Bioassay for Determining Itraconazole Levels in Blood

Introduction

Serum concentrations of itraconazole should be measured in patients receiving this drug to ensure that therapeutic concentrations are being achieved. This is necessary as drug absorption can be variable, and levels may be lowered by interactions with other drugs. The assay will give an indication of whether suitable blood levels have been achieved.

Hazards

Normal microbiological technique is adequate for safety whilst preparing the bioassay plate. Gloves should be worn whilst handling all patient samples, and in cases of high risk specimens, a class I safety cabinet should be used.

Specimens

Serum - minimum volume 200µl.

Plasma - minimum volume 200µl.

Samples may also be in the form of clotted blood (with or without anticoagulant), from which plasma/serum should be obtained by spinning the samples at 3000 rpm in a centrifuge for 10 minutes.

Only a post dose sample is required - this should be taken 4 hours after an oral dose of itraconazole. The first level should be determined one week after commencement of therapy, as this period of time is needed to build up to a steady state level. Levels should be determined weekly.

As itraconazole is highly lipophilic it is rarely detectable in aqueous body fluids such as urine and cerebrospinal fluid, therefore these samples need not be assayed. If it is necessary for pharmacokinetic purposes to measure these fluids, then a HPLC method should be used.

Materials (see Appendix for preparation of media)

• Yeast nitrogen base
• Trisodium citrate
• Base agar No. 1
• Itraconazole - Janssen Pharmaceuticals, Beerse, Belgium.
• Pooled negative serum
• Acetone
• Hydrochloric acid

Equipment

• 60°C water bath
• Haemocytometer
• Plastic bioassay plate (manufactured by NUNC)
• Cork borer No. 3 (8mm holes)
• Template for spacing holes
• Dial calipers
• Semi-logarithmic graph paper
• Levelling table
• Spirit level
• Digital pipettes and sterile pipette tips
• Sterile bijou bottles

Quality Control

Internal control procedure

1. Control samples with known amounts of itraconazole are placed on each bioassay plate.
2. Internal controls should give a value within a 20% range of the known value of itraconazole.

Procedure

1. Prepare stock solution of itraconazole (10,000mg/l). Weigh out 0.1g of pure itraconazole powder into a glass universal and add 5ml of acetone. Then, and only after adding the acetone, add 5ml of 0.2M hydrochloric acid. Vortex vigorously and place in a water bath at 60°C until dissolved, vortexing occasionally. Dispense into 1ml amounts and store in -20°C freezer.
2. Put 10ml of YNBG + trisodium citrate solution into a water bath at 60°C to heat before adding to the agar.
3. Melt 90ml amount of base agar.
4. Make suspension of sensitive Candida kefyr San Antonio strain (2 large loopfuls) in 5ml of sterile distilled water. Count cells using a haemocytometer, and adjust to 1x 10⁷ cells per ml.
5. Label orientation of bioassay plate.
6. Using levelling table ensure plate is completely flat.
7. When the molten agar has cooled to 60°C add 10ml of YNBG + trisodium citrate solution and 5ml of yeast suspension. Mix well without generating any bubbles. Pour bioassay plate ensuring no bubbles are present. Leave to solidify (Minimum time of 30 minutes).
8. Take itraconazole stock solution (10,000mg/l) and pooled negative serum out of the freezer, and allow to reach room temperature.
9. When the stock solution of the drug has reached room temperature, dilute, using a fresh sterile pipette tip for each concentration, in sterile distilled water to give three concentrations:
   1. 1000mg/l - label as concentration A (100µl of the stock solution and 900µl of sterile distilled water)
   2. 100mg/l - label as concentration B (100µl of concentration A and 900µl of sterile distilled water)
   3. 10mg/l - label as concentration C (100µl of concentration B and 900µl of sterile distilled water)Vortex each vial well.
10. Prepare the standards as follows:
    1. Dispense 1ml of plasma/serum into eight bijou bottles.
    2. Add to plasma/serum the amount of drug indicated below, using a digital pipette and sterile tips. Use a fresh sterile pipette tip for each standard. Vortex well.
    Standards:
    1. - 0.2mg/Ladd 20µl of concentration C
    2. - 0.4mg/Ladd 40µl of concentration C
    3. - 0.8mg/Ladd 8µl of concentration B
    4. - 1.6mg/Ladd 16µl of concentration B
    5. - 3.2mg/Ladd 32µl of concentration B
    6. - 6.4mg/Ladd 6.4µl of concentration A
    7. - 12.8mg/Ladd 12.8µl of concentration A
    8. - 25.6mg/Ladd 25.6µl of concentration A
    
    
11. When the agar has solidified, dry the plate, inverted with the lid open, at 37°C.
12. Once the agar surface has dried, cut out 36 wells of 8mm in diameter with sterile cork borer No. 3. (6 rows of 6)
13. Using the template, place 40µl of standard or patient specimen into the appropriate wells. Always test in duplicate or triplicate using a randomised pattern.
14. Allow the drug to pre-diffuse by leaving the plate to stand at room temperature for 30 minutes.
15. Incubate the plate at 37°C overnight (approximately 18 hours)
16. Measure the diameters of the zones of inhibition around each well using dial calipers, to the nearest 0.1mm, and record on the results form.

Interpretations/Calculations

1. Calculate the mean diameter for each standard and patient sample.
2. Plot the mean diameters of standards against itraconazole drug concentrations on semi-logarithmic paper, with the drug concentrations on the logarithmic ordinate. Draw the best fit straight line.
3. Use the graph to estimate the concentration of drug in the patient specimens and internal standards.

Limitations

1. Care must be taken when specimen details suggest that the patient is receiving another antifungal drug in addition to itraconazole. In such cases a HPLC assay may be necessary, or a bioassay using an organism resistant to the second antifungal.

Troubleshooting

1. Observation: a good standard curve is not obtained.
   Cause: standards are made up incorrectly/bioassay plate prepared incorrectly/plate read incorrectly.
   Action required: disregard results and repeat bioassay after a careful review of all steps in the protocol. If possible use new batches of medium, plasma/serum and antifungal drug stock.
2. Observation: internal control is not within 20% of the expected value.
   Cause: as above, or internal standard has deteriorated.
   Action required: check previous internal standard results for signs of deterioration. If none apparent, take action as above.

Reporting

1. Report as the value of itraconazole obtained in mg/l.
2. Provide target blood levels for guidance. Target level = >5 mg/l.
3. Levels above 15 mg/L are considered high and should be reported as such, although high levels are not normally associated with adverse side effects.

Timetable

Interpretations/Calculations (Steps 1 - 3): 30 minutes

Appendix

Preparation of Yeast Nitrogen Base with Glucose (YNBG) +Trisodium citrate

1. Dissolve 6.7g of yeast nitrogen base, 10g of glucose and 5.9g of trisodium citrate in 100ml of distilled water. Stir until completely dissolved.
2. Filter sterilize this solution.
3. Dispense into 10ml aliquots in sterile universal bottles.
4. Store at 4°C.

Preparation of Base Agar

1. Suspend 20g of Base agar No. 1 in 900ml of cold distilled water.
2. Heat to boiling to dissolve the agar and dispense into 90ml amounts.
3. Sterilize in an autoclave for 15 minutes at 121°C (15psi).
4. This agar can be stored at room temperature for up to 3 months.
5. The supplier of itraconazole was:
   Research Diagnostics Inc, Pleasant Hill Road, Flanders NJ 07836, USA.
   Tel (US) 973-584-7093
   Fax (US) 973-584-0210
   E-mail http://www.researchd.com

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