1. INTRODUCTION Chemical and metabolic stability of lactoferricin-based cationic antimicrobial tripeptides Valentijn Vergote 1, Johan Svenson 2, Christian Burvenich 1, Rasmus Karstad 2 and Bart De Spiegeleer 1, * 1 Drug Quality & Registration (DruQuaR) group, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium. 2 Department of Chemistry, Faculty of Science, University of Tromsø, N-9037 Tromsø, Norway. *Corresponding author: bart.despiegeleer@ugent.be (O.Ref.: 2009–250b)bart.despiegeleer@ugent.be DruQuaR Drug Quality & Registration (DruQuaR) format Man06.001.006-v01 The widespread overuse and misuse of antibiotics throughout the world has led to bacterial resistance to many commercially available antibiotics. Hence, the development of novel classes of antibiotics is required to address this pressing public health problem. Cationic antimicrobial peptides (CAPs) are considered to be a promising new class of drugs against multi-resistant bacteria [1]. While their minimum antimicrobial motif has been defined [2], the limited success of CAPs so far is partially due to unresolved chemical and metabolic stability issues. In order to obtain comprehensive information regarding the metabolic fate, these peptides and derivatives were tested in vitro by incubation in buffer/denatured plasma, plasma and organ homogenates. A general screening procedure was used [3], where aliquots are withdrawn at pre-determined time points and analyzed by HPLC-UV/PDA- ESI/MS for determination of CAP degradation kinetics and identification of the degradation metabolites. Based on this information, QSPR (Quantitative Structure-Property Relationships) models can be built to predict the stability as a function of the peptide sequence (i.e. which peptide bonds are most unstable). Such a model can then be used to suggest directions for new CAPs with improved pharmaceutical drugability characteristics. EXPERIMENTAL The general structure of the four CAPs tested is given in Figure 1. The CAPs are derived from lactoferricin, with the hydrophobic bulk provided by the central amino acid R 1 and the positive charges via the guanidine groups and the N-terminus. In vitro metabolic stability testing CAPs are incubated at 37°C in a standardized dilution of a biological matrix (e.g. mouse plasma) with Krebs-Henseleit buffer pH 7.4. Aliquots are withdrawn at pre-determined time points, and analyzed (after acidification, heating to 95°C for 5 min, and centrifugation) using HPLC with PDA detection. Results are expressed as half-life times, calculated from the curves obtained. HPLC analysis The tripeptide CAPs and their metabolites were separated using a single HPLC method. An Alltima C 18 HP AQ (250 mm × 4.6 mm, 100 Å, 5 µm) column (Grace) was applied at 30°C, with (A) 0.1% TFA in water, and (B) 0.1% TFA in acetonitrile. The gradient used for the separation was: 98% A + 2% B from 0 to 5 minutes, followed a linear ramp from 5 to 60 min going to 50% A + 50% B. The flow rate was set at 1.0 ml/min. RESULTS AND DISCUSSION CONCLUSIONS The procedure was found to be suitable for its intended purpose, with metabolite identification (e.g. Trp; also present in blank mouse plasma) based upon retention time. C-terminal protection with a benzylamide group results in improved in vitro mouse plasma metabolic stability of the CAP tested. Arg-Bip-Arg-NH 2 was found to be the least stable CAP when incubated with mouse plasma. REFERENCES [1]Svenson J, Stensen W, Brandsdal BO, Haug BE, Monrad J, Svendsen JS. Antimicrobial peptides with stability toward tryptic degradation. Biochemistry 47 (2008) 3777- 3788. [2]Strom MB, Haug BE, Skar ML, Stensen W, Stiberg T, Svendsen JS. The pharmacophore of short cationic antibacterial peptides. J Med Chem 46 (2003) 1567-1570. [3]Vergote V, Van Dorpe S, Peremans K, Burvenich C, De Spiegeleer B. In vitro metabolic stability of obestatin: Kinetics and identification of cleavage products. Peptides 29 (2008) 1740-1748. ACKNOWLEDGEMENTS: We thank Nadia Lemeire for experimental help. Figure 1. General CAP structure (R 1 = Trp or Bip; R 2 = NH 2 or NHBn) Typical chromatograms are shown in Figure 2. The degradation of the 4 CAPs in mouse plasma is shown in Figure 3, with calculated half-life values in Table 1. The performance and validity of the procedure applied was demonstrated by blanks, two types of control solutions (i.e. peptide recoveries without plasma being present, and peptide recoveries in presence of acid/heat-inactivated plasma) and the mass balances obtained for each time point (see Table 2). Figure 2. Arg-Trp-Arg-NHBn in mouse plasma: Overlay of chromatograms (UV at 280 nm) [RT native peptide = 37.2 min; RT Trp = 26.3 min] Table 1. Half-life values of CAPs in mouse plasma CAPHalf-life (min) [95% C.I.] Arg-Trp-Arg-NH 2 50.0 [48.9,51.2] Arg-Trp-Arg-NHBn72.5 [69.4,75.8] Arg-Bip-Arg-NH 2 29.5 [28.0,31.1] Arg-Bip-Arg-NHBn97.7 [90.9,105.6] Figure 3. In vitro metabolic stability of CAPs in mouse plasma Table 2. Typical mass balance results (Arg-Trp-Arg-NH 2 ) T#Peak area sum (% vs. Native peptide peak area at T0) 1598 (incl. 3 metabolites: 11.0%, 3.7% & 5.0%) 30102 (incl. 3 metabolites: 13.4%, 4.7% & 17.0%) 45101 (incl. 3 metabolites: 13.0%, 4.9% & 29.8%) 60100 (incl. 3 metabolites: 11.5%, 4.5% & 41.2%)