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.

Application of capillaries with minimized electroosmotic flow to the electrokinetic study of acidic drug-beta-oleoyl-gamma-palmitoyl-L-alpha-phosphatidyl choline liposome interactions.

Interaction of a model set of common drugs varying widely in their polarity as well as in their chemical structure (salicylic acid, acetylsalicylic acid, ketoprofen, phenytoin and propranolol) with beta-oleoyl-gamma-palmitoyl-L-alpha-phosphatidyl choline (POPC) liposomes was investigated by means of capillary electrophoresis. Two phosphate buffers differing in their pH (50 mM, pH 7.5 and 9.2) were used both for liposome reconstitution and as background electrolytes for capillary electrophoresis using capillaries with minimised electroosmotic flow (EOF). The liposomes showed practically no electrophoretic mobility and formed a stable plug in the capillary. At alkaline pH (9.2), the polyimide coated capillary exhibited residual endoosmotic flow (the EOF marker appeared before the detection window around 40 min as compared to 2.2 min in the untreated capillary; attempts to reveal endoosmotic flow at pH 7.5 were unsuccessful). The concentration of the mixture of the test compounds was 50 microg/ml (except for ketoprofen concentration of which was 5 microg/ml due to the lower solubility of the drug), i.e. large enough to exceed the binding capacity of the injected liposome plug at least at the neutral pH (7.5) which consequently resulted in two regions in the electropherogram, namely that which contained the unbound species and that corresponding to the liposome (lipid)-bound fraction. On the other hand in runs done at high pH of the background electrolyte (9.2) the whole amount injected interacted with the liposomes. Acidic drugs and phenytoin were run with negative polarity at the injection site. It was documented that both at pH 7.5 and 9.2 the investigated solutes interacted with POPC liposomes, though at pH 7.5 the equilibrium between the bound and unbound drugs was in favor of the unbound species. On the contrary, at pH 9.2 binding was considerably stronger and only the liposome bound fraction was seen upon electrophoresis. The well-known instability of phenytoin at room temperature resulted in the formation of an acidic hydrolytic product which was strongly bound to liposomes at the higher pH value. While no binding of phenytoin could be established at pH 7.5, at pH 9.2 this compound was degraded (hydrolyzed) and its degradation product was clearly bound to liposomes. It has to be emphasized that binding experiments must be done separately for acidic/neutral and basic drugs; binding of acidic/neutral drugs must be done at reversed polarity, while in order to reveal binding of basic drugs, positive polarity at the injection site must be used.

Affinity electrochromatography of acidic drugs using a liposome-modified capillary.

Liposomes can be effectively deposited on the inner surface of a capillary wall by flushing the electrophoretic system with a liposome suspension followed by air-drying of the capillary and removal of the excess of loosely bound liposomes by a 0.1 M NaOH wash. It was demonstrated that capillaries prepared in this way could be used for studies of analyte (drug)-liposome binding. The results were expressed as free binding energy changes [delta(deltaG0)] relatively to an arbitrarily selected standard (acetylsalicylic acid). The results were compared to [delta(deltaG0)] changes obtained from binding studies effected by capillary electrophoresis using a stable liposome plug in a capillary with minimized endoosmotic flow. Good agreement of data reported in the literature (without correction for the residual endoosmotic flow), our previous data obtained in a similar way (however, after the correction for the residual endoosmotic flow) and data obtained by the immobilized liposome affinity electrochromatography reported in this communication was achieved.

Interlaboratory reproducibility of mobility parameters in capillary electrophoresis for substance identification in systematic toxicological analysis.

An interlaboratory pilot study was performed to determine the reproducibility of mobility parameters in capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). The study was performed by an intended small number of laboratories (three) that used different brands of instruments (two). The effective mobility was corrected using standards by a method that was recently introduced to obtain a more reproducible migration parameter. A test set of 20 acidic test compounds and 5 reference compounds were analyzed during five days in each laboratory using CZE and MEKC. Buffers used consisted of 90 mM borate set at pH 8.4 (CZE) and 20 mM phosphate, 50 mM sodium dodecyl sulfate set at pH 7.5 (MEKC). Analyses were carried out using fused-silica capillaries at an electric field strength of either 52.6 kV/m or 37.5 kV/m. The interlaboratory reproducibility (mean RSD) of the effective mobility was 3.0% for CZE and 6.7% for MEKC. After applying the correction method, these values became 3.0% for CZE and 3.3% for MEKC, which is adequate for systematic toxicological analysis (STA) applications. A significant improvement of reproducibility for the calculated corrected effective mobility mu(eff)c was observed when variations are high. Therefore, it is recommended to use the correction method in interlaboratory situations, especially when instruments and capillaries from different manufacturers are used.

Dermal nitrate: an old marker of firearm discharge revisited with capillary electrophoresis.

The present work describes a capillary electrophoretic method for nitrite and nitrate determination to be used as a screening tool for investigating the residues of firearm discharge. The use of capillary electrophoresis allowed the rapid determination of nitrite and nitrate, which are major inorganic components of gunshot residues, offering a quantitative and selective alternative to the traditional paraffin test (dermal nitrate test). The method is simpler, cheaper, and faster than the modern approaches to gunshot residue analysis based on the determination of barium, lead and antimony by using flameless absorption spectrometry, inductively coupled plasma-mass spectrometry (ICP-MS), or scanning electron microscopy. The analysis was carried out in a bare fused-silica capillary (75 microm inner diameter) with a 100 mM borate buffer (pH 9.24). The detection was by UV absorption at 214 nm. Separation took place under reversed voltage of 15 kV. Bromide was used as the internal standard. Sensitivity was about 1 mM for both nitrite and nitrate. Reproducibility (intraday and day-to-day) was also good with relative standard deviations (RSDs) < 1.0% for relative migration times and < 4.5% for peak areas in both standard solutions and real matrix. Hair and skin samples from a victim shot in the head were successfully analyzed for the presence of nitrite and nitrate.