In Vitro DMPK

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The liver is the most important site of drug metabolism in the body. Hepatocytes contain the entire set of hepatic drug metabolizing enzymes (both phase I and phase II) maintained within the intact cell.
Hepatocyte stability assay monitors the ability of a compound to be metabolized by enzymes through biotransformation and is also the base for metabolite profiling.
The assay uses cryopreserved primary hepatocytes in suspension incubated in the presence of test compounds. The amount of compound left at different time points is quantified using LC-MS/MS.
Measurement of intrinsic clearance rates can inform the pharmacokinetic profile of an investigative compound and allow drug developers to select a species with the closest metabolic profile to humans for nonclinical testing to better predict behavior in patients.
Pooling of multiple donors offers more consistent results by minimizing single donor variation of enzymatic and uptake activity.

Low clearance compounds are increasingly prevalent in drug discovery, with the emphasis on reducing the metabolic clearance of new chemical entities in order to minimize dose, improve exposure and prolong the half-life.
Determining intrinsic clearance rates of such compounds in human hepatocyte suspensions may not be possible due to a short-term viability/activity of these cells.
For longer term studies, plated cryopreserved human hepatocytes overlayed with matrix extend cell viability to up to 5 days.
Even longer studies (up to 4 weeks) can be done using co-culture systems (HµREL® or HEPATOPAC®) of primary hepatocytes (donor pool) and non-parenchymal stromal cells which maintain hepatocyte function in long term culture.

Intrinsic clearance can be influenced by several processes including hepatic uptake, efflux, biliary excretion, and drug metabolism.
The predominant transporters involved in human hepatic uptake (OATPs, NTCP, OCTs and OATs3) determine intracellular concentrations which can influence clearance as well as potential DDI and hepatotoxicity.
The hepatic uptake assay utilizes the media loss approach and determines the hepatic uptake intrinsic clearance.
Inter-individual variability in hepatic uptake is also likely for substrates of hepatic uptake transporters which exhibit polymorphisms.
Substrates of transporters which exhibit polymorphisms will likely result in inter-individual variability.
This assay accurately determines the intrinsic clearance for metabolically stable compounds for which a traditional suspension assay fails to quantify.

Liver microsomes are subcellular fractions which contain membrane bound drug metabolizing enzymes such as cytochrome P450 (CYP), that metabolize approximatively 60 % of marketed compounds.
Microsomes can be used to determine the in vitro intrinsic clearance of a compound, but compared to whole cell models, they are more cost efficient as they are easy to prepare, use and store.
Microsomes can be pooled from multiple donors to minimize the effect of interindividual variability and fully characterized by using probe substrates to ensure activity is maintained between batches.
The use of species-specific microsomes can be used to enable an understanding of interspecies differences in drug metabolism.

The liver S9 fraction is a preparation containing both the microsomal and cytosolic fractions of the cell, therefore it incorporates both the majority of phase I (mainly microsomal) and phase II (often cytosolic) enzymes, allowing a relatively complete metabolite profile to be achieved.
Subcellular fractions are also easy to prepare, use and store enabling cost efficiencies over whole cell models.

Determination of the stability of new chemical entities in plasma is important as compounds which rapidly degrade in plasma generally show poor in vivo efficacy.
Plasma stability is also very useful for screening of prodrugs and antedrugs, where rapid conversion in plasma is desirable.
Ø Instability in plasma can result in misleading in vitro data and a be challenge for storage and analysis of clinical samples.
Compounds with the following functional groups tend to be more susceptible to hydrolysis in plasma: esters, amides, lactones, lactams, carbamides, sulfonamides, and peptic mimetics.
Compounds may exhibit interspecies differences in their stability in plasma.

Induction of cytochrome P450 enzymes is associated with an increased prevalence of clinical drug-drug interactions.
Cytochrome P450 induction assay identifies the potential of test compounds to induce CYP1A2, CYP2B6 or CYP3A4 in cultured human hepatocytes or HEPARG by evaluating mRNA levels and/or catalytic activity using specific substrates.
Test drug concentrations should be based on the expected human plasma drug concentrations and dose, and compound solubility, cytotoxicity and plasma protein binding should be taken into consideration.
Cytochrome P450 induction assay delivers fold-induction data normalized to vehicle control which can be compared to positive control responses.