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Lab techniques

Concentration of total drugs and metabolites

The basic requirement for a research laboratory in clinical pharmacokinetics is the availability of sensitive, specific and reproducible analytical methods for the measurement of total or unbound concentration of drugs and their major metabolites. Every assay, either chromatography or immunoassay based, must be validated according to the guidelines set forth by the FDA and preferably published in a peer reviewed journal. The requirement is so fundamental that a clinical study performed using non-validated assays is usually not publishable in a reputable journal.  Because of this requirement, my lab devotes considerable effort to the development and validation of analytical methods using HPLC-UV and LC-MS/MS.  The following assays are now available in the laboratory, all of which have been validated according to the FDA “Guidance for Industry:Bioanalytical Method Validation” and were published as manuscript or conference abstract:

• Acetaminophen concentration for determination of gastric emptying time (HPLC-UV)
• Acetaminophen glucuronide and sulfate concentrations (LC-MS/MS)
• Atorvastatin and metabolites [atorvastatin (ATV) lactone, o-hydroxy ATV, o-hydroxy ATV lactone, p-pydroxy ATV, p-hydroxy ATV lactone (LC-MS/MS)
• AZT (3’-azido-3’-deoxythymidine) glucuronidation as a probe for UGT2B7 activity (LC-MS/MS
• Chlorzoxazone hydroxylation as a probe for P450 2E1 activity (LC-MS/MS)
• Cortisol metabolite (6 beta hydroxy cortisol) and free cortisol concentration in urine (HPLC-UV)
• Cyclosporine; total concentration (LC-MS/MS)
• Estradiol-glucuronidation as a probe for UGT1A1 activity (HPLC-UV)
• Iodixanol concentration for determination of GFR (HPLC-UV)
• Iohexol concentration for determination of Glomerular Filtration Rate (GFR) (HPLC-UV)
• Ketoconazole concentration in human plasma (LC-MS/MS)
• Midazolam, 1’-OH and 4-OH midazolam (LC-MS/MS)
• Mifepristone concentration in rabbit plasma
• Mycophenolic acid (MPA) metabolites; concentration of MPAG and Acyl MPAG (HPLC-UV)
• Mycophenolic acid (MPA), total concentration (HPLC-UV)
• Oseltimivir and carboxylic acid (LC-MS/MS)
• PF-05190457 concentration in human and rat plasma and rat brain
• Pioglitazone and keto pioglitazone metabolite in human plasma (LC-MS/MS)
• Prednisolone, prednisone, cortisol and cortisone concentrations (LC-MS/MS)
• Propofol glucuronidation as a probe for UGT1A9 activity (HPLC-UV)
• Rosuvastatin acid, n-desmethayl rosuvastatin and rosuvastain lactone in plasma (LC-MS/MS)
• Sirolimus; total concentration (LC-MS/MS)
• Tacrolimus; total concentration (LC-MS/MS)
• Testosterone and 6-beta-testostrone (HPLC)
• Thyroid hormones, T3 and T4 (LC-MS/MS and ICP-mass spec.)

Unbound (free) concentration of drugs

• Cyclosporine; unbound concentration (equilibrium dialysis using [3H] cyclosporine as tracer)
• Mycophenolic acid, unbound concentration (ultrafiltration followed by LC-MS/MS)
• Tacrolimus; unbound concentration (equilibrium dialysis using [3H] tacrolimus as tracer)
• Prednisone, prednisolone and cortisol concentrations (ultrafiltration followed by LC-MS/MS)

Concentration of drugs in saliva

• Cyclosporine, saliva concentration (LC-MS/MS)
• Mycophenolic acid, saliva concentration (LC-MS/MS)
• Tacrolimus, saliva concentration (LC-MS/MS)
• Sirolimus, saliva concentration (LC-MS/MS) (under development)

Pharmacodynamic assays for immunosuppressive agents

• Measurement of inosine 5’-monophosphate dehydrogenase type–II (IMPDH-II) activity in peripheral blood mononuclear cells; this method is pharmacodynamics marker for mycophenolic acid (enzymatic reaction followed by LC-MS/MS)
• mRNA expression using Taqman and SYBR green methods (rtPCR).
• Measurement of intracellular cytokine (IL-2, TNF-α, IFN-gamma) production in mitogen stimulated peripheral blood T-lymphocytes (intracellular immunostaining followed by flow cytometer; this method is one of the Pharmacodynamics assays to access the immunosuppressive activity of calcineurin inhibitors at T-cell level).
• Measurement of phenotypic markers (CD86, CD54 and CD95) expression in mitogen stimulated peripheral blood B-lymphocytes (CD19+ cells); immunostaining followed by flow cytometer.
• ImmuKnow™ test (Cylex Inc):  this test detects cell-mediated immunity by measuring the concentration of ATP from CD4+ cells following stimulation. The assay is the only FDA-cleared assay that directly assesses the cell-mediated immune response.

Preclinical studies

To explain many observations originated from clinical PKPD studies, one has to utilize various techniques commonly used in the basic biomedical science or the preclinical stages of drug development.  Among these techniques employed in my laboratory are the followings:
• Characterization of metabolic capacity of human liver microsomes in metabolizing CYP or UGT substrates.
• Western blot and rtPCR.
• Pharmacokinetics and drug-drug interaction studies in Sprague Dawley rat.
• Isolation and fractionation of lipoproteins from plasma to access plasma distribution of drugs among lipoprotein fractions.
• Plasma protein studies of new drugs and binding of drugs to different blood cells (i.e. red blood cells and lymphocytes).
• Transwell assay using MDCK cell lines transfected with human MDR1 (ABCB1) or MRP2 (ABCC2) genes.  This assay is employed to characterize the drug-drug interaction at transporter level.
• Caco-2 transwell assay
• HepG2 culture to evaluate the effect of diabetes