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Microsoft word - eurpotec fulhold report in vitro efficacy of chd-fa against candida july 2008.doc

AGENT CHD-FA (CarboHydrate Derived - Fulvic Acid) IN FLUCONAZOLE RESISTANT CANDIDA ISOLATES Fulhold Ltd.
PHYSICAL PROPERTIES OF CHD-FA ALSO KNOWN AS FULVIC ACID Euprotec received 1 bottle of CHD-FA (henceforth called CHD-FA) reconstituted to a 4% solution. The solution has been stored at room temperature in the dark since delivery. The 4% CHD-FA solution is a yellow/brown slightly viscous solution with a strong METHODS - Preliminary Experiment
Susceptibility tests were performed on 5 isolates of Candida albicans all clinical isolates (all with reduced susceptibility to fluconazole). [Each culture was grown on Sabouraud agar at 37°C for 48 h to ensure purity.] RPMI-1640 (Sigma, Dorset, UK) supplemented to 2% glucose (Sigma), buffered with morpholinopropanesulfonic acid (MOPS), (Sigma) and adjusted to pH 7.0 was used as recommended in the European Committee on Antimicrobial Susceptibility Testing (E.Dis. 7.1) (Rodriguez-Tudela, J. L., F. Barchiesi, J. Bille, E. Chryssanthou, M. Cuenca- Estrella, D. Denning, J. P. Donnelly, B. Dupont, W. Fegeler, C. Moore, M. Richardson, P. E. Verweij, and the Subcommittee on Antifungal Susceptibility Testing (AFST) of the ESCMID European Committee for Antimicrobial Susceptibility Testing (EUCAST). 2003. Method for the determination of minimum inhibitory concentration (MIC) by broth dilution of fermentative yeasts. Clinical Microbiology and Infection 9:I–VIII.) EUPROTEC Ltd.
CONFIDENTIAL
Euprotec Fulhold Report In vitro efficacy of CHD-FA against Candida July 2008 All yeasts were cultured in ambient air at 35–37 °C on recovery medium (Sabouraud dextrose agar) for 18–24 h before testing. The inoculum was prepared by picking five distinct colonies from 18 to 24 h cultures and suspending them in 5 mL of sterile distilled water. The inoculum was completely suspended by vigorous shaking on a vortex mixer for 15s. The cell density was adjusted to the density of a 0.5 McFarland standard by adding sterile distilled water and measuring absorbance in a spectrophotometer at a wavelength of 530 nm. This gave a yeast suspension of 1–5 × 106cfu/mL. A working suspension was prepared by a further dilution of 1 in 10 further in 4 X RPMI to give 1–5 × 105cfu/mL (The RPMI medium used in the plates was prepared at 4 x the final strength to allow for a 75% dilution once the inoculum and diluted CHD-FA was added). Sterile plastic, disposable, microtitration plates with 96 flat-bottom wells were used. Column 1 of the microtitration tray was filled with 100µL of sterile water containing four times the final drug concentration (512 mg/L fluconazole). Columns 2–12 were filled with 50µL of distilled water 50µL amounts were taken from wells in column 1 and diluted two-fold by transferring them to column 2 with a multichannel pipette (± 2% coefficient of variation). 50 µL samples were then removed from column 2 and transferred to EUPROTEC Ltd.
CONFIDENTIAL
Euprotec Fulhold Report In vitro efficacy of CHD-FA against Candida July 2008 column 3, and so on through to column 10. The last 50 µL of diluted drug is then Thus, each well in columns 1–10 will contain 50µL of water containing four times the Stock solutions of CHD-FA were produced containing 4%, 2%, 1%, 0.5% and 0.25% of 100µL of the diluted CHD-FA was added to microtitration trays in so that row A contains a final dilution of 2% row B, 1%, row C 0.5%, row D 0.25% row E 0.125% and row F STEP 3 Addition of Candida albicans 50µL of the diluted Candida albicans suspension in 4 x RPMI was added to all wells. This produces a well containing 200 µL final volume (made up of 50µL diluted fluconazole, 100µL diluted CHD-FA or diluent, 50µL of 4 X RPMI containing Candida All plates were incubated at 37oC in air in a darkened incubator. Plates were read visually with the endpoint taken as the lowest concentration of drug that inhibited growth by 50% of that of the drug free control. EUPROTEC Ltd.
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Euprotec Fulhold Report In vitro efficacy of CHD-FA against Candida July 2008 2.2.1 RESULTS – Preliminary Experiment
Initial tests demonstrated that CHD-FA at 4%, 2% and 1% inhibited the growth of Candida albicans for at least 24 hours when combined with 1 X RPMI 1640 culture medium, growth in 0.5% CHD-FA occurs at similar levels to solvent controls. The inhibition of growth (4%, 2% and 1%) was possibly due to the strongly acidic pH. At the native pH of CHD-FA (pH 2.1) no synergy or antagonism with fluconazole could be The pH of CHD-FA was adjusted to pH7.0 using 10M NaOH solution (the ideal pH for fluconazole activity). This resulted in a brown slightly viscous solution with a strong characteristic odour. Susceptibility assays were repeated as above: As previously noted with native CHD-FA, 4%, 2% and 1% solutions inhibited the growth of Candida albicans for at least 24 hours when combined with 1 X RPMI 1640 culture medium, growth in 0.5% CHD-FA occurs at similar levels to solvent controls. No synergy or antagonism with fluconazole could be detected. The MICs of the C. albicans strains with and without CHD-FA are listed in Table 1 (the Table 1: Minimum Inhibitory Concentrations of Fluconazole (mg/L) combined with EUPROTEC Ltd.
CONFIDENTIAL
Euprotec Fulhold Report In vitro efficacy of CHD-FA against Candida July 2008 METHODS - Main Experiment
Susceptibility tests were performed on 40 isolates of Candida all clinical isolates (38 with reduced susceptibility to fluconazole). The group comprised 25 C. albicans, 11 C. glabrata (all with reduced susceptibility to fluconazole), and 4 C. tropicalis (all with reduced susceptibility to fluconazole). [Each culture was grown on Sabouraud agar at Sterile plastic, disposable, microtitration plates with 96 flat-bottom wells were used. Column 1 of the microtitration tray was filled with 100µL of sterile water containing four times the final drug concentration (512 mg/L fluconazole). Columns 2–12 were filled with 50µL of distilled water 50µL amounts were taken from wells in column 1 and diluted two-fold by transferring them to column 2 with a multichannel pipette (± 2% coefficient of variation). 50 µL samples were then removed from column 2 and transferred to column 3, and so on through to column 10. The last 50 µL of diluted drug is then EUPROTEC Ltd.
CONFIDENTIAL
Euprotec Fulhold Report In vitro efficacy of CHD-FA against Candida July 2008 Thus, each well in columns 1–10 will contain 50µL of water containing four times the Stock solutions of CHD-FA were produced containing 4% and 2% (also 1% and 0.5% for Candida parapsilosis and Candida krusei). 100µL of the diluted CHD-FA was added to microtitration trays in so that rows contain For Candida albicans and tropicalis: 1st row of pair 0.5% CHD-FA, 2nd row of pair solvent only 1st row of triplet 1% CHD-FA, 2nd row of triplet 0.5% CHD-FA, 3rd row of triplet solvent For Candida parapsilosis and Candida krusei rows included 2%, 1%, 0.5%, 0.25 and 0.125% CHD-FA and a row of solvent only. All the above concentrations were mixed with fixed concentrations of fluconazole at 128, 32, 8, 2, 0.5mg/L and solvent for 50µL of the diluted Candida sp suspension in 4 x RPMI was added to all wells. This produces a well containing 200 µL final volume (made up of 50µL diluted fluconazole, 100µL diluted CHD-FA or diluent, 50µL of 4 X RPMI containing Candida) All plates were incubated at 37oC in air in a darkened incubator. EUPROTEC Ltd.
CONFIDENTIAL
Euprotec Fulhold Report In vitro efficacy of CHD-FA against Candida July 2008 Plates were read visually with the endpoint taken as the lowest concentration of drug that inhibited growth by 50% of that of the drug free control. 4.1 RESULTS – Main Experiment
No regrowth of Candida occurred on prolonged incubation (96 hours) at 37oC or room Table 2 Efficacy of CHD-FA (0.5%) in combination with fluconazole against Candida albicans EUPROTEC Ltd.
CONFIDENTIAL
Euprotec Fulhold Report In vitro efficacy of CHD-FA against Candida July 2008 Table 3 Efficacy of CHD-FA (0.5%) in combination with fluconazole against Candida glabrata Table 4 Efficacy of CHD-FA (1%) in combination with fluconazole against Candida glabrata EUPROTEC Ltd.
CONFIDENTIAL
Euprotec Fulhold Report In vitro efficacy of CHD-FA against Candida July 2008 Table 5 Efficacy of CHD-FA (0.5%) in combination with Table 6 Efficacy of CHD-FA (0.5%) in combination with fluconazole against Candida parapsilosis Table 7 Efficacy of CHD-FA (0.5%) in combination with fluconazole against Candida krusei EUPROTEC Ltd.
CONFIDENTIAL
Euprotec Fulhold Report In vitro efficacy of CHD-FA against Candida July 2008 • CHD-FA is equally effective as an antifungal agent in vitro whether examined at it’s • CHD-FA inhibits the growth of Candida albicans, Candida tropicalis, Candida parapsilosis and Candida krusei when used as a 0.5% in vitro • CHD-FA inhibits the growth of Candida glabrata when used as a 1% solution • CHD-FA does not demonstrate synergistic of antagonistic activity when used in • The combination of 1% CHD-FA with fluconazole (0.25-128mg/l) inhibits the growth of • CHD-FA is highly effective against strains of C. krusei that are intrinsically resistant to EUPROTEC Ltd.
CONFIDENTIAL
Euprotec Fulhold Report In vitro efficacy of CHD-FA against Candida July 2008

Source: http://fulhold.com/pics/In_vitro_Candida_data.pdf

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