WO2002051415A2

WO2002051415A2 - Use of sequestrants for treating prion diseases

Info

Publication number: WO2002051415A2
Application number: PCT/IB2001/002572
Authority: WO
Inventors: Michel Xilinas; Claude Hannoun
Original assignee: Michel Xilinas; Claude Hannoun
Priority date: 2000-12-22
Filing date: 2001-12-17
Publication date: 2002-07-04
Also published as: WO2002051415B1; FR2818551A1; AU2002216305A1; FR2818551B1; WO2002051415A3

Abstract

The invention concerns the use of sequestrants for treating prion diseases. Particularly, it concerns sequestrants used for preparing a pharmaceutical composition for treating or preventing prion diseases in human and animal and, more generally, diseases mediated by non-conventional transmissible agents. Clioquinol and phanquone sequestrants are preferred sequestrants for preparing said composition. The invention also concerns methods for treating or preventing prion diseases.

Description

Use of chelators in the treatment of prion diseases.

Technical area.

The present invention relates to the use of two known compounds used together or separately for the preparation of a pharmaceutical composition intended for the treatment or prevention of diseases caused by prions. The invention describes a pharmaceutical composition for such treatment. The invention also relates to methods of treatment and prevention of prion diseases.

State of knowledge.

Diseases caused by Unconventional Communicable Agents (NCTAs), or prions, especially subacute transmissible spongiform encephalopathies

(ESST), reach humans and animals. They are very serious because they are almost always disabling and quickly fatal when they occur. There are many uncertainties as to their pathogenesis and various hypotheses have been put forward, but the most likely is that known as "prion", a term which designates a protein agent presenting with viruses certain analogies, especially the ability to replicate, but without the intervention of nucleic acids. If this theory holds true, it is a new type of infectious agent for which the virological concepts do not apply. These diseases have so far not been able to be treated or prevented and they represent a serious danger for human and animal health.

Prion diseases (from Prusiner, 1998, Proc Natl Acad Sci USA, 95, 13363-13383.).

Scrapie Sheep and Goat

Communicable mink encephalopathy Mink Chronic wasting disease Mule Deer & Elk

Bovine Spongiform Encephalopathy (BSE) Cattle

Feline Cat Spongiform Encephalopathy

Exotic Encephalopathy of Ungulates Nyala and Grand Kudu Kuru Male Creutzfeld-Jakob Disease, classic or sporadic (CJD) Male

Creutzfeld-Jakob disease, new variant (MCJnv) Male

Gerstmann-Straussler-Scheinker disease Man

Fatal familial insomnia Male

The most disturbing problem is that of Creutzfeld-Jakob Disease (CJD) in its new form. There are indeed two types of CJD. One, known as classic or sporadic, CJDc, is rare and affects mainly the elderly. The other, of recent appearance, is most likely due to human contamination from animal pathogens (cattle) and affects people of all ages. The epizootic of “mad cow disease” in Great Britain or Bovine Spongiform Encephalopathy (BSE), since 1995, has been followed by the appearance of a new human disease, identifiable by certain clinical and anatomo-pathological characters, designated by the term MCJnv (for new variant). It then also appeared in several European countries including France. For a few years, human disease remained infrequent, only a few human cases, and therefore did not generate much interest in the development of preventive or curative drugs. In animals, the problem was quantitatively greater (scrapie in sheep; BSE in cattle: more than 180,000 cases in Great Britain in 10 years), but it is difficult to consider treating sick animals since they are intended for human consumption and a cured animal could not be consumed due to the risk of persistence or latency of the agent despite clinical cure. Estimates of the risk of developing a human epidemic of CJDv in relation to bovine disease (Bovine Spongiform Encephalopathy, BSE) have given very variable results because the forecast models include several parameters whose empirical evaluation is very difficult. According to English estimates at the beginning of 2000, the number of cases in the next 20 years could be in the range of 100 to 20,000 cases. In the absence of any treatment, the prospect was therefore already worrying.

The evolution of human disease is very slow (up to 20 years of incubation) and studies are very difficult due to the absence of fully satisfactory animal models. The rate of appearance of new cases constantly modifies the parameters of the forecast and the estimated values are constantly modifiable. However, for a few months, the morbidity curve has been dangerously turning up and it is rising rapidly in the United Kingdom:

1999: 17 human cases of CJDnv 2000: in one semester only, 24 cases

The modeling now gives lower and upper limit values in a range of 63,000 to 136,000 cases in total (low and high values of the range). Some experts are even more pessimistic and envisage 500,000 cases. These results will be refined over time depending on the actual development of morbidity (-mortality). The introduction of new screening tests will allow a better estimate and above all to measure the extent of the danger more quickly.

The facts of the problem have therefore changed recently. It is now considered to be a relatively common disease, affecting all ages. It becomes much more urgent to develop solutions. It will take several years to know the fate of this epidemic, but in the meantime, the need for therapeutic agents is now more pressing. STATE OF THE ART: PATHOLOGICAL MECHANISMS IN PRION DISEASES, CONFORMATIONAL MODIFICATION.

The disease is associated with the presence and accumulation of a modified protein, derived from a normal PrPc protein, in the brains of affected individuals. The PrPc protein exists in all the species where it has been sought and its interspecific variation is low. Similar proteins have been found in mammals, birds and fish.

The transformation of PrPc into PrPsc is done by a simple change in the three-dimensional structure of the normal molecule. This modification is transmissible to new PrPc molecules and the transformation mechanism is obscure. It is a conformational transition from the normal form PrPc consisting mainly of alpha helical structures into a pathological protein, absent in healthy subjects composed of a majority of pleated beta sheets (Cohen, 1999, J. Mol.Biol ., 293: 313-320). Although their sequences are identical, PrPsc is resistant to proteinase K while PrPc is sensitive, which allows their identification.

The abnormal form of the prion protein, designated by the term PrPsc, has physicochemical properties different from the normal form. It resists digestion by proteolytic enzymes and has one of its two components, beta sheets, in excess. Prion replication appears to be linked to the interaction of the structure of the normal prion protein of the PrPc host organism with the modified PrPsc protein. Most transmissible spongiform encephalitis (ESST) are linked to the structure of this abnormal protein: they are accompanied by an accumulation and a deposition, in the brain, of PrPsc molecules polymerized in the form of fibrils.

Wadswort et al. (Nat Cell Biol, 1999, 1: 55-59) have shown that the two types of human PrPsc, encountered in the two subtypes of Creutzfeld-Jakob disease, can be transformed into one another by l alteration of the metal ion content. The dependence of the conformation of PrPsc and the attachment of metal ions such as copper or zinc represents a new mechanism of post-translational modification making it possible to generate multiple strains of prions with all the implications that this has for both the molecular classification and pathogenesis of prion diseases.

Several studies have shown that the PrPc protein is a metalloprotein capable of binding, at the level of a repetition of an octapeptide, a copper (II) molecule. It is precisely at this level of the protein that the change in conformation that leads to the prion occurs. It is therefore possible to think that copper is involved in the transformation of PrPc into a PrPsc prion.

Hornshaw et al. (Biochem Biophys Res Comm, 1995, 207, 621-629) have shown that the PrPc protein, present in mammals, which transforms, by modification of the secondary and tertiary molecular structures, into infectious prion protein (PrPsc), contains 3 or 4 copies of an octopeptide sequence (PHGGGWGQ ¹ ). This sequence is localized in a very conserved domain of the N-terminal part and it binds copper ions preferentially. This bond is dependent on the concentration of copper. In chicken, a hexapeptide sequence (NPGYPH ² ) has the same copper binding property. In a second publication, they showed an effect of the binding of Cu2 + ions on the secondary structure of synthetic peptides Octa4 and Hexa4 corresponding to the natural peptides of PrP in mammals and chicken (Hornshaw et al., BBRC, 1995, 214, 993- 999). The addition of copper induces an increase in the "random coil". The fluorescence of these two peptides is quenched by copper, while this property does not appear with other divalent cations.

Similar results were obtained by Stockel et al. (Biochem, 1998, 37, 7185- 7193). Using the prion protein of the Syrian hamster (SHaPrP), they show by circular dichroism spectroscopy in near UV and by tryptophan fluorescence a shift of the spectra towards the shortest wavelengths and

¹ Proline-histidme-glycme-glycine-glycm ^

² Asparagine-proline-glycine-tyrosine-proline-histidine. the induction of changes in the fluorescence emission of tryptophan, which confirms that Cu2 + modifies the structure of the molecule. This effect is specific to copper because there is no effect with Ca2 +, Co2 +, Mg2 +, Mn2 +, Ni2 +, Zn2 +. The bond with copper leads to the conformational modification of a predominantly alpha-helical form into a beta sheet structure. Equilibrium dialysis indicates that approximately 2 molecules of copper attach per physiological copper concentrations per PrP molecule. According to the study of the influence of pH, histidine could play a role in the ligation phenomenon. These results suggest that each copper molecule would attach to two octopeptides (PHGGGWGQ) with a histidine and perhaps a glycine that chelate the ion. The binding of two molecules would produce a change in the molecular structure of this region.

Miura et al. (Biochem, 1999, 38, 11560-11569) confirmed the preferential binding of Cu2 + in the vicinity of the octopeptide sequence. The study by Raman spectroscopy and absoφtion shows that at neutral pH, the octopeptide forms a 1: 1 complex with the Cu2 + ion by histidine and the triglycine peptide. At low acid pH, the binding is modified and the binding site passes from the Npi of histidine to Ntau and a cupric ion is attached to two histidine residues of different peptide chains. The microenvironment in the brain cell regulates the affinity for the Cu2 + ion during the transfer between the cell surface and the endosomes. Thus, it is conceivable that the His (Ntau) -Cu2 + - His (Ntau) bridge is involved in the aggregation of PrPc.

It is possible that the functions of PrP as a transporter of Cu (II) ions are to bind the copper ions from the extracellular medium under physiological conditions and to release all or part of them upon exposure to a pH acid in endosomes or secondary lysosomes (Whittal et al., Protein Sci, 2000, 9: 332-343).

Recent results show that the prion protein plays an important role in the central nervous system. Aronoff-Spencer et al. (Biochem, 2000, 39, 13760-13771) to study the site and the specific mode of copper fixation used a series of Cu (II) -peptide complexes composed of 1-, 2- or 3- octopeptides by paramagnetic resonance and dichroic circular spectroscopy. They have shown that copper is linked to the nitrogen of the histidine-imidazole side chain and to two nitrogen of the sequential glycine amide.

Viles et al. showed by visible circular dichroism the structural modification of the region of the four octapeptides in tandem following the fixation of Cu2 + (Viles et al., PNAS, 1999, 96, 2042-2047). These results are also confirmed by NMR proton spectroscopy: two peptides form a bridge between the nitrogen atoms of the imidazole rings of two histidine residues (Nepsilon2 and Ndeltal).

A possible mode of action for copper binding has been suggested by Shiraishi et al (Shiraishi et al., BBRC, 2000, 268, 398-402) who have shown that the catalytic activity of Cu2 + is completely suppressed when it was sequestered by binding with PrP. Its intervention in cellular oxidative processes is then impossible.

STATE OF THE ART: THERAPEUTIC APPROACHES.

Many approaches have been explored to find agents capable of preventing or stopping the molecular transformation leading to the disease. Encouraging effects have been obtained in certain experimental studies with products discovered empirically, but so far without concrete practical results allowing applications. The main classes of drugs considered to inhibit the development of NCTAs were polyanions, Congo red (anionic compound that stains amyloid), polyene antibiotics (amphotericin, MS 8209), anthracycline, dapsone.

More recently, it has also been envisaged to inhibit or reverse the transformation of the normal protein into a prion using synthetic peptides which can break beta sheets (C. Soto, Lancet, 2000, 355, 192-197). However, difficulties have arisen which prevent the use of these compounds. First, they are rapidly degraded by cellular peptidases, then they cause inappropriate immunological effects because they are antigenic, and finally, they hardly cross the barrier. hemato-meningeal which isolates the brain from the general blood circulation to protect it from infections and they cannot reach the place where their action would be useful.

The process we are considering is quite different in principle. It is extremely simple and uses drugs already known for other properties.

BODY OF THE INVENTION: USE OF A COPPER AND ZINC CHELATOR.

We have two chemical agents, well known and already used for other uses, clioquinol and phanquinone. These compounds have the property of easily passing the blood-brain barrier and they are ligands of copper and zinc. They have already been proposed in indications which have certain analogies, in the treatment of degenerative diseases of the nervous system, in particular Alzheimer's disease (AD) where zinc probably intervenes in an analogous configuration. Based on current knowledge of the pathogenesis of prion neurodegenerative diseases, it appears that the sequestration of copper and possibly zinc in brain tissue may prove beneficial in inhibiting the formation of beta sheets of PrP and limiting its transfromation. in PrPsc which is the form found in these diseases.

We offer the use of these products for the treatment of diseases due to unconventional agents whose pathogenesis involves copper.

Prion encephalopathies, including scrapie and bovine spongiform encephalopathy (BSE) in cattle, kuru and Creutzfeld-Jakob disease and Gerstmann-Strausseler-Scheinker syndrome in humans are neurodegenerative conditions characterized by an early loss of synaptic proteins, astrocytosis, a progressive loss of neurological functions and, often, aggregation and brain proteins with the formation of deposits. All these diseases appear to be caused by a conformational post-translational transition from a normal isoform of the prion protein (PrPc) to abnormal and pathogenic (PrPsc). Copper is directly associated with the pathophysiology of the prion. Until now, little has been known about the normal functions of the PrP protein in the brain and there is now a body of evidence for the intervention of PrP in copper metabolism. PrP deficient mouse brain cells have also been shown to be more sensitive to oxidative stress and copper deficiency.

The basis of the invention is based on the search for molecules endowed with the following properties.

Demonstrate a chelating activity with respect to copper and zinc; Have lipophilic properties;

Having a penetration capacity made it easy to pass the blood-brain barrier; Have pharmacological properties allowing oral administration in appropriate preparations;

Present a safety profile for human administration documented by clinical experience giving them an acceptable benefit / risk ratio for the treatment of prion diseases; Have a low chelating power vis-à-vis other metals of physiological importance (iron, calcium, magnesium, ...); Present a satisfactory distribution in the brain; Present a satisfactory distribution in the parenchymal cells Demonstrate a satisfactory reduction of oxidative stress in the brain.

Our research has led us to the discovery of two known chelators, clioquinol and phanquinone, which satisfy the criteria set out above, these two molecules have in common a strong chelating power on copper and zinc: however, clioquinol s 'associates more strongly with zinc and phanquinone associates more strongly with copper. Their chelating effects on iron, calcium and magnesium are weak or negligible. In the case of prion diseases, copper will be the main target. Clioquinol ³ administered intravenously penetrates the nervous system very effectively, which indicates that there is no meningeal barrier for this type of product (Shiraki, 1979, Handbook of Clinical Neurology, 37, 115, 139) . Administration of 250 mg of clioquinol orally in humans gives plasma concentrations of 1 to 5 μg / ml. In other experiments, the doses of 7.4 to 9.3 mg / kg / day gave concentrations of 2.7 to 5.8 μg / ml in plasma (Shiraki, 1979, loc. Cit.).

Phanquinone ⁴ has also been administered at doses of 50 mg three times a day in the past, inducing plasma levels and satisfactory clinical effects.

It is the lipophilic nature of these molecules that allows them to be well absorbed orally, to pass the blood-brain barrier and to be found in the brain. The data obtained in mice show that 18 hours after the administration of Zn65 intraperitoneally, a large number of grains containing zinc accumulated on the terminations of the granular cells and their extensions as well as on the pyramidal cells of the hippocampus. . The zinc grains were found mainly in the synaptic vesicles and less frequently on the mitochondria of the presynapses of the “mossy” endings, but not on the protosynapses of the granular and pyramidal cells or their extensions in the hippocampus.

The amyloid fibrils of amyloid beta, amylin and calcitonin have been shown previously to be neurotoxic and that in prion diseases the accumulation of PrPsc and / or deposition of amyloid is associated , in time and space, with neuronal degeneration and astrogliosis. (Rainer & Good, 2000, J Neurochem, 75, 2536-2545.) moreover, it has been demonstrated in scrapie that the titles

³ Clioquinol 30-26-7, C9H5C1INO, iodochlorohydroxyquinol, iodoc lorohydroxyquinoline, chloroiodoquine, 7-iodo-5-chloro-8-hydroxyquinoline, 5-Chloro-7-iodo-8-hydroxyquinoline, 5-Chloro-7- iodo-8 -quinolinol, 5-Chloro-8- ydroxy-7-iodoquinoline,

⁴ Phanquinone 84-12-8, C12H6N202, dihydro-5,6 ρhenanfhroline-4J dione-5,6, phanquone, phenanthroline quinone, phenantrolinquone, pheninquone the highest in infectious PrPsc were associated with membrane fractions and that PrPsc increased the micro membrane villi.

Recent reports (Wong, 2000, Bioch Biophys Res Comm, 275, 249-252) suggest that one of the mechanisms of the pathogenesis of prion diseases may be the imbalance of the reactions catalyzed by metals, which are the common denominator of several neurodegenerative diseases. These imbalances cause a deterioration of antioxidant functions which has the effect of increasing oxidative stress.

Examples of the brain activity of phanquinone and clioquinol have already been presented in another patent (WO 99/09981). In the presence of increasing concentrations of phanquinone from 0.1 μg / ml, the toxic effect of amyloid A beta

(25-35) is greatly reduced and even completely abolished at the concentration of

1 μg / ml of phanquinone.

Data from WO 99/09981 published under the PCT treaty show that both clioquinol and phanquinone reduce neurotoxicity in the ceveau by sequestering zinc and copper. The chelation of these metals is the basis of the treatment described here against human prion diseases.

An ideal treatment for prion diseases requires a molecule capable of reducing oxidative stress in the brain, decreasing the His (Ntau) -Cu-His (N tau) bond and limiting the formation of beta sheets. All these activities are linked to copper in the brain, the chelation of this metal and the decrease in the quantity available locally must act on the reduction of the conformational change which ensures the transition from PrPc to PrPnv. In addition, the chelation of copper and zinc will have an antioxidant effect in the brain by reducing the cleavage of reactive oxygen which is dependent on copper. DETAILED DESCRIPTION OF THE INVENTION.

The use of phanquinone and / or clioquinol is claimed for the treatment and / or prevention of prion diseases in humans or animals. These two molecules have already been used for other indications in humans and are relatively safe to use.

These two molecules are metal chelators including copper. Phanquinone is more effective at fixing copper than clioquinol. These two products have been proposed for the treatment of Alzheimer's disease because of this chelating property of zinc and copper and for reducing oxidative reactions in the brain. The amyloid of Alzheimer's disease is aggregated in the presence mainly of zinc, but also of copper and clioquinol and phanquinone, used for the chelation of the two metals, block the process of aggregation of the amyloid as well as the formation of plates. We therefore propose here the use of clioquinol and phanquinone separately or in combination in the treatment of diseases caused by non-conventional transmissible agents (NCTA) involving physiopathological mechanisms in which copper (and / or zinc) is involved. The use of clioquinol and phanquinone is the subject of this patent application. The use of clioquinol and phanquinone is justified by their different spectra of chelation of zinc and copper. The combination of the two could provide maximum chelation capacity because the two metals may be involved in the process of conformational change from the PrPc mpolecule to the pathological PrPsc molecule. In addition, an antioxidant effect will be obtained in the brain by the sequestration of copper and the reduction of reactive oxygen.

Chloroquinol is a drug known for its anti-amoebic and antibacterial properties and it has been used widely in the past for these indications. It is known to have activity of chelating metals such as zinc, iron, manganese, copper, mercury, nickel, and cobalt. The physicochemical properties of clioquinol have confirmed its chelating activity (Tateishi et al., 1973, Psychiatr Neurol, 75, 187-196)

Clioquinol injections have been reported to allow effective passage of the product through the brain by crossing the blood-brain barrier and reaching concentrations in the brain of the order of 20 μg / ml after intravenous injection of 20 mg / kg (Tateishi et al, 1973, loc. Cit., Tamura, 1975, Jap J Med Se Biol, Suppl 28, 69-77). The concentration of clioquinol is also high in regions of the brain like the hippocampus which are directly involved in Alzheimer's disease.

When radioactive labeled clioquinol is administered orally to the mouse, radioactivity can be detected for as little as three hours in the central nervous system. Experimental results show that clioquinol injected intravenously quickly penetrates the nervous system and can also be excreted quickly (Shiraki, 1979, loc. Cit). This fact justifies the use of clioquinol for the inhibition of the precipitation of amyloid and the clearance of zinc or other metals of the brain and in particular of the areas affected by Alzheimer's disease and prion diseases.

Clioquinol has been administered in the past in doses of 250 mg three times a day for periods of up to three months in the treatment of intestinal amebiasis, and it has been widely used to treat acute diarrhea. The only toxicity reported after several million treatments over several decades has been the observation made in Japan after administration of excessive doses for periods of up to two years. The dosage used included administration of 23 to 50 mg / kg / day for periods of 13 to 313 days. Many subjects have presented a manifestation of subacute neurotoxicity with demyelination of the optic nerve (subacute myelo-optic neurotoxicity = SMON). This neurotoxicity has recently been attributed to interference with the metabolism and transport of vitamin B12. To prevent these undesirable effects, a protective agent or a prosthetic group, such as vitamin B12, administered before, during or after the active products. The quantity should be sufficient to prevent the undesirable effects of the administration of clioquinol or phanquinone, at doses of 5 μg to 1 mg per day for vitamin B12. The ability of clioquinol to abolish the aggregation of amyloid A beta has been tested experimentally. In the presence of zinc (30 μM) the solubility of A beta is reduced (2.5 μM in TBS at pH 7.4) from 95% to 43%. When clioquinol (120 µM) is added to the zinc-A beta complex, the aggregation of A beta is reduced from 43% to 70%. Thus, the administration of clioquinol must sequester zinc and other heavy metals in the brain thanks to its lipophilic nature which allows it to induce relatively high concentrations in the brain and cerebrospinal fluid.

Phanquinone has been used in humans to fight against various agents: amoebas, trichomonas, giardia, and more generally as an antiseptic and antiparasitic. This drug has been widely used alone as an antidiarrheal and also in combination with clioquinol for the same indications.

Phanquinone has shown no toxic effects after dosing 150 mg daily for periods ranging from three weeks to six months.

Recently, extensive research has been conducted to study the effect of phanquinone and clioquiniol for the treatment of Alzheimer's disease. Experimental results show that clioquinol has the property of sequestering several heavy metals in vivo and in the brain, especially copper and zinc. Likewise, phanquinone has the ability to sequester zinc and copper in the brain. The capacity to bind copper is three times stronger for phanquinone than for clioquinol. In contrast, clioquinol binds zinc at least twice as well as phanquinone. It has also been shown that phanquinone, like clioquinol, have the same favorable properties for preventing the formation of amyloid and for ensuring the resolubilization of the amyloid in the brain.

Based on studies showing that clioquinol and phanquinone were potent zinc and copper chelators, and recent results showing that in the disease Alzheimer's, the precipitation of the amyloid takes place only in the presence of zinc and copper, research is carried out to study the effects of these two molecules in the treatment of this disease. The ability of the two molecules to prevent precipitation of amyloid in the brain and also to redissolve precipitated amyloid deposits in the brains of Alzheimer's patients is being demonstrated.

There is ample evidence that the accumulation of amyloid A beta in the brain is linked to the cause of Alzheimer's disease. A beta, a normal component of biological fluids, accumulates in a variety of ways, ranging from poorly soluble plaques to deposits that can be removed from post-mortem tissue by saline buffers. The factors responsible for this accumulation are mostly unknown, but this situation has certain analogies with that of amyloid deposits in prion diseases. Amyloid A beta is vulnerable to three factors: the presence of zinc, copper or a slightly acidic pH (Bush, 1994). Thus, the precipitation of A beta by copper is very exaggerated under pH conditions at 6.9.

The conclusions of these experiments can be interpreted in the following way. Divalent metals, most probably zinc in particular, keep A beta in an insoluble state in both healthy and sick subjects, which suggests a role for A beta in homeostasis. Other metals such as calcium or magnesium can act to keep A beta in the soluble state. A gentle treatment of tissues originating from subjects suffering from the disease and containing deposits of A beta by the chelating agent allows rapid dissolution of these deposits. It may be thought that ephemeral deposits of aggregated A beta would be present both in patients with the disease and in healthy subjects and that they are permanently precipitated and resolubilized. They are probably only detectable by background immunohistological staining. Cerebral lactic acidosis which complicates Alzheimer's disease could be one of the factors which contribute to the precipitation of A beta and this phenomenon could be dependent on the presence of copper. We also know that the regulation of zinc and copper concentration is abnormal in Alzheimer's disease. These results suggest that metal chelators will have a therapeutic effect in Alzheimer's disease and will serve as prophylactic agents by reducing the A beta load in the clinic. Clioquinol is one of the only chelators that is enriched in the brain due to its hydrophobicity, making it an excellent candidate for the development of clinical trials.

Recently, these metals have been shown to be an integral part of the beta deposits in the brains of people with Alzheimer's disease. It has also been shown that specific zinc and copper chelators can dramatically redissolve a significant proportion of A beta in tissues from post-mortem samples from subjects with Alzheimer's disease compared to the amount extracted by a control buffer not containing chelators. Finally, the first clinical trials conducted in patients suffering from Alzheimer's disease using clioquinol at a dose of 10 and 40 mg twice a day have shown biochemical and clinical modifications and improvements. These results allow an extension of the applications of the chelators concerned for the prevention and treatment of prion diseases and subacute spongiform encephalitis since the results obtained with Alzheimer's disease show that clioquinol does indeed penetrate the brain, at least in patients. studied and that their chelating activity caused significant biochemical changes.

Phanquinone has already been used widely in the past for the treatment of infectious diarrhea and for the treatment of giardiasis. This product has been shown to have biological activity on the brain by causing behavioral changes. It has also been used in combination with clioquinol for the same indications as clioquinol. It is capable of sequestering zinc and copper in the brain. The chelation of copper is three times more effective than that of clioquinol. the inhibitory properties of amyloid formation and redissolution in the brain have also been demonstrated. Clinical trials of the application of phanquinone to the treatment of Alzheimer's disease have recently been undertaken. Phanquinone has no toxic effects at doses up to 150 mg per day for three weeks to six months. We propose the use of these chelating products for the treatment of a human or animal subject presenting or being able to present a pathological condition which can relate to the action of prions, consisting in administering to the abovementioned subjects a quantity of clioquinol or phanquinone or d '' a combination of the two sufficient to prevent the action of prions, as well as a compound or a mixture of compounds selected from metal salts or prosthetic groups different from clioquinol and phanquinone having another activity against the disease prion in sufficient quantity to prevent undesirable effects from the administration of clioquinol. These products, like vitamin B12 will be used at a rate of 5μg to lmg of 5 μg to 250 mg for others.

Clioquinol, phanquinone and related compounds will be presented in a single pharmaceutical preparation or will be administered simultaneously, or sequentially in various orders. When administered sequentially, the administration periods may be three or four weeks. Clioquinol and phanquinone may be formulated for oral or parenteral use together or separately.

According to the considerations set out above, we propose the use of clioquinol in doses of 1 to 1000 mg or of phanquinone in doses of 1 to 1000 mg for the prevention and / or treatment of spongiform encephalopathies in humans and animal. Clioquinol can also be used as monotherapy in doses of 250 to 500 mg four times a day with the possibility of increasing up to 1000 mg three times a day. Similarly, phanquinone can be used as monotherapy in doses of 10 to 250 mg which can be increased to 500 mg three times a day. The pharmaceutical preparations can be administered by oral, parenteral or intradermal route.

A combination of the two molecules can provide a more powerful effect on the copper and zinc ligand of PrPc in order to block the molecular transition leading to the pathological state. The proposed combination is 250 mg clioquinol and 50 mg phanquinone three times daily by oral administration. In the combination, the clioquinol can be given in doses of 10 to 1000 mg and phanquinone in 10 to 250 mg.

Example 1

Preparation of a pharmaceutical composition containing clioquinol, phanquinone and vitamin B 12.

250 g of phanquinone and 250 g of clioquinol are mixed with 200 g of N '- (4-stéatoryl- aminophenyl) -trimethylammonium methyl sulfuric acid and 1025 g of lactose monohydrate for 5 minutes. 300 g of boiling water will be added all at once to a mixture of 100 corn starch suspended in 100 g of cold water. The starch suspension cooled to 40 ° C. is added to the mixture of phanquinone and clioquinol powders with continuous stirring. Then an aqueous solution of 5 g of vitamin B 12 is added to the mixture. The mixture is granulated using a 2.5 mm mesh screen and dried for 18 hours at 40 ° C. The dry granules are mixed with 400 g of corn starch and 20 g of magnesium stearate. The final mixture is formulated into tablets with a diameter of 8.0 mm and a weight of 500 mg.

There are other metal chelators that could be used in the treatment and prevention of spongiform encephalitis by similarly fixing zinc and copper and / or other metals in the brain in addition to phanquinone and clioquinol , to which we extend the claim and whose list follows. Hydroxyquinoline (oxine), 5-methyl-oxine (5-methyl-8-hydroxyquinoline), 2- mercaptopyridine-N-oxide, (2-mercapto-quinoline-N-oxide), 2-mercapto-4-n-propyl- pyridine-N-oxide, Nitroxoline, 2-hydroxypyridine-N-oxide, 2-hydroxyquinoline-N- oxide, 2-Hydroxy-4-methyl-5: 6-benzoquinoline-N-oxide, 5-methyl-8-hydroxy- quinoline, 8-hydroxy-quinoline-7-carboxylic acid, Hydroxy-quinoline sulfate, Chlor-hydroxy-quinoline, 8-hydroxy-quinoline-5-sulfonic acid, (dichloro-acetyl) -6- (2- fiιroyloxy) -l , 2,3,4-tetrahydro-quinoline, 2-heptyl-4-hydroχyquinoline-N-oxide or 2-n- heptyl-4-hydroxyquinoline-N-oxide, 4-hydroxy-quinoline-3-carboxylic acids (7 substituted benzyl-oxy, phenethyl-oxy or phenoxy-ethoxy), 6-hydroxy-quinoline, 6-nitroquinoline, 8-nitroquinoline, 6-methylquinoline, 8-methylquinoline, 8-hydroxyquinoline, 5,7-dibromo-quinoline, 2-n-nonyl-4-hydroxy [3-3H] quinoline, N- oxide2n-nonyl-4-hydroxyguinoline-N-oxide, 7-nitroso-8-hydroxyquinoline-5-sulfonic acid sodium sait, 5,7-dichloro-8-hydroxyquinoline, 2,2 ', 2 "-teφyridine, 8- hydroxyquinoline 5-sulfonic acid, 5-formyl-, 5- iodo-, 5-fluoro-, 5-acetyl-, and 5- methoxymethyl-8-hydroxyquinoline, Methyl-5 (8-hydroxyquinolyl) acetate, Ethyl 5- (8- hydroxyquinolyl) acetate-trihydroxyquinoline, 5-nitro-8- hydroxyquinoline, 4-hydroxyquinoline, 5J-dichloro-8-hydroxyquinaldine, 3-acetaldehyde to 4- hydroxyquinoline, 4-hydroxyquinoline-3-carboxylates, 5-phenylethyl-4- hydroxyquinoline-3-carboxylic acids, 2-n-alkyl-4 -hydroxyquinoline, 2-methyl-8- hydroxyquinoline, dibromo-8-hydroxyquinoline-5,7 dibromo-8-bezoil-o xyquinoldine, Arylglyoxal N-7-amino-5-substituted 8-hydroxyquinoline hemiacetals, 5- phenylglyoxylidenamin-8-hydroxyquinolines, Ethylenediaminetetraacetic acid, O- phenanthroline, 1,10-phenanthroline, 2,9-dimethyl-l, 10-phenanthroline, alpha, alpha '- dipyridyl, 2,2', 2 "-tripyridine, (2'-hydoxyphenyl) pyridine, (2'-Hydoxyphenyl) imidazoline, (2'-Hydoxynaphthyl-3 '-) imidazoline, Hydrazine-N: N -diacetic acid, Quinoline derivatives carrying carbon, sulfur or phosphorus substituents, and furaquinolines obtained from clioquinol by radical chain nucleophilic substitution reactions, Dehydracetic acid (3-acetyl-2-hydoxy-6-methyl- ³ -pyrone), 3-propionyl- 2- hydroxy-6-ethyl- ³ -pyrone), 3-butyryl-2-hydroxy-6-propyl- ³ -pyrone), 2-aminophenol, 4-butyl-2-aminophenol, Rhodotorullic acid, Mycobactin P, 8- hydroxyquinoline-7- carboxylate, 5-substituted 8-hydroxyquinolines, 5-formyl-, 5-iodo-, 5-fluoro-, 5-acetyl-, and 5-methoxymethyl-8-hydroxyquinoline, Methyl-5 (8-hydroxyquinolyl) acetate, Ethyl 5- (8-hydro xyquinolyl) acetate, 2,6-dihydroxyquinoline, 2, 5, 6 -trihydroquinoline, 5- formyl- 1 -methoxycarbonyl-4,6,8-trihydroxyphenazine, 5J-dichloro-8- hydroxyquinoline, 5-chlor-8-hydroxyquinoline, 4-hydroxyquinoline, 5- or 7-methylthio- 8 -hydroxyquinoline, 6-, 7-, or 8-substituted-4-hydroxyquinoline-3-carboxylic acids, 4- hydroxyquinoline-2 and -3-carboxylic acids, 5-phenylethyl -4-hydroxyquinoline-3- substituted carboxylic acids, 2-n-alkyl-4-hydroxyquinoline derivatives, 5-7 dibromo-8- hydroxyquinoline, O-acetyl-8-hydroxyquinoline, 2,2'-bipyridine, S-acetyl-8-mercaptoquinoline, 5,7 dibromo-8-benzoil-oxyquinoldine, Arylglyoxal N-7-amino-5- substituted 8-hydroxyquinoline hemiacetals , 5-phenylglyoxylidenamin-8-hydroxyquinolines, 8-hydroxyquinoline ester, Ethyl 6J-di-isobutoxy-4-hydroxyquinoline-3-carboxylate, Halogenated derivatives of salicylanilide, 8-hydroxyquinoline, 5-chloro-8-hydroxyquinoline (chloroxine) esters carboxylic acid, 2,2'-bipyridine, 8-hydroxyquinoline, 8-hydroxyquinoline and m-phenylenediamine, 1-aH-oxazirino [2,3-a] quinoline 1-a-carbonitrile and its substituted compounds of derivatives 3 - corresponding hydroxyquinoline, 6,7-dimefhoxy-4-hydroxyquinoline hydrochloride, 4nitroquinol-ine 1-oxide, 4-nitroquinoline 1-oxide, 4-aminoquinoline 1-oxide, batimastat, hydroxamic acids, fenamates

The invention being described here, it is obvious that it can be adapted, developed and extended in various ways. As cannot be overlooked by those skilled in the art, such variations cannot be considered to differ from the spirit and scope of the invention and such modifications, and will be included in the scope of possible subsequent claims.

In summary: * - A protein widely distributed in cells, PrPc is a regulator of the activity of an enzyme, super-oxide dismutase, involved in cellular resistance to oxidative stress.

* - PrPc is a ligand of copper and zinc.

* - The fixation of copper takes place at a precise level of the molecule, on the amino acids of a repeated octapeptide.

* - The transformation of the normal protein PrPc into PrPnv, a pathological protein takes place at the octapeptide level.

* - Copper (II) ions play a role in this process.

* - Removing the copper must prevent transformation. * - We have a family of known molecules already used for other uses which sequester copper and zinc and which easily pass the blood-brain barrier.

* - We propose the use of clioquinol and phanquinone or other chelating molecules for the treatment and prevention of transmissible subacute spongiform encephalopathies and of all diseases due to unconventional agents or prions.

Claims

claims

1 The use of clioquinol and phanquinone for the preparation of a pharmaceutical composition intended for the treatment or prevention of prion diseases in humans or animals and, more generally, diseases caused by non-communicable agents conventional (ATNC).

2 Use according to claim 1 when the disease is influenced by the action of diseases involving prions, in humans the various forms of Creutzfeld-Jakob disease, Gerstman-Straussler-Scheinker syndrome, kuru and in animals scrapie (scrapie) and bovine spongiform encephalopathy (BSE).

3 The use according to claims 1 or 2 of clioquinol in doses of 10 mg to 1 g.

4 The use according to claims 1 or 2 of phanquinone in doses of 10 mg to 500 mg.

5 The use according to claims 1 or 2 of a combination of clioquinol and phanquinone in doses of 10 mg to 1 g of clioquinol and 10 to 250 mg of phanquinone in a single pharmaceutical preparation.

6 The use according to one of claims 1 to 5 of clioquinone or phanquinone or their combination at a rate of one to four times a day.

7 The use according to one of the preceding claims of a metal salt or a prosthetic group administered before, during or after the administration of clioquinol and phanquinone.

8 The use according to claim 7 of vitamin B 12 as a prosthetic group.

9 The use according to one of the preceding claims of an amount of vitamin B 12 sufficient to prevent an undesirable effect of the administration of clioquinol and phanquinone.

10 The use according to claim 9 of vitamin B 12 at a dose of 5μg to 2 mg per day.

11 The use according to claim 9 of vitamin B 12 at a dose of 0.5 μg to 1 mg per day. 12 Use according to one of the preceding claims of the pharmaceutical preparation formulated for oral, parenteral or intradermal administration.

13 The use of clioquinol and / or phanquinone for the preparation of a pharmaceutical composition intended for the treatment of a human or animal subject presenting or being able to present a pathological condition which can be modified by the chelating action on copper and / or zinc.

14 Use of clioquinol and / or phanquinone for the preparation of a pharmaceutical composition intended for the treatment of a human or animal subject presenting or being able to present a pathological condition which may arise from the action of prions.

The use according to claim 14, consisting in administering to the aforementioned subjects: a) an amount of clioquinol or phanquinone or a combination of clioquinol and phanquinone to treat the pathological condition caused by prions, and b) an amount of a compound or a mixture of compounds selected from the group comprising metal salts or prosthetic groups different from clioquinol and phanquinone, having another activity with respect to prion disease. 16 Use according to claims 13, 14 or 15 wherein the daily dose of clioquinol is from 1 mg to 1 g.

17 Use according to claims 13 14 or 15 wherein the daily dose of clioquinol is from 1 mg to 250 mg, administered one to four times a day.

18 Use according to claims 13, 14 or 15 wherein the daily dose of phanquinone is from 1 mg to 1 g.

19 Use according to claims 13, 14 or 15 wherein the daily dose of phanquinone is from 1 mg to 250 mg, administered one to four times a day.

Use according to claim 15 wherein the amount of the compounds in (b) is 5 μg to 250 mg. 21 Use according to claim 15 wherein the compound different from clioquinol and phanquinone and having another activity is vitamin B 12. 22 Use according to claim 21 wherein the amount of vitamin B 12 is 5 μg to 2 mg.

23 Use according to claim 21 wherein the amount of vitamin B 12 is 0.5 to 1 mg. 24 Use according to claim 14 consisting in administering to said subjects: a) an amount of clioquinol effective for treating or preventing the action of prions, and b) a mixture of phanquinone and vitamin B 12 comprising an amount of phanquinone sufficient to prevent the action of prions and an amount of vitamin B 12 sufficient to prevent the undesirable effects of the administration of clioquinol.

25 Use according to claims 15 or 24 wherein a) clioquinol and b) the compound (s) are present in a single pharmaceutical preparation.

26 Use according to claims 15 or 24 wherein a) clioquinol and b) the compound (s) are administered simultaneously. 27 Use according to claims 15 or 24 wherein a) clioquinol and b) the compound (s) are administered sequentially.

28 Use according to claims 15 or 24 wherein a) clioquinol and b) vitamin B 12 are administered sequentially.

29 Use according to claims 15 or 24 wherein a) phanquinone and b) vitamin B 12 are administered sequentially.

30 Use according to claims 15 or 24 wherein the phanquinone is administered first followed by the administration of clioquinol second.

31 Use according to claims 15 or 24 wherein the clioquinol is administered initially followed by the administration of phanquinone in a second step. 32 Use according to claims 30 and 31 wherein the first period is from one to three weeks and the second from one to four weeks.

33 Use according to claims 13 and 15 wherein the clioquinol is formulated for oral administration.

34 Use according to claims 13 and 15 wherein the clioquinol is formulated for parenteral administration. Use according to claims 13 and 15 wherein the phanquinone is formulated for oral administration.

36 Use according to claims 13 and 15 wherein the phanquinone is formulated for parenteral administration.

37 Use according to claims 13 and 15 wherein the combination of clioquinol and phanquinone is formulated for oral administration.

38 Use according to claims 13, and 15 wherein the combination of clioquinol and phanquinone is formulated for parenteral administration.