PK/ADME Studies - Protocols

The following are standard sets of protocols.
Pharmacokinetics/ADME studies
Comparative metabolism and in vitro toxicity studies
Drug-drug interaction studies
Peroxisome proliferation studies
Cytotoxicity assays

Pharmacokinetics/ADME Studies

P101 Material Balance or Accountability Study*

• The objective of this study is to investigate the excretion rate and tissue distribution of the chemical.
• Groups of 4 male and 4 female rats at a single dose level
• Daily collection of urine and feces for 7 days
• Collect blood and major internal organs, and retain carcass upon necropsy
• Total radioactivity level in each sample is assayed and the recovered dose is compared with the administered dose. The percentage of distribution of the administered dose in urine, feces, and each major internal organ and the half-time for excretion are calculated.

P101-R Optional Studies

• Analysis of CO₂ in expired air
• Analysis of injection site (e.g., if the dosing route is percutaneous or intramuscular)
• Two dose levels instead of one
• Species other than rats (e.g., rabbits, dogs, primates)
• Repeated-dose study (unlabeled compound is administered daily for 13 days; on the 14th day a radioactive dose is administered)
• Major metabolites - isolation, characterization

P102 Plasma Level and Bioavailability Study*

• The objective of this study is to examine the pharmacokinetic profiles of the chemicals under test.
• These studies can be conducted by using unlabeled chemicals provided that appropriate analytical procedures (e.g., HPLC) are available.
• Groups of 4 male and 4 female rats per route of dosing
• One group is dosed intravenously and the second group is dosed by an extravascular route (e.g., oral, percutaneous). The dosages for the 2 groups need not be identical
• Blood is sampled at frequent intervals (e.g., 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, and 48 hr), and plasma levels of the chemical are assayed. The plasma- concentration-time data are analyzed to obtain the pharmacokinetic profile, including the area under the curve (AUC). Comparison of the AUCs after extravascular and intravenous dosing, after correcting for the dosage, provides a measure of bioavailability. Alternatively, if a radioactive chemical is used, comparison of the administered doses excreted in urine can be used to calculate the approximate bioavailability of the orally dosed chemical.

P102-R Optional Studies

• Species other than rats
• Plasma protein-binding study

Pharmacokinetics Studies of Nucleotide Therapeutics

• We can conduct tissue distribution studies of nucleotide-based materials using quantitative polymerase chain reaction (PCR), capillary electrophoresis, or in situ hybridization methods. Studies are designed to fit the needs of each particular test article.

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Comparative Metabolism and In Vitro Toxicity Studies

Incubation with In Vitro Preparations*

• Liver, kidney, small intestine from humans, dogs, monkeys, or rodents
• Isolated cells, tissue slices, or subcellular fractions (S9, microsomes, or cytosol, with cofactors)
• Two test chemical concentrations, 3 time points
• Protein assay for standardization
• Cytochrome P450 activity and isozyme profile available

Subcellular Fractions

Y301-R Rodent

Y301-D Dog/Monkey

Y301-H Human

Isolated Cells/Tissue Slices

Y302-R Rodent

Y302-D Dog

Y302-M Monkey

Y302-H Human

Complete analysis and metabolite identification are available.

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Drug-Drug Interaction Studies

Y303 Determination of Cytochrome P450 Form-Specific Metabolism · Measure metabolism of form-specific substrate · Correlation assay with six form-specific activities · Confirmation with microsomes from cells expressing single form of human P450

Y304 Cytochrome P450 Inhibition · Three drug concentrations · Pooled human liver microsomes from six donors · Six P450 form-specific assays for CYP1A, 2A6, 2C9, 2C19, 2D6, and 2E1

Cytochrome P450 Induction · Hepatocytes in primary culture · Preliminary range finding experiment · Definitive experiment with 5 concentrations · Measurement of marker enzyme activities, confirmed by Western blotting · Available in rat or human hepatocytes

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Peroxisome Proliferation Studies

Palmitoyl CoA-Oxidation · Hepatocytes in primary culture · Preliminary cytotoxicity experiment · Definitive experiment with 5 concentrations of the test agent and solvent and positive controls · Palmitoyl CoA-oxidation as endpoint · Four independent cultures per concentration in each experiment · Concurrent measurement of DNA or protein levels in each culture

Y201 Rat Hepatocytes

Y202 Human Hepatocytes

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Cytotoxicity Assays

Cytotoxicity Assays In Primary Cultures

Screening Assays · Hepatocytes or renal proximal tubules · Two experiments with 6 concentrations tested with positive, solvent, and media controls · Four replicate samples, 3 time points (conducted in 96-well dishes) · FIVE COMPOUNDS MINIMUM.

Y101 Enzyme Release (lactate dehydrogenase)

Y102 MTT Conversion (Mitochondrial Function)

Available Species · Human · Rat · Dog · Rabbit · Mouse · Guinea pig · Hamster · Nonhuman primates

Specific functional assays · Available for determining chemical mechanism of action and include urea synthesis, protein synthesis, lipid peroxidation, and oxygen consumption, among others.

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     Last Updated Jul 17, 2008