The study, to be published online Nov. 17 by the Proceedings of the National Academy of Sciences, demonstrates the first effective, non-toxic method of transporting drugs across multiple membrane barriers and even into cysts of Toxoplasma gondii, the single-celled microorganism that causes toxoplasmosis. It also describes a new therapeutic target within this common parasite.
"This is a major step forward in developing ways to treat one of mankind's most common chronic infections," said Rima McLeod, M.D., professor of ophthalmology and visual sciences at the University of Chicago and director of the study. "For the first time, we have access to this microbe in its latent stage, a part of its life cycle that was previously inaccessible."
"Better approaches to treating toxoplasmosis are needed," note the study authors. This disease, spread by cats and by eating undercooked meat, can cause devastating problems for those with weakened immune systems or when transmitted from mother to unborn child. About 3,000 infants are born each year in the U.S. with toxoplasmosis, which causes severe eye damage, mental retardation and death. The cost of caring for these children is thought to exceed $500 million per year.
In addition to active infections, T. gondii in its latent stage infects the nervous system of an estimated 3 billion people, including about 30 percent of Americans. Although it has long been considered harmless, it causes lifelong infections. The effects of those infections on physical and mental health are still being researched.
The new delivery system uses a short chain made up eight connected arginines, a naturally occurring amino acid, to ferry a drug across membranes. In 1996, scientists led by Paul Wender and Jonathan Rothbard at Stanford, discovered that short sequences of arginine could slip easily through biological membranes, either alone or attached to active molecules.
While Wender's team -- working with colleagues at Cell Gate, a California biotech firm -- refined this delivery system, McLeod and colleagues searched for new drug targets within T. gondii and related parasites from the apicomplexan family, which includes the causes of malaria and cryptosporidia. This family of microbes relies on enzymes that are not present in animals. Because the microbes require these enzymes to live and animals don't, they make ideal targets for treatment with minimal toxicity.
One such target is enoyl reductase, an enzyme discovered by McLeod, Michael Kirisits and Sarah Wernimont from McLeod's lab. The parasites require this enzyme to synthesize fatty acids, necessary for growth and reproduction.
In 2001, a research team (McLeod, Sean Prigge of Johns Hopkins, David Rice of Sheffield and Craig Roberts of Strathclyde) showed that triclosan, a common antiseptic used in toothpaste, skin creams and mouthwash, can kill the parasites responsible for toxoplasmosis and malaria. In the PNAS paper, they show that triclosan's antimicrobial effect comes, in part, from its ability to inhibit enoyl reductase.
The problem, however, has been how to get triclosan to the parasite. Even in its active stages, T. gondii live in the host's cells and are inaccessible to drugs. But soon after infection, many of the parasites enter a latent stage, called bradyzoites, causing chronic infection. Bradyzoites infect the central nervous system, including the eyes, often hiding within cysts inside the host's cells.
The researchers (including Benjamin Samuels, Doug Mack and Ernest Mui in McLeod's laboratory and Brian Hearn in the Wender laboratory) found that the short chains of arginine could deliver attached triclosan to every stage of the parasite's life cycle, including active parasites outside host cells, active parasites sequestered inside host cells, latent parasites outside cells and, most challenging, bradyzoites within cysts inside host cells.
The arginine chains, linked to triclosan, could cross multiple animal and microbial membranes to enter the host cell, pass through its internal barriers, enter cysts, which consist of densely packed animal and parasite constituents, enter the parasite, cross into the specialized organelles within, then release the triclosan, which inhibited the target enzyme.
The entry process is rapid. The effect on the parasite enzyme parallels the release of the antimicrobial from the carrier. The released antimicrobial inhibits the parasite in cells in tissue culture as well as in mice.
"We found this quite remarkable," said McLeod. "No current antimicrobial compound can cross the cyst wall, and development of new small-molecule medicines is hampered considerably by our inability to deliver them inside cells and the organism."
The discovery raises the possibility of treating active and latent infection in the eye by applying a lotion containing arginine-bound triclosan outside the patient's eye.
Additional authors of the paper include Stephen Muench from the University of Sheffield; and A.B. Law from Johns Hopkins University.
The National Institutes of Health, Research to Prevent Blindness, the Biotechnology and Biological Sciences Research Council and the Wellcome Trust supported this research.
Journal
Proceedings of the National Academy of Sciences