Efficient inhibition of human leukocytic elastase by means of α1-antitrypsin/peptide complexes.

α1 -Antitrypsin (AT), a serine protease inhibitor that specifically targets hydrolytic enzymes, plays a significant role in the termination of tissue inflammation and can therefore represent a key factor in chronic incidences as chronic obstructive pulmonary disease (COPD) or chronic hepatitis. A local and low-dose therapy for the treatment of acquired chronic inflammatory processes which are characterized by insufficient AT amounts but also of genetically conditioned AT deficiencies is supposed to be more effective and less cost-intensive compared to current therapies. In this study, a noncovalent complex formation between the cell-penetrating peptide carrier hCT(18-32)-k7 and AT was performed. The complex was applied to HEK293T/17 cells, as proof-of-principle, and polymorphonuclear leukocytes (PMN), which are responsible for tissue destruction and the perpetuation of inflammation in chronic processes. Both cell species show a successful uptake and subsequently both, an intracellular dot-shaped and homogeneous distribution of the complex demonstrating phagolysosomal as well as cytoplasmic availability. Furthermore, a decreased human leukocytic elastase (HLE) activity was observed after the direct complex administration to PMN. Since the application did not cause an enhanced vitality loss, the complex could facilitate an improvement in direct, local and low-dose treatment of chronically proceeding processes in order to attenuate protease-mediated tissue destruction.

Inhibition of human neutrophil elastase by α1-antitrypsin functionalized colloidal microcarriers.

Layer-by-layer (LbL)-coated microcarriers offer a good opportunity as transport systems for active agents into specific cells and tissues. The assembling of oppositely charged polyelectrolytes enables a modular construction of the carriers and therefore an optimized integration and application of drug molecules. Here, we report the multilayer incorporation and transport of α(1)-antitrypsin (AT) by colloidal microcarriers. AT is an anti-inflammatory agent and shows inhibitory effects toward its pro-inflammatory antagonist, human neutrophil elastase (HNE). The highly proteolytic enzyme HNE is released by polymorphonuclear leukocytes (PMNs) during inflammatory processes and can cause host tissue destruction and pain. The high potential of this study is based on a simultaneous intra- and extracellular application of AT-functionalized LbL carriers. Carrier application in PMNs results in significant HNE inhibition within 21 h. Microcarriers phagocytosed by PMNs were time dependently decomposed inside phagolysosomes, which enables the step-by-step release of AT. Here, AT inactivates HNE before being released, which avoids a further HNE concentration increase in the extracellular space and, subsequently, reduces the risk of further tissue destruction. Additionally, AT surface-functionalized microcarriers allow the inhibition of already released HNE in the extracellular space. Finally, this study demonstrates the successful application of LbL carriers for a concurrent extra- and intracellular HNE inhibition aiming the rebalancing of protease and antiprotease concentrations and the subsequent termination of chronic inflammations.

Maintenance of α(1)-antitrypsin activity by means of co-application of hypochlorous acid-scavengers in vitro and in the supernatant of polymorphonuclear leukocytes: as a basis for a new drug delivery approach.

Tissue destruction, pain and loss of function in chronically inflamed tissues can result from noxious agents released from myeloperoxidase (MPO) and its highly reactive product hypochlorous acid (HOCl) or proteases such as neutrophil elastase (NE). Currently there exists a high demand for medications that provide gentle treatments, free from side effects inherent in those prescribed today. One method to circumvent side effects is through the use of locally applied drug delivery. In contrast to systemic therapy, the main advantages of transport systems are the low dosages of drug with a time-controlled delivery. The aim of this study was to ascertain interactions of NE and its inhibitor α(1)-antitrypsin (AT), the influence of hypochlorous acid (HOCl), as well as its scavengers, in order to define an effective mixture of drugs acting in a synergistic way which can be applied by means of drug delivery systems. These investigations determine the effective amounts of AT/HOCl-scavengers that drug mixtures need for delivery under inflammatory conditions in order to prevent tissue damage. AT was shown to inhibit NE in a dose-dependent manner, whereas a physiological concentration of 1.14 µM AT caused a significant NE inhibition (78%, pH 7.5). The concomitant existence of MPO/HOCl inactivated AT in a dose-dependent manner as well. To regain AT efficacy, HOCl-scavengers, such as L-methionine, α-aminosalicylic acid and cefoperazone were additionally applied. Finally, AT was assembled as surface layer onto layer-by-layer biopolymer-coated microcarriers and carrier phagocytosis by polymorphonuclear leukocytes could be shown.

Interaction, uptake, and processing of LbL-coated microcarriers by PMNs.

Functionalized microcarriers or hollow capsules transporting active agents offer the opportunity for drug delivery inside cells. A promising application of these drug delivery systems is the direct transport as well as the release of immobilized antiinflammatory substances (AIS) into polymorphonuclear leukocytes (PMNs), which play a key role in the course of inflammatory processes. The intended delivery of AIS into the inflamed tissue could alleviate tissue destruction taking place during chronic inflammation, as well as facilitate the termination of these processes. In this study, the capability of functionalized CaCO(3) microcarriers as AIS transporter system targeted at PMNs is investigated. The time-dependent interaction of protamine sulfate and dextran sulfate multilayer-coated 5 µm ± 1 µm CaCO(3) carriers with PMNs, in comparison with the usage of SiO(2) carriers as monodisperse model system of defined sizes (1, 3, and 5 µm), reveals a sufficient carrier/cell interaction and uptake for coincubation periods between 2 and 24 h. Furthermore, the microcarriers are exposed to an environment simulating primary granule/phagosomal conditions after phagocytosis by means of PMN stimulation. The incubation of CaCO(3) microcarriers with cell supernatant demonstrates a partial multilayer decomposition (three to five layers) within 24 h, allowing the gradual release of AIS within the short PMN life span. This observation suggests a potential application for this drug delivery system inside immunologically active cells and may open the way to new alternatives in the treatment of chronic processes.

Impact of metabolic regulators on the expression of the obesity associated genes FTO and NAMPT in human preadipocytes and adipocytes.

BACKGROUND: FTO and NAMPT/PBEF/visfatin are thought to play a role in obesity but their transcriptional regulation in adipocytes is not fully understood. In this study, we evaluated the transcriptional regulation of FTO and NAMPT in preadipocytes and adipocytes by metabolic regulators. METHODOLOGY AND PRINCIPAL FINDINGS: We assessed FTO mRNA expression during human adipocyte differentiation of Simpson-Golabi-Behmel syndrome (SGBS) cells and primary subcutaneous preadipocytes in vitro and evaluated the effect of the metabolic regulators glucose, insulin, dexamethasone, IGF-1 and isoproterenol on FTO and NAMPT mRNA expression in SGBS preadipocytes and adipocytes. FTO mRNA levels were not significantly modulated during adipocyte differentiation. Also, metabolic regulators had no impact on FTO expression in preadipocytes or adipocytes. In SGBS preadipocytes NAMPT expression was more than 3fold induced by dexamethasone and isoproterenol and 1.6fold by dexamethasone in adipocytes. Complete glucose restriction caused an increase in NAMPT mRNA expression by more than 5fold and 1.4fold in SGBS preadipocytes and adipocytes, respectively. CONCLUSION: FTO mRNA expression is not significantly affected by differentiation or metabolic regulators in human adipocytes. The stimulation of NAMPT expression by dexamethasone, isoproterenol and complete glucose restriction may indicate a regulation of NAMPT by metabolic stress, which was more pronounced in preadipocytes compared to mature adipocytes.