2005 ANNUAL SCIENTIFIC MEETING 195 PEPTIDES - MORE THAN ANTI-AGING: A NOVEL ANTIMICROBIAL PEPTIDE FOR COSMETIC APPLICATIONS Durant Scholz, C. Murphy and R. Rivera Active Concepts, LLC, 238 Nicholas Avenue, South Plainfield, NJ 07080 A casual survey of the latest edition of the CFTA dictionary shows a listing of more than 100 different peptides. These range from small peptides with well established sequences to less well defined materials isolated from grains or soy, although these are not well differentiated from simple hydrolysates. A clear majority of these products are designed to support some type of anti-aging claim although the proposed mechanisms vary considerably. But that of course, is clearly what the consumer has come to expect from peptides. It may well be that peptides, like elastomers before them have become the next AHA, a raw material that is de rigueur in modem cosmetics. In molecular biology peptides function ubiquitously as cellular messengers. It is this role that much of the focus of research within the Cosmetic Industry has focused on. The role we will be looking at revolves around the ability of certain peptides to function as a component of an organism's innate immune system. Research in both food and medicine has flirted with this area for over 50 years, although it has only been the past 5 years that have brought a dramatic innovation. Much of this interest was driven by the high degree of sequence homology between antimicrobial peptides (AMP) across species lines. Our particular interest was sparked by the lack of incidence on the Korean peninsula relating to the SARS outbreak. It was postulated at that time, that consumption ofKimchi, a form of cabbage fermented with Leuconostoc sp., was responsible for the apparent disease resistance. Regardless of the veracity of this h yp othesis we began looking for AMP produced by the various species of Leuconostoc typically present in Kimchi. Our research ultimately focused on Leuconostoc kimchii. A BLAST (Basic Local Alignment Search Tool) search· of the genome revealed sequences for several potential antimicrobial peptides. We were able to isolate the peptides from culture and identify the corresponding genomic sequence. The genes were isolated and inserted into a tagged expression vector in E. coli. Once the expression of the recombinant peptide was confirmed the expressed peptides where fractionated and purified. The resulting peptides where screened for their antimicrobial efficacy. One of the candidate peptides PF l 056 showed excellent stability and strong antimicrobial efficacy making it an ideal candidate for cosmetic application. PFI 056 was tested for kill rate, MIC and its ability to preserve cosmetic systems. PF l 056 is a cationic peptide made up of 29 amino acids, with a molecular weight of approximately 3,000. The MIC studies where conducted as follows. Inoculate 5 ml LB in tubes with test strains from LB plates and incubate overnight at 37°C on a shaker ( l 80 rpm). Make serial dilutions of test samples at 10 times the required test concentrations. Dissolve test samples in distilled H 2 0 at 20 times the required maximal concentration 640,320, 160 ... 2.5 µg/ml. Dilute overnight bacterial cultures in LB to give 1 x 106 cfu/ml (Yeast l x l 05 cfu/ml). Dispense 100 µI of bacterial suspension in each well from column l to column 11. Do not add bacteria to column 12, and instead dispense 100 µI of LB (sterility control and blank for the plate scanner). Add 100 µl of PF1056 (lOx) to each well from column l to column 10 (column 11 isa control for bacteria alone, with no sample). Incubate the plates at 37°C for 18-24 hours. Plates are checked again at 40- 48 hours. When satisfactory growth is obtained (18-36 hours) scan the plates with an ELISA reader (or read by eye). MIC can be taken as the lowest concentration of drug that reduces, by more than 50% or 90% for MIC SI or MIC 90 respectively. [Figure 1.] Kill rates were performed using a standard agar dilution method. [Flgure2.] To evaluate temperature stability a l % solution of PFI 056 was held at either 3 7°C or 100°C for a period between 20 and 120 minutes. The heat treated solution was then used to conduct a zone of inhibition study. The study confirmed that PF1056 is unaffected by heat treatment within the range tested. [Figure 3.] A fresh l % solution of PFI056 was prepared and divided in to aliquots. The aliquots were adjusted to pH ranging from 3 - l O using either citric acid, or sodium hydroxide. [Figure 4.] Again zone of inhibition studies were conducted to confirm activity. Activity was seen at a pH range of3 - 5 and again at a pH of 8 indicating that the material would be suitable in most skin care and some surfactant systems.

196 JO URN AL OF COSMETIC SCIENCE A simple lotion formula was preserved with I% PFI 056 and challenge tested using S. aureus, B. subtilis, E.coli, P aeriginosa, C. albicans, and A. niger [Figure 5.]. Plates were counted at 24 hours, 48 hours, and 21 days. [Figure 6.] The results clearly demonstrate the potential PF1056 as a viable part of a cosmetic preservative system. 100 Figure 1 50 25 12.5 6.25 3.13 1 56 0 76 0.39 mwmt •-Bacill;.. - -Escherichia - S�lmon ua I 1 -Shigella -Vibrio -Gandida . -- - --- Figure3 Figure 5 Test Formulation Water 91.2 % Yeast Extract 2.8 % Propylene Glycol 5.0% PF1056 1.0 % 100% BO"k 60% 40% 20% Organism Gram+ Gram- Yeast Mold Figure2 6 9 12 15 18 21 24 Figure4 Figure6 Challenge Test 24Hour 48Hour 21 Day 10 IO 10 10 10 10 10 10 10 8 X 10 1 IO IO