NMR Study of the Secondary Structure and Biopharmaceutical Formulation of an Active Branched Antimicrobial Peptide.

The synthetic antimicrobial peptide SET-M33 is being developed as a possible new antibacterial candidate for the treatment of multi-drug resistant bacteria. SET-M33 is a branched peptide featuring higher resistance and bioavailability than its linear analogues. SET-M33 shows antimicrobial activity against different species of multi-resistant Gram-negative bacteria, including clinically isolated strains of Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumanii and Escherichia coli. The secondary structure of this 40 amino acid peptide was investigated by NMR to fully characterize the product in the framework of preclinical studies. The possible presence of helixes or ß-sheets in the structure had to be explored to predict the behavior of the branched peptide in solution, with a view to designing a formulation for parenteral administration. Since the final formulation of SET-M33 will be strictly defined in terms of counter-ions and additives, we also report the studies on a new salt form, SET-M33 chloride, that retains its activity against Gram-negative bacteria and gains in solubility, with a possible improvement in the pharmacokinetic profile. The opportunity of using a chloride counter-ion is very convenient from a process development point of view and did not increase the toxicity of the antimicrobial drug.

Intermolecular interactions play a role in the distribution and transport of charged contrast agents in a cartilage model.

The transport and distribution of charged molecules in polyelectrolyte solutions are of both fundamental and practical importance. A practical example, which is the specific subject addressed in the present paper, is the transport and distribution of charged species into cartilage. The charged species could be a contrast agent or a drug molecule involved in diagnosis or treatment of the widespread degenerative disease osteoarthritis, which leads to degradation of articular cartilage. Associated scientific issues include the rate of transport and the equilibrium concentrations of the charged species in the cartilage and the synovial fluid. To address these questions, we present results from magnetic resonance micro-imaging experiments on a model system of articular cartilage. The experiments yield temporally and spatially resolved data on the transport of a negatively charged contrast agent (charge = -2), used in medical examinations of cartilage, into a polyelectrolyte solution, which is designed to capture the electrostatic interactions in cartilage. Also presented is a theoretical analysis of the transport where the relevant differential equations are solved using finite element techniques as well as treated with approximate analytical expressions. In the analysis, non-ideal effects are included in the treatment of the mobile species in the system. This is made possible by using results from previous Monte Carlo simulations. The results demonstrate the importance of taking non-idealities into account when data from measurements of transport of charged solutes in a system with fixed charges from biological polyelectrolytes are analyzed.

Electrostatic interactions are important for the distribution of Gd(DTPA)(2-) in articular cartilage.

PURPOSE: The delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC) method can be used to assess the content of glycosaminoglycan in cartilage. In in vitro and model studies, the content of glycosaminoglycan is often expressed in terms of a fixed charge density (FCD). Values of the fixed charge density obtained using the dGEMRIC method differs from values obtained using other methods. The purpose of this work was to further clarify the origin of this discrepancy. METHODS: dGEMRIC experiments were performed in a µMRI setup on a custom-designed, well-defined model system capturing the relevant ionic features of cartilage. The model system allows for good control over and systematic variation of relevant parameters. The experimental data was compared with results from Monte Carlo simulations on a coarse-grained model. RESULTS: Application of ideal Donnan theory on data obtained from experiments as well as simulations lead to underestimation of the fixed charge density, in agreement with previous studies. CONCLUSION: To obtain more accurate estimates of the fixed charge density using the dGEMRIC method, interionic interactions need to be taken into account in the Donnan analysis. Furthermore, the results suggest that the combination of µMRI dGEMRIC experiments and Monte Carlo simulations are useful tools for an improved understanding of these effects. Magn Reson Med 76:500-509, 2016. © 2015 Wiley Periodicals, Inc.

Monte Carlo simulations of Donnan equilibrium in cartilage.

23Na magnetic resonance imaging and the delayed gadolinium-enhanced magnetic resonance imaging methods to investigate cartilage can be used to determine the fixed charge density of cartilage. The methods give results that differ by a factor of 2. In this study, we use Monte Carlo simulations on a model system of cartilage and find that the difference originates from the Coulombic intermolecular interactions between the ions in the cartilage, and in the synovial fluid. Those interactions are neglected in the standard Donnan analysis that generally is adopted to evaluate magnetic resonance imaging data.