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The goal of treating diabetes with an effective, noninvasive oral delivery of insulin may one day be achieved through the use of a B12-insulin system.

Molecular images rendered by Damian Alis, SU Department of Chemisrty

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Chemistry professor Robert Doyle leads
a team of student researchers to new territories
in the fights against diabetes and ovarian cancer
 
By Judy Holmes | Photos by Susan Kahn
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In his chemistry lab, Professor Robert Doyle explores ways to attach drugs and other therapeutic agents to B-complex vitamins.
Villegas
Doctoral student Nerissa Villegas (above) focuses her research on developing a new method for detecting ovarian cancer
cells at an early stage.

In less than two years since arriving at Syracuse University, Robert Doyle has guided his team of young researchers on a journey of discovery, resulting in groundbreaking research that may lead to new treatments for diabetes and ovarian cancer.

For Doyle, a chemistry professor in the College of Arts and Sciences, discovery and innovation boil down to asking a simple question. “Sometimes a question arises in the literature, and you realize that you would like to be the first to answer it,” Doyle says. “It’s even better if no one else is trying to answer the question the way you are.”

In fact, it’s those unanswered questions that Doyle finds most intriguing. That may be the reason his lab is one of only a handful of university-based research labs worldwide that is exploring new ways to diagnose and treat diseases by attaching drugs and other therapeutic agents to B-complex vitamins, such as B12. Doyle’s research team—composed of 12 graduate and undergraduate students—discovered a way to administer insulin orally by coaxing vitamin B12 to give insulin a ride through the digestive system. His team is also making significant progress in attaching vitamin B9 (folic acid) to agents that can identify and kill drug-resistant ovarian cancer cells. The work may someday enable doctors to detect ovarian cancer at earlier stages than currently possible and to treat tumors more effectively with fewer side effects.

Folic acid and B12 are essential ingredients in the cellular production of deoxyribonucleic acid (DNA), which governs all cell processes. Because B vitamins are essential to cell survival, researchers believe the vitamins can be used to transport drugs and other therapeutic agents directly into cells before the active pharmaceutical agents are destroyed in the digestive system or excreted by the kidneys.

It’s an exploration that Doyle has immersed himself in for the past several years. A native of Ireland, Doyle earned a Ph.D in chemistry in 2002 at Trinity College in Dublin and spent the next year as a postdoctoral fellow at Australian National University. In 2003, he joined the research team of Professor Ann Valentine at Yale University and was appointed a Rudolph Anderson Foundation Postdoctoral Fellow in 2004, allowing him to work on metallocylase enzymes and iron transport proteins. He was recruited by Syracuse University because of his expertise and interest in pursuing these new areas of research. “Often the trick is to make drugs that are not destroyed before they reach their target,” Doyle says. “Human cells can only get vitamins from the food we eat; therefore, vitamins tend to get a special pass through the digestive system, so they can be absorbed by the cells.”

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Professor Robert Doyle and doctoral student Amanda Petrus are part of a team that created a method for binding insulin (red) to vitamin B12 (yellow). The molecular image represents the B12-insulin conjugate bound to a B12 uptake protein.
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New Way to Take Insulin
More than 100 million people worldwide are afflicted by diabetes, a disease that inhibits the body’s ability to effectively use glucose, which provides energy for all life processes. One form of diabetes is caused by the destruction of cells in the pancreas that produce the hormone insulin. The body uses insulin to transport glucose into cells, where it is used for energy or stored. This form of diabetes, known as insulin-dependent diabetes, requires patients to inject insulin into their bodies at least once a day.

In the past no one had been completely successful at finding a way for insulin to be effective when taken orally. The problem was twofold: Researchers had to find a mechanism that would both prevent insulin from being destroyed in the digestive system and enable it to be absorbed from the digestive system into the bloodstream, where it could then interact with its receptor. Vitamin B12 proved to be the answer to both problems. Because B12 is so critical to life processes, it passes through the digestive system unharmed and enters the bloodstream through specialized cells in the small intestine. Doyle’s team, led by doctoral student Amanda Petrus, decided to investigate how insulin could hitch a ride with B12 as it travels through the digestive system and into the bloodstream. “We asked ourselves, ‘Would B12 deliver insulin orally?’” Doyle says. “It turns out that it could, but we didn’t know that at the time. However, we could have told you a million reasons why it wouldn’t work.”

The first step was to examine previous research to determine what had already been done to answer the question. There wasn’t a lot to go on, but earlier studies had demonstrated it was possible. The key was to attach the agents so the body would still recognize the new compound as B12. “In some ways, you are standing on the shoulders of giants,” Petrus says. “You look at what others have done and pull out pieces and processes that might work for you.”

Petrus spent almost a year combining those bits and pieces with processes she developed to create a method for successfully binding insulin to B12. One particularly difficult task was finding a way to isolate the individual insulin molecules. “Insulin molecules like to hang out with each other,” Petrus says. “I found a way to prevent them from sticking together. Down the road, someone might apply the process I developed to his or her project.”

Once the oral insulin compound was successfully created, the Doyle group collaborated with Timothy Fairchild, an exercise science professor in the School of Education, to determine if the compound was effective in lowering blood glucose in diabetic rats. Fairchild, who met Doyle when they both arrived at SU in fall 2005, has been investigating the effect of exercise on blood sugar (glucose) levels. Researchers in his lab also are studying the effect of high-fructose corn syrup on blood sugar and insulin concentrations in the bloodstream. In short, Fairchild’s research involves looking at blood sugar and insulin on a human scale, whereas Doyle’s research examines the same substances at a cellular level—a perfect match from the researchers’ perspective.

Fairchild helped Petrus test her oral insulin compound in his laboratory. The test results were impressive from both a research perspective and for their broader implications. There is much work to do before researchers will know if Petrus’s oral insulin compound will be effective for humans. However, the early results, published in the peer-reviewed journal ChemMedChem in December 2007, look promising.

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Doctoral student Anthony Vortherms leads the first university-based research team in the country to attach the cancer-killing drug AZT to a B9-polymer compound.

“The chemistry to make the B9-PEG compound is relatively simple and well worked out. But no one had used AZT before to target ovarian cancer cells.”

 

Doyle’s research team will continue to refine the compound to increase its effectiveness. The team is also collaborating with Robert Smith of the N.C. Brown Center for Ultrastructure Studies at the SUNY College of Environmental Science and Forestry, on a series of specialized experiments that use a technique called gold-based electron microscopy to take pictures of insulin binding to glucose. “We’ve proved you can use B12 to deliver insulin orally,” Doyle says. “We know it works because we lowered blood glucose levels. Following the insulin-binding act on a cellular level to show that it works is just good, careful science.”

Search-and-Destroy Mission
Each year, more than 25,000 women in the United States are diagnosed with ovarian cancer, the fifth leading cause of cancer deaths in women. Ovarian cancer is difficult to detect and even more difficult to treat because the vast majority of cases are diagnosed after the cancer has spread beyond the ovaries. Currently, 75 percent of women with ovarian cancer diagnosed in stage three or four die within five years of diagnosis.

Two research teams in Doyle’s lab hope to help turn the tide in the fight against ovarian cancer by finding ways to send vitamin B9 (folic acid), tethered to some unique hitchhikers, on a mission to seek out and destroy ovarian cancer cells. While all cells need B9 to survive, cancer cells need particularly large amounts of the vitamin because they grow and reproduce at such rapid rates. To take in large amounts of B9, some types of cancer cells, including ovarian ones, continue to rely on a system present in normal cells only during fetal development and early infancy. Doyle’s research teams are using this system, called the B9 (or folate) receptor system, as a targeted entry route into the cancer cells. Because this B9 receptor doorway is not present in normal cells, researchers hope it can be used to deliver drugs and other agents directly to the cancer cells without harming normal tissue. “In essence, what we want to do is tell an imaging agent or drug to only find and/or kill the cancer cells,” Doyle says. “If successful, we will be able to detect ovarian cancer at an earlier stage and then kill it more effectively using lower dosages of a drug or radiation and with fewer side effects.”

The problem both research teams had to first overcome was finding a way to prevent the B9 “Trojan horse” from being excreted by the kidneys before it gets through the back door. B9 is a water-soluble vitamin. Excess quantities of the vitamin found circulating in the bloodstream are easily absorbed and excreted by the kidneys. To solve this problem, the teams explored previous research and developed a process for attaching B9 to a polyethylene glycol (PEG) polymer. The polymer enlarges the B9 molecule, making it harder for the kidneys to excrete and allowing larger quantities of the B9 Trojan horse to get past the kidneys and reach the intended target. The imaging agents and drugs are attached to the B9-PEG unit so they, too, can get by the kidneys.

Killing Cancer Cells
Using this B9-PEG system, a group led by doctoral student Anthony Vortherms was the first university-based research team in the country to attach the cancer-killing drug AZT to the compound. They then successfully targeted and killed drug-resistant ovarian cancer cells grown in Doyle’s lab. Although AZT is currently used to treat people infected with the human immunodeficiency virus (HIV), it was originally designed as an anticancer agent, but proved too toxic. Vortherms’s research was published as the lead article in the January 2008 issue of Nucleosides, Nucleotides, and Nucleic Acids, a peer-reviewed journal. “The chemistry to make the B9-PEG compound is relatively simple and well worked out,” Vortherms says. “But no one had used AZT before to target ovarian cancer cells.”  

Vortherms continues to refine and test his compound on ovarian cancer cells. He and Doyle will collaborate with Dawn Post, a neurosurgery professor at SUNY Upstate Medical University’s Institute for Human Performance, to determine the compound’s potential for further development as a drug to treat ovarian cancer.

Illuminating Cancer Cells
Another research team in Doyle’s lab, led by doctoral student Nerissa Villegas, is working to develop a new method of detecting ovarian cancer cells by attaching the metals rhenium and technetium to the B9-PEG compound. When B9 and its metal hitchhikers enter cancer cells, the cells light up when scanned by an imaging device. Different isotopes or forms of the metals also have the potential to kill the cancer cells.  “Currently, ovarian cancer is virtually undetectable in the early stages,” Doyle says. “Our work is showing a lot of promise in this area.”   

Villegas’s team collaborates on the rhenium research with chemistry professor and department chair Jon Zubieta, and on the technetium work with researchers at McMaster University in Hamilton, Ontario, which has facilities for handling radioactive metals. A third promising metal, gallium, presented especially difficult control problems, which Villegas had to solve before it could be tested in combination with the B9-PEG compound. That research, which took two years, was published in Polyhedron (December 2006), a peer-reviewed journal. “In that paper, we actually described the structure of the cage that traps the gallium and makes it behave,” Doyle says. “Gallium 67 [a radioactive form that emits gamma rays] is potentially a potent killer of ovarian cancer cells. The trick is being able to handle the gallium and to show that it goes where it is supposed to go.”

Amy Rabideau ’09 is among the undergraduates who work on
research teams in Professor Robert Doyle’s lab.

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During the past year, Villegas has been building on her earlier research to develop a method to attach gallium to the lab’s B9-PEG compound to target ovarian cancer cells. The hope was that the compound would kill the cells. Instead, the compound successfully targeted the cancer cells, but did not kill them. This work, however, showed that these systems have great potential for use as non-toxic, controllable, imaging agents to detect ovarian cancer cells. The results of Villegas’s research will be published in an upcoming issue of Drug Targets Insight, a peer-reviewed journal. “Even though we didn’t get the expected results, we have contributed to the field,” Villegas says. “The good news is we proved we could use gallium to target cancer cells. This work shows when you are careful and follow through on your idea that sometimes unexpected—but exciting—results can occur.”

The struggle to find new ways to fight ovarian cancer is both a personal and a professional quest for Villegas, whose mother died of the disease in 2005, two days before Villegas was accepted into SU’s Ph.D. program. “My goal when I came here was to do cancer research,” she says. “When I learned about Professor Doyle’s research, I decided to switch from organic chemistry to inorganic, so I could work on ways to target ovarian cancer.”  Villegas says working with rhenium and technetium is “a breeze” compared to gallium, but she is not giving up on gallium. “Not many labs work with gallium because it is so difficult,” she says. “The early successes with the rhenium and technetium experiments are giving me an emotional break from gallium.”

As work continues on the B9 and B12 avenues of research, Doyle is always looking for that next unanswered question. He plans to expand his research to include an exploration of vitamin B1 (thiamine) to see how it might be used as a delivery vehicle. “B1 is an interesting system,” he says. “We’re going to see where it takes us.” And that, Doyle says, is the essence of fundamental research. “You want to do research that will have an impact 20 years from now,” he says. “What we learn through fundamental research stays around for a long time and has an impact on the future of science and discovery.” 

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