Cyclodextrin derivatives in the treatment or prevention of lysosomal neurodegenerative diseases

Abstract

The present invention generally relates to the use of single isomer chemically modified cyclodextrins, i.e., S-(carboxyalkyl)-thio-cyclodextrin salts in medicaments for preventing or treating lysosomal storage diseases. More specifically, the present invention relates to isomerically pure, single isomer hexa-S-(carboxyalkyl)-hexathio-α-cyclodextrin sodium salt, heptath-S-(carboxyalkyl)-heptathio-β-cyclodextrin sodium salt, and octa-S-(carboxyalkyl)-octathio-γ-cyclodextrin sodium salt in medicaments for preventing or treating lysosomal storage diseases.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Application No. 17 / 039,483, filed September 30, 2020, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention generally relates to the use of single isomer chemically modified cyclodextrins, ie, S-(carboxyalkyl)-thio-cyclodextrin salts, in medicaments for preventing or treating lysosomal storage diseases.

[0004] More specifically, the present invention relates to isomerically pure, single isomer hexa-S-(carboxyalkyl)-hexathio-α-cyclodextrin sodium salt, heptas-S-(carboxyalkyl)-heptasthio-β-cyclodextrin sodium salt and octa-S-(carboxyalkyl)-octathio-γ-cyclodextrin sodium salt in a medicament for preventing or treating lysosomal storage diseases. Background Art

[0005] Lysosomal storage disease (LSD) is a term for about 50 rare inherited metabolic disorders caused by defects in lysosomal function. Lysosomes are organelles of enzymes within cells that digest macromolecules and pass the fragments to other parts of the cell for recycling. This process requires several enzymes. If one of these enzymes is defective (for example, due to a mutation), macromolecules accumulate within the cell, eventually making it dysfunctional. Lysosomal storage diseases are usually caused by lysosomal dysfunction caused by a deficiency of a single enzyme required for lipid metabolism.

[0006] U18666A is an inhibitor of intracellular cholesterol transport that, due to its multiple actions, has led to important discoveries in lipid research and helped to understand the pathophysiology of a variety of diseases, including LSD. U18666A inhibits oxidosqualene cyclase, leading to the discovery of a pathway for the formation of polar sterols, which have been shown to be important regulators of lipid metabolism. It was recognized that U18666A inhibits cholesterol export from late endosomes and lysosomes, greatly advancing the understanding of the major pathways of cholesterol transport within cells. U18666A inhibition of cholesterol transport mimics the loss of functional protein that causes LSD disease, providing a model for these conditions. U18666A subsequently became a tool for evaluating the importance of molecular transport through the lysosomal pathway in a variety of disease states, such as atherosclerosis, Alzheimer's disease, and prion infection. U18666A also provides animal models for two important diseases: petit mal (absence) epilepsy and cataracts. This compound is the first chronic model of absence epilepsy. U18666A has also been used to address the role of oxidative stress in apoptosis. Therefore, pathological model conditions triggered by U18666A may provide a tool to test various possible therapeutic strategies.

[0007] Cyclodextrin (CD) is a class of cyclic oligosaccharides, which are obtained from the enzymatic conversion of starch, for example, under the action of the bacterium Bacillus macerans, by the action of the enzyme CD glycosyltransferase. CD forms a host-guest complex with a variety of compounds and is commonly used as an excipient. These enzyme-modified starch derivatives are cyclic oligosaccharides in the shape of a torus with a hydrophobic inner cavity and a hydrophilic outer portion. There are three unmodified ("parent") types, namely α-, β- and γ-CD, which are composed of 6, 7 and 8 glucose units, respectively, and have an increased inner cavity diameter. Chemical derivatization of the parent CD is used to change the solubility curve, complexing properties, biodegradability and toxicity.

[0008] Coisne et al. reviewed the use of CD for the treatment of cholesterol-related neurodegenerative diseases (Molecules 2016, 21, 1748). The use of β-CD, differently methylated β-CD, 2-hydroxypropyl β-cyclodextrin (HPBCD), per-6-alkylamino-β-CD, and sulfobutyl ether β-cyclodextrin were discussed. The article constructed a professional bias that the single isomer γ-CD derivative called Sugammadex is not suitable for such treatment: "β-CD has proven to be very useful in treatment because they do not show any hypersensitivity reactions, unlike Sugammadex. This modified single isomer γ-CD used in anesthesia to reverse the effects of neurovascular blocking drugs has caused allergic reactions in some patients."

[0009] Among them, patent application WO2019067269 describes a method for preventing or treating a lysosomal disease or condition in a subject in need thereof, comprising administering an effective amount of CD to the subject. The specification does not provide guidance on the potential uses of isomerically pure S-(carboxyalkyl)-thio-CD, and the disclosed potential therapeutic uses are only demonstrated in the examples of natural (unmodified) α-, β-, γ-CD, 2-hydroxypropyl α-cyclodextrin, HPBCD, and methyl β-cyclodextrin.

[0010] Most of the information related to the use of CD to improve LSD symptoms is the application of cyclodextrins to treat Niemann–Pick disease type C (NPC), a multiorgan storage disorder characterized by the accumulation of unesterified cholesterol (UC) and other lipids in lysosomes. Central nervous system (CNS) neurons widely display multimembranous cytoplasmic storage bodies, and in addition to UC, GM2 and GM3 gangliosides accumulate intracellularly. Patients show progressive neurological decline. Mutations in the NPC1 (approximately 95% of patients) or NPC2 genes lead to the same disease phenotype (Vanier MT. Complex lipid trafficking in Niemann-Pick disease type CJ Inherit Metab Dis 2015;38:187-199). It is believed that these two encoded proteins, i.e., transmembrane NPC1 and soluble luminal NPC2, interact with UC and / or other lipids in a collaborative manner to promote their discharge from late endosome / lysosome (LE / LY) compartments (Infante RE, Wang ML, Radhakrishnan A et al., NPC2 facilitates bidirectional transfer of cholesterol between NPC1 and lipid bilayers, a step in cholesterol egress from lysosomes. Proc Natl Acad Sci USA 2008; 105: 15287-15292). The therapeutic strategy for NPC disease includes drugs to inhibit substrate accumulation, increase the function of defective proteins, and for downstream sequelae such as inflammation and oxidative stress (Rosenbaum AI, Maxfield FR. Niemann-Pick type C disease:molecular mechanisms and potential therapeutic approaches. J Neurochem 2011; 116: 789-795).The most effective therapy to date is HPBCD, which delays clinical onset, prolongs lifespan, and reduces UC and glycolipid accumulation in the CNS and other organs, even after subcutaneous administration to NPC1- or NPC2-deficient mice (Davidson CD et al., PLoS ONE 2009;4:e6951.; Liu B et al., J Lipid Res 2008;3:663-669; Liu B et al., Proc Natl Acad Sci USA 2009;106:2377-2382.; Liu B et al., J Lipid Res 2010;51:933-944).

[0011] Several mechanisms have been proposed for therapeutic correction of Niemann-Pick type C disease by CD, but the main idea is that CD directly replaces the function of NPC proteins in the LE / LY compartment (Chen FW et al., PLoS ONE 2010; 5: e15054, Ramirez CM et al., J Lipid Res 2011; 52: 688-698). Supporting this idea, HPBCD treatment was also found to be effective in mice lacking these two NPC proteins, but not in other diseases with secondary lysosomal storage of functional NPC proteins and cholesterol. Exactly how CD mimics NPC protein function or otherwise mediates CNS correction remains unclear. Almost all treatment-related studies on NPC animal models have used HPBCD, a multi-complex statistically derived β-CD with hydroxypropyl side groups, but little attention has been paid to how different possible chemical derivatizations of CD may affect efficacy. Furthermore, few studies have been conducted on the potential efficacy of any other CDs, and since studies have shown that HPBCD is ototoxic (Ward S et al., Pediatr Res 2010; 68: 52-56, Crumling MA et al., PLoS ONE 2012; 7: e53280), there is a great need to identify safer and more effective alternative CDs (e.g., as shown in Example 1 of the present invention, the superior therapeutic effect of the subject compound was demonstrated even at a reduced concentration of one-tenth compared to the therapeutic effect of HPBCD). One of the most likely reasons for the current use of high-dose CD derivatives (all of which are complex isomer mixtures) is because of the complex nature of these CD derivatives, the truly effective ingredients are unknown and diluted by the close isomeric homologues of the CD derivatives. The use of single isomer CD derivatives provides the possibility of achieving the desired efficacy by administering lower doses. A screening study involving methyl-β-CD, 2-hydroxypropyl α-, β-, and γ-CD, and sulfobutyl ether α-, β-, and γ-CD showed that CDs other than HPBCD can improve disease without ototoxicity and are worthy of long-term treatment studies, with 2-hydroxypropyl γ-CD and sulfobutyl ether γ-CD in particular being found to be effective in the mouse NPC1 model (Davidson, CD et al., Annals of Clinical and Translational Neurology, Vol. 3, No. 5, pp. 366-380, 2016).

[0012] In a Phase III clinical trial, therapeutic doses of HPBCD ( Cyclo TM-CTD Inc.), with a dose of 1500-2500 mg / kg body weight. HPBCD is an orphan drug in the EU and the U.S. Currently, all of these commercially available HPBCD derivatives are complex isomer mixtures, i.e., highly composite materials consisting of thousands of positional isomers, geometric isomers, and even optical isomers.

[0013] Vtesse, Inc. has obtained a patent for a HPBCD composition with a narrower substituent distribution curve isolated from commercial pharmacopoeia grade HPBCD, where the starting material commercial HPBCD meets the requirements of the European Pharmacopoeia and the United States Pharmacopoeia (WO2016201137). The claimed product is a mixture of β-cyclodextrin molecules substituted with hydroxypropyl groups at one or more hydroxyl positions. The mixture may include unsubstituted β-cyclodextrin molecules, wherein the mixture contains less than 1% of unsubstituted β-cyclodextrin ("DS-0") and β-cyclodextrin substituted with one hydroxypropyl group ("DS-1"); the mixture contains at least 85% of β-cyclodextrin substituted with three hydroxypropyl groups ("DS-3"), β-cyclodextrin substituted with four hydroxypropyl groups ("DS-4"), β-cyclodextrin substituted with five hydroxypropyl groups ("DS-5"), and β-cyclodextrin substituted with six hydroxypropyl groups ("DS-6"); the mixture contains less than 1% of β-cyclodextrin substituted with nine hydroxypropyl groups ("DS-9") and β-cyclodextrin substituted with ten hydroxypropyl groups ("DS-10"), as determined by peak height in an electrospray MS spectrum. The inventors propose to use a pharmaceutical composition containing this designated narrow distribution HPBCD (Adrabetadex, VTS-270) for the treatment of Niemann-Pick Type C disease via intrathecal or intracerebroventricular administration. A Phase 2 / 3 clinical trial is ongoing with VTS-270 in individuals aged 2 to 25 years diagnosed with NPC1.

[0014] In addition to HPBCD, the following cyclodextrin derivatives and complexes have been evaluated in preclinical studies for their therapeutic effects on NPC1:

[0015] Oraxion Therapeutics has developed a linear polymer (ORX-301) based on β-cyclodextrin with a molecular weight of about 33 kDa and a biodegradable ketal-type linker. The study showed that subcutaneous injection of ORX-301 at a dose of one-fifth of the HPBCD dose (over 1500 mg / kg) that has been shown to be effective can extend the average lifespan of mice with NPC1 (Kulkarni, A. et al., Sci Rep 8, 9547 (2018)). ORX-301 is in preclinical development.

[0016] Japan Maize Products Co Ltd, Nihon Shokuhin Kako Co Ltd and Kumamoto University NUC filed a patent application (EP3078379A1) for a pharmaceutical composition for treating or preventing lysosomal diseases, the composition comprising hydroxypropyl-γ-cyclodextrin as an active ingredient.

[0017] To overcome the disadvantages of systemic HPBCD therapy, i.e., rapid renal clearance of the therapeutic agent, Egele et al. designed an anionic HPBCD polyrotaxane as a sustained-release formulation based on a polyalkylene phosphate core to improve pharmacokinetics (Egele et al., J Mater Chem B. 2019, 28; 7(4): 528-537). Polyalkylene phosphates contain hydrophobic decamethylene spacers linked by biodegradable anionic phosphodiester bonds. HPBCD was first threaded to the polymer, and then α-CD was threaded to the polymer to prevent sudden release of the threaded HPBCD. The results showed that HPBCD was slowly released from the water-soluble polyrotaxane. Polyrotaxanes sustained a 20% reduction in cholesterol levels in NPC1 cells relative to untreated material. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The effects of different treatments on locomotor activity of 5-day-old zebrafish larvae are shown. *p<0.05; **p<0.01; ***p<0.001. Summary of the invention

[0019] The present disclosure provides a more effective and safer composition as an alternative to the statistically substituted isomer mixtures of different HPBCD species or HPBCD compositions currently used to improve lysosomal storage diseases. The disadvantages associated with the use of HPBCD composites are their high dose / exposure levels (over 1500 mg / kg) and the known ototoxicity associated with their use as a side effect. It is not difficult to understand that this randomly substituted cyclodextrin derivative with a complex isomer mixture, which was originally developed as an excipient and is now also used as an active pharmaceutical ingredient, will produce adverse side effects because it needs to be used at very high doses. Lysosomal storage diseases treated by the present invention include, but are not limited to, the following diseases which can be modeled by a condition triggered with U18666A: aspartylglucosaminuria, Wolman disease, cystinosis, Danon disease, Fabry disease, Farber disease, fucosidosis, Gaucher disease, GM1-gangliosidosis type I / II / III, GM2-gangliosidosis, α-mannosidosis type I / II, β-mannosidosis, metachromatic leukodystrophy, sialidosis type I / II, mucolipidosis type IV, Scheie syndrome, Hunter syndrome, Sanfilippo syndrome A, Sanfilippo syndrome B, Sanfilippo syndrome, Silippo syndrome C, Sanfilippo syndrome D, galactosialidosis type I / II, Krabbe disease, Sandhoff disease, Vogt-Spielmeyer disease, Hurler syndrome, Niemann-Pick disease type C, I-cell disease (mucolipidosis type II), pseudo-Hurler polydystrophy, Morquio syndrome, Maroteaux-Lamy syndrome, Sly syndrome, mucopolysaccharidosis type IX, multiple sulfatase deficiency, Batten disease, Tay-Sachs disease, Pompe disease, Batten disease, Batten disease, late infantile, northern epilepsy type, pycnodystrophy, Schindler disease, sialiduria, and Salla disease.

[0020] We have surprisingly found that the following types of cyclodextrin derivatives are suitable for the purpose of ameliorating the above mentioned malignant conditions:

[0021] S-(Carboxyalkyl)-thio-cyclodextrin salts:

[0022] -6A,6B,6C,6D,6E,7F-hexa-S-(2-carboxyethyl)-6A,6B,6C,6D,6E,6F-hexathio-α-cyclodextrin sodium salt (Sualfadex sodium)

[0023] -6A,6B,6C,6D,6E,7F,6G-Septa-S-(2-carboxyethyl)-6A,6B,6C,6D,6E,6F,6G-heptathio-β-cyclodextrin sodium salt (Subetadex sodium)

[0024] -6A,6B,6C,6D,6E,7F,6G,6H-Octathio-S-(2-carboxyethyl)-6A,6B,6C,6D,6E,6F,6G,6H-octathio-γ-cyclodextrin sodium salt (Sugammadex sodium)

[0025] Based on previous findings in the literature (L. Booij et al., Anaesthesia 2009 Mar;64 Suppl 1:38-44; Anton Bom et al., J Crit Care. 2009 Mar;24(1):29-35; and G Della Rocca et al., Minerva Anestesiol. 2009 May;75(5):349-51), we unexpectedly discovered that chemically pure single isomer carboxyethyl-thio-cyclodextrin, originally designed as an artificial receptor for administering neuromuscular blockade, exhibits significant potential in treating cholesterol and lipid storage disorders in animal models.

[0026] Example 1 shows the significant inhibitory activity of Sualfadex sodium, Subetadex sodium and Sugammadex sodium on cholesterol accumulation in the zebrafish (Danio rerio) LSD disease model compared to HPBCD as a positive control. Example 2 shows that the improvement of LSD disease is manifested by improved motor activity of the test animals due to treatment with Sualfadex sodium, Subetadex sodium and Sugammadex sodium. Example 3 shows the efficacy of the investigated S-(carboxyalkyl)-sulfo-cyclodextrin salts in effectively improving LSD disease despite their low cholesterol affinity compared to HPBCD, a known drug against LSD disease. DETAILED DESCRIPTION

[0027] Example 1

[0028] Several cyclodextrin derivatives were evaluated in a zebrafish model of Niemann-Pick disease type C1 (administered with U18666A). Cholesterol accumulation in the brain was quantified by filipin staining of Casper mutant zebrafish (albino strain) larvae. Results were compared with the reference compound hydroxypropyl-β-cyclodextrin (HPBCD).

[0029] In this study, the best performing cyclodextrins were as follows: heptas(2,6-di-O-methyl)-β-cyclodextrin, octa(2,3,6-tri-O-methyl)-γ-cyclodextrin, Sualfadex sodium, Subetadex sodium, Sugammadex sodium. Filipin staining was studied in five-day-old zebrafish larvae (Danio rerio, Casper strain) pretreated with U18666A.

[0030] The fertilized eggs were hatched and stored in an incubator at 28 °C.

[0031] Cyclodextrin (CD) was dosed from a stock solution (0.1%) in standard E3 medium. E3 medium was prepared as follows:

[0032] Element

[0033] -34.8g NaCl

[0034] -1.6 g KCl

[0035] -5.8 g CaCl 2 ·2H 2 O

[0036] -9.78 g MgCl 2 6H 2 O

[0037] To prepare 60X stock solution, dissolve the ingredients in water to a final volume of 2 L. Adjust pH to 7.2 with NaOH and autoclave. To prepare 1X medium, dilute 6.5 mL of 60X stock solution to 1 L and add 100 μL of 1% methylene blue.

[0038] The stock solution was diluted with E3. Compounds were administered in the swimming water. Each concentration was tested in 25 larvae. The dosage regimen is shown in Table 1.

[0039] Table 1: Dosage regimen for treatment

[0040] Group Day 3 (17.00 hours) Day 4 (9.00 hours) Day 5 U18666A-placebo E3 E3 analyze U18666A-Cyclodextrin U18666A 0.25 μg / mL Cyclodextrin analyze

[0041] The experimental compounds were tested at a concentration of 0.05%. As a reference, hydroxypropyl-β-cyclodextrin (Sigma) was tested at a concentration of 0.5%.

[0042] Filipin staining

[0043] Treated 5 dpf (5 days post fertilization) Casper larvae were fixed in 4% paraformaldehyde for 30 minutes. After washing with PBS (2 times), they were stained with 50 μg / mL Filipin (Sigma) in PBS for 30 minutes. The larvae were then washed twice (with PBS).

[0044] Whole larvae were embedded and images were captured on a Dino-Lite digital USB microscope (AM4115T4) for GFP / FITC recording. Images were evaluated by an independent assessor who was blinded to the treatments. The assessor scored fluorescence in the head region as absent, mild, or severe.

[0045] Statistical analysis

[0046] Data were analyzed by Chi-square test. All conditions were compared to the U18666A placebo treatment group.

[0047] Effect on Filipin Staining

[0048] Fluorescence was measured as an indicator of the amount of filipin staining. The higher the filipin staining, the more cholesterol accumulated in the brain. Individual larvae were scored (by a blinded assessor) as being darkly stained, lightly stained, or not stained at all. The number of larvae in each category is shown in Table 2.

[0049] Table 2: Filipin staining analysis of test groups.

[0050]

[0051] Animals treated with U18666A (U18666A-placebo) were predominantly darkly stained (17 of 25 animals), suggesting that this compound induces cholesterol accumulation in the brain. HPBCD significantly reduced this accumulation (only 9 of 24 animals showed dark staining, and 2 juveniles showed no staining at all). Heptadex (2,6-di-O-methyl)-β-cyclodextrin and octa(2,3,6-tri-O-methyl)-γ-cyclodextrin showed only a small, non-significant reduction in U18666A-induced cholesterol accumulation. Sualfadex sodium, Subetadex sodium, and Sugammadex sodium (0.05%) were very active; these single isomer compounds resulted in a very strong reduction in cholesterol accumulation. This compound was more active than the reference HPBCD (even when used at a 10-fold concentration: 0.5%).

[0052] Example 2

[0053] Mobility test

[0054] On the day of testing, zebrafish larvae (strain AB, Casper) at 5 days post fertilization (dpf) were treated with U18666A dissolved in standard E3 medium. Different concentrations (0.5-5%) of different CDs were tested for reversal of the effects of U18666A (1 mg / ml) on the overall activity of zebrafish larvae at 5 days post fertilization. Larvae were pretreated with U18666A for 16 hours and then treated with CDs for 24 hours. Statistical analysis of each experiment was performed using a two-way ANOVA, followed by a Tukey post-hoc test (**p<0.01). CDs were dissolved in standard E3 medium.

[0055] The behavior of the test species was observed in 48-well plates in the Danio Vision (Noldus IT, Wageningen). The Danio Vision observation chamber is a complete system designed for high-throughput testing of zebrafish larvae in 48-well plates. It includes the observation chamber and the well-known EthoVision XT video tracking software to quantify the distance moved by the animals.

[0056] The results are Figure 1 It is represented graphically in (distance moved - arbitrary units).

[0057] Example 3

[0058] Interaction between cyclodextrin and unesterified cholesterol

[0059] Phase solubility studies were performed in 5 ml solutions at room temperature, where discrete concentrations of cyclodextrin solutions were weighed and excess cholesterol was added. After 24 hours of equilibrium at 25 ± 3 ° C (using a magnetic stirrer at 500 RPM), the equilibrium cholesterol concentration dissolved was determined by HPLC after filtering through a syringe filter with a nominal pore size of 0.45 micron polysulfone membrane. The experimentally determined cholesterol concentrations are listed in Table 3 in the presence of different concentrations of HPBCD, Sualfadex sodium, Subetadex sodium, and Sugammadex sodium.

[0060] Table 3: Equilibrium concentration of cholesterol dissolved in cyclodextrin

[0061]

Claims

1. Use of S-(carboxyalkyl)-thio-cyclodextrin or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating or preventing Niemann-Pick disease type C1, wherein the S-(carboxyalkyl)-thio-cyclodextrin is selected from 6A, 6B, 6C, 6D, 6E, 7F-hexa-S-(2-carboxyethyl)-6A, 6B, 6C, 6D, 6E, 6F-hexathio-α-cyclodextrin, 6A, 6B,6C,6D,6E,7F,6G-hepta-S-(2-carboxyethyl)-6A,6B,6C,6D,6E,6F,6G-heptathio-β-cyclodextrin and 6A,6B,6C,6D,6E,7F,6G,6H-octa-S-(2-carboxyethyl)-6A,6B,6C,6D,6E,6F,6G,6H-octathio-γ-cyclodextrin.

2. The use according to claim 1, wherein the S-(carboxyalkyl)-thio-cyclodextrin is 6A, 6B, 6C, 6D, 6E, 7F-hexa-S-(2-carboxyethyl)-6A, 6B, 6C, 6D, 6E, 6F-hexathio-α-cyclodextrin.

3. The use according to claim 1, wherein the S-(carboxyalkyl)-thio-cyclodextrin is 6A, 6B, 6C, 6D, 6E, 7F, 6G-hepta-S-(2-carboxyethyl)-6A, 6B, 6C, 6D, 6E, 6F, 6G-heptathio-β-cyclodextrin.

4. The use according to claim 1, wherein the S-(carboxyalkyl)-thio-cyclodextrin is 6A, 6B, 6C, 6D, 6E, 7F, 6G, 6H-octa-S-(2-carboxyethyl)-6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H-octathio-γ-cyclodextrin.

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