Development of a Scintillation Proximity Assay for [3H]epibatidine binding sites of Tetronarce californica muscle-type nicotinic acetylcholine receptor.

The therapy of intoxication with distinct organophosphorus (OP) compounds is still limited today. Especially chemical warfare agents like tabun and soman as well as novichok intoxications are difficult to address using established oxime therapeutics. These neurotoxins inhibit acetylcholinesterase (AChE), a pivotal enzyme in the synaptic cleft. The following accumulation of acetylcholine in the synaptic cleft leads to a dysfunctional, desensitized state of nicotinic acetylcholine receptors (nAChR). Without adequate treatment, the resulting cholinergic crisis leads to death by respiratory arrest. Consequently, the research approach for new therapeutic options needs to be expanded. A promising option would be substances interacting directly with nAChRs. Therefore, screening methods for new drug candidates are needed, with affinity assays playing an important role. In the present work, a saturation and competition scintillation proximity assay (SPA) for binding studies at [3H]epibatidine binding sites, conventionally classified as orthosteric binding sites of the muscle type nAChR was developed. This method offers several advantages over other assay technologies because no separation as well as washing steps are required to remove unbound ligands. Assay precision and solvent tolerance were validated according to the guidelines for validation of bioanalytical methods of the Food and Drug Administration (FDA) and European Medicines Agency (EMA). The newly developed binding assay was successfully implemented on an automated pipetting platform and is suitable for high-throughput-screening receptor-ligand interactions at the nAChR. Furthermore, it allows countering the affinity at the orthosteric binding site in the presence of highly toxic agents such as nerve agents or structurally similar pesticides. Related to further pharmacological results, the affinity to [3H]epibatidine binding sites can provide additional information on whether potential drug candidates would be suitable for treatment of nerve agent poisoning.

Transglutaminase-catalyzed covalent anti-myostatin peptide depots.

Nature realizes protein and peptide depots by catalyzing covalent bonds with the extracellular matrix (ECM) of tissues. We are translating this natural blueprint for the sustained delivery of a myostatin-inhibiting peptide (Anti-Myo), resulting in an enzyme depot established from injectable solutions. For that, we fused Anti-Myo to the D-domain of insulin-like growth factor I, a transglutaminase (TG) substrate. TG catalyzed the covalent binding of the D-domain to ECM proteins, such as laminin and fibronectin, on bioengineered ECM and in mice. ECM decorated with Anti-Myo suppressed myostatin activity and pathway activation and reduced the differentiation of preconditioned bone marrow-derived macrophages into osteoclasts in vitro.

An Introductory Guide to Protease Sensitive Linker Design Using Matrix Metalloproteinase 13 as an Example.

Proteases play a crucial role, not only in physiological, but also in pathological processes, such as cancer, inflammation, arthritis, Alzheimer's, and infections, to name but a few. Their ability to cleave peptides can be harnessed for a broad range of biotechnological purposes. To do this efficiently, it is essential to find an amino acid sequence that meets the necessary requirements, including interdependent factors like specificity, selectivity, cleavage kinetics, or synthetic accessibility. Cleavage sequences from natural substrates of the protease may not be optimal in terms of specificity and selectivity, which is why these frequently require arduous and sometimes unsuccessful optimization such as by iterative exchange of single amino acids. Hence, here we describe the systematic design of protease sensitive linkers (PSLs)─peptide sequences specifically cleaved by a target protease─guided by the mass spectrometry based determination of target protease specific cleavage sites from a proteome-based peptide library. It includes a procedure for identifying bespoke PSL sequences, their optimization, synthesis, and validation and introduces a program that can indicate potential cleavage sites by hundreds of enzymes in any arbitrary amino acid sequence. Thereby, we provide an introduction to PSL design, illustrated by the example of matrix metalloproteinase 13 (MMP13). This introduction can serve as a guide and help to greatly accelerate the development and use of protease-sensitive linkers in diverse applications.

PEGylated Recombinant Aplysia punctata Ink Toxin Depletes Arginine and Lysine and Inhibits the Growth of Tumor Xenografts.

In recent years, a novel treatment method for cancer has emerged, which is based on the starvation of tumors of amino acids like arginine. The deprivation of arginine in serum is based on enzymatic degradation and can be realized by arginine deaminases like the l-amino acid oxidase found in the ink toxin of the sea hare Aplysia punctata. Previously isolated from the ink, the l-amino acid oxidase was described to oxidate the essential amino acids l-lysine and l-arginine to their corresponding deaminated alpha-keto acids. Here, we present the recombinant production and functionalization of the amino acid oxidase Aplysia punctata ink toxin (APIT). PEGylated APIT (APIT-PEG) increased the blood circulation time. APIT-PEG treatment of patient-derived xenografted mice shows a significant dose-dependent reduction of tumor growth over time mediated by amino acid starvation of the tumor. Treatment of mice with APIT-PEG, which led to deprivation of arginine, was well tolerated.

In situ setup for screening of drug permeation by NMR spectroscopy.

There are various commercially available setups for studying drug permeation, which differ in cost and manual labor. We explore an artificial membrane in an NMR tube to assess drug permeation with automated measurements. NMR-based concentrations were validated with HPLC and compared to a conventional setup. Setup-specific challenges and workarounds as well as future setup-designs for this and other applications are discussed.

Matrix Metalloproteinase-Responsive Delivery of PEGylated Fibroblast Growth Factor 2.

Attachment of polyethylene glycol (PEG) chains is a common, well-studied, and Food and Drug Administration-approved method to address the pharmacokinetic challenges of therapeutic proteins. Occasionally, PEGylation impairs the activity of pharmacodynamics (PD). To overcome this problem, disease-relevant cleavable linkers between the polymer and the therapeutic protein can unleash full PD by de-PEGylating the protein at its target site. In this study, we engineered a matrix metalloproteinase (MMP)-responsive fibroblast growth factor 2 (FGF-2) mutant that was site-specifically extended with a PEG polymer chain. Using bioinspired strategies, the bioconjugate was designed to release the native protein at the desired structure/environment with preservation of the proliferative capacity in vitro on NIH3T3 cells. In vivo, hepatic exposure was diminished but not its renal distribution over time compared to unconjugated FGF-2. By releasing the growth factor from the PEG polymer in response to MMP cleavage, restored FGF-2 may enter hard-to-reach tissues and activate cell surface receptors or nuclear targets.

Bioresponsive Cytokine Delivery Responding to Matrix Metalloproteinases.

Cytokines are regulated in acute and chronic inflammation, including rheumatoid arthritis (RA) and myocardial infarction (MI). However, the dynamic windows within which cytokine activity/inhibition is desirable in RA and MI change timely and locally during the disease. Therefore, traditional, static delivery regimens are unlikely to meet the idiosyncrasy of these highly dynamic pathophysiological and individual processes. Responsive delivery systems and biomaterials, sensing surrogate markers of inflammation (i.e., matrix metalloproteinases - MMPs) and answering with drug release, may present drug activity at the right time, manner, and place. This article discusses MMPs as surrogate markers for disease activity in RA and MI to clock drug discharge to MMP concentration profiles from MMP-responsive drug delivery systems and biomaterials.

PEGylation of Human Vascular Endothelial Growth Factor.

Vascular endothelial growth factor A-165 (VEGF-A165) positively modulates neointimal hyperplasia, lumen stenosis, and neovascularization. One challenge for the use of VEGF-A165 for potential therapy is its short serum half-life. Therefore, we are designing VEGF-A165 bioconjugates carrying polyethylene glycol (PEG). The purity of the recombinantly expressed human VEGF-A165 exceeded 90%. The growth factor had a half-maximal effective concentration of 0.9 ng/mL (EC50) and induced tube formation of human umbilical vein endothelial cells. PEGylation was conducted by Schiff base reaction followed by reductive amination. After purification, two species were obtained, with one or two PEG attached per VEGF-A165 dimer. Both resulting bioconjugates had a purity exceeding 90%, wild-type bioactivity, and increased hydrodynamic radii as required for prolonging the half-life.

Bile Is a Selective Elevator for Mucosal Mechanics and Transport.

Mucus mechanically protects the intestinal epithelium and impacts the absorption of drugs, with a largely unknown role for bile. We explored the impacts of bile on mucosal biomechanics and drug transport within mucus. Bile diffused with square-root-of-time kinetics and interplayed with mucus, leading to transient stiffening captured in Brillouin images and a concentration-dependent change from subdiffusive to Brownian-like diffusion kinetics within the mucus demonstrated by differential dynamic microscopy. Bile-interacting drugs, Fluphenazine and Perphenazine, diffused faster through mucus in the presence of bile, while Metoprolol, a drug with no bile interaction, displayed consistent diffusion. Our findings were corroborated by rat studies, where co-dosing of a bile acid sequestrant substantially reduced the bioavailability of Perphenazine but not Metoprolol. We clustered over 50 drugs based on their interactions with bile and mucin. Drugs that interacted with bile also interacted with mucin but not vice versa. This study detailed the dynamics of mucus biomechanics under bile exposure and linked the ability of a drug to interact with bile to its abbility to interact with mucus.

On the reproducibility of extrusion-based bioprinting: round robin study on standardization in the field.

The outcome of three-dimensional (3D) bioprinting heavily depends, amongst others, on the interaction between the developed bioink, the printing process, and the printing equipment. However, if this interplay is ensured, bioprinting promises unmatched possibilities in the health care area. To pave the way for comparing newly developed biomaterials, clinical studies, and medical applications (i.e. printed organs, patient-specific tissues), there is a great need for standardization of manufacturing methods in order to enable technology transfers. Despite the importance of such standardization, there is currently a tremendous lack of empirical data that examines the reproducibility and robustness of production in more than one location at a time. In this work, we present data derived from a round robin test for extrusion-based 3D printing performance comprising 12 different academic laboratories throughout Germany and analyze the respective prints using automated image analysis (IA) in three independent academic groups. The fabrication of objects from polymer solutions was standardized as much as currently possible to allow studying the comparability of results from different laboratories. This study has led to the conclusion that current standardization conditions still leave room for the intervention of operators due to missing automation of the equipment. This affects significantly the reproducibility and comparability of bioprinting experiments in multiple laboratories. Nevertheless, automated IA proved to be a suitable methodology for quality assurance as three independently developed workflows achieved similar results. Moreover, the extracted data describing geometric features showed how the function of printers affects the quality of the printed object. A significant step toward standardization of the process was made as an infrastructure for distribution of material and methods, as well as for data transfer and storage was successfully established.

Bioconjugation of a Fibroblast Activation Protein Targeted Interleukin-4.

Interleukin-4 (IL-4) is an immune-modulating therapeutic with growing potential for the treatment of inflammatory diseases. Current challenges of IL-4 therapy include a low serum half-life and pleiotropic activity, suggesting effective targeting of IL-4. To develop an interleukin-4 bioconjugate with rapid targeting to inflammatory disease sites, we report the chemical synthesis, bioconjugation, and in vitro characterization of a murine interleukin-4 (mIL-4) conjugate decorated with a fibroblast activation protein inhibitor (FAPI). The FAPI targeting moiety features 2,2',2″,2‴-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid (DOTA) to allow future biodistribution and imaging studies of the FAPI-mIL-4 bioconjugate. We demonstrated site-specific coupling of mIL-4 and FAPI-DOTA deploying chemo-enzyme and enzyme chemistries with a high purity exceeding 95%. The FAPI-DOTA modified mIL-4 was bioactive with polarization of murine macrophages into the M2 state while maintaining specific binding to FAP on fibroblast cells. Together, these results point to future in vivo use of the FAPI-mIL-4 bioconjugate to assess biodistribution and biological effects in animal models of inflammatory joint disease.

Harnessing Bile for Drug Absorption through Rational Excipient Selection.

Bile solubilization and apparent solubility at resorption sites critically affect the bioavailability of orally administered and poorly water-soluble drugs. Therefore, identification of drug-bile interaction may critically determine the overall formulation success. For the case of the drug candidate naporafenib, drug in solution at phase separation onset significantly improved with polyethylene glycol-40 hydrogenated castor oil (RH40) and amino methacrylate copolymer (Eudragit E) but not with hydroxypropyl cellulose (HPC) in both phosphate-buffered saline (PBS) and PBS supplemented with bile components. Naporafenib interacted with bile as determined by 1H and 2D 1H-1H nuclear magnetic resonance spectroscopy and so did Eudragit E and RH40 but not HPC. Flux across artificial membranes was reduced in the presence of Eudragit E. RH40 reduced the naporafenib supersaturation duration. HPC on the other side stabilized naporafenib's supersaturation and did not substantially impact flux. These insights on bile interaction correlated with pharmacokinetics (PK) in beagle dogs. HPC preserved naporafenib bile solubilization in contrast to Eudragit E and RH40, resulting in favorable PK.

An exploratory study on the effect of mechanical stress on particle formation in monoclonal antibody infusions.

Monoclonal antibody infusions (mAb-i) are administered for the treatment of various diseases. They are often transported over long distances from the compounding site to the site of administration. However, transport studies are typically carried out with the original drug product but not with compounded mAb-i. To address this gap, the impact of mechanical stress on the formation of subvisible/nanoparticles in mAb-i was investigated by dynamic light scattering and flow imaging microscopy. Different mAb-i concentrations were subjected to vibrational orbital shaking and stored at 2-8°C up to 35 days. The screening revealed that pembrolizumab and bevacizumab infusions show the highest propensity for particle formation. Especially bevacizumab at low concentrations exhibited an increase in particle formation. Because of the unknown health risks associated with the long-term application of subvisible particles (SVPs)/nanoparticles in infusion bags, stability studies carried out in the frame of licensing application procedures should also focus on SVP formation in mAb-i. In general, pharmacists should minimize the time of storage and mechanical stress during transport, especially in the case of low-concentrated mAb-i. Moreover, if siliconized syringes are used, they should be washed once with saline solution to minimize particle entry.

Nature-inspired synthesis of antibacterial glucovanillin derivatives.

The ongoing threat of Antimicrobial Resistance (AMR) complicated by the rise of Multidrug-Resistant (MDR) pathogens calls for increased efforts in the search for novel treatment options. While deriving inspiration from antibacterial natural compounds, this study aimed at using synthetic approaches to generate a series of glucovanillin derivatives and explore their antibacterial potentials. Among the synthesized derivatives, optimum antibacterial activities were exhibited by those containing 2,4- and 3,5-dichlorophenylamino group coupled to a glucovanillin moiety (compounds 6h and 8d respectively). In those compounds, the Minimum Inhibitory Concentrations (MIC) of 128-256 µg/mL were observed against reference and MDR strains of Klebsiella pneumoniae, Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE). Moreover, these findings emphasize the claims from previous reports on the essence of smaller molecular size, the presence of protonatable amino groups and halogens in potential antibacterial agents. The observed moderate and broad-spectrum activities of the stated derivatives point to their suitability as potential leads towards further efforts to improve their antibacterial activities.

Transdermal carbon monoxide delivery.

Overuse injuries or acute trauma in joints often lead to painful tendinopathy, and pharmacological treatment effects are limited. The site of the disease is hard to reach with drugs, both systemically and through the skin. Therapeutic gases may close this gap, as they permeate easier through tissues than conventional small molecules. We present a patch device releasing the anti-inflammatory gas carbon monoxide (CO) through the skin to the subcutaneous tendons and tissues. CO is chemically generated upon device activation and its design maximizes CO exposure to the underlying skin and protects the patient from all side and degradation products. The patch delivered CO successfully through the intact skin, granting lasting, subcutaneous CO exposure for up to 16 h. Furthermore, the released CO induced the proliferation of fibroblasts and the polarization of monocytes into anti-inflammatory M2 macrophages. In conclusion, the CO-releasing device might open an entirely new treatment option against tendinopathies in case of a positive outcome of future in vivo studies.

Solid microemulsion preconcentrates on pH responsive metal-organic framework for tableting.

Poorly water-soluble drugs are frequently formulated with lipid-based formulations including microemulsions and their preconcentrates. We detailed the solidification of drug-loaded microemulsion preconcentrates with the acid-sensitive metal-organic framework ZIF-8 by X-ray powder diffraction and solid-state nuclear magnetic resonance spectroscopy. Adsorption and desorption dynamics were analyzed by fluorescence measurement, high-performance liquid chromatography, dynamic light scattering and 1H-DOSY experiments using the model compounds Nile Red, Vitamin K1, and Lumefantrine. Preconcentrates and drugs were successfully loaded onto ZIF-8 while preserving its crystal structure. The solid powder was pressable to tablets or 3D-printed into oral dosage forms. At low pH, colloidal solutions readily formed, solubilizing the poorly water-soluble compounds. The use of stimuli-responsive metal organic frameworks as carriers for the oral delivery of lipid-based formulations points towards solid dosage forms readily forming colloidal microemulsions.

Cyano(fluoro)borate and cyano(hydrido)borate ionic liquids: low-viscosity ionic media for electrochemical applications.

The synthesis and detailed characterization of low-viscosity room-temperature ionic liquids (RTILs) and [BnPh3P]+ salts with the cyano(fluoro)borate anions [BF(CN)3]- (MFB), [BF2(CN)2]- (DFB), and [BF3(CN)]- as well as the new mixed-substituted anion [BFH(CN)2]- (FHB) is described. The RTILs with [EMIm]+ or [BMPL]+ as countercations were obtained in yields of up to 98% from readily available alkali metal salts and in high purities that allow application in electrochemical devices. Trends in thermal stability, melting and freezing behavior, density, electrochemical stability, dynamic viscosity, specific conductivity and ion diffusivity have been assessed and compared to those of the related tetracyanoborate- and cyano(hydrido)borate-RTILs. The crystal structure analysis of the [BnPh3P]+ salts of [BFn(CN)4-n]- (n = 0-4), [BHn(CN)4-n]- (n = 1-3) and [BFH(CN)2]- provided experimental access to anion volumina that together with ion molecular mass, electrostatic potential, shape and chemical stability have been correlated to physicochemical properties. In addition, the cytotoxicity of the [EMIm]+-ILs and potassium or sodium salts was studied.

Design, synthesis and biological evaluations of quinolone amides against African trypanosomiasis with improved solubility.

The human African trypanosomiasis is a devastating parasitic infection, which is caused by the protozoan Trypanosoma brucei and transmitted by the bite of the tsetse fly. An untreated infection usually results in death and only few drugs with significant drawbacks are currently available for treatment. Previous investigations revealed the quinolone amide MB007 as a lead compound with an excellent selectivity for T. b. brucei. Here, new quinolone amides were synthesized for deeper insights into the structure-activity relationship. Furthermore, the aqueous solubility of the compounds was analyzed, as the poor solubility of previous quinolone amides impeded in vivo studies for target identification. The biological evaluation led to the new lead structure 9f, which exhibits a promising in vitro activity against T. b. brucei (IC50 = 22 nM) and showed no cytotoxicity against macrophages. Moreover, compounds 10b and 10c were discovered, which possessed an improved solubility combined with a decent selectivity.

Oral Use of Therapeutic Carbon Monoxide for Anyone, Anywhere, and Anytime.

Carbon monoxide (CO) is a therapeutic gas with therapeutic potential in intestinal bowel disease. Therapeutic efficacy in the gastrointestinal tract (GIT) must be paired with safe and convenient use. Therefore, we designed an oral CO releasing system (OCORS) pairing tunable CO release into the GIT while preventing the release of any other molecule from within the device, causing safety concerns. The dimensions of the device, which is manufactured from 3D printed components, are within compendial limits. This is achieved by controlling CO decarbonylation from a molybdenum complex with a FeCl3 solution. OCORS' surrounding silicon membranes control release rates, as does the loading with carbonylated molybdenum complex and FeCl3 solution. Herein we describe the development of the system, the characterization of the CO releasing molecule (CORM), and the CO release kinetics of the overall system. Neither the CORM nor isocyanoacetate as a potential reaction byproduct were cytotoxic. Finally, we demonstrated by design validation in an in vivo porcine model that, except for the release of the therapeutic CO, OCORS isolates all components during transit through the stomach. We could show that OCORS generated and released CO locally into the stomach of the animals without systemic exposure, measured as the carboxyhemoglobin content in the blood of the pigs. In conclusion, OCORS derisks oral development by limiting patient exposure to (desirable) CO while preventing contact with any further (undesirable) chemical, by-, or degradation products. CO generating devices come in reach, which now can be used by anyone, anywhere, and anytime.