It was almost three decades ago, but Kim Brouwer, PharmD, PhD, still remembers a young patient she met as a pharmacy resident at University of Kentucky’s Albert B. Chandler Medical Center .

“There was a little boy who came in with Reye’s Syndrome, a rare condition characterized by swelling in the liver and the brain that can affect children recovering from a viral infection, which is often triggered by aspirin,” Brouwer says. “You should never give aspirin to children, but thirty years ago they didn’t know that.

“This little boy ended up dying because his liver just shut down. I remember thinking what a shame it was that we didn’t have a better understanding of what caused drug-associated liver injury, and why we couldn’t have done a better job predicting who would be susceptible to that sort of reaction.

“This incident certainly caught my attention with respect to drug disposition and the liver. I was interested in pharmacokinetics, so how the liver handled drugs was a natural fit. I developed a strong interest in this area because of many experiences as a pharmacy resident.”

During graduate school, Brouwer studied pharmacokinetics and liver metabolism/disposition of drugs. She later completed her postdoctoral training with a professor who was internationally recognized for her research in liver impairment associated with pregnancy and drug therapy.

“During my training, I realized that there was a career’s worth of work to be done in understanding how the liver handles drugs, and why some drugs cause liver damage,” says Brouwer, chair of the School’s Division of Pharmacotherapy and Experimental Therapeutics.

She has indeed made a career out of studying drugs and the liver, and her knowledge on the subject is something of a rare commodity.

“Very few labs in the United States have focused on these important research questions,” she says. “Worldwide there are just a handful of investigators that have expertise in this area.”

The Liver’s Drainage Compartment

After a drug is taken orally and is absorbed into the bloodstream, it first goes the liver, where it comes in contact with hepatocytes, cells that contain the liver’s metabolic machinery. If the drug is taken up by the hepatocytes, it can be metabolized and/or excreted back into the bloodstream or into bile, which Brouwer calls the drainage compartment of the liver.

While the blood carries its portion of the drug to other parts of the body, the bile drains into the intestines, where some drug may be reabsorbed. Brouwer says researchers have only a limited understanding of the processes by which drugs are excreted into bile and the extent of reabsorption back into the blood.

“That’s because it’s not easy to reach this drainage system,” she says. “It’s all internal. It’s only in people that have liver disease where they have exteriorized bile flow or some type of surgery that we even have access to that drainage compartment.

“One of the major obstacles to progress in this field was the abilty to access, or at least quantitate in some way, the excretion of drugs in bile.”

About five years ago, Brouwer’s lab developed a simple method to collect bile from healthy individuals. A volunteer swallows an multi-channel tube which is localized to the region of the intestine just below the stomach, where the bile drain dumps from the gall bladder into the intestines. A balloon at the end of the tube is positioned in the intestine below this drainge site. The balloon can be inflated to block the bile from flowing futher down the intestine. When the volunteer is given the naturally-occurring hormone cholecystokinen, the gall bladder contracts and expels bile into the intestine where the bile is trapped by the inflated balloon. Researchers can then siphon out the bile in the intestine through aspiration ports in the multi-channel tube and examine its contents, including the amount of drug excreted in the bile.

Being able to measure how much of a drug is taken up by the liver and drained into bile may be important in determining the efficacy or toxicity of the compound. Drugs and/or metabolites that are active in another part of the body need to get back into the blood from the liver in order to be effective. In other cases, the metabolites that are formed in the liver could cause toxicity if they are secreted into the intestines via bile.

“What’s happened over the last several decades is that medicinal chemists have learned to design molecules that are metabolically stable, so they are not extensively metabolized by the liver,” Brouwer says. “But their structural features make them more likely to be excreted in bile, so there’s the need to be able to really understand the extent of biliary excretion and, in some cases, to try and minimize that uptake and excretion process which removes drug from the circulation.”

An In Vitro Simulation

Obtaining biliary excretion data in humans using the novel approach that Brouwer developed is expensive. In addition, because in vivo methods involve giving a drug to humans, the compound has to be fairly far along in development before it can meet the safety guidelines for a clinical trial.

In an effort to predict hepatic uptake and biliary clearance using an in vitro system, Brouwer and her research team, in collaboration with Ed LeCluyse, PhD, developed B-CLEAR. Isolated hepatocytes from rat or human liver are cultured in a sandwich configuration, between two layers of collagen. Within several days, the cells develop a connecting network of bile ducts, simulating the structure of the liver.

Using this system, researchers can examine the disposition of a drug in side-by-side culture dishes. In one dish, the bile ducts are sealed so that the bile secreted from the cells is trapped. In the other dish, the ducts are open, allowing the bile to drain out of the system. Calculating the difference between the amount of drug in the two cultures provides a measurement of how much of the drug is drained via bile.

“It’s a pretty straightforward way to get an estimate of biliary excretion,” Brouwer says. “We’ve done a lot of work showing that the data correlates very well. So what you measure in vitro predicts what you measure in vivo.

“The pharmaceutical companies need to know whether a candidate compound will exhibit low, intermediate, or high biliary clearance in humans. The compound may not be a very useful drug if biliary clearance is really high, meaning that the compound might be excreted in the bile before any measurable concentrations could be measured in the blood of patients.”

With B-CLEAR, researchers can test for biliary clearance relatively early in a drug’s development instead of waiting until the compound is deemed safe for clinical trials. Based on the in vitro results, they might tweak the chemical structure of the compound to optimize uptake and excretory properties.

“To do that effectively, you have to have good in vitro screening systems,” Brouwer says. “Right now, that’s where the limitation is in the science. Other than this system and transfection systems, which have some limitations, good screening systems are not available. But the goal is to have a screen so the chemist can look at a series of analogs and can measure how adding a ring structure or a hydroxyl group could affect the extent of uptake and/or biliary excretion.”

Going into Business

In 2001, UNC-Chapel Hill licensed B-CLEAR to Qualyst, a start-up company co-founded by Dhiren Thakker, PhD, Gary Pollack, PhD and Brouwer. The company uses the technology to screen drug compounds for pharmaceutical companies and also sells B-CLEAR kits.

“The pharmaceutical industry is very excited about this technology,” Brouwer says. “There’s a lot of interest right now in how to use this system, and how much information can be extrapolated to humans from it. It’s a very exciting time.”

Brouwer credits Thakker for taking the lead in making the company a reality despite challenges that arose after the 9/11 terrorist attacks, which occurred when the business was just getting started. Brouwer chairs Qualyst’s scientific advisory board but says she prefers to stay hands-off on a day-to-day basis and focus more on the research aspect instead.

“For me, it’s about the science—how we can develop tools to make better predictions about potential drugs. To develop the right tools, you have to understand the mechanisms, so I continue to be very focused on how can we use the system to understand what’s going on,” she says.

Brouwer says companies using B-CLEAR usually start off by testing it with compounds for which they have in vivo data, in order to gauge the accuracy of predictions. Once satisfied with the system’s accuracy, companies then use it to screen compounds for which they have no in vivo data.

“Initially there was some skepticism, but as more and more data have been generated, with all different types of compounds from many different companies and academic laboratories, it’s looking to be very predictive,” Brouwer says.

She says one of the challenges is that it takes a while for a new technology to catch the attention of the pharmaceutical industry. On the other hand, companies are quick to abandon a technology if they are not satisfied with the results.

“There’s kind of a narrow window of time that we have to establish the methodology,” she says. “There’s a lot that needs to be done. We’ve focused mostly on rats and humans. Pfizer, in collaboration with us, generated some monkey and dog hepatocyte data. I see a lot of applications for the technology, way beyond what we’re currently using it for.”

Refining the System

Brouwer is currently working to optimize the B-CLEAR in vitro system she helped develop, as well as in the process of obtaining two additional patents for the technology. She would like to be able to apply this system to examine how disease states and drug interactions affect the biliary clearance of compounds in humans.

Her research is also looking at what determines whether a drug will be drained into bile or back into blood. If researchers can find a way to direct the metabolite into one or the other, it could be a powerful tool.

“You might be able to minimize toxicity,” Brouwer says. “If the compound was causing toxicity in the intestinal track when excreted into the gut, you might be able to divert it into blood for urinary excretion. There’s a lot of work to be done in that area.”

Brouwer is also trying to understand whether some drugs accumulate in certain regions of the cell, and whether that could be the reason for their toxicity. Currently, when researchers measure the amount of drug in the body, they usually measure the concentration in blood, urine, or bile, but they can’t measure the concentration inside the cells.

“It might be that the cells are accumulating large amounts of some drugs, and that might cause toxicity in some patients,” Brouwer says. “But we can’t see that because we’re not measuring that compartment. So developing tools like imaging methods to look at cellular accumulation of drugs is also an area we’re looking at with this system.”

Drug-Induced Liver Injury

Another topic that Brouwer is studying with her in vitro system is drug-induced liver injury.

“You wonder, as sophisticated as our technology is, how could we get drugs to humans and not realize we have a problem until a number of people die,” Brouwer says.

“It turns out that this model system may be useful in helping us predict some of that toxicity early in the development process, or at least give us indications that there may be certain people, certain disease states, people with certain genetic expression levels of their proteins that might handle the drug differently.”

Brouwer says that many cases of drug-associated liver injury involve a normal, healthy individual having a bad reaction to a common drug. Many such instances, she added, have been attributed more to unusual circumstances—such as a combination of medications that produced a reaction—rather than to the drug.

“But if you go back and look at the literature, the most common cause of fatal liver injury is Tylenol,” she says. “The statistics are pretty impressive. It’s clearly a drug that would never make it to market today because of the safety margin. There have just been so many deaths reported with it.

“But it is an effective drug. With drugs, it’s always a balance between the dose that gives you efficacy and the dose that gives you toxicity. Sometimes it’s a very fine line.”

Other Projects

Brouwer is also conducting a couple of clinical studies. One is aimed at predicting a more accurate dose of the anti-cancer drug sorafenib. Currently the drug is dosed based on body weight. Brouwer hopes to be able to use imaging agents and pharmacokinetic models to predict how a patient’s liver handles the drug, making it possible to tailor the dose to better fit each individual. The other clinical study looks at the effects of ritonavir, an anti-retroviral drug used in HIV therapy. In addition, Brouwer is part of an international group that is writing an FDA white paper on drug transporters, which is expected to be published in fall 2009.