Marine bacterial exopolysaccharide derivatives and their use in the treatment of mucopolysaccharidosis

Abstract

The present invention provides low molecular weight persulfated polysaccharides having anti-heparanase activity, prepared from marine natural exopolysaccharides secreted by mesophilic marine bacteria from deep-sea hydrothermal environments, and relates to the use of these low molecular weight persulfated polysaccharides for the prevention or treatment of mucopolysaccharidosis, particularly mucopolysaccharidosis type III (San Filippo syndrome). [Solution] The inventors have shown that low molecular weight persulfated polysaccharides obtained from marine natural exopolysaccharides (EPS) secreted by strain GY785 (Alteromonas infernus species of the genus Alteromonas) or from strain HE800 (Vibrio diabolicus species of the genus Vibrio) exhibit anti-heparanase (HPSE) activity.

Description

[Technical Field]

[0001] Related applications This application claims priority to European Patent Application No. EP 20 183 661.6, filed on 2 July 2020, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Mucopolysaccharidosis (MPS) is a group of rare hereditary metabolic disorders within the larger family of lysosomal storage disorders (LSDs). The overall incidence is 1 in 25,000–30,000 births. However, mucopolysaccharidosis, particularly the milder forms of the disorder, is often unrecognized, leading to underdiagnosis or misdiagnosis and making it difficult to determine their true frequency in the general population. MPS is characterized by a deficiency (absence or dysfunction) of one of 11 specific lysosomal enzymes involved in the metabolism (catabolism or degradation) of glycosaminoglycans (GAGs)—long, unbranched polysaccharides that play essential roles in connective tissue biology and cellular crosstalk. Such lysosomal enzyme deficiencies lead to the accumulation of undegraded and partially degraded GAGs within lysosomes, resulting in permanent, progressive cytotoxicity that causes multisystem disease. Individuals with MPS disorder share many similar symptoms, such as the involvement of multiple organs, characteristic "rough" facial features, and skeletal abnormalities, particularly joint problems. Additional findings include short stature, cardiac abnormalities, respiratory irregularities, hepatomegaly and splenomegaly, and / or neurological abnormalities. The severity of different MPS disorders varies greatly among affected individuals, even among individuals with the same type of MPS, and even among individuals of the same family. While each MPS is clinically distinct, most patients generally experience a period of normal development followed by a decline in physical and / or mental function. MPS includes the following different types: MPS IH / S (Hurler / Scheyé syndrome), MPS IH (Hurler syndrome), MPS IS (Scheyé syndrome), MPS II (Hunter syndrome), MPS III (Sanfilippo syndrome), MPS IV (Morcchio syndrome), MPS IX (Hyaluronidase deficiency or Natowicz syndrome), MPS VII (Sly syndrome), and MPS VI (Maroto-Lamy syndrome). Currently, there are no specific treatments available for supportive care and management of complications, and treatment strategies are needed for the large subgroups of MPS patients.

[0003] Among MPS disorders, mucopolysaccharidosis type III, also known as Sanfilippo syndrome, is the most common. MPS III consists of four distinct subtypes: A, B, C, and D, each subtype caused by a deficiency in one of four enzymes involved in the breakdown of heparan sulfate. The overall incidence of MPS III is 0.28 to 4.1 per 100,000 births. The incidence of the different subtypes has a very uneven geographical distribution; however, types A and B are always more common than types C and D. In all MPS III subtypes, central nervous system (CNS) involvement is prominent (neurodegeneration, progressive dementia, hyperactivity, seizures, and behavioral disorders), but other symptoms may also be present, such as skeletal conditions that affect growth and cause degenerative joint disease, hepatosplenomegaly, macrocraniosis, and hearing loss. Except for attentive patients, death usually occurs in the 20s, and the survival rate of children with MPS III subtype A is shorter. Currently, there are no established treatments or standard procedures for patients with Sanfilippo syndrome. In the absence of effective therapies, patient care is limited to symptom management and elective support. MPS III disorder is debilitating enough to justify attention and research, and is challenging for parents and caregivers. Gene therapy, bone marrow transplantation, chaperone molecules, substrate deprivation therapy, and subarachnoid enzyme therapy are among the most active areas of therapeutic research.

[0004] While some developments offer hope that therapeutic interventions to halt destructive mental and behavioral exacerbations may be feasible at some point in the future, there are currently no effective therapies available for MPS III and other MPS conditions. Therefore, there remains a need in the art for therapeutic options in the management of mucopolysaccharidosis disorders. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] WO 2006 / 003290 [Patent Document 2] WO 2007 / 066009 [License 3] WO 02 / 02051 [License 4] European Licensed Vow EP 0 221 977 [Non-licensed literature]

[0006] [Non-licensed Document 1] "Remington's Pharmaceutical Sciences", EW Martin, 18th edition, 1990, Mack Publishing: Easton, PA [Non-licensed Document 2] Rehm et al., Rev. Microbiol., 2010, 8: 578-592 [Non-licensed Document 3] Colliec-Jouault et al., Handbook of Exp. Pharmacol., 2012, pp. 423-449. [Non-licensed Document 4] Delbarre-Ladrat et al., Microorganisms, 2017, 5(3): 53 pages [Non-licensed Document 5] Raguenes et al., Int. J. Syst. Bacteriol., 1997, 47: 989-995 [Non-licensed Document 6] Rougeaux et al., J. Carbohydr. Res., 1999, 322: pages 40~45 [Non-licensed Document 7] Raguenes et al., J. Appl. Microbiol., 1997, 82: 422-430 [Non-licensed Document 8] Roger et al., Carbohydr. Res., 2004, 339: 2371-2380. [Non-licensed Document 9] Guezennec et al., Carbohydr. Polym., 1998, 37: 19-24 [Non-licensed Document 10] Colliec-Jouault et al., Biochim. Biophys. Acta, 2001, 1528: 141 - 151

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Summary of the Invention

Means for Solving the Problems

[0007] The inventors have shown that low molecular weight persulfated polysaccharides obtained from marine natural exopolysaccharides (EPS) secreted by strain GY785 (Alteromonas infernus) or from strain HE800 (Vibrio diabolicus) exhibit anti-heparanase (HPSE) activity. They demonstrated that low molecular weight persulfated polysaccharides can affect heparan sulfate (HS) turnover, thus leading to incomplete degradation of intracellular HS in fibroblasts derived from a mouse model of type IIIA mucopolysaccharidosis. Heparanase is the first enzyme to degrade heparan sulfate in its catabolic pathway, cleaving large segments of the HS chain that are subsequently depolymerized within lysosomes by exoglycosidase. Although the mechanism is not fully understood, it is thought that intact HS chains that are not cleaved by HPSE (due to the inhibitory effect of low molecular weight persulfated polysaccharides) cannot enter the lysosomal degradation pathway and are instead redirected to the extracellular space for clearance in the blood and urine. Therefore, cells can be preserved from toxic lysosomal HS accumulation. Treatment with low molecular weight persulfated polysaccharides may alleviate symptoms caused by lysosomal overload due to incompletely degraded heparan sulfate.

[0008] Therefore, in the first aspect, the present invention relates to a low molecular weight persulfated polysaccharide having antiheparanase activity for use in the prevention or treatment of mucopolysaccharidosis in a subject, which is a derivative of a natural exopolysaccharide (EPS) secreted by mesophilic marine bacteria from deep-sea hydrothermal environments, and the following steps: (a) A process comprising free radical depolymerization of marine native EPS from Alteromonas strain GY785 or Vibrio diabolix strain HE800 to obtain depolymerized EPS having a molecular weight of 5,000 to 100,000 g / mol; (b) A subsequent step consisting of sulfation of the depolymerized EPS to obtain over-sulfated and depolymerized EPS, the method comprising adding at least one sulfating agent to the depolymerized EPS in an amount sufficient to obtain a sulfated polysaccharide having a degree of sulfate group substitution of 10% to 55% by mass compared to the total mass of the over-sulfated and depolymerized EPS; and (c) A subsequent step consisting of isolating a low molecular weight over-sulfated polysaccharide from the over-sulfated and depolymerized EPS, the low molecular weight over-sulfated polysaccharide having a molecular weight of about 5,000 to about 16,000 g / mol Relates to a low molecular weight over-sulfated polysaccharide obtained using a method comprising the above.

[0009] In certain embodiments, in step (a) of the preparation method defined above, the free radical depolymerization is carried out on natural GY785 EPS secreted by strain GY785, and the low molecular weight over-sulfated polysaccharide having anti-heparanase activity has a molecular weight of about 6 kDa to about 10 kDa or about 7 kDa to about 9 kDa, and a degree of sulfate group substitution of about 30% to about 40% by mass compared to the total mass of the over-sulfated polysaccharide.

[0010] For example, the low molecular weight over-sulfated polysaccharide having anti-heparanase activity may be GYS8 having a molecular weight of about 8 kDa and a degree of sulfate group substitution of about 36% by mass compared to the total mass of the over-sulfated polysaccharide.

[0011] In certain embodiments, in step (a) of the preparation method defined above, the free radical depolymerization is carried out on natural HE800 EPS secreted by strain HE800, and the low molecular weight over-sulfated polysaccharide having anti-heparanase activity has a molecular weight of about 3 kDa to about 7 kDa or about 4 kDa to about 6 kDa, and a degree of sulfate group substitution of about 45% to about 55% by mass compared to the total mass of the over-sulfated polysaccharide.

[0012] For example, the low molecular weight persulfated polysaccharide having anti-heparanase activity may be HE5.1, which has a molecular weight of approximately 5.1 kDa and a degree of sulfate group substitution of approximately 50% by mass compared to the total mass of the persulfated polysaccharide.

[0013] In certain embodiments, the step of isolating low molecular weight persulfated polysaccharides from persulfated and depolymerized EPS is carried out by fractionation, particularly by size exclusion chromatography.

[0014] In certain embodiments, mucopolysaccharidosis is mucopolysaccharidosis type III. Mucopolysaccharidosis type III may be subtype A, subtype B, subtype C, or subtype D.

[0015] In another aspect, the present invention provides a pharmaceutical composition comprising a low molecular weight persulfated polysaccharide having a therapeutically effective amount of antiheparanase activity as defined herein, and at least one pharmaceutically acceptable carrier or excipient, for use in the prevention or treatment of mucopolysaccharidosis in a subject.

[0016] In certain embodiments, mucopolysaccharidosis is mucopolysaccharidosis type III. Mucopolysaccharidosis type III may be subtype A, subtype B, subtype C, or subtype D.

[0017] These and other objects, advantages and features of the present invention will become apparent to those skilled in the art after reading the following detailed description of preferred embodiments. [Brief explanation of the drawing]

[0018] [Figure 1] This figure shows a schematic model of heparan sulfate proteoglycan and heparanase trafficking. [Figure 2]This figure shows the effect of treatment on total proteoglycans. The graph shows the total amount of proteoglycans in control and treated cells (fibroblasts derived from a mouse model of MPSIIIA). For each exopolysaccharide derivative (HES5.1(A5_3) and GYS8(A5_4)), the mean of two independent experiments is shown in ±SD. [Figure 3] This figure shows the distribution of proteoglycans in control and treated cells. The graph shows the radioactivity measured in the extracellular and intracellular compartments corresponding to all proteoglycans after the first purification step. For each exopolysaccharide derivative, the mean of two independent experiments is shown in ±SD. [Figure 4] This figure shows the distribution of heparan sulfate in control and treated cells. The graph shows the percentage of heparan sulfate (HS) in the corresponding proteoglycans (PGs) in the extracellular and intracellular compartments. PGS in each compartment is considered to be 100%. For each compound ((A) A5_4 (GYS8) and (B) A5_3 (HES5.1)), the mean of two independent experiments is shown in ±SD. [Figure 5] This figure shows the PAGE-NaCl profiles of intracellular and extracellular heparan sulfate (HS) in MPSIIIA cells. HS was isolated from control cells and analyzed by PAGE. The standard was detected with Azure A 0.08%, and HS from MPSIIIA cells was detected by autoradiography. The left side shows the intracellular and extracellular HS profiles obtained using ImageJ software, and the corresponding gels are shown on the right side. [Figure 6] This figure shows the profiles obtained by GFC. (A) The first graph shows the standard curve. Standard molecular weights are shown. (B) The second graph shows the intracellular and extracellular HS profiles in control MPSIIIA cells. [Figure 7]This figure shows the PAGE-NaCl profiles of intracellular and extracellular hemoglobin (HS) in MPSIIIA cells treated with A5_3 (HES5.1). HS were isolated from control and treated cells, analyzed by PAGE NaCl, and detected by autoradiography. [Figure 8] This figure shows the PAGE-NaCl profiles of intracellular and extracellular hemoglobin (HS) in MPSIIIA cells treated with A5_4(GYS8). HS were isolated from control and treated cells, analyzed by PAGE NaCl, and detected by autoradiography. [Figure 9] This figure shows the GFC profiles of hematopoiesis (HS) from control (untreated) cells and cells treated with 20 μg / ml A5_3 (HES5.1). (A) Overlay of extracellular HS profiles from control and treated cells. (B) Overlay of intracellular HS profiles from control and treated cells. Treatment causes a shift in the extracellular HS to its corresponding molecular weight (MW). [Figure 10] This figure shows the GFC profiles of hemoglobin (HS) from control (untreated) cells and cells treated with 20 μg / ml A5_4 (GYS8). (A) Overlay of extracellular HS profiles from control and treated cells. (B) Overlay of intracellular HS profiles from control and treated cells. Treatment causes a shift in the extracellular HS towards its corresponding molecular weight (MW). [Figure 11] This figure shows the PAGE profiles of hematopoiesis (HS) from MPSIIIA cells treated with A5_3 (HES5.1). (A) Intracellular HS from cells treated with 0, 20, 50, and 100 μg / ml of A5_3. (B) Extracellular HS from cells treated with 0, 20, 50, and 100 μg / ml of A5_3. [Modes for carrying out the invention]

[0019] definition As used herein, the term “subject” refers to a human or other mammal (e.g., primates, dogs, cats, goats, horses, pigs, mice, rats, rabbits, etc.) that can develop mucopolysaccharidosis, but may or may not have the disease. Non-human subjects may be transgenic or modified animals. In many embodiments of the present invention, the subject is human. In such embodiments, the subject is often referred to as “individual” or “patient.” These terms do not represent a specific age and therefore encompass neonates, children, teenagers and adults. The term “patient” more specifically refers to an individual suffering from the disease. Thus, the term “mucopolysaccharidosis patient” refers to an individual suffering from (i.e., diagnosed with) mucopolysaccharidosis.

[0020] As used herein, the term “inhibit” means to prevent something from happening, to delay the occurrence of something, and / or to reduce the degree or likelihood of something happening. Accordingly, as used interchangeably herein, the terms “heparanase inhibitor” and “HPSE inhibitor” refer to a molecule, compound, or agent that inhibits (i.e., blocks, reduces, and / or delays) the normal function of heparanase. Where used herein to characterize a molecule, compound, or agent, the term “having anti-heparanase activity” refers to a molecule, compound, or agent that is a heparanase inhibitor.

[0021] The term “treatment” is used herein to characterize a method or process aimed at (1) delaying or preventing the onset of a disease or condition (in this case, mucopolysaccharidosis); (2) slowing or halting the progression, exacerbation, or worsening of the symptoms of a disease or condition; (3) improving the symptoms of a disease or condition; or (4) treating a disease or condition. A treatment may be administered after the onset of a disease or condition for a therapeutic effect. Alternatively, a treatment may be administered before the onset of a disease or condition for a prophylactic or preventive effect. In this case, the term “prevention” is used.

[0022] "Pharmaceutical composition" is defined herein as comprising an effective amount of a low molecular weight (LMW) persulfated polysaccharide derivative having anti-heparanase activity according to the present invention, and at least one pharmaceutically acceptable carrier or excipient.

[0023] As used herein, the term “therapeutic effective dose” means any amount of a molecule, compound, drug or composition sufficient to satisfy its intended purpose, e.g., a desired biological or therapeutic response in a cell, tissue, system or subject.

[0024] The term “pharmaceutically acceptable carrier or excipient” refers to a carrier medium that does not interfere with the efficacy of the biological activity of the active ingredient and is not excessively toxic to the host at the concentration in which it is administered. This term includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and adsorption retarders, etc. The use of such media and agents for pharmaceutically active substances is well known in the art (see, for example, “Remington's Pharmaceutical Sciences,” E.W. Martin, 18th edition, 1990, Mack Publishing: Easton, PA, which is incorporated herein by reference in its entirety).

[0025] As used herein with respect to numbers, the terms “approximately” and “about” generally include numbers within a 10% range (greater than or less than) of the number in either direction, unless otherwise specified or evident from the context (unless such a number exceeds 100% of the possible value).

[0026] As described above, the present invention provides low molecular weight persulfated polysaccharides having antiheparanase activity, which are derivatives of natural exopolysaccharides secreted by mesophilic marine bacteria from deep-sea hydrothermal environments, and relates to the use of these low molecular weight persulfated polysaccharides in the prevention or treatment of mucopolysaccharidosis, particularly in the prevention or treatment of type III mucopolysaccharidosis.

[0027] I-Low molecular weight persulfated exopolysaccharide derivative The low molecular weight persulfated polysaccharides used in this invention are derivatives of two natural exopolysaccharides (EPS), HE800 EPS and GY785 EPS, secreted by mesophilic marine bacteria from deep-sea hydrothermal environments. In recent years, there has been growing interest in the isolation and identification of novel marine-derived polysaccharides that may have new applications in diverse industries. They compete with polysaccharides from other sources such as seagrasses, crustaceans, animals, or plants. There has been considerable interest in the mass cultivation of microorganisms from marine environments, representing an innovative approach to the biotechnological use of underutilized resources. Marine bacterial EPS and its derivatives have several significant advantages as therapeutic compounds, as they can be produced at a viable economic cost under controlled conditions in accordance with Good Manufacturing Practice standards, and patients exhibit a very low risk of infection with non-conventional infectious agents, such as prions or emerging viruses, due to a large "species barrier."

[0028] Marine bacteria from deep-sea hydrothermal vent environments, belonging to three main genera (Vibrio, Alteromonas, and Pseudoalteromonas), demonstrated their ability to produce unusual extracellular polymers in aerobic, carbohydrate-supplemented media. The secreted exopolysaccharides present original structural features that can be modified to design bioactive compounds and enhance their specificity (Rehm et al., Rev. Microbiol., 2010, 8: pp. 578-592; Colliec-Jouault et al., Handbook of Exp. Pharmacol., 2012, pp. 423-449; Delbarre-Ladrat et al., Microorganisms, 2017, 5(3): p. 53). In particular, the first EPS-producing species of the genus Vibrio isolated from active deep-sea hydrothermal vent samples was named Vibrio diabolix (Raguenes et al., Int. J. Syst. Bacteriol., 1997, 47: pp. 989-995). It is a high molecular weight (>10) EPS called HE800 EPS. 6g / mol (Rougeaux et al., J. Carbohydr. Res., 1999, 322: pp. 40-45) produces an exopolysaccharide, which consists of linear tetrasaccharide repeat units: two glucuronic acid residues, one N-acetylated glucosamine residue and one N-acetylated galactosamine residue. Another bacterium, named Alteromonas infernus—a novel species of the genus Alteromonas—was also isolated from deep-sea sediments of Guaymas Inner Bay (Gulf of California) (Raguenes et al., J. Appl. Microbiol., 1997, 82: pp. 422-430). The bacterium Alteromonas infernus produces a water-soluble EPS called GY785 EPS, which is a branched heteropolysaccharide with non-saccharide repeat units containing four glucose residues, two galactose residues, two glucuronic acid residues, and one galacturonic acid residue with a sulfate group at the C2 position (Roger et al., Carbohydr. Res., 2004, 339: pp. 2371-2380; Guezennec et al., Carbohydr. Polym., 1998, 37: pp. 19-24).

[0029] Low molecular weight (LMW) persulfated polysaccharide derivatives from the marine natural exopolysaccharides HE800 EPS and GY785 EPS were prepared by the present inventors (Colliec-Jouault et al., Biochim. Biophys. Acta, 2001, 1528: 141-151; WO 2006 / 003290; WO 2007 / 066009; WO 02 / 02051; Guezennec J. et al., Carbohydrate Polymers 1998, 37(1): pp. 19-24; Senni et al., Mar. Drugs, 2013, 11: pp. 1351-1369; Merceron et al., Stem Cells, 2012, 30: pp. 471-480; Senni et al., Mar. Drugs, 2011, 9: 1664-1681; This has been previously prepared by Heymann et al. (Molecules, 2016, 21:309) using a first step of radical depolymerization followed by a sulfation reaction, thereby producing bioactive molecules with a molecular weight of <30 kg / mol (30,000 Da). In the embodiment of the present invention, low molecular weight persulfated polysaccharides are prepared using a similar method, the said method comprising the following steps: (a) A process comprising free radical depolymerization of marine natural exopolysaccharide (EPS) from Alteromonas strain GY785 or Vibrio strain HE800 to obtain depolymerized EPS having a molecular weight of 5,000 to 100,000 g / mol; (b) A subsequent step comprising sulfation of a depolymerized EPS to obtain a persulfated and depolymerized EPS, comprising adding at least one sulfating agent to the depolymerized EPS in an amount sufficient to obtain a sulfated polysaccharide having a degree of sulfate substitution of about 10% to about 55% by mass compared to the total mass of the persulfated and depolymerized EPS; and (c) A subsequent step comprising isolating a low molecular weight persulfated polysaccharide from persulfated and depolymerized EPS, wherein the low molecular weight persulfated polysaccharide has a molecular weight of approximately 5,000 to approximately 16,000 g / mol.

[0030] In a particular embodiment, the depolymerized EPS obtained after step (a) is freeze-dried.

[0031] In other embodiments, the dialysis step follows step (b) of the process.

[0032] During the first depolymerization step, the natural EPS can be used in liquid form, since it is secreted into the culture medium by bacteria. Preferably, the culture medium is centrifuged and only the supernatant containing the natural EPS and free of bacterial debris is collected. The natural EPS can be collected by any suitable technique known to those skilled in the art, such as membrane ultrafiltration, and then freeze-dried as is or in the form of added salts, as needed.

[0033] The step of free radical depolymerization of natural EPS is preferably carried out by adding a solution of an oxidizing agent to a reaction mixture containing natural EPS, preferably in the presence of a metal catalyst. The oxidizing agent is preferably selected from peroxides, particularly hydrogen peroxide, and peracids, particularly peracetic acid and 3-chloroperbenzoic acid. The addition is preferably carried out continuously and with stirring for 30 minutes to 10 hours. The reaction mixture is preferably maintained at a pH of 6 to 8 and a temperature of approximately 30°C to 70°C throughout the entire free radical depolymerization reaction by adding a basicizing agent, such as sodium hydroxide.

[0034] In a specific embodiment of the present invention, in this step, natural EPS is present in the reaction mixture at a concentration of about 2 mg to about 10 mg per ml of the reaction mixture.

[0035] In a preferred embodiment, the oxidizing agent is a solution of hydrogen peroxide (H2O2) having a concentration of about 0.1% to about 0.5% by mass, preferably about 0.1% to 0.2% by mass, and is added at a flow rate of about V1 / 1000 to V1 / 10 ml / min, preferably V1 / 50 to V1 / 500 ml / min, more preferably V1 / 100 ml / min, where V1 is the amount of reaction medium containing marine exopolysaccharide (EPS) to which the hydrogen peroxide solution is added.

[0036] Metal catalysts that can be used during the depolymerization process include, in particular, Cu, as described in European Patent Application EP 0 221 977. 2+ Fe 2+ and Cr 3+ Ions and Cr2O7 2- Preferably selected from anions. Depending on the specific embodiment, the metal catalyst is about 10 -3 M ~ about 10 -1 It is present in the reaction mixture at a concentration of M, preferably about 0.001 M to about 0.05 M.

[0037] The free radical depolymerization process according to the present invention and described above makes it possible to obtain a homogeneous low molecular weight polysaccharide derivative in a single step and with excellent yield. In connection with the present invention, the term “homogeneous derivative” means a derivative that exhibits a single major peak representing a dominant population of polysaccharide chains, which is homogeneous in size as characterized by a polydispersity index I(Mw / Mn) of <5 when investigated using high-speed size exclusion chromatography, where Mw is the mass-average molecular weight and Mn is the number-average molecular weight.

[0038] In certain embodiments, when the depolymerization reaction is complete, the resulting polysaccharide derivative is reduced using a reducing agent to stabilize the chain, and its reduced end is highly reactive, particularly to avoid chain hydrolysis by a "peeling" reaction. The properties of the reducing agent that can be used for this effect are not essential. In particular, the reducing agent may be sodium borohydride.

[0039] The metal catalyst used in the depolymerization step may be removed at the end of the depolymerization reaction (or at the end of the reduction reaction, if a reduction step is performed) by any preferred method, for example, by ion exchange chromatography, preferably by a weak cation exchange resin that has been passedivated beforehand, or by treatment with EDTA (ethylenediaminetetraacetic acid).

[0040] Polysaccharide derivatives obtained from depolymerization and / or reduction can be recovered, if necessary, by any suitable technique well known to those skilled in the art, for example, by membrane ultrafiltration or dialysis. They are then lyophilized and fractionated by size exclusion chromatography to increase their purity as required to improve the subsequent sulfation steps. Finally, the purified polysaccharide derivatives are prepared in the form of salts by adding a weak or strong base, which can be selected from, for example, pyridine, triethylamine, tributylamine, tetrabutylammonium hydroxide, and sodium hydroxide. These lyophilized salts can be prepared, for example, by eluting an aqueous solution of the polysaccharide derivative at a concentration of 1-8 mg / ml on an ion exchange resin column, for example, one sold by Dow Chemical under the name DOWEX®. The eluate is collected as long as the pH remains acidic, for example, less than 5, and then the pH is subsequently adjusted to approximately 6.5 with the desired base specified above. The polysaccharide derivatives in salt form are then ultrafiltered and lyophilized.

[0041] The lyophilized polysaccharide derivative is preferably dissolved in an anhydrous solvent at the beginning of the sulfation step, possibly in the form of an addition salt. The solvent is preferably selected from dimethylformamide (DMF), dimethyl sulfoxide (DMSO), formamide, and mixtures thereof. The amount of polysaccharide derivative present in the anhydrous solvent may be approximately 1 mg / ml to 10 mg / ml, preferably about 1 mg / ml to about 5 mg / ml, and more preferably about 2.5 mg / ml. The dissolution of EPS in the anhydrous solvent is preferably carried out at room temperature for about 1 to 2 hours with stirring under argon or nitrogen using a molecular sieve, and then at a temperature of 40°C to 50°C, preferably about 45°C, for about 2 hours.

[0042] One or more chemical sulfating agents used during the sulfation process can be added to the lyophilized or solution-form depolymerized and / or reduced EPS.

[0043] The sulfating agent is preferably selected from pyridine sulfate (free or polymer-bound), dimethylformamide sulfate, triethylamine sulfate, and a complex of trimethylamine sulfate. One or more chemical sulfating agents are added to the solution of the polysaccharide derivative in an amount preferably about 4 to about 6 times, more preferably about 5 times, the mass of the polysaccharide derivative in the solution. The chemical sulfation reaction is then preferably carried out with stirring for 2 to 24 hours, depending on the desired degree of sulfation. When the desired degree of sulfation is reached, the sulfation reaction is stopped after cooling of the reaction medium as follows: - Precipitation of sodium chloride in the presence of saturated acetone or methanol, followed by dissolution of the precipitate in water; - Alternatively, preferably, water is added in a proportion equal to 1 / 10 of the reaction amount, and the pH of the reaction medium is adjusted to 9 with a basicizing agent such as sodium hydroxide (3M).

[0044] In certain embodiments, the solution of the sulfated polysaccharide derivative is preferably dialyzed to remove various salts and then freeze-dried. The final product (i.e., low molecular weight persulfated polysaccharide) generally having a precise molecular weight and a low polydispersity index is obtained by isolation from the resulting low molecular weight depolymerized EPS. Isolation can be carried out by any suitable method known in the art. Preferably, isolation is carried out by fractionation performed by size exclusion chromatography.

[0045] The low molecular weight persulfated polysaccharides according to the present invention have a low polydispersity index of less than 5, preferably 1.5 to 4, and more preferably less than 2. The polydispersity index (PDI), as used herein, is a measure of the distribution of molecular mass of an EPS derivative. The PDI to be calculated is the mass-average molecular weight divided by the number-average molecular weight. PDI is commonly measured by size exclusion chromatography.

[0046] The low molecular weight persulfated polysaccharides according to the present invention have a degree of sulfate group substitution of 10% to 55% by mass compared to the total mass of the sulfated polysaccharide derivative. In certain embodiments, the degree of sulfate group substitution is 10% to 40%, 20% to 45%, or 20% to 40%. In other embodiments, the degree of sulfate group substitution is 30% to 60%, 40% to 55%, or about 50%.

[0047] In certain embodiments, the low molecular weight persulfated polysaccharide is prepared from natural GY785 EPS secreted by strain GY785 (Alteromonas infernus of the genus Alteromonas), having a molecular weight of about 6 kDa to about 10 kDa or about 7 kDa to about 9 kDa, and a degree of sulfate substitution of about 30% to about 40% by mass compared to the total mass of the persulfated polysaccharide. In certain embodiments, the low molecular weight persulfated polysaccharide is GYS8, which is prepared from natural GY785 EPS, has a molecular weight of about 8 kDa, and a degree of sulfate substitution of about 36% by mass compared to the total mass of the persulfated polysaccharide.

[0048] In other embodiments, the low molecular weight persulfated polysaccharide is prepared from natural HE800 EPS secreted by the HE800 strain (Vibrio diabolix of the genus Vibrio), having a molecular weight of about 3 kDa to about 7 kDa or about 4 kDa to about 6 kDa, and a degree of sulfate substitution of about 45% to 55% by mass compared to the total mass of the persulfated polysaccharide. In a particular embodiment, the low molecular weight persulfated polysaccharide is HES5.1, prepared from natural HE800 EPS, having a molecular weight of about 5.1 kDa, and a degree of sulfate substitution of about 50% by mass compared to the total mass of the persulfated polysaccharide.

[0049] II. Uses of low molecular weight persulfated polysaccharides 1. Efficacy The inventors have demonstrated that the low molecular weight persulfated polysaccharides described herein are heparanase (HSPE) inhibitors (i.e., exhibit anti-heparanase activity). Therefore, due to their anti-HSPE activity, the above low molecular weight persulfated polysaccharides, particularly HES5.1 and GYS8, can be used in the treatment or prevention of mucopolysaccharidosis, preferably type III mucopolysaccharidosis, in subjects. The terms “mucopolysaccharidosis” and “MPS” are used interchangeably herein. They refer to a subgroup of lysosomal storage disorders characterized by the accumulation and storage of glycosaminoglycans (GAGs) in lysosomes. Mucopolysaccharidosis may be MPS IH / S (Hurler / Scheyet syndrome), MPS IH (Hurler syndrome), MPS IS (Scheyet syndrome), MPS II (Hunter syndrome), MPS III (Sanfilippo syndrome), MPS IV (Morcchio syndrome), MPS IX (hyaluronidase deficiency or Natowicz syndrome), MPS VII (Sly syndrome), or MPS VI (Maroto-Lamy syndrome). Preferably, the mucopolysaccharidosis is MPS III (Sanfilippo syndrome). MPS III is characterized by the presence of undegraded heparan sulfate due to the deficiency of one of the four enzymes necessary for its catabolism, which are responsible for one of the four subtypes of MPS III: type IIIA (heparan sulfamidase), type IIIB (alpha-N-acetylglucosaminidase), type IIIC (alpha-glucosaminide-N-acetyltransferase), and type IIID (N-acetylglucosamine-6-sulfatase).

[0050] The treatment methods of the present invention can be achieved using low molecular weight persulfated polysaccharides or their pharmaceutical compositions described herein. These methods generally involve administering an effective amount of a low molecular weight persulfated polysaccharide (the above, particularly HES5.1 or GYS8) or its pharmaceutical composition to a subject requiring such administration. Administration can be carried out using any method known to those skilled in the art. In particular, low molecular weight persulfated polysaccharides or their compositions can be administered by any of a variety of routes, including, but not limited to, spray, parenteral, oral, or topical routes.

[0051] Preferably, the subjects are MPS III patients. In the embodiment of the present invention, the MPS III disorder may be subtype A, B, C, or D. In certain embodiments, the MPS III disorder is MPS IIIA or MPS IIIB.

[0052] Generally, low molecular weight persulfated polysaccharides or compositions thereof are administered in an effective dose, i.e., a sufficient amount to serve its intended purpose. The exact amount to be administered of low molecular weight persulfated polysaccharides or pharmaceutical compositions varies depending on the subject, such as the subtype of MPS III disorder, the age, sex, weight, and general health status of the person being treated, and the desired biological or medical response. In certain embodiments, the effective dose is one that blocks, delays, and / or reduces the possibility of at least one symptom associated with MPS disorder. For example, in the case of MPS III disorder, the symptoms may be behavioral problems (tantrums, hyperactivity, destructive behavior, aggressive behavior, pica, sleep disturbances, seizures), gait problems, stiff joints, visual and / or hearing impairments, difficulty communicating, etc. The effects of the treatment according to the present invention can be monitored using any of the diagnostic assays, tests, and procedures known in the art.

[0053] In certain embodiments, the low molecular weight persulfated polysaccharides or compositions thereof described herein are administered alone by the method according to the present invention. In other embodiments, the low molecular weight persulfated polysaccharides or compositions thereof are administered in combination with at least one additional therapeutic agent or therapeutic procedure. The low molecular weight persulfated polysaccharides or compositions thereof may be administered before the administration of the therapeutic agent or procedure, simultaneously with the therapeutic agent or procedure, and / or after the administration of the therapeutic agent or procedure.

[0054] Therapeutic agents that can be administered in combination with the low molecular weight persulfated polysaccharides or compositions thereof described herein can be selected from a variety of bioactive compounds known to have beneficial effects in the management of MPS disorders, particularly MPS III disorders (e.g., anti-inflammatory agents, immunomodulators, analgesics, antimicrobial agents, antibacterial agents, antibiotics, antioxidants, bactericidal agents, antiseizure drugs, pharmaceuticals for heart disease, and combinations thereof). Therapeutic procedures that can be performed in combination with the administration of low molecular weight persulfated polysaccharides or compositions thereof include, but are not limited to, orthopedic surgery to correct joint abnormalities, corneal transplants for visual impairments, and correction of hearing impairments. Other therapies used to manage the symptoms of Sanfilippo syndrome include speech therapy, occupational therapy, physiotherapy, and behavioral therapy.

[0055] 2-Administration The desired dosage of the low molecular weight persulfated polysaccharides described herein (after formulation with one or more suitable pharmaceutically acceptable carriers or excipients as needed) can be administered to the subject requiring it by any preferred route. Various delivery systems are known and can be used to administer the exopolysaccharide derivatives of the present invention, including tablets, capsules, injectable solutions, liposomes, microparticles, microcapsules, etc. Methods of administration include, but are not limited to, transdermal, intradermal, intramuscular, intraperitoneal, intrafocal, intravenous, subcutaneous, intranasal, pulmonary, epidural, ocular, and oral routes. The low molecular weight persulfated polysaccharides or compositions thereof described herein can be administered by adsorption through the epithelium or cutaneous mucosa (e.g., mouth, mucosa, rectal, and intestinal mucosa) by any convenient or other suitable route, e.g., by infusion or bolus injection. Administration may be systemic or topical. Parenteral administration can be directed to a given tissue of the patient, for example, by catheter introduction. As will be understood by those skilled in the art, in embodiments in which a low molecular weight persulfated polysaccharide is administered together with an additional therapeutic agent, the exopolysaccharide derivative and the therapeutic agent may be administered by the same route (e.g., orally) or by different routes (e.g., orally and intravenously).

[0056] 3. Dosage The administration of the low molecular weight persulfated polysaccharides (or compositions thereof) described herein is such that the amount delivered is effective for the intended purpose. The route of administration, formulation, and dosage administered depend on the desired therapeutic effect, the severity of the disorder being treated, the presence of any infections, the patient's age, sex, weight, and general health status, as well as the potency, bioavailability, and in vivo half-life of the low molecular weight persulfated polysaccharide, the use (or non-use) of concurrent therapy, and other clinical factors. These factors can be readily determined by the attending physician during therapy. Alternatively, or additionally, the dosage administered can be determined from studies using animal models. Adjusting the dose to achieve maximum efficacy based on these or other methods is well known in the art and is within the capabilities of a skilled physician. As studies are performed using the low molecular weight persulfated polysaccharides described herein, further information will emerge regarding appropriate dosage levels and durations of treatment.

[0057] The treatment according to the present invention may consist of a single dose or multiple doses. Accordingly, the administration of the low molecular weight persulfated polysaccharide or composition thereof described herein may be constant for a certain period of time or periodic, and may be at specific intervals, e.g., every hour, daily, weekly (or any other multi-day interval), monthly, yearly (e.g., in a time-release form). Alternatively, delivery may occur multiple times during a given period, e.g., twice a week or more, twice a month or more, etc. Delivery may be continuous for a period of time, e.g., intravenous delivery.

[0058] III-Pharmaceutical Composition As noted above, the low molecular weight persulfated polysaccharides described herein can be administered on their own or as part of a pharmaceutical composition. Accordingly, the present invention provides a pharmaceutical composition comprising an effective amount of a low molecular weight persulfated polysaccharide (particularly HES5.1 or GYS8) and at least one pharmaceutically acceptable carrier or excipient. In some embodiments, the composition further comprises one or more additional biological agents.

[0059] The low molecular weight persulfated polysaccharides or their pharmaceutical compositions described herein may be administered in any amount and using any route of administration effective to achieve the desired prophylactic or therapeutic effect. The optimal pharmaceutical formulation can be varied depending on the route of administration and the desired dosage. Such formulations can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the administered active ingredient.

[0060] The pharmaceutical compositions of the present invention can be formulated in dosage units for ease of administration and uniformity of dosage. As used herein, the term "dosage unit" refers to a physically individual unit for a patient being treated. However, the total daily dose of the composition is understood to be determined by the attending physician within the bounds of sound medical judgment.

[0061] 1. Formulation Injectable formulations, such as sterile injectable aqueous or oily suspensions, can be formulated by known techniques using suitable dispersants or wetting agents and suspending agents. Sterile injectable formulations may be sterile injectable solutions, suspensions, or emulsions in non-toxic, parenterally acceptable diluents or solvents, such as solutions in 2,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's Solution USP, and isotonic sodium chloride solutions. Furthermore, sterile non-volatile oils are conventionally used as solutions or suspensions. For this purpose, any non-irritating non-volatile oil, including synthetic mono- or diglycerides, can be used. Fatty acids such as oleic acid can also be used in the preparation of injectable formulations. Sterile liquid carriers are useful in sterile liquid compositions for parenteral administration.

[0062] Injectable formulations can be sterilized, for example, by filtration using a bacterial-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium before use. Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be administered, for example, by intravenous, intramuscular, intraperitoneal, or subcutaneous injection. The injection may be by a single push or by stepwise infusion. If necessary or desired, the composition may contain a local anesthetic to reduce pain at the injection site.

[0063] To extend the effects of the active ingredient, it is often desirable to slow down the absorption of the ingredient from subcutaneous or intramuscular injection. Slowing down the absorption of parenterally administered active ingredients can be achieved by dissolving or suspending the ingredient in an oily vehicle. Injectable depot formulations are prepared by forming a microencapsulation matrix of the active ingredient in a biodegradable polymer, such as polylactide-polyglycolide. The rate of ingredient release can be controlled by the ratio of the active ingredient to the polymer and the properties of the specific polymer used. Other examples of biodegradable polymers include poly(orthoesters) and poly(anhydride). Depot injection formulations can also be prepared by incorporating the active ingredient into liposomes or microemulsions that are compatible with body tissues.

[0064] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, elixirs, and compressed compositions. In addition to the low molecular weight persulfated polysaccharides described herein, liquid dosage forms may include inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoic acid, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed, peanut, maize, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, oral compositions may also include adjuvants, such as wetting agents, suspending agents, preservatives, sweeteners, flavorings and fragrances, thickeners, colorants, viscosity modifiers, stabilizers, or osmotic modifiers. Suitable liquid carriers for oral administration include water (which may contain the above-mentioned additives, e.g., cellulose derivatives such as carboxymethylcellulose sodium solution), alcohols (monohydric and polyhydric alcohols, e.g., glycols) and their derivatives, and oils (e.g., coconut oil and peanut oil). For compressed compositions, the liquid carrier may be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.

[0065] Solid dosage forms for oral administration include, for example, capsules, tablets, pills, powders, and granules. In such solid dosage forms, the low molecular weight persulfated polysaccharides described herein are accompanied by at least one inert, pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate, and: (a) fillers or bulking agents, such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and gum arabic; (c) humectants such as glycerol; and (d) disintegrants, such as agar and carbonate. (e) Dissolution retarders such as paraffin; absorption enhancers such as quaternary ammonium compounds; (g) Wetting agents such as cetyl alcohol and glycerol monostearate; (h) Absorbents such as kaolin and bentonite clay; and (i) Lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate and mixtures thereof, may be mixed with one or more of these. Other excipients suitable for solid formulations include surface modifiers, such as nonionic and anionic surface modifiers. Representative examples of surface modifiers include, but are not limited to, poloxamer 188, benzalkonium chloride, calcium stearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, colloidal silicon dioxide, phosphates, sodium dodecyl sulfate, magnesium aluminum silicate and triethanolamine. In the case of capsules, tablets, and pills, the dosage form may also include a buffering agent.

[0066] Similar types of solid compositions can also be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycol. Solid dosage forms of tablets, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, controlled-release coatings, and other coatings well known in the pharmaceutical technology field. They may optionally contain opacifiers, and they may be compositions that release only the active ingredient, preferably at a specific site in the intestinal tract, and optionally with a delay. Examples of embedding compositions that can be used include polymers and waxes.

[0067] In certain embodiments, it may be desirable to administer the composition of the invention topically to a specific area. This can be achieved, for example, by topical injection, topical application, injection, catheter, suppository, or skin patch, stent, or other implant, but is not limited to these.

[0068] For topical administration, the composition is preferably formulated as a gel, ointment, lotion, or cream containing a carrier such as water, glycerol, alcohol, propylene glycol, fatty alcohol, triglycerides, fatty acid esters, or mineral oil. Other topical carriers include liquid petroleum, isopropyl palmitate, polyethylene glycol, ethanol (95%), polyoxyethylene monolaurate in water (5%), or sodium lauryl sulfate in water (5%). Other substances such as antioxidants, humectants, viscosity stabilizers, and similar agents may be added as needed.

[0069] Furthermore, in certain cases, it is anticipated that the compositions of the invention may be placed in transdermal devices positioned on, in, or beneath the skin. Such devices include patches, implants, and injections that release the active ingredient by a passive or active release mechanism. Transdermal administration includes all administrations that cross the body surface and the inner lining of passages in the body, including epithelial and mucous membrane tissues. Such administrations can be carried out using the compositions in the form of lotions, creams, foams, patches, suspensions, liquids, and suppositories.

[0070] Transdermal administration can be achieved through the use of transdermal patches containing the active ingredient (i.e., low molecular weight persulfated polysaccharides as described herein) and a skin-safe carrier, enabling delivery of the ingredient to the bloodstream for systemic absorption through the skin. The carrier can take any number of forms, such as creams and ointments, pastes, gels, and occlusion devices. Creams and ointments may be oil-in-water or water-in-oil viscous liquids or semi-solid emulsions. Pastes consisting of absorbent powder dispersed in petroleum or hydrophilic petroleum containing the active ingredient may be preferred. Various occlusion devices, such as semipermeable membranes covering a reservoir containing the active ingredient or a matrix containing the active ingredient, can be used to release the active ingredient into the bloodstream, with or without a carrier.

[0071] Suppository formulations can be made from traditional materials containing cocoa butter, with or without the addition of waxes and glycerin to alter the melting point of the suppository. Water-soluble suppository bases such as polyethylene glycol of various molecular weights can also be used.

[0072] Substances and methods for producing various formulations are known in the art and can be adapted to carry out the invention in question. Suitable formulations for antibody delivery can be found, for example, in "Remington's Pharmaceutical Sciences," E.W. Martin, 18th edition, 1990, Mack Publishing: Easton, PA.

[0073] 2. Additional biological activators In certain embodiments, the low molecular weight persulfated polysaccharides described herein are the sole active ingredient in the pharmaceutical composition of the present invention. In other embodiments, the pharmaceutical composition further comprises one or more biological activators. Examples of suitable biological activators include, but are not limited to, anti-inflammatory agents, immunomodulators, analgesics, antimicrobial agents, antibacterial agents, antibiotics, antioxidants, bactericides, antiseizure agents, pharmaceuticals for heart disease, and combinations thereof.

[0074] In such pharmaceutical compositions, the low molecular weight persulfated polysaccharides and at least one additional therapeutic agent described herein can be combined into one or more formulations for simultaneous, separate, or sequential administration of the low molecular weight persulfated polysaccharides and therapeutic agents. More specifically, the compositions of the invention can be formulated in such a way that the low molecular weight persulfated polysaccharides and therapeutic agents can be administered together or independently of each other. For example, the low molecular weight persulfated polysaccharides and therapeutic agents can be formulated together in a single composition, or they can be kept (e.g., in different compositions and / or containers) and administered separately.

[0075] 3. Medical pack or kit In another embodiment, the present invention provides a pharmaceutical pack or kit comprising one or more containers (e.g., vials, ampoules, test tubes, flasks, or bottles) containing one or more components of the pharmaceutical composition of the invention, enabling the administration of the low molecular weight persulfated polysaccharide described herein.

[0076] The different components of a pharmaceutical pack or kit can be supplied in solid (e.g., lyophilized) or liquid form. Each component is generally suitable for being divided equally into its respective container or provided in a concentrated form. The pack or kit according to the present invention may include a medium for reconstituting the lyophilized components. The individual containers of the kit are preferably kept sealed for commercial purposes.

[0077] In certain embodiments, the pack or kit includes one or more additional therapeutic agents. If necessary, a cautionary note or package insert in the form prescribed by the government agency regulating the manufacture, use, or sale of the pharmaceutical or biological product may be attached to the container, and such cautionary note shall reflect the approval by the agency for manufacture, use, or sale for human administration. The cautionary note in the package insert may include instructions for the use of the pharmaceutical composition according to the treatment methods disclosed herein.

[0078] Identifiers, such as barcodes, high-frequency signals, ID tags, etc., may be present in or on the kit. These identifiers can be used to specifically identify the kit for purposes such as quality control, inventory management, and tracking between workstations.

[0079] Further aspects and advantages of this invention are disclosed in the following drawings and examples, which are illustrative and should be considered not to limit the scope of this application. [Examples]

[0080] The following examples describe some preferred modes for fabricating and carrying out the present invention. However, it should be understood that these examples are for illustrative purposes only and do not limit the scope of the present invention. Furthermore, unless the descriptions in the examples are presented in the past tense, the text, as with the rest of the specification, does not imply that the experiments were actually performed or that the data were actually obtained.

[0081] Working hypothesis Figure 1 presents a schematic model of heparan sulfate (HS) proteoglycan and heparanase (HPSE) trafficking. In this scheme: 1. In the Golgi apparatus, HS chains are polymerized, and pre-HPSE is processed to produce pro-HPSE by elimination of the N-terminal signal peptide. 2. The newly biosynthesized HSPGs are then transferred to the cell membrane where they can interact with pro-HPSE. 3. The complex is rapidly internalized by endocytosis and then 4. accumulated in late endosomes. 5. Upon fusion of late endosomes with lysosomes, pro-HPSE is activated and cleaves HS chains, which are then completely degraded by lysosomal hydrolase. 6. HPSE and HSPGs can be recycled from endosomes to the cell surface. Activated HPSE appears to pursue other pathways within the cell. 7. Trimming of HS from syndecane by activated HPSE present in late endosomes leads to the formation of the syndecane-syntenin-ALIX complex. Endosomal membrane invagination subsequently leads to the formation of intraluminal vesicles (ILVs) and then multivesicular bodies (MVBs). The MVBs release ILVs as exosomes as a result of fusion with the cell membrane, delivering their cargo to recipient cells. In the presence of high levels of HPSE, the enzyme can be found on the surface of exosomes and modulates the tumor microenvironment. 8. Lysosomal exocytosis has been observed in malignant cells. 9. HPSE also regulates autophagy by promoting the fusion of lysosomes with autophagosomes, which break down macromolecules into monomeric units. 10. Perinuclear lysosomal HPSE can also translocate to the nucleus and regulate gene transcription and cell differentiation.

[0082] The working hypothesis was that inhibition of HPSE by exopolysaccharide derivatives could alter the ebb and flow of HS catabolism in Sanfilippo syndrome by preventing lysosomal degradation of HPSE cleavage fragments of HS in the presence of defective degrading enzymes.

[0083] Exopolysaccharide derivatives As previously mentioned, bacterial GY785 and HE800 exopolysaccharides (EPS) were produced, purified, and characterized (Guezennec et al., Carbohydr. Polym., 1998, 37: pp. 19-24). The preparation, purification, and characterization of low molecular weight persulfated EPS derivatives were carried out as previously described (Ruiz Velasco et al., Glycobiology, 2011, 21: 781-795; WO 2006 / 003290; Colliec-Jouault et al., Biochim. Biophys. Acta, 2001, 1528: pp. 141-151; WO 2007 / 066009; WO 02 / 02051; Guezennec J. et al., Source: Carbohydrate Polymers 1998, 37(1): pp. 19-24; Senni et al., Mar. Drugs, 2013, 11: pp. 1351-1369; Merceron et al., Stem Cells, 2012, 30: pp. 471-480; Senni et al., Mar. Drugs, 2011, 9: pp. 1664-1681; Heymann et al., Molecules, 2016, 21:309). Briefly, natural high molecular weight GY785 EPS and HEP800 were first depolymerized using a free radical depolymerization process to obtain low molecular weight derivatives of different molecular weights. These low molecular weight GY785 and HEP800 EPS derivatives were then sulfated in dimethylformamide (DMF) using pyridine sulfate as a sulfating agent to yield low molecular weight persulfated polysaccharides. The molecular weight (MW) before and after sulfated polysaccharides was determined by HPSEC-MALS and sulfur content (mass %) by HPAEC chromatography. ATR-FTIR and NMR spectroscopy were used to investigate the efficiency of the sulfated reaction. Heparin sodium salt H4784 from porcine intestinal mucosa was purchased from Sigma.

[0084] (Example 1) Anticoagulant and antiheparanase activity Endoglucuronidase heparanase (HPSE) is the first enzyme to break down heparan sulfate in its catabolic pathway, cleaving large segments of the HS chain that are subsequently depolymerized within lysosomes by exoglycosidase. HS-6-O-sulfatases 1 and 2 (Sulf1 and Sulf2) remove sulfate groups from glucosamine units, modifying the charge distribution in HS and influencing their interactions with ligand proteins such as growth factors. The goal of the first experiment was to determine whether low molecular weight persulfated EPS derivatives could inhibit the enzymes heparanase and sulfatase that modify HS chains after synthesis.

[0085] result Anticoagulant and antiheparanase activity of exopolysaccharide derivatives. In collaboration with Dr. Jin-ping Li (Uppsala University, Sweden), the inventors determined the antiheparanase (anti-HPSE) activity of different exopolysaccharide (EPS) derivatives. The results are presented in Table 1 below. Anti-HPSE activity was found to be charge-dependent of the EPS derivative, but was not significantly affected by the size of the EPS derivative contained in 5,000–16,000 Da. IC of EPS derivatives 50 The value was determined to be 1–5 μM (see Table 1). As a result, the tested EPS derivatives show potent potential as HPSE inhibitors.

[0086] Furthermore, in collaboration with Dr. Romain Vives (Structure and Activites des Glycosaminoglycanes, Institut de Biologie Structurale de Grenoble), the inventors found that EPS derivative studies also inhibit 6-O-endosulfatases (Sulf1 and Sulf2), which are enzymes that modify the sulfation pattern of HS and thus modulate their affinity for ligand proteins.

[0087] [Table 1]

[0088] (Example 2) Marine bacterial exopolysaccharide derivatives as possible treatments for Sanfilippo syndrome Two low molecular weight persulfated polysaccharides, HES5.1 (A5_3) and GYS8 (A5_4), which were found to exhibit the most interesting properties in Example 1, were further investigated to explore their ability to act as treatments for Sanfilippo treatment. Based on the working hypothesis that inhibition of the initial cleavage of the HS chain prevents lysosomal degradation of the fragment, exopolysaccharide derivatives should be active in treating all forms of Sanfilippo syndrome (MPSIII). The inventors studied MPS III subtype A (MPSIIIA), one of the MPSIII disorders in which the enzyme HS-sulfamidase is inactivated by mutation. According to the hypothesis, inhibition of HPSE by low molecular weight persulfated polysaccharides should modify the balance of HS catabolism by preventing lysosomal degradation of HPSE cleavage fragments in the context of defective catabolic enzymes.

[0089] The inventors studied in vitro using fibroblasts derived from a mouse model of MPSIIIA (in collaboration with Dr. K. Hemsley, Adelaide, Australia) (Crawley et al., Brain Res., 2006, 1104: 1-17). They purified HS in the extracellular compartment (medium plus surface) and intracellular compartment and characterized their size and charge by gel electrophoresis (PAGE) and gel filtration (GFC). They analyzed the effect of treatment with low molecular weight persulfated polysaccharides on these parameters. For each of the exopolysaccharide derivatives, two independent experiments were performed starting from seeding of the cells to isolation of HS and final analysis of the size (length / molecular weight) of HS.

[0090] Results Analysis of the amount of heparan sulfate (HS). In each experiment, the cells were counted and the results were normalized to the total number of cells. To further normalize the results, 35A correction was made to the SO4 incorporation level, which was calculated as the ratio between the total radioactivity recovered and measured and the initial radioactivity administered to the cells. In this experiment, incorporation levels were found to range from 1.2% to 5.6%.

[0091] Observation of the effect on proteoglycans: The first purification step in size exclusion chromatography is free Na2 35 This enabled the isolation of high molecular weight species from SO4. The total amount of sulfated proteoglycans (PGs) was considered as the material recovered from the first purification step and was expressed in cpm (radioactivity). For both exopolysaccharide derivatives (HES5.1(A5_3) and GYS8(A5_4)), the total amount of PGs was found to be similar in untreated control MPSIIIA cells and treated MPSIIIA cells (see Figure 2).

[0092] By observing the distribution of PG in more detail, the inventors observed a greater amount of proteoglycans in the extracellular compartment than in the corresponding intracellular compartment (see Figure 3). The variability between control and treated cells was not significant, meaning that EPS derivative treatment did not affect the total amount and distribution of PG produced by the cells.

[0093] Observation of the effect of heparan sulfate: Heparan sulfate was isolated from both extracellular and intracellular fractions, and it represented a small portion of the total glycosaminoglycans present in the cells. In fact, the percentage of HS in total PG was found to range from 12% to 37% in control cells. The inventors calculated the percentage of HS relative to PG in the corresponding fraction, assuming the amount of PG to be 100% (see Figure 4). In control cells, HS was determined to correspond to (29±11)% of intracellular PG, (16±4)% of extracellular PG, and (20±5)% of total PG (n=8). In general, the distribution of HS and the total percentage of HS differed, but there were no statistically significant differences between control and treated cells. There was a tendency toward an increase in HS in the extracellular compartment when cells were treated with A5_4 (GYS8) rather than A5_3 (HES5.1).

[0094] In summary, it can be concluded that treatment of MPSIIIA cells with exopolysaccharide derivatives does not alter the total amount and distribution of proteoglycans. While the percentage of total heparan sulfate fluctuates, it is not significantly affected by EPS derivative treatment.

[0095] The following steps in the analysis involved examining the dimensions (length / molecular weight) of heparan sulfate, based on the idea that treatment with low molecular weight persulfated polysaccharides would affect the first step of HS degradation, and therefore higher molecular weight HS chains would be found in the intracellular compartment.

[0096] Analysis of the molecular weight of heparan sulfate (HS) in control cells. Wild-type cells produce full-size HS with a molecular weight exceeding 20 kDa, which is assembled in the intracellular compartment and expressed on the cell surface as a proteoglycan (Colliec-Jouault et al., J. Biol. Chem., 1994, 269: pp. 24953-24958). Lysosomal catabolism of HS is rapid, and the low molecular weight (LMW) intermediate is transient and undetectable (Yanagishita and Hascall, Proteoglycan metabolism by rat ovarian granulosa cells in vitro. Source: Wight, Mecham RP (eds.), Biology of the proteoglycans, 1st ed. Orlando: Academic Press; 1987: pp. 105-128). In MPSIIIA cells, intracellular HS showed a widely diffused distribution in the LMW region and partially degraded HS fragments accumulating in lysosomes due to dysfunctional sulfamidase, as confirmed by PAGE-NaCl electrophoresis (see Figure 5). By comparing the sample with glycosaminoglycan standards (Mulloy et al., Thromb Haemost. 1997, 77(4): pp. 668-674), it was possible to estimate the molecular weight of extracellular HS concentrated in the high molecular weight region (>20 kDa). In contrast, widespread HS fragments accumulating in the LMW region (<20 kDa) could be observed in intracellular HS, resulting in smears on the gel.

[0097] The inventors then decided to use a different technique, choosing gel filtration chromatography (GFC) on CL6B resin equilibrated in PBS, 0.15 M NaCl, pH 7.4. While molecular separation by PAGE depends not only on molecular weight but also on the overall negative charge, separation by GFC depends largely on the molecular size. Figure 6 shows examples of calibration curves obtained using standards, as well as profiles of intracellular and extracellular HS for control. A clear peak in intracellular HS was observed with GFC. Molecular weights and distributions are presented in Table 2. The mean MW of extracellular HS calculated by PAGE (n=8) was found to be 37.1 kDa, ranging from 24 to 50.7 kDa, while that calculated by GFC (n=3) was 43 kDa, ranging from 17.7 to 116.7 kDa. Intracellular hematopoiesis (HS) was well determined by GFCs alone, with an average MW of 7.9 kDa, but fragments ranged from 3.5 to 28 kDa (n=5).

[0098] [Table 2]

[0099] Analysis of the molecular weight of heparan sulfate (HS) in treated cells. The effect of treatment with low molecular weight persulfated polysaccharides on MPSIIIA cells was that HS remained undegraded from treated cells, while MW blocked the partial degradation of intracellular HS, making it similar to extracellular species (>20 kDa). Three concentrations of EPS derivatives were tested: 20, 50, and 100 μg / ml. Cells were treated for 4 days before adding the radioactive sulfate donor and collected after 24 hours.

[0100] Figures 7 and 8 show examples of PAGE NaCl gels obtained from control and A5_3(HES5.1) treated cells, and control and A5_4(GYS8) treated cells, respectively. Control cells exhibit low intracellular HS and high extracellular HS. Treated cells clearly show higher intracellular HS (>20 kDa) compared to untreated cells. The molecular weight of extracellular HS is the same for treated and untreated cells. The observed effects were similar across the three concentrations used.

[0101] Figure 9 shows the GFC profiles of HS from control and treated (20 μg / ml A5_3(HES5.1)) cells. As observed by the superimposed GFC profiles, extracellular HS were unaffected by the treatment. The treatment affected intracellular HS: a clear shift to higher MW (>20 kDa) was observed due to the treatment, and the MW of treated HS was similar to that of extracellular high molecular weight HS.

[0102] Figure 10 shows the GFC profiles of HS from control and treated (20 μg / ml A5_4(GYS8)) cells. The effect of A5_4 treatment is similar to that of A5_3: extracellular HS are unaffected by treatment with low molecular weight persulfated polysaccharides, but intracellular HS shift to higher MW (>20 kDa).

[0103] Tables 3 and 4 summarize the results of PAGE and GFC analysis. Both techniques demonstrate the specificity of the treatment, which affects only intracellular hemoglobin (HS). The effects are similar across all three concentrations tested.

[0104] [Table 3]

[0105] [Table 4]

[0106] Another way to represent the MW distribution of HS is to look at the percentage of chains before and after a certain threshold. For simplicity, the inventors defined this as the peak of extracellular HS in the PAGE profile corresponding to the peak of intracellular HS after treatment, as shown in Figure 11 and Table 5. Since the effects were found to be similar at the three concentrations tested, the results are expressed as the average of the three concentrations. Clearly, the percentage of high molecular weight HS after treatment with A5_3 (HES5.1) or A5_4 (GYS8) reaches the value of extracellular control HS.

[0107] [Table 5]

[0108] conclusion Both A5_3(HES5.1) and A5_4(GYS8) were shown to affect HS turnover and thus lead to incomplete degradation of intracellular HS. Similar effects were observed when cells were treated with different concentrations of each low molecular weight persulfated polysaccharide, indicating that 20 μg / ml of the EPS derivative is effective, and possibly even lower concentrations are possible. According to the initial working hypothesis, intact HS chains not cleaved by HPSE cannot enter the lysosomal degradation pathway and are redirected to the extracellular space for clearance in the blood and urine. Thus, cells can be protected from toxic lysosomal HS accumulation. As a result, both A5_3(HES5.1) and A5_4(GYS8) have potent potential for the treatment of patients with Sanfilippo syndrome. Treatment with these EPS derivatives may alleviate symptoms caused by lysosomal overload due to incompletely degraded heparan sulfate.

[0109] Throughout this application, various references describe the latest technology relating to the present invention. The information disclosed in these references is incorporated herein by reference.

Claims

1. A low molecular weight persulfated polysaccharide having anti-heparanase activity for use in the prevention or treatment of mucopolysaccharidosis in the subject, which is a derivative of a natural exopolysaccharide (EPS) secreted by mesophilic marine bacteria from deep-sea hydrothermal environments, and the following steps: (a) A process comprising free radical depolymerization of marine native EPS from Alteromonas strain GY785 or Vibrio diabolix strain HE800 to obtain depolymerized EPS having a molecular weight of 5,000 to 100,000 g / mol; (b) A subsequent step comprising sulfation of depolymerized EPS to obtain persulfated and depolymerized EPS, comprising adding at least one sulfating agent to the depolymerized EPS in an amount sufficient to obtain a sulfated polysaccharide having a degree of sulfate substitution of 10% to 55% by mass relative to the total mass of the persulfated and depolymerized EPS; and (c) A subsequent step comprising isolating low molecular weight persulfated polysaccharides from persulfated and depolymerized EPS, wherein the low molecular weight persulfated polysaccharides have a molecular weight of approximately 5,000 to approximately 16,000 g / mol. Low molecular weight persulfated polysaccharides obtained using a method including [a specific method].

2. The low molecular weight persulfated polysaccharide according to claim 1, wherein in step (a), the free radical depolymerization is carried out with natural GY785 EPS secreted by the GY785 strain, and the molecular weight is about 6 kDa to about 10 kDa or about 7 kDa to about 9 kDa, and the degree of sulfate group substitution is about 30% to about 40% by mass compared to the total mass of the persulfated polysaccharide.

3. The low molecular weight persulfated polysaccharide according to claim 2, wherein GYS8 has a molecular weight of approximately 8 kDa and a degree of sulfate group substitution of approximately 36% by mass compared to the total mass of the persulfated polysaccharide.

4. The low molecular weight persulfated polysaccharide according to claim 1, wherein in step (a), the free radical depolymerization is carried out with natural HE800 EPS secreted by the HE800 strain, and the molecular weight is about 3 kDa to about 7 kDa or about 4 kDa to about 6 kDa, and the degree of sulfate group substitution is about 45% to about 55% by mass compared to the total mass of the persulfated polysaccharide.

5. The low molecular weight persulfated polysaccharide according to claim 4, which is HE5.1 having a molecular weight of approximately 5.1 kDa and a degree of sulfate group substitution of approximately 50% by mass compared to the total mass of the persulfated polysaccharide.

6. The low molecular weight persulfated polysaccharide according to any one of claims 1 to 5, wherein the step of isolating the low molecular weight persulfated polysaccharide from the persulfated and depolymerized EPS is carried out by fractionation, in particular by size exclusion chromatography.

7. The low molecular weight persulfated polysaccharide according to any one of claims 1 to 6, wherein the mucopolysaccharidosis is mucopolysaccharidosis type III.

8. The low molecular weight persulfated polysaccharide according to claim 7, wherein the mucopolysaccharide type III is subtype A, subtype B, subtype C, or subtype D.

9. A pharmaceutical composition comprising a therapeutically effective amount of a low molecular weight persulfated polysaccharide having anti-heparanase activity according to any one of claims 1 to 6, and at least one pharmaceutically acceptable carrier or excipient, for use in the prevention or treatment of mucopolysaccharidosis in a subject.

10. The pharmaceutical composition according to claim 9, wherein the mucopolysaccharidosis is mucopolysaccharidosis type III.

11. The pharmaceutical composition according to claim 10, wherein the mucopolysaccharide type III is subtype A, subtype B, subtype C, or subtype D.

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