The Efficacy of Tetracyclines in Peripheral and Intracerebral Prion Infection

et al. (2008) The Efficacy of Tetracyclines in Peripheral and Intracerebral Prion Infection. PLoS ONE 3(3): e1888. doi:10.1371/journal.pone.0001888 The Efficacy of Tetracyclines in Peripheral and Intracerebral Prion Infection Ada De Luigi 0 Laura Colombo 0 Luisa Diomede 0 Raffaella Capobianco 0 Michela Mangieri 0 Claudia Miccolo 0 Lucia Limido 0 Gianluigi Forloni 0 Fabrizio Tagliavini 0 Mario Salmona 0 Mark R. Cookson, National Institutes of Health, United States of America 0 1 Department of Molecular Biochemistry and Pharmacology, Istituto di Ricerche Farmacologiche ''Mario Negri'' , Milano , Italy , 2 Department of Neuroscience, Istituto di Ricerche Farmacologiche ''Mario Negri'' , Milano , Italy , 3 Fondazione I.R.C.C.S. Istituto Neurologico ''Carlo Besta'' , Milano , Italy 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 1024 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 1024 dilution of the 263K scrapie strain. A single intracerebroventricular infusion of 25 mg/ 20 ml 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. - Funding: This work was supported by the European Union within the frame of Neuroprion and ExpTheraprion, Fondazione Cariplo (Grant NOBEL-Guard), the Italian Ministry of University and Research (FIRB, RBNE03PX83), and the Italian Ministry of Health. Competing Interests: The authors have declared that no competing interests exist. Transmissible spongiform encephalopathies are fatal neurodegenerative diseases that cause extensive loss of cerebral neurons with formation of vacuoles, giving a sponge-like appearance to the tissue. They include several diseases with different causes (infectious/iatrogenic, sporadic or genetic origin), recognized both in humans and animals as variations of the same disorder [1,2]. Although the precise pathogenic mechanism is still not understood, prion diseases involve the deposition of aggregates of disease-related isoforms (PrPSc) of a host-encoded protein (PrPC) in the central nervous system. During the progression of the disease, a portion of the a-helix and random coil structure in PrPC is refolded into a bpleated sheet in PrPSc, a conformational change that renders PrPSc poorly soluble and resistant to protease digestion [3]. Consequently, PrPSc aggregates accumulate around neurons in affected brain areas, a process thought to lead to neuronal dysfunction and death, and subsequently the clinical symptoms of infection. Many classes of molecules have been screened in experimental therapy [47], some showing promising anti-prion activity, but most of them suffer the limitation of poor passage through the bloodbrain barrier (BBB) and severe toxicity. Currently, there is no therapy for prion diseases in humans. Three drugs, pentosan polysulfate [8,9], quinacrine [10,11] and flupirtrine [12], are currently used in the clinical management of human prion diseases, although none of them have yet been proved to be effective, reinforcing the importance of identifying new treatment strategies. Based on structural analogies with Congo red, tetrapyrroles and acridine derivatives we hypothesized that tetracyclines might interact with PrPSc and interfere with PrP amyloid formation [13]. These old drugs have well-characterized pharmacological and safety profiles, low toxicity, and some cross the blood-brain barrier efficiently when a suitable route of administration is used. In addition, minocycline and doxycycline have neuroprotective properties in different neurodegenerative diseases [14] due to their anti-inflammatory and anti-apoptotic effects. Our studies indicated that tetracyclines are good candidate antiprion drugs. Their ability to bind to fibrillary assemblies, monomeric and oligomeric forms of PrP peptides, homologous to residues 82 to 146 (PrP82-146) and 106 to 126 (PrP106-126) of human PrP, and disrupt peptide aggregates, was previously reported [13]. NMR spectroscopy indicated that tetracyclines achieved through-space interactions with the hydrophobic peptide domains. In cell-free studies the various tetracyclines analogues showed marked differences in facilitating proteinase K (PK) digestion of PrP peptides [15,7]. In cell culture studies tetracyclines inhibited neuronal death and astroglial proliferation induced in vitro by the PrP peptides [13]. Incubation of 263K scrapie-infected brain homogenate with 1 mM tetracycline or doxycycline resulted in more than 90% reduction in the PK-resistant core of PrPSc. It was also reported [16] that these compounds can interact with partially purified PrPSc from patients with the new variant of CreutzfeldtJakob disease, and cattle with bovine spongiform encephalopathy. In vivo there was significant delay in the onset of clinical signs of disease and prolonged survival in hamsters injected with 1 mM tetracycline-pre-treated inocula. When tetracyclines were preincubated with highly diluted scrapie-infected inocula one third of animals did not develop disease [16]. At the time of the onset of symptoms in controls PrPSc accumulation was less abundant in the brain of tetracycline-treated animals, as was the severity of spongiform changes and astrogliosis in the cerebral cortex and subcortical gray structures [16]. We report the efficacy of tetracycline, doxycycline and minocycline in prolonging the survival of hamsters infected intramuscularly (im), subcutaneously (sc) or intracerebrally (ic) with the 263K scrapie strain and showed that the drugs are also active when administered at the appearance of the first symptoms. Effect of tetracyclines followi (...truncated)