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NTP Scientists Help New Treatment Enter Clinical Trials

By Eddy Ball
December 2006

Richard Irwin
NTP Chemist Richard Irwin (Photo courtesy of Steve McCaw)

Molly Vallant
NTP Biologist Molly Vallant (Photo courtesy of Steve McCaw)

In a study published in the November issue of Human Gene Therapy, National Toxicology Program Chemist Richard Irwin, Ph.D., and Biologist Molly Vallant collaborated in a detailed toxicity and biodistribution analysis that has moved a novel gene transfer treatment protocol closer to clinical trial. In earlier studies, the protocol demonstrated promise for reducing side effects from radiation therapy for head and neck cancer.

As part of the pre-clinical approval process needed to progress to phases1 and 2 of human clinical studies, the Food and Drug Administration (FDA) required scientists from the National Institute of Dental and Craniofacial Research (NIDCR) to submit animal toxicity studies on the protocol. To get the data and exhaustive analysis needed, researchers from NIDCR and BioReliance Invitrogen Bioservices partnered with experts at NTP to produce a "gold standard" study using Good Laboratory Practices.

Approximately 40,000 patients are diagnosed with head and neck cancers in the United States each year. While survival rates have improved dramatically, the treatment causes severe damage to the fluid-secreting portion, or acinar cells, of the salivary glands that are within the field of radiation. Without enough saliva to lubricate and cleanse the teeth, mouth and throat, patients can experience dry mouth, damage to upper gastrointestinal tract tissues, tooth cavities, inflammation of mucus membranes in the mouth and frequent infections.

People also can have difficultly swallowing, speaking or tasting food, sometimes leading to a significant loss of appetite, considerable discomfort and a marked decline in quality of life. At the present time, there is no approved corrective treatment for the condition, known as radiation-induced salivary hypofunction.

Although radiation destroys the fluid-producing acinar cells, the ductal cells, which do not produce saliva, are usually not damaged. This observation led NIDCR Chief, Gene Therapy and Therapeutics Branch Bruce Baum, D.M.D., Ph.D., to collaborate in studies of salivary gland repair with then Johns Hopkins University Professor of Medicine Peter Agre, M.D. Agre discovered the water transport protein aquaporin in 1991 and was awarded the Nobel Prize in Chemistry in 2003 for his work.

The scientists hypothesized that an effective treatment could be developed using a recombinant adenoviral vector, similar to a cold virus, to transfer the gene for human aquaporin-1, which forms pores in cell membranes, to ductal cells, turning them into fluid producers. In a series of experiments with radiated animals, including rats, miniature pigs and non-human primates, the investigators found that treatment resulted in a dose-dependent increase in salivary flow to 80 percent of normal, a two to three fold increase over post-radiation levels.

Irwin, Vallant and colleagues conducted detailed and careful studies of vector safety and biodistribution of the vector beyond the oral cavity. Animals were housed individually, treated humanely and anesthetized in accordance with the guidelines during vector administration and follow-up.

Researchers monitored the health of 200 adult male and female rats, divided into four experimental groups per gender, over a 92-day period. Animals in the test groups received injections of the virus containing the gene for aquaporin-1 into the submandibular duct. For toxicity determination purposes, the treatment dose was approximately ten times the corresponding lowest and highest doses proposed for clinical study. Researchers collected saliva, blood and salivary glands from five animals in each staggered start group 48 hours after vector administration.

Researchers observed no clinical or gross pathological signs of adverse toxicological effects in animals after gene transfer. There were no treatment-associated losses of animals. Animals in all groups continued to thrive after treatment, with normal patterns of weight gain and food and water consumption. Except for local, dose-dependent inflammatory changes in the targeted gland, animals showed no severe or permanent damage to the salivary gland and limited vector distribution elsewhere in the body. Despite some gender differences in response to treatment, clinical chemistry indicators of major organ function were normal for all animals.

Citation: Zheng C, Goldsmith CM, Mineshiba F, Chiorini JA, Kerr A, Wenk ML, Vallant M, Irwin RD, Baum BJ. 2006. Toxicity and biodistribution of a first-generation recombinant adenoviral vector, encoding aquaporin-1, after retroductal delivery to a single rat submandibular gland. Hum Gene Ther 17:1122-1133.



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