BREAKING THROUGH THE BBB: Some researchers are engineering bispecific antibodies (yellow and green molecules), with one arm serving to help the molecule cross the blood-brain barrier (BBB) while the other arm dictates the drug’s function in the brain.COURTESY OF RYAN WATTS/GENENTECH. FROM SCIENCE TRANSLATIONAL MEDICINE VOL 3, ISSUE 84 (25 MAY 2011). REPRINTED WITH PERMISSION FROM AAAS.When neuroscientist Ryan Watts talks about receptor-mediated transcytosis, he sounds like an orchestra conductor describing his favorite piece of music. To him, the passage of a molecule through a cell membrane via a receptor-assisted vesicle is an art form, and, more importantly, a way into the brain.

In 2004, Watts formed the neuroscience unit at pharmaceutical company Genentech. Right away, he organized a program to develop antibodies against the protein fragment amyloid beta, a component of brain plaques associated with Alzheimer’s disease. But as soon as he began, Watts, like many...

Antibodies actually can get into the brain, just not very efficiently. For every thousand antibodies injected into the blood, only one will find its way into the brain, likely by slipping unnoticed into vesicles crossing the barrier. Unfortunately, that dramatic concentration decrease prevents researchers from developing effective drug treatments, because using higher doses would cause harmful effects in the rest of the body. “We needed a way to get more antibodies into the brain,” says Watts.

Some small, lipid-soluble drugs do cross the BBB simply by diffusion through the cell membrane, and others, like caffeine, enter successfully via specialized transporter proteins. Many larger molecules, such as antibodies and enzymes, however, can’t get through unless one uses a needle or catheter to puncture the BBB with brute force. Not surprisingly, however, such methods often result in dangerous complications, such as infections and tissue damage. So Watts, along with Genentech biochemist Mark Dennis, devised a far more subtle solution to get antibodies to cross the BBB—receptor-mediated transcytosis. Their success surprised neuroscientists and caught the attention of the rest of the pharma industry, which has become eager to identify new ways to breach the BBB.

“Two years ago, if you talked about the blood-brain barrier, you’d hear, ‘Eh, okay, another failure.’ People criticized all sorts of approaches, showing no interest,” says Jean-Paul Castaigne, CEO of AngioChem, a Montreal-based biotech company developing treatments for brain diseases. Today, however, “we are seeing a major shift,” he says. “As often happens, one starts and the others follow.”

Genentech’s early achievements breaching the BBB have opened the floodgates. These days crossing the barrier is in vogue, with numerous companies and academics devising diverse, creative, and sometimes downright wacky ways to pry open a window into the brain. In doing so, they hope to deliver drugs that will treat Alzheimer’s disease, multiple sclerosis, Parkinson’s disease, and many more brain illnesses that are currently intractable.

Ski lift to the skull

A WEB OF VESSELS: The brain is replete with blood vessels—some 500 miles of them—but getting drugs from the blood into brain tissue is limited by the blood-brain barrier (BBB), comprised of tight junctions between the endothelial cells lining brain capillaries. © TO-BBBTo successfully get into the brain to treat Alzheimer’s, Watts and Dennis designed one arm of their antibody to sneak the drug into the brain by binding to the transferrin receptor on BBB endothelial cells, which typically allows the passage of iron. Once inside the brain, the antibody’s second arm binds and inhibits β-secretase (BACE1), an enzyme that processes an amyloid beta precursor, thereby cutting off production of the harmful protein fragment.

But the first antibody they designed to bind to the transferrin receptor was not successful in crossing the barrier. The antibody was being trapped within the capillaries of the BBB, nearly reaching the brain, but unable to break free from the receptor to complete its therapeutic duties—as if a chairlift hauled passengers to the top of a ski slope, but then the passengers failed to jump off.

The researchers wondered if the antibody’s high affinity for the transferrin receptor might be the problem. What if, they thought, the antibody bound the receptor more loosely, so that it could easily let go and fall into the brain? They tested a slew of low-affinity antibodies and found that the strategy worked like a charm. This time, the antibodies hopped off at the top of the ski lift.

Today, Genentech, now a member of the Roche Group, is conducting primate studies with the two-armed antibody, and Watts hopes to soon move the therapy to clinical trials. “It looks really interesting,” he says, with unrestrained glee in his voice. “That’s all I can say.”

Also in the hunt to cross the BBB is AngioChem, which has developed another approach based on receptor-mediated trancytosis. Instead of targeting the transferrin receptor, however, AngioChem’s technology utilizes a receptor in the BBB called lipoprotein receptor-related protein, or LRP-1, a promiscuous protein that binds more than 40 ligands and can transport molecules up to 700 kilodaltons in molecular weight—about 4 times the size of an antibody—across the BBB. By analyzing several of LRP-1’s ligands, the company identified a 19-amino-acid sequence that, like a zip code, homes a package to its destination. Now, the company is adding that zip-code sequence to proteins and therapeutics to deliver them through the LRP-1 door into the brain. The company’s proof-of-concept drug—a treatment for brain cancer that combines paclitaxel, an oft-used drug for brain cancer, with the zip-code peptide—is currently in Phase 2 trials.

The Netherlands-based biotech company to-BBB takes yet another receptor approach: hiding drugs in little balls of lipids, called liposomes, then decorating those liposomes with molecules of glutathione (GSH), a three-pronged peptide that is rapidly taken up into the brain through specialized transporters. The identity of those transporters is currently unknown, says the company’s chief scientific officer, Pieter Gaillard, but they effectively transport the GSH-coated liposomes across the BBB. The company has one treatment for brain cancer based on the widely used chemotherapeutic drug doxorubicin that recently entered Phase 2 trials, and is also actively pursuing treatments for multiple sclerosis and stroke. “There’s no limit of what you can get into a liposome,” says Gaillard. “We’ve gotten whole antibodies, enzymes, and other large molecules inside.”

Nosing around

SPRINGING A LEAK: One strategy for allowing drugs to penetrate brain tissue is to create a semipermeable window into the brain by using nasal mucosa to patch a surgically introduced hole in the BBB. Here, fluorescent microscopic images of mouse brains demonstrate the increase in area and intensity of uptake of a fluorescent marker following the surgical procedure.PLoS ONE 8(4): e61694As a sinus surgery specialist at the Massachusetts Eye and Ear Infirmary and Harvard Medical School in Boston, Benjamin Bleier often assists colleagues with surgeries that go in through the nose to excise brain tumors. To do so, he cuts a hole in the lining of the brain, through both the BBB’s mesh of blood vessels and the protective cerebrospinal fluid barrier, just above the sinus cavity. “That part is easy. It’s always easy to destroy tissue,” says Bleier. “The part that is difficult is to close the hole.” Over the past seven years, however, Bleier and colleagues have optimized a closing technique that uses a patient’s own nasal lining to cover the hole made by the incision.

At the same time, Bleier studies drug delivery through the nose and has learned that the nasal lining is highly permeable to drugs. Many topical and aerosol treatments are delivered to the bloodstream through the nose, including pain and nausea medications and even insulin.

One day, Bleier put two and two together. “On one hand, we had this very permeable barrier that allows a lot of drugs through in a very efficient matter. And here we are putting this barrier directly against the brain” to patch the holes we’d made, he says. “We’d essentially created a large window in the blood-brain barrier. So could we use that to deliver drugs to the brain?”

In a proof-of-concept study published this year, Bleier and colleagues performed the nasal surgery on mice, then delivered molecules of up to 500 kilodaltons in size—1,000 times larger than those that can cross an intact BBB—through the nasal lining. They are now performing the same procedure in a mouse model of Parkinson’s disease, this time delivering an experimental treatment for the motor neuron disorder.

Skeptics challenge that creating a permanent hole in the BBB would expose the brain to the risk of infection, but Bleier notes that the nasal lining is armed with immune cells to defend against invaders, such as bacteria and viruses, and that the lining’s cell membranes actively pump out toxins that diffuse into the cells. “It’s a very active barrier, not just a fence,” he says. Because of this, the nasal-lining patch does not increase the brain’s risk of infection, Bleier argues. Indeed, surgeons have been repairing surgical incisions into the brain with the nasal lining for several years, and patients do not get postoperative meningitis or other brain infections, so the technique has a strong safety track record.

Permeating the BBB

Of course, entering the brain through transplanted nasal tissue is not the only option. Bleier’s method is just one of several creative techniques to cross the BBB emerging from academic labs. At Columbia University in New York City, bioengineer Elisa Konofagou is also applying an established medical technique to the BBB problem: the use of ultrasound and microbubbles. Tiny bubbles, made of lipid shells and a gas core, are injected into the bloodstream, then triggered to expand and contract using waves of sound. Quick ultrasound pulses can help researchers image an organ, such as the heart, while longer ultrasound pulses have been used to damage tumor tissue as a treatment for cancer.

More recently, however, it has been observed that the force of the bubbles’ movement causes endothelial cells of the BBB to temporarily separate, creating a momentarily permeable barrier. “It’s very safe,” she says. “With the right number of pulses and the right pressure, you can get a drug in and get the barrier to recover.” Last year, her lab used the method to deliver brain-derived neurotrophic factor (BDNF) to mice, which resulted in hippocampal neurons taking up the drug and activating downstream signaling pathways. Her team has also tested the technique in nonhuman primates, and hopes to move it into the clinic in the next two years.

At Johns Hopkins University, nanomedical researcher Rangaramanujam Kannan and collaborator Sujatha Kannan are also taking advantage of a compromised BBB. During neuroinflammatory illnesses, such as cerebral palsy and Alzheimer’s, the BBB becomes impaired just enough for nanoparticles to slip through. But designing a nanoparticle to penetrate the brain once it is past the BBB is easier said than done, he notes. “There have been many studies reporting that when there is sufficient breakdown of the blood-brain barrier, nanoparticles can get in,” says Kannan. “The problem has been that once they cross the blood-brain barrier, they don’t move into the brain tissue or get taken up by target cells.”

To overcome this hurdle, Kannan and his colleagues developed tiny tree-like synthetic nanoparticles called dendrimers, each only about 4 nanometers in size. For some unknown reason, these molecules move into the brain and travel to activated inflammatory cells. In a paper last year, the team attached dendrimers to an anti-inflammatory drug called N-acetyl-L-cysteine (NAC) to successfully treat rabbits with cerebral palsy. The dendrimers delivered the NAC straight to rampant inflammatory astrocytes and microglia in the brain, and the rabbits responded with improvement in coordination and motor control, nearly reaching the motor skill level of healthy controls. The team also used the technique to arrest retinal degeneration in rats.

Other techniques push, prod, and pressure the BBB to allow bigger and bigger drugs to enter the brain. A team at National Taiwan University recently showed that heparin, a common anticoagulant, used in conjunction with focused ultrasound, enhances delivery of molecules across the barrier. At Cincinnati Children’s Hospital Medical Center, a team is attaching pieces of the fatty protein apolipoprotein E (apoE) that bind to fat receptors on BBB endothelial cells to successfully deliver an enzyme into neurons of mice with a lysosomal storage disorder and quell their symptoms. And at Johns Hopkins School of Medicine in Baltimore, molecular biologists have demonstrated that a protein located on the surface of BBB endothelial cells, called frizzled-4, plays an important role in the arrangement of cerebral blood vessels and can be mutated to cause a leaky BBB without destroying the overall integrity of the barrier.

The list goes on. Which method is most successful will likely depend on what type of molecule one is trying to get into the brain. “This is a growing field,” says Watts. “There are a lot of cool things that are happening. It’s fun to be a part of it.” 

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