Patent Application
20080058249
Stable peptide-galactolipid complexes in aqueous solution are for instance formed by the following general procedure: The galactolipid material in an amount of about 60 mg is weighed in a 100 ml glass flask. The peptide in an amount of about 3 mg is dissolved in 30 ml PBS (10 mM sodium phosphate, 150 mM NaCl, pH 7.4) and this solution is added to the galactolipid material. The sample is vigorously shaken, using a suitable shaker at high speed, for 2 h after which the mixture has become almost clear, and is then allowed to equilibrate and settle for about 30 min at room temperature. Optionally, the almost clear solution is subjected to extrusion through a polycarbonate membrane with a pore size of 100 nm or less, in order to remove or reduce the size of large complexes. Alternatively, the almost clear solution is subjected to filtration through a sterile filter with a pore size of 0.22 μm or less, in order to make the solution sterile.
The LL-20, LL-25, LL-37 and LL-38 peptides were synthesized using solid phase synthesis with the 9-fluorenylmethoxycarbonyl / tert-butyl strategy. The crude peptides, as the trifluoroacetate salts, were purified by HPLC and finally isolated by lyophilization. The purity was determined by means of HPLC. Analysis of composition of amino acids showed that the relative amounts of each amino acid corresponded with the theoretical values for the respective peptide. The antimicrobial activity of the peptides was tested using an inhibition assay.
To prepare the solution of the complex the peptide and CPL-Galactolipid are weighed in a 100 ml glass flask and then PBS (10 mM sodium phosphate, 150 mM NaCl, pH 7.4) is added. The sample is vigorously shaken, using a suitable shaker at high speed, for 1-2 h or until the mixture has become clear, and is then allowed to equilibrate and settle for about 30 min at room temperature. Samples of LL-20, LL-25, LL-37 and LL-38 as trifluoroacetate salts and CPL-Galactolipid were prepared using the amounts stated in Table 1 below. The peptide mixtures all contained 0.20% CPL-Galactolipid.
Ocular inspections were made after 2 h and 2 days of storage at room temperature of the mixtures. These inspections revealed that LL-25, LL-37 and LL-38 in the concentration ranges of 68-98 ppm, 98-213 ppm and 50-102 ppm, respectively, resulted in clear or almost clear solutions. The LL-20 mixture showed large sediments in the investigated concentration range. The molecular weight of LL-20 was calculated to be 2.4 kDa.
The interactions between the charged antimicrobial peptide and the neutral lipid are supposed to be sufficiently strong to accomplish a stabilization of the peptide but weak enough to release the peptide from the complex once it has been delivered to the site of action as shown in wound healing experiments.
The data thus demonstrate that stable complexes are formed between the cationic peptide and CPL-Galactolipid only if the peptide has a molecular weight >2.5 kDa. A preferred peptide:galactolipid weight ratio can be 1:10-1:27.
Test of antimicrobial activity of LL-20 and LL-25 complexes The antimicrobial activity was tested using an inhibition zone assay. As a test bacterium, Bacillius megaterium was used. The following data was obtained.
The data shows that LL-25 showed an antimicrobial activity at a concentration of 68 ppm. It was also shown that LL-25 exhibit activity using the complex with CPL-Galactolipid. The complex with LL-20 had no antimicrobial activity.
From a drug development point of view it would be advantageous if the enzymatic degradation of a peptide could be hampered or blocked since this would increase the half-life of the intact peptide, which then could exert its biological functions over an extended period of time.
Pseudomonas aeruginosa is a common wound pathogen that produces elastase, a hydrolytic enzyme, with capacity to rapidly degrade antimicrobial peptides produced by an infected host, in its efforts to combat bacterial infections. In humans, LL-37 is the most important antimicrobial peptide and its degradation by elastase from Pseudomonas aeruginosa has been studied previously (A. Schmidtchen et al., Molecular Microbiology (2002) 46 (1), 157-168).
In this study we compared the enzymatic degradation rate of LL-37 in an aqueous buffered system to that of a colloidal solution of LL-37 in a galactolipid complex. The experimental procedures for enzymatic degradation were essentially as described using a reverse phase HPLC system (C-18) operating at 210 nm.
In brief: Two stock solutions A and B were prepared. Solution A, “the reference”, contained 100 μg/ml of LL-37 in PBS, pH 7.4. Solution B, “the complex”, contained in addition to 100 μg/ml LL-37 in PBS, pH 7.4, 0.2% of galactolipids (w/w). Two sets of samples were prepared in Eppendorf tubes, 8 tubes from each stock solution. One tube in each set of samples was kept as a negative control (no enzyme added) and to the remaining samples were added an effective amount of elastase from Psuedomonas aeruginosa, giving a final ratio of enzyme to substrate (peptide) of approximately 1:2500. The reactions were kept at 37° C. and samples were withdrawn at predetermined intervals. The reactions were stopped by heating the samples to 100° C. for 5 minutes. After stopping, the reactions were stored at −18° C. prior analysis.
All samples were analysed in duplicates and the peak area for LL-37 was normalized to that of the negative control, which was set to 1.00. Retention times are given as relative retention time (RRT) to LL-37, which is set to RRT=1.00. During degradation of LL-37 in the buffered solution, several different peaks, representing fragments of LL-37 were detected. All fragments eluted at shorter retention times to that of LL-37, indicating their lower molecular weights. From chromatography it is evident that the degradation of LL-37 in a buffered aqueous solution is fast and that the peptide degrades in a step wise manner, first giving a relatively large fragment, at RRT=0.88, which is further degraded to a fragment at RRT=0.66. After 20 h, almost all material had been degraded to low molecular weight fragments having short retention times in the chromatographic system. However, when LL-37 is in the form of a galactolipid complex no detectable amounts of degradation products were found in any of the samples. The results are given in Table 3 below.
From the table above it is evident that LL-37 in a buffered aqueous solution is rapidly degraded when treated with elastase from Pseudomonas aerogunosa. However, when LL-37 in the form of a galactolipid complex is subjected to identical experimental conditions no such degradation is observed, clearly demonstrating the protective effect of the galactolipid formulation.
The present invention is not limited in scope by these described examples. It is thus anticipated that it should be possible to form similar complexes based on galactolipids using other bioactive compounds having molecular weights less than 30 kDa, and being amphiphilic with a net charge. The optimal conditions, that is, the weight ratio of peptide to galactolipid material and the total concentration of the two ingredients in the solution can be obtained by experiments. The aqueous solution should have an appropriate composition, ionic strength and pH as described above. The best composition for each unique peptide and galactolipid mixture is thus established and validated by means of the technically simple procedure described above.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0401942-8 | Jul 2004 | SE | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/SE05/00252 | 2/23/2005 | WO | 00 | 12/22/2006 |
| Number | Date | Country | |
|---|---|---|---|
| 60546966 | Feb 2004 | US |
