sPLA(2). We have tested the cytotoxicity of the thio-ester pro anticancer ether lipids toward cancer cells, and the results showed that the cytotoxicity is indeed maintained upon sPLA(2) exposure. To further understand the origin for the observed different hydrolysis rates for the esters, we have applied molecular dynamics simulations and density functional theory. The combination of these theoretical methods has given valuable insight into the molecular mechanism for sPLA(2) action on sulfur-containing phospholipids. It appears that the enzyme-catalyzed hydrolysis of thio-esters follow a different pathway compared to the hydrolysis pathway of the free thio-ester.

Special delivery: Liposomal drug-delivery systems in which prodrugs are activated specifically by disease-associated enzymes have great potential for the treatment of severe diseases, such as cancer. A new type of phospholipid-based prodrug has the ability to form stable small unilamellar vesicles (see picture). Activation of the prodrug vesicles by the enzyme sPLA(2) initiates a cyclization reaction, which leads to the release of the drug.