Ionisk selvsamling med farvestoffet hydroxynaphtol blue og tensidet benzyldimethyldodecylammonium- chlorid

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G_s protein peptidomimetics as allosteric modulators of the β₂-adrenergic receptor

A series of G(s) protein peptidomimetics were designed and synthesised based on the published X-ray crystal structure of the active state β(2)-adrenergic receptor (β(2)AR) in complex with the G(s) protein (PDB 3SN6). We hypothesised that such peptidomimetics may function as allosteric modulators that target the intracellular G(s) protein binding site of the β(2)AR. Peptidomimetics were designed to mimic the 15 residue C-terminal α-helix of the G(s) protein and were pre-organised in a helical conformation by (i, i + 4)-stapling using copper catalysed azide alkyne cycloaddition. Linear and stapled peptidomimetics were analysed by circular dichroism (CD) and characterised in a membrane-based cAMP accumulation assay and in a bimane fluorescence assay on purified β(2)AR. Several peptidomimetics inhibited agonist isoproterenol (ISO) induced cAMP formation by lowering the ISO maximal efficacy up to 61%. Moreover, some peptidomimetics were found to significantly decrease the potency of ISO up to 39-fold. In the bimane fluorescence assay none of the tested peptidomimetics could stabilise an active-like conformation of β(2)AR. Overall, the obtained pharmacological data suggest that some of the peptidomimetics may be able to compete with the native G(s) protein for the intracellular binding site to block ISO-induced cAMP formation, but are unable to stabilise an active-like receptor conformation

Formulation and Characterization of Insulin Nanoclusters

Biopharmaceuticals (e.g., therapeutic proteins and peptides) are characterized by having a high specificity, potency, and biocompatibility, which give them a potential for the treatment of a range of diseases. The structure of biopharmaceuticals challenges their formulation and administration. Most proteins and peptides are degraded in the gastro-intestinal (GI) tract, which is why they are administered via invasive methods such as intravenous (IV) and subcutaneous (SC) injection. The successful development of new formulations can overcome the mental and physical challenges associated with invasive drug administration. Oral formulations remain the ultimate goal due to the convenience of this route of administration. Meanwhile, there is still room for improvement when it comes to the invasive formulations. For instance, a reduced dosing frequency is expected to significantly improve patient compliance and treatment of disease.There is a growing interest in nanoparticles (NP) as carriers of therapeutics. NPs can change the pharmacokinetics and pharmacodynamics of drugs, for example via increased absorption, a protection from degradation in physiological fluids, and an enhancement of the therapeutic effect. Nanoclusters (NCs) is a type of formulation in which the biopharmaceutical is reversibly clustered to acts as both the therapeutic and the vehicle. Studies have shown that NCs can improve the cellular uptake and extend the duration of action of biopharmaceuticals and deliver high doses via small injection volumes.In this project, insulin NCs have been formulated using desolvation and crosslinking. The project illustrates general and important formulation considerations and challenges in the synthesis and characterization of biopharmaceutical NCs. For example, strategies that can provide control over the process of clustering may negatively impact the protein’s biological activity or challenge the output of biophysical characterization techniques. The insulin NCs have a mean size of about 200 nm in water, 10 mM sodium chloride, and 5 % glucose, and are stable in these diluents for at least three months. In physiologically relevant media, the NCs partially dissociate. Crosslinking slightly decreases the potency and the efficacy of insulin because of a reduced flexibility and steric hindrance upon binding to the receptor. The efficacy of the crosslinked protein increases when it is clustered into NCs, which may be due to an increased cell uptake and intracellular receptor stimulation. The NCs are not toxic to cells. Upon SC administration in rats, high insulin NC doses can decrease the blood glucose levels in a controlled manner. The NCs forms the basis for the development of new insulin formulations that can deliver high doses via a reduced dosing volume and frequency to extend the duration of action and minimize the risk of hypoglycemia. Insulin formulations with these characteristics are of great interest due to the increasing number of diabetic and insulinresistant patients