Therapeutic compositions comprising iminosugars for the treatment of diseases involving heparan sulfate accumulation - Patents.com

Abstract

The compositions disclosed herein are intended for the treatment and prevention of diseases caused by the accumulation of heparan sulfate, including mucopolysaccharidosis, Alzheimer's disease and cancer. These compositions contain iminosugars and their derivatives belonging to the L-stereotype as active ingredients. The L-iminosugars of the present invention can reduce the level of heparan sulfate in cells of patients suffering from mucopolysaccharidosis and cancer, and can also reduce the accumulation of amyloid plaques in models of neurodegenerative diseases. Therefore, the use of these compounds prevents the onset of symptoms associated with these diseases, thus improving the quality and life span of patients suffering from diseases characterized by the accumulation of heparan sulfate.

Description

[Technical field]

[0001] The present invention relates to certain iminosugars belonging to the L-stereotype and pharma- ceutically acceptable salts thereof for use in the treatment and prevention of diseases caused by accumulation of heparan sulfate, in particular mucopolysaccharidoses, Alzheimer's disease and cancer.

[0002] 2. Background of the Invention Mucopolysaccharidoses (MPS) are inherited metabolic disorders caused by the loss or deficiency of lysosomal enzymes required for the catabolism of the glycosaminoglycans (GAGs), heparan sulfate (HS), dermatan sulfate (DS), keratan sulfate (KS), chondroitin sulfate (CS) and hyaluronic acid (HA) [Neufeld, EF and Muenzer, EF The mucopolysaccharidoses, in: Scriver, CR et al (Eds), The Metabolic and Molecular Bases of Inherited Diseases, McGraw-Hill, 2001, pp. 3421-3452]. The lack of these lysosomal enzymes leads to the accumulation of non-degraded GAGs in various intracellular compartments and dysfunction of multiple organs and systems, with different clinical manifestations depending on the defective enzyme and the type of GAGs accumulated. MPS is therefore classified into 11 different disorders (MPS I, II, IIIA, IIIB, IIIC, IIID, IVA, IVB, VI, VII and IX) depending on the defective lysosomal enzyme and into 7 subtypes considering the accumulated products: MPS I-heparan and dermatan sulfate; MPS II-heparan and dermatan sulfate; MPS III-heparan sulfate; MPS IV-keratan sulfate and chondroitin 6-sulfate; MPS VI-dermatan sulfate; MPS VII-heparan sulfate, dermatan sulfate and chondroitin 6-sulfate; MPS IX-hyaluronic acid. The accumulation of GAGs on cell membranes and within lysosomes, together with other pathological mechanisms, leads to various clinical outcomes with a wide phenotypic diversity [Mehta, A. and Winchester, B. Lysosomal Storage Disorders: A Practical Guide, First Edition 2012].Typical clinical manifestations of the disease include neurological disorders, cardiovascular dysfunction, skeletal, joint, airway, hearing and vision disorders, and death in the second or third decade of life [Oussoren E. et al. (2011). Biochim. Biophys. Acta 1812, 1542;Schiattarella GG et al. (2015). PLoS One 10, e0131662;Costa, R. et al. (2017). Hum. Mol. Genet. 26, 1643;Bellettato, CM and Scarpa, MJ (2010). Inherit. Metab. Dis. 33, 347].

[0003] Timely diagnosis is essential for MPS patients, and the few currently available therapeutic strategies are variable and limited in efficacy [Hollak CEM, Wijburg FA. (2014) J Inherit metab dis. 37, 587]. Current treatment options for MPS include enzyme replacement therapy (ERT), substrate synthesis inhibition therapy (SRT), pharmacological chaperone therapy (PCT), gene therapy (GT) and hematopoietic stem cell transplantation (HSCT) [Fecarotta, S. et al. (2018). Ital. J. Pediatr. 44, 124;Poswar, F. et al. (2017). Expert Opin. Investig. Drugs 26, 1331]. Many of these strategies are described in US2013302308, WO2012177778, US8623910, US2012190642, US2011008810, EP2081023, KR100762945, KR20040084881, US8105788, US2009092996, WO02055064, RU2196988, RU2083205, JP2008102114, JP4965999, JP2003265196. These therapeutic treatments have some limitations. In particular, the most commonly used ERT is unable to correct all defects associated with these pathologies, especially those related to the central nervous system, due to the inability of the recombinant enzyme to overcome the blood-brain barrier. Treatment with stem cells is also ineffective, but is extremely risky, above all due to the uncertain fate of the stem cells after administration to the patient, including the possibility that these cells may acquire a tumor phenotype. GT is not yet used in the clinic today due to the high immunogenicity of the vector and the risk associated with the integration of the viral genome into the genome of the treated patient, despite its inactivity. Due to the limitations of such strategies, scientific research continues to investigate the pathophysiology of MPS in order to identify new therapeutic strategies.

[0004] Based on these considerations, it can be concluded that MPS currently does not have a well-suited treatment to reduce or eliminate the severe symptoms of patients affected by these diseases.

[0005] The availability of GAG biosynthesis inhibitors has also been tested in MPS disease [Fecarotta, S. et al. (2018). Ital. J. Pediatr. 44, 124;Poswar, F. et al. (2017). Expert Opin. Investig. Drugs 26, 1331]. This approach, known as "substrate synthesis suppression therapy" (SRT), employs small molecules that can cross the blood-brain barrier and therefore have the potential to treat the neurological phenotype of the disease. The first molecule identified as a promising drug for SRT in MPS patients with neurological symptoms was genistein, a soy-derived isoflavone with structural similarity to 17β-estradiol that inhibits GAG synthesis by affecting epidermal growth factor (EGF)-dependent molecular signaling pathways [Jakobkiewicz-Banecka, J. et al. (2009). J. Biomed. Sci.16, 26]. However, genistein was shown to be ineffective in clinical trials in patients with MPS III. Identification of novel molecules interfering with GAG synthesis may provide a useful tool to improve the neurological phenotype of MPS patients. On the other hand, manipulation of GAG synthesis to modulate the biological function of HSPGs (HS proteoglycans) has been performed in several diseases using synthetic xylosides that lower GAGs bound to proteoglycans (especially HS) [Chua, JS and Kuberan, B. (2017). Acc. Chem. Res. 50, 2693].

[0006] Due to the ability of HSPGs to regulate multiple cellular functions, including cell proliferation, differentiation, adhesion, migration, survival and signal transduction, these complex molecules have emerged as promising therapeutic targets for the treatment of several diseases, including cancer, inflammation, infection, wound closure, pulmonary diseases, Alzheimer's disease and other diseases [Varki, A. et al., Essentials of Glycobiology, 2nd ed., Cold Spring Harbor Laboratory Press, New York, 2009].

[0007] For the last decades, HSPGs have been fascinating research subjects due to their complex structural features, their exquisitely regulated biosynthetic machinery, and the diverse functions they play in the organism from development to adulthood. These studies have revealed the important role of HSPGs in cancer development and progression, and they are currently being investigated as promising biomarkers and therapeutic targets for cancer. The pleiotropic nature of HSPGs structure / activity gives rise to their ability to act as inhibitors or promoters of tumor growth and invasion, depending on the tumor type. Dysregulation of the structural and functional properties of HSPGs resulting in malignancies can be attributed both to altered expression levels and to changes in their structure and function as a result of altered activity of their biosynthetic enzymes or modifiers. Indeed, in the tumor microenvironment, HSPGs undergo structural changes through translocation of proteoglycan ectodomains from the cell surface or fragmentation and / or desulfation of HS chains, which affect the function of HSPGs and have significant consequences on the molecular interactions between tumor cells and their microenvironment and on the behavior of the tumor cells themselves.

[0008] Among their functions, HSPGs help many viruses enter host cells at various stages of their life cycle. Viruses utilize HSPGs for attachment to host cells, internalization, intracellular trafficking, release and spread. Recently, the involvement of HSPGs in the pathogenesis of SARS-CoV-2 infection has been established [De Pasquale, V. et al. (2021). Int J Mol Sci. 22, 6574].

[0009] The common thread among all the different causes of neuropathology in the Alzheimer's disease brain is the early accumulation of HSPGs and HS glycosaminoglycans. All these events further suggest that HSPGs / HSGAGs play an important role in the pathogenesis of Alzheimer's disease neuropathology.

[0010] Recently, iminosugars have shown remarkable pharmacological potential in the management of lysosomal storage diseases (LSDs) as a result of their ability to interact with carbohydrate processing enzymes [Nash, RJ et al. (2011). Future Med. Chem. 3, 1513;Platt, FM et al. (2018). Nat. Rev. Dis. Prim. 4, 27;Compain, P.;Martin, O. (2007). “Iminosugars: From Synthesis to Therapeutic Applications” (John Wiley & Sons, Ltd);Butters, TD et al. (2003), Curr. Top. Med. Chem. 3, 561]. These glycomimetics have been applied in the treatment of LSD due to their inhibition of substrate accumulation in lysosomes (SRT) [Platt, FM and Jeyakumar, M. (2008). Acta Paediatr. 97, 88;Coutinho, MF et al. (2016), Int. J. Mol. Sci. 17, 1065] and their ability to reversibly bind to lysosomal glycosidases at subinhibitory concentrations and improve the function of mutant enzymes (PCT) [Sanchez-Fernandez, EM, et al. (2016) Chem. Commun. 52, 5497;Cox, TM et al., (2008) “Medicinal use of Iminosugars” in “Iminosugars: From Synthesis to Therapeutic Applications” (John Wiley & Sons, Ltd), pp. 295-326].

[0011] Two iminosugars have entered the market for LSD therapy: miglustat (also known as D-NBDNJ), approved for the treatment of type I Gaucher disease [Cox, TM et al. (2000). The Lancet 355, 1481] and type C Niemann-Pick disease (as SRT therapy) [Pineda, M. et al. (2018). Orphanet J. Rare Dis. 13, 140], and migalastat (also known as DGJ), which is currently the only pharmacological chaperone approved and used in Fabry disease [Benjamin, ER et al. (2009). J. Inherit. Metab. Dis. 32, 424; Markham, A. (2016) Drugs 76, 1147].

[0012] In addition to these iminosugars, a wide variety of other derivatives are being evaluated for use as drug candidates in several LSDs, including Pompe disease and MPS [Parenti, G. et al. (2021). EMBO Mol. Med. 13, e12836;Diaz, JCL et al. (2020), Int. J. Mol. Sci. 21, 1]. In this context, promising activity has been observed for some iminosugars as pharmacological chaperones for the treatment of MPS II, III and IV [Zhu, S. et al. (2021). Chem. - A Eur. J. 27, 11291;Fantur, K. et al. (2010). Mol. Genet. Metab. 100, 262;Thonhofer, M. et al. (2016). Carbohydr. Res. 429, 71; Takai, T. et al. (2013). Mol. Ther. 21, 526].

[0013] Furthermore, an interesting application of iminosugars in MPS concerns the hypothesis that secondary accumulation of gangliosides could be a therapeutic target for patients with neurological disorders. Based on this, the iminosugar D-NBDNJ (miglustat) was evaluated as a substrate inhibitor for MPS type III due to its ability to interfere with glycosphingolipid metabolism [Fecarotta, S. et al. (2018). Ital. J. Pediatr. 44, 124]. Despite promising results obtained in preclinical studies [Kaidonis, X. et al. (2016). Mol Gen. Metab. 118, 110], no beneficial effects were observed in MPS III patients treated with D-NBDNJ (miglustat) [Guffon, N. et al. (2011). J. Pediatrician. 159, 838]. These data clearly suggest that iminosugars are attractive candidates for MPS treatment.

[0014] Within the framework of our studies aimed at investigating the role of chirality on the pharmacological activity of iminosugars and other bioactive compounds [Esposito, A. et al. (2020). Chem. - A Eur. J. 26, 2597; Esposito, A. et al. (2020) Mar. Drugs 18, 572; Esposito, A. et al., (2019) RSC Adv. 9, 21519], L-iminosugars, which have very promising potential in the treatment of rare diseases, have recently come into the spotlight. In particular, L-NBDNJ (D-NBDNJ, the enantiomer of miglustat), unlike its D-enantiomer, has been shown to have interesting potential as a candidate for combination therapy of Pompe disease without acting as an inhibitor of most glycosidases [D'Alonzo, D. et al. (2017). J. Med Chem. 60, 9462].

[0015] Even more interesting results were obtained when L-iminosugars were examined for application in cystic fibrosis (CF) [Esposito, A. et al. (2020). Int. J. Mol. Sci. 21, 3353]. Indeed, L-NBDNJ and its congeners exhibited anti-inflammatory and antibacterial properties in vitro and in vivo, highlighting the promise of these compounds as therapeutic candidates for the treatment of CF lung disease [De Fenza, M. et al. (2019). Eur. J. Med. Chem. 175, 63;De Gregorio, E. et al. (2020). Antibiotics 9, 362].

[0016] Description of the Invention It has now been found that L-iminosugars having the following structural formula, identified by the abbreviations L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ, and their pharma- ceutically acceptable salts, exhibit a remarkable ability to inhibit heparan sulfate accumulation and are therefore useful tools for the treatment of mucopolysaccharidoses I, II, III or VII and their subtypes, in particular Sanfilippo syndrome and its subtypes A, B, C and D, as well as for the treatment of other pathologies characterized by heparan sulfate accumulation, such as Alzheimer's disease and tumors. [ka]

[0017] The synthesis of L-NBDNJ (N-butyl-L-deoxynojirimycin, the unnatural enantiomer of miglustat) was described by D'Alonzo D. et al. (2017). J. Med. Chem. 60, 9462; L-NBDNJ was shown to be a candidate drug for combination therapy of Pompe disease.

[0018] The synthesis of L-iminosugars L-DNJ (the unnatural enantiomer of deoxynojirimycin or duvoglustat) and L-AMPDNM (N-adamantanomethoxypentyl L-DNJ) has been described by D'Alonzo D. et al. (2017). J. Med. Chem. 60, 9462 and De Fenza M. et al. (2019). Eur. J. Med. Chem. 175, 63.

[0019] The compound L-MONDNJ (N-methoxynonyl L-DNJ) is novel and constitutes a further object of the present invention.

[0020] For anticipated therapeutic use, the iminosugars L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ or pharma- ceutically acceptable salts thereof are formulated into pharmaceutical compositions suitable for oral or parenteral administration, such as capsules, tablets, liquids and the like, containing suitable excipients.

[0021] The dosage is determined by the specialist based on the patient's condition, weight, sex and age, as well as by the pharmacokinetic and toxicological properties of the compound. In principle, the dosage can be equivalent to the dosage of already used drugs (miglustat and migalastat), for example, 10-1000 mg per day, in one or multiple doses.

[0022] The pharmacological activity observed for compounds L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ or their pharma- ceutically acceptable salts is highly interesting in comparison with other structurally similar iminosugars (compounds L-NNDNJ, L-HPDNJ and L-NPDNJ, whose formulas are reported below), which did not show any activity when evaluated under the same conditions.

[0023] The structural formulae of the compounds according to the invention and comparative compounds are reported below. [ka]

[0024] The invention is explained in detail in the experimental section below.

[0025] Example 1: Synthesis of L-MONDNJ (N-methoxynonyl L-DNJ) and the corresponding hydrochloride derivative Engineering Synthesis of 1,9-diiodiononane. Iodine (2.6 g, 10.2 mmol) was added to a stirred suspension of polymer-triphenylphosphine (PS-TPP; 100-200 mesh, ~3 mmol / g triphenylphosphine) (3.4 g, 10.5 mmol) in anhydrous dichloromethane (25 mL) under an argon atmosphere. After 10 min, 1,9-nonanediol (0.41 g, 2.56 mmol) was added to the suspension and the reaction was stirred at room temperature for 1 h. The suspension was then filtered by washing with dichloromethane to remove the triphenylphosphine oxide anchored to the polymer. The filtrate was washed with saturated Na 2 S 2 O 3 The mixture was washed with saturated NaCl solution and extracted with dichloromethane.

[0026] The organic phase was dried (Na 2 SO 4 ), which was evaporated under reduced pressure to give the desired 1,9-diiodiononane (oil, 0.9 g, 95% yield). 1 H NMR (400 MHz, CDCl 3 ): δ 1.21-1.47 (m, 10H), 1.76-1.88 (m, 4H), 3.19 (t, J = 7.0 Hz, 4H). In this reaction, exchange of the polymeric triphenylphosphine with triphenylphosphine gives similar results in terms of reaction time and yield; however, this procedure requires a purification step by precipitation of triphenylphosphine oxide or by chromatography.

[0027] Engineering b: Synthesis of 1-iodo-9-methoxynonane. NaH (60% dispersion in mineral oil, 0.10 g, 2.55 mmol) was added to a solution of methanol (0.12 mL, 2.95 mmol) in dry THF (3.5 mL) under magnetic stirring at 0° C. and argon atmosphere. The reaction mixture was stirred at the same temperature for 1 h; then a solution of 1,9-diiodiononane (0.75 g, 1.95 mmol) in THF (3.5 mL) was added. The solution was allowed to warm to room temperature and stirred at the same temperature for 48 h. Dichloromethane was then added and the solution was first washed with aqueous NH 4 The organic phase was washed with saturated NaCl solution. 2 SO 4 The mixture was dried at 40° C. and the solvent was evaporated under reduced pressure. Silica gel chromatography of the crude residue (hexane:EtOAc=95:5) gave pure 1-iodo-9-methoxynonane (oil, 0.42 g, 75% yield). 1 H NMR (400 MHz, CDCl 3 ): δ 1.21-1.47 (m, 8H), 1.51-1.63 (m, 4H), 1.76-1.88 (m, 2H), 3.19 (t, J = 7.0 Hz, 2H), 3.30 (s, 3H), 3.36 (t, J = 6.6 Hz, 2H).

[0028] Project c: Synthesis of L-MONDNJ (N-methoxynonyl L-DNJ). A solution of L-DNJ (0.20 g, 1.22 mmol) in dehydrated DMF (4 mL) was stirred at room temperature under argon atmosphere with K 2 CO 3 (0.5 g, 3.6 mmol) was added. A solution of 1-iodo-9-methoxynonane (0.42 g, 1.46 mmol) in DMF (4.0 mL) was added dropwise and the reaction mixture was heated to 80° C. and stirred for 16 h. The solvent was removed under reduced pressure and subjected to silica gel chromatography (acetone:MeOH=8:2) to give pure L-MONDNJ.

[0029] Project D:Preparation of L-MONDNJ·HCl (N-methoxynonyl L-DNJ·HCl). L-MONDNJ·HCl hydrochloride was obtained by addition of 1 M HCl (1.22 mmol) followed by evaporation under reduced pressure (0.30 g, 75% yield). 1 H NMR (500 MHz, CD 3 OD): δ 1.28-1.49 (m, 10H), 1.52-1.63 (m, 2H), 1.67-1.88 (m, 2H), 2.95-3.11 (m, 2H), 3.13-3.27 (m, 1H), 3.31 (s, 3H), 3.40 (t, J = 6.5 Hz, 4H), 3.47 (dd, J = 4.9, 11.8 Hz, 1H), 3.61 (t, J = 11.8 Hz, 1H), 3.65-3.77 (m, 1H), 3.91 (d, J = 11.6 Hz, 1H), 4.14 (d, J = 11.6 Hz, 1H).

[0030] Example 2: Treatment with L-deoxyminosugars (L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ) as hydrochloride salts reduces lysosomal defects in a cellular model of Sanfilippo disease B (MPS IIIB). To study the effects of compounds on lysosomal dysfunction in a neuronal model of Sanfilippo B disease (MPS IIIB), we recently generated stable clones of the human neuroblastoma cell line SK-NBE in which the NAGLU gene, responsible for MPS IIIB, was silenced [De Pasquale V, et al. (2021). Biochim Biophys Acta Mol Cell Res. 1868, 119113.]. Mimicking the characteristics of Sanfilippo B disease, silencing of NAGLU causes heparan sulfate accumulation and lysosomal accumulation in the cytoplasm of stable SK-NBE clones compared to control (WT) clones.

[0031] Therefore, a NAGLU-silenced clone (cl5) and a control clone (WT) were chosen to test the effect of L-deoxyaminosugars in hydrochloride salt form (L-DNJ, L-NBDNJ, L-NNDNJ, L-HPDNJ, L-NPDNJ, L-AMPDNM and L-MONDNJ) on the lysosomal phenotype of our Sanfilippo B (MPS IIIB) cell model.

[0032] Clone 5 was cultured under normal growth conditions in the presence of 20 μM of each L-deoxyaminosugar, and after 48 h, lysosomal accumulation was assessed by immunofluorescence by using a specific antibody against Lamp1 (lysosomal marker). Untreated clone 5 shows enlarged positive Lamp1 lysosomal structures in the cytoplasm compared to the WT control clone (Table 1). Treatment with L-DNJ, L-NBDNJ, L-AMPDNM, and L-MONDNJ causes a dramatic reduction in lysosomal enlargement and accumulation in the clone 5 (cl5) model system of Sanfilippo B (MPS IIIB) (Table 1). Furthermore, upon treatment with active L-deoxyaminosugars, lysosomes are no longer concentrated in the perinuclear region of the cell, as seen in some lysosomal diseases, but are physiologically distributed throughout the entire cytoplasm. Interestingly, the L-deoxyaminosugars L-NNDNJ, L-HPDNJ and L-NPDNJ did not show any activity against the lysosomal phenotype of the Sanfilippo B model clone 5 (cl5) (Table 1).

[0033] [Table 1]

[0034] The physiological distribution of lysosomes in the cytoplasm in L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ treated cells is more evident when comparing these results with those obtained by immunofluorescence of Lamp1 protein in WT control normal clones.

[0035] Treatment with any of the seven L-iminosugars did not cause any changes in size and lysosomal distribution in non-diseased WT model cells.

[0036] Taken together, these results show for the first time that treatment with selected L-iminosugars (L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ) can reduce lysosomal defects in a cellular model of Sanfilippo disease B (MPS IIIB).

[0037] Example 3: Treatment with L-deoxyaminosugars (L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ) as hydrochloride salts reduces HS accumulation in a cellular model of Sanfilippo disease B (MPS IIIB). Clone 5 was grown under normal growth conditions in the presence of 20 μM of each L-iminosugar, and after 48 h, heparan sulfate (HS) accumulation was evaluated by immunofluorescence staining for HS. Untreated clone 5 showed accumulation of HS on the cell membrane compared to the WT control clone (Table 2). However, a dramatic reduction in HS staining was observed in the Sanfilippo B model system (MPS IIIB) cl5 in the presence of compounds L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ (Table 2). Also in this case, the L-iminosugars L-NNDNJ, L-HPDNJ and L-NPDNJ did not show any activity in reducing HS accumulation in the Sanfilippo B model tested (Table 2). These results are consistent with those obtained with Lamp1 staining of lysosomes.

[0038] [Table 2]

[0039] Furthermore, treatment with any of the seven L-iminosugars did not cause any changes in the amount of HS in non-diseased WT model cells.

[0040] Taken together, these results show for the first time that treatment with selected L-iminosugars L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ is able to reduce HS accumulation in a cellular model of Sanfilippo B disease (MPS IIIB) generated in our laboratory.

[0041] Example 4: Treatment with L-deoxyaminosugars (L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ) as hydrochloride salts reduces lysosomal defects and HS accumulation in fibroblasts from patients with Sanfilippo A and B (MPS IIIA and IIIB). To verify whether the selected iminosugars could exert the same effect on fibroblasts from patients affected by Sanfilippo disease, human adult dermal fibroblasts HDFa (purchased from Sigma-Aldrich) were used as a control and fibroblasts from patients affected by Sanfilippo A and B (MPS IIIA and IIIB) were used as disease models.

[0042] The human cell lines, i.e. fibroblasts, from patients with MPS (Sanfilippo disease) used in the examples were obtained from the G. Gaslini Institute "Cell Line and DNA Biobank from Patients Affected by Genetic Diseases" -Telethon Genetic Biobank Network -Telethon research service in Genoa. These cells are classified by identification code and by type of disease so that the patient cannot be identified. The cells were obtained at the Gaslini Institute, with the informed consent of the patient for their collection and for the extension of their possible use, for diagnostic and / or research purposes, in accordance with the current legislation required by the guidelines followed by Telethon biobanks. The Network operates in accordance with all applicable Italian privacy and data protection laws (including the Italian Data Protection Authority, Personal Data Protection Code, Legislative Decree no. 196, 30th June 2003, published in Official Gazette No. 174 of the Italian Republic, 29th July 2003; Italian Data Protection Authority, General Authorization for the processing of genetic data, 24th June 2011, published in Official Gazette No. 159 of the Italian Republic, 11th July 2011).

[0043] Cells were cultured in the presence of a 20 μM dose of selected L-iminosugars and processed after 48 h for immunofluorescence of HS and Lamp1. Treatment with L-iminosugars had no effect on control HDFa (Table 3). On the other hand, the same L-iminosugars L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ caused a strong reduction in HS and lysosomal accumulation in fibroblasts from patients affected by Sanfilippo A and B (MPS IIIA and MPS IIIB) (Table 3).

[0044] [Table 3]

[0045] Also in this case, the L-iminosugars L-NNDNJ, L-HPDNJ and L-NPDNJ did not show any activity in reducing the accumulation of HS and Lamp1 in fibroblasts from Sanfilippo A and B patients (Table 3).

[0046] Taken together, these results show for the first time that treatment with selected L-iminosugars L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ is able to prevent HS and lysosomal accumulation in fibroblasts from patients affected by Sanfilippo A and B diseases.

[0047] Example 5: Treatment with D-deoxyaminosugars as hydrochlorides (D-DNJ or DNJ, D-NBDNJ or NBDNJ or D-AMPDNM or AMPDNM, or D-AMPDNM o AMPDNM, e D-MONDNJ o MONDNJ) has no effect on lysosomal defects and HS accumulation in fibroblasts from patients with Sanfilippo A and B (MPS IIIA and IIIB). To demonstrate that iminosugars (D-DNJ, also known as duvoglustat, and D-NBDNJ, also known as miglustat) are currently available for the treatment of other lysosomal diseases, and that the stereoisomers D-AMPDNM and D-MONDNJ do not show the same efficacy as the subject compounds of the present invention, human adult dermal fibroblasts HDFa (purchased from SIGMA) were used as a control, and fibroblasts from patients with Sanfilippo A and B (MPS IIIA and IIIB) were used as disease models. Cells were grown in the presence of iminosugars D-DNJ, D-NBDNJ, D-AMPDNM, and D-MONDNJ at a dose of 20 μM and processed for immunofluorescence of HS and Lamp1 after 48 hours. Treatment with D-iminosugars had no effect on HS and lysosomal accumulation in both HDFa and fibroblasts from patients with Sanfilippo A and B (MPS IIIA and MPS IIIB) (Table 4).

[0048] [Table 4]

[0049] Taken together, these results indicate that selected L-iminosugars L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ are effective in preventing HS and lysosomal accumulation in cellular models of mucopolysaccharidoses, e.g., fibroblasts from patients affected by Sanfilippo A and B.

[0050] Example 6: Treatment with L-deoxyaminosugars in the form of hydrochlorides (L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ) induces a reduction in the amount of HS in HeLa tumor epithelial cell lines and a reduction in their growth. To investigate whether treatment with selected L-deoxyaminosugars (L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ) could interfere with HS synthesis, we chose a cell line in which NAGLU was not mutated and whose cell membrane was highly modified with heparan sulfate. For this purpose, HeLa tumor epithelial cells (purchased from ATCC) were cultured for 48 h in the presence of selected L-iminosugars and the amount of HS was evaluated by immunostaining. Treatment with the same L-iminosugars L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ caused a strong reduction in the accumulation of HS on the HeLa cell membrane (Table 5).

[0051] [Table 5]

[0052] Also in this case, the L-iminosugars L-NNDNJ, L-HPDNJ and L-NPDNJ did not show any activity in reducing HS accumulation in HeLa tumor epithelial cells (Table 5).

[0053] Furthermore, treatment with the L-deoxyaminosugars L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ caused a decrease in the number of HeLa cells after 48 hours, as shown by the cell proliferation assay reported in Table 6.

[0054] [Table 6]

[0055] The results demonstrate that treatment with L-DNJ L-iminosugars, L-NBDNJ, L-AMPDNM and L-MONDNJ caused a reduction in HS in HeLa cancer cells and subsequently suppressed tumor cell growth as HS is essential to maintain the proliferation of cancer epithelial cells.

[0056] The data reported in Table 6 also demonstrate that iminosugars belonging to the D-type, D-DNJ or duvoglustat, D-NBDNJ or miglustat, D-AMPDNM and D-MONDNJ, do not show the same efficacy in reducing cancer cell proliferation compared to the subject compounds of the present invention.

[0057] These data demonstrate that L-iminosugars act by interfering with HS synthesis, a completely novel mechanism compared to that of other commercially available iminosugars. Furthermore, these results indicate that the present invention is applicable not only to mucopolysaccharidoses, where there is accumulation of HS, but also to cancer diseases, where HS is essential to support the growth of tumor cells and their metastatic machinery.

[0058] Example 7: Treatment with L-deoxyaminosugars in the form of hydrochlorides (L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ) induces a reduction in the amount of amyloid beta fibrils in a cellular model of Sanfilippo disease B (MPS IIIB). To demonstrate the therapeutic application of selected iminosugars L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ against neurodegenerative diseases, we used the Sanfilippo B (cl5) neuronal model (Example 2) recently generated in our laboratory.

[0059] A clone (cl5) in which the causative gene NAGLU of MPS IIIB was stably silenced accumulates beta-amyloid fibrils in the cytoplasm, mimicking the characteristics of neurodegenerative diseases. In fact, Sanfilippo syndrome is also defined as "pediatric Alzheimer's".

[0060] Disease clones (cl5) were grown in the presence of L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ at a dose of 20 μM and the corresponding enantiomers D-DNJ, D-NBDNJ, D-AMPDNM and D-MONDNJ, and processed after 48 hours by immunofluorescence staining for beta-amyloid peptide 1-42. The data reported in Table 7 show that treatment with L-deoxyaminosugars results in an overall reduction in the accumulation of amyloid fibrils compared to untreated cells (sham). In contrast, treatment with the corresponding D-enantiomers shows no effect on the reduction of the accumulation of amyloid fibrils compared to untreated clones (sham) (Table 7).

[0061] [Table 7]

[0062] These results show for the first time that the claimed L-iminosugars L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ are effective in reducing amyloid plaque accumulation and treating neurodegenerative processes such as Alzheimer's. The data reported in Table 7 also demonstrate that the D-iminosugars D-DNJ, D-NBDNJ, D-AMPDNM and D-MONDNJ do not show the same efficacy in reducing neurodegenerative markers as the subject compounds of the present invention.

Claims

1. A pharmaceutical composition for treating or preventing diseases involving heparan sulfate accumulation, selected from mucopolysaccharidosis, Alzheimer's disease, and tumors, comprising the formula: 【Transformation 3】 A pharmaceutical composition comprising an imino sugar represented by or a pharmaceutically acceptable salt thereof.

2. The pharmaceutical composition according to claim 1, wherein the disease accompanied by the accumulation of heparan sulfate is mucopolysaccharidosis type I, II, III, or VII.

3. The pharmaceutical composition according to claim 2, wherein the disease involving the accumulation of heparan sulfate is Sanfilippo syndrome or its subtype (A, B, C, or D).

4. The pharmaceutical composition according to claim 1, wherein the disease accompanied by the accumulation of heparan sulfate is Alzheimer's disease.

5. The pharmaceutical composition according to claim 1, wherein the disease accompanied by the accumulation of heparan sulfate is cancer.

6. formula: 【Chemistry 4】 The compound indicated by or a pharmaceutically acceptable salt thereof.

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