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Combination therapy in Type 2 Diabetes Mellitus is necessary to achieve tight glycaemic control and reduce complication risk. Current treatment plans require patients to take several drugs concomitantly leading to low therapy adherence. This study describes the development and characterisation of a stable parenteral co-formulation of a sodium glucose co-transporter 2 inhibitor (dapagliflozin) and a therapeutic lipidated peptide, using hydroxypropyl-β-cyclodextrin as an enabling excipient. Using NMR, calorimetry, computational modelling and spectroscopic methods, we show that besides increasing the solubility of dapagliflozin, cyclodextrin prevents self-association of the peptide through interaction with the lipid chain and amino acids prone to aggregation including aromatic groups and ionisable residues. While those interactions cause a dramatic secondary structure change, no impact on potency was seen in vitro. A subcutaneous administration of the co-formulation in rat showed that both drugs reach exposure levels previously shown to be efficacious in clinical mono-therapy studies. Interestingly, a faster absorption rate was observed for the peptide formulated within the cyclodextrin vehicle with respect to the buffer vehicle, which could trigger an earlier onset of action. The cyclodextrin based co-formulation is therefore a promising approach to develop a fixed dose combination of a therapeutic peptide and a small molecule drug for increased patient adherence and better blood glucose control.

Lipidation is a powerful strategy to improve the stability in vivo of peptide drugs. Attachment of a lipid chain to a hydrophilic peptide leads to amphiphilicity and the potential for surfactant-like self-assembly. Here, the self-assembly and conformation of three lipidated derivatives of the gastrointestinal peptide hormone PYY3-36 is examined using a comprehensive range of spectroscopic, scattering, and electron microscopy methods and compared to those of the parent PYY3-36 peptide. The peptides are lipidated at Ser(11), Arg(17), or Arg(23) in the peptide; the former is within the β-turn domain (based on the published solution NMR structure), and the latter two are both within the α-helical domain. We show that it is possible to access a remarkable diversity of nanostructures ranging from micelles to nanotapes and fibrillar hydrogels by control of assembly conditions (concentration, pH, and temperature). All of the lipopeptides self-assemble above a critical aggregation concentration (cac), determined through pyrene fluorescence probe measurements, and they all have predominantly α-helical secondary structure at their native pH. The pH and temperature dependence of the α-helical conformation were probed via circular dichroism spectroscopy experiments. Lipidation was found to provide enhanced stability against changes in temperature and pH. The self-assembled structures were investigated using small-angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (cryo-TEM). Distinct differences in nanostructure were observed for lipidated and unlipidated peptides, also depending on the position of lipidation. Remarkably, micelles containing lipopeptides with α-helical peptide conformation were observed. Gelation was observed at higher concentrations in certain pH intervals for the lipidated peptides, but not for unlipidated PYY3-36. Thus, lipidation, in addition to enhancing stability against pH and temperature variation, also provides a route to prepare PYY peptide hydrogels. These findings provide important insights into the control of PYY3-36 conformation and aggregation by lipidation, relevant to the development of future therapeutics based on this peptide hormone, for example, in treatments for obesity.

The use of peptides as therapeutic agents is undergoing a renaissance with the expectation of new drugs with enhanced levels of efficacy and safety. Their clinical potential will be only fully realised once their physicochemical and pharmacokinetic properties have been precisely controlled. Here we demonstrate a reversible peptide self-assembly strategy to control and prolong the bioactivity of a native peptide hormone in vivo. We show that oxyntomodulin, a peptide with potential to treat obesity and diabetes, self-assembles into a stable nanofibril formulation which subsequently dissociates to release active peptide and produces a pharmacological effect in vivo. The subcutaneous administration of the nanofibrils in rats results in greatly prolonged exposure, with a constant oxyntomodulin bioactivity detectable in serum for at least 5 days as compared to free oxyntomodulin which is undetectable after only 4 h. Such an approach is simple, cost-efficient and generic in addressing the limitations of peptide therapeutics., The clinical potential of peptide therapeutic agents can only be fully realised once their physicochemical and pharmacokinetic properties are precisely controlled. Here the authors show a reversible peptide self-assembly strategy to control and prolong the bioactivity of a native peptide hormone in vivo.

The human peptide hormone Oxyntomodulin (Oxm) is known to induce satiety, increase energy expenditure, and control blood glucose in humans, making it a promising candidate for treatment of obesity and/or type 2 diabetes mellitus. However, a pharmaceutical exploitation has thus far been impeded by fast in vivo clearance and the molecule‘s sensitivity to half-life extending structural modifications. We recently showed that Oxm self-assembles into amyloid-like nanofibrils that continuously release active, soluble Oxm in a peptide-deprived environment. S.c. injected Oxm nanofibrils extended plasma exposure from a few hours to five days in rodents, compared to s.c. applied soluble Oxm. Here we show that Oxm fibril elongation kinetics and thermodynamics display a uniquely low temperature optimum compared to previously reported amyloid-like peptide and protein assemblies. Elongation rate is optimal at room temperature, with association rates 2–3 times higher at 25 °C than at ≥37 °C or ≤20 °C. We deduce from a combination of Cryo electron microscopy and spectroscopic methods that Oxm fibrils have a double-layered, triangular cross-section composed of arch-shaped monomers. We suggest a thermodynamic model that links the necessary molecular rearrangements during fibrillation and peptide release to the unique temperature effects in Oxm self-assembly and disassembly.

Peptide therapeutics have the potential to self-associate, leading to aggregation and fibrillation. Noncovalent PEGylation offers a strategy to improve their physical stability; an understanding of the behavior of the resulting polymer/ peptide complexes is, however, required. In this study, we have performed a set of experiments with additional mechanistic insight provided by in silico simulations to characterize the molecular organization of these complexes. We used palmitoylated vasoactive intestinal peptide (VIP-palm) stabilized by methoxy-poly(ethylene glycol)(skDa)-cholane (PEG-cholane) as our model system. Homogeneous supramolecular assemblies were found only when complexes of PEG-cholane/VIP-palm exceeded a molar ratio of 2:1; at and above this ratio, the simulations showed minimal exposure of VIP-palm to the solvent. Supramolecular assemblies formed, composed of, on average, 9-11 PEG-cholane/VIP-palm complexes with 2:1 stoichiometry. Our in silico results showed the structural content of the helical conformation in VIP-palm increases when it is complexed with the PEG-cholane molecule; this behavior becomes yet more pronounced when these complexes assemble into larger supramolecular assemblies. Our experimental results support this: the extent to which VIP-palm loses helical structure as a result of thermal denaturation was inversely related to the PEG-cholane:VIP-palm molar ratio. The addition of divalent buffer species and increasing the ionic strength of the solution both accelerate the formation of VIP-palm fibrils, which was partially and fully suppressed by 2 and >4 mol equivalents of PEG-cholane, respectively. We conclude that the relative freedom of the VIP-palm backbone to adopt nonhelical conformations is a key step in the aggregation pathway.

Because nonalcoholic steatohepatitis (NASH) is associated with impaired liver regeneration, we investigated the effects of G49, a dual glucagon-like peptide-1/glucagon receptor agonist, on NASH and hepatic regeneration. C57Bl/6 mice fed chow or a methionine and choline–deficient (MCD) diet for 1 week were divided into 4 groups: control (chow diet), MCD diet, chow diet plus G49, and M+G49 (MCD diet plus G49). Mice fed a high-fat diet (HFD) for 10 weeks were divided into groups: HFD and H+G49 (HFD plus G49). Following 2 (MCD groups) or 3 (HFD groups) weeks of treatment with G49, partial hepatectomy (PH) was performed, and all mice were maintained on the same treatment schedule for 2 additional weeks. Analysis of liver function, hepatic regeneration, and comprehensive genomic and metabolic profiling were conducted. NASH was ameliorated in the M+G49 group, manifested by reduced inflammation, steatosis, oxidative stress, and apoptosis and increased mitochondrial biogenesis. G49 treatment was also associated with replenishment of intrahepatic glucose due to enhanced gluconeogenesis and reduced glucose use through the pentose phosphate cycle and oxidative metabolism. Following PH, G49 treatment increased survival, restored the cytokine-mediated priming phase, and enhanced the proliferative capacity and hepatic regeneration ratio in mice on the MCD diet. NASH markers remained decreased in M+G49 mice after PH, and glucose use was shifted to the pentose phosphate cycle and oxidative metabolism. G49 administered immediately after PH was also effective at alleviating the pathological changes induced by the MCD diet. Benefits in terms of liver regeneration were also found in mice fed HFD and treated with G49. Conclusion: Dual-acting glucagon-like peptide-1/glucagon receptor agonists such as G49 represent a novel therapeutic approach for patients with NASH and particularly those requiring PH. (Hepatology 2017;65:950-968)., Supported by MINECO (SAF2015-65267-R) cofunded by the Fondos Europeos de Desarrollo Regional de la Unión Europea (FEDER), CIBERdem (ISCIII, Spain), INFLAMES (PIE-14/00045 (PIE-14/00045 ISCIII, Spain)), and the European Foundation for the Study of Diabetes (to A.M.V.). Financial support was also received from MedImmune (MA-416191) as a research agreement with the Instituto de Investigaciones Biomedicas Alberto Sols.

The purpose of this study was to design microspheres combining sustained delivery and enhanced intracellular penetration for ocular administration of antisense oligonucleotides. Nanosized complexes of antisense TGF-beta2 phosphorothioate oligonucleotides (PS-ODN) with polyethylenimine (PEI), and naked PS-ODN were encapsulated into poly(lactide-co-glycolide) microspheres prepared by the double-emulsion solvent evaporation method. The PS-ODN was introduced either naked or complexed in the inner aqueous phase of the first emulsion. We observed a marked influence of microsphere composition on porosity, size distribution and PS-ODN encapsulation efficiency. Mainly, the presence of PEI induced the formation of large pores observed onto microsphere surface. Introduction of NaCl in the outer aqueous phase increased the encapsulation efficiency and reduced microsphere porosity. In vitro release kinetic of PS-ODN was also investigated. Clearly, the higher the porosity, the faster was the release and the higher was the burst effect. Using an analytical solution of Fick's second law of diffusion, it was shown that the early phase of PS-ODN and PS-ODN-PEI complex release was primarily controlled by pure diffusion, irrespectively of the type of microsphere. Finally, microspheres containing antisense TGF-beta2 nanosized complexes were shown, after subconjunctival administration to rabbit, to significantly increase intracellular penetration of ODN in conjunctival cells and subsequently to improve bleb survival in a rabbit experimental model of filtering surgery. These results open up interesting prospective for the local controlled delivery of genetic material into the eye.

Peptides are ideal drug candidates due to their potency and specificity, but suffer from a short half-life and low membrane permeability. Acylation can overcome these limitations but the consequences to stability under different formulation conditions and stresses are largely unreported. Using synchrotron radiation circular dichroism (SRCD), we show that palmitoylation of a 28 amino acid peptide hormone (pI 9.82) induced a structural transition from 310-helix to α-helix, irrespective of buffer type and pH investigated (5.5-8.0) when compared to the non acylated analogues. These conformational preferences were retained in the presence of non-ionic micelles but not anionic micelles, which induced an α-helical structure for all peptides. Palmitoylation promoted an irreversible peptide denaturation under thermal stress at pH ≥ 6.5 and increased the propensity for loss of helical structure under high photon flux (here used as a novel accelerated photostability test). The presence of either ionic or non-ionic micelles did not recover these conformational changes over the same irradiation period. These results demonstrate that acylation can change peptide conformation and decrease thermal-/photo-stability, with important consequences for drug-development strategies.