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Science & Technology

June 4, 2007
Volume 85, Number 23
Web Exclusive

Appraising Penetration

Drug Developers Rely On A Variety Of Tools To Figure Out Whether Their Lead Gets Into The Brain And Then To Its Target

Sarah Everts

TO COMBAT the formidable challenge of developing drugs that can treat neurological diseases and cross the blood-brain barrier (BBB), those in the research trenches employ a variety of tools—from in vitro models of the BBB to imaging techniques—to design and assess the merits of new leads.

Reproduced from J. Cell. Biol./© Rockefeller University Press
Left Out A red dye injected into a normal mouse cannot cross its blood-brain barrier (left). Transgenic mice lacking a protein required to create the tight junctions between BBB cells cannot sequester the brain from the dye.
View Enlarged Image

At the outset, most medicinal chemists in industry use computer modeling algorithms that predict BBB permeability to assess the promise of their small-molecule leads. The software, either purchased or developed in-house, considers the compounds' physicochemical properties, such as molecular size, hydrophobicity, hydrogen-binding potential, and solubility.

But many nonphysicochemical factors also can interfere with successful delivery of a brain drug. For example, a lead might get into the brain but be recognized by so-called protein efflux transporters that pump it back out. Or the active compound might not be the lead but rather some metabolite of the lead that comes out of the liver, thereby confounding BBB penetration predictions based on the lead. So although newer algorithms strive to include these complicating factors, in silico assessments are generally used to guide—but not dictate—design, says Stephen Hitchcock, director of medicinal chemistry at Amgen.

With high-throughput screening a mainstay of modern drug development, a good in vitro model of the BBB is essential. The challenge for such model makers is that the tight-knit endothelial cells that form the BBB have very picky tastes. In the niche of the brain, nearby astrocyte and pericyte cells provide chemical cues that tell BBB endothelial cells to express the proteins that make the tight junctions of the barricade.

"The problem is, when you take endothelial cells out of the brain and grow them in the petri dish, there's a profound down regulation of almost all the BBB-specific genes," says William Pardridge, director of the Blood-Brain Barrier Research Laboratory at the University of California, Los Angeles. This means a model BBB can be leakier than its real-life counterpart, allowing leads that might not penetrate the BBB in vivo to cross it in vitro.

To further complicate things, drugs that actually do cross the BBB can be missed in the lab if the expression of certain BBB-specific genes is reduced in a model BBB. For example, reduced expression of BBB-genes in vitro could mean a lack of nutrient transporters that some drugs, such as the Parkinson's disease treatment L-dopa, rely on for crossing the BBB.

"If you screen L-dopa as a potential drug to cross the BBB with an in vitro model, you would conclude it doesn't cross," Pardridge notes. Pardridge and others challenge the industry's formerly widespread reliance on a BBB in vitro model comprised of dog kidney endothelial cells. Although the kidney cells do have some of the tight junctions of brain BBB cells, they do not originate from the brain and are not exposed to the chemical cues in that niche that lead to the BBB.

There's a potpourri of strategies to improve in vitro models. For example, Eric Shusta, a biomedical engineer at the University of Wisconsin, Madison, is at the early stages of developing a BBB in vitro model. Shusta notes that the model "combines brain endothelial cells with differentiating neural stem cells in a dish to help provide some of the necessary chemical cues found in vivo."

Others trying to develop improved BBB models, like the start-up Cellial Technologies, based in Lens, France, have in vitro models based on brain endothelial cells, which express efflux as well as nutrient transporters. Researchers there initially developed a model made from bovine brain endothelial cells that required culturing them for 12 days with other brain cells to elicit expression of BBB-specific proteins. This initial model was too labor-intensive for the pharma clients they were seeking, says Romeo Cecchelli, a pharmacologist at the University of Artois, whose lab spun off the start-up. The company has recently come up with a way to get the brain endothelial cells to form the tight junctions of the BBB in vitro in only four days. The method relies on a cocktail of chemicals instead of the coculturing of several cell types. Cecchelli hopes this approach will be more user-friendly in the high-throughput screening setting favored by the pharmaceutical industry.

ANOTHER STRATEGY is to side-step the living cell model altogether. For example, one of the in vitro models used by Wyeth Research medicinal chemists is a membrane composed of brain lipids, says Edward Kerns, the company's associate director of chemical and screening science.

Instead of trying to find an in vitro model that wholly emulates the BBB, Kerns and his colleagues aim to develop in vitro screens that accurately test for a specific characteristic of molecules that tend to cross the BBB—for example, lipophilicity or the absence of recognition by efflux pumps.

"We use in vitro models diagnostically," Kerns says, "not to entirely reproduce the BBB in a dish." He says they use the in vitro models to assess what is going right or wrong in the in vivo models.

Scientists have developed several animal models in order to evaluate some BBB characteristics. For example, drug developers can check whether a lead is getting cleared by an efflux pump in vivo by comparing the distribution of a drug in the brain of a normal mouse to that of a knockout mouse engineered to lack the BBB efflux pump.

Another major issue for researchers is assessing whether a lead is traveling further than just over the BBB. "We want to make sure a lead is reaching its target in the brain," says Richard Hargreaves, vice president of imaging and proteomics at Merck Research Laboratories. When testing whether a lead makes it to its neural target, researchers send across the BBB a radiolabeled positron emission tomography (PET) tracer designed to bind to the same target. Then they deliver their putative drug and hope that it finds its target, which they measure by watching the PET signal decrease as the tracer is displaced by the drug.

Hargreaves says the technique reveals whether the drug lead is hitting the target, as well as whether this interaction has the ideal pharmacological impact. For example, an antiobesity lead Merck scientists were testing did successfully cross the BBB and hit the right target, staying there "24–7" as revealed by PET imaging, says Hargreaves. But it showed poor efficacy in early clinical trials. Merck subsequently abandoned the lead because the imaging data indicated that improvements in BBB penetration or targeting were unlikely to help.

The combination of these tools, from in vitro screening to PET imaging, is helping researchers gauge whether a given drug lead is crossing the BBB to reach the brain and also whether it is hitting the right therapeutic target.

more on this topic

  • Brain Barricade
  • Researchers are tackling the grueling challenge of getting brain therapies across the blood-brain barrier
  • WEB EXCLUSIVE: Appraising Penetration
  • Drug Developers Rely On A Variety Of Tools To Figure Out Whether Their Lead Gets Into The Brain And Then To Its Target
Chemical & Engineering News
ISSN 0009-2347
Copyright © 2011 American Chemical Society

more on this topic

  • Brain Barricade
  • Researchers are tackling the grueling challenge of getting brain therapies across the blood-brain barrier
  • WEB EXCLUSIVE: Appraising Penetration
  • Drug Developers Rely On A Variety Of Tools To Figure Out Whether Their Lead Gets Into The Brain And Then To Its Target

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