Bioassays for itraconazole blood levels: an interlaboratory collaborative study

F. C. Oddsa,*, B. Dupontb, M. G. Rinaldic, D. A. Stevensd, D. W. Warnocke and R. Woestenborghsa

a Janssen Research Foundation, B2340 Beerse, Belgium; b Hôpital de l' Institut Pasteur, 75724 Paris, France; c Fungus Testing Laboratory, The University of Texas Health Science Center, San Antonio, TX 78284-7750; d Division of Infectious Diseases, Santa Clara Valley Medical Center and Stanford University Medical School, San Jose, CA 95128-2699, USA; e Mycology Reference Laboratory, Public Health Laboratory Service, Bristol BS2 8EL, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Duplicate bioassays for itraconazole and hydroxy-itraconazole were run with 30 serum samples in five laboratories, each using a different method. Both itraconazole and hydroxy- itraconazole were used as standards. Despite quantitative variations, the results of the bioassays correlated sufficiently to indicate the relative level of antifungal activity in the test samples.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Blood levels of the broad-spectrum antifungal agent itraconazole and its active hydroxy metabolite can be measured by high-performance liquid chromatography (HPLC) 1,2,3,4,5 or by inhibition zone bioassay. 1,2,3,4,6,7 The correlation between bioassay measurements and the sum of itraconazole and hydroxy-itraconazole levels determined by HPLC does not show a one-to-one relationship. Average levels determined by bioassay can be 0.5- 7.3 times the levels of one or both active triazoles determined by HPLC, depending on the method and antifungal standard used. 1,2,3,8

No standardized method for itraconazole bioassays exists, and laboratories differ in the experimental conditions and indicator organisms they use. The considerable variation in reported ratios of bioassay:HPLC levels suggests that interlaboratory reproducibility in bioassay test results may be less than ideal. We therefore undertook a collaborative comparison of itraconazole plus hydroxy-itraconazole level determinations in bioassays done with a standard panel of serum samples.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the first phase of the study, 15 samples of pooled human serum were tested. Six of the pools came from patients who had received oral itraconazole monotherapy. Seven pools of serum from healthy volunteers were spiked with itraconazole and hydroxy-itraconazole to give samples with a concentration range (itraconazole + hydroxy-itraconazole) of 0.01- 10.05 mg/L. Two further pooled serum samples from patients with no detectable antifungal agent were included as negative controls. Each of the 15 samples was divided into two aliquots and the 30 lots of serum were randomly coded for blinded bioassay in the five participating laboratories (designated laboratories A- E in this paper). A single lot of itraconazole powder was supplied for preparation of bioassay standards. A sixth set of the sample panel was tested for itraconazole and hydroxy- itraconazole levels by HPLC as previously described. 5,8

The design of the second phase of the study matched that of the first, except that 15 new serum samples were all spiked with itraconazole, hydroxy-itraconazole, or both, and the total (itraconazole + hydroxy-itraconazole) concentrations were chosen to cover the range 0.38- 9.5 mg/L. Each spiked serum pool was again divided into two randomly coded aliquots. The bioassays were run as before except that results were generated with both itraconazole and hydroxy-itraconazole standard curves.

The bioassay methods used in four of the laboratories were as previously described 1,4,6,9 with, respectively, the following indicator organisms: Candida kefyr ATCC 46764, Candida albicans 3153A, C. kefyr isolate SA, and C. albicans NCPF 3281. In the fifth laboratory, the bioassay was performed as follows. A solution containing 3.0 g Bacto agar (Difco, Basingstoke, UK) in 200 mL Eagle' s minimal essential medium (EMEM cat. No. 14-100-49; Flow Laboratories, Irvine, UK) was autoclaved, cooled to approximately 60°C and poured into square plastic dishes, 23 cm x 23 cm (Nunc Biologicals, Roskilde, Denmark). To a solution containing 1.5 g Bacto agar in 30 mL EMEM, autoclaved and cooled to 56°C in a water bath, was added 3 mL of a suspension of C. albicans isolate RV4688 containing 4 x 10 7 cells/mL. This mixture was poured as a seed layer on top of the EMEM agar. The test serum samples and standard solutions in pooled human serum were pipetted in 50 µL volumes on to sterile 1.3 cm filter paper discs which were placed on the surface of the seed layer. The plate was left to stand at 4°C for 24 h then incubated at 30°C for 24 h and the inhibition zone diameters were measured.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
HPLC

The HPLC measurements (all from phase 1 of the study) were accurate to within 10% of the nominal itraconazole and hydroxy-itraconazole concentrations in the seven spiked serum samples. Concentrations as low as 0.005 mg/L were measurable by HPLC and the mean coefficient of variation for all 15 duplicate samples was 2.3%. The four serum samples containing no active compounds were correctly reported as ‘not detectable’ .

Bioassay— first round of tests

The lowest detection limits reported in the bioassays ranged from 0.156 to 0.63 mg/L, and 13 of the 30 samples were reported as below the level of detectability in all five laboratories (Table I). Only seven of the 13 positive samples contained sufficiently high concentrations of azoles for levels to be determined in both duplicates of the samples in all five laboratories. For two of these samples the result was reported as ‘>10 mg/L’ by one laboratory. The degree of correlation and interlaboratory agreement for the seven measurable samples was good. Mean coefficients of variation for duplicate assays ranged from 5.1% to 12.5%. The measurements, expressed as bioassay activity, indicated an antifungal concentration in the samples that was an average of 2.25 times higher than the total azole level as determined by HPLC.


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Table I. HPLC and bioassay results for 15 paired serum samples containing hydroxy-itraconazole and itraconazole
 
Bioassay— second round of tests

The concentrations of itraconazole and hydroxy-itraconazole spiked in the samples in the second phase were chosen to give on-scale bioassay results. Nevertheless, some laboratories still reported results for these samples that were above or below the range of bioassay measurements (Table II). The measured levels of antifungal activity by bioassay varied considerably with the relative content of itraconazole and hydroxy-itraconazole in the samples. With an itraconazole standard curve, laboratories A, B, D and E all obtained results close to the spiked concentrations for the samples containing only itraconazole (Table II), but when hydroxy-itraconazole was present, alone or combined with itraconazole, the bioassay result from these laboratories was several-fold higher than the total spiked azole concentration. In laboratory C, the ratio of bioassay level:spiked level decreased with increasing spiked level, regardless of whether the sample contained itraconazole alone, hydroxy-itraconazole alone, or both azoles.


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Table II. Bioassay results for 15 paired serum samples spiked with itraconazole alone, hydroxy-itraconazole alone, or both compounds
 
With a hydroxy-itraconazole standard, bioassay results from laboratories A, B, D and E closely matched the spiked concentrations for samples containing hydroxy-itraconazole alone (Table II), but the results from these laboratories underestimated the azole concentrations for samples containing itraconazole alone and itraconazole + hydroxy-itraconazole. The results from laboratory C were considerable underestimates of the spiked azole concentrations for all samples tested when a hydroxy-itraconazole standard was used.

The results from laboratory A, which showed the highest variation between duplicate samples in both phases of the study, nevertheless gave the closest estimate of total spiked azole concentrations in phase 2, regardless of the relative spiked concentrations of itraconazole and hydroxy- itraconazole in the sample.

The average coefficients of variation for the bioassay results with the 15 paired test samples ranged from 5.6% to 21.6% with the itraconazole standard and from 5.7% to 16.7% with the hydroxy-itraconazole standard (Table II).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Measurement of itraconazole and hydroxy-itraconazole blood levels constitutes a useful part of patient management, as it provides reassurance that itraconazole given by mouth is being absorbed sufficiently to provide adequate concentrations for treatment of a fungal infection. The concentrations of hydroxy-itraconazole and itraconazole in blood samples from patients receiving oral treatment with itraconazole capsules vary considerably. 1,2,3 HPLC is undoubtedly the most sensitive, specific and reproducible method for determining levels of individual azole antifungal agents in blood samples, as confirmed in this study by the low coefficients of variation for duplicate samples tested by HPLC and by the greater number of samples with low azole concentrations that could be assayed by HPLC.

Our study has reconfirmed that bioassay methods for estimation of serum levels of itraconazole and its hydroxy- metabolite tend both to overestimate and underestimate the total concentrations of these components in a sample, depending on the experimental conditions used. Despite the interlaboratory variations, the correlative trend between bioassay results and total (itraconazole + hydroxy-itraconazole) concentrations was unequivocal (Tables I and II), showing that bioassay can indicate the relative level of antifungal azole present in a serum sample even when different laboratories use different bioassays rather than a single, standardized assay. Differences in susceptibility of marker isolates to the two agents 1,2 might account for some of the discrepancies between bioassay and HPLC results.

Use of multiplication constants for converting bioassay data to match HPLC results 3 is a valid approach only if the ratio of bioassay result to total (HPLC) azole concentration is constant or nearly so. The results of the present study show that bioassay:HPLC ratios vary between laboratories and must therefore first be established locally if attempts are to be made to express bioassay results as ‘equivalent’ to a ‘ total azole’ concentration. In practice it is probably better to quote results as ‘ bioactivity equivalent to’ a concentration of azole, rather than to suggest that bioassay directly measures azole concentrations.


    Acknowledgments
 
This study was supported by a grant from the Janssen Research Foundation. We gratefully acknowledge the technical help of Olivier Ronin and Filip Woestenborghs.


    Notes
 
* Corresponding author. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Hostetler, J. S., Heykants, J., Clemons, K. V., Woestenborghs, R., Hanson, L. H. & Stevens, D. A.(1993). Discrepancies in bioassay and chromatography determinations explained by metabolism of itraconazole to hydroxyitraconazole: studies of interpatient variations in concentrations. Antimicrobial Agents and Chemotherapy 37, 2224–7.[Abstract]

2 . Hulsewede, J. W., Dermoumi, H. & Ansorg, R.(1995). Determination of itraconazole and hydroxy-itraconazole in sera using high-performance-liquid-chromatography and a bioassay. Zentralblatt für Bakteriologie 282, 457- 64.[ISI][Medline]

3 . Law, D., Moore, C. B. & Denning, D. W.(1994). Bioassay for serum itraconazole concentrations using hydroxyitraconazole standards. Antimicrobial Agents and Chemotherapy38 , 1561–6.[Abstract]

4 . Warnock, D. W., Turner, A. & Burke, J.(1988). Comparison of high performance liquid chromatographic and microbiological methods for determination of itraconazole. Journal of Antimicrobial Chemotherapy 21, 93–100.[Abstract]

5 . Woestenborghs, R., Lorreyne, W. & Heykants, J.(1987). Determination of itraconazole in plasma and animal tissues by high–performance liquid chromatography. Journal of Chromatography B: Biomedical Applications 413 , 332–7.

6 . Dupont, B. & Drouhet, E.(1987). Early experience with itraconazole in vitro and in patients: pharmacokinetic studies and clinical results. Reviews of Infectious Diseases 9, Suppl. 1, S71–6.[ISI][Medline]

7 . Perfect, J. R., Savani, D. V. & Durack, D. T.(1986). Comparison of itraconazole and fluconazole in treatment of cryptococcal meningitis and candida pyelonephritis in rabbits. Antimicrobial Agents and Chemotherapy 29, 579–83.[ISI][Medline]

8 . Heykants, J., Van Peer, A., Van de Velde, V., Van Rooy, P., Meuldermans, W., Lavrijsen, K. et al. (1989). The clinical pharmacokinetics of itraconazole: an overview. Mycoses 32, Suppl. 1, 67–87.[ISI][Medline]

9 . Bodet, C. A., Jorgensen, J. & Drutz, D. J.(1985). Simplified bioassay method for the measurement of flucytosine or ketoconazole. Journal of Clinical Microbiology 22 , 157–60.[ISI][Medline]

Received 20 May 1998; returned 8 July 1998; revised 26 October 1998; accepted 24 December 1998