Therapeutic composition for treating heparan sulfate accumulation diseases

Abstract

The compositions disclosed herein are contemplated for use in the treatment and prevention of diseases caused by heparan sulfate accumulation, including mucopolysaccharide storage disorders, Alzheimer's disease, and cancer. These compositions include iminosugars belonging to the L-stereoseries and derivatives thereof as active ingredients. The L-iminosugar of the present invention can reduce the level of heparan sulfate in patient cells affected by mucopolysaccharide storage diseases and cancers, and reduce the accumulation of amyloid plaques in a neurodegenerative disease model. Thus, the use of these compounds prevents the occurrence of symptoms associated with these diseases, thereby improving the quality of life and longevity of patients suffering from diseases characterized by heparan sulfate accumulation.

Description

[0001] The present invention relates to specific iminosugars belonging to the L-steric series and their pharmaceutically acceptable salts for use in treating and preventing diseases caused by heparan sulfate accumulation, in particular mucopolysaccharidosis, Alzheimer's disease and cancer. Background of the Invention

[0003] Mucopolysaccharidoses (MPS) are inherited metabolic diseases caused by the absence or deficiency of lysosomal enzymes necessary for the degradation of glycosaminoglycans (GAG), 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 undegraded GAGs in various cellular compartments and dysfunction of various organs and systems, with different clinical manifestations depending on the type of defective enzymes and accumulated GAGs. Thus, MPS is divided into 11 different diseases (MPS I, II, IIIA, IIIB, IIIC, IID, IVA, IVB, VI, VII, and IX) based on the defective lysosomal enzymes, and into 7 subtypes if the accumulated products are considered: MPS I - heparan sulfate and dermatan sulfate; MPS II - heparan sulfate and dermatan sulfate; MPS III - heparan sulfate; MPS IV - keratan sulfate and 6-chondroitin sulfate; MPS VI - dermatan sulfate; MPS VII - heparan sulfate, dermatan sulfate, and 6-chondroitin sulfate; MPS IX - hyaluronan. GAGs accumulate on cell membranes and within lysosomes, leading to a variety of clinical consequences along with other pathogenic mechanisms, and with a wide range of phenotypic variability [Mehta, A. and Winchester, B. Lysosomal Storage Disorders: A Practical Guide, First Edition 2012].Typical clinical symptoms of the disease include neurological disorders, cardiovascular dysfunction, defects in bones, joints, respiratory tract, hearing and vision, 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].

[0004] Timely diagnosis is crucial for MPS patients. Currently, only a few treatment strategies with unstable and limited efficacy are available [Hollak CEM, Wijburg FA. (2014) J Inherit metab dis. 37, 587]. Current treatment options for MPS include enzyme replacement therapy (ERT), substrate reduction 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]. Most of these strategies are described in US2013302308, WO2012177778, US8623910, US2012190642, US2011008810, EP2081023, KR100762945, KR20040084881, US8105788, US2009092996, WO02055064, RU2196988, RU2083205, JP2008102114, JP4965999, JP2003265196. These treatments have several limitations. In particular, the most commonly used ERT cannot correct all defects associated with these pathologies, especially those related to the central nervous system, because the recombinant enzyme cannot overcome the blood-brain barrier. Treatment with stem cells is also ineffective, but most importantly, extremely dangerous due to the unknown fate of the stem cells after administration to the patient, including the possibility that these cells acquire a tumor phenotype. GT is still not currently used clinically due to the high immunogenicity of the vector and the risk of integration of the viral genome (albeit inactive) into the genome of the patient being treated. Due to the limitations of such strategies, scientific research continues to study MPS pathophysiology in order to identify new treatment strategies.

[0005] Based on these considerations, it can be inferred that MPS currently has no fully adequate treatment to alleviate or eliminate the severe symptoms in patients affected by these diseases.

[0006] The potential use of GAG biosynthesis inhibitors has also been tested in MPS diseases [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-reduction therapy" (SRT), employs small molecules that are able to cross the blood-brain barrier and therefore have the potential to treat the neurological phenotype of the disease. The first molecule identified as a potential drug for SRT in MPS patients with neurological manifestations was genistein, a soy-derived isoflavone with a structure similar to 17β-estradiol that inhibits GAG synthesis by affecting epidermal growth factor (EGF)-dependent molecular signaling pathways [Jakóbkiewicz-Banecka, J. et al., (2009). J. Biomed. Sci. 16, 26]. However, genistein has been shown to be ineffective in clinical trials of MPS III patients. The identification of novel molecules that interfere with GAG synthesis may provide useful tools for improving the neurological phenotype of MPS patients. On the other hand, synthetic xylosides have been used to manipulate GAG ​​synthesis in a variety of diseases, which can reduce the binding of GAGs to proteoglycans (especially HS), thereby regulating the biological functions of HSPG (HS proteoglycan) [Chua, JS and Kuberan, B. (2017). Acc. Chem. Res. 50, 2693].

[0007] Because HSPGs are able to regulate a variety of cellular functions, including cell proliferation, differentiation, adhesion, migration, survival, and signaling, these complex molecules have emerged as potential therapeutic targets for the treatment of a variety of diseases, including cancer, inflammation, infection, wound healing, lung disease, Alzheimer's disease, and others [Varki, A. et al., Essentials of Glycobiology, 2nd ed., Cold Spring Harbor Laboratory Press, New York, 2009].

[0008] Over the past decades, HSPGs have become an interesting subject of investigation due to their complex structural features, finely regulated biosynthetic mechanisms, and the wide range of functions they perform in organisms from development to adulthood. From these studies, key roles for HSPGs in cancer initiation and progression have emerged and they are currently being explored as potential biomarkers and therapeutic targets for cancer. The multifaceted nature of HSPG structure / activity enables them to act as inhibitors or promoters of tumor growth and invasion, depending on the tumor type. Dysregulation of HSPG structural and functional features leading to malignancy may result from altered expression levels as well as changes in their structure and function due to altered activities of their biosynthetic enzymes or modifiers. Indeed, in the tumor microenvironment, HSPGs undergo structural changes through displacement of the proteoglycan ectodomain from the cell surface or cleavage and / or desulfurization of HS chains, which in turn affect the function of HSPGs, with significant consequences for the molecular interactions between tumor cells and their microenvironment, as well as for the behavior of the tumor cells themselves.

[0009] Among their functions, HSPGs help many viruses invade host cells at various stages of their life cycle. Viruses use HSPGs for host cell attachment, internalization, intracellular transport, egress, and dissemination. Recently, it has been determined that HSPGs are involved in the pathogenesis of SARS-CoV-2 infection [De Pasquale, V. et al., (2021). Int J Mol Sci. 22, 6574].

[0010] The common link between all the different causes of neuropathology in the Alzheimer's disease brain is the early accumulation of HSPGs and HS glycosaminoglycans. All of these events further indicate that HSPGs / HSGAGs are key players in the pathogenesis of Alzheimer's disease neuropathology.

[0011] In recent years, iminosugars have shown considerable pharmacological potential in the treatment of lysosomal storage diseases (LSDs) due to 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 used in the treatment of LSDs, both by inhibiting the accumulation of substrates 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 by reversibly binding to lysosomal glycosidases at sub-inhibitory concentrations, improving the function of mutant enzymes (PCT) [Sánchez-Fernández, 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].

[0012] Two iminosugars have entered the LSD therapeutic market, Miglustat, also known as D-NBDNJ, which has been approved for the treatment of Gaucher disease type I [Cox, TM et al., (2000). The Lancet 355, 1481] and Niemann-Pick disease type C (as SRT therapy) [Pineda, M. et al. (2018). Orphanet J. Rare Dis. 13, 140], and Migalastat, also known as DGJ, is currently the only pharmacological chaperone approved and used to treat Fabry disease [Benjamin, ER et al., (2009). J. Inherit. Metab. Dis. 32, 424; Markham, A. (2016) Drugs 76, 1147].

[0013] In addition to these iminosugars, a variety of other derivatives have been evaluated for use as candidate drugs in several LSDs, including Pompe disease and MPS [Parenti, G. et al., (2021). EMBO Mol. Med. 13, e12836; Díaz, JCL et al., (2020), Int. J. Mol. Sci. 21, 1]. In this context, some iminosugars have promising activity as pharmacological chaperones for the treatment of MPS II, III, and IV [Zhu, S. et al., (2021). Chem.-AEur. 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].

[0014] In addition, an interesting application of iminosugars in MPS involves the hypothesis that secondary storage of gangliosides may represent a therapeutic target for patients with neurological involvement. On this basis, the iminosugar D-NBDNJ (Miglustat) was evaluated as a substrate-reducing agent for MPS type III because it is able to interfere with glycosphingolipid metabolism [Fecarotta, S. et al., (2018). Ital. J. Pediatr. 44, 124]. Despite promising results 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 indicate that iminosugars are attractive drug candidates for the treatment of MPS.

[0015] In the framework of our research aimed at exploring the role of chirality on the pharmacological activity of iminosugars and other bioactive compounds [Esposito, A. et al., (2020). Chem.–AEur. J. 26, 2597; Esposito, A. et al., (2020) Mar. Drugs 18, 572; Esposito, A. et al., (2019) RSC Adv. 9, 21519], the very promising potential of L-iminosugars in the treatment of rare diseases has recently been highlighted. In particular, L-NBDNJ, the enantiomer of D-NBDNJ (Miglustat), has shown interesting potential as a candidate for combination therapy for Pompe disease and, unlike its D-enantiomer, is not an inhibitor of most glycosidases [D'Alonzo, D. et al., (2017). J. Med Chem. 60, 9462].

[0016] More interesting results were obtained when considering the application of L-iminosugars to cystic fibrosis (CF) [Esposito, A. et al., (2020). Int. J. Mol. Sci. 21, 3353]. In fact, L-NBDNJ and their congeners exhibited anti-inflammatory and antibacterial properties both in vitro and in vivo, highlighting the potential 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; DeGregorio, E. et al., (2020). Antibiotics 9, 362]. Summary of the invention

[0017] It has now been found that L-iminosugars (abbreviated as L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ) having the following structural formula and their pharmaceutically acceptable salts exhibit a significant ability to inhibit the accumulation of heparan sulfate and are therefore useful tools for treating mucopolysaccharidosis type I, II, III or VII and subtypes thereof, in particular Sanfilippo syndrome and its A, B, C and D subtypes, as well as for treating other disorders characterized by heparan sulfate accumulation, such as Alzheimer's disease and tumors.

[0018]

[0019] D'Alonzo D. et al., (2017). J. Med. Chem. 60, 9462; (2017) described the synthesis of L-NBDNJ (N-butyl-L-deoxynojirimycin, a non-natural enantiomer of Miglustat); L-NBDNJ has been shown to be a candidate drug for the treatment of Pompe disease.

[0020] D'Alonzo D. et al., (2017). J. Med. Chem. 60, 9462 and De Fenza M. et al., (2019). Eur. J. Med. Chem. 175, 63 describe the synthesis of L-iminosugars L-DNJ (non-natural enantiomer of deoxynojirimycin or Duvoglustat) and L-AMPDNM (N-adamantanylmethoxypentyl L-DNJ).

[0021] The compound L-MONDNJ (N-methoxynonyl L-DNJ) is novel and constitutes a further object of the present invention.

[0022] For the intended therapeutic use, the iminosugars L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ or their pharmaceutically acceptable salts will be formulated into pharmaceutical compositions suitable for oral or parenteral administration, such as capsules, tablets, solutions, etc., containing suitable excipients.

[0023] The dosage will be determined by experts based on the patient's condition, weight, sex and age, as well as the pharmacokinetic and toxicological properties of the compound. In principle, the dosage can be similar to that of the drugs already in use (Miglustat and Migalastat), for example 10 to 1000 mg per day, administered once or several times.

[0024] The observed pharmacological activities of compounds L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ or their pharmaceutically acceptable salts are very interesting compared to other structurally similar iminosugars (compounds L-NNDNJ, L-HPDNJ and L-NPDNJ, whose molecular formulas are shown below), which did not show any activity when evaluated under the same conditions.

[0025] The structural formulas of compounds according to the invention and comparative compounds are reported below.

[0026]

[0027] The invention is described in detail in the following experimental section.

[0028] Example 1: Synthesis of L-MONDNJ (N-methoxynonyl L-DNJ) and its corresponding hydrochloride derivatives.

[0029] Step a: 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 minutes, 1,9-nonanediol was added to the suspension (0.41 g, 2.56 mmol) and the reaction was stirred at room temperature for 1 hour. The suspension was then filtered and washed with dichloromethane to remove polymer-anchored triphenylphosphine oxide. The filtrate was washed with saturated Na2S2O3 and saturated NaCl solutions and extracted with dichloromethane.

[0030] The organic phase was dried (Na2SO4) and evaporated under reduced pressure to give the desired 1,9-diiodononane (oil, 0.9 g, 95% yield). 1 H NMR (400 MHz, CDCl3): δ 1.21-1.47 (m, 10H), 1.76-1.88 (m, 4H), 3.19 (t, J = 7.0 Hz, 4H). In this reaction, the use of triphenylphosphine instead of polymeric triphenylphosphine gave similar results in terms of reaction time and yield; however, this process requires a purification step by precipitation of triphenylphosphine oxide or by chromatography.

[0031] Step 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) at 0°C and under argon atmosphere with magnetic stirring. The reaction mixture was stirred at the same temperature for 1 hour; then, a solution of 1,9-diiodononane (0.75 g, 1.95 mmol) in THF (3.5 mL) was added. The solution was warmed to room temperature and stirred at the same temperature for 48 hours. Subsequently, dichloromethane was added, and the solution was washed first with aqueous NH4Cl solution and then with saturated NaCl solution. The organic phase was dried with Na2SO4, and the solvent was evaporated under reduced pressure. Chromatography of the crude residue on silica gel (hexane: EtOAc = 95:5) gave pure 1-iodo-9-methoxynonane (oil, 0.42 g, yield 75%). 1 H NMR (400MHz, CDCl3): δ1.21-1.47(m,8H), 1.51-1.63(m,4H), 1.76-1.88(m,2H), 3.19(t,J=7.0Hz,2H), 3.30(s,3H), 3.36(t,J=6.6Hz,2H).

[0032] Step c: Synthesis of L-MONDNJ (N-methoxynonyl L-DNJ). To a solution of L-DNJ (0.20 g, 1.22 mmol) in dry DMF (4 mL) was added K2CO3 (0.5 g, 3.6 mmol) at room temperature under argon atmosphere with magnetic stirring. 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 hours. The solvent was removed under reduced pressure, and chromatography was performed on silica gel (acetone: MeOH = 8:2) to obtain pure L-MONDNJ.

[0033] Step d: Preparation of L-MONDNJ·HCl (N-methoxynonyl L-DNJ·HCl) L-MONDNJ HCl hydrochloride (0.30 g, yield 75%) was obtained by adding 1M HCl (1.22 mmol) followed by evaporation under reduced pressure. 1 H NMR (500MHz, CD3OD): δ1.28-1.49(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=6.5Hz,4H),3.47(dd,J=4.9,11.8Hz,1H),3.61(t,J=11.8Hz,1H),3.65-3.77(m,1H),3.91(d,J=11.6Hz,1H),4.14(d,J=11.6Hz,1H).

[0034] Example 2: Treatment with hydrochloride salts of L-deoxyaminosugars (L-DNJ, L-NBDNJ, L-AMPDNM, and L-MONDNJ) reduces lysosomal defects in a cell model of Sanfilippo B disease (MPS IIIB).

[0035] To investigate the effects of these 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 causing 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 NAGLU resulted in cytoplasmic and lysosomal accumulation of heparan sulfate in stable SK-NBE clones (compared to control (WT) clones).

[0036] Therefore, a NAGLU-silenced clone (cl5) and a control clone (WT) were chosen to test the effects of L-deoxyaminosugars (L-DNJ, L-NBDNJ, L-NNDNJ, L-HPDNJ, L-NPDNJ, L-AMPDNM, and L-MONDNJ) in their hydrochloride form on the lysosomal phenotype of our Sanfilippo B (MPS IIIB) cell model.

[0037] Clone 5 was cultured in the presence of 20 μM of each L-deoxyamino sugar under normal growth conditions, and lysosomal accumulation was assessed by immunofluorescence after 48 h using a specific antibody against Lamp1 (lysosomal marker). Untreated clone 5 showed enlarged Lamp1-positive lysosomal structures within the cytoplasm compared to the WT control clone (Table 1). Treatment with L-DNJ, L-NBDNJ, L-AMPDNM, and L-MONDNJ significantly reduced lysosomal enlargement and accumulation in the clone 5 (cl5) model system of Sanfilippo B (MPS IIIB) (Table 1). In addition, after treatment with active L-deoxyamino sugars, lysosomes were no longer concentrated in the perinuclear region of the cells, as occurs in several lysosomal diseases, but were physiologically distributed throughout the cytoplasm. Interestingly, the L-deoxyamino sugars L-NNDNJ, L-HPDNJ, and L-NPDNJ did not show any activity on the lysosomal phenotype of the Sanfilippo B model clone 5 (cl5) (Table 1).

[0038] Table 1

[0039]

[0040] Comparing these results with those obtained by immunofluorescence of Lamp1 protein in WT control normal clones, the physiological distribution of lysosomes in the cytoplasm was more obvious in L-DNJ, L-NBDNJ, L-AMPDNM, and L-MONDNJ-treated cells.

[0041] Treatment with any of the seven L-iminosugars did not result in any changes in the size and distribution of lysosomes in non-lesional WT model cells.

[0042] Overall, these results demonstrate for the first time that treatment with selected L-iminosugars (L-DNJ, L-NBDNJ, L-AMPDNM, and L-MONDNJ) is able to reduce lysosomal defects in a cell model of Sanfilippo B disease (MPS IIIB).

[0043] Example 3: Treatment with hydrochloride salts of L-deoxyaminosugars (L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ) reduces HS accumulation in a cell model of Sanfilippo B disease (MPS IIIB).

[0044] Under normal growth conditions, clone 5 was grown in the presence of 20 μM of each L-imino sugar, and the accumulation of heparan sulfate (HS) was assessed by immunofluorescence staining of HS after 48 hours. Untreated clone 5 showed accumulation of HS on the cell membrane compared to the WT control clone (Table 2). However, in the presence of compounds L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ, a sharp reduction in HS staining was observed in the Sanfilippo B model system (MPS III B) cl5 (Table 2). Also in this case, L-imino sugars L-NNDNJ, L-HPDNJ and L-NPDNJ did not show any activity in reducing HS accumulation in the tested Sanfilippo B model (Table 2). These results are consistent with the results obtained using Lamp1 staining of lysosomes.

[0045] Table 2

[0046]

[0047] Furthermore, treatment with any of the seven L-iminosugars did not result in changes in the amount of HS in non-lesional WT model cells.

[0048] Overall, these results demonstrate for the first time that treatment with the selected L-iminosugars L-DNJ, L-NBDNJ, L-AMPDNM, and L-MONDNJ is able to reduce HS accumulation in a cell model of Sanfilippo B disease (MPS IIIB) generated in our laboratory.

[0049] Example 4: Treatment with hydrochloride salts of L-deoxyaminosugars (L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ) reduces lysosomal defects and HS accumulation in fibroblasts from patients affected by Sanfilippo A and B (MPS IIIA and IIIB).

[0050] To verify whether the selected iminosugars could also 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.

[0051] The human cell line fibroblasts from MPS (Sanfilippo disease) patients used in the examples were obtained from the G. Gaslini Institute in Genoa, "Biobank of Cell Lines and DNA of Patients Affected by Genetic Diseases" - Telethon Genetic Biobank Network - Telethon Research Services. The cells were classified according to an identification code and the type of disease, without identifying the patient. The cells of the patients were collected at the Gaslini Institute and the collection was extended to the preservation and its possible use, for diagnostic and / or research purposes, with informed consent, in accordance with the current regulations required by the guidelines followed by the Telethon Biobank. The network complies with the current Italian privacy and data protection laws, including: Italian Data Protection Authority, Personal Data Protection Law, Legislative Decree No. 196, June 30, 2003, published in the Official Gazette of the Italian Republic No. 174 of July 29, 2003; Italian Data Protection Authority, General Authorization for the Processing of Genetic Data, June 24, 2011, published in the Official Gazette of the Italian Republic No. 159 of July 11, 2011.

[0052] Cells were cultured in the presence of the selected L-iminosugars (at a dose of 20 μM) and processed for HS and Lamp1 immunofluorescence 48 hours later. Treatment with L-iminosugars had no effect on the 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 of patients affected by Sanfilippo A and B (MPS IIIA and MPS IIIB) (Table 3).

[0053] Table 3

[0054]

[0055] Also in this case, the L-iminosugars L-NNDNJ, L-HPDNJ and L-NPDNJ did not show any activity on the reduction of HS and Lamp1 accumulation in fibroblasts of Sanfilippo A and B patients (Table 3).

[0056] Overall, these results show for the first time that treatment with the selected L-iminosugars L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ is able to prevent the accumulation of HS and lysosomes in fibroblasts from patients affected by Sanfilippo A and B diseases.

[0057] Example 5: Treatment with hydrochloride of D-deoxyamino sugars (D-DNJ or DNJ, D-NBDNJ or NBDNJ or D-AMPDNM or AMPDNM, or D-AMPDNM or AMPDNM, or D-MONDNJ or MONDNJ) has no effect on lysosomal defects and HS accumulation in fibroblasts from patients affected by Sanfilippo A and B (MPS IIIA and IIIB).

[0058] To demonstrate that iminosugars (D-DNJ, also known as Duvoglustat, and D-NBDNJ, also known as Miglustat) currently available for the treatment of other lysosomal diseases, as well as stereoisomers D-AMPDNM and D-MONDNJ, do not have the same efficacy as the compounds of the present invention, adult dermal fibroblasts HDFa were used as a control (purchased from SIGMA), 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 20 μM doses of iminosugars D-DNJ, D-NBDNJ, D-AMPDNM, and D-MONDNJ, and processed for HS and Lamp1 immunofluorescence after 48 hours. Treatment with D-iminosugars had no effect on HDFa and fibroblasts from patients affected by Sanfilippo A and B (MPS IIIA and MPS IIIB), either on HS or lysosomal accumulation (Table 4).

[0059] Table 4

[0060]

[0061] Overall, these results demonstrate the efficacy of selected L-iminosugars L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ in preventing the accumulation of HS and lysosomes in cellular models of mucopolysaccharidosis (e.g., fibroblasts) from patients affected by Sanfilippo A and B.

[0062] Example 6: Treatment with L-deoxyaminosugars (L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ) in the form of their hydrochloride salts induces a decrease in the HS content and a reduction in the growth of the HeLa tumor epithelial cell line.

[0063] To test whether treatment with selected L-deoxyamino sugars (L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ) would interfere with HS synthesis, we selected a cell line that did not undergo NAGLU mutation and that was highly modified with heparan sulfate on the cell membrane. For this purpose, HeLa tumor epithelial cells (purchased from ATCC) were cultured for 48 hours in the presence of selected L-imino sugars and the amount of HS was assessed by immunostaining. Treatment with the same L-imino sugars L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ caused a strong reduction in HS accumulation on the HeLa cell membrane (Table 5).

[0064] Table 5

[0065]

[0066] Also in this case, the L-iminosugars L-NNDNJ, L-HPDNJ and L-NPDNJ did not show any activity on the reduction of HS accumulation in HeLa tumor epithelial cells (Table 5).

[0067] Furthermore, as shown in the cell proliferation assay reported in Table 6, treatment with L-deoxyaminosugars L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ caused a decrease in the number of HeLa cells after 48 hours.

[0068] Table 6

[0069]

[0070] The results suggest that treatment with L-DNJ L-iminosugar, L-NBDNJ, L-AMPDNM, and L-MONDNJ induces a decrease in HS in HeLa cancer cells and subsequently inhibits tumor cell growth, as HS is essential for maintaining the proliferation of cancer epithelial cells.

[0071] The data reported in Table 6 also demonstrate that the iminosugars belonging to the D series, D-DNJ or Duvoglustat, D-NBDNJ or Miglustat, D-AMPDNM and D-MONDNJ do not have the same efficacy in reducing cancer cell proliferation compared to the compounds of the present invention.

[0072] These data suggest that L-iminosugars interfere with HS synthesis in a novel mechanism compared to the mechanism of action of other commercially available iminosugars. Furthermore, these results suggest that the present invention may be applied not only to mucopolysaccharidoses where HS accumulates, but also to cancer diseases where HS is essential for supporting tumor cell growth and its metastatic mechanism.

[0073] Example 7: Treatment with L-deoxyaminosugars (L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ) in the hydrochloride form induces a reduction in the amount of amyloid-β fibrils in a cell model of Sanfilippo B disease (MPS IIIB).

[0074] To demonstrate the therapeutic application of selected iminosugars L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ for neurodegenerative diseases, we used the Sanfilippo B (cl5) neuron model recently generated in our laboratory (Example 2).

[0075] Clone (cl5) stably silencing NAGLU (the causative gene of MPS IIIB) is able to mimic the characteristics of neurodegenerative diseases, as it accumulates beta-amyloid fibrils in the cytoplasm. In fact, Sanfilippo syndrome has also been defined as "childhood Alzheimer's disease".

[0076] The diseased clone (cl5) was grown in the presence of L-DNJ, L-NBDNJ, L-AMPDNM and L-MONDNJ and the corresponding enantiomers D-DNJ, D-NBDNJ, D-AMPDNM and D-MONDNJ at a dose of 20 μM and processed after 48 hours by immunofluorescence against β-amyloid peptide 1-42. The data reported in Table 7 show that treatment with L-deoxyamino sugars leads to an overall reduction in the accumulation of amyloid fibrils compared to untreated cells (mock). In contrast, treatment with the corresponding D-enantiomers had no effect on the reduction of amyloid fibril accumulation compared to untreated clones (mock) (Table 7).

[0077] Table 7

[0078]

[0079] 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 the accumulation of amyloid plaques and treating neurodegenerative processes such as Alzheimer's disease. The data reported in Table 7 also show that, unlike the compounds of the present invention, the D-iminosugars D-DNJ, D-NBDNJ, D-AMPDNM and D-MONDNJ do not have the same efficacy in reducing neurodegenerative markers.

Claims

1. An iminosugar of the following formula or a pharmaceutically acceptable salt thereof: It is used for the treatment and prevention of diseases in which heparan sulfate accumulates, the diseases being selected from mucopolysaccharidosis, Alzheimer's disease and cancer.

2. The iminosugar for use according to claim 1, wherein the heparan sulfate storage disease is mucopolysaccharidosis type I, II, III or VII.

3. Iminosugar for use according to claim 2, wherein the disease is Sanfilippo syndrome and its subtypes (A, B, C, D).

4. The iminosugar for use according to claim 2, wherein the disease is Alzheimer's disease.

5. The iminosugar for use according to claim 2, wherein the disease is a tumor.

6. Compounds of the following formula and pharmaceutically acceptable salts thereof:

Images