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ADME PK

Microsomal stability assay

Understand the metabolism of your compounds by using our microsomal stability assay to measure in vitro intrinsic clearance or to identify metabolites formed.

Microsomal stability is one of Cyprotex's in vitro ADME screening services. Cyprotex deliver consistent, high quality data with cost-efficiency that comes from a highly automated approach.

Measurement of in vitro intrinsic clearance using microsomes

  • The liver is the most important site of drug metabolism in the body. Approximately 60 % of marketed compounds are cleared by hepatic CYP-mediated metabolism1.
  • Liver microsomes are subcellular fractions which contain membrane bound drug metabolizing enzymes.
  • Microsomes can be used to determine the in vitro intrinsic clearance of a compound.
  • The use of species-specific microsomes can be used to enable an understanding of interspecies differences in drug metabolism.
  • Easy to prepare, use and store enabling cost efficiencies over whole cell models.
  • Microsomes are pooled from multiple donors to minimize the effect of interindividual variability.
  • Microsomes are fully characterized using probe substrates to ensure activity is maintained between batches.
The liver microsomal in vitro T1/2 approach can be a suitable approach to measure in vitro CLint, which can be scaled up to the in vivo situation and used in the prediction of human clearance.

1Obach RS (1999) Drug Metab Dispos 27(11); 1350-135

Protocol

Microsomal stability assay protocol

Test Compound Concentration 3 μM (different concentrations available)
Microsome Concentration 0.5 mg/mL (different concentrations available)
Time Points 0, 5, 15, 30, 45 minutes
Cofactor NADPH
Final DMSO Concentration 0.25%
Compound Requirements 50 μL of 10 mM solution
Controls 0 μM (blank);
Minus cofactor (45 min only);
Positive control compounds with known activity
Analysis method LC-MS/MS
Data Delivery Intrinsic clearance
Standard error of intrinsic clearance
Half life

Follow on metabolite profiling and structural elucidation

Cyprotex's microsomal stability assay can be extended to profile the metabolites that are formed. Cyprotex’s biotransformation services are supported by high resolution, accurate mass spectrometry. These services can provide information on an individual species’ metabolite profile, or a cross-species comparison to identify potential differences in metabolism which could in turn help to interpret pharmacology and toxicity data. Structural elucidation can also be performed on the potential metabolites’ MS/MS fragmentation data. All biotransformation studies are performed by a dedicated team of experts.

Please refer to our Metabolite Profiling and Identification section for further details.

Data

Data from Cyprotex's Microsomal Stability assay

A set of known drugs were screened in Cyprotex's Microsomal Stability assay in quadruplicate on 4 separate occasions. The data show reproducibility over a range of intrinsic clearance values (Figure 1). Figure 2 shows that data generated in Cyprotex's Microsomal Stability compare well with literature data.

 
Figure 1
Mean intrinsic clearance data for 13 compounds obtained using Cyprotex's Microsomal Stability assay (error bars represent the standard deviation from quadruplicate incubations within each run of the assay).

The graphs shows consistency of data both within the assay and between separate runs of the assay.
Figure 2
Comparison of Cyprotex's Human Microsomal Stability intrinsic clearance data with literature data.

The inter-laboratory variability in literature data can be considerable as shown by the error bars (mean ± standard deviation) on the graph. Literature data taken from Riley et al. (2005)3

Q&A

Questions and answers on microsomal stability

Explain the benefits of using liver microsomes for drug metabolism studies?

The liver is the main organ of drug metabolism in the body. Subcellular fractions such as liver microsomes are useful in vitro models of hepatic clearance as they contain many of the drug metabolizing enzymes found in the liver. Microsomes are easy to prepare and can be stored for long periods of time. They are easily adaptable to high throughput screens which enable large numbers of compounds to be screened rapidly and inexpensively.

Please provide an overview of Cyprotex's Microsomal Stability assay.

The microsomes are incubated with the test compound at 37°C in the presence of the co-factor, NADPH, which initiates the reaction. The reaction is terminated by the addition of methanol containing internal standard. Following centrifugation, the supernatant is analyzed on the LC-MS/MS. The disappearance of test compound is monitored over a 45 minute time period. An example of a typical depletion profile is shown in Figure 3.

Figure 3
Graph shows test compound disappearance with time in the presence of liver microsomes.

The ln peak area ratio (compound peak area/internal standard peak area) is plotted against time and the gradient of the line determined.

How do I interpret the data from the microsomal stability assay?

There are several ways in which the data can be used. Firstly, the compounds can be ranked in terms of their intrinsic clearance values. Unless the compound is a pro-drug, very highly cleared compounds are generally considered to be unfavorable as they are likely to be rapidly cleared in vivo resulting in a short duration of action. Classification bands can be used to categorize compounds into low, medium or high clearance. The CLint classification bands for each species in Table 1 are calculated from a rearrangement of the well stirred model4 detailed in the following equation assuming an extraction ratio (E) of 0.3 and 0.7 for the low and high boundaries, respectively. This can then be scaled to intrinsic clearance (µL/min/mg protein) using the relevant liver weights5 and microsomal protein concentration6, 7 obtained from the literature. Due to lack of literature information the monkey and mouse microsomal protein concentration was assumed at 50mg microsomal protein/g liver.

CLint =

Where CLH = E x QH

QH = liver blood flow (mL/min/kg)5
E = Extraction Ratio
CLH = Hepatic Clearance (mL/min/kg)
fu = fraction unbound in plasma (assumed at 1)

Clearance CategoryIntrinsic Clearance (µL/min/mg protein)
HumanMonkeyDogRatMouse
Low < 8.6 < 12.5 < 5.3 < 13.2 < 8.8
High > 47.0 > 67.8 > 28.9 > 71.9 > 48.0

Table 1: Classification bands typically used for categorizing compounds into low, medium or high clearance.

Secondly, the data can be used in conjunction with other in vitro parameters to predict the pharmacokinetics of a compound in vivo using the simulation software Cloe PK. Thirdly, species specific differences in drug metabolism can be investigated. This may be useful in identifying an appropriate species for pre-clinical development.

Why would I screen my compounds in the microsomal stability assay rather than the hepatocyte stability assay?

Microsomes are adaptable to high throughput screening and enable large numbers of compounds to be screened inexpensively. Our clients tend to use this assay as an initial screen to rank order compounds of interest in terms of their metabolic stability, and then perform a secondary screen on a smaller number of selected compounds using hepatocytes.

What controls are used in the assay?

• Two positive control compounds are included for each species. These compounds are known to be metabolized by liver microsomes.

SpeciesControl Compounds
Human Dextromethorphan
Verapamil
Rat Diazepam
Diphenhydramine
Mouse Diazepam
Diphenhydramine
Monkey Quinidine
Verapamil
Dog Dextromethorphan
Verapamil

• A control incubation is performed in the absence of NADPH to reveal any chemical instability or non-NADPH dependent enzymatic degradation.

• A control is included that contains all reaction components with the exception of the test compound. This control identifies any potential interfering component which may affect the analysis.

Can I investigate Phase II metabolism in liver microsomes?

The microsomal stability assay is primarily used to investigate Phase I metabolism using NADPH as the enzyme co-factor. However liver microsomes can also be used to study Phase II metabolism if the correct incubation conditions are used. We have recently validated this using the pore forming agent alamethicin in conjunction with appropriate Phase II cofactors for example UDPGA. This allows you to gain an understanding as to the contribution Phase II metabolism has on the overall metabolism of a test compound. It is also possible to study coupled Phase I and Phase II metabolism by using all of the relevant co-factors in the incubation.

References

1 McGinnity DF et al. (2004) Drug Metab Dispos 32; 1247-1253
2 Obach RS. (1999) Drug Metab Dispos 27(11); 1350-1359
3 Riley RJ et al. (2005) Drug Metab Dispos 33; 1304-1311
4 Houston JB. (1994) Biochem Pharmacol 47(9); 1469-1479
5 Davies B. and Morris T. (1993) Pharma Res 10(7); 1093-1095
6 Barter ZE et al. (2007) Curr Drug Metab 8(1); 33-45
7 Iwatsubo T et al. (1997) JPET 283(2); 462-469

Cloe® is a registered trade mark owned by Cyprotex PLC
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