The efficacy of tetracyclines in peripheral and intracerebral prion infection

Abstract

We have previously shown that tetracyclines interact with and reverse the protease resistance of pathological prion protein extracted from scrapie-infected animals and patients with all forms of Creutzfeldt-Jakob disease, lowering the prion titre and prolonging survival of cerebrally infected animals. To investigate the effectiveness of these drugs as anti-prion agents Syrian hamsters were inoculated intramuscularly or subcutaneously with 263K scrapie strain at a 10(-4) dilution. Tetracyclines were injected intramuscularly or intraperitoneally at the dose of 10 mg/kg. A single intramuscular dose of doxycycline one hour after infection in the same site of inoculation prolonged median survival by 64%. Intraperitoneal doses of tetracyclines every two days for 40 or 44 days increased survival time by 25% (doxycycline), 32% (tetracycline); and 81% (minocycline) after intramuscular infection, and 35% (doxycycline) after subcutaneous infection. To extend the therapeutic potential of tetracyclines, we investigated the efficacy of direct infusion of tetracyclines in advanced infection. Since intracerebroventricular infusion of tetracycline solutions can cause overt acute toxicity in animals, we entrapped the drugs in liposomes. Animals were inoculated intracerebrally with a 10(-4) dilution of the 263K scrapie strain. A single intracerebroventricular infusion of 25 microg/20 microl of doxycycline or minocycline entrapped in liposomes was administered 60 days after inoculation, when 50% of animals showed initial symptoms of the disease. Median survival increased of 8.1% with doxycycline and 10% with minocycline. These data suggest that tetracyclines might have therapeutic potential for humans.

Figure 1. Effect of tetracyclines following peripheral scrapie infection.

PrP^Sc immunohistochemistry of brain tissue (A), spinal cord (B), spleen (C), and Peyer's patches (D) of hamsters at the terminal stage of disease after intramuscular inoculation of 263K scrapie infected brain homogenate alone at 10⁻⁴ dilution. All samples were counterstained with hematoxylin. The pictures are representative of the immunohistochemical results for all terminal animals. Magnification scale bar: 1 mm (A), 250 µm (B-C-D). (E) Survival of hamsters injected intramuscularly with a 10⁻⁴ dilution of 263K scrapie-infected brain homogenate followed one hour later by a single intramuscular dose of doxycycline (10 mg/kg) at the same site. Untreated animals (□), Doxycycline (•). (F) Survival of hamsters injected intramuscularly with a 10⁻⁴ dilution of 263K scrapie-infected brain homogenate followed one hour later by an intraperitoneal dose of 10 mg/kg of tetracycline, doxycycline or minocycline, then every 2 days up to 40 days post-infection. Untreated animals (□), Tetracycline (▵), Doxycycline, (•), Minocycline (○). (G) Survival of hamsters injected subcutaneously with a 10⁻⁴ dilution of 263K scrapie-infected brain homogenate followed four days later by an intraperitoneal dose of 10 mg/kg of doxycycline, then every 2 days up to 44 days post-infection. Untreated animals (□), Doxycycline, (•).

Figure 2. Effect of tetracyclines following intracerebral scrapie infection.

(A) Immunoblot analysis of 263K scrapie infected brain homogenate (1%) after incubation in the absence (lane 1), or presence of doxycycline (lanes 2, 3), liposome-containing doxycycline (lane 4) and empty liposomes (lane 5), followed by proteinase K digestion. The blots were probed with the antibody 3F4 (1∶5,000). Molecular mass markers are indicated to the left. (B) Cerebral cortex of hamster four days after an intracerebroventricular infusion of 25 µg/20 µl liposome-containing doxycycline. Doxycycline-related fluorescence appears to be partly diffused in the neuropil and partly associated with nerve cell bodies and processes (arrows) and glial cells. Magnification scale bar: 50 µm. (C) Survival of hamsters injected intracerebrally with a 10⁻⁴ dilution of 263K scrapie-infected brain homogenate followed 60 days later by a single intracerebroventricular infusion of 25 µg/20 µl doxycycline or minocycline-containing liposomes. By this time 50% of infected animals showed initial clinical symptoms of disease, i.e. hyperreactivity to tactile and acoustic stimulations. Untreated animals (□), LipoDoxycycline, (•), LipoMinocycline (○).