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Blood-Brain Barrier Bridged By Molecular Packaging Technique

• Attaching "targetor" and cholesterol moieties to peptides enables conjugates to slip past brain9sprotective barrier Stu Borman, C&EN Washington

A research group at the University / % of Florida has found a way to

J L j L deliver higher levels of peptides to the brain, a development that could lead to potential treatments for Alzhei­mer's disease, Parkinson's disease, and other afflictions of the central nervous sys­tem (CNS). The new technique is based on a molecular packaging concept that permits peptides to slip past the brain's natural protective barrier and then pre vents them from slipping back out.

The technique was developed by Nicholas S. Bodor and coworkers at the Center for Drug Discovery of the Uni­versity of Florida, Gainesville [Science, 257, 1698 (1992)]. The university has filed for a patent on it.

The major impedi­ment to development of CNS-active peptides is the blood-brain barrier, a set of specialized endo­thelial cells that forms the lining of blood capil­laries in the brain. Tight junctions between these cells prevent the indis­criminate passage of sub­stances from the blood into the brain.

To enter the brain, peptides must therefore pass through these en­dothelial cells, rather than around them. Sub­stances must be lipo­philic (lipid-soluble) to diffuse 'into endothelial cells. Hydrophilic (wa­

ter-soluble) nutrients, like glucose and essential amino acids, can enter the cells, but only by mediated transport, a pro­cess by which carrier molecules and channel-forming ionophores shepherd desired substances across cell mem­branes. Because most peptides are hy­drophilic, they cannot cross endothelial membranes on their own, and specific transport systems to carry them across are limited.

If peptides do manage to pass through the blood-brain barrier, they face peptide-degrading enzymes that will readily chew them up. And if they escape that fate, they face another im­pediment—the fact that, by an equilibri­um process, they may simply diffuse back out of endothelial cells as quickly as they diffused in.

Researchers have tried several strate­gies to get peptide drugs past these ob­structions. These include:

• Injecting peptides directly into the intraventricular region of the CNS.

• Using drugs to temporarily loosen the tight junctions between capillary endothelial cells.

Packaged enkephalin peptide (Tyr-Ala-Gly-Phe-Leu) passes through blood-brain barrier and produces prolonged analgesic effects

• Covalently linking small peptide drugs to larger peptides, creating "chi­meric" peptides for which specific re­ceptor-mediated transport systems ex­ist.

• Creating peptide "prodrugs"— lipophilic esters or amides capable of entering the brain and then being hy-drolyzed to active forms.

However, all these strategies have drawbacks that have prevented their widespread implementation. Direct in­jection of drugs into the brain or spinal cord is invasive and tends to deliver peptides only to the surface of the brain (because of rapid breakdown by peptidolytic enzymes). Procedures in which the spaces between endothelial cells are opened have been associated with some severe side effects, including inflammation, encephalitis, and sei­zures. It can be difficult to get pharma­cologically significant amounts of chi­meric peptides into the brain because of the inherently limited capacity of some receptor-based transport systems. And once prodrugs are past the blood-brain barrier they can diffuse right out

again, or can be rapidly cleaved by peptidases.

Now, Bodor and co­workers have demon­strated the brain deliv­ery and in-vivo activity of two enkephalin pep­tides by "packaging" each peptide with two molecular groups. A cholesterol moiety dress­es the peptide wolf in sheep's clothing by dis­guising its essentially hydrophilic nature. And a "targetor" moiety (sep­arated from the peptide by a "spacer" group) locks the peptide in the brain and throws away the key. These modifica­tions not only increase the lipid solubility of a

OCTOBER 5,1992 C&EN 57

SCIENCE/TECHNOLOGY

Reactions in brain produce active drug

Cholesterol group

"Targetor"

CT' Ν I

CH3

Spacer

CH3

Peptide

CO —NH—CH—CO 4-YAGFL-f-O

I CH3

CH3

CO—NH—CH—CO

CH3

• Passive transport through the blood-brain barrier

• Oxidation

-YAGFLH-0

Enzymatic removal of cholesterol

CO—NH—CH—C0 4-YAGFL

*N+

I CH3

Enzymatic release of free peptide

YAGFL

Note: Targetor"-spacer-peptide conjugate and free peptide are believed to be active forms of drug. In peptide, Υ = tyrosine, A = alanine, G = glycine, F = phenylalanine, and L = leucine

peptide—thus permitting it to pass through the blood-brain barrier—but they also target the drug specifically to brain tissue and impart strong resistance to breakdown by peptidases.

The large, lipophilic cholesterol group both increases the lipid solubility of the drug and protects the peptide from be­ing recognized by peptidases inside the blood-brain barrier. The researchers are investigating the possibility that the bulky lipophilic group may also protect peptides from breakdown in the diges­tive system, which would enable them to be administered as oral drugs.

Specific targeting to brain tissue is ac­complished by the 1,4-dihydrotrigonel-linate targetor. A unique feature of the blood-brain barrier is that lipid-soluble neutral species can flow in, but positive­ly charged species can't flow out. After the packaged drug passes through the blood-brain barrier, the targetor is oxi­dized enzymatically to a positively charged trigonellinate salt, trapping the drug on the brain side of the barrier. The targetor also tends to concentrate drugs specifically in brain tissue because its ox­idation in peripheral (non-CNS) tissues leads to its rapid elimination by the kid­ney and bile—a process that doesn't oc­cur in the brain.

Upon intravenous administration of the packaged peptide to rats, Bodor and coworkers observe a significant and long-lasting delay in the tail-flick re­sponse, a test of sensitivity to pain— indicating that the conjugate reaches the brain and produces prolonged analgesic effects. No analgesic effects are ob­served, however, after intravenous ad­ministration of free enkephalins or en­kephalins that are only partially conju­gated (with either the targetor or the cholesterol group).

Some time after a packaged drug passes the blood-brain barrier, the ester linkage with the cholesterol group un­dergoes enzymatic hydrolysis, leaving the targetor-peptide conjugate. The free peptide may also be released slowly from the targetor-peptide conjugate. "The activity we see in the brain is prob­ably a combination of the intrinsic activ­ity of the locked-in form [the targetor-peptide conjugate] and the activity of the released peptide," says Bodor.

The targetor concept was first devel­oped in the early 1980s, also by Bodor and coworkers. However, it took 10 years to figure out just the right way to put the targetor, spacer, and lipophilic

group together in a package that works reliably.

What is the upper limit on the size of the peptide unit in a packaged peptide? The researchers don't know exactly. However, says Bodor, "Based on the ease with which we could deliver the enkephalin peptides—which have five amino acids—I think we could go up to 15 residues. That would cover a large number of very important peptides."

Bodor's group has found that, in addi­tion to enkephalin peptides, the packag­ing technique works on peptides related to Alzheimer's disease. Bodor says: "Many other peptides are active in the CNS— affecting blood pressure, the ac­tivities of various hormones, and analge­sia, for example. There are many aspects of brain function that could benefit from this work."

At least one researcher remains un­convinced that all pertinent questions on passage of peptides through the blood-brain barrier have been answered. Neuroendocrinologist William A. Banks of Veteran Affairs Medical Center, New Orleans, who specializes in techniques for passage of peptides across the barri­er, calls the study by Bodor and cowork­ers "novel and interesting," but believes the researchers "have overstated the de­

gree to which the blood-brain barrier is impermeable." He says his lab and oth­ers have shown that if s possible to en­hance the CNS activity of peptides "without a lot of fancy chemistry" just by making the peptides more lipid solu­ble or by taking advantage of peptide transport systems.

"It's true that lipidization of peptides can somewhat enhance penetration," replies Bodor, "but lipidization is not enough. If we put just cholesterol or another lipid function on the peptides, we don't see any activity."

Banks also contends that Bodor and coworkers should have done key tests with opiate antagonists that would have unequivocally proven that the analgesic effects observed were caused by passage of enkephalin analogs into the brain. "Many believe that opiate peptides can induce analgesia by acting outside the brain, at peripheral sites," he says.

However, Bodor says his group ac­counted for this possibility by making direct comparisons with unmodified peptides. "We have used mass spec­trometry to identify specific peptide derivatives in the brain and have seen long-term activity," he states. "This dif­ferentiates our work from all previous attempts." Π

58 OCTOBER 5,1992 C&EN