Uroporphyrin I as a therapeutic compound for prion diseases

Abstract

The present invention relates to the use of uroporphyrin I (UROI) or a pharmaceutically acceptable salt or metal complex thereof for the treatment and / or prevention of transmissible spongiform encephalopathies (TSEs) or prion diseases.

Description

[Technical Field]

[0001] The present invention relates to methods, compounds and compositions that can be used for the treatment and / or prevention of prion diseases (transmissible spongiform encephalopathies). [Background technology]

[0002] Transmissible spongiform encephalopathies (TSEs), or prion diseases, are rapidly progressive, always fatal neurodegenerative disorders that affect humans, among other mammals, for which no treatment is available. In humans, the best-known prion diseases include Creutzfeldt-Jakob disease (CJD), fatal familial insomnia (FFI), and Gerstmann-Straussler-Scheinker syndrome (GSS), while in other mammals, scrapie in sheep and goats, bovine spongiform encephalopathy in cattle, and chronic wasting disease in deer are the most common.

[0003] The main features of these diseases are spongy changes in the brain, widespread neuronal death resulting in significant vacuolization, and the presence of amyloid plaques or deposits, which are large protein aggregates that accumulate in the central nervous system. The latter is a common feature of amyloidogenic or protein misfolding-related neurodegenerative diseases. In the case of TSE, these protein deposits are the cellular prion protein, or PrP. C It is composed almost exclusively of misfolded isoforms of the endogenous prion protein, also known as PrP. This is a glycoprotein normally associated with the cell membrane and is most abundantly expressed in cells of the central nervous system. Its physiological role is not precisely understood, but recent studies implicate it in the maintenance of myelin. Less well-known misfolding events can occur, resulting in the formation of PrP. C undergoes a conformational change, and PrP Sc This abnormal isoform exhibits a high β-sheet content, is protease-resistant, and tends to aggregate, and is not identical to the native PrP fold. CPrP forms amyloid fibrils with the ability to induce the same conformation as PrP, and thus propagates and diffuses through a self-template polymerization process. Sc Besides being protease-resistant and self-propagating, prions are neurotoxic and their accumulation leads to neurodegeneration. Considering the protein nature of prions and their propagation mechanism, TSEs can arise from three different sources: 1) in acquired or infectious forms of the disease, PrP Sc PrP is acquired from exogenous sources via ingestion of prion-contaminated tissues or fluids, or via medical procedures using prion-contaminated materials. Sc then transfers PrP from the new host C 2) In familial or hereditary TSEs, PrP C Mutations in the gene encoding PrP enhance its misfolding ability, ultimately resulting in PrP Sc PrP is formed, which initiates the transmission process and makes this type of TSE inherited in an autosomal dominant manner. 3) Finally, in idiopathic or sporadic TSEs, wild-type PrP is probably present in the absence of a pathologically associated mutation. C Misfolding of PrP occurs rarely, Sc Furthermore, two notable phenomena closely related to the prion propagation mechanism have been reported in TSEs: the existence of a transmission barrier and the existence of different prion strains. Sc PrP from different species or polymorphic variants is defined as a conformer, which exhibits significantly different biochemical and biological properties and thereby causes different disease forms. Given that the amino acid sequence of a protein influences its conformation, Sc It is not uncommon for different strains to form, each exhibiting different conformations with characteristic properties. However, in the case of prions, this strain phenomenon is not unique to PrPs formed by the same PrP sequence. Sc This phenomenon also occurs in PrP Sc Conformation of PrP CThis phenomenon is based on the structural compatibility between PrP from a species or polymorphic variant, allowing the PrP to acquire the exact same structure, even if the sequence differs and maintains the characteristics of the defined strain. The structural compatibility required for the transmission of a particular prion strain leads to a second phenomenon: the existence of a transmission barrier. Sc But the same PrP C Once acquired by a new host with the sequence, it spreads without any barriers in the new host, and its conformation is easily induced, so that the host-derived PrP C However, the PrP of the new host C Sequence acquired PrP Sc If the sequence differs, this PrP C If transmission is not possible at all, PrP Sc and PrP C A strong transmission barrier exists between the PrP and the PrP, the latter of which cannot adopt the same conformation. Nevertheless, this barrier can be overcome more easily in other cases, and strain characteristics may be maintained despite an initial delayed disease progression, or new strains may emerge in the host, possibly due to incomplete template formation. Thus, determined prion strains may be expressed as different PrP C The ability to consume sequences and induce their conformation or other misfolding determines the host range of the strain. Because the precise molecular mechanisms that strengthen or weaken the transmission barrier are completely unknown, the transmissibility of a prion strain to a new host is currently unpredictable, and new strains may emerge during transmission, making it difficult to predict the transmissibility of a particular PrP. Sc -PrP C Each pair needs to be assessed for the presence of such barriers.

[0004] Given the specific mechanism of prion transmission, the presence of natively folded or intracellular PrP isoforms capable of adopting a prion conformation as a conversion substrate is essential for prion propagation and spread, and ultimately for the development of neurodegenerative pathologies. This fact has been demonstrated in various ways, most notably by the generation of PrP knockout transgenic mouse models that are completely resistant to prion disease, even after intracerebral inoculation with exogenous prions, which is always fatal in wild-type animals. Thus, the development of TSEs requires the presence of self-templated PrP, which acts as a nucleus or seed for propagation. Sc and PrP, which acts as a misfolding reaction substrate. C Both, as well as some interaction between the two, are necessary.

[0005] Furthermore, the physical nature of prion misfolding and propagation allows replication in vitro in cell-free systems, where trace amounts of PrP Sc The addition of a seed (PrP) C induces the conversion of PrP C -PrP Sc This further demonstrates that the interaction between prions is necessary for prion pathogenesis. Several methods have been developed for in vitro prion propagation, faithfully recapitulating the autologous template-mediated propagation that occurs in the central nervous system of affected individuals. Among these systems, the Protein Misfolding Cyclic Amplification (PMCA) method, which involves alternating sonication and incubation, is one of the most successful, as it preserves strain characteristics and reproduces transmission barriers. The development of this method initially involved brain homogenates from healthy animals as a source or substrate of PrP and PrP from the brains of affected individuals as seeds. Sc This reaction was based on the use of exogenous PrP, but it has now become possible to reproduce spontaneous prion misfolding. ScIt is performed in the absence of seeds and allows the de novo generation of distinct strains of infectious prions. Finally, the latest development in this regard has led to the Protein Misfolding Shaking Amplification (PMSA) assay, which is derived from PMCA and uses sonication instead of shaking and can use recombinant PrP (rec-PrP) produced in bacteria as the reaction substrate, thus offering unprecedented possibilities to evaluate the misfolding capacity of all imaginable PrP sequences and artificial mutants.

[0006] Prion diseases are always fatal, and because of their transmissibility and potential for zoonotic transmission, there is a possibility that the prion responsible for bovine spongiform encephalopathy could be transmitted to humans and cause an epidemic, such as new Creutzfeldt-Jakob disease, making the development of a treatment for TSEs urgently needed. This disease is caused by PrP C from PrP Sc This is caused by the conversion of PrP Sc The search for compounds that can block this process is an obvious therapeutic approach. C For example, molecular chaperones can be used to enhance the stability of the native conformation or PrP Sc targeting PrP, either to promote clearance of aggregates or C and PrP Sc Compounds that can inhibit the interaction of prions limit the propagation and spread of prions.

[0007] As mentioned above, to date, no treatments are available for prion diseases, and only a few clinical attempts have been made to treat these diseases or at least provide neuroimaging agents. A wide variety of compounds have been evaluated in vitro and in vivo, but none have been found to be able to sufficiently extend survival in animal models of prion disease. These substances include anionic molecules conjugated to polymers such as pentosan polysulfate (used in clinical trials via intracerebroventricular administration), sulfated glycosaminoglycans and their derivatives with lipophilic substituents, and polyacrylamides substituted with sulfated acetylglycosamine units. Another class of polymers with anti-prion activity is comprised of dendrimers. In any case, because macromolecules have a major drawback—their inability to penetrate the blood-brain barrier—most research on anti-prion compounds has focused on small molecules, including, for example, cyclic tetrapyrroles (such as porphyrins and phthalocyanines), aminothiazoles and related compounds; acridines, quinolines, and analogues; compounds with chaperone activity targeting the native prion protein; inhibitors of the protein-folding activity of ribosomal receptors (PFAR); N-(aryl or heteroaryl)amides; various simple aromatic and heterocyclic compounds; and antibiotics, cholesterol-lowering drugs, and steroids (see Mustazza C, Sbriccoli M, Minosi P, Raggi C. Small Molecules with Anti-Prion Activity. Curr Med Chem. 2020;27(33):5446–5479. doi: 10.2174 / 0929867326666190927121744).

[0008] The anti-prion activity of cyclic tetrapyrroles such as porphyrins and phthalocyanines was first reported by W.S. Caughey's group in 1998 (Caughey W.S., Raymond L.D., Horiuchi M., Caughey B. Inhibition of protease-resistant prion protein formation by porphyrins and phthalocyanines. Proc. Natl. Acad. Sci. USA, 1998, 95, 12117-12122. doi: 10.1073 / pnas.95.21.12117; Priola, S.A.; Raines, A.; Caughey, W.S. Porphyrin and phthalocyanine antiscraphie compounds. Science 2000, 287, 1503-1506. doi: 10.1126 / science.287.5457.1503; WO0009111). Although these compounds were shown to inhibit prion propagation to some extent in vitro and in vivo, their potential efficacy when administered to animals along with prion seeds was called into question. Furthermore, despite their promising antiprion effects, the porphyrins and phthalocyanines initially tested were not further developed as therapeutic agents for prion diseases, primarily due to the difficulty and toxicity of administration.

[0009] Porphyrin is a compound of porphyrin (C2OH 14 N4), in which four modified pyrrole rings are interconnected at the α-carbon atom via methine bridges (=CH-) to form a closed ring overall called a tetrapyrrole.

[0010] Porphyrins exist in many organisms, exhibiting a high degree of diversity and forming part of abundant cellular components such as chlorophyll, vitamin B12, or the heme group (an important part of hemoglobin). In some cases, these porphyrins form complexes with metal ions, forming part of myoglobin and hemoglobin, while porphyrins complexed with iron ions are known as metalloporphyrins. The most representative natural compounds of the latter group, all of which are part of the heme biosynthetic pathway, are uroporphyrin, coproporphyrin, pentaporphyrin, hexaporphyrin, and heptaporphyrin. [ka]

[0011] The functionality of porphyrins varies greatly depending on the groups or radicals that can be attached to the pyrrole ring or the alpha carbon atom that bears these groups.

[0012] A unique group of natural porphyrins is the uroporphyrins, which are intermediates in the biosynthesis of heme. In animals, they are synthesized from glycine and succinyl-CoA via a complex multienzyme pathway involving several intermediates, including δ-aminolevulinic acid, porphobilinogen, uroporphyrinogen, coproporphyrinogen, and protoporphyrinogen. These are ultimately converted to protoporphyrins, which are complexed with iron to form heme. Given the strict regulation of this biosynthetic pathway, almost all natural porphyrins in animals are part of hemoglobin, myoglobin, or respiratory fermentation. However, some of these compounds, namely uroporphyrin, coproporphyrin, and protoporphyrin, are found in small amounts in free form as by-products of the heme biosynthetic pathway.

[0013] Porphine derivatives can also be divided into two major groups based on the radical attachment site: those in which the side group is directly attached to the pyrrole ring, and those in which it is attached to the α-carbon atom connecting the pyrrole groups.

[0014] Examples of Group 1 compounds: [ka]

[0015] Examples of compounds in Group 2: [ka]

[0016] However, since Caughey's pioneering work over 20 years ago, porphyrins and phthalocyanines have not received much attention as potential therapeutic compounds for prion diseases, except for ASOS (antisense oligonucleotides) [Raymond GJ, Zhao HT, Race B, Raymond LD, Williams K, Swayze EE, Graffam S, Le J, Caron T, Stathopoulos J, O'Keefe R, Lubke LL, Reidenbach AG, Kraus A, Schreiber SL, Mazur C, Cabin DE, Carroll JB, Minikel EV, Kordasiewicz H, Caughey B, Vallabh SM. Antisense oligonucleotides extend survival of prion-infected mice. JCI Insight. 2019 Jul 30;5(16):e131175. doi: 10.1172 / jci.insight.131175] and other agents have emerged as more promising candidates, including antibody-based immunotherapy, of which the fully humanized prion protein monoclonal antibody PRN100 was the first agent used in human clinical trials for CJD patients and has shown very promising results (Mead S, Khalili-Shirazi A, Potter C, Mok T, Nihat A, Hyare H, Canning S, Schmidt C, Campbell T, Darwent L, Muirhead N, Ebsworth N, Hextall P, Wakeling M, Linehan J, Libri V, Williams B, Jaunmuktane Z, Brandner S, Rudge P, Collinge J. Prion protein monoclonal antibody (PRN100) therapy for Creutzfeldt-Jakob disease: evaluation of a first-in-human treatment program.Lancet Neurol. 2022;21(4):342-354. doi: 10.1016 / S1474-4422(22)00082-5). .

[0017] Overall, there are currently no effective treatments for prion diseases. With the exception of PRN100, human clinical trials have not been successful, hindered by the rarity of prion diseases. Currently, treatments are primarily focused on providing supportive care. These treatments include: Medications, such as antidepressants or sedatives to relieve psychological symptoms, opiates to relieve pain, or drugs such as sodium valproate and clonazepam to relieve muscle spasms. Assistance. As the disease progresses, many people need help with personal care and performing daily activities. Fluid and nutritional support. In advanced stages of the disease, intravenous or tube feeding may be required.

[0018] Thus, there remains a great need to find effective treatments for prion diseases. Summary of the Invention

[0019] Herein, we describe a novel treatment for transmissible spongiform encephalopathies (TSEs) or prion diseases (both terms are used interchangeably). In particular, we demonstrate that uroporphyrin I (UROI) is the most effective anti-TSE porphyrin described by Caughey and coworkers, TMPyP-Fe. 3+We demonstrate that uroporphyrin I can inhibit prion propagation (propagation) in vitro using mouse brain homogenates to a much greater extent than human prion propagation (see Example 1, Figure 1). Furthermore, the inhibitory effect of uroporphyrin I occurs strain-independently (see Example 1, Figure 2). Advantageously, UROI can also inhibit human prion propagation (see Example 2, Figure 3). The anti-prion effect found in vitro was confirmed in vivo using transgenic mouse strains genetically engineered to accumulate high levels of systemic UROI. These mice were challenged with different prion strains, and in all cases, disease onset was delayed relative to wild-type animals (see Example 3, Figures 5 and 6).

[0020] Thus, the present invention relates to uroporphyrin I or a pharmaceutically acceptable salt or metal complex thereof for use in the treatment and / or prevention of prion diseases.

[0021] A further embodiment is a pharmaceutical composition for use in the treatment and / or prevention of prion diseases, said composition comprising uroporphyrin I, or a pharmaceutically acceptable salt or metal complex thereof, and a pharmaceutically acceptable excipient.

[0022] Also disclosed is a method for treating and / or preventing a prion disease in a subject, said method comprising administering to said subject a therapeutically effective amount of uroporphyrin I, or a pharmaceutically acceptable salt or metal complex thereof. In a preferred embodiment, said subject is a human.

[0023] In certain embodiments of the present invention, the prion disease is selected from the group consisting of Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSS), fatal familial insomnia (FFI), sporadic fatal insomnia (sFI), diverse protease-sensitive prion disease (VPSPr), kuru, scrapie, transmissible mink encephalopathy (TME), chronic wasting disease (CWD), bovine spongiform encephalopathy (BSE), and feline spongiform encephalopathy (FSE).

[0024] These aspects and their preferred embodiments are also further detailed below in the detailed description and claims.

[0025] All features described in this specification (including the claims, description and drawings) may be combined in any combination except for mutually exclusive feature combinations.

[0026] For a better understanding of the present invention, its objects and advantages, the following drawings are attached hereto: [Brief explanation of the drawings]

[0027] [Figure 1] Figure 1. Comparative evaluation of the in vitro prion propagation inhibitory potential of uroporphyrin I and TMPyP (Fe3+). To evaluate the in vitro prion propagation inhibitory potential of UROI and TMPyP (Fe3+), a 24-hour PMCA reaction was performed using wild-type mouse brain homogenate as the substrate and the mouse prion strain RML as the seed. The seed was serially diluted with substrate (1:10, 1:50, 1:250, 1:1250, 1:6250, and 1:31250). Each compound was added to the sample at 20, 10, and 5 μM for UROI and 50, 20, 10, and 5 μM for TMPyP (Fe3+). DMSO, used as the diluent for both compounds, was also added to the dilution set as a positive control for prion propagation. All PMCA products were analyzed for misfolded PrP by PK digestion, electrophoresis, and Western blotting using Saf-83 (1:400) as the primary anti-PrP antibody. Both UROI and TMPyP demonstrated the ability to inhibit prion propagation, and the highest dilution at which PrPSc was detected was lower than the DMSO control in both cases. Nevertheless, UROI inhibited prion propagation at the lowest concentrations of both compounds tested, making it 125-fold more effective as an inhibitor. PK: Proteinase K; MW: Molecular weight. See Example 1. [Figure 2]Figure 2. Determining the potential prion strain specificity of uroporphyrin I in its prion transmission inhibitory effect. To confirm whether the prion transmission inhibitory activity observed with UROI was strain-dependent, PMCA was performed using wild-type mouse brain homogenate as the substrate and three different mouse prion strains, RML, 22L, and 301C, as seeds, serially diluted in the substrate (1:10, 1:50, 1:250, 1:1250, 1:6250, and 1:31250). For each strain, UROI was added to the serial dilution set at three different concentrations (20, 10, and 5 μM) to confirm dose-dependent effects. A dilution set using DMSO instead of UROI was also subjected to PMCA as a positive control for the prion transmission efficiency of each strain. After a single 24-hour PMCA run, all PMCA products were analyzed for PrPSc by PK digestion, electrophoresis, and Western blotting (primary antibody Saf83 at 1:400). The highest dilution at which PrPSc was detectable was noted as a measure of prion propagation efficiency. Compared with the highest dilution at which PrPSc was detectable in the DMSO control, UROI exhibited similar inhibitory potency across the three strains, suggesting a strain-independent mechanism of action. UROI inhibited prion propagation 125-fold at 5 μM, approximately 3125-fold at 10 μM, and almost completely at 20 μM. PK: proteinase K; MW: molecular weight. See Example 1. [Figure 3]Figure 3. Evaluation of the ability of uroporphyrin I to inhibit human prion propagation in vitro. To examine whether UROIs can also inhibit human prion propagation, recombinant PrP harboring two major human polymorphic variants (M129 and V129) was produced in Escherichia coli (E. coli) and used as PMCA substrates with PrP knockout brain homogenate. Previously generated recombinant human prion (M129) was used as a seed, and serial dilutions of 1:10 from 10-3 to 10-9 were performed with each substrate. UROIs were added to the dilution series at 50, 20, and 10 μM. DMSO controls were also included as positive controls for prion propagation efficiency in the absence of UROIs. After 24 h of PMCA reaction, all products were analyzed for PrPSc by PK digestion, electrophoresis, and Western blotting (primary antibody 3F4 at 1:10,000). The highest dilution at which PrPSc was detectable was determined as a measure of prion propagation efficiency. In the case of rec-human PrP with the M129 mutant, the inhibitory effect of UROI was reduced compared to the V129 mutant, reaching a 5-log reduction at 50 μM, a 4-log reduction at 20 μM, and a 2-log reduction at 10 μM. These results suggest that UROI exerts different effects on the M129 mutant and the V129 mutant due to their different binding affinities, suggesting a mechanism of action dependent on UROI-PrP binding. PK: proteinase K; MW: molecular weight. See Example 2. [Figure 4]Figure 4. Uroporphyrin I levels in the brain and blood of wild-type mice were compared with those of TgUro and TgVole-TgUro mice, confirming that the in vitro prion propagation inhibition could be due to elevated uroporphyrin I levels. Wild-type, TgUro, and TgVoleTgUro mice were sacrificed to examine UROI levels in the blood and brain, demonstrating the correlation between elevated UROI levels in vivo and potential prion propagation inhibition. Blood was extracted by submandibular puncture the day before sacrifice; the brain was thoroughly perfused prior to extraction to avoid contamination of this tissue with blood-borne UROI. Both types of samples were processed for intracellular UROI extraction, and UROI concentrations were measured by HPLC coupled to a fluorescence detector. A) UROI levels ranged from 0 to 2 μM in the blood of wild-type animals, whereas TgUro mice showed levels of 40 to 160 μM, two orders of magnitude higher on average in these models. In contrast, blood UROI levels in TgVole-TgUro mice ranged from 10 to 50 μM. B) Regarding UROI levels in the brain, due to the low permeability of UROI across the blood-brain barrier, the levels were 0.3 to 1.5 μM in wild-type animals, 1.1 to 2.7 μM in TgUro, and 0.8 to 1.9 μM in TgVole-TgUro. Although the mean values ​​in this model were significantly higher than those in wild-type animals, given the efficacy of UROI in inhibiting prion propagation in vitro, these values ​​were sufficient to detect differences in survival time following prion inoculation. See Example 3. [Figure 5]Figure 5. Evaluation of the effect of elevated uroporphyrin I levels on inhibiting mouse prion transmission and extending survival or delaying disease onset in vivo. To assess whether the elevated UROI levels observed in TgUro mice were sufficient to inhibit prion transmission in vivo and extend survival or delay disease onset, and to validate the anti-prion effects observed in vitro, wild-type and TgUro animals were inoculated intracerebrally with three different mouse prion strains: RML, 22L, and 301C. Prion-infected animals were followed daily for clinical signs of neurological disease and then sacrificed, after which blood and brain samples were extracted for analysis. For each prion strain, survival times of wild-type and TgUro animals were compared, and Kaplan-Meier survival curves were plotted to represent survival times as days post-inoculation (dpi) (corresponding to time of clinical onset). TgUro mice inoculated with the RML strain showed the highest survival benefit, with a 30% increase in survival compared to wild-type animals, whereas inoculation with 22L and 301C resulted in 10% and 12% increases in survival, respectively, demonstrating that high levels of URO can inhibit prion propagation in vivo and delay disease onset (see Example 3). [Figure 6]Figure 6. Evaluation of the effect of elevated uroporphyrin I levels on inhibiting prion transmission from various species in vivo and extending survival or delaying disease onset. To determine whether elevated UROI levels in the brain and blood in vivo could inhibit prion transmission from species other than mice, we chose the TgVole-TgUro hybrid model, which expresses vole PrPC instead of mouse PrPC. Because they are susceptible to infection by prions from different species (including human prions) and exhibit elevated UROI levels, albeit lower than those seen in TgUro, we inoculated them: A) intracerebrally with the Vole-CWD prion strain, a vole prion derived from serial inoculations of deer chronic wasting disease prions and therefore distinct from previously used mouse prions. Furthermore, this prion strain inoculated into voles exhibits the fastest reported prion disease onset in any animal model, making it suitable for testing the inhibitory effects of UROI in vivo. B) Human prion strains causing Gerstmann-Straussler-Scheinker syndrome (GSS) were inoculated intraperitoneally. In this case, brain homogenate containing human GSS prions was inoculated intraperitoneally. This administration method better mimics the slower disease progression believed to occur in humans, as prions require a neuroinvasive process to establish effective infection. Prion-infected animals were followed daily for clinical signs of neurological disease and sacrificed, at which time blood and brain samples were extracted for analysis. Kaplan-Meier survival curves comparing the survival of TgVole and TgVole-TgUro hybrid animals show survival times (equivalent to time of clinical onset) as days postinoculation (dpi). A) Animals inoculated with Vole-CWD had an 11% increase in survival compared to TgVole animals, and UROI levels were comparable to those in wild-type mice. These results confirm the effectiveness of increased UROI levels in delaying the onset of prion disease and demonstrate a strain- and species-independent inhibitory effect. B) In animals inoculated with human prions (GSS), an unprecedented increase in survival of over 200% was observed compared to TgVole animals, with two of the inoculated animals now free of neurological signs.Thus, although elevated UROI levels proved effective in delaying the onset of human prion disease, the greater efficacy is likely explained by elevated systemic UROI levels, which may impede peripheral prion propagation and neuroinvasion (see Example 3). [Figure 7] Figure 7. Determination of the interaction site and affinity of uroporphyrin I with human PrP M129 by nuclear magnetic resonance. To determine whether UROI exerts its prion propagation inhibitory effect through binding to PrP, and if so, the affinity and interaction site, recombinant human PrP carrying the M129 mutant was produced in Escherichia coli (E. coli), purified, and mixed with different concentrations of UROI. Heteronuclear Single Quantum Coherence (HSQC) spectra were obtained, which allow detection of the chemical shifts of interacting residues. The signals corresponding to each UROI:PrP ratio are shown in the figure using the following gray code: strong 1:0, high 1:0.2, medium 1:0.5, and low 1:0.75. Zooming in on specific regions of the spectrum reveals regions with more pronounced chemical shifts, indicating that those residues in the protein preferentially interact with UROI (i.e., residues 23K, 24K, 25R, and 104K of human PrP). A three-dimensional model of the PrP-UROI interaction is also shown. See Figure 4. DETAILED DESCRIPTION OF THE INVENTION

[0028] In 2000, W.S. Caughey's group (Science 2000, 287, 1503-1506) synthesized phthalocyanine (PcTS) and two different metalloporphyrins (DPG2-Fe 3+ y TMPyP-Fe 3+(2013) reported the anti-prion activity of a series of compounds tested in vitro and in vivo. PcTS was the most effective compound. Despite the significant delay in time to death observed, the actual efficacy of the coadministration of the compound and the inoculation of the prion at the same site was questioned, and the toxicity of the compound at therapeutic doses precluded its development as a viable treatment strategy at the time. Furthermore, antibodies and their derivatives are currently on the list of most promising candidates for the treatment of prion diseases, and research efforts are currently focused in this direction.

[0029] However, based on Caughey's work, we explored the potential anti-prion effects of other natural porphyrins that may be less toxic than those previously tested. Uroporphyrin I (UROI), a by-product of the heme biosynthetic pathway found in excess in some forms of porphyria, was selected as an anti-prion candidate because of the availability of animal models naturally expressing high levels of this compound and its potent in vitro anti-prion activity. Therefore, the prion propagation inhibitory abilities of the selected porphyrins for the development of therapeutic strategies for TSE were first evaluated in the cell-free prion propagation systems PMCA and PMSA, as well as in various animal models. In both cases, UROI demonstrated exceptional ability to inhibit or reduce prion propagation. Furthermore, unexpectedly, the inhibitory ability of UROI was significantly greater than that of TMPyP-Fe, the most active porphyrin proposed by Caughey. 3+ It was found to be significantly 125 times higher than the

[0030] There are four isomers of uroporphyrin, which Fischer designated I, II, III, and IV to indicate the four possible arrangements of acetate (Ac) and propionate (Pr) groups around the eight β-positions of the porphyrin macrocycle. Their chemical structures are shown in Scheme 4. [ka]

[0031] The present invention relates to uroporphyrin I (UROI) or a pharmaceutically acceptable salt or metal complex thereof.

[0032] "Pharmaceutically acceptable," as used herein, means a substance that is not biologically or otherwise undesirable, i.e., that can be incorporated into a pharmaceutical composition administered to a patient without causing undesired biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0033] The present invention also contemplates "salts" of uroporphyrin I. For example, the salts may be acid addition salts, base addition salts, or metal salts. Specific examples of acid addition salts include inorganic acid addition salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, and phosphate, and organic acid addition salts such as acetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulfonate, p-toluenesulfonate, trifluoroacetate, and camphorsulfonate. Specific examples of base addition salts include inorganic base salts such as ammonium salts, and organic base salts such as ethylenediamine, ethanolamine, N,N-dialkyleneethanolamine, triethanolamine, glutamine, and amino acid base salts. Specific examples of metal salts include sodium, potassium, calcium, magnesium, aluminum, and lithium salts.

[0034] According to a particular embodiment, the salt of uroporphyrin is uroporphyrin I dihydrochloride (CAS 68929-06-6).

[0035] Metal complexes of uroporphyrin I are formed by replacing the central hydrogen of the porphyrin ring with a metal, metalloid, or metal compound. The central nitrogen of uroporphyrin I can bind and form complexes with any metal or metalloid. At least one of the four central nitrogens of the tetrapyrrole macrocycle can participate in complex formation. The metal or metalloid can be Fe. 3+ The metals or metalloids that can form uroporphyrin complexes include Li, Na, Mg, B, Al, Si, P, K, Ca, Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Ge, As, Rb, Sr, Y, Zr, Nb, Mo, Tc, Ru, Rh, Pd, Ag, Cd, In, Sn, Sb, Cs, Ba, La, Hf, Ta, W, Re, Os, Ir, Pt, Au, Hg, Tl, Pb, Bi, Th, and lanthanides. Complexes in which the metal or metalloid is in a specific oxidation state are also included. For example, uroporphyrin I can be formed with Co. 2+ and Co 3+ Also included are uroporphyrin I complexes in which the metal, metalloid, or metal compound bears additional ligands. As used herein, the prefix "metallo" includes complexes formed with metalloids, unless otherwise specified.

[0036] Typical metals that can be incorporated into the uroporphyrin I structure are iron (Fe), cobalt (Co), gallium (Ga), tin (Sn), zinc (Zn), chromium (Cr), magnesium (Mg), and various elements of the lanthanide series.

[0037] To the inventors' knowledge, the usefulness of uroporphyrin (or a pharmaceutically acceptable salt or metal complex thereof) in the treatment of prion diseases has not been disclosed.

[0038] The present invention relates to the use of uroporphyrin I or a pharmaceutically acceptable salt or metal complex thereof for the manufacture of a medicament or pharmaceutical composition for the treatment and / or prevention of prion diseases.

[0039] As used herein, the term "prion disease" refers to transmissible (transmissible) spongiform encephalopathies. This group of neurological disorders affects humans and many animal species and causes a "sponge-like" degeneration of brain tissue. Another unique feature of all of these diseases is the accumulation of abnormal forms of prion protein within nerve cells, ultimately leading to the death of the host. All prion diseases can be transmitted from one host to another, but there is still controversy as to whether it is an infectious agent, such as a virus, that causes the conversion of normal protein to abnormal protein, or the abnormal prion protein itself, i.e., the prion.

[0040] Prion diseases affect probably most mammalian species. Examples of (non-human) animals include: Scrapie: sheep, goats TME (Transmissible Mink Encephalopathy): Mink CWD (Chronic Wasting Disease): Mule deer, deer, and elk BSE (Bovine Spongiform Encephalopathy): Dairy cows, cattle FSE (Feline Spongiform Encephalopathy): Cats and other felines

[0041] Humans are also susceptible to several prion diseases, including: CJD (Creutzfeldt-Jakob disease) GSS (Gerstmann-Straussler-Scheiker syndrome) ·FFI (Fatal Familial Insomnia) ·sFI (sporadically fatal insomnia) ·Various protease-sensitive prion diseases (VPSPr) Kuru

[0042] More specifically, human prion diseases include kuru, sporadic Creutzfeldt-Jakob disease (sCJD), familial CJD (fCJD), iatrogenic CJD (iCJD), Gerstmann-Straussler-Scheinker (GSS) disease, fatal familial insomnia (FFI), sporadic fatal insomnia (sFI), diverse protease-sensitive prion disease (VPSPr), and more recently, novel CJD (nvCJD or vCJD). In addition to these human diseases, prion-related diseases have been recognized in several animal hosts. Scrapie, a naturally occurring disease in sheep and goats, causes ataxia, behavioral changes, and severe pruritus, leading to scratching behavior, hence the name. Other prion diseases in animals include transmissible mink encephalopathy (TME), chronic wasting disease of deer and elk (CWD), feline spongiform encephalopathy (FSE), and bovine spongiform encephalopathy (BSE).

[0043] According to the present invention, any of the above-mentioned prion diseases can be treated and / or prevented with uroporphyrin I (UROI) or a pharmaceutically acceptable salt or metal complex thereof.

[0044] In more particular embodiments, the prion disease is selected from the group consisting of Creutzfeldt-Jakob disease (CJD), fatal familial insomnia (FFI), sporadic fatal insomnia (sFI), diverse protease-sensitive prion disease (VPSPr), Gerstmann-Straussler-Scheinker syndrome (GSS), scrapie, bovine spongiform encephalopathy (BSE), and chronic wasting disease (CWD).

[0045] Examples of pharmaceutical compositions include any solid composition (eg, tablets, pills, capsules, granules) or liquid composition (eg, solution, suspension, lotion, or emulsion).

[0046] These compositions can be administered, for example, orally, topically, transdermally, nasally, intravenously, intramuscularly, intraperitoneally, intracerebrospinal, intracranially, intraspinal, subcutaneously, intraarticularly, intrasynovially, or intrathecally. Other modes of administration are not excluded. In certain embodiments, the compositions are administered intracerebrospinal or intracranially.

[0047] Each composition may contain one or more excipients known to those skilled in the art, depending on its route of administration. The compositions or medicaments of the invention can be produced according to standard procedures known to those skilled in the art.

[0048] The term "excipient" refers to components of a drug compound other than the active ingredient (as defined by the European Medicines Agency (EMA)). Excipients preferably include "carriers, adjuvants, and / or vehicles." Carriers are the form into which a substance is incorporated to improve drug delivery and efficacy. Drug carriers are used in drug delivery systems, such as controlled-release technologies, to prolong in vivo drug action, decrease drug metabolism, and mitigate drug toxicity. Carriers are also used to design drugs to enhance the effectiveness of drug delivery to target sites of pharmacological action (U.S. National Library of Medicine, National Institutes of Health). Adjuvants are substances added to drug products that affect the action of the active ingredient in a predictable way. Vehicles are excipients used as a vehicle to provide bulk for the administration of a drug product, or preferably substances with no therapeutic activity (Stedman's Medical Spellchecker, 2006 Lippincott Williams & Wilkins). Such pharmaceutical carriers, adjuvants or vehicles can be sterile liquids, excipients, fractionators, wetting agents or diluents such as water and oils (including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.). Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin. The choice and amount of these excipients to be used will vary depending on the application form of the pharmaceutical composition.

[0049] Also disclosed is a method for treating a patient suffering from or suspected of suffering from a prion disease, comprising administering to a patient in need of such treatment or prevention an effective amount of uroporphyrin or a pharmaceutically acceptable salt or metal complex thereof.

[0050] The terms "subject," "patient," or "individual" preferably refer to mammals, particularly humans or ruminants, including humans and other mammals (such as sheep, goats, mink, mule deer, deer, elk, dairy cows and other cattle, cats and other felines, etc.). Ruminants include, for example, mule deer, elk, dairy cows, cattle, sheep, goats, deer, or buffalo. Mink is an example of a mammal that does not belong to the suborder Ruminantia.

[0051] As used herein, the term "treatment" generally includes eradication, elimination, reversal, mitigation, correction, or control of a prion disease after its onset.

[0052] As used herein, the term "prevention" refers to the ability of a given substance, composition or pharmaceutical to avoid, minimize or make it more difficult for a prion disease to develop or progress before it occurs.

[0053] An "effective" or "therapeutically effective" amount of a drug or pharmacologically active agent refers to a non-toxic but sufficient amount of the drug or agent to provide the desired effect. In the treatment of the present invention, an "effective amount" of uroporphyrin I or its pharmaceutically acceptable salt or metal complex is an amount of the compound effective to provide the desired effect. The "effective" amount will vary from subject to subject, depending on the individual's age and health, the specific active agent(s), and the like. Therefore, it is not always possible to specify an exact "effective amount." However, an appropriate "effective" amount in a particular case can be determined by one of ordinary skill in the art using routine experimentation.

[0054] Uroporphyrin I or a pharmaceutically acceptable salt or metal complex thereof can be used in combination with other additional useful drugs in the prevention and / or treatment of prion diseases. The additional drugs may form part of the same pharmaceutical composition or may be provided in the form of a separate composition from the pharmaceutical composition comprising uroporphyrin I or a pharmaceutically acceptable salt or metal complex thereof for simultaneous or sequential administration.

[0055] According to a particular embodiment of the present invention, uroporphyrin I or a pharmaceutically acceptable salt or metal complex thereof is intended to inhibit, block or reduce the propagation of prions.

[0056] According to another embodiment of the present invention, uroporphyrin I or a pharmaceutically acceptable salt or metal complex thereof is intended to prolong survival and / or delay the onset of disease.

[0057] According to another embodiment of the present invention, uroporphyrin I or a pharmaceutically acceptable salt or metal complex thereof is intended to inhibit, prevent or reduce spongy changes in the brain.

[0058] According to another embodiment of the present invention, uroporphyrin I or a pharmaceutically acceptable salt or metal complex thereof is intended to inhibit, prevent or reduce the significant vacuolization due to widespread neuronal cell death.

[0059] According to another embodiment of the present invention, uroporphyrin I or a pharmaceutically acceptable salt or metal complex thereof is intended to inhibit, prevent or reduce the accumulation or deposition of amyloid plaques in the central nervous system.

[0060] According to another embodiment of the present invention, uroporphyrin I or a pharmaceutically acceptable salt or metal complex thereof is C from PrP Sc The present invention is intended to inhibit, prevent or reduce the conversion of

[0061] The following examples are provided as evidence in support of the present invention to demonstrate that UROIs have demonstrated significant ability to prevent or reduce prion propagation both in vitro and in vivo. The following examples are merely illustrative of certain embodiments of the present invention and are not intended to limit the present invention in any way. [Example]

[0062] Example 1. Evaluation of the ability of uroporphyrin I to inhibit prion propagation in vitro by PMCA based on brain homogenate To assess whether uroporphyrin I (UROI) can be a good inhibitor of prion propagation, its ability to reduce or prevent prion propagation was examined using brain homogenates (PrP) from healthy animals as a substrate. C source) and brain homogenate (PrP) from end-stage prion-infected animals as seeds. Sc The compounds were tested using a cell-free prion amplification system, PMCA, based on the prion gene (as a source of prion signaling). Specifically, a 10% (w / v) homogenate of perfused brain from a C57B16 wild-type mouse homogenized in conversion buffer was used as the reaction substrate, and three different mouse prion strains (RML, 22L, and 301C) were used as seeds to determine potential strain-specific effects of the compounds. The latter were obtained from 10% brain homogenates of mice pre-inoculated with each prion strain and sacrificed after the onset of overt neurological signs of disease.

[0063] First, the inhibitory ability of UROI was examined by comparing it with TMPyP-Fe, the most effective porphyrin described by Caughey and coworkers (Science 2000, 287, 1503-1506). 3+To compare the inhibitory potential of the compounds, they were added to the PMCA substrate at different concentrations (5, 10, 20, and 50 μM). Because both compounds were diluted in DMSO, an equal volume of this solvent was added to control reactions without inhibitors. Next, brain homogenate containing the RML prion strain was serially diluted with the substrate. To quantify the inhibitory potential, dilutions of 1:10, 1:50, 1:250, 1:1250, 1:16250, and 1:31250 (species:substrate) were performed for each compound concentration and the DMSO control. All samples were then subjected to a 24-h PMCA round using a horn-cup sonicator (Misonix Q-700, Qsonica) programmed to alternate between 30-minute incubations and 20-second sonications at 80% amplitude. The temperature was maintained constant at 38°C throughout the 24-h PMCA round. For the detection of misfolded PK-resistant PrP, prion propagation or amplification was monitored via proteinase K (PK) digestion of all PMCA products, electrophoresis, and Western blotting. Sc The highest dilution of seed at which prion is detected provides a measure of the prion propagation efficiency, and its reduction compared to the limiting dilution of the DMSO control provides a measure of the inhibitory effect of the test compound.

[0064] Both UROI and TMPyP showed the ability to inhibit prion propagation, which is due to the Sc This is because the highest dilution at which UROI was detected was lower than the DMSO control in both cases. Nevertheless, UROI was 125-fold more efficient as an inhibitor, since it reduced prion propagation at the lowest concentrations of both compounds tested (see Figure 1).

[0065] Using a similar method, we also evaluated the strain specificity of UROI inhibition using three different mouse prion strains as seeds. Using the same PMCA substrate prepared from perfused brains of C57Bl6 mice, we tested the inhibitory effects of 20, 10, and 5 μM UROI. For each compound concentration, three serial dilutions of prion seeds (1:10, 1:50, 1:250, 1:1250, 1:16250, and 1:31250) were performed. To determine the propagation efficiency of each prion strain in the absence of UROI, DMSO (as a vehicle for the UROI solution) was added to the propagation control. All samples underwent a 24-h PMCA round under the same conditions as before. PrP propagation was monitored by PK digestion electrophoresis and Western blotting. The PrP levels after PMCA for each strain were compared with the DMSO control. Sc We focused on the highest dilution at which the virus could be detected.

[0066] As shown in Figure 2, DMSO control showed no significant difference in PrP Sc Compared with the highest dilution at which prion was detectable, UROI showed similar inhibitory potency against the three strains, suggesting a strain-independent mechanism of action. UROI inhibited prion propagation by 125-fold at 5 μM, approximately 3125-fold at 10 μM, and almost completely at 20 μM.

[0067] Example 2. Evaluation of the ability of uroporphyrin I to inhibit human prion propagation in vitro by recombinant PrP-based PMCA Due to limitations in testing the inhibitory effects of compounds in vitro using human brain homogenate, we evaluated the human prion-specific effects of UROI using human recombinant PrP (rec-PrP) produced in Escherichia coli as the substrate and recombinant human prion prepared in the laboratory as the seed. To this end, human recombinant PrP containing the two major polymorphic variants (M129 and V129) was bacterially expressed and purified by immobilized metal affinity chromatography (IMAC). The protein was dialyzed to obtain soluble, natively folded protein, and then mixed with 10% (w / v) perfused brain homogenate from PrP knockout transgenic mice to provide intracerebral cofactors that promote prion propagation in vitro. Different concentrations of UROI (50, 20, and 10 μM) were added to these substrates, consisting of brain homogenates supplemented with human rec-PrP M129 and V129, and DMSO was added as a propagation control for each. Recombinant prion seeds were then serially diluted 1:10 into each substrate for each UROI concentration and DMSO control (10 -1 ~10 -9 All samples then underwent a 24-h PMCA round using a horn-cup sonicator (Misonix Q-700, Qsonica) programmed to alternate between 30-min incubations and 20-s sonications at 80% amplitude, with the temperature maintained constant at 38°C using a circulating water bath. Prion propagation was again monitored by PK digestion, electrophoresis, and Western blotting of all PMCA products, focusing in each case on the highest dilution at which misfolded, protease-resistant rec-PrP was detected.

[0068] For rec-human PrP with the M129 mutant, the inhibitory effect of UROI was reduced compared to that of the V129 mutant, reaching a 5-log reduction at 50 μM, a 4-log reduction at 20 μM, and a 2-log reduction at 10 μM. These results suggest that UROI exerts differential effects on rec-human PrP due to its different binding affinity to the M129 or V129 mutant, suggesting a mechanism of action dependent on UROI-PrP binding (see Figure 3).

[0069] Example 3. Mouse model used to evaluate the ability of uroporphyrin I to inhibit prion propagation in vivo Two different mouse models were used to evaluate the efficacy of uroporphyrin I in inhibiting prion propagation in vivo and whether this could extend the survival time of prion-infected animals. Because UROI has low solubility in aqueous solutions and it is difficult to achieve high circulating UROI concentrations over long periods in wild-type mice, an animal model that naturally produces large amounts of this porphyrin was used as proof of concept. This transgenic mouse strain, called TgUro, was genetically modified to introduce a deleterious mutation in its uroporphyrin III synthase (UROSIII), resulting in dysregulation of the heme biosynthetic pathway and the accumulation of high levels of systemic UROI. Therefore, endogenous mouse PrP C This model, which reproduces a form of porphyria, exhibits red urine, red bones and teeth, and skin photosensitivity due to excessive porphyrin excretion, among other symptoms, but exhibits a lifespan similar to that of wild-type mice if protected from intense UV exposure. C Since UROI is identical to wild-type mice in terms of expression of prion markers, if UROI cannot inhibit prion propagation in vivo, it should be equally susceptible to mouse prion infection.

[0070] Endogenous mouse PrP CAs a mouse model expressing endogenous mouse PrP, TgUro is susceptible to a limited range of prions, namely mouse prions, which hinders the evaluation of the efficacy of prion strains from other species. To overcome this limitation and evaluate the efficacy of UROI in inhibiting transmission from multiple different prion strains in vivo, a second mouse model was developed. C Instead of a vole ( Myodes glareolus )PrP C The researchers used transgenic mice expressing the specific PrP gene, which they called TgVole. C In prion research, these rodents are known as universal acceptors of prions because of their susceptibility to many different prion strains from different mammals, including human prion strains. To obtain continuously high systemic concentrations of UROI in TgVole mice, they were crossed with TgUro mice, and vole PrP was obtained. C We obtained a hybrid mouse strain that expresses urinary bladder cancer (UROI) and exhibits high levels of circulating UROI due to the same UROSIII enzyme deficiency. This strain was designated TgVole-TgUro. This hybrid mouse strain not only exhibits high susceptibility to prion infection but also has slightly lower circulating UROI levels, making it more difficult to demonstrate the in vivo inhibitory effect of UROI on prion propagation than the TgUro model.

[0071] HPLC-mediated monitoring of uroporphyrin I levels in the blood and brain of wild-type, TgUro, and TgVole-TgUro animal models The majority of UROIs are found in the cytoplasm of cells, particularly red blood cells in blood, and therefore their extraction is necessary for accurate measurement of UROI levels.

[0072] To analyze UROI levels in the brain, brains were thoroughly perfused to remove blood before extraction to avoid contamination with the significantly higher levels of UROI in the blood. These samples were thoroughly homogenized in 6 M HCl solution and then sonicated to achieve complete lysis of the cells. After incubating the samples at 37 °C for 30 min, they were centrifuged to remove all cellular debris, and the supernatant containing soluble UROI was filtered through a 0.22 μm pore membrane several times as necessary to obtain a clear flow-through. After processing, they were stored at -20 °C until measurement by high-performance liquid chromatography (HPLC). UROI extraction from blood samples was performed in a similar manner; blood samples were diluted 1:10 with 6 M HCl solution, vigorously homogenized to ensure complete lysis of the cells, and then sonicated. The samples were again incubated at 37°C for 30 minutes, then centrifuged to remove all cellular debris. The supernatant containing soluble UROI was filtered through a 0.22-μm pore membrane as many times as necessary to obtain a clear flow-through. After processing, the samples were stored at -20°C until analyzed by the same HPLC method. In each case, each sample was processed in triplicate.

[0073] UROI was detected by HPLC (ALLIANCE 2695, Waters) coupled to a fluorescence detector (Waters 474 Scanning Fluorescence Detector) utilizing its intrinsic fluorescence. Samples were loaded onto a KROMASIL 100 C18 250 x 4.6 mm chromatography column (Teknokroma, Thermo Scientific) using a gradient of two solvents (solution A: 0.1 M ammonium acetate, pH 5.16; solution B: 100% acetonitrile) as the mobile phase. 5–20 μl of each sample was injected onto the column, and measurements were performed at emission wavelengths of 405 nm and excitation wavelengths of 610 nm. UROI levels were determined based on a standard curve prepared with pure UROI.

[0074] To correlate elevated UROI levels with the potential for in vivo prion propagation inhibition, wild-type, TgUro, and TgVoleTgUro mice were sacrificed and UROI levels in the blood and brain were examined. Blood was collected by submandibular puncture the day before sacrifice, and the brain was thoroughly perfused prior to extraction to avoid contamination of this tissue with blood-borne UROI. Both types of samples were processed for intracellular UROI extraction, and UROI concentrations were measured using HPLC coupled to a fluorescence detector. While UROI levels in the blood of wild-type animals ranged from 0 to 2 μM, TgUro mice showed levels of 40 to 160 μM, representing average levels two orders of magnitude higher in these models. Meanwhile, UROI levels in the blood of TgVole-TgUro animals ranged from 10 to 50 μM (see Figure 4A and B). Regarding UROI levels in the brain, the differences were smaller due to the low permeability of UROI across the blood-brain barrier (0.3–1.5 μM in wild-type animals, 1.1–2.7 μM in TgUro, or 0.8–1.9 μM in TgVole-TgUro). The mean values ​​in this model were significantly higher, but given the efficacy of UROI in inhibiting prion propagation in vitro, these differences were sufficient to detect differences in survival time following prion inoculation (see Figure 4B).

[0075] Bioassay to Evaluate the Efficacy of Prion Propagation Inhibition by Uroporphyrin I in Vivo Groups of 5–8 TgUro or TgVole-TgUro transgenic mice were used for intracerebral prion inoculation, with wild-type and TgVole mice serving as controls, respectively. Briefly, mice were inoculated intracerebrally with 1% brain homogenate from individuals previously inoculated with each prion strain and sacrificed after the onset of overt neurological signs of disease. Specifically, TgUro and wild-type mice were inoculated with three different mouse prions (RML, 22L, and 301C) to assess the potential for strain-specific effects on UROI. TgVole-TgUro hybrids and their respective TgVole controls were similarly inoculated with CWD-vole prions and prions causing human Gerstmann-Straussler-Scheinker syndrome. The initial inoculation consisted of sequential infection of voles with deer prions (a strain that causes chronic wasting disease in calves) and the CWD-vole strain. This prion is the earliest and therefore most aggressive prion strain described in an experimental model to date, and was selected because it causes neurological signs as early as 100 days postinoculation (dpi), compared with the typical incubation period of >150 dpi in rodent prion diseases. Furthermore, this hybrid model and its controls were also intraperitoneally inoculated with 10% brain homogenate from a deceased GSS patient, taking advantage of the model's sensitivity to human prions. Intraperitoneal inoculation was chosen in this case as it closely resembles the natural prion disease that occurs in humans, due to its longer incubation period, potential neuroinvasive process (transport of prions from the periphery to the brain), and slower disease progression.

[0076] All prion-inoculated animals housed in a BSL-3 animal facility were monitored daily for neurological signs, including kyphosis, gait disturbances, changes in hair coat, depressed mental status, flattened back, eye discharge, hyperactivity, poor physical condition, and incontinence. They were sacrificed when at least three of the listed signs, indicative of a neurodegenerative process, were simultaneously observed. The brains of sacrificed animals were removed, and both hemispheres were divided. One half was fixed in formalin for histopathological examination, while the other half was cryopreserved for biochemical studies to confirm prion infection and disease-modifying effects. The parameter used to determine the efficacy of UROI inhibition was the survival time of the animals, expressed as days after inoculation. Blood samples were also collected at terminal stages to determine UROI levels. Furthermore, UROI levels in the blood and brain were determined using non-prion-infected wild-type, TgUro, and hybrid mice of different ages, as detailed in the next section.

[0077] TgUro mice inoculated with the RML strain showed the greatest survival time extension of 30% compared to wild-type mice, whereas inoculation with 22L and 301C resulted in survival extensions of 10% and 12%, respectively, demonstrating that high levels of UROI can inhibit prion propagation in vivo and delay disease onset (see Figure 5).

[0078] To determine whether increasing UROI levels in the brain and blood could inhibit the transmission of prions from species other than mice in vivo, we investigated mouse PrP C Instead of vole PrP CWe selected the TgVole-TgUro hybrid model, which expresses the genotype TgVole (see Figure 6). These models are susceptible to prions from different species (including human prions) and exhibit increased UROI levels, albeit lower than those observed in TgUro. Therefore, we used: A) a Vole-CWD prion strain, derived from serial inoculations of cervid-derived chronic wasting disease prions, was inoculated intracerebrally. This prion strain, therefore, is distinct from the mouse prion strain previously used. Furthermore, inoculation of voles with this prion strain results in the most rapid onset of prion disease reported in any animal model, making it ideal for testing the in vivo inhibitory effects of UROI. B) a human prion strain causing Gerstmann-Straussler-Scheinker syndrome (GSS) was inoculated intraperitoneally. In this case, brain homogenate containing human GSS prions was inoculated intraperitoneally. This administration method better mimics the slow disease progression likely to occur in humans, as prions require a neuroinvasive process to establish effective infection. Prion-infected animals were followed daily for clinical signs of neurological disease and then sacrificed, after which blood and brain samples were extracted for analysis. Kaplan-Meier survival curves comparing the survival times of TgVole and TgVole-TgUro hybrid animals were plotted, showing survival time (corresponding to the time of clinical disease onset) as days post-inoculation (dpi). A) Animals inoculated with Vole-CWD showed an 11% increase in survival compared to TgVole animals, with UROI levels comparable to those in wild-type mice. This confirms the effectiveness of elevated UROI levels in delaying the onset of prion disease and demonstrates a strain- and species-independent inhibitory effect. B) Animals inoculated with human prions (GSS) showed an unprecedented >200% increase in survival compared to TgVole animals, with two of the inoculated animals currently free of neurological signs. Thus, the effectiveness of increasing UROI levels in delaying the onset of human prion disease was demonstrated, and this high efficacy may be explained by the increased systemic UROI levels, which may interfere with peripheral prion propagation and neuroinvasion.

[0079] Example 4. NMR characterization of the potential mechanism of action of uroporphyrin I as a prion propagation inhibitor To determine the potential mechanism by which UROI inhibits prion propagation, we investigated its ability to bind natively folded PrP. The high binding affinity suggests that UROI acts as a chemical chaperone, inhibiting PrP. C by stabilizing the conformation of PrP or by blocking the relevant interaction site. C and PrP Sc For this purpose, nuclear magnetic resonance (NMR) was the technique of choice.

[0080] Produced in E. coli, purified through IMAC, and dialyzed for accurate native folding, N 15 Labeled human recombinant PrP (M129 polymorphic variant) was mixed with different amounts of UROI solution (2 mM stock solution) for titration and binding affinity determination. A Bruker Advance III de 800 MHz RMN spectrometer with a 298 K cryoprobe was used for all titration experiments. HSQC spectra were obtained using the following pulse program: fhsqcf3gpph [fast-HSQC, phase-sensitive ge 2D 1H-15N HSQC with watergate (3-9-19)], which was then processed with TopSpin 4.0.6. Assignments were made using CCP NMR and referenced to chemical shifts in the Biological Magnetic Resonance Data Bank (BMRB) (accession number 4402). Chemical shift analysis was completed using an in-house script in MatLab to automatically determine potential UROI-PrP interaction sites. All information was used to calculate an in silico molecular model of the interaction using the CHASSYSDOCK platform (AutoDock VINA, including Autodock 4.2 y AutoDock Tools). The structure of PrP from which the model was developed has the PDB code 1QLZ.

[0081] UROI is PrP CTo evaluate whether UROI exerts its prion propagation inhibitory effect through binding with PrP and, if so, to determine the affinity and interaction site, recombinant human PrP with the M129 mutant was produced in E. coli, purified, and mixed with different concentrations of UROI. Heteronuclear Single Quantum Coherence (HSQC) spectra were obtained, which allow detection of the chemical shifts of interacting residues. Signals corresponding to each UROI:PrP ratio are shown in the following gray code: strong 1:0, high 1:0.2, medium 1:0.5, and low 1:0.75. Zooming in on specific regions of the spectrum reveals regions with more pronounced chemical shifts, indicating that those residues in the protein preferentially interact with UROI (i.e., residues 23K, 24K, 25R, and 104K of human PrP). A three-dimensional model of the PrP-UROI interaction is also shown in Figure 7.

Claims

1. A pharmaceutical composition for use in the treatment and / or prevention of prion diseases, comprising uroporphyrin I (UROI) or a pharmaceutically acceptable salt or metal complex thereof.

2. The pharmaceutical composition according to claim 1, wherein the prion disease is selected from Creutzfeldt-Jakob disease (CJD), Gerstmann-Streussler-Scheinker syndrome (GSS), fatal familial insomnia (FFI), sporadic fatal insomnia (sFI), diverse protease-sensitive prion disease (VPSPr), kuru, scrapie, transmissible mink encephalopathy (TME), chronic wasting disease (CWD), bovine spongiform encephalopathy (BSE), and feline spongiform encephalopathy (FSE).

3. A pharmaceutical composition for use in the treatment and / or prevention of prion diseases, comprising uroporphyrin I (UROI), a pharmaceutically acceptable salt or metal complex thereof, and a pharmaceutically acceptable excipient.

4. The pharmaceutical composition according to claim 3, wherein the prion disease is selected from Creutzfeldt-Jakob disease (CJD), Gerstmann-Streussler-Scheinker syndrome (GSS), fatal familial insomnia (FFI), sporadic fatal insomnia (sFI), diverse protease-sensitive prion disease (VPSPr), kuru, scrapie, transmissible mink encephalopathy (TME), chronic wasting disease (CWD), bovine spongiform encephalopathy (BSE), and feline spongiform encephalopathy (FSE).

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