1 Institut de Génétique Humaine du CNRS, 141 rue de la Cardonille, 34396 Montpellier, France
2 Laboratoire de Chimie de Coordination du CNRS, 205 route de Narbonne, 31077 Toulouse, France
3 Service de Neurovirologie, CEA, CRSSA, EPHE, BP 6, 92265 Fontenay aux Roses cedex, France
4 Laboratoire de Biochimie, Hôpital St Eloi, 80 av. A. Fliche, 34295 Montpellier Cedex 5, France
Correspondence
Sylvain Lehmann
Sylvain.Lehmann{at}igh.cnrs.fr
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ABSTRACT |
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Contributed equally to this work.
In memory of Professor Dominique Dormont.
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INTRODUCTION |
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In the present work, we tested cationic phosphorus-containing dendrimers (P-dendrimers) for anti-prion activity. These new classes of branched-polyamines differ from the molecules described previously by Supattapone et al. (1999), principally by their protonated tertiary amine end-groups (Loup et al., 1999
). They have a hydrophilic surface and a hydrophobic backbone which allows very efficient membrane penetration (Loup et al., 1999
). Here we show that P-dendrimers were able to clear PrPSc rapidly in ScN2a cells with an IC50 in the nM range. They can interact with PrP, and are effective against pre-existing PrPSc, as was observed when incubated with brain homogenates infected with different prion strains. Using a rapid in vivo screening model we show that these drugs are able to inhibit PrPSc accumulation in the spleen of mice intraperitoneally inoculated with scrapie. P-dendrimers represent, therefore, new molecules for TSE therapy, or at least for post-exposure prophylaxis, since no pre-clinical diagnosis is available to date.
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METHODS |
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P-dendrimers.
P-dendrimers were synthesized by the Laboratoire de Chimie de Coordination du CNRS and were selected following the study by Supattapone et al. (1999) with the aim of obtaining new anti-prion agents that could be useful in vivo. P-dendrimers are characterized by the presence in their backbone of aminothiophosphates at each branching point which may enhance biocompatibility. The repetitive controlled divergent growth technique which forms the P-dendrimer backbone used these phosphorus atoms at each branching point. Phosphorus-containing dendrimers pd-G3, C624H1104N183Cl48O42P45S42 (generation 3); pd-G4, C1296H2256N375Cl96O90P93S90 (generation 4) and pd-G5, C2640H4560N759Cl192O186P183S186 (generation 5) were synthesized and purified as described previously (Loup et al., 1999
). They have protonated terminal tertiary amines (Fig. 1
a). P-dendrimers were dissolved in sterile distilled water at a stock concentration of 10 mg ml1 and filtered through a 0·22 µm Millipore filter before use. A plot of the effect of the pH of the medium on the charge carried by P-dendrimers was obtained by polyelectrolyte titration (Fig. 1b
), which consisted of a stoichiometric reaction of a polycation with a polyanion. The titration end point was determined by means of a particle charge detection apparatus from Mütek, München, Germany.
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Incubation of brain homogenates with P-dendrimers.
Brain homogenates of 263K, Chandler and 22L strains at 10 % (w/v) were kindly provided by R. Carp (New York State Institute for Basic Research, USA) and BSE homogenates by T. Baron (AFSSA, Lyon, France). Eighty micrograms of brain homogenate in 40 µl PBS (2 % w/v) was incubated with 200 µg P-dendrimers ml1 or PBS for 2 h at 37 °C with constant shaking. After incubation, samples were made up to 0·5 % NP-40, 0·5 % sodium deoxycholate, and were digested with PK as described for cell lysates (see below). The samples were then mixed with an equal volume of 2x SDS sample buffer and analysed by Western blotting for the presence of PrPSc.
Cell culture and detection of PrP.
The ScN2a cells used in this study correspond to clone N2a#58 infected with the mouse-adapted scrapie strain 22L (Nishida et al., 1999). Cells were cultured in MEM containing 10 % fetal calf serum, 300 µg geneticin ml1, 100 U penicillin ml1 and 100 ng streptomycin ml1 in an atmosphere of 5 % CO2. After incubation in the presence of P-dendrimers for the indicated time, cells were collected in PBS and lysed for 20 min at 4 °C in PBS containing 0·5 % NP-40, 0·5 % sodium deoxycholate, 1 µg pepstatin and leupeptin ml1 and 2 mM EDTA. After 1 min of centrifugation at 10 000 g, the supernatant was collected and the total protein concentration was measured using the BCA protein assay kit (Pierce). Samples were adjusted for protein concentration and were treated with 16 µg PK (mg total protein)1 for 30 min at 37 °C. Digestion was stopped by the addition of 1 mM Pefabloc and incubation for 5 min on ice. After centrifugation (20 000 g for 45 min, 4 °C), the supernatants were discarded and the pellets were resuspended in reducing SDS sample buffer. Proteins were electroblotted onto Immobilon membranes. PrPSc was detected using SAF-mix antibodies and a peroxidase-conjugated goat anti-mouse secondary antibody. PrPC was detected in the cell lysate before PK digestion with rabbit polyclonal antibody P4566 and a peroxidase-conjugated goat anti-rabbit secondary antibody. Blots were developed using enhanced chemiluminescence (ECL). Films were analysed using Sigma Scan image analysis software.
Cell-to-cell ex vivo transmission.
Cells were collected from a confluent 175 cm2 flask under sterile conditions and resuspended in 100 µl cold PBS with 5 % glucose. Cell suspensions were submitted to four cycles of freeze-thawing in liquid nitrogen to prepare the inoculum. The extracts were then passed through a 27-gauge needle several times. Twenty microlitres of the extracts diluted in 1 ml Opti-MEM were added to uninfected N2a#58 cells for 2 days. Cells were passaged every 34 days at confluence and tested for the presence of PrPSc.
Interaction of P-dendrimers with PrP.
To study the possible interaction between PrP and P-dendrimers, 15 ml Amberlite IR-120 (plus) ion exchange resin (sodium form) was immersed in pentane and mechanically stirred for 1 h, and then filtered and rinsed with distilled tetrahydrofuran and water (until the filtrate remained colourless). Ten millilitres of this wet resin was introduced into a flask containing a solution of 342·5 mg pd-G4 in 15 ml distilled water. The mixture was mechanically stirred for 96 h. The resin was then filtered and washed with distilled water. The filtrate was evaporated, weighed and analysed by 31P NMR in D2O to control for the absence of P-dendrimer (Loup et al., 1999). For pull-down experiments, beads were rinsed several times in a buffer containing 150 mM NaCl, 0·5 % Triton X-100, 0·5 % sodium deoxycholate, 50 mM Tris/HCl (pH 7·5) and protease inhibitors (1 µg pepstatin and leupeptin ml1, 0·5 mM PMSF, 2 mM EDTA). Twenty micrograms of beads coated, or not, with pd-G4 were added to 50 µl N2a#58 cell lysate at 0·5 mg protein ml1 for 2 h at 4 °C under constant agitation. After a rapid centrifugation (10 000 g for 30 s), the beads were collected, washed several times with the same buffer and boiled for 5 min with 30 µl SDS loading buffer. After centrifugation, the supernatant was collected, subjected to SDS-PAGE and Western blotted as described above.
Infectivity assay.
Experiments were performed in C57BL/6 mice acquired from R. Janvier (Charles Rivers, L'Arbresle, France). Three groups of eight 3-week old female mice were inoculated with strain C506M3 by the intraperitoneal (i.p.) route with 100 µl 2 % brain homogenate in 5 % glucose. The murine strain C506M3 is derived from a natural case of sheep scrapie (a kind gift from Corinne Lasmezas, CEA, Fontenay aux Roses, France). Strain C506M3 has been characterized in intracerebral and i.p. infection models (Lasmezas et al., 1996).
For the treatment with pd-G4, mice were intraperitoneally injected every 2 days, from day 2 after the inoculation to day 30, with either 50 or 100 µg per mouse. The untreated group were injected with physiological serum. The mice were sacrificed 30 days after infection, the time when spleen PrPSc reaches a plateau in untreated animals.
PrPSc detection in the spleen of C57BL/6 mice by Western blot.
Tissues were homogenized at 10 % (w/v) in the same lysis buffer used for PrPSc detection in cell culture. PrPSc was purified by centrifugation in the presence of detergents, after adapted PK digestion (10 µg PK for 100 mg tissue). Samples were loaded on a 12 % polyacrylamide SDS gel and then transferred onto a nitrocellulose membrane. Immunoblotting was performed as described for the cell culture detection of PrPSc. For each experiment, the brain of a C57BL/6 mouse at the terminal stage of the disease was used as a positive control.
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RESULTS |
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Cell-to-cell transmission experiment
A cell-to-cell transmission experiment was performed to evaluate the possibility of infectivity remaining following P-dendrimer treatment of ScN2a. This alternative approach to animal inoculation is now used by several laboratories and illustrates the possibility of detecting infectivity by means of highly susceptible cell lines (Bosque & Prusiner, 2000; Nishida et al., 2000
; Klohn et al., 2003
). We have previously demonstrated that prion transmission to N2a#58 was possible over a four logs range of infectivity (Lehmann et al., 2001
). Cell extracts from ScN2a treated with 10 µg P-dendrimer ml1 for 3 weeks, and from untreated cells were therefore prepared and used to infect N2a#58. The presence of PrPSc was subsequently assayed after several passages (Fig. 4b
). Even after up to eight passages (lanes 26), PrPSc could not be detected in the N2a#58 cells inoculated with treated cells, while cells inoculated with control ScN2a accumulated significant levels of PrPSc after only three passages (lanes 711).
P-dendrimers modified protease resistance of PrPSc from brain homogenates at neutral pH
The effect of P-dendrimers on PrPSc from 263K, BSE, Chandler and 22L brain homogenates from different species was tested (Fig. 5a). An average decrease of 50 % in the PrPSc level from infected 22L brain was observed following incubation of the homogenate with pd-G4 at neutral pH. pd-G4 was also able to induce a significant decrease of PrPSc in Chandler, BSE and even a disappearance in 263K samples.
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Inhibition of prion replication in C57BL/6 mice
To confirm the potential of P-dendrimers as anti-prion agents in vivo, we applied a protocol allowing for a rapid estimation of the peripheral accumulation of PrPSc during the incubation period. C57BL/6 mice were challenged intraperitoneally (i.p.) with C506M3 scrapie brain homogenate derived from terminally ill mice. Two different groups of eight mice were treated with 50 or 100 µg of P-dendrimer per mouse, every 2 days by i.p. injection from day 2 to day 30 post-inoculation. Control animals were injected with the vehicle only (physiological saline). The mice were sacrificed 30 days after infection, the time when spleen PrPSc reaches a plateau in untreated animals. Western blot analysis of mouse spleens revealed that treatment with 50 or 100 µg P-dendrimers inhibited PrPSc accumulation significantly by up to 66 or 88 %, respectively (P<0·05; MannWhitney U-test) (Fig. 6 bc).
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DISCUSSION |
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In this study, P-dendrimers were able to remove PrPSc from ScN2a cells infected with the 22L strain, and inhibited PrPSc replication in the spleen of C57BL/6 mice that had been intraperitoneally infected. Therefore, P-dendrimers can be considered relevant candidates for prion therapy. Indeed, cell culture studies showed that P-dendrimers were able to inhibit PrPSc replication in a dose-dependent manner (Fig. 2). Dendrimers were also able to clear prion infectivity, since PrPSc did not reappear 5 weeks after treatment was stopped and no infectivity was detected in cell-to-cell transmission assays (Fig. 4b
). When the different P-dendrimer generations were compared, the generation 4 (pd-G4) and generation 5 (pd-G5) species, which have higher surface densities than pd-G3, were the most efficient. These data also confirmed the potency of branched polyamines for denaturating PrPSc as observed by Supattapone et al. (1999)
, and showed that branching architecture and high surface density of terminal tertiary amines were required for dendrimers to denature PrPSc in vitro. This suggested that an optimum balance exists between the size of the compounds and the number of end-groups presented on their surface.
The possible mechanism by which P-dendrimers could interfere with prion replication was also investigated. In particular, P-dendrimers can bind to PrP molecules as shown by the retention of PrP on dendrimer-coated amberlite beads. These molecules also reduced PrPSc PK resistance in different animal brain tissues from several sources (experimentally infected rodents and BSE-infected cow). This suggests that preformed PrPSc aggregates are disrupted by P-dendrimers. Moreover, time-course experiments demonstrated that P-dendrimers are rapid in action, as PrPSc clearance occurred within 4 h of treatment. The low IC50 of these molecules allows them to be well tolerated, with no apparent cytotoxicity. This was confirmed by the fact that the levels of total protein, PrPC and GAPDH remained unchanged in the cultures during treatment (Fig. 4). Toxic concentrations of P-dendrimers in culture were above 25 µg ml1, which is much lower than those observed for other dendrimers such as PAMAM (Malik et al., 2000
). In our study, in vivo experiments with C57BL/6 mice showed that P-dendrimers were well tolerated at the concentration used during the 1 month of treatment. No significant changes in behaviour or body weight of the animals were observed. Significant haemolysis, granuloma or abscess reaction were also absent from the injection site and histopathology analysis of several organs was unaltered.
Importantly, dendrimers appear to have a wide biodistribution throughout the body, as they can be detected after peripheral injection in various organs such as liver, intestine, stomach, lung muscle, kidney and bone (Malik et al., 2000). In our experiments, P-dendrimers were most likely able to reach the spleen where they inhibited PrPSc replication. With regard to the pathogenesis of TSEs, the role of the lympho-reticular system organs in the early stage of infection has been demonstrated in a number of studies (Bruce et al., 2000
). This prompted us to develop a protocol based on a rapid estimation of peripheral accumulation of PrPSc. In particular, the spleen of C57BL/6 mice is infectious and exhibits PrPSc accumulation long before the agent reaches the central nervous system after intraperitoneal or oral inoculation of strain C506M3, thus more probably mimicking the natural route of contamination by prions (Lasmezas et al., 1996
). Since therapies to control brain pathology are very challenging, it is essential to develop strategies that can inhibit prion replication before or during neuroinvasion. It has been demonstrated that blocking replication in the spleen with different compounds increased the incubation time (Beringue et al., 2000
; Heppner et al., 2001
; Mabbott et al., 2003
). In our in vivo experiments, we observed a significant but variable inhibition of PrPSc generation in the spleen of infected mice treated with P-dendrimers (Fig. 6
). It is possible that higher doses of P-dendrimers would have completely blocked PrPSc accumulation in the spleen. Recent data obtained following the same protocol using quinacrine and MS-8209, on C57BL/6 animal models with the identical strain C506M3, have shown that quinacrine was not effective on peripheral PrPSc accumulation, while MS-8209, which is an analogue of amphotericin B, was able to inhibit PrPSc replication with efficacies between 70 % and 90 %. Since we observed an average efficacy of the P-dendrimer close to the one observed for MS8209, and considering the fact that this latter molecule was described in the past as an anti-prion agent delaying the incubation time by a factor of two in hamsters (Barret et al., 2003
), we can confidently predict that P-dendrimers will delay the incubation time. Although dendrimers are well biodistributed, there are no data reporting their entry into the central nervous system through the bloodbrain barrier. In this regard, it might be possible to consider P-dendrimer treatment at least as a post-exposure prion prophylaxis as has been proposed for other molecules (Sethi et al., 2002
). Whether treatment with P-dendrimers would also be effective when given later after exposure is not yet known. We will investigate this in the future and are optimistic for the outcome, since P-dendrimer also induced the degradation of pre-existing PrPSc aggregates in vitro. Indeed, as stated above, once aggregates exist in brain homogenates, dendrimers are effective and can reduce PrPSc PK resistance.
Interestingly, high molecular mass polymers, like dendrimers, have been widely used as soluble drug carriers to improve drug targeting and therapeutic efficacy (Padilla De Jesus et al., 2002). They could thus be designed to possess cavities which can accommodate guest molecules. In this context, it might be possible to synthesize hybrid molecule to combine the natural anti-prion activity of P-dendrimers with that of other molecules such as porphyrins or tricyclic derivatives (Korth et al., 2001
; Priola et al., 2000
). Targeting the dendrimers to particular tissues might also be possible by having different types of end-groups, as demonstrated by several projects aiming to treat infectious diseases (Bourne et al., 2000
; Nishikawa et al., 2002
) or cancer (Vincent et al., 2003
).
In summary, the results reported here indicate that P-dendrimers exert an inhibitory effect on PrPSc generation and an anti-infectivity action as observed in both cell and mice models, thus offering a therapeutic approach to prion diseases. In particular, P-dendrimers were effective on different prion strains including that responsible for BSE. This is of a particular interest, given the unique properties of the latter strain and its involvement in the appearance of variant CJD (Will et al., 1996).
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ACKNOWLEDGEMENTS |
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Received 17 October 2003;
accepted 12 February 2004.
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