In the Lab | Microsomes Jump Into the Testing Pool

Pooled human liver microsomes help predict safety

Obtaining accurate and reliable results when building a house or fixing a car requires the appropriate tools. While this is also true for drug discovery and development, many of the tools that worked accurately and reliably a few years ago are no longer the best tools for the job. Technology changes rapidly, and scientists must change along with it. High performance liquid chromatography, for example, has given way to liquid chromatography/mass spectrometry, which yields more sensitive and accurate measurements. Until recently, inhibition and clearance testing of new chemical entities (NCEs) were conducted using a mixed pool of microsomes. But other tools have been developed, including "purpose-pooled" human liver microsomes.

Over the past decade, the prediction of drug-drug interactions (DDIs) from in vitro studies has become a rapidly expanding field of research. Any NCE introduced to the human body may increase or decrease drug metabolism either by induction or inhibition of enzymes. This is a major contributing factor to adverse drug interactions, because changes in enzyme activity may affect the clearance of various drugs. If one drug inhibits the body’s ability to metabolize another drug, the second drug could potentially accumulate within the body to toxic—and sometimes fatal—levels.

For example, terfenadine (Seldane; Marion Merrell Dow, Inc., Kansas City, Mo.), when administered with cytochrome P450 (CYP) CYP3A4 inhibitors like diltiazem, can cause adverse cardiovascular events, including a life-threatening cardiac arrhythmia called torsade de pointes. Due to these unforeseen side effects, Seldane was removed from the market in 1998. Known DDIs do not necessarily rule out the efficacy of a drug, but they affect labeling to account for dosage adjustments or alternate drug selections that enable concomitant medications without adverse interactions.

Celsis In Vitro Technologies (Baltimore, Md.) has developed the purpose-pooling of InVitroCYP human liver micro- somes (HLMs) into distinct classes of high and moderate cytochrome CYP activity, delivering more sensitive metabolism data for researchers.

Time for Change

Any NCE introduced to the human body may increase or decrease drug metabolism either by induction or inhibition of enzymes. This is a major contributing factor to adverse drug interactions, because changes in enzyme activity may affect the clearance of various drugs. If one drug inhibits the body’s ability to metabolize another drug, the second drug could potentially accumulate within the body to toxic—and sometimes fatal—levels.

The Food and Drug Administration (FDA) requires that all NCEs be tested for possible DDIs. To do this, researchers need a physiologically relevant biologic model in which to test for possible interactions and assess effects. Currently, CYP enzymes are responsible for about 80% of oxidative drug metabolism and about 50% of the overall elimination of commonly used drugs. For this reason, the FDA and the pharmaceutical industry focus on CYPs above all other metabolic enzymes.

Although these enzymes are present in many tissues of the body, the liver contains more CYP enzymes than any other organ. Liver microsomes are membrane vesicles derived from hepatic endoplasmic reticulum containing CYP and other critical drug metabolizing enzymes. They have long been the workhorse for researchers studying absorption, distribution, metabolism, and elimination (com- monly referred to as ADME).

In conjunction with the development of more sensitive analytical methods, HLMs have been established as key reagents for screening NCEs in an efficient, interpretable, and cost-effective manner. In particular, HLMs allow researchers to perform the inhibition and clearance screenings that are crucial for the clinical safety profile of an NCE during the discovery, development, and post-marketing phases. However, a critical issue has emerged that challenges inhibition studies—namely, the impact of blind-pooling microsomes with low-expressed CYPs.

The FDA’s draft guidance recommends that the metabolism of an NCE be defined during drug development and that its interactions with other drugs be explored as part of an adequate assessment of its safety and effectiveness—with specific and defined assay conditions and acceptance criteria.1 NCEs are often characterized by their IC50 (concentration of inhibitor required for 50% enzyme inhibition). Yet, IC50 values are easily affected by different assay conditions, such as pH level and substrate concentration. For this reason, results from different laboratories cannot be compared reliably. Because the alternative—characterizing a compound by its Ki, an immutable affinity constant—entails significant work, its use in drug discovery has been limited.

Figure 1. Purpose-Pooled InVitroCYP Microsomes: Enzyme Activity Levels by Class, km Values

Recognizing the lack of a standard methodology across NCE studies, the FDA’s guidance document suggests several ways to increase data consistency, beginning with the use of specific probe substrates at or below Km concentrations "if metabolic rate [of the reaction] is sufficient." This allows for closer correlation between a compound’s IC50 and Ki. IC50 determinations require sufficient enzyme activity, however; this can be an issue with CYPs that have low expression in the liver, such as CYP 2C19 and CYP 2D6. To overcome this challenge and to comply with the FDA guidance, either analytical methods must become more sensitive to better measure the lower amount of metabolite formed, or the CYP activities of the HLMs must be increased to a level that allows for accurate detection of inhibition.

Increasing CYP activity of all clinically relevant CYPs in HLMs has not been an option in the past. Microsomes have traditionally been available in a "one-size-fits-all" paradigm regardless of the testing objectives. Pooled microsome products that are available on the market today are blended blindly: The tissue is collected and enzyme activity is tested retrospectively. The manufacturer does not determine CYP activity until after the lot is produced. As a rule, this method yields lots with "average" CYP activity, which may not provide sufficient enzyme activity levels for some assays, like inhibition.

Microsome Selection

Celsis has developed a new approach, purpose-pooling, in which individual donor HLMs are characterized for CYP activity levels prior to pooling. Based on specified criteria, target activities are entered into a proprietary algorithm, resulting in the selection of particular HLMs from the donor library. Because purpose-pooling predicts activity levels prior to manufacturing, the method produces more consistent and reliable microsomes with the desired CYP activity. This approach can be used, for example, to create a pool or class of HLMs with distinctly higher activities of clinically relevant CYP enzymes to meet the unique requirements of inhibition and certain metabolism studies (see Figure 1).

A key requirement of HLMs in the pharmaceutical industry is consistent and reproducible results between lots. This reduces the need for validating new lots or introducing new variables when screening an NCE and, for large pharmaceutical development operations, better harmonization among sites. By utilizing a purpose-pooled approach, InVitroCYP HLMs can be produced within defined ranges or classifications, reducing lot-to-lot variations.

Pooled human liver microsomes ready for the research lab.

Purpose-pooling generates a variety of microsomal products suited for specific uses and divided into three classes: high activity, moderate activity, and customized microsomes. InVitroCYP H-class HLMs are prepared from tissues that are screened and selected for high activity across the panel of relevant CYP enzymes, including those CYPs that are traditionally low in activity. For example, 2D6 is the second most important enzyme in terms of the number of drugs CYP enzymes metabolize, yet it has especially low activity.

When the uninhibited activity is near the lower limit of quantitation, there is a reduced range that can be inhibited. This limitation may prohibit the researcher’s ability to determine IC50. H-class HLMs, however, offer a larger dynamic range of activity. For inhibition studies, results can be determined within both the higher and lower inhibitor concentrations, producing the requisite sigmoidal curve central to determining accurate IC50. Enlarging the target activity range makes a more complete and accurate assessment possible.

Other Research Benefits

H-class HLMs are currently being evaluated for two additional research benefits. In some assays, the objective is to identify the metabolite(s) produced through drug metabolism. In order to make metabolite identifications, the researcher hopes to produce as many of the metabolites as possible in a single reaction. High-activity microsomes have been shown to yield greater volumes of these metabolites. Another potential benefit of H-class HLMs is in high-throughput screening. Because activity is higher, it may be possible to lower the concentration of microsomal protein—reducing both cost per sample and nonspecific protein binding—while obtaining the same end result.

In conjunction with the development of more sensitive analytical methods, human liver microsomes have been established as key reagents for screening new chemical entities in an efficient, interpretable, and cost-effective manner.

Just as H-class microsomes are formulated for activity within a specific range, the InVitroCYP M-class HLMs are designed to reveal the average oxidative drug metabolism of compounds. For traditional stability testing, M-class HLMs are ideal, because they are produced to represent an average human response and, indeed, the enzyme activity levels fall within the midrange. Critical research information can be derived from standard equations and existing in vitro-in vivo extrapolation (IVIVE) models.

Finally, for studies that require specialized microsomes, InVitroCYP C-class HLMs can be designed to match specific research criteria. A C-class preparation can produce microsomes with specific donor demographic requirements such as age, gender, race, or activity level for a particular CYP.

With a multi-class microsome system, researchers have new, better tools to meet specific ADME requirements. Today, researchers are using the new InVitroCYP H-class HLMs for their greater sensitivity in measuring inhibition without compromising the relevance of the data. M-class HLMs are well suited for stability and clearance studies. Both classes can be manufactured in large lots with high lot-to-lot consistency. With time, the process of purpose-pooling may yield even greater specificity in reagent production, allowing for testing methodologies and efficiencies not yet imagined.

Moeller and Dr. Lee are scientific advisors for Celsis In Vitro Technologies. For more information, call (312) 476-1200 or go to www.celsis.com/microsome.

References

1. United States Food and Drug Administration. Department of Health and Human Services. Guidance for Industry: Drug Interaction Studies—Study Design, Data Analysis, and Implications for Dosing and Labeling. Washington, D.C.: FDA; 2006. Available at: http://www.fda.gov/ cber/gdlns/interactstud.htm. Accessed November 17, 2008.