a Laboratoires de Technologie Pharmaceutique and b Laboratoire de Microbiologie Médicale, Institut de Pharmacie, Université de Liège, Belgium
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Abstract |
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Introduction |
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Unfortunately, miconazole is practically insoluble in water (<1.03 mg/L) and was consequently formulated with a non-ionic surfactant, polyoxyl 35 castor oil (Cremophor EL), for parenteral administration. Polyoxyl 35 castor oil is associated with several side effects, most notably anaphylactoid reactions.36 The use of this solution was limited because of the toxic effects related to the presence of this surfactant.2 The Daktarin IV solution was withdrawn from the Belgian market in 1997.
Fungal infections caused by common, new and emerging yeasts and moulds (filamentous fungi) have recently increased, partially as a result of the growing number of immunocompromised patients. Infections due to filamentous fungi are less common than those due to yeasts, but new filamentous species have emerged in this immunosuppressed population.7,8 Unfortunately, the number of parenteral solutions available to treat fungal invasive infections is limited. The increasing number of systemic fungal infections and the limited number of injectable solutions are all arguments in favour of a new parenteral solution containing miconazole without polyoxyl 35 castor oil.
We have shown previously that a combination of lactic acid and hydroxypropyl-ß-cyclodextrin (HP-ßCD) has a synergic effect on the aqueous solubility of miconazole and allows solubilization of >10 mg of miconazole per millilitre, which is the concentration of the formerly marketed solution.9,10 Among the different cyclodextrins (CDs) available, HP-ßCD was chosen owing to its high solubilizing power and its low systemic toxicity in comparison with ßCD.1113
It has also been shown that in vivo, this CD does not interfere with the release of miconazole. The HP-ßCD intravenous solution and the surfactant solution are bioequivalent in sheep.14
HP-ßCD in combination with lactic acid was proposed as an alternative to the use of surfactants for the parenteral administration of miconazole.
The aim of this work was to compare the in vitro antimycotic activity of the surfactant solution (Daktarin IV) with that of the proposed new formulation containing HP-ßCD and lactic acid.
In this report, we compared the two solutions with dilutions of miconazole pure substance by determining their MIC for 67 yeasts and 50 filamentous fungal strains.
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Materials and methods |
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Miconazole, HP-ßCD and lactic acid were obtained from Janssen Pharmaceutica (Beerse, Belgium), Janssen Biotech (Olen, Belgium) and Merck Belgolabo (Overijse, Belgium), respectively. All other products were of analytical grade.
The Daktarin IV solution was obtained from Janssen Pharmaceutica just after its withdrawal from the Belgian public market.
Preparation of the miconazoleHP-ßCD solution
The miconazoleHP-ßCD solution was prepared by dissolving miconazole (10 mg/mL) in a solution of HP-ßCD (100 mM), lactic acid (50 mM) and NaCl (4.48 mg/mL). The solution was filtered through a 0.22 µm filter.
Test organisms
One hundred and six clinical isolates (67 yeasts and 39 filamentous fungi) corresponding to 106 different patients were used in this study. The clinical strains studied are given in the Table. All strains were tested in duplicate. Variations equal to or less than one dilution were observed between duplicates. This variation was not statistically significant.
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Susceptibility testing method
We used many of the test conditions of the NCCLS microdilution method for yeasts15 and of the NCCLS microdilution method for filamentous fungi.16 These NCCLS methods were devised for the testing of pure antifungal substances against yeasts and moulds but we considered the conditions described for these tests were suitable for our comparison of miconazole formulations in vitro; they do not deal with the testing of miconazole. We adapted these methods to miconazole as proposed by Makimura et al.17 for yeasts. Endpoints were determined spectrophotometrically instead of visually as proposed by the NCCLS. However, a visual control was also introduced for the filamentous fungi.
A stock solution of miconazole was prepared in DMSO then diluted as recommended by the NCCLS guidelines for antifungal agents of limited solubility. The Daktarin IV solution and the HP-ßCD solution were diluted eight-fold with 0.9% NaCl in purified water. These solutions were then diluted in test medium to twice the final concentration, which was a series from 0.025 to 12.5 mg/L. The final concentration of DMSO did not exceed 1% and did not affect the growth of fungi.17 From here on, the dilutions prepared from the DMSO stock solution will be referred to as miconazole DMSO solutions.
RPMI 1640 medium (Gibco-BRL, Paisley, UK) with l-glutamine and without sodium bicarbonate, and buffered to pH 7.0 with 0.165 M morpholinepropanesulphonic acid (MOPS) was used for both yeasts and filamentous fungi as recommended by NCCLS guidelines.
Volumes of 100 µL of the double-concentration drug substance were inoculated into the wells of sterile flat bottomed 96-well microplates, with the first well of each row serving as sterility control and the last one serving as growth control.
As recommended by the NCCLS, the yeast inoculum suspensions were prepared from 24 h old cultures on Sabouraud dextrose agar at 35°C. The cell densities of the resulting suspensions were adjusted with a spectrophotometer by adding sufficient sterile saline to increase the transmittance to that produced by a 0.5 McFarland standard at a wavelength of 530 nm. These working suspensions were diluted 1:50 then 1:20 with RPMI 1640 broth medium, which results in 0.5 x 103 to 2.5 x 103 cells/mL.
The mould inoculum suspensions were prepared from 7-day-old cultures on potato dextrose agar at 35°C. The suspensions diluted 1:50 (or 1:25 for Scedosporium apiospermum isolates) in the standard medium correspond to twice the density needed of approximately 0.4 x 104 to 5.0 x 104 cfu/mL.
One hundred microlitres of the suspensions were added to each well, except for row 1 (sterility control), resulting in the desired final drug concentration (0.02512.5 mg/L) and inoculum size.
As recommended by the NCCLS, the microplates were incubated at 35°C (or 28°C for the dermatophytes) in a humid box and read after 48 h for most yeasts and moulds except Rhizopus spp. (24 h), and C. neoformans and S. apiospermum (72 h).
The endpoint was determined spectrophotometrically when the positive control gave a turbidity 0.2 at 630 nm. The endpoint was recorded as a reduction of growth below 20% of the positive control (80% inhibitory concentration: IC80).
Statistical analysis
The MICs of the three solutions were compared using analysis of variance (ANOVA). Results were considered to be significant at the 5% critical level.
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Results |
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Discussion |
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The difference observed between the miconazole DMSO solution and the other two concerning the activity against A. fumigatus is difficult to explain. However, this difference is not relevant for clinical interpretation if we refer to the proposed breakpoints for miconazole (8 mg/L = susceptible,
16 mg/L = resistant).21 Concerning the activity against R. oryzae, only one strain was tested so no conclusion can be drawn.
As shown by the P value, the CD solution has the same antimycotic activity as the surfactant solution. Compared with the surfactant and the miconazole DMSO solution, HP-ßCD does not interfere with the activity of miconazole. This can be explained by the low affinity constant of the miconazoleHP-ßCD complex in an acidic medium.14 Because of the dilution of the solution, miconazole is released from the CD cavity and is available in the free, active form. Thus, no significant difference was found between the solutions.
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Notes |
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References |
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2 . Kauffman, A. & Carver, P. (1997). Use of azoles for systemic antifungal therapy. Advances in Pharmacology 39, 14789.
3 . Brewster, M. E., Estes, K. S. & Bodor, N. (1989). Development of a non-surfactant formulation for alfaxalone through the use of chemically modified cyclodextrins. Journal of Parenteral Science and Technology 43, 2625.[ISI][Medline]
4 . Hopkins, C. S. (1988). Adverse reaction to a cremophor containing preparation of intravenous vitamin. Intensive Therapeutic Clinical Monitoring 9, 2545.
5 . Howrie, D. L., Ptachcinski, R. J., Griffith, B. P., Hadersty, R. J. & Venkataramanan, R. (1984). Anaphylactoid reactions associated with parenteral cyclosporine use: possible role of cremophor EL. Drug Intelligence and Clinical Pharmacy 19, 4257.
6 . Reynolds, D. J. & Aronson, J. K. (1992). Selected side effects Part 8. Anaphylactoid reaction to intravenous vitamin K. Prescribers' Journal 32, 16770.
7 . Cormican, M. G. & Pfaller, M. A. (1996). Standardization of antifungal susceptibility testing. Journal of Antimicrobial Chemotherapy 38, 56178.[Abstract]
8 . Pfaller, M. A., Marco, F., Messer, S. A. & Jones, R. N. (1998). In vitro activity of two Echinocandin derivatives, LY3033366 and MK-0991 (L-743,792), against clinical isolates of Aspergillus, Fusarium, Rhizopus, and other filamentous fungi. Diagnostic Microbiology and Infectious Disease 30, 2515.[ISI][Medline]
9 . Piel, G., Evrard, B. & Delattre, L. (1997). Complexes à multicomposants de miconazole avec différents acides et cyclodextrines. Journal de Pharmacie de Belgique 52, 124.
10 . Piel, G., Evrard, B., Fillet, M., Llabres, G. & Delattre, L. (1998). Development of a non-surfactant parenteral formulation of miconazole by the use of cyclodextrins. International Journal of Pharmaceutics 169, 1522.[ISI]
11 . Brewster, M. E., Estes, K. S. & Bodor, N. (1990). An intravenous toxicity study of hydroxypropyl-ß-cyclodextrin, a useful drug solubilizer, in rats and in monkeys. International Journal of Pharmaceutics 59, 23143.[ISI]
12 . Fromming, K. H. & Szejtli, J. (1994). Cyclodextrins in Pharmacy, (Davies, J. E. O., Ed.). Kluwer Academic Publishers, Dordrecht.
13 . Yoshida, A. (1988). Pharmaceutical evaluation of hydroxyalkylethers of ß-cyclodextrin. International Journal of Pharmaceutics 46, 21722.[ISI]
14 . Piel, G., Evrard, B., Van Hees, T. & Delattre, L. (1999). Comparison of the IV pharmacokinetics in sheep of miconazoleHP-ßCD and SBE7-ßCD solutions and a micellar solution. International Journal of Pharmaceutics 180, 415.[ISI][Medline]
15 . National Committee for Clinical Laboratory Standards. (1997). Reference Method for Broth Dilution Antifungal Susceptibility Testing for Yeasts: Approved Standard M27-A. NCCLS, Wayne, PA.
16 . National Committee for Clinical Laboratory Standards. (1998). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Conidium-Forming Filamentous Fungi: Proposed Standard M38-P. NCCLS, Wayne, PA.
17 . Makimura, K., Sudo, T., Kudo, M., Uchida, K. & Yamaguchi, H. (1998). Development of reference procedures for broth microdilution antifungal susceptibility testing of yeasts with standardized endpoint determination. Microbiological Immunology 42, 559.[ISI][Medline]
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Davey, K. G., Holmes, A. D., Johnson, E. M., Szekely, A. & Warnock, D. W. (1998). Comparative evaluation of FUNGITEST and broth microdilution methods for antifungal drug susceptibility testing of Candida species and Cryptococcus neoformans. Journal of Clinical Microbiology 36, 92630.
19 . Espinel-Ingroff, A., Dawson, K., Pfaller, M., Anaissies, E., Breslin, B., Dixon, D. et al. (1995). Comparative and collaborative evaluation of standardization of antifungal susceptibility testing for filamentous fungi. Antimicrobial Agents and Chemotherapy 39, 3149.[Abstract]
20 . Manavathu, E. K., Alangaden, G. J. & Terner, S. A. (1996). A comparative study of the broth micro- and macro-dilution techniques for the determination of the in vitro susceptibility of Aspergillus fumigatus. Canadian Journal of Microbiology 42, 9604.[ISI][Medline]
21 . Sutton, D. A., Fothergill, A. W. & Rinaldi, M. G. (1998). Clinically Significant Fungi. Williams and Wilkins, Baltimore, MD.
Received 23 November 2000; returned 7 February 2001; revised 16 March 2001; accepted 19 April 2001