Microsomal Stability Assay
Creative Bioarray is committed to providing microsomal stability assay service with high effectivity and reliability to accelerate drug development. We provide microsomal stability assays for all small compounds such as pharmaceuticals, industrial chemicals, and clients' products.
Microsomal Stability Assay Introduction
- Drugs are metabolized in the liver
- The drug's metabolism in the body and whether it forms metabolites are also important parameters in assessing the bioavailability, toxicity, and dosing potential of a compound.
- Metabolic transformation of a drug in the liver can significantly change its efficacy, which may be harmful to the body.
- On this account, drug candidates are tested early in the discovery process for metabolic stability.
- Why Microsomal Stability Assay?
- Metabolism stability refers to the susceptibility of drugs to bio-transformational enzymes such as CYPs and UGTs, which are abundant in the liver. Microsomes from the liver are generally used to study in vitro drug metabolism.
- The metabolic stability assays offer a method to calculate the rate of clearance of a test compound over time in microsomal incubations, and these data are used to evaluate intrinsic clearance.
- Microsomal assays primarily assess metabolism by the cytochrome P450 system (phase I enzymes).
- Microsomes are easy to prepare and can be stored for a long time. They are easily adaptable to high-throughput screening, allowing large numbers of compounds to be screened quickly and inexpensively.
- Data from microsomal assays allow clients to identify metabolic properties earlier and focus on improving drug candidates through structure-activity relationships.
Brief Protocol
- In the presence of the co-factor NADPH, which initiates the reaction, the microsomes are incubated with the test compound at 37°C.
- The reaction was terminated by adding methanol containing an internal standard.
- After centrifugation, the supernatant was analyzed on LC-MS/MS. The disappearance of the test compound is monitored over 45 minutes.
- The ln peak area ratio (compound peak area/internal standard peak area) is plotted against time, and the gradient of the line is determined.
| Microsomal Stability Assay | |
| Test system | Human liver microsomes (pooled) |
| Test Compound Concentration | 3 µM (or custom) |
| Microsome Concentration | 0.5 mg/mL |
| Time Points | 0, 5, 15, 30, 45, 60 minutes |
| Cofactor | NADPH |
| Number of Replicates | 3 |
| Negative Control | Vehicle (0.1% DMSO) Heat inactivated microsomes |
| Positive Controls | Verapamil (rapid clearance) Diazepam (low clearance) |
| Compound Requirements | 50 µL of 10 mM solution |
Case Study
- Preparation of rat liver microsomes
Rats were sacrificed, and the liver was collected and perfused with saline. Liver homogenized using PBS. The liver was homogenized using PBS. The homogenate was centrifuged at 9000 x g for 20 min at 4 °C. Protein concentration was determined and stored at -80 °C until use.
- Methods and Results
As seen in the protocol above, dispense 30 µL of 1.5 µM test compound solution containing 0.5 mg/mL microsomes solution to the assay plates designated for different time points (0, 5, 15, 30, 45 min). After the incubation, transfer the supernatant from each well into a 96-well sample plate for LC/MS analysis.
| Microsomal Stability Assay Results | ||||||
| Sample Name | HLM 0.5 | |||||
| R2 | T1/2 (min) | CLint(mic) (μL/min/mg) | CLint(liver) (mL/min/kg) | Remaining (T=60min) | Remaining (NCF=60min) | |
| Testosterone | 0.9554 | 15 | 92.6 | 83.3 | 6.40% | 81.30% |
| Diclofenac | 0.9994 | 4.2 | 332.1 | 298.9 | 0.00% | 87.50% |
| Propafenone | 0.9173 | 5 | 277.3 | 249.5 | 0.00% | 113.20% |
| Notes: NCF: abbreviation of no co-factor. No NADPH is added to NCF samples (replaced by buffer) during the 60-minute incubation. If the NCF remaining is less than 60%, then possibly non-NADPH dependent metabolism occurs R2: correlation coefficient of the linear regression for the determination of the kinetic constant T1/2: half life T1/2 = 0.693 / K, where K is the rate constant from a plot of ln [concentration] vs. incubation time. CLint(mic): intrinsic clearance CLint(mic) = 0.693 / T1/2 / mg microsome protein per mL CLint(liver) = CLint(mic) × mg microsomal protein/g liver weight × g liver weight/kg body weight | ||||||
Quotation and Ordering
Creative Bioarray's microsomal stability assay can be further extended to metabolite profiling and give out more information about the metabolism of your compound. S9 stability assay and hepatocyte stability assay are also available at Creative Bioarray. Liver S9 fraction (Cat. CSC-C4093X) as a product can be used in genotoxicity tests such as the Ames test as a metabolic activator. To find out more about our services and products, please feel free to leave a message below, or contact us.
Reference
- LeCluyse, Edward L., and Eliane Alexandre. "Isolation and culture of primary hepatocytes from resected human liver tissue." Hepatocytes. Humana Press, 2010. 57-82.
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