实验概要
Background
The pharmaceutical and biotechnology industry’s goal is to discover therapeutic agents that are both safe and effective at treating or preventing diseases. Compounds identified as selective and potent in the early drug discovery phase are progressed to preclinical drug development for further evaluation. It is estimated that over 10% of drugs fail in clinical trials due to pharmacokinetic reasons , and the US Food and Drug Administration (FDA) guidelines emphasize the identification of metabolic pathways, relevant metabolites and potential drug-drug interactions for new chemical entities (NCEs) where metabolism is the principal route of elimination Consequently, drug metabolism studies are a critical constituent of any drug development program.
The primary site of metabolism for many drugs is the liver. Liver-derived systems such as liver slices, sub-cellular liver fractions, and intact hepatocytes are typically utilized to assess the metabolism of NCEs. Intact hepatocytes contain the cytochrome P450’s (CYPs), other non-P450 enzymes, and phase II enzymes such as sulfo- and glucuronosyltransferases, and thus represent a prime model system for studying drug disposition in vitro. NCEs can be screened and rank-ordered according to metabolic half-life estimates or in vitro intrinsic clearance values (Clint,in vitro) obtained from metabolic stability studies. Moreover, metabolic screening assays enable drug developers to focus on the improvement of compounds through structural activity relationships (SAR) and prevent the progression of labile compounds to more costly in vivo studies. Given that cryopreserved hepatocytes retain enzymatic activities similar to those of fresh hepatocytes and offer convenience to the end user , the utility of cryopreserved hepatocytes in efforts to define a drug’s disposition in vitro is advantageous as compared to other model systems.
Important notes
Review this protocol to ensure y ou have all the necessary reagents and equipment prior to starting the procedure. Once thawed, cryopreserved hepatocytes must be used immediately and will not maintain viability if refrozen.
Use universal safety precautions and appropriate biosafety cabinet when handing primary hepatocytes.
主要试剂
Cryopreserved hepatocytes for suspension use
Williams’ Medium E, 500 mL
Hepatocyte Maintenance Supplement Pack (Serum-free)
12-well non-coated plates15-mL conical tubes (1 per compound)
Compound stocks: test articles (TA) and positive controls (PC). Suitable positive controls may include:
Stop solution
实验步骤
1. In separate conical tubes, add the test compounds and positive control(s) to warm Incubation Medium to yield the desired working concentration(s). For example, prepare 2 μM by adding 10 μL of 1 mM test article stock solution to 5 mL of Incubation Medium. Note: if DMSO is used as a solvent, the concentration should not exceed 0.1%, with a maximum of 1% in the final Incubation Medium.
2. Pipette 0.5 mL of Incubation Medium containing the test article or positive control into respective wells of a 12-well non-coated plate. Note: Final substrate concentration will be 1 μM once step 6 is complete.
3. Place the plate in the incubator on an orbital shaker to allow the substrates to warm for approximately 5-10 min prior to initiation of the reactions.
4. For the negative control, boil 1.0 x 106 viable hepatocytes/mL for 5 min to eliminate enzymatic activity. Use enough volume to cover the number of negative controls desired.
5. Remove the 12-well non-coated plate containing the substrates from the incubator.
6. Start reactions by adding 0.5 mL of 1.0 x 106 viable cells/mL in each appropriate well of the plate to yield a final cell density of 0.5 x 106 viable cells/mL. Pipette 0.5 mL of the inactivated hepatocytes into the negative control wells.
7. Return the plate to the orbital shaker in the incubator and adjust the shaker speed to 90-120 rpms.
8. Remove well contents in 50 μL aliquots at time points 0, 15, 30, 60, 90 and 120 min.
Additional time points 180 min and 240 min may be included, but should not be necessary for healthy and metabolically competent hepatocytes to detect high turnover compounds.
In abbreviated screening assays where only two time points are used to assess NCE metabolic stability, it is strongly advised that a 0 time point and a secondary time point up to 120 min is selected.
9. Stop the incubations by addition of sample aliquots (e.g. 50 μL) to tubes containing the appropriate quenching solvent and either freeze at -70°C, or directly extract as per analytical methods.
10. Determine the in vitro half-life (t1/2) of the parent compound by regression analysis of the percent parent disappearance vs. time curve.
11. Intrinsic clearance in vitro (Clint in vitro) can be calculated according to the equation: Clint in vitro = kV/N, where k = 0.693/t1/2, V = incubation volume (1 mL) and N = number of hepatocytes per well (0.5 x 106 viable cells).
12. Clint in vitro may be scaled to in vivo predictions.
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