Testing to assess the metabolic fate of an investigational compound has moved away from animal models for two reasons: it’s increasingly seen as unethical and it’s increasingly obvious that it doesn’t work.
"There are too many species differences," says Albert Li, PhD, president and CEO of In Vitro ADMET Laboratories, Columbia, Md. "I realized some time ago that testing with animals simple wasn’t adequate," and that’s when he set out to create in vitro systems that could mimic the in vivo activities required. To that end, Li, while working at Monsanto, headed the team that invented the so-called artificial liver. Later, post-Monsanto, he refined the methods for the cryopreservation of hepatocytes.
Turning to the application at hand, drug metabolism, Li helped pioneer methods for the use of microsomes enriched with enzymes from the endoplasmic reticulum, the cellular site of most metabolic activity.
"That went on for about 10 years, but we came to discover that you don’t always get the right answer because we were only observing oxidation, and not phase two conjugation reactions, which can occurs simultaneously," he says. So, after tearing the system apart, Li decided to put it all back together.
"The gold standard is intact liver cells," says Li. "But really, the next challenge is to design a system with multiple organs—metabolism is not just in the liver," thus, the design of the Integrated Discrete Multiple Organ Culture plate (IdMOC).
Simply put, IdMOC is a big well with smaller wells inside, with each well having cells from different organ. "This way a drug is mixed with the media and is then metabolized by all the different cell types simultaneously," Li explains.
Li’s remaining work is to optimize the assay. "Liver aside, other cell types are not well represented. We need heart, lung, kidney, neurons…," he continues. IdMOC will expand as the desired cell lines become available.
A potential source for any cell type is an embryonic stem cell (ESC). Investigators in Europe—where the ethical issues with ESCs are not quite so tangled—have recently published on using ESCs to produce and study cardiomyocytes and hepatocytes in metabolism assays.1
As with any system, there are pros and cons. The most obvious pro in the approach is the elimination of a liver donor. "I can get millions of cells this way," says Professor Carl-Fredrik Mandenius, PhD, Linköping University, Sweden. Further, this approach does away with the use of immortalized cells that may not behave as cells do de novo.
ESCs also allow for cells banks of individuals of a specific genetic/health profile. Certainly it would be advantageous to have a ready source of hepatocytes from a diabetes patient.
The drawback is the milieu—the cells, regardless of source, are out of physical place. "That’s the challenge, recreating the architecture, the environment of the organ," he says. The current focus of his work is the construction of a microorgan device, complete with permeability for perfusion.
"So far the progress in this is quite promising," says Mandenius, pointing out that what he’s trying to enhance is not just place, but time. "We want cultures we can test for 3 to 4 months, really compare how cells are responding after repeated exposure." This aspect is key in many areas of drug testing where long-term exposure to drug is difficult to assess. "After all," says Mandenius, "you have your liver for your whole life."
StemCell Technologies from Vancouver uses stem cells isolated from bone marrow to test drugs for hemotoxicity. The utility of this is to observe if the active pharmaceutical ingredient in question hinders stem cells’ ability to differentiate to all the different cell types found in the blood.
"What you have are primary cells that are immature, that can grow in a semi-solid matrix that allows for differentiation into distinct cell types that can then be distinguished as clones," says Jackie Damen, PhD, director of contract assay services. The company’s bread and butter is the testing of chemotherapeutics, but their analysis has expanded to include antimicrobials and environmental toxins.
While the stem cell method may not give you detailed information on a metabolite, it does inform on the effect of a drug’s metabolism on differentiation pathways. The assay in question is a microscopy study of the cell types present in culture.
"A customer once submitted a compound that we found inhibited erythroid colony development," says Damen. "When I told him, he was ecstatic." The drug was already in the clinic and patients had become anemic, and the client had surmised a secondary effect on erythropoiesis (EPO), rather than EPO progenitors themselves. This information had a direct impact on patient management.
Cells fit to print, print to fit
The gremlin working to skew the results of any cell-based system is architecture, given that a collection of any cells, in situ, is not flat. There is much research underway to construct 3D cultures, and one approach involves cell printing. Wei Sun, PhD, department of mechanical engineering, Drexel University, Philadelphia, explains. "With the cell printing method, it makes it more feasible to build a 3D model. It gives you greater control, especially if you want to make use of different cell types together." Ink jets, ultrasound, or lasers can be used to deposit cells. "We use an extruder and a micronozzle," says Sun. "This way we’re able to exert a specific force on the cell and put it exactly where we want it to go within a 3D structure."
As mentioned, the challenge once cells are deposited is perfusion and viability. Sun believes this can be partially overcome by using adult pluripotent stem cells. He describes one effort underway at Massachusetts Institute of Technology that is building a structure with stem cells, to which a particular growth factor will be added. "What they propose is a device that starts out at T=0," Sun explains. "The device is alive, so essentially we’re looking at 4D," an ability that Sun believes will be useful in a number of ways, including the study of drug release profiles.2
Cells that qualify
Regardless of the cell source, you have to know what it can do. "There’s much more of a demand these days for reagents, which are specifically qualified for an application," relates Charles Crespi, PhD, vice president, BD Biosciences, Bedford, Mass. The characteristics of a particular hepatic cell line were once established in-house in Big Pharma, but these days, Crespi is called on to do it. "We have cells that are prequalified for transport assays, metabolic stability assays …" he says. Knowing this saves the investigator a great deal of time.
The need has to do with the donor. "Every donor is different, old versus young, smoker versus non," says Crespi. The source of the cells determines its relative enzymatic activity.
Customers are also now looking at different types of activity. Generally, for metabolism, investigators are used to looking at CYP450. "Medicinal chemists have actually become very adept at making molecules which are not substrates or inhibitors of P450," Crespi explains. Focus has now shifted to other enzymes.
"We now offer a series of carboxylesterases—they hydrolyze ester groups, which can be important for a lot of prodrugs," says Crespi. A further class of enzymes now available is uridine glucuronyl acyl transferases (UGT). "Looking at UGT is one more way to understand just exactly how your drug is being metabolized, by what individual enzyme," he says. Unlike intact cells, these assays are carried out as purified subcellular fractions. The assays can be purchased, or BD Biosciences can perform the assay for customers.
Crespi declined to be specific about future offerings from BD, but stated that there were still gaps in understanding how a drug is metabolized, "and we’re working to fill those gaps."
About the Author
Neil Canavan is a freelance journalist of science and medicine based in New York.
1. Mandenius, et al. Cardiotoxicity testing using pluripotent stem cell-derived human cardiomyocytes and state-of-the-art bioanalytics: a review. J Appl Toxicol. 2011;31(3):191-205.
2. Chang RC, et al. Microprinting of liver micro-organ for drug metabolism study. Methods Mol Biol. 2011;671:219-38.