Microsomes are now available in different activity levels, giving ADME researchers more options when screening new drugs.
For half a century, researchers have relied on microsomes as key reagents for identifying and screening new chemical entities (NCEs) for drug development. Yet, the full potential of these vesicular fragments of endoplasmic reticulum has not been realized. New processes of pooling human liver microsomes for application-based studies are expanding the research horizon. Researchers may now elect to use reagents of varying activity levels appropriate to their specific objectives in order to expedite ADME-tox screening in the laboratory. The same microsomes from days gone by are tomorrow’s hope for a new era marked by greater choice—an era that will better serve the increasingly specific requirements of ADME professionals.
Yesterday: The downsides of random sampling
Traditionally, research microsomes have been derived using a method that draws from a random sampling of donors to create a pool presumably representing an “average” population. Once pooled, the microsomes are tested for their activity levels. More active microsomes will metabolize chemical entities at a greater rate than will less active reagents. The consistency of activity levels from one product lot to another is dependent on the uniformity of the donors over time.
Pools of “average” microsomes present some known challenges for ADME testing. For one, it can be difficult, or even impossible, to measure inhibition values when CYP activities are insufficient to accurately gauge using available analytical methods. Also, when the number of individual donors is limited, the introduction of new donors over time can cause pooled activity levels to fluctuate. These fluctuations require researchers to re-validate new reagents in comparison to previous lots. As a result, laboratory productivity is reduced and accurate data capture becomes more challenging.
Today: The benefits of purpose-pooled microsomes
To overcome the challenges of using average microsomes for ADME testing, a new approach has been developed. Testing the CYP activities of microsomes prior to pooling allows the manufacturer to customize the blend per specific research objectives and ensure consistency across lots. Customizing, or “purpose-pooling”, microsomes for their eventual application begins by targeting specific enzyme activity levels and may be refined to mimic the variations that are prevalent in different populations. Lots may be tailored to represent demographically-defined qualities such as gender and ethnicity, or to control for population characteristics such as smoking and alcohol use. Customization is the future of ADME testing because some populations demonstrate genetically-defined levels of CYP activity that are not matched by the “average” microsome pool commonly used in research today.1
The diversity generated from purpose-pooling microsomes is advantageous in the context of the various in vitro applications. High-activity microsomes exhibit higher rates of drug metabolism, while moderate-activity microsomes yield a more “average” metabolism. Depending on the objectives of the research and the NCE in question, either pool may be appropriate. For example, high-activity microsomes may be beneficial for studying inhibition or metabolic profiling, where large amounts of metabolites and activities are desirable. Moderate-activity microsomes may be best utilized for in vitro-in vivo extrapolation (IVIVE), where “average” activities are required.
However, the use of purpose-pooled microsomes should not be defined so narrowly. Indeed, moderate-activity lots may not always be appropriate for every stability study. Each blended lot has a legitimate role in metabolism and drug-drug interaction assays based on research objectives and expectations. Researchers are quickly developing a proficiency for selecting purpose-pooled microsomes as they come to appreciate the benefits of customization.
Purpose-pooled microsomes also facilitate high-throughput screening (HTS). Via HTS, researchers can quickly conduct thousands, or even millions, of tests using automation. The consistency of lot activity levels made possible by the purpose-pooling methodology offers an ideal reagent for large-scale HTS projects. Additionally, the high-activity microsomes may be diluted to lower total protein concentration while maintaining sufficient activities, thereby reducing nonspecific protein binding of compounds. Furthermore, dilution of high-activity microsomes effectively decreases the cost per sample. These benefits are harbingers of the great potential of purpose-pooling.
Tomorrow: The future of cyropreserved hepatocytes
With research professionals facing more choices when it comes to microsomes, the industry is looking to the next logical steps. All eyes are focused on purpose-pooling cryopreserved hepatocytes. Providers of in vitro and ADME-tox products are further refining production methods of both microsomes and hepatocytes for more granular population-based studies. With the promise of increased productivity in the laboratory, greater attention is being given to new data management capabilities, including in silico modeling tools and better IVIVE models.
Experts are increasingly espousing the need for more information and data related to hepatocyte use in ADME testing. Many believe that hepatocytes provide a more in vivo-like result. Currently, most research utilizes individual donors for hepatocytes, but the desire for pooled hepatocytes that can mimic an “average” population response drives the creation of new purpose-pooled products. The obvious progression, then, is for companies that provide hepatocytes to produce hepatocyte pools with targeted enzyme activities, just as seen with microsomes.
While hepatocytes will play an important role, microsomes continue as the current gold standard of most ADME testing.2 The proven benefits of the purpose-pooling method offer a future vision of new contributions yet to come. Soon, highly-customized microsomes will be used for specific population-based studies. For example, the schizophrenic patient population includes a disproportionately high number of individuals who smoke cigarettes. Smoking induces CYP1A2. Because many commonly prescribed schizophrenia drugs are metabolized by CYP1A2, smokers metabolize their antipsychotic medication at higher rates than non-smokers, thus reducing drug efficacy.3
In order to understand the effect of an NCE on this, or any, specific population, there is a need for customized microsomes because the average pool is less relevant. Likewise, there is a growing need to steer research towards customized microsomes for polymorphisms defined by genetic pools. With made-to-order reagents, research could be targeted to the African-American, western European, or any other genetically-distinct population.
The future for ADME research looks especially bright when aligned with the recent developments in the area of in silico data analysis. Once researchers have obtained in vitro data, in silico data mining uses a computer modeling platform to simulate responses in virtual populations. Via in silico analysis, researchers can augment their own in vitro data with databases containing human physiological, genetic and epidemiological information to forecast pharmacokinetic behavior in “real world” population.
More robust and customized tools for ADME research are paving faster, more cost-effective roads toward life-changing pharmaceutical therapies. Purpose-pooled microsomes and hepatocytes are chief among these tools. When used to perform HTS and augmented by the power of in silico modeling, a new generation of purpose-pooled reagents holds promise for more targeted, cost-efficient results.
1. Murray M. Pharmacogenetic variation in drug oxidizing CYPs: impact on drug therapy, drug safety and drug interactions. Current Pharmacogenomics. 1(3):159-173, 2003.
2. Dutton G. Using ADME data to cut drug toxicology failures. Genetic Engineering and Biotechnology News. 27(14), 2007.
3. Bozikas VP, Papakosta M, Niopas I, Karavatos A, Mirtsou-Fidani V. Smoking impact on CYP1A2 activity in a group of patients with schizophrenia. European Neuropsychopharmacology. 14(1):39-44, 2004.
About the Author
Timothy A. Moeller is a scientific advisor at Celsis In Vitro Technologies. He has been with Celsis for six years, in sales and marketing, research and development, CRO and product operations.
Ji Young Lee, Ph.D. is a scientific advisor at Celsis In Vitro Technologies. Her expertise spans sales, marketing and product operations, and she has served in these roles at Celsis for the past three years.
This article was published in Drug Discovery & Development magazine: Vol. 12, No. 1, January, 2009, pp. 18-20.
Filed Under: Drug Discovery