Primary and Secondary Binding of Exenatide to Liposomes.

The interactions of exenatide, a Trp-containing peptide used as a drug to treat diabetes, with liposomes were studied by isothermal titration calorimetry (ITC), tryptophan (Trp) fluorescence, and microscale thermophoresis measurements. The results are not only important for better understanding the release of this specific drug from vesicular phospholipid gel formulations but describe a general scenario as described before for various systems. This study introduces a model to fit these data on the basis of primary and secondary peptide-lipid interactions. Finally, resolving apparent inconsistencies between different methods aids the design and critical interpretation of binding experiments in general. Our results show that the net cationic exenatide adsorbs electrostatically to liposomes containing anionic diacyl phosphatidylglycerol lipids (PG); however, the ITC data could not properly be fitted by any established model. The combination of electrostatic adsorption of exenatide to the membrane surface and its self-association (Kd = 46 µM) suggested the possibility of secondary binding of peptide to the first, primarily (i.e., lipid-) bound peptide layer. A global fit of the ITC data validated this model and suggested one peptide to bind primarily per five PG molecules with a Kd ≈ 0.2 µM for PC/PG 1:1 and 0.6 µM for PC/PG 7:3 liposomes. Secondary binding shows a weaker affinity and a less exothermic or even endothermic enthalpy change. Depending on the concentration of liposomes, secondary binding may also lead to liposomal aggregation as detected by dynamic light-scattering measurements. ITC quantifies primary and secondary binding separately, whereas microscale thermophoresis and Trp fluorescence represent a summary or average of both effects, possibly with the fluorescence data showing somewhat greater weighting of primary binding. Systems with secondary peptide-peptide association within the membrane are mathematically analogous to the adsorption discussed here.

Do interactions between protein and phospholipids influence the release behavior from lipid-based exenatide depot systems?

The release mechanism for proteins and peptides from vesicular phospholipid gels (VPGs) is very complex. Drug release proceeds via a combination of erosion of the gel and diffusion of the drug out of it. This diffusion can be retarded by a slow permeation of the drug across the lipid bilayers in the gel as well as by its direct binding or adsorption to the lipid bilayers. Finally, the viscosity and homogeneity of the formulation may affect the release behavior. So far a direct correlation between one of these parameters and the release kinetics is not possible. In the present study, we aimed to investigate the contribution of drug-membrane interactions to the release kinetics of exenatide from differently composed VPGs (POPC, POPG and mixtures of both). To this end, in vitro release of exenatide as well as in vitro release of the phospholipids was monitored. Binding affinities were determined by microscale thermophoresis (MST). The sustained release behavior of exenatide could not simply be correlated to high viscosity of the VPG formulation. Release of exenatide from VPGs of anionic membranes containing POPG proceeded with a half-life of the order of 5 days and it seems to be controlled by the erosion of the gel. Its rate is unaffected by the initial pH inside the gel, independently of the strong impact of pH on exenatide binding to the membrane. At pH 4.5, exenatide is cationic and binds to membranes containing anionic POPG with a high affinity (Kd ≈ 10-30 µM). No high affinity membrane binding of exenatide is detected in this at pH 7.4, where exenatide is anionic, and to zwitterionic membranes composed of POPC. Exenatide release from the latter has a significantly longer half-life of 30 to 55 days. That means, these VPGs are much more resistant to erosion and show a very slow diffusional release. In this case, diffusion should be slowed down by the barrier function of the membranes rather than membrane affinity. In conclusion, erosion of the VPG matrix and membrane permeability of the drug are the major parameters influencing the release of exenatide from VPGs of POPC-POPG, whereas drug binding to the membranes had a minor effect only.

Vesicular phospholipid gels as drug delivery systems for small molecular weight drugs, peptides and proteins: State of the art review.

Lipid-based drug delivery has been investigated for a long time when it comes to liposomes and solid-lipid implants or solid-lipid nanoparticles. The promising, characteristic properties of these systems have led to the development of newer lipid-based drug delivery systems for the sustained release of drugs like liposomes for sustained delivery of substances, DepoFoam™ technology, phospholipid-based phase separation gels and vesicular phospholipid gels. Vesicular phospholipid gels (VPGs) are highly concentrated, viscous dispersions of high amounts of phospholipids in aqueous drug solution. The easy, solvent-free manufacturing process, high biocompatibility and various applications, as depot formulation for the sustained delivery of drugs and as a storage form of small unilamellar vesicles make VPGs highly attractive as drug carriers. Over the last years, the solvent free preparation process has advanced from high pressure homogenization to dual centrifugation (DC). Thereby a very simple one step process has been established for the preparation of VPGs. The semisolid VPG was first described in 1997 by Brandl et al. Since then, many formulations have been developed, encapsulating small molecular weight drugs like 5-FU (2003), cetrorelix (2005), cytarabine (2012) and exenatide (2015). In 2010, the first pharmaceutical protein, erythropoietin, was encapsulated in VPGs and sustained release of the substance was shown in vitro. In 2015, G-CSF was encapsulated in VPGs and tested in vivo for rotator cuff repair in a rat model and for PEGylated IFN-ß-1b sustained release from vesicular phospholipid gels was demonstrated in vitro. Further, a very elegant administration technique for VPGs via needle-free injection was established. However this promising drug delivery system does still leave space for improvement and optimization. This review summarizes information about lipid-based depot systems in general and focuses on the historical development of VPGs. It emphasizes the advantages and drawbacks of VPGs as drug delivery device. Additionally, novel preparation methods and applications of VPGs will be discussed. A focus will be set on delivery of pharmaceutical proteins and peptides.

Pharmacological Modulation of the Psychiatric Risk Factor FKBP51 Alters Efficiency of Common Antidepressant Drugs.

Despite a growing body of research over the last few decades, mental disorders, including anxiety disorders or depression, are still one of the most prevalent and hardest to treat health burdens worldwide. Since pharmacological treatment with a single drug is often rather ineffective, approaches such as co-medication with functionally diverse antidepressants (ADs) have been discussed and tried more recently. Besides classical ADs, there is a growing number of candidate targets identified as potential starting points for new treatment methods. One of these candidates, the FK506 binding protein 51 (FKBP51) is linked to a number of psychiatric disorders in humans. In this study, we used SAFit2-a newly developed modulator of FKBP51, which has shown promising results in rodent models for stress-related disorders delivered in a depot formulation. We combined SAFit2 with the commonly prescribed selective serotonin reuptake inhibitor (SSRI) escitalopram and performed basic behavioral characterization in a mouse model. Remarkably, co-application of SAFit2 lowered the efficacy of escitalopram in anxiety-related tests but improved stress coping behavior. Given the fact that mental diseases such as anxiety disorders or depression can be divided into different sub-categories, some of which more or less prone to stress, SAFit2 could indeed be a highly beneficial co-medication in very specific cases. This study could be a first, promising step towards the use of FKBP51 modulators as potent and specific enhancers of AD efficiency for subclasses of patients in the future.

The stress regulator FKBP51: a novel and promising druggable target for the treatment of persistent pain states across sexes.

It is well established that FKBP51 regulates the stress system by modulating the sensitivity of the glucocorticoid receptor to stress hormones. Recently, we have demonstrated that FKBP51 also drives long-term inflammatory pain states in male mice by modulating glucocorticoid signalling at spinal cord level. Here, we explored the potential of FKBP51 as a new pharmacological target for the treatment of persistent pain across the sexes. First, we demonstrated that FKBP51 regulates long-term pain states of different aetiologies independently of sex. Deletion of FKBP51 reduced the mechanical hypersensitivity seen in joint inflammatory and neuropathic pain states in female and male mice. Furthermore, FKBP51 deletion also reduced the hypersensitivity seen in a translational model of chemotherapy-induced pain. Interestingly, these 3 pain states were associated with changes in glucocorticoid signalling, as indicated by the increased expression, at spinal cord level, of the glucocorticoid receptor isoform associated with glucocorticoid resistance, GRß, and increased levels of plasma corticosterone. These pain states were also accompanied by an upregulation of interleukin-6 in the spinal cord. Crucially, we were able to pharmacologically reduce the severity of the mechanical hypersensitivity seen in these 3 models of persistent pain with the unique FKBP51 ligand SAFit2. When SAFit2 was combined with a state-of-the-art vesicular phospholipid gel formulation for slow release, a single injection of SAFit2 offered pain relief for at least 7 days. We therefore propose the pharmacological blockade of FKBP51 as a new approach for the treatment of persistent pain across sexes, likely in humans as well as rodents.

Stress-responsive FKBP51 regulates AKT2-AS160 signaling and metabolic function.

The co-chaperone FKBP5 is a stress-responsive protein-regulating stress reactivity, and its genetic variants are associated with T2D related traits and other stress-related disorders. Here we show that FKBP51 plays a role in energy and glucose homeostasis. Fkbp5 knockout (51KO) mice are protected from high-fat diet-induced weight gain, show improved glucose tolerance and increased insulin signaling in skeletal muscle. Chronic treatment with a novel FKBP51 antagonist, SAFit2, recapitulates the effects of FKBP51 deletion on both body weight regulation and glucose tolerance. Using shorter SAFit2 treatment, we show that glucose tolerance improvement precedes the reduction in body weight. Mechanistically, we identify a novel association between FKBP51 and AS160, a substrate of AKT2 that is involved in glucose uptake. FKBP51 antagonism increases the phosphorylation of AS160, increases glucose transporter 4 expression at the plasma membrane, and ultimately enhances glucose uptake in skeletal myotubes. We propose FKBP51 as a mediator between stress and T2D development, and potential target for therapeutic approaches.

Needle-Free Injection of Vesicular Phospholipid Gels-A Novel Approach to Overcome an Administration Hurdle for Semisolid Depot Systems.

Vesicular phospholipid gels (VPGs) are depot formulations for the sustained release of drugs which are characterized by a high amount of phospholipids in the formulation. They consist of physiological excipients only and therefore display high biocompatibility. Their manufacture is simple, cheap, solvent free, and ideal for the processing of proteins and peptides because of the low stress on the molecule, for example, by elevated temperatures. One major hurdle of VPGs is their high viscosity which makes them hard to almost impossible to inject with conventional, thin needles used for subcutaneous administration. However, so far no data are published to overcome this administration challenge. In the present study, needle-free injection was investigated and successfully applied as a technology for the easy and elegant administration of VPGs. VPGs with different phospholipid content were injected with a Biojector 2000 into gelatin blocks and full thickness pig skin postmortem as in vitro models and the injection depth was determined after injection. The release behavior was tested after shearing the VPG with the device to evaluate the effect of shearing on the drug release from the formulation. No differences were observed when compared to an ejection with needle and syringe.