1. Abstract Occidiofungin is a glycolipopeptide isolated from the liquid culture of the soil bacterium Burkholderia contaminans MS14. Occidiofungin is rapidly fungicidal at submicromolar concentrations and has distinct bioactivities from currently used therapeutic antifungals. Previous studies on occidiofungin led to the characterization of the covalent structure, spectrum of activity, and toxicity in mice. In current work, we embark upon a series of in vitro and in vivo studies to determine whether it possesses pharmacological properties that would make it a potential drug candidate for the treatment of systemic fungal infections. We report our progress toward the development of an extraction method for occidiofungin from plasma, and toward the development of an LCMS method for quantifying the level of occidiofungin in mouse plasma following intravenous administration. Background Occidiofungin is a 1200 Da, non-ribosomally synthesized glycolipopetide that has a wide spectrum of antifungal activity. The covalent structure of occidiofungin is represented in Figure 1. Occidiofungin was found to be active against several strains of clinically pathogenic fungi. The strains of fungi and the Minimum Inhibitory Concentration (MIC) of occidiofungin are listed in Table 1. Figure 1: Covalent structure of occidiofungin; R1= H or OH, R2= H or Cl (2) Table 1: MICs of occidiofungin against different strains of Candida(1) Further, time kill assays performed using occidiofungin on Candida albicans indicated that occidiofungin was rapidly fungicidal (Figure 2a). Postantifungal effect (PAFE) assay suggested that occidiofungin either has an intracellular target or binds tightly to the fungal cell, because the antifungal activity was observed even after three washes following treatment (Figure 2b). Figure 2: Kill curves for time-kill (A) and postantifungal effect (B) experiments on Candida albicans ATCC 66027. Symbols are as follows: □, 8× MIC (16 μg/ml); ●, 2× MIC (4 μg/ml); ▴, 1× MIC (2 μg/ml), 0.5x MIC (1ug/ml) and control (0ug/ml) Development of a quantitative LC-MS/MS method for the novel antifungal compound Occidiofungin using TraceFinder 3.1 Wanjin Tang 1 ; Jamie K. Humphries 2 ; Lawrence J. Dangott 3 ; Akshaya Ravichandran 1 ;Leif Smith 1 1 Dept of Biology, Texas A&M University, College Station, TX; 2 ThermoFisher Scientific, Austin, TX; 3 Protein Chemistry Laboratory, Texas A&M University, College Station, TX Methods Occidiofungin, at a concentration of 2.5mg/kg of the body weight, was injected intravenously into the mice. 1% β-cyclodextrin was used as the vehicle. Blood was drawn from the mice from the lateral saphenous vein and the tail vein at 1, 3, 5, 7, 9 and 12 hours post injection (hpi) and spun at 13000rpm for ten minutes to obtain plasma, which was stored at -20°C until use. The samples were analyzed using the conditions described in Figure 3. Figure 3: The LC and the MS components of the methods used for detection of occidiofungin. Protocol Development Extraction of occidiofungin from plasma was attempted using 1:2.5 of plasma to 100% acetonitrile concentration and 1:1 ratio of plasma to 100% methanol concentration. Azithromycin was used as the internal standard. TraceFinder 3.1 enables the user to compare multiple extraction methods while optimizing the protocol for sample preparation. The panel seen in Figure 4 provides a clear comparison of different methods at the click of a single button. Figure 4: Using Comparative View with no group settings allows for the display of data from multiple extraction techniques to be compared side by side with a fixed Y-axis. The power of comparison helps the user to choose the best technique. Extraction results can be filtered in such a way that the internal standard (Azithromycin) and the compound of interest (occidiofungin) can be compared separately between sample preparation methods. 2ug/ml of occidiofungin in plasma was extracted using several solvents. The efficiency of extraction can be compared as in Figure 5a and 5b. Based on the comparison made between the peak characteristics of the internal standard, the 1:1 methanol extraction was utilized and standard curves were made. The limit of detection (LOD) for occidiofungin using the optimized extraction method is 10ng/ml. This can be seen in Figure 5c. a) c) b) c) Figure 5: Optimization of solvent extraction methods a) Comparison of internal standard peaks using different solvent extraction methods; b) Comparison of occidiofungin peaks (the selected method is highlighted in red); c) Detection of internal standard and occidiofungin at 10ng/ml using 1:1 methanol extraction procedure Results A calibration curve was set up by analyzing a series of known concentrations of occidiofungin in mouse plasma, using the 1:1 methanol extraction method. The concentrations of occidiofungin used were 50ng/ml, 100ng/ml, 200ng/ml, 400ng/ml, 800ng/ml and 1000ng/ml. The curve obtained had an R value of 0.9963, as seen in Figure 6. Figure 6: Calibration curve of occidiofungin Plasma samples obtained at different time-points and extracted using the 1:1 methanol method were analyzed. Plasma was obtained from six mice and pooled into two groups of three mice each. Concentration of occidiofungin increases from the 1 hpi time-point up to the 5 hpi time-point. There is then a decrease in the concentration until 12 hpi. This is represented by Figure 7. Figure 7: Calculated concentrations of OF in plasma samples obtained over a time period. The trendline indicates the average concentration between the two groups Conclusion A working method for the extraction and detection of occidiofungin in plasma has been developed. Further routes of administration of drug, such as pulmonary, oral and intramuscular, can now be performed and analysis will be done using the method developed. The results obtained in this study indicate that occidiofungin peak plasma concentration occurs at 5 hpi. It is interesting to note that even at 12 hpi, the concentration of occidiofungin is similar to 1 hpi. This suggests that occidiofungin has a slow clearance rate from blood. Further studies in which we collect plasma at later time points will provide a better understanding of the clearance rate for occidiofungin. The concentration of occidiofungin in plasma at 12 hpi is higher than the Minimum Inhibitory Concentration for several strains listed in Table 1. This plasma concentrations for this duration suggests that occidiofungin may be effective for the treatment of systemic fungal infections. Efficacy studies will be performed to determine whether occidiofungin can treat a systemic fungal infection in mice. TraceFinder 3.1, through the use of comparative data review capabilities and multiple chromatographic display options, allowed a complex multiple channeled HPLC method to be developed, reviewed and corrections made in rapid succession. By utilizing the abilities of data filtering and flagging, results were obtained in only minutes versus hours within the application. The user never had to pass the data to Excel or external application to review data to make corrections to the experimental procedure. Acknowledgements This work was funded by Cancer Prevention and Research Institute of Texas (CPRIT RP121002) References 1. Dayna Ellis, Jiten Gosai, Charles Emrick, Rachel Heintz, Lanette Romans, Donna Gordon, Shi-En Lu, Frank Austin and Leif Smith. Occidiofungin's Chemical Stability and In Vitro Potency against Candida Species. Antimicrob. Agents Chemother. February 2012 vol. 56 no. 2 765-769. 2. Ravichandran A, Gu G, Escano J, Lu SE, Smith L. The presence of two cyclase thioesterases expands the conformational freedom of the cyclic Peptide occidiofungin. J Nat Prod. 2013 Feb 22;76(2):150-6. 80% Methanol70% Methanol 50% Methanol 70% MeOH; 2N HCl70% ACN; 0.1% AcOH70% ACN; 2N HCl 80% Methanol70% Methanol50% Methanol 70% MeOH; 2N HCl70% ACN; 0.1% AcOH 70% ACN; 2N HCl 70% MeOH; 0.1% AcOH R value = 0.9963