Novel delivery system and new target show promise for toxoplasmosis treatment
November 17, 2003
A multi-center research team has discovered how to deliver antimicrobial medications directly to the infectious parasites that cause diseases such as toxoplasmosis, even when the parasites lay hidden and inactive within cysts, where they have been untreatable by any available medicines.
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, MD, professor of ophthalmology and visual sciences at the University of Chicago, who led 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. We also have a better means of delivering medicines to its active stage, as well a new target for treatment."
"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 U.S. patients with toxoplasmosis is thought to exceed $5 billion 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, causing 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, 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 cryptosporidiosis.
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 T. gondii enoyl reductase, an enzyme first identified in McLeod's lab. The parasites require this enzyme to synthesize fatty acids, necessary for survival.
In 2001, a research team led by McLeod, David Rice of Sheffield and Craig Roberts of Strathclyde showed that triclosan, a common antiseptic used in toothpaste, skin creams and mouthwash, and known to affect the bacterial version of this enzyme, can kill the parasites responsible for toxoplasmosis and malaria.
In the PNAS paper, they show that triclosan's antimicrobial effect comes from its ability to inhibit T. Gondii 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. 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 host cells.
The researchers 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 inside host cells, latent parasites outside cells and, most challenging, bradyzoites within cysts inside host cells.
The arginine chains, linked to triclosan, could rapidly cross multiple animal and microbial membranes. They were able to enter the host cell, pass through its internal barriers and cross into cysts, which are surrounded by densely packed animal and parasite constituents.
Within a cyst, the arginine compound could enter the parasite, cross into its specialized organelles, then release the triclosan in a way that inhibited the target enzyme. Learning the basic structure of this enzyme enabled the team to attach triclosan to the arginines in the most effective way.
The system inhibits the parasite in mice and in tissue culture.
"We found this quite remarkable," said McLeod. "No current antimicrobial compound can cross the cyst wall. 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 triclosan or other antimicrobials bound to a transporter which would carry it into the eye.
The National Institutes of Health, Research to Prevent Blindness, the Biotechnology and Biological Sciences Research Council, and the Wellcome Trust supported this research.
Note to Editors: A multi-center team collaborated on this project. McLeod, Ernest Mui, and Douglas Mack of Chicago showed that triclosan can kill the parasites responsible for toxoplasmosis and malaria. Michael Kirisits, McLeod, and Sarah Wernimont from Chicago and Craig Roberts from Strathclyde University identified the target enzyme in Toxoplasma and produced the recombinant enzyme. Sean Prigge and Anthony Law at Johns Hopkins University and David Rice and Stephen Muench at Sheffield University characterized the enzyme and with McLeod and Paul Wender designed the triclosan-arginine combination. McLeod, Benjamin Samuels, Doug Mack, and Ernest Mui of Chicago and Wender, Rothbard, and Brian Hearn of Stanford found that the short chains of arginine could deliver triclosan to the parasite. Brian Hearn, with Wender, designed and synthesized the compounds tested.
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