Required from Customer

• A study design—the customer must specify the value of several assay variables listed in the Options section
• Test compound in powder form or preformulated
• Exact molecular mass of the test compound and its salt form
• MSDS or handling and storage information, e.g., light sensitive, store at -20°C, stability, solubility, purity, and activity
• If applicable, instruction for dosing vehicle preparation

Deliverables

• In-life observations on each animal
• A table containing test compound concentrations in plasma samples at each time point
• A table listing relative exposure (bioavailability) based on the  AUC for each compound,  C_max and T_max for each compound
• Half life, volume of distribution, clearance after IV dosing
• Bioavailability after OG administration
• An appendix containing the bioanalytical data

Substrate

• Test compound dissolved in an appropriate dosing vehicle

Assay System

• Conscious, fasted NHPs typically weighing between 4 and 7 kg.  Water is offered ad libitum

Assay Conditions

• Three NHPs (N=3) per dose group
• For each route of administration:
  • Dose at a concentration specified by the customer
  • Sample blood from the saphenous vein at time points specified by the customer
  • Prepare plasma samples using anticoagulant specified by customer—no anticoagulant is used when the  customer requests serum sample only
  • Pre-study work is done to establish the calibration range and method reproducibility
  • Determine the concentrations of test compound using a LC-MS/MS method with a minimum 8-point calibration curve
  • Non-compartmental analysis is used to determine PK parameters for each test compound

Assay QC

• Adverse reactions, if any, are reported to the customer
• Analytical data are accepted only when at least two-thirds of the analytical control samples and 60% of the standards used to create the calibration curve have a back calculated accuracy of ±15%  (at LLOQ, ±20%)

Notes

1. The amount of test compound or formulation that is required depends upon the dose, i.e., mg test compound per kg of NHPs, and the number of NHPs.
2. The standard breed is Cynomolgus. The customer can specify the use of other commercially available breeds.
3. Absorption Systems can develop the dose vehicle for this study.
4. The customer can elect to perform the bioanalysis while Absorption System only does the dosing and sampling.
5. The standard anticoagulant is sodium heparin. The customer can specify that another anticoagulant be used.
6. A variety of administration routes in addition to oral and intravenous dosing are available.
7. A complete analytical method development includes:
   1. Optimization of MS: Initial conditions will be established by infusing a solution containing the test compound into an appropriate mass spectrometer. The goals in MS optimization are to maximize detector response for the test compound and determine the approximate lower limit of quantitation (LLOQ) of the test compound.
   2. Optimization of HPLC: HPLC parameters will be optimized using an appropriate selection of LC column(s) and mobile phase. The goal in HPLC optimization is to develop appropriate chromatographic run-times while maintaining adequate levels of sensitivity.
   3. Optimization of Sample Preparation: Various means of sample preparation, e.g., direct injection, dilution with appropriate solution, protein precipitation, solid phase extraction etc., will be evaluated for their efficiency in extracting the test compound from the biological matrix. The goal in extraction optimization is to achieve the required lower limit of quantitation (LLOQ) while alleviating matrix suppression effects.
8. A validation is performed using calibration standards and quality control (QC) samples containing the test compound at known concentrations. In order for the analytical method to be considered acceptable the results from at least one method validation run must meet the following criteria:
   1. The standard curve must include a minimum of six standards with their concentration back calculated to ±15% (±20% at the LLOQ) of their theoretical concentration when the correlation coefficient of the regression model is ≥ 0.990.
   2. The calculated concentration of at least two-thirds of all QC samples and at least one-third of the QC replicates must fall with ±15% of their respective theoretical concentration.
   3. The intra-assay coefficient of variation (CV) of the replicate determinations of the low, medium and high QC samples must not exceed ±15%, and the average concentration for each set of QC replicates as calculated using the standard curve must be within ±15% of their theoretical concentration.
   4. The lowest standard is accepted as the lower limit of quantitation if the typical response at this concentration is at least 5 times greater than any interference in the blank matrix at the retention time of the analyte(s).
   5. The response ratio of any interfering peak(s) in the matrix blanks at the retention time of the analyte(s) must be <20% of the response to the LLOQ standard.

Options

1. The customer must specify:
   • the use of either the standard or a custom report format
   • the dose vehicle(s)
   • the routes of administration
   • the breed of non-human primates—typically we use Cynomolgus
   • the number of NHPs per dosing route/treatment
   • crossover or non-crossover
2. The customer can request:
   • a different anticoagulant be used
   • that Absorption Systems determine a suitable dosing vehicle
   • that Absorption Systems prepare the dosing vehicle using the customer’s instructions
   • a more complete bioanalytical method validation, e.g. inter-day variability, and assessment of stability samples
   • that Absorption Systems only dose and sample the NHPs (in-life only)
   • that Absorption Systems analyze the plasma collected by others
   • that Absorption Systems perform the PK analysis on data collected by others