1a. Objectives (from AD-416):
To verify antifungal chemosensitizing agents discovered in in vitro bioassays are also functional in a mammalian animal model system using BALB/c mice.
1b. Approach (from AD-416):
Chemosensitizing agents will be determined by PMR scientists using standard in vitro (Petri dish, microtitre plate) bioassays. These assays will include commercial antifungal agents co-applied with candidate chemosensitizing agents. A number of fungi pathogenic to animals and humans will be included such as Aspergillus flavus, A. fumigatus, A. terreus and Candida species and Cryptococcus neoformans. Agents found to be effective under in vitro assays will be provided to a collaborator at Montana State University. Animal testing will use neutropenic mice, BALB/c. The mice are treated with cyclophosphamide i. p. prior to infection, then with antifungal drug and chemosensitizing agent. The mice will be infected, nasally. The following day, treatments will be administered in five cohorts, a Tween control, one receiving only fungi, one receiving only antifungal drug, one receiving only chemosensitizing agent and one will receive both antifungal drug and chemosensitizer.
3. Progress Report:
Based on prior research, and published by the ARS scientists in the parent project, thymol was identified as an effective chemosensitizing agent, in vitro. This is to say that in Petri dish experiments, adding thymol to fluconazole, greatly reduced, by more than 10-fold, the amount of fluconazole needed to kill a fungus, Asperigllus fumigatus. This fungus is the causative agent of tragic disease (invasive aspergillosis) of humans and animals, including farm animals and poultry. We next examined if what was found in the in vitro experiments were translatable to treatment of the disease by itself by using an animal model, mice. Two experiments were performed. The first involved five cohorts of five BALB/c mice, each. One cohort, non-treatment control, received only an injection of TWEEN (used for dissolving drug and chemosensitizer (thymol). The remaining cohorts were all infected with lethal doses of Aspergillus fumigatusI. These cohorts included one receiving no drug or chemosensitizer, another receiving only thymol (40mg/kg), another receiving only the antifungal drug fluconazole (40 mg/kg) (at a non-effective dose), and one receiving the same dose of fluconazole plus the thymol chemosensitizer. All mice in the TWEEN control survived. Four of the five mice in the fluconazole plus thymol cohort survived. All mice in the other cohorts perished within a week after infection. In the second experiment, a higher dose of fluconazole (200mg/kg) was tested with and without thymol (at the same dose as the first experiment, 40 mg/kg). The effort, here, was to attempt to see if all mice within the fluconazole plus thymol cohort could survive the infection. So, the number of mice in the test cohort was doubled from five to 10. Unfortunately, the batch of BALB/c mice provided by the supplier was unhealthy and even mice in the control group (receiving only TWEEN) perished. Hence, the results of this experiment were unreliable, despite the fact that more mice in the fluconazole plus thymol treatment survived than in the fluconazole only cohort. This experiment is planned to be repeated.