TETRACYCLINES

Tetracyclines are antibiotics used to treat various conditions in humans today.  Doxycycline in particular has shown promise in reducing prion propagation in mice.  Human doxycycline trials  are underway in Europe and scheduled to be initiated at Case Western Reserve University in Cleveland, OH within the next year or two.

“Innovation of therapeutics and prophylaxis for prion diseases,” Shinkeigaku (2009)
http://www.ncbi.nlm.nih.gov/pubmed/20030256

There is no established treatment for prion diseases; however, recently several drug candidates, including pentosan polysulfate and doxycycline, have been clinically used on a trial basis to prevent accumulation of abnormal prion protein in the brain. So far the outcome of the trials is still very far from the goal where a complete cure of the diseases is expected. In order to bridge the gap between the reality and the ideal, the followings are suggested. First, combination therapy needs to be developed against multi-targets: inhibition of prion replication; degradation and scavengery of prion; inhibition of prion-related neurodegeneration. Secondly, preclinical diagnostic means, by which healthy prion-carriers can be revealed before the onset of the diseases, should be explored for earlier therapeutic interventions. The last is to disclose intrinsic disease susceptibility factors and environmental factors, both of which could solely or jointly facilitate in suppressing prion replication and disease progress. Exploitation of these items should be tough but will be deserved for overcoming the fatal diseases.

 

“Doxycycline control of prion protein transgene expression modulates prion disease in mice,” Tremblay et. al., inc. Prusiner, (1998)
http://www.pnas.org/content/95/21/12580.full

Conversion of the cellular prion protein (PrPC) into the pathogenic isoform (PrPSc) is the fundamental event underlying transmission and pathogenesis of prion diseases. To control the expression of PrPC in transgenic (Tg) mice, we used a tetracycline controlled transactivator (tTA) driven by the PrP gene control elements and a tTA-responsive promoter linked to a PrP gene [Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, 5547–5551]. Adult Tg mice showed no deleterious effects upon repression of PrPC expression (>90%) by oral doxycycline, but the mice developed progressive ataxia at ≈50 days after inoculation with prions unless maintained on doxycycline. Although Tg mice on doxycycline accumulated low levels of PrPSc, they showed no neurologic dysfunction, indicating that low levels of PrPSc can be tolerated. Use of the tTA system to control PrP expression allowed production of Tg mice with high levels of PrP that otherwise cause many embryonic and neonatal deaths. Measurement of PrPSc clearance in Tg mice should be possible, facilitating the development of pharmacotherapeutics.

On this background, we undertook development of a system where the level of PrP expression could be regulated to modulate the rate of prion formation. We chose the tetracycline-responsive gene system that was developed by using the Escherichia coli tetracycline resistance Tn10 operon (19). It makes use of a transactivator (tTA) obtained by fusing the tetracycline repressor with the transactivation domain of the herpes simplex virus VP16 transcription factor. The tTA binds specifically with high affinity to the tetracycline operator (tetO) and activates transcription from a minimal promoter linked to the target gene. Binding of doxycycline, a tetracycline analog, to tTA prevents the protein from binding to the tetO region, thereby preventing target gene expression.

The brains from untreated Tg(tTA:PrP)3 mice exhibited extensive neuronal loss in the hippocampal pyramidal cell layer (Fig. 3G) and dentate gyrus and focal loss of Purkinje cells and granular cells in the cerebellum (Fig. 3C). These changes were accompanied by moderate to severe astrocytic gliosis in all regions examined, including the neocortex, hippocampus, entorhinal cortex, thalamus, caudate nucleus, and substantia nigra, as well as cerebellar granular and molecular layers. As noted above, the doxycycline-treated Tg(tTA:PrP)3 mice did not show signs of CNS dysfunction. When these mice were sacrificed ≈200 days after inoculation with prions, their brains showed no signs of neurodegeneration.

Doxycycline administered to adult Tg(tTA:PrP) mice acutely repressed the expression of PrPC but did not produce any recognizable adverse effects in the mice over a 30-day period. Neither the viability nor the neurological status of the mice was compromised, and histological examination of the brains did not reveal any abnormalities. These results indicate that high levels of PrPC are not essential for short-term neuronal survival, as its expression can be repressed over 20-fold without adverse effects. It is noteworthy that adult Tg(tTA:PrP)3 mice were placed on oral doxycycline to repress their PrPC expression and have remained well for >380 days with continual administration of doxycycline (Table 5).

With the production of Tg(tTA:PrP) mice, it is possible to examine the effects of low or intermediate levels of PrPSc in the CNS. We found that low levels of PrPSc did not produce any deleterious clinical or histological effects up to 380 days after inoculation of RML prions in Tg(tTA:PrP)3 mice (Fig. 3C). Studies of Prnp+/0 mice with one functional PrP allele show greatly prolonged incubation times (15) but at a higher accumulation of PrPSc than was anticipated (17). Studies with Tg(tTA:PrP) mice where the levels of PrPC expression are held at different levels throughout the incubation time should help to clarify this issue.

The findings reported here clearly show that repression of PrPC expression in young adult Tg(tTA:PrP) mice is not deleterious, whereas accumulation of PrPSc in the same line of animals is lethal (Table 5). Even though Purkinje cell degeneration in 70-week-old Prnp0/0 mice has been found (9), our data continue to argue that the accumulation of PrPSc and not a loss of PrPC function is responsible for the pathogenesis of prion disease.


Reversing the course of prion diseases by blocking the production of PrPSc through repression of PrPC expression will allow us to measure the removal of PrPSc. Such clearance studies, which were not previously possible, are a prelude to the development of effective therapies where drugs that block PrPSc formation are administered at the earliest onset of symptoms to patients with sporadic Creutzfeldt–Jakob disease. At present, we have no understanding of how much PrPSc can be tolerated by the CNS and how rapidly it will disappear once synthesis of its precursor, PrPC, is repressed.

 

“Tetracyclines and Prion Infectivity,” Forloni et. al., Infectious Disorders — Drug Targets (2009)
http://www.ingentaconnect.com/content/ben/iddt/2009/00000009/00000001/art00004

In the last decade information has accumulated on the potential anti-prion activity of polycyclic compounds. Initially we showed that the antitumoral idodoxorubicin reduced the infectivity in experimental scrapie. On the basis of the chemical homology with anthracyclines, we rapidly moved to tetracyclines, compounds that are safer and widely used as antibiotics in clinical practice. The tetracyclines, essentially doxycycline and minocycline, were characterized as a therapeutical tool in transmissible spongiform encephalopathies (TSE) through the cell-free condition, in cellular and animal models and they are now being investigated clinically with this indication. Tetracyclines interact with aggregates obtained by synthetic PrP peptides or pathological PrP (PrPsc) extracted from TSE brains, and they destabilize the structure of amyloid fibrils, reducing their resistance to digestion by proteinase K. Tetracyclines also interact with peptide oligomeric structures and inhibit the protein misfolding associated with PrPsc formation. These activities have been accompanied by a reduction of infectivity, verified by doxycycline treatment in experimental scrapie, and some curative effects after either peripheral or intracerebral infection. The anti-amyloidogenic activity of tetracyclines was tested in other forms of peripheral and central amyloidosis, with interesting results. This article analyzes the development of tetracyclines as a therapeutic tool in TSE in the light of recent results obtained in our laboratories.

 

“The efficacy of tetracyclines in peripheral and intracerebral prion infection,” De Luigi et. al., Plos One (2008)
http://www.ncbi.nlm.nih.gov/pubmed/18365024

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.