MONITORING CYCLOSPORINE IN SALIVA

• US 20080255765A1
• Filed: 11/16/2007
• Published: 10/16/2008
• Est. Priority Date: 05/19/2005
• Status: Abandoned Application

• Current Assignee: State of Rhode Island and Providence Plantations
• Original Assignee: State of Rhode Island and Providence Plantations
• Inventors: Akhlaghi, Fatemeh, Mendonza, Anisha E.
• Application Number:
  US11/941,509
• Publication Number:
  US 20080255765A1
• Time in Patent Office: Days
• US Class Current: 702/19
• CPC Class Codes: A61K 38/13 Cyclosporins
• CPC Class Codes: G01N 1/38 Diluting, dispersing or mixing samples
• CPC Class Codes: G01N 33/9493 Immunosupressants

Abstract

Saliva offers an alternative specimen for the therapeutic monitoring of cyclosporine (CsA) in children and patients with difficult venous access. For a highly protein-bound drug such as CsA, saliva provides a practical approach for measuring the unbound concentration. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is ideally suited for the measurement of drugs in saliva. A solid-phase extraction technique, analytic liquid chromatography over an Aqua Perfect column, maintained at 65° C., and electrospray tandem mass spectrometry were used to quantify CsA in saliva. The method used cyclosporine C (CsC) as the internal standard. Mobile phase comprised of a 97:3 voL mixture of methanol and 30 mmol/L ammonium acetate at a flow rate of 0.5 mL/min. Chromatograms using mass transitions of m/z 1219.9→m/z 1202.9 for CsA and m/z 1235.9→m/z 1218.9 for CsC were obtained. The calibration curve was linear from 1 to 300 μg/L with correlation coefficient values ranging from 0.9732 to 0.9968). The lower limit of quantification was 1 μg/L and limit of detection was 0.6 μg/L with an average extraction recovery of 84.7±2.6% for CsA and 93.7±4.4% for CsC from the saliva matrix. The accuracy of the method ranged from 92% to 104.7%, and the intra- and interim coefficients of variation were 6.9-12.2% and 8.3-12.1%, respectively. The correlation coefficient value between the CsA concentration measurements in 15 paired blood-saliva samples from kidney transplant recipients was 0.695 (P=0.006). The noninvasive and simple method of saliva collection coupled with the LC-MS/MS quantification technique for CsA analysis would generate novel data that could benefit patients undergoing CsA therapy.

Claims

1. a method of monitoring cyclosporine in saliva, said method comprising:

obtaining a saliva sample;

freezing and thawing said saliva sample;

sonicating said saliva sample;

adding said saliva sample to a solvent and water mixture to determine the concentration of cyclosporine in said saliva.

2. The method of claim 1 wherein the solvent is acetonitrile.

3. The method of claim 1, wherein monitoring of cyclosporine is saliva is a substitute for the monitoring cyclosporine in blood.

4. The method of claim 1, wherein the measurement of cyclosporine concentration in human saliva is sensitive with a lower limit of quantification of 1 ng/mL allowing a measurement of very low concentrations.

5. The method of claim 1 wherein the method is specific for parent cyclosporine molecule such that the measured concentrations represent the concentration of parent drug not inactive metabolites.

6. The method of claim 1 wherein the saliva is added to the solvent.

7. The method of claim 1, wherein saliva pre-treatment with sonication improved recovery and consistency of cyclosporine extraction from saliva.

8. A prediction equation calculated from testing of multiple samples of cyclosporine in saliva versus cyclosporine in blood from the same person, such that by knowing either the amount of cyclosporine in either blood or saliva, the amount of cyclosporine in the other can be calculated.

9. A method of determining the correlation of concentration of cyclosporine in a blood sample and the amount of cyclosporine in a saliva sample from the same person to determine the amount of cyclosporine in a person'"'"'s blood by evaluating their saliva, said method includes evaluating numerous saliva samples and blood samples from various individuals;

plotting the amounts of cyclosporine in the saliva versus the amount of cyclosporine in the blood of an individual on a plot such that once a plot is prepared, one only needs to determine the amount of cyclosporine in a saliva sample, place the amount on the plot to be able to determine the equivalent amount of cyclosporine in the person'"'"'s blood.