ST1859 reduces prion infectivity and increase survival in experimental scrapie
On the basis of the structural homologies between ST1859 (1[(2-hydroxy-1-naphtyl)methyl]-2-naphthol) and the anti-prion agents and its anti-amyloidogenic activity, we tested whether this molecule altered the biochemical properties of aggregates formed in vitro by synthetic prion peptides and affected prion infectivity in experimental scrapie. Co-incubation of ST1859 with the peptides PrP 106–126 and PrP 82–146 reduced their fibrillogenic capacity and their resistance to digestion with protease K. Hamsters inoculated with the ST1859-treated homogenate showed a significant delay in the onset of clinical signs of disease and longer survival. Survival was also significantly longer in infected hamsters treated peripherally with ST1859 for the whole post-inoculation period until the onset of clinical symptoms. Similar results were found with the analogue ST1745. Our data indicate that ST1859 reduces prion infectivity and can exert a therapeutic effect in experimental scrapie.
Prion diseases are transmissible neurodegenerative disorders (TSE) of humans and animals for which no effective treatment is available. Conformationally altered, protease-resistant forms of the prion protein (PrP) termed PrPsc appear to be critical for disease transmissibility and pathogenesis, offering a primary target for therapeutic strategies . PrPsc has some abnormal chemico-physical properties such as insolubility and protease resistance, and accumulates in the brain as amorphous aggregates and amyloid fibrils. Numerous molecules have been tested for their ability to reduce PrPsc infectivity in vitro [3, 10, 23] using infected cell lines or in vivo experimental scrapie, but until now, the therapeutic approach to TSE remains a challenge .
In the past, starting from the anti-amyloidogenic activity of iododoxorubicin , we demonstrated its anti-prion activity . Based on chemical analogy, we tested tetracyclines, a class of antibiotics with a better safety profile. The anti-amyloidogenic activity of the tetracyclines was verified in vitro, and treatment ex-vivo with these drugs strongly prolonged the survival in experimental scrapie and in some cases completely cured the infected animals [6, 22]. More recently, the efficacy of tetracyclines was confirmed by peripheral treatments [4, 11]. On the basis of these observations, doxycycline, a tetracycline that can pass the blood-brain barrier (BBB), was administered for compassionate reasons for several years to a group of CJD subjects. Retrospective analysis of the results supported the possibility of a double-blind placebo/doxycycline clinical trial in CJD subjects, which is now in progress. We also showed that tetracyclines interfere with the spontaneous assembly of β amyloid 1-42 (Aβ1-42) . The chemical analogy of tetracyclines and other drugs, like phenothiazines, which may interfere with prion infectivity , suggested that polycyclic structures might exert anti-amyloidogenic activity. We proved the anti-amyloidogenic activity against Aβ1-42 of a di-naphthol derivative, ST1859 [1,1-methylene-di-(2-naphthol)], a compound with anthelminthic and anti-inflammatory activity. In vitro ST1859 binds specifically to Aβ1-42 and prevents its aggregation and fibril formation. 14C-labeled ST1859 rapidly crosses the BBB in healthy and transgenic mice, achieving concentrations several times higher in the brain than in plasma . The molecule was also tested in volunteers and Alzheimer patients for positron emission tomography (PET) analysis as a means of identifying Aβ deposits in human brain .
In the present study, we tested ST1859, and its analogue ST1745, as anti-prion agents in vitro and in vivo. We evaluated their effects on the biochemical properties of aggregates formed in vitro by synthetic PrP peptides and on prion infectivity in experimental scrapie.
Materials and methods
Peptide synthesis and anti-aggregation activity
Peptides corresponding to region 106–126 (KTNMKHMAGAAAAGAVVGGLG) or 82–146 (GQPHGGGWGQGGGTHSQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIHFGSDYE) of the human prion protein sequence were synthesized using solid-phase chemistry, with a 433A instrument (Applied Biosystems, [9, 18]). PrP 106–126 and PrP 82–146 were dissolved at a final concentration of 1 mM in 100 mM Tris–HCl, pH 7.4, containing 20% DMSO (buffer solution) and added to the same volume of buffer solution only (control samples) or buffer solution containing ST1859 or ST1745 (treated samples) at a final concentration of 0.25, 0.50, or 1 mM. The samples were incubated at 37°C for 72 h. After centrifugation at 13,000g for 15 min at 4°C, the supernatant was discarded and the pellet was dissolved in formic acid and analyzed by reverse-phase HPLC. The relative amount of non-sedimentable peptide was calculated as a percentage of the peptide in the initial solution.
Proteinase K digestion
PrP 106–126 and PrP 82–146 were dissolved as described for anti-aggregation activity and added to the same volume of buffer solution only (control samples) or buffer solution containing ST1859 or ST1745 (treated samples) at a final concentration of 0.25, 0.50, or 1 mM. The samples were incubated at 37°C for 72 h and after incubation, CaCl2 was added in buffer solution to a final concentration of 1 mM. Prion protein was digested with proteinase K (37°C, 30 min) at a 1:100 (w/w) enzyme-to-substrate ratio. Proteolysis was terminated by the addition of EGTA (5 mM final concentration). After centrifugation at 13,000g for 15 min at 4°C, the supernatant was discarded, and the pellet was dissolved in formic acid and analyzed by reverse-phase HPLC. Parallel samples were run without ST1859 or ST1745 and analyzed as above with or without proteinase K digestion. The extent of proteolysis was calculated from the peptide present in the pellet as a percentage of the total amount originally present.
Male golden Syrian hamsters, 8–10 weeks old, were housed under conventional laboratory conditions at room temperature (20°C) and 40% humidity with a 12/12 h light/dark cycle (7:00 a.m.–7:00 p.m.). The animals were allowed free access to food and water.
Brain homogenate infected with the 263K strain of scrapie at a dilution of 10−3 was incubated for 24 h at 37°C with either the test compounds (1 mM) or PBS. The hamsters were injected intraperitoneally (i.p., 50 μL, 10−3) or intracerebrally (i.c. 20 μL, 10−5) with infected homogenate pre-incubated with the test compounds or PBS. All of the preparations of inoculum, with or without the ST compounds, contained 20% DMSO. Two groups of animals infected i.c. and two groups infected i.p. were treated peripherally with ST1859 or ST1745 three times a week until clinical symptoms were seen in the inoculated controls. All of the animals, control and treated, received an injection of the vehicle (PBS with 20% DMSO) or compound dissolved in the same solution. ST1859, 25 mg/kg, was well tolerated, but some animals treated with ST1745 at the same dosage died, so the dose was reduced to 10 mg/kg from the third week of treatment. After this, no more deaths were recorded before scrapie symptoms appeared.
The animals were observed twice a week to record their weight, and the onset and progression of clinical signs of disease. Behavioral analysis included their reactivity to tactile and acoustic stimulation, posture, balance and coordination, and tremors, as described previously . At the terminal stage of disease, the animals were euthanased, and 6–10 animals per group were used for statistical analysis.
Procedures involving animals and their care were conducted in conformity with national and international laws and policies (EEC Council Directive 86609, OJ L358, 1, 12 December 1987; Italian Legislative Decree 116/92, Gazzetta Ufficiale della Repubblica Italiana n.10, 18 February 1992; Guide for the Care and Use of Laboratory Animals, US National Research Council, 1996).
These findings prompted us to examine whether the physicochemical changes in PrP peptides induced by ST1859 or ST1745 were associated with a decrease in prion infectivity. 263K scrapie-infected brain homogenates from hamsters at the terminal stage of disease were incubated with 1 mM ST1859 or ST1745 or vehicle for 24 h and then inoculated into Syrian hamsters.
Effect of treatment with ST1859 and ST1745 on the survival of Syrian hamsters inoculated with homogenate infected with 263K
Median survival (days)
Significance. (Logrank test)
(A) Intracerebrally i.c.
Co-incub. with ST1859
P < 0.001
Co-incub with ST1745
P < 0.001
P < 0.03
(B) Intraperitoneally i.p.
Co-incub with ST1859
P < 0.001
Co-incub with ST1745
P < 0.001
P < 0.001
P < 0.001
After intraperitoneal inoculation (10−3), the median survival time was longer, 152 days (Table 1B), under control conditions, and the treatment with ST1859 and ST1745 prolonged it 51% (230 days, P < 0.001 vs. control, Logrank test) and 23% (187 days, P < 0.001 vs. control, Logrank test), respectively. The appearance of symptoms was also delayed, so both drugs significantly inhibited prion infectivity.
In another group of animals, we tested the therapeutic effects of ST1859 and ST1745 administered three times per week at 25 mg/kg i.p., starting from the day after the inoculation and continuing until symptoms appeared in the control group. The ST1745 dosage had to be reduced to 10 mg/kg after the first 3 weeks because of mortality among the animals treated with this drug, but at the lower dosage, no more deaths were recorded during the treatment.
The systemic treatments had only a modest effect on survival when the infection was intracerebral (Table 1A). Only ST1745 induced a significant activity (P < 0.03, Logrank test), and in this case, median survival increased about 8% (129 days) compared to the control condition (119 days, Table 1A).
When the inoculum was applied intraperitoneally (Table 1B), ST1859 or ST1745 induced a significant increase in the median survival time (12%) (controls 152 days, ST1859 170*, ST1745, 170*; *P < 0.001 vs. controls, Logrank test). In both cases, the appearance of symptoms was delayed to a similar extent.
ST1859 interacts directly with PrP peptides, reducing their fibrillogenic capacity and resistance to protein K digestion. The effect under cell-free conditions was evident using the biologically active peptide PrP 106–126, and also with the long peptide 82–146, which is homologous to the sequence identified in GSS brain [5, 17, 18, 22]. Hypothetically, the reduction in resistance to proteinase digestion should be accompanied by an effect on prion infectivity. In fact, incubation of 263K scrapie-infected brain homogenate with ST1859 and its analog ST1745 before inoculation did delay the appearance of clinical symptoms and prolonged survival. We tested the effect on prion infectivity using inoculation directly into the brain or peripheral application at two different concentrations, and in both cases, there was a substantial effect on survival with both ST1859 or ST1745.
The pharmacokinetics of ST1859 is extremely favorable in terms of BBB passage, as shown in humans in a PET pilot study . Its chemical characteristics are similar to those of other compounds that interfere with PrPsc aggregates, like tetracyclines or tetrapyrroles [3, 15]. These data led us to investigate the therapeutic potential of ST1859 in prion diseases as well. The direct interaction with PrPsc might explain the anti-prion effect, and the ex-vivo studies in experimental scrapie confirmed this possibility. Similar results were obtained with doxycycline [22, 6], but we also investigated the efficacy of ST1589 and ST1745 in 263K-infected hamsters treated peripherally after the inoculation. When the inoculum was applied intracerebrally, ST1859 had a modest and non-significant effect; the result was similar with ST1745, but in this case, treatment significantly prolonged survival. When the infection was induced by an intraperitoneal inoculum, the results were a little better. Both drugs significantly increased the median survival time by about 12%, while the comparison of the mean survival times showed an increase of 14% for ST1745 and 18% for the ST1859. These results are promising; both drugs not only reduced prion infectivity when incubated with the inoculum, but also induced a partial therapeutic effect. This could be improved with a different schedule of treatment, considering the rapid metabolism of ST1859 in the body . Details of the pharmacokinetics of the drug were not available when we started the experiments, so the decision to treat the hamsters only three times per week was in fact not optimal, and treatment once or even twice a day would be more appropriate to ensure a high level of the drug in the brain for long enough to be effective.
The possibility of combining different pharmacological activities in a single molecule steered the synthesis of ST1745, which contains bisnaphthol and acetylcarnitine structures. The neuroprotective activity of acetylcarnitine has been studied in numerous models, and a positive result was found in AD patients [14, 19]. Thus, treatment with ST1745 should improve the efficacy of ST1859 with the addition of protection against neurodegeneration. There were no substantial differences between ST1859 and ST1745 in either the ex-vivo experiment or with systemic treatment. However, the absence of a difference in the inhibition of prion infectivity is encouraging, since ST1745 maintained a level of anti-prion activity similar to that of ST1859. It is important to consider the mortality observed in some animals treated with 25 mg/kg ST1745 in the early period that obliged us to reduce the dosage to 10 mg/kg. This does not improve the therapeutic perspective of this drug, though in terms of biological mechanism, it is interesting that the effects with ST1745 were obtained at a lower concentration than with ST1859. When the inoculum was applied peripherally, both drugs had exactly the same effect on the median survival time of the hamsters.
For ST1745 too, the schedule of treatment could be improved, for instance, treatment was stopped at the onset of symptoms in the control group, but the drug may still have neuroprotective activity. In any case, initial evidence did not indicate any substantial improvement in the efficacy of ST1745 over ST1859 in experimental scrapie, but since the anti-prion activity of ST1859 is conserved, interest in this compound has not faded. Together with more appropriate treatment conditions, other investigations needed to clarify the mechanism of action of these drugs. ST1859 and ST1745 confirmed their potential as a therapeutic tool for neurodegenerative disorders involving protein misfolding. In prion-related encephalopathies, where it is particularly difficult to identify active molecules, they are candidates for a prophylactic strategy to inactivate prions and prevent acquired forms of disease.
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