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>The Treatment of Transmissible Spongiform Encephalopathies

The Treatment of Transmissible Spongiform Encephalopathies


Treatment: review


S. Dealler, Consultant Microbiologist, Burley General Hospital, Burnley BB10 2PQ.  01282 701501

This particular page discusses the chemicals that have been shown to have a useful effect on the development of disease signs either in vitro on in vivo.  Links to chemicals that can be used for clinical purposes will lead to other pages

Index:


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Polysulphonated polyglycosides


Initially the in vitro experiment of Caughey et al (56) that demonstrates that these have some activity on the infective agent is considered. Then each of them will be discussed individually. Individual activity: see Table 1 and Figure 1.

Table 1.  In vitro tests on polysulphonated glycosides and congo red

 Potent inhibitors of PrPsc production:

Intermediate inhibitors of PrPsc production: No effect or not tested:


Introduction


The effect of congo red and pentosan polysulphate (PPS) were seen even if the inhibitor was used transiently, and washed from the culture vessel.  The idea that this could be due to the chemical association with the cell walls was felt unlikely as the serial passages would have diluted this also.  The effect therefore seemed to be due to some association remaining between both compounds and the infective agent and that this remained despite cellular culture passage.

The effect of stopping the build up of PrPsc was considered to be possibly due to the chemicals causing the increase in its breakdown of it either as a produced form or as the infective agent itself. An attempt was made to look for destabilisation of PrPsc but none was found.

The mechanism by which the compounds appeared to work were discussed in the article (56) and considered to be their interaction with the infective form of the agent inside the cell and inhibition of its multiplication.  The fact that the successful substances were high molecular weight polysulphates except congo red, which has a molecular weight 697, suggested to the authors that the CR might build up in a polymerising form.  Also, the lower sulphation of the less active kappa-carrageenan as opposed to the iota or lambda forms suggested that this was important.  The low molecular weight of dextran sulphate 8 was associated with much lower activity than dextran sulphate 500 (DS500) suggesting that MW is also an important factor.  The knowledge that the formation of amyloid took place in vivo with the interaction of glycoso-aminoglycans (such as heparin) suggested also that PrPsc would interact with these compounds in a relatively reliable way.

No claims have been made for the use of this type of compound for the treatment of animals with symptoms.  There is no penetration of them into the CNS.  They tend to be toxic but little data is available.

It has now been shown that the polysulphonated polyglycans all did interact with the PrPc in vitro and the exact strength of this interaction was shown to be highest for pentosan polysulphate (293).  In this work is seemed that the copper ligand activity of the PrPc altered the PPS binding.   It is interesting in that the heparins that are associated with scrapie in vivo and extracted in vitro could be shown to have relatively low levels of sulphation (294).  The significance of this is unclear except that it would be expected that low sulphation forms would permit the amyloid to come apart easiest and so be found in the study, whereas high sulphation GAGs may not permit this to take place.

It has now been shown that GAGs, dextran and PPS can be used to increase the in-vitro alteration of PrPc to PrPsc in a non-cellular format (288).  The exact mechanism is unclear except that this is not surprising if the molecules supply some of the energy required for the PrPc alteration.

We are now sure that GAGs are found in decreased amounts in the brain stems of animals infected with BSE (and all other TSEs when tested) (327) although it makes up a significant fraction of amyloidal plaque itself (328) and this is present through out the development of the disease.  We know that heparin actually increases the production of PrPsc in vitro (cell free) from PrPc, and that it acts in compettition to both other GAGs/polyanions and the dyes.

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Pentosan polysulphate (PPS)

Major information on pentosan polysulphate and a prophylactic  is on another web site.

Pentosan is obtained from beechwood shavings and as such is effectively polyxylose with a molecular weight of approximately 5,000, being derived from relatively pure lignin derivatives, but of short enough chains to be extracted by the processes involved.  After sulphation, pentosan becomes pentosan polysulfate (PPS), a highly sulphated, semi-synthetic polysaccharide somewhat similar to heparin or dextran sulphate (57) (figure 7). Amazingly it can be given orally in capsules (Elmaron, Norton Health Care, Harlow. 01279 424425) and it seems to be excreted intact in the urine. As such it is found on the surface of the bladder and the prostate and has been used for many months as continuous courses.  Increased blood transaminases are found in 1.2% of patients and this takes place several months after the start of the course.
  Since 1962 PPS has been used to prevent postoperative thromboembolism in France.  At equal doses it has 8% of the activity of heparin.  The dose used was 50 milligrams intramuscularly or subcutaneously for 2 days.  PPS or heparin are probably equally effective in general surgery with a similar chance of haemorrhage (57).  PPS, like heparin, catalyses the inactivation of thrombin through antithrombin III.  It has recently been shown to be of value in the treatment of interstitial cystitis (58) and abacterial chronic prostatitis, where irritation of the of the organs is prevented by the presence of pentosan.
  Pentosan may have tumoricidal potential (59) in vitro and animal (mice) studies show that PPS inhibits autocrine and paracrine growth stimulation by Kaposi's sarcoma-derived fibroblast growth factor,  thereby repressing tumour.  But in mice, as tumour size increased, efficacy of pentosan decreased.  This may be due to not enough PPS given to inhibit the higher activity of growth factor in larger tumour.
  PPS inhibits reverse transcriptase (57,60) and inhibits virus absorption onto cells (61) at 1 microgram/ml for virus replication and 16 micrograms/ml for giant cell formation; giant cells are believed to be important in the depletion of CD4+ T-lymphocytes (61).  PPS has been shown to inhibit RNAase H activity (62) and this may also be involved in its in-vitro ability to inhibit HIV replication. This type of compound, a polyanion seems to bind to factor H, which is involved in the stimulation of the alternative compliment pathway.
  Its action against scrapie has been demonstrated fairly early (63) and it was considered initially because of the lower toxicity when 50mg per mouse was inoculated intraperitoneally.   Later experiments showed that inoculation with pentosan polysulphate 50, 60 or 70 days before infection with scrapie decreased the effective dose that was given in that there was a significant increase in the incubation period and a higher dose was required to cause infection to take place (64).  It was expected that the pentosan would be found in the spleen phagocytosing cells after inoculation and that this could be stained for with toluidine blue.  However (unlike the effect seen with dextran 500) the pentosan was not apparently present (64) and the authors felt that this might suggest that a different mechanism of action should be considered for the two chemicals.  PPS was the most effective agent when inoculated into hamsters (vs DS500 and suramin) in that it increased incubation period by a wide range of days (from nil to 100% of the normal incubation period) (65).  It was also found that the delay in symptom onset did not seem to be dependent on whether the compound was injected 2hr or 24hr after the scrapie agent (65) and that the effective dose of scrapie inoculated (as measured by incubation period) seemed to drop to a similar lower dose no matter what the actual dose inoculated was.  The reason for this is unclear.  The author discusses the effects that the incubation period in mice is increased even when the PPS is inoculated weeks after the scrapie (but not in hamsters) in terms of the possibility that the agent enters the nervous ends in hamsters before the PS has its effect; although no adequate evidence for this taking place is available (65).  The intraperitoneal, intacerebral and oral treatment of mice inoculated (i.p.) with various scrapie strains showed the drug to be active as a prophylactic against the development of disease but that the oral rout was not as effective (326).
  PPS was found to be the most potent of chemicals tested in vitro against the scrapie agent growing in neuroblastoma cells (56) with MIC of around 10 ng/ml (see above).


Dextran Sulphate

Dextran sulphate 500, a polysulphated (1,6)-D-glucopyranoside chain with a molecular weight of approximately 500,000 (Figure 7).  Although the 1,6 linkage is predominant, there is a certain degree of 1,4 linkage present in the commercial compound.  Sulphonation takes place via ester linkages at the 2,3, and 4 points on the D-glucopyranosyl ring but the level of sulphonation on any particular ring is variable.   Sulphonic acid residues make up approximately 17% of the molecular weight of the molecule indicating that approximately 2 of the 3 sites are occupied by sulphonic acid per glucopyranosyl ring (Sigma).  The pharmacology of the compound is poorly investigated except that it is not expected to enter the central nervous system and excretion will be very slow with break down of the compound into D-glucose and local metabolism taking place.
  It was demonstrated that a single inoculation administered 72 days before infection still had a  statistically significant effect on the survival of mice challenged with the 139A strain of scrapie (1)(Table 13).  When injected intravenously it was taken up by macrophage cytoplasm in the spleen and lymph nodes (66) and could be found there for more than 100 days.  the mode of action was through inhibition of the replication of the agent (63) and other polyanions with lower toxicity caused a similar effect when administered around the time of infection (63).  Notably this was true either if the compound was inoculated either before or after the injection of scrapie (67).  The finding that DS500 prevented the uptake of the agent into splenic cells or perhaps into nerve endings (65) may be of significance. Work by Kimberlin and Walker (68) indicated that the dose of DS500 was important in the level by which  the incubation period of the disease in mice was increased, but that the route of inoculation (i.p vs i.v.) was of little significance. When given intracerebrally with the agent no effect was seen (63). The incubation period was increased in hamsters by a single dose of DS500 in a dose dependent manner up to 32mg/kg. The incubation period of the disease was increased by around 20% (maximum) when given within 2 hrs of the inoculation of the scrapie agent and to rhave less effect when given further before or after the agent inoculation (Table 13).  Higher doses were found, however to be acutely toxic with a TD50  of 85.4 mg/kg of body weight.
  The mode of action is not clear.  Initially it was suggested that the action of DS500 as a B-cell mitogen might be involved but that has not been born out (68) and its activity as an immunostimulant are unlikely to be of significance.

Carrageenan

This is the hydrocolloid obtained by extraction with water or aqueous alkali from some members of the class of Rhodophyceae (red seaweeds).  It consists chiefly of a mixture of the ammonium, calcium, magnesium, potassium and sodium salts of sulphonated galactose and 3-6-anhydrogalactose  (and  other) copolymers.  The prevalent copolymers in the hydrocolloids are k-, i- and l-carrageenans.

Chemistry:  Idealised repeating units of carrageenans i.e. if pure and if perfect chains.  In biological products this is unlikely to be perfect. (structures shown for i,k and l in Figure 8, Table 14)

Table 2.  Idealised carrageenan structure
 

*in the original definition only 70% of the 3-linked b-D-galactopyranose were sulphated

This 'idealised structure' is unrealistic in that the rate of sulphonation and the ratio of one pyranose residue to another are not perfect in the biological product (69,70,71).  However the repeating disaccharide residues give rise to a reasonable ability to estimate the structure of the compound in solution (71,72).  Using computer models it is possible predict, even when building in to the structure extra monosaccharides that are dissimilar to the repeating disaccharides, stable single right handed helices.  One author has found double helices composed of parallel 3-fold right handed single helices (73). It is agreed that the size of the molecule (possible 200,000-residue polymer groups) does not permit X-ray diffraction to give a good determination of structure and hence the various computer models may be the only current indication that we can get.  The calculations showed variations in the predicted shape of the helices of  i,k and l carrageenan and that these differences depended to a large degree on the sulphonation of the glycopyranose monomers (72). This viewpoint is probably simplistic in that carrageenan exists as a network of fibres that interact in the format of a loose lattice (71).  The interesting results showing that k carrageenan, the least sulphonated of the group was also least active  against PrPsc production might suggest that a further compound, furcellaran, which is similar to k carrageenan but with only half the number of sulphated sites (71), could be tested.

Carrageenans are used in the pharmacy and food industries as a suspending and gelling agents (74).  Chondrus was used similarly.  A degraded form of carrageenan was used to be given in gastro-intestinal disorders but it was associated with lesions in animals and is no longer used.  After considering further animal data it was decided that an acceptable daily intake was  'not specified' to be allocated to refined non-degraded carrageenan.  This means that on the basis of available data, the total daily intake of this form, arising from its use at the levels necessary to achieve the desired effect and from its acceptable background in food, does not represent a hazard to health.  The establishment of an acceptable daily intake was not deemed necessary.
  Carageenan is known as a powerful inhibitor of macrophage activity (75-80), which is odd in that polyanions are also used as stimulators of macrophages.  The reason for both of these activities is that it will stimulate macrophages in such a way that they will apparently no longer respond to further stimulation from specific antigens. As a result it causes a decrease in non-specific resistance to related antigens in mice (82).    It has commonly been used as an adjuvant with an antigen for the rapid and effective production of antibodies (82) and the experimental production of hyperalgesia (83).  The actual significance of carrageenans in this experiment was possibly their ability to inhibit the presentation of particulate antigens to lymphocytes and presumably to other cells also (84), and with high levels of infusion to effectively ablate the activity of macrophage (Kupfer) cells in the liver (85).   The pharmacology of the products is poorly known following parenteral administration but it is expected to be taken up by reticuloendothelial cells in a similar manner to heparin.
  Little data is available except that of Caughey et al (56) as to the action of carrageenans as anti-TSE agents.



 

Heparins

A linear polysaccharide of MW 60,000 to 100,000, covalently attached to a core protein that is found in mast cell secretory granules.  It is synthesised as alternating D-glucuronic and irregularly sulphonated N-acetyl-D-glucosamine residues (Figure 7).  Epimerisation of the glucuronic acid to iduronic acid may take place with sulphonation of the 2 position of either.   Heparan sulphate  is chemically similar but has less epimerisation of the glucuronic acid (Table 15) and dermatan sulphate is a similar compound.  Heparin acts as an anticoagulant by stimulating antithrombin III, is given parenterally, distributed in the extracellular body fluid outside the central nervous system and is cleared mainly by first order kinetics into the reticuloendothelial system (half life around 8 hrs).
  Its effect on the production of PrPsc in vitro was relatively low compared with pentosan (56) (see above).
 

Table 15.  Idealised structures of heparan and dermatan sulphate
 
Sulphated Glycoside 1 Sulphated Glycoside 2
Heparan sulphate D-glucuronic acid N-acetyl-D-glucosamines 
Dermatan sulphate  L-iduronic acid N-acetyl-D-galactosamine
    2-sulphate  4,6-sulphate

NB Heparan sulphate has less iduronic 2-sulphate  N-sulphate acid than 3,6-sulphate heparin

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Heteropolyanion-23


(ammonium,5-tungsto-2-antimonate) (HPA-23) is a tungstoantimonate which has a sodium ion in the centre of a cage structure and three unoccupied sites on the outside. It is discussed under this section although it is not a polysulphonated polyglycoside because its mode of action is felt to be similar. Its molecular weight is approximately 6800 daltons.  It is similar to HPA-57, which has the same structure with a cobalt ion at each external binding site.  HPA-39 is identical to HPA-23 except that the central sodium ion is replaced by potassium (68,86).  When an intravenous dose of HPA-23 was given to a mouse within 4 hrs of the inoculum of scrapie it would decrease the apparent infective dose given by more than 99%.  As a result of this, if 100 lethal dose units of scrapie had been given peripherally, then the animal would survive (87,88).  The effect decreased rapidly, however, after the 4 hr limit with the effect seen to tail off after this in a similar manner to dextran 500 (Table 13).  The effect does not depend on the site of injection but when the HPA 23 is inoculated s.c. after being mixed with the infection before hand it is apparently ineffective; hence suggesting that it has no direct anti-TSE effect (89). Little data is available as to the pharmacokinetics of the compound.  The effect of HPA-39 and HPA-57 were less than that of HPA-23 (68). In an experiment by a different author, similarly inoculating mice (and later in a marmoset monkey (90)) but with CJD,  HPA-23 was found to be ineffective (89), and clinical reports of its use in patients with symptoms showed no apparent effect (89,91).  Also the treatment failure was associated with intravascular coagulation which is assumed to be due to the drug (89).

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Diazo dyes etc

This has been put as a list of dyes.  However the chemicals involved do not all act as dyes and are not necessarily such.  In this condition it should be understood why Congo red interacts with prions and hence why these other molecules of similar structure do also


Congo red
Interaction between Congo red and amyloidal proteins: This is a di-sulphonated di-diazo dye (Figure 10) that was originally manufactured for the cotton dying industry (formerly called cotton red B or C).  It is known as a pH indicator, changing from blue to red as the pH passes from 3 to 5.  Although it has been used as a dye for many factors, it has been well known in histopathology as a specific stain for amyloid (92) although other factors do take up the stain to a lesser extent.  The specificity of the stain can be increased by using it in alkaline alcoholic solvents (93,94) and in high salt solution (93,95).  The effect is to produce red staining amyloid visible under white light  and to produce a green birefringence when viewed under polarised light.  The effect was unexplained in that such a polarisation of the dye would require that the there was some polarisation of the amyloid (Figure 11). This gives some idea as to the structure of amyloid deposits, which themselves have been investigated using electron microscopy (96). It is now well established that the attachment is through non-polar hydrogen bonds as opposed to the electrochemical bonds formed between Congo red and many other tissue components and that the alcoholic alkaline stain and the salt solution (used to optimise the stain differential of amyloid from other tissues) are a result of this.  It is also found to be almost impossible to over-differentiate by washing the dye out in part of a histopathological staining procedure.  Other factors in cells also stain; e.g. elastic tissue and eosinophil granules will also stain red.  However, the birefringence is only seen with amyloid, as is a fluorescence (orange to red) (92) seen under ultraviolet light but at a low level. Two factors are important in the Congo red reaction; the linearity of the dye molecule and the b-pleated sheet configuration of the protein involved. If the spatial configuration of either is altered, even though the chemical groupings are left intact, the reaction fails (97).  The decisions on exactly how the CR interacts with amyloid have been going on for some time but occaisionally important factors appear.  For instance the interaction of a single molecule of CR to one of amyloid (268)

Congo red interaction with PrP.  Action against TSE infection by Congo red.  It had been known for some time that PrPsc concentrates would aggregate in vitro to form birefringent rods when stained with congo red (99) but its anti-TSE effect was initially demonstrated in vitro by Caughey and Race (100), who used mouse neuroblastoma cell cultures infected with mouse-passaged scrapie.  They demonstrated that PrPc continued to be produced but was no longer converted into PrPsc even though the cells themselves were infected. This appeared to be totally effective at concentrations of less than 1.4 micromolar  (note that the stains used in histology are saturated alcoholic solutions and probably in mM range).  No toxicity was seen on the cellular cultures.  Similar techniques were used to show the action of various poly-anions and Congo red on the quantitative build up of PrPsc (Figure 14).  This showed that pentosan sulphate and iota-carrageenan were more active than Congo red but all showed peak activity was being reached at around 10 ng/ml (14 nmolar), approximately the same level as the MIC of penicillin for streptococci.  Again, no decrease in the amount of PrPsc was found that had been in the culture prior to the addition of the agents.  This would suggest that although these factors could prevent further PrPsc being formed it could not induce the breakdown of what was there already.  It has now been shown that Congo red increases the life expectancy of scrapie-infected hamsters (101) but the toxicity of the compound is currently unknown or unavailable.
  Attempts were made to use different forms of Congo Red (263) but it was found that even small changes in the structure prevented all major action as an inhibitor of PrPsc production.  When attempts were made to biotinylate Congo red by modifying the amine group, whis also prevented all ability to stain amyloid (Dealler, unpublished).
  Inoculation of mice with scrapie and treatment with Congo red appeared to prevent the breakdown of the PrPsc during the initial phase of infection and therefore to permit the animal to die earlier.  It was as if the dose given was higher because much of the inoculum was not destroyed (264). This was explained (265) n that the CR actually prevented the breakdown and this could be shown in vitro by the effect of CR treatment of CR maintaining the structure of the PrPsc even when boiled with SDS.  In fact the only things that they could find that would alter CR-PrPsc was acidic 3M guanidine thiocyanate.  Further studies in which the CR was given later than the scrapie to the mice showed an improvement in the incubation period (266).  Indeed CR seemed to permit the aggregation of PrPsc in suspension and to maintain this afterwards (267): it was as if this was an irreversible step. It was as if the CR overstabilised the aggregation step and that this was actually prevented the multiplication in cell culture.  However other compounds that were thought to interact with the PrPsc in the same site did not cause this....and so it was felt that CR was particularly over interactive with the PrPsc.  However the potential toxicity of CR and its carcinogenicity made it difficult to use.  Also, the fact that it was a dye made it difficult to penetrate the central nervous system and hence it was unlikely to affect the disease once it had penetrated there.

Other diazo dyes that inhibit the produciton of PrPsc in vitro

Sirius Red F3A and Trypan blue (287).  It must not be forgotten that Sirius red can be used as a replacement for Congo red in the amyloid staining technique (although Paneth cell granules also stain red) (98), and that various di-sulphonated di-diazo dyes exist that could be of significance (Figure 12) (92).   At this time no amyloid dyes other than Congo red have been tested for activity against TSE infection. However trypan blue (Figure 13), which is chemically fairly similar was found to be marginally active against scrapie (98) in one experiment but inactive in another (89).  In a cell free experiment Trypan Blue, Evans Blue, Sirius Red F3B, Primuline and Thioflavin-S were all more active than CR in the inhibition of PrPsc production. In cell culture experiments, however, Sirius red  F3B was found to be active (287) but Trypan Blue, Evans Blue,  Primuline and Thioflavin-S were less active than CR.

Other diazo compounds.  It was realised that there was no reason why the they should have to be coloured in order to permit inhibition of the production of PrPsc in cell culture.  Griffiths group at Cardiff (262) produced a group of compounds and their relative ability co inhibit PrPsc production but in a different cell culture from the neuroblastoma used by Caughey.  It was found quickly that the compounds were often less than a 100 times less active in this cell line and that almost all of the compounds had some activity although it was highly variable between the groups.  Chrysamine G is a carboxylic acid analogue of CR which will act as a histological dye that stains specifically amyloid.  it binds to the beta amyloid of Alzheimers disease in vitro and partitions into the brain of normal mice.   One group showed that it would bind into the Alzheimer's amyloid of the brain at autopsy by passing dye through the arteries. It was showed to be a good stain in this way of cerebrovascular amyloid (270).

Fluorescent brighteners and fluorescent amyloid stains.  There is a small group of these dyes that are thought to interact with amyloid in the same way as CR.  This has been difficult to repeat (Dealler, not published) and the dyes themselves difficult to obtain.  The chemical Blankophor BBU (Bayer UK)  (also called Phorwhite BBU) could not be shown to have any effect despite its indication in the scientific literature (269).  No attempts have been made currently to use the fluorescent amyloid stain thioflavin T .  It should be noted that also thioflavine S can work but is a mixture of dyes probably containing some thioflavin T.  Its action in vitro in cell free and to some degree cellular systems (287) was therefore interesting.  In histopathology thioflavin T is less specific than CR and can show no circular dichroism.  No attempts have been made to test its action against PrPsc production.

Porphyrins and phthalocyanines.  This is a group of much larger compounds that can be created as sulphonated compounds. Because of their structure (cyclic tetrapyrroles), however they are not necessarily prevented from penetration into the central nervous system.  The best appeared to be phthalocyanine tetrasulphate when it was given early after the inoculation of scrapie into mice (hamster forms).  However the 263k scrapie is known to penetrate the CNS early and hence any effect seen would have to be following early effect outside the CNS. The hope that drugs that penetrated the CNS would have some major effect was damned (273,274) although if given early enough the compounds would more than double the life expectancy of the animal.  At the moment we must assume that these compounds may be of greater use in research than in clinical treatment.

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Discussion after dyes and polyanions


All of these compounds have specific carbohydrate chains with relatively variable O-sulphonation.  Despite this variation they share the ability to inhibit the production of PrPsc in vitro (HPA-23 has not been tested in this way).  It is presumed from the molecular structure that their sulphonation apart from the HPAs is essential and that the size of the molecule is also important.   The relative shape of the molecules, which appears to be well known for the carrageenins and heparin may give some insight into the way to optimise activity.  The structure of pentosan would fit in the molecular modelling of  Urbani et al (70) but the variation of the sulphonation that seems to be present in all of these structures may make full elucidation of structure difficult.  More specific levels of sulphonation on each glycoside residue and purer products being used would give a more accurate indication as to the ideal compound to be used.   The effect in vitro (but only in vivo in peripheral inoculation and when given within a certain period of the injection of infection) has suggested to some researchers that the effect is complex and not just by the interaction with the agent before it reaches a cell to infect (89) (Figure 9).  The blockage of macrophage activity with silica injections also did not seem to have a great effect in vitro (68) and hence the drugs' effect in preventing the development of the disease may not be due to an effect on the reticuloendothelial system.

One of the factors that gives the greatest insight into the compounds and their action appears to be their uptake and storage within cells.  The full pharmacokinetics of PPS is not known but its uptake and then storage within RE cells would explain the long term effect of the drug in prophylaxis.

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Suramin

This is a trisodium salt of a sulphonated organic acid (Figure 15) (benzamido-methylbenzamido-naphthalene trisulphonic acid) of urea: a large polar anion.  It contains 6 sulphonic acid groups per molecule. The drug is used as a systemic human antiparasitic agent, is given perenterally and is known to have a long half life in the body (around 48 hrs following an initial phase).  It is excreted via the urine unchanged and has a range of toxic side effects.
  When inoculated into hamsters the week before or the week after scrapie a small increase (2-5 days) in the incubation period resulted (control: around 115 days) (65).  There was no difference between 15mg/kg and 40mg/kg.  Inadequate precision of the incubation period for the control mice (infected with scrapie but not given suramin) was given in the article,  and so the findings with suramin must be considered as doubtful.  Further research (305) attempted to showthat the effect that they saw was due to the intracellular rerouting of the PrPsc as the infectious agent within the cell.  Work in cellular culture showed that suramin did decrease the production of PrPsc at around 12 micromolar concentrations in the suspension growth fluid (283).

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Polyene antibiotics

Amphotericin B (a heptaene) (AB) was initially considered for treatment, mainly because the agent for CJD was unknown and so this agent, generally only active against fungi, could possibly be of value.  Its mechanism of action is debated but the binding of the compound to ergosterol, the major sterol found in the membranes of eurkaryotic cells is considered to be of significance.  Because of the interaction of it with cholesterol (the major sterol of mammalian cell walls) it was considered that it may also be found in the viral envelope of certain viruses (e.g. herpes) as some of this is derived from the cells that are virally infected.  Indeed antiviral activity against herpes virus and HIV have been reported (102,103)
  Initial tests showed an increase in the incubation period of CJD when inoculated into an African green monkey (89,104).
  Tests were carried out on hamsters, which were inoculated intracerebrally with 263K strain of scrapie and had an incubation period of approximately 54 days.  When inoculated with 1mg/kg for 6 days a week the incubation period could be extended to 99 days (105). An effect was also seen after intraperitoneal inoculation.  The effect was further looked into by investigating the rise in the level of infectivity in brain tissue and splenic tissue in hamsters following i.c. inoculation (106). This showed that as of 20 days after inoculation the rate of rise of infectivity in the brain was lower in AB treated than untreated hamsters.  Infectivity in the spleen was relatively low in control animals and less than 2 x 103 IU/g by the use of AB.  It also showed that AB, given 6 days per week at up to 1mg/kg or as a single 60mg dose administered 2 hr after i.c. inoculation, delayed the onset of symptoms.  The later finding that the effect of AB was limited to the 263K strain of scrapie and the C506 murine strain of scrapie (107,108,109) was extended when it was also shown inactive on the 139H strain of scrapie (110) but active on the KFu strain of CJD when inoculated into mice (110).  It became more complex when it was found that AB had no effect on the incubation period of 263K scrapie in Chinese hamsters but it had a clear effect on neither Syrian nor Armenian hamsters (110).
  The mode of action of AB was considered by Pocchiari's group to be either:
 a.  AB prevents scrapie agent penetration of cell walls.
 b.  inhibits scrapie multiplication mechanism
 c.  delays the build up of PrPsc.
  It is suggested by Hope (111) that AB may work by a membrane-related inhibition of a proteolytic processing of PrPc that may lead to the formation of PrPsc.  It is suggested that AB interacts with the scrapie agent in a competitive manner from the shape of the dose/response curve (106) but this is not certain as AB may be in fact acting at other possible points in the pathogenic chain.   Pocchiari is also quite determined that the spleen and lymph organs are obligatory organs of replication for the scrapie agent before it reaches the brain following peripheral inoculation (106), whereas this has not been adequately demonstrated.  AB induces the phagocytic ability of macrophages (112) and similar induction is seen following vaccinia virus or BCG inoculation, both of which facilitate scrapie infection (113,114).  It is suggested by Pocchiari that AB may simply not work in scrapie through activity on macrophages, or the action that it has may be more specific on the macrophages than that seen with the other two methods.  The possibility that the mode of transport of the TSE agent to the brain is actually via the slow circulation of specific forms of macrophages and the interaction with microglia has been suggested (115).  Further light was thrown on the mechanism of AB action when it was found to decrease the rate of rise of infectivity in brain tissue but not to alter the amount
 

of PrPsc that was building up; as such it was claimed to separate the scrapie agent from PrPsc (108)
  Despite the good in-vitro effects, AB (1mg/kg ) failed to ameliorate clinical CJD (116).

Mepartricin (Methyl partricin, a tetraene).  Minimal effect was seen with this drug when tested on hamsters with scrapie (106) and only after scrapie was inoculated i.p.  This drug is used generally in Europe for the treatment of deep seated mycoses and has no conspicuous advantages over AB.

MS-8209. This is the hydrosoluble N-methylglucamine salt of 1-deoxy-1-amino-4,6- -O-benzylidine-D-fructosyl-amphotericin B (117) (Mayoly-Spindler Laboratories, Chatou, France). This was tested by inoculations 6 days a week into the hamster peritoneum.  The hamster had been infected intracerebrally with 263K scrapie agent and the levels of PrPsc, and infectivity measured during the incubation period.  It was found that both infectivity,  PrPsc and glial fibrilliary acidic protein were delayed in their build up but they both reached the same level as was found in control animals by the time of death. Delay in death was by 57 days with AB, and 84 days with MS-8209 (117). Unlike amphoteracin B, the agent appeared to be able to slow the progression of disease in the mouse model even if the drug was with held until after symptoms had appeared (118) and increased incubation period when the infective inoculum was given orally, i.c. or intraperitonealy. This suggested that to the authors that the PrPsc levels were involved in some way with pathogenesis and the parallel rise suggested that the prion theory was reasonable (107,119). One odd seen effect when the drug was started late in the disease was that the PrPsc accumulation kinetics continued to take place but seemed to be less associated with the survival (120).  MS-8209 was also tested in mice on a mouse adopted strain of BSE and C506M3 scrapie strain and found to be active in both but efficacy was greater in scrapie than in BSE (120). This agent was considered because of its low toxicity, apparently one fifth of AB (119).    Repeated research showing that the drugs increased the incubation period in vivo have been carried out (295-302) but it is still unclear as to the mode of action.

The exact site of action was unclear for these antibiotics in that one group indicated that some action took place before the penetration of the CNS (299,301), whereas others suggested that at least MS-8209 was able to act on the development of the disease after symptoms had already appeared.  The lack of any effect in cellular cultures was further investigated and it was shown that amphitericin B could interfere with the production of PrPsc in cell cultures (325).  The action seems related to a modification of prP trafficking through the association of this GPI anchored protein with detergent resistant microdomains.  To some degree Amph B is a detergent and to this may make sense.

Amphoteracin B lipid complex was tested in hamster scrapie and seemed to have a similar effect to AB using 263K.  A greater effect was seen on the build up of PrPsc in the spleen than with AB but a similar effect was seen in the brain (120)

Pharmacology: Amphotericin B is an insoluble fermentation product of Streptomyces nodus, dissolved with desoxycholate in water.  It has a wide spectrum from complex fungi to Leishmania spp, Naegleria genus Prototheca spp, and Hartmanella spp.  Its selective action is through the interaction with ergosterol in the wall of the organism in contrast to the cholesterol found in mammalian cells walls. Poorly absorbed from oral administration, serum infusion concentrations  should not exceed 2mg/l.  A half life of 1-2 days followed by a slow elimination  phase of about 15 days and it is cleared more rapidly in children resulting in lower serum concentrations.  Being highly protein bound in serum gives it poor penetration to certain body sites including the CSF, urine, eye etc.  Not removed significantly by haemodialysis. Side effects: fever, rigors, headache, backache, vomiting and thrombophlebitis.  Hypokalaemia and anaemia are common and some degree of renal damage almost invariable with effective courses and to some extent irreversible (121).

Other polyenes:

There are around 100 polyenes (121) to be considered but few offer any advantage when considering mycoses and fungal infections.  However, as can be seen with the different response to AB than to mepartricin, there may be an advantage with scrapie or other TSEs.  Few of these are available obtained:

Of these mepartricin, AB and nystatin have very similar spectra but variotin is more active against dematophytes and trichomycin more active against protozoa.

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Ouabain

A cardiac glycoside, plant derived, that is no longer used in widespread chemotherapy of cardiac failure. Its structure (Figure 17) shows it to be neither an anion nor sulphonated.  It has been shown to increase the incubation period by 44 days (untreated mean; 308 +/- 48 days) of mice inoculated intramuscularly with CJD (89) but little further research has been carried out on this.

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Iododoxorubicin

This is a halogenated anthracycline anticancer agent (4'-deoxy-4'-iododoxorbicin) (Figure 18) that is currently under Phase II clinical trials.  The aim of the drug was to have a lower cardiotoxicity compared to doxorubicin or daunorubicin but has a slightly altered range of anticancer activity as a result (122).  Doxorubicin is not stable in gastric acid and must be given i.v. after which it is quickly taken into tissues and bound to cellular components.  Presumably from first pass through the liver it is metabolised and so there is a rapid fall in blood concentrations initially.  However, as a result from the prolonged cell fragment attachment of doxorubicin and its metabolites, it has a long half life with less than 2% of the agent appearing in the urine and predominant excretion being in the bile. The findings from iododoxorubicin are similar and would suggest similar pharmacokinetics (123).
  It has been tested in a similar way to amphotericin B and shown in hamsters (i.c. 263K agent) to cause an increase in the incubation period by approximately 20%  (124,118) and decreases the build up of PrPsc (Forloni, G. in press).  It appears have some effect even though the scrapie inoculation was directly into the brain.  No study has been published as yet on other strains of disease or other species.  Apparently the agent has been shown to interact with natural amyloid fibrils of different biochemical composition and induce amyloid resorption in patients with systemic amyloidosis and inhibit amyloid formation in a murine model of reactive amyloidosis.  In the study with hamsters less PrPsc was shown to build up, less amyloid and less spongiosis and astrogliosis at specific parts of the incubation period but at the time of death both the control and test animal brains were similar in these factors.

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Antibodies to PrP

Brain grafts from tga20 mice which over-expressed the PrP gene were put into knock-out mice brains (125).  The grafts were found able to develop prion disease following i.c. inoculation although the animal itself showed few symptoms; in fact the cell tissue was destroyed to a much greater degree than at the time of death of a normal mouse with scrapie.  However, the tissue that surrounded the graft was also marginally affected by the laying down of PrP plaques but this appeared to cause no histopathological damage.  Prion infectivity administered to peripheral sites or orally did not cause the development of spongiform change or infectivity in the brain graft (125).  This was considered to be because antibodies were produced against the PrP in the inoculum; as the animal produced none of its own, this was seen by the immune system as an antigenic protein and the antibodies had appeared soon after the brain graft had taken place. When a lck-PrP trans-gene was added to this strain of mouse it no longer produced antibodies and infection of the CNS graft after intraperitoneal inoculation became successful. (125).  The therapeutic relevance of this finding is currently conjectured but seems to suggest that either antibodies are significant or the lck-PrP gene was also expressed by other tissues, which were themselves significant in the penetration of the agent to the CNS. The report that anti-PrP antibodies neutralised infectivity of scrapie in hamsters must indicate that immune effectors can delay the onset of disease (126)  The finding that the agent appears to be taken up by dendritic cells (127) and that they are of significance in the transfer of disease is odd in that antibody-agent complexes would have been taken up by dendritic cells.   Studies  of protective immunity against isolates of TSE containing foreign PrP have been attempted and this experiment should be repeated by others (128).

Specific experiments in Amyloid-beta  positive transgenic mice could be shown, in familial forms of disease to progressively have an increased brain loading of the amyloid.  One group showed that not only did amyloid immunisation stop the production of the plaques but it also stopped the development of clinical symptoms and may actually get rid of plaques that had already formed (329,330).

A similar experiment was carried out but by using the antibodies against PrPc epitopes present in the culture medium of scrapie-infected neutoblastoma cultures.  It was found that these antibodies would abolish the production of any new PrPsc by the cell culture and the PrPsc already present would decrease in the cells to 10 percent of the original amount within 4 days. The authors (331) noted that the action depended on the epitope to which the antibody was directed and considered that this may may permit the development of specific drugs that would be active against the disease.   The reason why this is most important is that the antibodies were active only outside the cell, as the PrPc was produced and attached to the cell wall with GPI.  It is only at this point that the antibodies could link to it.  Clearly, as the PrPc was brought into the cell, the antibody continued to have action and this prevented the changing of the PrPc to PrPsc.

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Steroids

One article has suggested that there is a reduced susceptibility to scrapie in mice after prednisolone administration (129). Other articles suggest that neither ACTH (130), cortisone (130), nor prednisolone (89) made any difference to the incubation period of scrapie in vivo.

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Arachis oil

When injected into a mouse at the time of the infection this decreases the effectiveness of the inoculum (131).  The effect is thought to be due to the anti-inflammatory effect of the compound and the interaction with lymphoid tissue. However another researcher could not repeat this result (89).

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NMDA antagonists

Much of the work into N-Methyl-D-Alanine antagonists has been carried out concerning HIV infection of lymphcytes.  NMDA is a calcium channel and required for the intracellular stimulation of apoptosis, which is followed by the break up of cellular DNA into relatively small fragments.
The apoptosis takes place after an early rise in the calcium ion level inside cells and can be pregented  using NMDA antagonists such as:

 memantine (1-amino-3,5-dimethyladamantane;MEM)
 MD-Ada (1-N-methylamino-3,5-dimethladamantane)
 MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine malate)

The original work demonstrated the presence of NMDA receptors (275) in cellular cultures but follow up work by Muller et al (276) was more important in that it demonstrated that PrPsc-producing neuroblastoma cells cultured in vitro were more likely to remain viable when a NMDA antagonist was present in the culture.  They also showed that the death that took place in the cellular cultures could be associated with apoptotic destruction of the DNA and that when the NMDA receptor inhibitors were present the production of PrPsc continued but damage was not seen.
  These findings are important in that they suggest that the cellular destruction that may take place in the brain may well involve the release of NMDA neurotoxins by macrophages in association with the production of PrPsc by the neurones, as, when either effect was missing, the cellular death rate was similar to controls.   This does not fit with the suggestion that the cellular death takes place via damage to lysosomal membranes but rather that the neuronal death is an apoptotic event.  Further research is needed into this subject.

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Heat shock protein 104


The demonstration of a prion-like phenomenon in yeast cells (277-281) has opened the possibility of testing certain systems for treatment. Two genetic factors in yeast (PSI+) and (URE3). It was found that a specific protein, Sup35, was converted into a form that would act as an inherited agent (PSI+) and this could be transferred to another yeast in the form of an infectious agent.

It was also found that a specific protein HSP-104, when in normal quantities would not prevent the formation of PSI form of Sup35.  It acted as a chaperone protein that was able to reform protein secondary and tertiary structure following heat shock.  However, when excess amounts of the protein was present in the cells, they would lose the prion-like agent and, when after this the HSP was removed, the cell did not become infective again.  i.e. it had been cured.  However, for a reason that is not clear,  when relatively low amounts of HSP-104 were present in the cell, it would lose its infectivity but when the HSP returned to normal levels the infectivity also returned.   The ability of an HSP to 'cure' yeast cells can be understood and its finding may be important in considering methods to affect mammalian cell cultures.

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Branched polyamines


These are compounds in which there are amine groups linked at several sites in a carbon chain but which have amine linking sites at one end.  As a result the end of one chain links to the amine of another and, if multiple links take place, then branches occur and the compound grows with multiple amine sites.

It was shown (282) that relatively large branched polyamines would prevent the production of PrPsc by mouse neuroblastoma cells and, after a week, cure the cells of most of the PrPsc that was present.  The PrPsc would actually return after around 3 weeks and so complete cure was probably uncertain.  In this way (i.e. in a similar manner to that seen with PPS) culture lines could be virtually cured of infection.  However, the concentrations required were approximately 1.5. micrograms per ml present in the growth fluid.  Although their molecular weight was high (generally 1000-14,000) and hence the molar concentration was less than 1.5nM the compounds were commonly found to be toxic to the cell lines and these contentrations were potentially much too high for clinical use.  It seemed as if high densities of amine groups was required on the surface of the compounds to be affective and that chloroquine, a lysosomotrophic agent, could prevent their effect (282).  It was because of this that the group felt that the branched polyamines were working in either endosomes or lysosomes.  It should be noted that the 100microgram/ml of chloroquine did not seem to inhibit the production of the PrPsc itself...as was found by much smaller concentrations of the drug later (283).  For some unclear reason, if the polyamine was added to the PrPsc at pH 4 or below, then the PrPsc became sensitive to proteinase K.  Whatever conformational change that had taken place did not seem to go away even if the pH was neutralised and then waiting for long periods.  What is suggested is that the polyamines are acting on the PrPsc in an acid compartment in the cell and that this permitted the clearance of the PrPsc.  Chloroquine is thought to cause an alkalinisation of the lysosome and so it is not surprising that it stopped  the polyamine effect.  Further work showed that when a fluoroceinated polyamine (polypropyleneimine PPI) was put with the cells the fluorocein concentrated in the lysosomes and that inoculation of cultured infected cells that had been treated with the PPI for a long period into mice showed no infection to remain (285,6).

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Lysosomotrophic drugs (including Acridines and Phenothiazines)

The initial work was done using quinacrine (see separate section), tilorone, chloroquine and suramin (283) and then a much large group of compounds was tried (284) although their lysosomotrophic ability was not tested.  All these compounds were either acridine, phenothiazine, or similar tricyclic molecules.  The ability of acridines to act specifically as lysocomotrophic drugs through their prevention of the breakdown of mucopolysaccharides (and hence them building up in lysosomes) has been a major direction of research (see Acridine section).  The phenothiazines could not be stated at this time to be active as in the same way and so the discussion by Korth (283) as to why the drugs work in cellular culture is difficult to certain of and more research is required.   The toxicity of tilorone, suramin's inability to affect prion production after a short period following scrapie inoculation into hamsters (305), and relatively high toxicity (to the cell cultures) of chloroquine made quinacrine the most useful drug to be found.  The comparison of the chemical structure of the acridines suggested that quinacrine's long di-ethylated side chain was important  and that its tricyclic ring structure was also necessary to permit the low MIC.   Quinacrine had no effect on the cell free conversion of PrPc to PrPsc (283).

The phenothiazines (284) that were tested showed dramatically greater activity than other neurotropic drugs and chlorpromazine, which has a long side chain was probably the most useful (even though it was about 10 fold less active than quinacrine).  

The greatest importance of the quinacrine and chlorpromazine are that they have been used as human therapeutic drugs for a long period and hence their toxicities are known.

The mode of action of the drugs was suggested to be due to the complex formation of GAGs in lysosomes with the compounds and hence in some way interfering with their formation of prions there.  The suggestion is that these compounds can penetrate cellular membranes and when present in the lysosomes, the pH changes will alter their charge and hence they will interact with GAGs and prevent their breakdown.

The lysosomal action of tilorone and acridines is well investigated (310-318).  As such it is clear that specific structural relations are found between the acridine and the effectivity in the mucopolysaccharidosis that can be demonstrated using dyes.  It is also fairly clear that acridines are easily taken up into cells, and retained there as can be seen with acridine orange.  One facinating effect is the ability to damage lysosomes by the UV light stimulation of the acridine.

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Cysteine protease inhibitors


The chemicals tested (283) were E-64d, E-64 and leupeptin.  E-64d, a membrane permeable form, with an inhibitory concentration of 0.5 micromoles, was approximately 4 times more active than E-64 and 50 times more active than leupeptin.   None of the compounds was significantly toxic to the cultured neuroblastoma cells.  The mode of action was unclear but  E-64d had no effect on the cell free conversion of PrPc to PrPsc (283).  No specific reason for the action of the drugs was put forward.

E-64d has been used with low toxicity in animals in an attempt to develop a drug that would be useful in the treatment of muscular dystrophy.  It is because of this that the potential use of the drug in prion disease is considered possible in the future (306).

It is known that lysosomal enzymes are involved in the degradation of AA and AL amyloid proteins and it has been considered that inhibitors may be involved in this (307).  The penetration of E-64d into cells and the intracellular action as a cysteine protease inhibitor is well described (308) and the structural basis for the action of the drugs has been well investigated (309).

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Beta-sheet breaker peptides


Small (5-residue) peptides can be used to break the beta-sheets of Abeta-amyloid (Alzheimer's) or a 13 residue peptide chain would do this to prions (TSE). In vitro PrPsc could be shown to reverted to a PrPc format to some degree but not completely.  In a cellular model of familial prion disease it could be shown that the peptide would prevent the production of more PrPsc and in the animal models the treatment of the infectious material (i.e. before inoculation) decreased infectivity by 90-95% (319,320).
Most of the research has been done in Alzheimer's models in rats.  In these, the beta-amyloid builds up but can be stopped using intravenous beta-sheet breaker peptide (321,322).  In this it can be shown that the peptide gives rise to a reversal of brain lesions and to a loss of fibrillar amyloid in the brain itself (324).
The major problems with these compounds is that they cannot be given orally, will be broken down by peptidases in the body (often found in the kidney or liver), and do not penetrate the central nervous system.  To some degree this has been solved by the design of the peptide of the use of peptidase inhibitors (323) in Alzheimer's model animals.  However this seems to be much more difficult with the larger peptides required for prion disease.


Tetracycline


This is a broad spectrum antibacterial antibiotic but which has action against many other organisms (e.g. malaria).  It acts through the inhibition of protein production at the RNA stage.

Some evidence appeared that it may be of significance in the inhibition of amyloidal plaques in Alzheimer's disease and this has been shown to be correct but a minor activity (332).

Tetracycline affects abnormal properties of synthetic PrP and  PrPsc.  It binds to amyloidal fibrils corresponding to residues 106-126 and 82-146 of humand PrP, it hinders assembly of these peptides into amyloid fibrils, it reverts the protease reisstance of PrP peptide aggregates and prPsc extracted from the brain tissues of people that have died of CJD, and prevents neuronal death and astrocyte proliferation induced by PrP peptides in vitro.  NMR spectroscopy showed that it interacted with specific sites on the PrP.

The major problem with all this is that tetracycline does not penetrate the CNS in humans.  However there are other forms of the class of chemical that may permit this.

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References (on another web site)


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