1 Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840, USA
2 Institute for Antiviral Research, Animal, Dairy, and Veterinary Sciences Department and Biotechnology Center, Utah State University, Logan, UT 84322-4700, USA
3 Chengdu Jinniu Institute, Food Bureau of Sichuan Province, Chengdu Sichuan, China
Correspondence
David A. Kocisko
DKocisko{at}niaid.nih.gov
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ABSTRACT |
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MAIN TEXT |
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Because PrP-res is associated with disease and infectivity, it has been a target of therapeutic intervention for TSEs (Aguzzi et al., 2001; Dormont, 2003
; Brown, 2002
). Murine N2a cells chronically infected with the RML (Chandler) strain of scrapie (Race et al., 1988
) have been used widely to test compounds for their ability to inhibit PrP-res formation (Caughey et al., 1999
; Beranger et al., 2001
; Kocisko et al., 2003
). Many compounds that inhibit PrP-res in cell culture have also delayed the onset of TSEs in animal models, but none has been curative. These compounds include porphyrins and phthalocyanins (Caughey et al., 1998
; Priola et al., 2000
), polyene antibiotics (Dormont, 2003
), Congo red (Caughey & Race, 1992
; Ingrosso et al., 1995
), suramin (Gilch et al., 2001
), sulfated glycans and other polyanions (Ehlers & Diringer, 1984
; Kimberlin & Walker, 1986
; Farquhar & Dickinson, 1986
; Caughey & Raymond, 1993
; Birkett et al., 2001
; Schonberger et al., 2003
; Gabizon et al., 1993
). We recently used N2a cell cultures infected with either the RML or the 22L strains of scrapie to screen PrP-res inhibitors from a library of 2000 drugs and natural products (Kocisko et al., 2003
).
In this study we tested a number of the most potent of these new cell culture PrP-res inhibitors against scrapie infection in transgenic mice (Tg7). Tg7 mice produce no mouse PrP, but express approximately 4- to 8-fold higher levels of hamster PrP than do hamsters. They have a short disease incubation period of 4550 days after intracerebral (i.c.) inoculation with a high dose of the 263K strain of hamster scrapie (Race et al., 2000
; Priola et al., 2000
).
Compounds were administered either to treat an established infection or to test for prophylaxis. To test for activity against an established infection, compound administration started 2 weeks after i.c. scrapie inoculation and continued for 56 weeks. The 2-week period after i.c. inoculation allowed time for the disease to progress before the compound was administered. To test for prophylaxis, administration of a compound began 2 weeks before and continued for 4 weeks after intraperitoneal (i.p.) scrapie inoculation. The rationale was to have a compound approaching a steady-state level in the mouse, enabling it to block a peripheral inoculation of scrapie infectivity from being established in the brain. The treatment following inoculation would allow time for the animal potentially to clear infectious material while the compound prevented further formation of PrP-res.
Compounds were administered either as an i.p. injection or in the drinking water. For i.p. injections, compounds were dissolved or suspended in an appropriate buffer and the dose volume was 10 ml kg1. Injections were given three times per week, on Monday, Wednesday and Friday. Solutions of compounds in drinking water were made to yield the desired dose based on the mean daily consumption of water by mice, 15 ml (100 g body wt)1. A solution of compound in the drinking water was the sole source of water for the mice during the dosing period. All 263K scrapie brain homogenates made up for inoculation in these studies were in physiological buffer supplemented with 2 % fetal bovine serum. Different control groups are presented because testing was not done all at once and mice were inoculated with different homogenate preparations. In these studies, Tg7 mice were euthanized when clinical signs of scrapie were present, which included ruffled fur, lethargy, ataxia and weight loss. All procedures were approved by the Institution's Animal Care and Use Committee and were designed to minimize the animals' pain and distress. Animals that died from causes other than scrapie, such as from inoculation, dosing and anaesthetizing procedures, have been excluded from the data.
Compounds evaluated in animals had IC50 (concentration of a compound inhibiting half of the production of PrP-res) values of 1 µM against both the RML and the 22L scrapie strains in cell culture. Since the in vivo testing involved hamster 263K scrapie, it was felt that compounds that inhibited multiple strains of mouse scrapie had a better chance of showing efficacy against PrP from another species. The inhibitors tested had been identified previously (Kocisko et al., 2003
) except for polyphenolic extracts of grape seed and pine bark (data not shown). In addition to their history of use in humans, the anti-psychotic drugs thioridazine, thiothixene and trifluoperazine were also selected for testing because they are known to cross the bloodbrain barrier of humans. Amodiaquine is an inexpensive anti-malarial drug that has been used extensively in humans. The polyphenol tannic acid, which is contained in many foods, was the most potent inhibitor in our test set with an IC50 of
100 nM in both the scrapie-infected neuroblastoma cells and a solid-phase cell-free hamster 263K conversion assay (Kocisko et al., 2003
). A tea extract containing
55 % epigallocatechin monogallate and other polyphenols was also tried because of its relatively low toxicity and use as a human food. Finally, tetrandrine, a Chinese herbal medicine with anti-malarial activity, was tested. Generally, the highest known tolerated dose of a compound in mice was given to maximize the chance of seeing an effect. For instance, 5 mg thioridazine kg1 dosed i.p. was used in this trial because 10 mg kg1 i.p. is not tolerated (Burke et al., 1990
). In our experiments, 10 mg trifluoperazine kg1 was mildly toxic but was tolerated, and 4500 mg tannic acid kg1 per day was not tolerated but 3000 mg kg1 per day had no apparent toxicity.
Table 1 contains the incubation period of each individual Tg7 mouse after i.c. inoculation of 263K scrapie brain homogenate and administration of compounds. No compound used as a treatment against established infection after i.c. inoculation significantly extended incubation periods. Nor was any compound protective when administered for a week prior to i.c. inoculation.
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Since many potent inhibitors of PrP-res formation in vitro are not efficacious against scrapie in animals, it is important to consider animal testing of inhibitors prior to clinical trials, especially in cases in which there might be negative side effects for the patient. Quinacrine, an anti-malarial drug used extensively in humans, was found to inhibit RML (Doh-ura et al., 2000; Korth et al., 2001
) and later 22L (Kocisko et al., 2003
) mouse scrapie strains in cell culture. Based on its PrP-res inhibitory activity in cell culture, as well as its previous human use, testing of quinacrine in human TSE trials was strongly advocated, even in the absence of any supportive animal data (Korth et al., 2001
). However, in subsequent in vivo testing, quinacrine was ineffective in scrapie-infected mice (Collins et al., 2002
; Barret et al., 2003
) and, unfortunately, only transiently beneficial in some CJD patients, albeit with some liver toxicity (Kobayashi et al., 2003
; Nakajima et al., 2004
). Thus, although many inhibitors of PrP-res in cell culture are known, testing in TSE-infected rodents should help to select those with the most promise for human clinical trials.
When testing the efficacy of compounds against scrapie infection in an in vivo system there are many complex variables. Some of these include the dose, the vehicle, the dosing regimen, when to initiate and terminate treatment, the routes of compound administration and scrapie inoculation, the animal model and the TSE strain. Part of the difficulty in deciding on these variables is a lack of understanding of TSE pathogenesis. We have tried to select reasonable options from among these variables, but many others might be considered. Although none of the inhibitors tested herein was effective in our in vivo tests, we report the results of these expensive and time-consuming experiments in the hope that future work with potential anti-TSE therapeutics and prophylactics can advance beyond our particular approaches rather than repeat them.
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ACKNOWLEDGEMENTS |
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Received 2 March 2004;
accepted 22 April 2004.